**Part 2**

**Cardiovascular Complications** 

154 Type 1 Diabetes Complications

Romano, G.; Moretti, G.; Di Benedetto, A.; Ruso, G et al. (1998) Skin lesion in diabetes

Saukkonen, T.; Savilahti, E.; Reijonen, H.; Ilonen, J.; Tuomilehto-Wolf, E.; Akerblom, HK.

Maki, M.; Holm, K.; Hallstrom, O.; (1995) Seroconversion of reticulin autoahmtibodies

Roldan, MB.; Barrio, R.; Roy, G.; Parra, C.; Alonso, M et al. (1998) Diagnostic value of

101-106.

13:464-70.

*Endocrinol Metab* 1998; 11: 751-756.

mellitus: prevalence and clinical correlations. *Diabetes Res Clin Pract* 1998; 39(2):

(1996) Celiac disease: Frequent occurrence after clinical onset of insulin-dependent diabetes mellitus: Childhood Diabetes in Finland Study Group. *Diabet Med* 1996;

predios coeliac disease in insulin dependent diabetes mellitus. *Gut* 1995; 36: 239-42.

serological markers for celiac disease in diabetic children and adolescent. *J Pediatr* 

**7** 

*Sweden* 

**Etiopathology of Type 1 Diabetes:** 

*Division of Paediatric Cardiology, Pediatric Heart Center, Lund University and Skåne University Hospital Lund* 

Petru Liuba and Emma Englund

**Focus on the Vascular Endothelium** 

Type 1, or insulin-dependent diabetes results from the destruction of insulin-producing cells in the pancreas. It typically occurs in previously healthy children, being one of the most common childhood diseases in modern time. Intriguingly, the diabetes morbidity continues to rise in most parts of the world, but the causes of this development remain elusive (the

Many of the patients with type 1 diabetes may develop particularly in adult life severe complications encompassing both the microvascular (retinopathy, nephropathy, and neuropathy) and macrovascular (myocardial infarction, stroke and peripheral artery disease) system. The main underlying mechanism in the large vessels is represented by accelerated atherosclerosis with subsequent narrowing of the vessels and potential risk for plaque rupture. In smaller, as in the large vessels, severely impaired function of the inner layer (so-called endothelium) has been detected, and correlated with the risk of developing vascular complications, suggesting it to play a major role in precipitating the vascular

It is now well established that this widespread vasculopathy develop many years before the onset of vascular complications, perhaps even before the outbreak of diabetic hyperglycemia. What exactly drives this process remains speculative, but increasing evidence suggest that factors thought to trigger type 1 diabetes may as well be harmful for the vessels. Furthermore, as delineated below, the microvascular network surrounding the islets in the pancreas appears to have important contribution to the injury process that ultimately leads to type 1 diabetes. Further multidisciplinary efforts are warranted in order

There is a widespread global variation in the incidence and prevalence of type 1 diabetes. In a report by Karvonen et al. from 2000, age-adjusted incidences ranged from a low of <1/100,000 per year in China and South America to a high of >20/100,000 per year in Sardinia, Finland, Sweden, Norway, Portugal, the United Kingdom, Canada, and New Zeeland. The incidence increased with age in most of the populations with the highest incidence observed in children 10-14 years of age. An updated report from 2006 (the

to better understand the mechanism of this disease and its complications.

**2. Epidemiology and pathogenesis of type 1 diabetes** 

**1. Introduction** 

disease.

Diabetes Mondial (DIAMOND) Project Group, 2006).

### **Etiopathology of Type 1 Diabetes: Focus on the Vascular Endothelium**

Petru Liuba and Emma Englund

*Division of Paediatric Cardiology, Pediatric Heart Center, Lund University and Skåne University Hospital Lund Sweden* 

#### **1. Introduction**

Type 1, or insulin-dependent diabetes results from the destruction of insulin-producing cells in the pancreas. It typically occurs in previously healthy children, being one of the most common childhood diseases in modern time. Intriguingly, the diabetes morbidity continues to rise in most parts of the world, but the causes of this development remain elusive (the Diabetes Mondial (DIAMOND) Project Group, 2006).

Many of the patients with type 1 diabetes may develop particularly in adult life severe complications encompassing both the microvascular (retinopathy, nephropathy, and neuropathy) and macrovascular (myocardial infarction, stroke and peripheral artery disease) system. The main underlying mechanism in the large vessels is represented by accelerated atherosclerosis with subsequent narrowing of the vessels and potential risk for plaque rupture. In smaller, as in the large vessels, severely impaired function of the inner layer (so-called endothelium) has been detected, and correlated with the risk of developing vascular complications, suggesting it to play a major role in precipitating the vascular disease.

It is now well established that this widespread vasculopathy develop many years before the onset of vascular complications, perhaps even before the outbreak of diabetic hyperglycemia. What exactly drives this process remains speculative, but increasing evidence suggest that factors thought to trigger type 1 diabetes may as well be harmful for the vessels. Furthermore, as delineated below, the microvascular network surrounding the islets in the pancreas appears to have important contribution to the injury process that ultimately leads to type 1 diabetes. Further multidisciplinary efforts are warranted in order to better understand the mechanism of this disease and its complications.

#### **2. Epidemiology and pathogenesis of type 1 diabetes**

There is a widespread global variation in the incidence and prevalence of type 1 diabetes. In a report by Karvonen et al. from 2000, age-adjusted incidences ranged from a low of <1/100,000 per year in China and South America to a high of >20/100,000 per year in Sardinia, Finland, Sweden, Norway, Portugal, the United Kingdom, Canada, and New Zeeland. The incidence increased with age in most of the populations with the highest incidence observed in children 10-14 years of age. An updated report from 2006 (the

Etiopathology of Type 1 Diabetes: Focus on the Vascular Endothelium 159

gland (autoimmune thyroiditis). These disorders also share some of the HLA DQ diabetes

It is well known that long-term exposure to hyperglycemia, hyperlipidemia and inflammation, all of which being important features of type 1 diabetes, is harmful to the endothelial cells, causing further endothelial dysfunction, and in the long run, accelerated atherosclerosis. Lymphocyte accumulation within the arterial wall is an important mechanistic component in the atherosclerotic process and contributes to endothelial cell injury and dysfunction. The endothelial injury, in turn, promotes additional immune events, including release of different chemokines and cytokines resulting in further transmigration of immune cells, and synthesis of C-reactive protein via liver activation by interleukin-6. Recent study, assessing the relationship of genetic susceptibility with endothelial dysfunction in young patients with type 1 diabetes, found significant correlation between HLA-DQ 2/8, which confers the highest risk for developing type 1 diabetes, and cutaneous microvascular dysfunction (Odermarsky et al., 2007). This could imply, although its does not prove, a role for HLA in the pathogenesis of type 1 diabetes. Further studies at our center are under way to investigate whether such changes could be present already before

**4. Exogenous risk factors and type 1 diabetes: Is vascular endothelium a** 

Putative environmental triggers include viruses, environmental toxins and foods, but it has been difficult to demonstrate a reproducible correlation between them and the development of type 1 diabetes (The environmental determinants of diabetes in the young (TEDDY) Study Group, 2008). Although appealing, there is no evidence to date of a direct interplay between infections and genetics in the causation of type 1 diabetes. the risk of developing T1D seems to increase with the number of infections experienced by an individual during the year preceding the onset of T1D. Although we currently lack the knowledge of the precise underlying mechanisms, there are other reports on similar associations between infectious recurrence and chronic diseases such as multiple sclerosis or rheumatoid arthritis. In some animal studies, the development of atherosclerotic plaque was accelerated by repeated infection. One possible mechanistic link between these chronic inflammatory diseases (e.g. atherosclerosis and T1D) and infection might be endothelial injury. Infections cause vascular endothelial dysfunction, which may persist for up to 1 year after the infectious illness. Mild respiratory infections ("common cold") seem to aggravate arterial endothelial dysfunction in young patients with T1D. Those with increased recurrence of infections of this type are more susceptible to decreased carotid artery elasticity. The latter was earlier shown to be in part dependent on the functional integrity of endothelial cells. In atherosclerosis-susceptible mice, the degree of endothelial vasomotor dysfunction in skin microcirculation correlates with the number of pathogen inoculations (Odermarsky M,

It has been shown that even mild viral infection causes vascular endothelial dysfunction, which may persist for up to 1 year after the infectious illness (Charakida et al., 2005). Infections promote the inflammatory environment needed for endothelial cell activation and upregulation of HLA. These changes could, if genetic susceptibility is present, contribute to homing, transmigration, and accumulation of inflammatory cells in certain tissues. In the

risk alleles. An activated endothelium could be the link.

the clinical onset of type 1 diabetes.

Liuba P unpublished data).

**link?** 

DIAMOND Project Group, 2006) showed trends of increased incidence annually across the world in the populations studied (4.0% in Asia, 3.2% in Europe, and 5.3% in North America) with the exception of Central America and the West Indies were the trend was a decrease of 3.6%. The rate of increase in incidence cannot be explained by genetic shift in such a short period of time. An increasing burden of environmental factors, probably along with increased immune-mediated sensitivity of pancreatic cells to gene-environment interaction, could account at least in part for the rising prevalence of type 1 diabetes.

Traditionally, interplay between genetic susceptibility and environmental factors is thought to provide the fundamental element for the disease. The major genetic susceptibility for developing type 1 diabetes is located on the human leukocyte antigen (HLA)-DQ region on chromosome 6. Two HLA-DQ haplotypes, DQA1 0301-DQB1 0302 (DQ8) and DQA1 0501- DQB1 0201 (DQ2) are associated with high risk for developing diabetes. Almost 90% of patients with type 1 diabetes have at least one of these two haplotypes, compared to 20% of the general population (Redondo et al., 2001). These susceptibility genes are important regulators of the immune response; the molecules they encode reside on the cell surface and have the ability to process and present antigens to autoreactive T-cells.

In genetically susceptible individuals there is a pathological presentation of autoantigens on the cell surface of the pancreatic islet cells. This, in combination with a failing negative selection of the T-cells in the thymus and decreased regulatory capacity of regulatory T-cells in peripheral blood (Lindley et al., 2005), leads to an inflammatory response within the pancreatic islets as well as to the production of antibodies against -cell antigens. Islet cell antibodies were the first ones described, but we now also recognize autoantibodies to insulin, glutamic acid decarboxylase and protein tyrosine phosphatase. Up to 90% of the patients with new onset type 1 diabetes have autoantibodies directed against one or more of these autoantigens It is yet to prove if the autoantibodies have an active role in the pathogenesis of type 1 diabetes but the presence and persistency of autoantibodies appear to increase the likelihood of developing type 1 diabetes. The inflammatory response leads to destruction of the pancreatic -cells and progressive loss of insulin secretion until reaching critically low levels or even complete insulin deficiency.

#### **3. Vascular endothelium in type 1 diabetes**

In children with diabetes risk HLA, signs of systemic endothelial cell activation can be seen already before the clinical onset of diabetes (Toivonen et al, 2004), suggesting it to be an early event in the disease process. Postmortem studies near the onset of type 1 diabetes have shown that class II HLA molecules may be abundantly expressed on vascular endothelial cells lining the capillaries and capillary sinusoids in the islets (Itoh et al., 1993). The upregulation of HLA is paralleled by strong expression of adhesion molecules (i.e. ICAM-1) in the same endothelial areas (Hanninen et al., 1992). These events, seemingly induced by circulating proinflammatory mediators, facilitate homing and migration of inflammatory cells such as T cells across the dysfunctional endothelium . Interaction between antigenspecific T-cells and antigen/HLA complexes on the endothelial cells surface induced a rapid transmigration of the T-cells across the endothelial cell layer (Greening et al., 2003). Similar changes may be found on the surface of endothelial cells of other vascular beds (Greening et al., 2003).This fits well with the clinical observation that type 1 diabetes is often comorbid with chronic autoimmune diseases in other organs such as gut (celiac disease) and thyroid

DIAMOND Project Group, 2006) showed trends of increased incidence annually across the world in the populations studied (4.0% in Asia, 3.2% in Europe, and 5.3% in North America) with the exception of Central America and the West Indies were the trend was a decrease of 3.6%. The rate of increase in incidence cannot be explained by genetic shift in such a short period of time. An increasing burden of environmental factors, probably along with increased immune-mediated sensitivity of pancreatic cells to gene-environment interaction,

Traditionally, interplay between genetic susceptibility and environmental factors is thought to provide the fundamental element for the disease. The major genetic susceptibility for developing type 1 diabetes is located on the human leukocyte antigen (HLA)-DQ region on

In genetically susceptible individuals there is a pathological presentation of autoantigens on the cell surface of the pancreatic islet cells. This, in combination with a failing negative selection of the T-cells in the thymus and decreased regulatory capacity of regulatory T-cells in peripheral blood (Lindley et al., 2005), leads to an inflammatory response within the pancreatic islets as well as to the production of antibodies against -cell antigens. Islet cell antibodies were the first ones described, but we now also recognize autoantibodies to insulin, glutamic acid decarboxylase and protein tyrosine phosphatase. Up to 90% of the patients with new onset type 1 diabetes have autoantibodies directed against one or more of these autoantigens It is yet to prove if the autoantibodies have an active role in the pathogenesis of type 1 diabetes but the presence and persistency of autoantibodies appear to increase the likelihood of developing type 1 diabetes. The inflammatory response leads to destruction of the pancreatic -cells and progressive loss of insulin secretion until reaching

In children with diabetes risk HLA, signs of systemic endothelial cell activation can be seen already before the clinical onset of diabetes (Toivonen et al, 2004), suggesting it to be an early event in the disease process. Postmortem studies near the onset of type 1 diabetes have shown that class II HLA molecules may be abundantly expressed on vascular endothelial cells lining the capillaries and capillary sinusoids in the islets (Itoh et al., 1993). The upregulation of HLA is paralleled by strong expression of adhesion molecules (i.e. ICAM-1) in the same endothelial areas (Hanninen et al., 1992). These events, seemingly induced by circulating proinflammatory mediators, facilitate homing and migration of inflammatory cells such as T cells across the dysfunctional endothelium . Interaction between antigenspecific T-cells and antigen/HLA complexes on the endothelial cells surface induced a rapid transmigration of the T-cells across the endothelial cell layer (Greening et al., 2003). Similar changes may be found on the surface of endothelial cells of other vascular beds (Greening et al., 2003).This fits well with the clinical observation that type 1 diabetes is often comorbid with chronic autoimmune diseases in other organs such as gut (celiac disease) and thyroid

0201 (DQ2) are associated with high risk for developing diabetes. Almost 90% of patients with type 1 diabetes have at least one of these two haplotypes, compared to 20% of the general population (Redondo et al., 2001). These susceptibility genes are important regulators of the immune response; the molecules they encode reside on the cell surface and

0301-DQB1

0302 (DQ8) and DQA1

0501-

could account at least in part for the rising prevalence of type 1 diabetes.

have the ability to process and present antigens to autoreactive T-cells.

chromosome 6. Two HLA-DQ haplotypes, DQA1

critically low levels or even complete insulin deficiency.

**3. Vascular endothelium in type 1 diabetes** 

DQB1

gland (autoimmune thyroiditis). These disorders also share some of the HLA DQ diabetes risk alleles. An activated endothelium could be the link.

It is well known that long-term exposure to hyperglycemia, hyperlipidemia and inflammation, all of which being important features of type 1 diabetes, is harmful to the endothelial cells, causing further endothelial dysfunction, and in the long run, accelerated atherosclerosis. Lymphocyte accumulation within the arterial wall is an important mechanistic component in the atherosclerotic process and contributes to endothelial cell injury and dysfunction. The endothelial injury, in turn, promotes additional immune events, including release of different chemokines and cytokines resulting in further transmigration of immune cells, and synthesis of C-reactive protein via liver activation by interleukin-6.

Recent study, assessing the relationship of genetic susceptibility with endothelial dysfunction in young patients with type 1 diabetes, found significant correlation between HLA-DQ 2/8, which confers the highest risk for developing type 1 diabetes, and cutaneous microvascular dysfunction (Odermarsky et al., 2007). This could imply, although its does not prove, a role for HLA in the pathogenesis of type 1 diabetes. Further studies at our center are under way to investigate whether such changes could be present already before the clinical onset of type 1 diabetes.

#### **4. Exogenous risk factors and type 1 diabetes: Is vascular endothelium a link?**

Putative environmental triggers include viruses, environmental toxins and foods, but it has been difficult to demonstrate a reproducible correlation between them and the development of type 1 diabetes (The environmental determinants of diabetes in the young (TEDDY) Study Group, 2008). Although appealing, there is no evidence to date of a direct interplay between infections and genetics in the causation of type 1 diabetes. the risk of developing T1D seems to increase with the number of infections experienced by an individual during the year preceding the onset of T1D. Although we currently lack the knowledge of the precise underlying mechanisms, there are other reports on similar associations between infectious recurrence and chronic diseases such as multiple sclerosis or rheumatoid arthritis. In some animal studies, the development of atherosclerotic plaque was accelerated by repeated infection. One possible mechanistic link between these chronic inflammatory diseases (e.g. atherosclerosis and T1D) and infection might be endothelial injury. Infections cause vascular endothelial dysfunction, which may persist for up to 1 year after the infectious illness. Mild respiratory infections ("common cold") seem to aggravate arterial endothelial dysfunction in young patients with T1D. Those with increased recurrence of infections of this type are more susceptible to decreased carotid artery elasticity. The latter was earlier shown to be in part dependent on the functional integrity of endothelial cells. In atherosclerosis-susceptible mice, the degree of endothelial vasomotor dysfunction in skin microcirculation correlates with the number of pathogen inoculations (Odermarsky M, Liuba P unpublished data).

It has been shown that even mild viral infection causes vascular endothelial dysfunction, which may persist for up to 1 year after the infectious illness (Charakida et al., 2005). Infections promote the inflammatory environment needed for endothelial cell activation and upregulation of HLA. These changes could, if genetic susceptibility is present, contribute to homing, transmigration, and accumulation of inflammatory cells in certain tissues. In the

Etiopathology of Type 1 Diabetes: Focus on the Vascular Endothelium 161

The ability to detect endothelial dysfunction, before it progresses to overt vasculopathy, could facilitate the early diagnosis and management of high-risk individuals in childhood. There are several techniques that can be used, though they are not in clinical use to any higher extent. Here we will present two methods; flow-mediated dilatation and laser

doppler flowmetry with iontophoresis, which are the ones used in our research.

**6.1 Assessment of arterial dysfunction via flow-mediated dilatation of the brachial** 

Blood vessels respond to an increase in blood flow, or more precisely shear stress, by dilating. This phenomenon is called flow-mediated dilatation. The principal mediator for this is endothelium-derived nitric oxide. Assessment of flow-mediated dilatation of the brachial artery safely and non-invasively provides a measure of the systemic endothelial function. The brachial artery response to increased shear stress has been shown to correlate significantly with invasive testing of brachial (Irace et al., 2001) and coronary endothelial function (Andersson et al., 1995), as well as with the extent and severity of coronary atherosclerosis (Neunteufl et al., 1997), and carotid artery intima-media thickness (Gaeta et

Several factors affect the response to the increase in shear stress, including temperature, food, drugs and sympathetic stimuli, female hormonal status, among others, and when conducting a study using this technique you must take these confounding factors into consideration. Ultra sound systems used must be equipped with software for twodimensional imaging, colour and spectral Doppler, an internal electrocardiogram monitor and a high-frequency vascular transducer. A straight segment of the brachial artery above the antecubital fossa is imaged in the longitudinal plane with the ultrasound probe securely fixed using a stereotactic clamp. A blood pressure cuff is then placed on the forearm and inflated to supra-systolic pressure. After cuff release, reactive hyperaemia results and is quantified using Doppler. The arterial diameter is recorded at end diastole using electrocardiographic gating to determine the response of the brachial artery to increase in flow. The flow-mediated dilatation is expressed as a percentage change of the arterial diameter from the baseline vessel size (Corretti et al., 2002; Thijssen et al., 2011). To control the smooth muscle cells ability to dilate the vessel, independently of the endothelium, and to determine maximum obtainable vasodilatation a dose of nitro-glycerine is administrated via

**6.2 Assessment of microvascular dysfunction via laser doppler flowmetry with** 

Iontophoresis is a non-invasive method of introducing charged substances across the surface of the skin by means of a small electric charge. The basic principle is that molecules of drugs in a solution that are positively or negatively charged will migrate across the skin under influence of an applied current according to the rule that like charges repel each other. The amount of drug delivered is dependent on the magnitude and duration of the current applied. The response in the skin vasculature is measured via a laser doppler device (Morris & Shore, 1996). The coherent light directed at the skin changes when it comes in contact with moving tissues (red blood cells) and the emerged light, i.e. skin perfusion, is measured by a

**6. Methods for evaluating endothelial function** 

**artery** 

al., 2000).

spray or sublingual tablet.

**iontophoresis in the skin** 

photodiode.

pancreas these microcirculatory changes could perhaps have a role in the pathogenesis of type 1 diabetes, but this is still hypothetical.

#### **5. Endothelial function and dysfunction**

The endothelial cells line the inner surface of all blood vessels, providing a metabolically active interface between blood and tissue. These cells modulate blood flow, nutrient delivery, coagulation and thrombosis, and leukocyte diapedesis. The endothelium synthesizes important bioactive substances. Of these, nitric oxide is the most potent vasodilator and protector of vascular function, inhibiting platelet activation and aggregation, preventing leukocyte adhesion and migration through the vessel wall, diminishing smooth muscle cell proliferation and migration, and counteracting adhesion molecule expression (Beckman et al., 2002). Nitric oxide is synthezised by the endothelial isoform of nitric oxide synthase. The process involves enzymatic conversion of L-arginine into nitric oxide and L-citrulline. The release of nitric oxide can be up- or downregulated by different factors. The hormone estrogen, physical exercise, and certain dietary factors are examples of upregulators, whereas smoking and oxidized low-density lipoproteins, via oxidative stress, are examples of downregulators (Michel & Vanhoutte, 2010).

Endothelial dysfunction is considered to be the first step in a long-lasting and complex development that leads to atherosclerosis. The dysfunction is a result of an imbalance in the redox-equilibrium towards oxidative stress leading to impaired nitric oxide bioavailability, either caused by its reduced synthesis or by increased breakdown via reactive oxygen species. (Versari et al., 2009). The dysfunctioning endothelium may produce other substances and mediators such as endothelin 1, thromboxane A2, prostaglandin H and reactive oxygen species, with vasoconstricting, pro-inflammatory, and proatherosclerotic effects (Virdis et al., 2010).

Given the excess of inflammatory and oxidative stress in type 1 diabetes, the endothelium in individuals with type 1 diabetes is continually exposed to factors promoting the development of endothelial dysfunction. The hyperglycemia, excess free fatty acid release, and insulin resistance leads to adverse events within the endothelial cell (Beckman et al., 2002). Recent findings support the concept that genetically susceptible individuals, i.e. diabetes high-risk HLA, are more prone to develop endothelial dysfunction (Odermarsky et al., 2007) and ongoing studies are investigating whether this dysfunction in fact may precede the clinical onset of type 1 diabetes.

The generalized microvascular dysfunction in type 1 diabetes is an important mechanism in the development of these microvascular complications. Nephropathy, retinopathy and neuropathy are all related to damage to the small vessels of the kidney, retina and nerves. Significant associations have been reported between the different microvascular complications of type 1 diabetes; patients with one complication often develops a second one, suggesting common risk factors and pathogenetic mechanisms (Girach & Vignati, 2006).

Overt microvascular disease is however rare during childhood and adolescence. Early signs, such as increases in albumin excretion rates and glomerular filtration rates, renal hypertrophy, changes in retinal microvasculature and impaired autonomic nervous system function may be detectable in kids with type 1 diabetes and often progress during puberty. Microalbuminuria is the earliest stage of clinical nephropathy and is predictive of progression to overt diabetes nephropathy and, notably, of cardiovascular disease (Rossing et al., 1996).

#### **6. Methods for evaluating endothelial function**

160 Type 1 Diabetes Complications

pancreas these microcirculatory changes could perhaps have a role in the pathogenesis of

The endothelial cells line the inner surface of all blood vessels, providing a metabolically active interface between blood and tissue. These cells modulate blood flow, nutrient delivery, coagulation and thrombosis, and leukocyte diapedesis. The endothelium synthesizes important bioactive substances. Of these, nitric oxide is the most potent vasodilator and protector of vascular function, inhibiting platelet activation and aggregation, preventing leukocyte adhesion and migration through the vessel wall, diminishing smooth muscle cell proliferation and migration, and counteracting adhesion molecule expression (Beckman et al., 2002). Nitric oxide is synthezised by the endothelial isoform of nitric oxide synthase. The process involves enzymatic conversion of L-arginine into nitric oxide and L-citrulline. The release of nitric oxide can be up- or downregulated by different factors. The hormone estrogen, physical exercise, and certain dietary factors are examples of upregulators, whereas smoking and oxidized low-density lipoproteins, via

Endothelial dysfunction is considered to be the first step in a long-lasting and complex development that leads to atherosclerosis. The dysfunction is a result of an imbalance in the redox-equilibrium towards oxidative stress leading to impaired nitric oxide bioavailability, either caused by its reduced synthesis or by increased breakdown via reactive oxygen species. (Versari et al., 2009). The dysfunctioning endothelium may produce other substances and mediators such as endothelin 1, thromboxane A2, prostaglandin H and reactive oxygen species, with vasoconstricting, pro-inflammatory, and proatherosclerotic

Given the excess of inflammatory and oxidative stress in type 1 diabetes, the endothelium in individuals with type 1 diabetes is continually exposed to factors promoting the development of endothelial dysfunction. The hyperglycemia, excess free fatty acid release, and insulin resistance leads to adverse events within the endothelial cell (Beckman et al., 2002). Recent findings support the concept that genetically susceptible individuals, i.e. diabetes high-risk HLA, are more prone to develop endothelial dysfunction (Odermarsky et al., 2007) and ongoing studies are investigating whether this dysfunction in fact may

The generalized microvascular dysfunction in type 1 diabetes is an important mechanism in the development of these microvascular complications. Nephropathy, retinopathy and neuropathy are all related to damage to the small vessels of the kidney, retina and nerves. Significant associations have been reported between the different microvascular complications of type 1 diabetes; patients with one complication often develops a second one, suggesting

Overt microvascular disease is however rare during childhood and adolescence. Early signs, such as increases in albumin excretion rates and glomerular filtration rates, renal hypertrophy, changes in retinal microvasculature and impaired autonomic nervous system function may be detectable in kids with type 1 diabetes and often progress during puberty. Microalbuminuria is the earliest stage of clinical nephropathy and is predictive of progression to overt diabetes nephropathy and, notably, of cardiovascular disease (Rossing

common risk factors and pathogenetic mechanisms (Girach & Vignati, 2006).

oxidative stress, are examples of downregulators (Michel & Vanhoutte, 2010).

type 1 diabetes, but this is still hypothetical.

effects (Virdis et al., 2010).

et al., 1996).

precede the clinical onset of type 1 diabetes.

**5. Endothelial function and dysfunction** 

The ability to detect endothelial dysfunction, before it progresses to overt vasculopathy, could facilitate the early diagnosis and management of high-risk individuals in childhood. There are several techniques that can be used, though they are not in clinical use to any higher extent. Here we will present two methods; flow-mediated dilatation and laser doppler flowmetry with iontophoresis, which are the ones used in our research.

#### **6.1 Assessment of arterial dysfunction via flow-mediated dilatation of the brachial artery**

Blood vessels respond to an increase in blood flow, or more precisely shear stress, by dilating. This phenomenon is called flow-mediated dilatation. The principal mediator for this is endothelium-derived nitric oxide. Assessment of flow-mediated dilatation of the brachial artery safely and non-invasively provides a measure of the systemic endothelial function. The brachial artery response to increased shear stress has been shown to correlate significantly with invasive testing of brachial (Irace et al., 2001) and coronary endothelial function (Andersson et al., 1995), as well as with the extent and severity of coronary atherosclerosis (Neunteufl et al., 1997), and carotid artery intima-media thickness (Gaeta et al., 2000).

Several factors affect the response to the increase in shear stress, including temperature, food, drugs and sympathetic stimuli, female hormonal status, among others, and when conducting a study using this technique you must take these confounding factors into consideration. Ultra sound systems used must be equipped with software for twodimensional imaging, colour and spectral Doppler, an internal electrocardiogram monitor and a high-frequency vascular transducer. A straight segment of the brachial artery above the antecubital fossa is imaged in the longitudinal plane with the ultrasound probe securely fixed using a stereotactic clamp. A blood pressure cuff is then placed on the forearm and inflated to supra-systolic pressure. After cuff release, reactive hyperaemia results and is quantified using Doppler. The arterial diameter is recorded at end diastole using electrocardiographic gating to determine the response of the brachial artery to increase in flow. The flow-mediated dilatation is expressed as a percentage change of the arterial diameter from the baseline vessel size (Corretti et al., 2002; Thijssen et al., 2011). To control the smooth muscle cells ability to dilate the vessel, independently of the endothelium, and to determine maximum obtainable vasodilatation a dose of nitro-glycerine is administrated via spray or sublingual tablet.

#### **6.2 Assessment of microvascular dysfunction via laser doppler flowmetry with iontophoresis in the skin**

Iontophoresis is a non-invasive method of introducing charged substances across the surface of the skin by means of a small electric charge. The basic principle is that molecules of drugs in a solution that are positively or negatively charged will migrate across the skin under influence of an applied current according to the rule that like charges repel each other. The amount of drug delivered is dependent on the magnitude and duration of the current applied. The response in the skin vasculature is measured via a laser doppler device (Morris & Shore, 1996). The coherent light directed at the skin changes when it comes in contact with moving tissues (red blood cells) and the emerged light, i.e. skin perfusion, is measured by a photodiode.

Etiopathology of Type 1 Diabetes: Focus on the Vascular Endothelium 163

gene-environment interaction on vascular endothelium and the timing and role of endothelial dysfunction in the development of type 1 diabetes and associated cardiovascular

Aburawi, E., Liuba, P., Pesonen, E., Ylä-Herttuala, S. & Sjoblad, S. (2004). Acute respiratory

Diabetes care, Vol. 27, No. 11, (November 2004), pp. 2733-5, ISSN 1935-5548 Akerblom, HK., Vaarala, O., Hyöty, H., Ilonen, J. & Knip, M. (2002). Environmental factors

Anderson, TJ., Uehata, A., Gerhard, MD., Meredith, IT., Knab, S., Delagrange, D.,

Beckman, JA., Creager, MA. & Libby, P. (2002). Diabetes and atherosclerosis: epidemiology,

Blom, L., Nyström, L. & Dahlquist, G. (1991). The Swedish childhood diabetes study.

Cines, DB., Pollak, ES., Buck, CA., Loscalzo, J., Zimmerman, GA., McEver, RP., Pober, JS.,

Corretti, MC., Anderson, TJ., Benjamin, EJ., Celermajer, D., Charbonneau, F., Creager, MA.,

EURODIAB ACE Study group. (2000). Variations and trends in incidence of childhood

Gaeta, G., Di Michele, M., Cuomo, S., Guarini, P., Foglia, MC., Gene Bond, M. & Trevisan,

Diabetologia, Vol. 34, No. 3, (March 1991), pp. 176–81, ISSN 0012-186X Charakida, M., Donald, AE., Terese, M., Leary, S., Halcox, JP., Ness, A., Davey Smith, G.,

viral infections aggravate endothelial dysfunction in children with type 1 diabetes.

in the etiology of type 1 diabetes. Am J Med Genet, Vol. 115, No. 1, (May 2002), pp.

Lieberman, EH., Ganz, P., Creager, M., Yeung, A. & Selwyn, AP. (1995). Close relation of endothelial function in the human coronary and peripheral circulations. J Am Coll Cardiol, Vol. 26, No. 5, (November 1995), pp. 1235-41,

patophysiology, and management. JAMA, Vol. 287, No. 19, (May 2002), pp. 2570-

Vaccinations and infections as risk determinants for diabetes in childhood.

Golding, J., Friberg, P., Klein, NJ. & Deanfield JE. for the Avon Longitudinal Study of Parents and Children study team. (2005). Endothelial dysfynction in childhood infection. Circulation, Vol. 111, No. 13, (March 2005), pp. 1660-65, ISSN

Wick, TM., Konkle, BA., Schwartz, BS., Barnathan, ES., McCrae, KR., Hug, BA., Schmidt, A-M. & Stern, DM. (1998). Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood, Vol. 91, No. 10, (May 1998), pp. 3527-

Deanfield, J., Drexler, H., Gerhard-Herman, M., Herrington, D., Vallance, P., Vita, J. & Vogel, R. for the International Brachial Artery Reactivity Task Force. (2002). Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery-a report from the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol, Vol. 39, No. 2, (January 2002), pp. 257-65,

diabetes in Europe. Lancet, Vol. 355, No. 9207, (March 2000), pp. 873-876, ISSN

M. (2000). Arterial abnormalities in the offspring of patients with premature

disease.

**9. References** 

18-29, ISSN 1552-4833

ISSN 0735-1097

81, ISSN 1528-0020

1524-4539

3561, ISSN 1528-0020

ISSN 0735-1097

0140-6736

Acetylcholine is the standard test drug for the assessment of endothelial function. The response to acetylcholine, using iontophoresis, correlates with diabetes duration and level of glycosylated haemoglobin (Khan et al., 2000). The mechanisms of acetylcholine-induced vasodilatation via ionphoresis remain debatable. In humans, nitric oxide appears to be the main mediator, but other endothelium-dependent vasodilators may contribute as well (Turner et al., 2008).

Sodium nitroprusside is a nitric oxide donor and acts directly, i.e. endotheliumindependent, as a control on the smooth muscle cells causing vasodilatation. Cathodal current is used for delivering sodium nitroprusside.

The current for both substances is set to 100 A for 20 seconds. For each of the substances to be tested five consecutive and equal doses are applied to generate dose-response curves. Baseline perfusion and changes in response to the substance are expressed as area under the curve.

#### **7. The "common soil" hypothesis-is this applicable in type 1 diabetes?**

In 1995, Michael Stern put forward the "common soil" hypothesis, which suggests a shared genetic and environmental origin for type 2 diabetes and atherosclerosis (Stern, 1995). According to this hypothesis, infections leading to chronic inflammation could pertain to the group of environmental etiological factors. Indeed, the risk of developing type 1 diabetes – a condition associated with significant morbidity in cardiovascular diseases in adult life – could rise during viral infections (Blom et al., 1991). Moreover, the risk for childhood diabetes seems to increase in accordance with a higher number of infections during the year preceding diagnosis. Studies in rodent models of atherosclerosis suggest similar dosedependent association between infection and vascular changes (Liuba et al., 2000; Tormakangas et al., 2005). Furthermore, diabetic patients are more vulnerable to viral infections due to defective lymphocyte-related immunity. In a previous cross-sectional study on diabetic children, we found that recurrent viral infections in the upper airways ("common cold") during the past year had cumulative adverse effects on the elastic properties (i.e. compliance) of carotid arteries. In a multivariate analysis, the number of viral infections, along with age and plasma levels of glycosylated hemoglobin, significantly and independently predicted the decrease in carotid artery compliance (Odermarsky et al., 2008a). Although impaired carotid elasticity is generally regarded as a marker of early atherosclerosis, these findings do not necessarily imply causality to accelerated atherosclerosis. Prospective studies in children are currently in progress.

#### **8. Prevention strategies in type 1 diabetes via vascular pathways?**

Should vascular endothelial dysfunction prove to play a pivotal role in the pathogenesis of both type 1 diabetes and its associated vascular disease, it is then conceivable that combined endothelium-targeting and immunoregulatory strategies already in diabetes-risk individuals without overt type 1 diabetes (i.e. diabetes high-risk HLA individuals) might reduce not only the cardiovascular burden but also the prevalence of type 1 diabetes later in life. Dietary supplementation with L-arginine (substrate for nitric oxide synthesis via nitric oxide synthase), or antioxidants, in order to improve nitric oxide bioactivity, could for instance be relatively simple, risk-free strategy with potential benefit on endothelial dysfunction. Further studies are needed to provide additional mechanistic insights into the gene-environment interaction on vascular endothelium and the timing and role of endothelial dysfunction in the development of type 1 diabetes and associated cardiovascular disease.

#### **9. References**

162 Type 1 Diabetes Complications

Acetylcholine is the standard test drug for the assessment of endothelial function. The response to acetylcholine, using iontophoresis, correlates with diabetes duration and level of glycosylated haemoglobin (Khan et al., 2000). The mechanisms of acetylcholine-induced vasodilatation via ionphoresis remain debatable. In humans, nitric oxide appears to be the main mediator, but other endothelium-dependent vasodilators may contribute as well

Sodium nitroprusside is a nitric oxide donor and acts directly, i.e. endotheliumindependent, as a control on the smooth muscle cells causing vasodilatation. Cathodal

The current for both substances is set to 100 A for 20 seconds. For each of the substances to be tested five consecutive and equal doses are applied to generate dose-response curves. Baseline perfusion and changes in response to the substance are expressed as area under the

In 1995, Michael Stern put forward the "common soil" hypothesis, which suggests a shared genetic and environmental origin for type 2 diabetes and atherosclerosis (Stern, 1995). According to this hypothesis, infections leading to chronic inflammation could pertain to the group of environmental etiological factors. Indeed, the risk of developing type 1 diabetes – a condition associated with significant morbidity in cardiovascular diseases in adult life – could rise during viral infections (Blom et al., 1991). Moreover, the risk for childhood diabetes seems to increase in accordance with a higher number of infections during the year preceding diagnosis. Studies in rodent models of atherosclerosis suggest similar dosedependent association between infection and vascular changes (Liuba et al., 2000; Tormakangas et al., 2005). Furthermore, diabetic patients are more vulnerable to viral infections due to defective lymphocyte-related immunity. In a previous cross-sectional study on diabetic children, we found that recurrent viral infections in the upper airways ("common cold") during the past year had cumulative adverse effects on the elastic properties (i.e. compliance) of carotid arteries. In a multivariate analysis, the number of viral infections, along with age and plasma levels of glycosylated hemoglobin, significantly and independently predicted the decrease in carotid artery compliance (Odermarsky et al., 2008a). Although impaired carotid elasticity is generally regarded as a marker of early atherosclerosis, these findings do not necessarily imply causality to accelerated

**7. The "common soil" hypothesis-is this applicable in type 1 diabetes?** 

atherosclerosis. Prospective studies in children are currently in progress.

**8. Prevention strategies in type 1 diabetes via vascular pathways?** 

Should vascular endothelial dysfunction prove to play a pivotal role in the pathogenesis of both type 1 diabetes and its associated vascular disease, it is then conceivable that combined endothelium-targeting and immunoregulatory strategies already in diabetes-risk individuals without overt type 1 diabetes (i.e. diabetes high-risk HLA individuals) might reduce not only the cardiovascular burden but also the prevalence of type 1 diabetes later in life. Dietary supplementation with L-arginine (substrate for nitric oxide synthesis via nitric oxide synthase), or antioxidants, in order to improve nitric oxide bioactivity, could for instance be relatively simple, risk-free strategy with potential benefit on endothelial dysfunction. Further studies are needed to provide additional mechanistic insights into the

(Turner et al., 2008).

curve.

current is used for delivering sodium nitroprusside.


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Odermarsky, M., Nilsson, A., Lernmark, A., Sjoblad, S. & Liuba, P. (2007). Atherogenic

Odermarsky, M., Lernmark, A., Truedsson, L. & Liuba, P. (2008b). Cutaneous microvascular

diabetes. Pediatr Res, Vol. 63, No. 4, (April 2008), pp. 420-2, ISSN 0031-3998 Odermarsky, M., Andersson, S., Pesonen, E. Sjoblad, S., Ylä-Herttuala, S. & Liuba, P. (2008a).

Redondo, MJ., Fain, PR. & Eisenbarth, GS. (2001). Genetics of type 1A diabetes. Recent Prog

Rossing, P., Hougaard, P., Borch-Johnsen, K. & Parving, HH. (1996). Predictors of mortality

Stern MP. (1995). Diabetes and cardiovascular disease. The "common soil" hypothesis.

TEDDY Study Group., (2008). The environmental determinants of diabetes in the young

The DIAMOND Project Group. (2006). Incidence and trends of childhood Type 1 diabetes

Thijssen, DHJ., Black, MA., Pyke, KE., Padilla, J., Atkinson, G., Harris, RA., Parker, B.,

Toivonen A, Kulmala P, Rahko J, Ilonen J, Knip M. (2004). Soluble adhesion molecules in

Toivonen, A., Turner, J., Belch, JJF. & Khan, F. (2008). Current concepts in assessment of

Tormakangas, L. ,Erkkila, L., Korhonen, T., Tiirola, T., Bloigu, A., Saikku, P. & Leinonen, O.

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vascular and lipid phenotypes in young patients with type 1 diabetes are associated with diabetes high-risk HLA genotype. Am J Physiol Heart Circ Physiol, Vol. 293,

dysfunction is associated with human leukocyte antigen-DQ in youths with type 1

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microvascular endothelial function using laser doppler imaging and iontophoresis. Trends Cardiovasc Med, Vol. 18, No. 4, (May 2008), pp. 109-16,

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myocardial infarction. N Engl J Med, Vol. 343, No. 12, (September 2000), pp. 840-6, ISSN 1533-4406


Girach, A. & Vignati, L. (2006). Diabetic microvascular complications – can the presence of

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Hanninen, A., Jalkanen, S., Salmi, M., Toikkanen, S., Nikolakaros, G. & Simell, O. (1992).

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Itoh N, Hanafusa T, Miyazaki A, et al.. Mononuclear cell infiltration and its relation to the

Karvonen, M., Viik-Kajander, M., Moltchanova, E., Libman, I., LaPorte, R. & Toumilehto, J.

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Lammi, N., Karvonen, M. & Tuomilehto, J. (2005). Do microbes have a causal role in type

Liuba, P., Karnani, P., Pesonen, E., Paakkari, I., Forslid, A., Johansson, L., Persson, K.,

Lindley, S., Dayan, CM., Bishop, A., Roep, BO., Peakman, M. & Tree, TIM. (2005). Defective

Michel, T. & Vanhoutte, PM. (2010). Cellular signalling and NO production. Pflugers Arch-Eur J Physiol, Vol. 459, No. 6, (May 2010), pp. 807-16, ISSN 1432-2013 Morris, SJ. & Shore, AC. (1996). Skin blood flow reponses to the iontophoresis of

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Macrophages, T cell receptor usage, and endothelial cell activation in the pancreas at the onset of insulin-dependent diabetes mellitus. J Clin Invest, Vol. 90, No. 5,

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(2000). Incidence of childhood type 1 diabetes worldwide. Diabetes Care, Vol. 23,

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Wadstrom, T. & Laurini, R. (2000). Endothelial dysfunction after repeated Chlamydia pneumoniae infection in apolipoprotein E-knockout mice. Circulation,

suppressor function in CD4+CD25+ T-cells from patients with type 1 diabetes.

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P. & Weidinger, F. (1997). Systemic endothelial dysfunction is related to the extent

and severity of coronary artery disease. Atherosclerosis, Vol. 129, No. 1, (February 1997), pp. 111-8, ISSN 0021-9150


**8** 

Yu-Long Li

*United States of America*

**Cardiovascular Autonomic Dysfunction in** 

**Diabetes as a Complication: Cellular and** 

*Department of Emergency Medicine, University of Nebraska Medical Center*

Diabetes is a major world health problem, which affects more than 23 million people in the US and an estimated 250 million worldwide. Diabetes mellitus is a metabolic disease characterized by high blood glucose levels resulted from an inability in pancreatic insulin secretion or insulin resistance. Usually diabetes mellitus is mainly divided into type 1 diabetes characterized by loss of the insulin production from beta cells of the pancreatic islets and type 2 diabetes characterized by insulin resistance (defective responsiveness of body tissues to insulin) and relatively reduced insulin secretion. Although type II diabetes is by far the most common affecting 90 to 95% of the US diabetic population, the studies focusing on the type 1 diabetes cannot be ignored because about 1 in every 400 to 600 children and adolescents has type 1 diabetes and about 2 million adolescents aged 12-19

Diabetes mellitus is chronic progressive disease that usually cannot be cured. Following the natural progression of disease, diabetes without proper treatments can cause many severe complications including diabetic ketoacidosis, cardiovascular disease, chronic renal failure, retinal damage. These complications obviously enhance the risk for diabetic patients. In these complications of the diabetes mellitus, cardiovascular autonomic dysfunction is a serious although poorly understood long term diabetic complication. Indeed, diabetic patients with cardiovascular autonomic dysfunction have consistently been shown to have an enhanced risk of premature death (Rosengard-Barlund *et al.*, 2009). More importantly, the age-adjusted relative risk for cardiovascular disease in type 1 diabetes far exceeds that of type 2 diabetes (Krolewski *et al.*, 1987; Libby *et al.*, 2005). Therefore, exploring the mechanisms responsible to the cardiovascular autonomic dysfunction can provide an important and new pharmacological and genetic target for improving the prognosis and

**2. Baroreflex dysfunction in type 1 diabetes and the contribution of the baroreflex dysfunction in prognosis and mortality of the type 1 diabetes** 

Cardiovascular autonomic function is the autonomic neural regulation of cardiovascular function, which presents the balance between sympathetic and parasympathetic innervation

**1. Introduction** 

have pre-diabetes in the US.

reducing the mortality in diabetic state.

**Molecular Mechanisms** 


### **Cardiovascular Autonomic Dysfunction in Diabetes as a Complication: Cellular and Molecular Mechanisms**

Yu-Long Li

*Department of Emergency Medicine, University of Nebraska Medical Center United States of America*

#### **1. Introduction**

166 Type 1 Diabetes Complications

Versari, D., Daghini, E., Virdis, A., Ghiadoni, L. & Taddei, S. (2009). Endothelial dysfunction

Virdis, A., Ghiadoni, L., Taddei, S. (2010). Human endothelial dysfunction: EDCFs. Pflugers Arch-Eur J Physiol, Vol. 459, No. 6, (May 2010), pp. 1015-23, ISSN 1432-2013

suppl 2, (November 2009), pp. S314-21, ISSN 1935-5548

as a target for prevention of cardiovascular disease. Diabetes care, Vol. 32, No.

Diabetes is a major world health problem, which affects more than 23 million people in the US and an estimated 250 million worldwide. Diabetes mellitus is a metabolic disease characterized by high blood glucose levels resulted from an inability in pancreatic insulin secretion or insulin resistance. Usually diabetes mellitus is mainly divided into type 1 diabetes characterized by loss of the insulin production from beta cells of the pancreatic islets and type 2 diabetes characterized by insulin resistance (defective responsiveness of body tissues to insulin) and relatively reduced insulin secretion. Although type II diabetes is by far the most common affecting 90 to 95% of the US diabetic population, the studies focusing on the type 1 diabetes cannot be ignored because about 1 in every 400 to 600 children and adolescents has type 1 diabetes and about 2 million adolescents aged 12-19 have pre-diabetes in the US.

Diabetes mellitus is chronic progressive disease that usually cannot be cured. Following the natural progression of disease, diabetes without proper treatments can cause many severe complications including diabetic ketoacidosis, cardiovascular disease, chronic renal failure, retinal damage. These complications obviously enhance the risk for diabetic patients. In these complications of the diabetes mellitus, cardiovascular autonomic dysfunction is a serious although poorly understood long term diabetic complication. Indeed, diabetic patients with cardiovascular autonomic dysfunction have consistently been shown to have an enhanced risk of premature death (Rosengard-Barlund *et al.*, 2009). More importantly, the age-adjusted relative risk for cardiovascular disease in type 1 diabetes far exceeds that of type 2 diabetes (Krolewski *et al.*, 1987; Libby *et al.*, 2005). Therefore, exploring the mechanisms responsible to the cardiovascular autonomic dysfunction can provide an important and new pharmacological and genetic target for improving the prognosis and reducing the mortality in diabetic state.

#### **2. Baroreflex dysfunction in type 1 diabetes and the contribution of the baroreflex dysfunction in prognosis and mortality of the type 1 diabetes**

Cardiovascular autonomic function is the autonomic neural regulation of cardiovascular function, which presents the balance between sympathetic and parasympathetic innervation

Cardiovascular Autonomic Dysfunction in

cholesterol) (Soedamah-Muthu *et al.*, 2008).

(O'Brien *et al.*, 1991).

**increased mortality rate** 

are involved in this clinical phenomenon.

Diabetes as a Complication: Cellular and Molecular Mechanisms 169

cardiovascular autonomic dysfunction is associated with a high risk of cardiac arrhythmias and with sudden death in the diabetic state. A longitudinal study by O'Brien et al (O'Brien *et al.*, 1991) has investigated 5-year survival in 506 randomly selected patients with insulindependent diabetes mellitus. In this study, the cumulative 5-year mortality rate in the diabetic patients with cardiovascular autonomic dysfunction (27%) is about 5-fold more than in the diabetic patients with normal cardiovascular autonomic function (5%). However, there is no difference in duration of diabetes between the deceased diabetic patients with and without cardiovascular autonomic dysfunction (O'Brien *et al.*, 1991). A meta-analysis (Maser *et al.*, 2003) and the epidemiology of diabetes complication study (Orchard *et al.*, 1996) also showed that cardiovascular autonomic dysfunction could contribute to the increased risk of mortality rate in the individuals with diabetes. In the recent EURODIAB prospective complications study, the researchers have found that cardiovascular autonomic dysfunction is an important risk marker for mortality rate, exceeding the effect of the traditional risk factors (such as age, waist-to-hip ratio, pulse pressure, and non-HDL

Since the diabetic patients are more likely to have many known diabetes-associated risk factors besides cardiovascular autonomic dysfunction (Soedamah-Muthu *et al.*, 2008), the question is whether cardiovascular autonomic dysfunction is an independent risk factor to predict the mortality rate of the diabetic patients. Some studies have addressed this question to minimize the potential interference of other risk factors (for example age, sex, height, smoking, diabetes duration, etc) by matching these variables in the diabetic patients with and without cardiovascular autonomic dysfunction (O'Brien *et al.*, 1991; Orchard *et al.*, 1996; Rathmann *et al.*, 1993). In Rathmann's study (Rathmann *et al.*, 1993), diabetic patients with and without cardiovascular autonomic dysfunction were matched for age, sex, and duration of diabetes. The 8-year survival rate estimate in patients with cardiovascular autonomic dysfunction was 77% compared with 97% in those with normal cardiovascular autonomic function in this study (Rathmann *et al.*, 1993). O'Brien et al. have also matched age, sex, and duration of diabetes in the diabetic patients with and without cardiac autonomic dysfunction in their study (O'Brien *et al.*, 1991). They found that the cardiovascular autonomic dysfunction was associated with the mortality rate of the type 1 diabetic patients

**2.3 Potential mechanisms responsible for cardiovascular autonomic dysfunction-**

Although many studies mentioned above have confirmed that the cardiovascular autonomic dysfunction is involved in increasing mortality rate of type 1 diabetic patients, we really don't know whether cardiovascular autonomic dysfunction is directly or indirectly responsible for the increased mortality rate. It is possible that several possible mechanisms

First, a few clinical studies have reported that some type 1 diabetic patients in good health the previous day are found dead in the morning in an undisturbed bed with no sign of the symptoms (such as sweating and struggle) and negative autopsy results, which is named as the 'dead in bed' syndrome (Tattersall & Gill, 1991; Weston & Gill, 1999). One recent clinical study has found that ECG abnormalities including QT prolongation, cardiac rhythm disturbance, and subsequent ventricular tachyarrhythmia appear in the ambulant patients with type 1 diabetes (Gill *et al.*, 2009). The ECG abnormalities can serve as

resulting in periodic fluctuation in heart rate and rhythm. Although there are many invasive and non-invasive methods to evaluate the cardiovascular autonomic function in diverse clinical and research settings, cardiovascular autonomic function typically is measured by a short-term evoked cardiovascular reflex, especially arterial baroreflex.

#### **2.1 Baroreflex dysfunction**

The arterial baroreflex normally acts to prevent wide oscillations in blood pressure and heart rate, acting on both sympathetic and parasympathetic limbs of the cardiovascular autonomic nervous system. Dysfunction of the arterial baroreflex control on the blood pressure and heart rate has been described in many studies not only in the type I diabetic patients, but also in experimental models of the type I diabetes.

In the diabetic patients, heart rate variability is the most widely used index of the arterial baroreflex function. Some studies in more heterogeneous groups of patients with type 1 diabetes have indicated that: (1) showing lower global heart rate variability; (2) relative increase in the low-frequency component (sympathetic activity) of the heart rate variability; (3) relative reduction in the high-frequency component (parasympathetic activity) of the heart rate variability; and (4) higher ratio of the low frequency to the high-frequency (Lishner *et al.*, 1987; Rosengard-Barlund *et al.*, 2009; Ziegler *et al.*, 2001). Clinical research data have confirmed that arterial baroreflex sensitivity is reduced in type 1 diabetic patients with a wide range of age and diabetic duration (Lefrandt *et al.*, 1999; Weston *et al.*, 1996;Weston *et al.*, 1998; Dalla *et al.*, 2007). More importantly, this attenuated arterial baroreflex function was found in the type 1 diabetic patients without the clinical complications, the alterations of the other autonomic function tests, or the overt autonomic neuropathy (Lefrandt *et al.*, 1999; Rosengard-Barlund *et al.*, 2009). Therefore, it is of note that the reduced arterial baroreflex sensitivity can be an earlier sensitive marker of the cardiovascular autonomic dysfunction in the type 1 diabetic patients.

In order to obtain new insights into human type 1 diabetes, animal models of the type 1 diabetes have been widely used in the biomedical studies focusing on the type 1 diabetes, such as alloxan-induced diabetic rabbits (McDowell *et al.*, 1994b), streptozotocin-induced diabetic rats (Hicks *et al.*, 1998; Maeda *et al.*, 1995; Van *et al.*, 1998; Chen *et al.*, 2008), and calmodulin transgenic OVE26 diabetic mice (Gu *et al.*, 2008). Streptozotocin (STZ)-induced diabetic rat is an animal model of insulin-dependent diabetes usually used to study the cardiovascular alterations including cardiovascular autonomic dysfunction caused by diabetes even if the changes of cardiovascular function in this animal model don't fully match the alterations observed under the clinical type 1 diabetic states (Hicks *et al.*, 1998). In the STZ-induced diabetic rats, the arterial baroreflex dysfunction is presented as early as 5 days after the STZ administration (Maeda *et al.*, 1995). Much evidence has documented that the arterial baroreflex is decreased in all kinds of type 1 diabetic models (Chen *et al.*, 2008; Dall'Ago *et al.*, 1997; De Angelis *et al.*, 2000; Gu *et al.*, 2008; Maliszewska-Scislo *et al.*, 2003; McDowell *et al.*, 1994b; Van *et al.*, 1998).

#### **2.2 Association of cardiovascular autonomic dysfunction with mortality rate**

30 years ago, Ewing et al (Ewing *et al.*, 1980) first reported that there was a mortality rate of 53% after 5 years in diabetic patients with abnormal autonomic function, compared with a mortality rate of about 15% over the 5 year period among diabetic patients without abnormal autonomic function. Thereafter the growing evidence has confirmed that

resulting in periodic fluctuation in heart rate and rhythm. Although there are many invasive and non-invasive methods to evaluate the cardiovascular autonomic function in diverse clinical and research settings, cardiovascular autonomic function typically is measured by a

The arterial baroreflex normally acts to prevent wide oscillations in blood pressure and heart rate, acting on both sympathetic and parasympathetic limbs of the cardiovascular autonomic nervous system. Dysfunction of the arterial baroreflex control on the blood pressure and heart rate has been described in many studies not only in the type I diabetic

In the diabetic patients, heart rate variability is the most widely used index of the arterial baroreflex function. Some studies in more heterogeneous groups of patients with type 1 diabetes have indicated that: (1) showing lower global heart rate variability; (2) relative increase in the low-frequency component (sympathetic activity) of the heart rate variability; (3) relative reduction in the high-frequency component (parasympathetic activity) of the heart rate variability; and (4) higher ratio of the low frequency to the high-frequency (Lishner *et al.*, 1987; Rosengard-Barlund *et al.*, 2009; Ziegler *et al.*, 2001). Clinical research data have confirmed that arterial baroreflex sensitivity is reduced in type 1 diabetic patients with a wide range of age and diabetic duration (Lefrandt *et al.*, 1999; Weston *et al.*, 1996;Weston *et al.*, 1998; Dalla *et al.*, 2007). More importantly, this attenuated arterial baroreflex function was found in the type 1 diabetic patients without the clinical complications, the alterations of the other autonomic function tests, or the overt autonomic neuropathy (Lefrandt *et al.*, 1999; Rosengard-Barlund *et al.*, 2009). Therefore, it is of note that the reduced arterial baroreflex sensitivity can be an earlier sensitive marker of the

In order to obtain new insights into human type 1 diabetes, animal models of the type 1 diabetes have been widely used in the biomedical studies focusing on the type 1 diabetes, such as alloxan-induced diabetic rabbits (McDowell *et al.*, 1994b), streptozotocin-induced diabetic rats (Hicks *et al.*, 1998; Maeda *et al.*, 1995; Van *et al.*, 1998; Chen *et al.*, 2008), and calmodulin transgenic OVE26 diabetic mice (Gu *et al.*, 2008). Streptozotocin (STZ)-induced diabetic rat is an animal model of insulin-dependent diabetes usually used to study the cardiovascular alterations including cardiovascular autonomic dysfunction caused by diabetes even if the changes of cardiovascular function in this animal model don't fully match the alterations observed under the clinical type 1 diabetic states (Hicks *et al.*, 1998). In the STZ-induced diabetic rats, the arterial baroreflex dysfunction is presented as early as 5 days after the STZ administration (Maeda *et al.*, 1995). Much evidence has documented that the arterial baroreflex is decreased in all kinds of type 1 diabetic models (Chen *et al.*, 2008; Dall'Ago *et al.*, 1997; De Angelis *et al.*, 2000; Gu *et al.*, 2008; Maliszewska-Scislo *et al.*, 2003;

**2.2 Association of cardiovascular autonomic dysfunction with mortality rate** 

30 years ago, Ewing et al (Ewing *et al.*, 1980) first reported that there was a mortality rate of 53% after 5 years in diabetic patients with abnormal autonomic function, compared with a mortality rate of about 15% over the 5 year period among diabetic patients without abnormal autonomic function. Thereafter the growing evidence has confirmed that

short-term evoked cardiovascular reflex, especially arterial baroreflex.

patients, but also in experimental models of the type I diabetes.

cardiovascular autonomic dysfunction in the type 1 diabetic patients.

McDowell *et al.*, 1994b; Van *et al.*, 1998).

**2.1 Baroreflex dysfunction** 

cardiovascular autonomic dysfunction is associated with a high risk of cardiac arrhythmias and with sudden death in the diabetic state. A longitudinal study by O'Brien et al (O'Brien *et al.*, 1991) has investigated 5-year survival in 506 randomly selected patients with insulindependent diabetes mellitus. In this study, the cumulative 5-year mortality rate in the diabetic patients with cardiovascular autonomic dysfunction (27%) is about 5-fold more than in the diabetic patients with normal cardiovascular autonomic function (5%). However, there is no difference in duration of diabetes between the deceased diabetic patients with and without cardiovascular autonomic dysfunction (O'Brien *et al.*, 1991). A meta-analysis (Maser *et al.*, 2003) and the epidemiology of diabetes complication study (Orchard *et al.*, 1996) also showed that cardiovascular autonomic dysfunction could contribute to the increased risk of mortality rate in the individuals with diabetes. In the recent EURODIAB prospective complications study, the researchers have found that cardiovascular autonomic dysfunction is an important risk marker for mortality rate, exceeding the effect of the traditional risk factors (such as age, waist-to-hip ratio, pulse pressure, and non-HDL cholesterol) (Soedamah-Muthu *et al.*, 2008).

Since the diabetic patients are more likely to have many known diabetes-associated risk factors besides cardiovascular autonomic dysfunction (Soedamah-Muthu *et al.*, 2008), the question is whether cardiovascular autonomic dysfunction is an independent risk factor to predict the mortality rate of the diabetic patients. Some studies have addressed this question to minimize the potential interference of other risk factors (for example age, sex, height, smoking, diabetes duration, etc) by matching these variables in the diabetic patients with and without cardiovascular autonomic dysfunction (O'Brien *et al.*, 1991; Orchard *et al.*, 1996; Rathmann *et al.*, 1993). In Rathmann's study (Rathmann *et al.*, 1993), diabetic patients with and without cardiovascular autonomic dysfunction were matched for age, sex, and duration of diabetes. The 8-year survival rate estimate in patients with cardiovascular autonomic dysfunction was 77% compared with 97% in those with normal cardiovascular autonomic function in this study (Rathmann *et al.*, 1993). O'Brien et al. have also matched age, sex, and duration of diabetes in the diabetic patients with and without cardiac autonomic dysfunction in their study (O'Brien *et al.*, 1991). They found that the cardiovascular autonomic dysfunction was associated with the mortality rate of the type 1 diabetic patients (O'Brien *et al.*, 1991).

#### **2.3 Potential mechanisms responsible for cardiovascular autonomic dysfunctionincreased mortality rate**

Although many studies mentioned above have confirmed that the cardiovascular autonomic dysfunction is involved in increasing mortality rate of type 1 diabetic patients, we really don't know whether cardiovascular autonomic dysfunction is directly or indirectly responsible for the increased mortality rate. It is possible that several possible mechanisms are involved in this clinical phenomenon.

First, a few clinical studies have reported that some type 1 diabetic patients in good health the previous day are found dead in the morning in an undisturbed bed with no sign of the symptoms (such as sweating and struggle) and negative autopsy results, which is named as the 'dead in bed' syndrome (Tattersall & Gill, 1991; Weston & Gill, 1999). One recent clinical study has found that ECG abnormalities including QT prolongation, cardiac rhythm disturbance, and subsequent ventricular tachyarrhythmia appear in the ambulant patients with type 1 diabetes (Gill *et al.*, 2009). The ECG abnormalities can serve as

Cardiovascular Autonomic Dysfunction in

and arterial blood pressure.

affect the arterial baroreflex function.

**diabetes** 

Diabetes as a Complication: Cellular and Molecular Mechanisms 171

The arterial baroreflex is a homeostatic mechanism that alters heart rate and blood pressure in response to changes in arterial wall tension detected by the baroreceptors in the carotid sinus and aortic arch. The arterial baroreflex arc includes an afferent limb, a central neural component and autonomic neuroeffector components. As the primary afferent limb of the baroreceptor reflex, baroreceptor neurons sense blood pressure by increasing their discharge (excitation) when arterial blood pressure rises. This excited signal in baroreceptor neurons reaches to the dorsal medial nucleus tractus solitarii (NTS, the first site of baroreceptor neuron contacting with central nervous system), in which the integrated input signal inhibits the efferent sympathetic outflow to the heart and peripheral vascular, and activates efferent parasympathetic activity to the heart those decrease peripheral vascular resistance, heart rate, and arterial blood pressure. Conversely, the baroreceptor afferent signal decreases when arterial blood pressure falls, which reflexly induces an increase in heart rate

As mentioned above, blunted arterial baroreflex sensitivity is observed in type 1 diabetic patients and animal models. What are the mechanisms responsible for the attenuated arterial baroreflex sensitivity in type 1 diabetes? Every site within the baroreflex arc may be responsible for the depressed baroreflex sensitivity in type 1 diabetes. Therefore, we will discuss the fact that the reduced baroreflex sensitivity results from functional and/or structural changes in the baroreceptors (including nerve terminals and neuron somata),

As the primary afferent limb of the arterial baroreceptor reflex, baroreceptor neurons are pseudo-unipolar neurons (T-shaped neurons) consisting of a cell body existing in the nodose or petrosal ganglia and an initial axon segment. This axon segment bifurcates near the soma into a peripheral process innervating aortic arch and carotid sinus for sensing the alteration of the arterial blood pressure and a central process terminating in the NTS for conveying the afferent signals to the central nervous system. The mechanisms responsible for mediating afferent sensitivity of barosensitive neurons to pressure are complex and not thoroughly understood. The process of translating changes in arterial wall tension into impulse traffic to the NTS involves 2 broad functional steps: 1) mechanotransduction which is governed by the properties of mechanosensitive ion channels in the nerve terminal and the mechanical properties of the coupling of the arterial wall to the sensory terminal; and 2) spike initiation which is governed by the excitability of membrane voltage sensitive ion channels that influence the electrical (cable) properties of the axonal process and cell body. All of these factors could be (and likely are) altered in type 1 diabetes, which can directly

central neural integration, and autonomic efferent component.

**3.1 Role of baroreceptor in the blunted arterial baroreflex in type 1 diabetes** 

**3.1.1 Changes of baroreceptor afferent nerve and terminal in type 1 diabetes** 

Although some studies have suggested that diabetes-induced postural hypotension results from impairments of afferent baroreceptors and of sympathetic neurons innervating the vascular wall and heart in diabetic patients (Low *et al.*, 1975; Iovino *et al.*, 2011), there is only fragmentary evidence to support this assumption because of the inability of clinical cardiovascular autonomic function tests to separate the role of the afferent, central, and

**3. Mechanisms responsible for the reduced baroreflex function in type 1** 

principal underlying causes of the 'dead in bed' syndrome (Gill *et al.*, 2009). Cardiovascular autonomic dysfunction itself can link to the QT prolongation and sudden death (Weston & Gill, 1999). In another study, type 1 diabetic adolescents with impaired cardiovascular autonomic function are associated with the possible development of cardiac arrhythmias and left-ventricular hypertrophy (Karavanaki *et al.*, 2007). In addition, decreased heart rate variability is also a predictive risk factor for ventricular arrhythmia and sudden cardiac death (Kleiger *et al.*, 1987). Loss of cardiac vagal drive combined with loss of baroreceptor reflex sensitivity is thought to mediate the decreased heart rate variability and autonomic instability that exacerbate arrhythmia susceptibility (Binkley *et al.*, 1991). These studies indicate that cardiovascular autonomic dysfunction (decreased heart rate variability and loss of baroreceptor reflex sensitivity) is correlated with the prognosis and mortality in patients with type 1 diabetes via increasing the susceptibility to the lethal arrhythmias.

Second, although cardiovascular autonomic dysfunction is an independent risk factor to predict the mortality rate of the diabetic patients described above, other abnormalities (such as increased stiffness of the vascular walls at the site of the arterial baroreceptors, left ventricular hypertrophy, endothelial dysfunction, renal failure, peripheral neuropathy, etc) usually coexist with cardiovascular autonomic dysfunction in type 1 diabetic patients (Toyry *et al.*, 1997; Lluch *et al.*, 1998; Lefrandt *et al.*, 2010). Therefore, it is possible that the interaction between cardiovascular autonomic dysfunction and other concomitant abnormalities is responsible for the increased mortality rate in type 1 diabetic patients. It has been shown that cardiovascular autonomic function is easily impaired in type 1 diabetic patients with microalbuminuria (renal dysfunction) (Lefrandt *et al.*, 1999; Clarke *et al.*, 1999). O'Brien et al have reported that renal failure-induced mortality rate is higher in type 1 diabetic patients with cardiovascular autonomic dysfunction than in those without cardiovascular autonomic dysfunction (O'Brien *et al.*, 1991). In a 23 year follow-up study, cardiovascular autonomic dysfunction may be involved in a higher mortality rate induced by microalbuminuria in type 1 diabetic patients (Messent *et al.*, 1992). Similarly, renal disease also can partially explicate the cardiovascular autonomic dysfunction-increased mortality rate in patients with diabetes mellitus (Weinrauch *et al.*, 1998; Kim *et al.*, 2009). In addition, using logistic regression analysis, one recent study has addressed the relationship between cardiovascular autonomic dysfunction and other abnormalities in 684 type 1 diabetic patients (Pavy-Le *et al.*, 2010). The research data have shown that retinopathy, peripheral neuropathy, and erectile dysfunction are closely correlated to the severity of the cardiovascular autonomic dysfunction (Pavy-Le *et al.*, 2010). Furthermore, some studies have also found a consistent association between cardiovascular autonomic dysfunction and silent myocardial ischemia, in which the patient's risk coefficient related to the cardiovascular autonomic dysfunction is higher in asymptomatic diabetic patients with silent myocardial ischemia than in those without silent myocardial ischemia (Valensi *et al.*, 2001; Vinik & Ziegler, 2007; Katz *et al.*, 1999).

Finally, several studies reported the involvement of cardiorespiratory arrest in the mortality of the diabetic patients with cardiovascular autonomic dysfunction (Page & Watkins, 1978; Bergner & Goldberger, 2010; Douglas *et al.*, 1981). The research data from Page et al. (Page & Watkins, 1978) have demonstrated that young diabetic patients with severe cardiovascular autonomic dysfunction can appear to have cardiorespiratory arrest due to the impairment of cardiorespiratory function. The cardiorespiratory arrest may be responsible for the mortality of these diabetic patients (Page & Watkins, 1978).

principal underlying causes of the 'dead in bed' syndrome (Gill *et al.*, 2009). Cardiovascular autonomic dysfunction itself can link to the QT prolongation and sudden death (Weston & Gill, 1999). In another study, type 1 diabetic adolescents with impaired cardiovascular autonomic function are associated with the possible development of cardiac arrhythmias and left-ventricular hypertrophy (Karavanaki *et al.*, 2007). In addition, decreased heart rate variability is also a predictive risk factor for ventricular arrhythmia and sudden cardiac death (Kleiger *et al.*, 1987). Loss of cardiac vagal drive combined with loss of baroreceptor reflex sensitivity is thought to mediate the decreased heart rate variability and autonomic instability that exacerbate arrhythmia susceptibility (Binkley *et al.*, 1991). These studies indicate that cardiovascular autonomic dysfunction (decreased heart rate variability and loss of baroreceptor reflex sensitivity) is correlated with the prognosis and mortality in patients with type 1 diabetes via increasing the

Second, although cardiovascular autonomic dysfunction is an independent risk factor to predict the mortality rate of the diabetic patients described above, other abnormalities (such as increased stiffness of the vascular walls at the site of the arterial baroreceptors, left ventricular hypertrophy, endothelial dysfunction, renal failure, peripheral neuropathy, etc) usually coexist with cardiovascular autonomic dysfunction in type 1 diabetic patients (Toyry *et al.*, 1997; Lluch *et al.*, 1998; Lefrandt *et al.*, 2010). Therefore, it is possible that the interaction between cardiovascular autonomic dysfunction and other concomitant abnormalities is responsible for the increased mortality rate in type 1 diabetic patients. It has been shown that cardiovascular autonomic function is easily impaired in type 1 diabetic patients with microalbuminuria (renal dysfunction) (Lefrandt *et al.*, 1999; Clarke *et al.*, 1999). O'Brien et al have reported that renal failure-induced mortality rate is higher in type 1 diabetic patients with cardiovascular autonomic dysfunction than in those without cardiovascular autonomic dysfunction (O'Brien *et al.*, 1991). In a 23 year follow-up study, cardiovascular autonomic dysfunction may be involved in a higher mortality rate induced by microalbuminuria in type 1 diabetic patients (Messent *et al.*, 1992). Similarly, renal disease also can partially explicate the cardiovascular autonomic dysfunction-increased mortality rate in patients with diabetes mellitus (Weinrauch *et al.*, 1998; Kim *et al.*, 2009). In addition, using logistic regression analysis, one recent study has addressed the relationship between cardiovascular autonomic dysfunction and other abnormalities in 684 type 1 diabetic patients (Pavy-Le *et al.*, 2010). The research data have shown that retinopathy, peripheral neuropathy, and erectile dysfunction are closely correlated to the severity of the cardiovascular autonomic dysfunction (Pavy-Le *et al.*, 2010). Furthermore, some studies have also found a consistent association between cardiovascular autonomic dysfunction and silent myocardial ischemia, in which the patient's risk coefficient related to the cardiovascular autonomic dysfunction is higher in asymptomatic diabetic patients with silent myocardial ischemia than in those without silent myocardial ischemia (Valensi *et al.*,

Finally, several studies reported the involvement of cardiorespiratory arrest in the mortality of the diabetic patients with cardiovascular autonomic dysfunction (Page & Watkins, 1978; Bergner & Goldberger, 2010; Douglas *et al.*, 1981). The research data from Page et al. (Page & Watkins, 1978) have demonstrated that young diabetic patients with severe cardiovascular autonomic dysfunction can appear to have cardiorespiratory arrest due to the impairment of cardiorespiratory function. The cardiorespiratory arrest may be responsible for the mortality

susceptibility to the lethal arrhythmias.

2001; Vinik & Ziegler, 2007; Katz *et al.*, 1999).

of these diabetic patients (Page & Watkins, 1978).

#### **3. Mechanisms responsible for the reduced baroreflex function in type 1 diabetes**

The arterial baroreflex is a homeostatic mechanism that alters heart rate and blood pressure in response to changes in arterial wall tension detected by the baroreceptors in the carotid sinus and aortic arch. The arterial baroreflex arc includes an afferent limb, a central neural component and autonomic neuroeffector components. As the primary afferent limb of the baroreceptor reflex, baroreceptor neurons sense blood pressure by increasing their discharge (excitation) when arterial blood pressure rises. This excited signal in baroreceptor neurons reaches to the dorsal medial nucleus tractus solitarii (NTS, the first site of baroreceptor neuron contacting with central nervous system), in which the integrated input signal inhibits the efferent sympathetic outflow to the heart and peripheral vascular, and activates efferent parasympathetic activity to the heart those decrease peripheral vascular resistance, heart rate, and arterial blood pressure. Conversely, the baroreceptor afferent signal decreases when arterial blood pressure falls, which reflexly induces an increase in heart rate and arterial blood pressure.

As mentioned above, blunted arterial baroreflex sensitivity is observed in type 1 diabetic patients and animal models. What are the mechanisms responsible for the attenuated arterial baroreflex sensitivity in type 1 diabetes? Every site within the baroreflex arc may be responsible for the depressed baroreflex sensitivity in type 1 diabetes. Therefore, we will discuss the fact that the reduced baroreflex sensitivity results from functional and/or structural changes in the baroreceptors (including nerve terminals and neuron somata), central neural integration, and autonomic efferent component.

#### **3.1 Role of baroreceptor in the blunted arterial baroreflex in type 1 diabetes**

As the primary afferent limb of the arterial baroreceptor reflex, baroreceptor neurons are pseudo-unipolar neurons (T-shaped neurons) consisting of a cell body existing in the nodose or petrosal ganglia and an initial axon segment. This axon segment bifurcates near the soma into a peripheral process innervating aortic arch and carotid sinus for sensing the alteration of the arterial blood pressure and a central process terminating in the NTS for conveying the afferent signals to the central nervous system. The mechanisms responsible for mediating afferent sensitivity of barosensitive neurons to pressure are complex and not thoroughly understood. The process of translating changes in arterial wall tension into impulse traffic to the NTS involves 2 broad functional steps: 1) mechanotransduction which is governed by the properties of mechanosensitive ion channels in the nerve terminal and the mechanical properties of the coupling of the arterial wall to the sensory terminal; and 2) spike initiation which is governed by the excitability of membrane voltage sensitive ion channels that influence the electrical (cable) properties of the axonal process and cell body. All of these factors could be (and likely are) altered in type 1 diabetes, which can directly affect the arterial baroreflex function.

#### **3.1.1 Changes of baroreceptor afferent nerve and terminal in type 1 diabetes**

Although some studies have suggested that diabetes-induced postural hypotension results from impairments of afferent baroreceptors and of sympathetic neurons innervating the vascular wall and heart in diabetic patients (Low *et al.*, 1975; Iovino *et al.*, 2011), there is only fragmentary evidence to support this assumption because of the inability of clinical cardiovascular autonomic function tests to separate the role of the afferent, central, and

Cardiovascular Autonomic Dysfunction in

sensitivity in STZ-induced diabetic rats.

function in response to pressure changes.

Diabetes as a Complication: Cellular and Molecular Mechanisms 173

Dworkin, 2007). The aortic depressor nerves (the peripheral process of the aortic baroreceptor neuron) are composed of both afferent A-type (myelinated) axons (about 25%) and C-type (unmyelinated) axons (about 75%) (Yamasaki *et al.*, 2004). There are very different dynamic sensory discharge characteristics between A-type and C-type baroreceptor afferents. C-type afferents are activated mainly at very high pressure and have lower firing frequencies, irregular discharge patterns (Thoren *et al.*, 1999), and appear to be the primary regulators of tonic baseline levels of arterial blood pressure besides regulating the baroreflex sensitivity (Seagard *et al.*, 1993). A-type afferents have lower pressure thresholds with very stable, proportional firing patterns (Thoren *et al.*, 1999), which are thought to regulate the baroreflex sensitivity but not baseline levels of arterial blood pressure (Seagard *et al.*, 1993). Electrical Stimulation of the rat aortic depressor nerve has several advantages to examine the baroreflex function. First, the rat aortic depressor nerve contains only baroreceptor afferent fibers and no chemoreceptor afferent fibers to transmit the chemoreceptor information (Fan *et al.*, 1996; Kobayashi *et al.*, 1999; Sapru & Krieger, 1977; Sapru *et al.*, 1981). Second, the baroreflex induced by stimulating rat aortic depressor nerve is measured without the aortic baroreceptor terminals in the reflex arc, which allows us to specifically examine the role of electrical excitability of aortic baroreceptor in the baroreflex function (second process mentioned above). Third, by varying the frequency of stimulus, one can differentially activate A- and C- afferent fibers, and thus evaluate the relative contribution of each to the altered aortic baroreceptor excitability and baroreflex function in STZ-induced diabetes. In our preliminary study, the baroreflex responses of blood pressure and heart rate to the electrical stimulation of the aortic baroreceptor nerve are significantly depressed in STZ-induced diabetic rats (Fig. 1). In addition, our study also found that microinjection of angiotensin II type 1 (AT1) receptor antagonist (20 µM L158,809) into the nodose ganglia significantly improved the baroreflex sensitivity induced by aortic depressor nerve stimulation in STZ-induced diabetic rats (Fig. 1). Simultaneously, AT1 receptor antagonist also normalized the depressed cell excitability in the aortic baroreceptor neurons of STZ-induced diabetic rats (Li & Zheng, 2011). The fact is that nodose neurons are found to influence the conduction and frequency of the electrical impulses in the baroreceptor central axons projecting to the central nervous system when electrical signals in the baroreceptor peripheral axons reach the nodose neurons (Ducreux *et al.*, 1993). One review paper has concluded that the excitability of vagal afferent neurons has dramatic consequences for the regulation and modulation of vago-vagal reflex (Browning, 2003). Furthermore, Devor (Devor, 1999) has reported that electrical excitability of the soma in the dorsal root ganglia may be required to insure the reliable afferent electrical impulses transmitted to the spinal cord. These results, taken together, demonstrate that the reduced cell excitability of the aortic baroreceptor neurons contributes to the blunted baroreflex

However, results from reflex experiments evoked by the electrical stimulation need to be tempered because the electrical stimulation technique does not represent a physiological substrate for baroreceptor activation. Thus, arterial baroreflex evoked by changes in arterial blood pressure should be done to further address the role of the aortic baroreceptor neurons in the arterial baroreflex in the type 1 diabetes. Of course, in this approach (blood pressuremediated baroreflex sensitivity), possible alterations in the mechanotransduction process at the baro-sensory nerve terminal may also play a role in the suppressed baroreceptor

efferent components of the arterial baroreflex. In general, the function of the baroreceptor afferent nerve and terminal is investigated by recording the single fiber or multifiber activity of the aortic depressor nerve or carotid sinus nerve in a perfused isolated aortic arch/carotid sinus preparation (do Carmo *et al.*, 2007; Doan *et al.*, 2004; Fazan, Jr. *et al.*, 1997; McDowell *et al.*, 1994b; Reynolds *et al.*, 1994; Reynolds *et al.*, 1999; Xiao *et al.*, 2007; Zhang *et al.*, 2004). However, the baroreceptor function in the diabetic state is studied only in the aortic depressor nerve (Fazan, Jr. *et al.*, 1997; Fazan, Jr. *et al.*, 1999; McDowell *et al.*, 1994b; Reynolds *et al.*, 1999) but not in the carotid sinus nerve (Salgado *et al.*, 2001). This may be because there are only baroreceptor afferent fibers and no chemoreceptor afferent fibers in rat aortic depressor nerve unlike the carotid sinus nerve (Fan *et al.*, 1996; Kobayashi *et al.*, 1999; Sapru & Krieger, 1977; Sapru *et al.*, 1981). Based on the results from some studies, there is no evidence to show the changes of the aortic depressor nerve activity in STZ-induced type 1 diabetic rats (Fazan, Jr. *et al.*, 1997; Reynolds *et al.*, 1999; Dall'Ago *et al.*, 2002) and alloxaninduced diabetic rabbits (McDowell *et al.*, 1994b), compared to the sham animals. In addition, Gu *et al* (Gu *et al.*, 2008) have found that the baroreceptor function of the aortic depressor nerve is preserved in the ascending phase of the arterial blood pressure but is blunted in the descending phase of the arterial blood pressure in type 1 diabetic mice. Nevertheless, the results obtained by a new approach, named as cross-spectral analysis, indicate that a significant decrease of the aortic baroreceptor nerve function is observed in anesthetized rats with either short term (10-20 days) or long term (12-18 weeks) STZinduced diabetes (Fazan, Jr. *et al.*, 1999). This new approach uses the magnitude of the transfer function obtained by analyzing the relationship between beat-by-beat time series of mean arterial blood pressure and aortic depressor nerve activity as the index of the aortic baroreceptor nerve function, whose advantage is to evaluate the aortic baroreceptor nerve function under more physiological conditions (Salgado *et al.*, 2001; Fazan, Jr. *et al.*, 1999; deBoer *et al.*, 1987) compared to the arterial blood pressure/aortic depressor nerve activity curve used in other studies (Dall'Ago *et al.*, 2002; Fazan, Jr. *et al.*, 1997; Gu *et al.*, 2008; McDowell *et al.*, 1994b; Reynolds *et al.*, 1999). In addition, Fazan et al have found that the morphological change in the aortic depressor nerve, an afferent arm of the baroreflex may result in the arterial baroreflex impairment in the STZ-induced diabetic rats (Fazan *et al.*, 2006). Therefore, the functional and structural alterations of the baroreceptor afferent nerve in type 1 diabetes still need to be further clarified in future study.

By light, electron, and confocal microscopies, some researchers have identified the aortic baroreceptor terminals in the adventitia of the aortic arch from dogs, rabbits, cats, rats, and mice (Aumonier, 1972; Cheng *et al.*, 1997; Krauhs, 1979; Li *et al.*, 2010). More importantly, Li et al have demonstrated that diabetes induces morphological atrophy of the aortic baroreceptor terminals in type 1 diabetic mice (Li *et al.*, 2010). However, there is no report on the functional role of the aortic baroreceptor terminals in sham and type 1 diabetic animals because it is difficult to separate aortic baroreceptor terminals to other tissues (such as smooth muscle and endothelium) in the aortic arch. It is possible that using gene and short hairpin RNA (shRNA) transfection can solve this problem in future study.

#### **3.1.2 Role of aortic baroreceptor neurons in the arterial baroreflex in the type 1 diabetes**

Many studies have used the responses of blood pressure and heart rate to electrical stimulation of baroreceptor-containing nerve (aortic depressor nerve) for the evaluation of the baroreflex sensitivity in rats (Fan & Andresen, 1998; Salgado *et al.*, 2007; Tang &

efferent components of the arterial baroreflex. In general, the function of the baroreceptor afferent nerve and terminal is investigated by recording the single fiber or multifiber activity of the aortic depressor nerve or carotid sinus nerve in a perfused isolated aortic arch/carotid sinus preparation (do Carmo *et al.*, 2007; Doan *et al.*, 2004; Fazan, Jr. *et al.*, 1997; McDowell *et al.*, 1994b; Reynolds *et al.*, 1994; Reynolds *et al.*, 1999; Xiao *et al.*, 2007; Zhang *et al.*, 2004). However, the baroreceptor function in the diabetic state is studied only in the aortic depressor nerve (Fazan, Jr. *et al.*, 1997; Fazan, Jr. *et al.*, 1999; McDowell *et al.*, 1994b; Reynolds *et al.*, 1999) but not in the carotid sinus nerve (Salgado *et al.*, 2001). This may be because there are only baroreceptor afferent fibers and no chemoreceptor afferent fibers in rat aortic depressor nerve unlike the carotid sinus nerve (Fan *et al.*, 1996; Kobayashi *et al.*, 1999; Sapru & Krieger, 1977; Sapru *et al.*, 1981). Based on the results from some studies, there is no evidence to show the changes of the aortic depressor nerve activity in STZ-induced type 1 diabetic rats (Fazan, Jr. *et al.*, 1997; Reynolds *et al.*, 1999; Dall'Ago *et al.*, 2002) and alloxaninduced diabetic rabbits (McDowell *et al.*, 1994b), compared to the sham animals. In addition, Gu *et al* (Gu *et al.*, 2008) have found that the baroreceptor function of the aortic depressor nerve is preserved in the ascending phase of the arterial blood pressure but is blunted in the descending phase of the arterial blood pressure in type 1 diabetic mice. Nevertheless, the results obtained by a new approach, named as cross-spectral analysis, indicate that a significant decrease of the aortic baroreceptor nerve function is observed in anesthetized rats with either short term (10-20 days) or long term (12-18 weeks) STZinduced diabetes (Fazan, Jr. *et al.*, 1999). This new approach uses the magnitude of the transfer function obtained by analyzing the relationship between beat-by-beat time series of mean arterial blood pressure and aortic depressor nerve activity as the index of the aortic baroreceptor nerve function, whose advantage is to evaluate the aortic baroreceptor nerve function under more physiological conditions (Salgado *et al.*, 2001; Fazan, Jr. *et al.*, 1999; deBoer *et al.*, 1987) compared to the arterial blood pressure/aortic depressor nerve activity curve used in other studies (Dall'Ago *et al.*, 2002; Fazan, Jr. *et al.*, 1997; Gu *et al.*, 2008; McDowell *et al.*, 1994b; Reynolds *et al.*, 1999). In addition, Fazan et al have found that the morphological change in the aortic depressor nerve, an afferent arm of the baroreflex may result in the arterial baroreflex impairment in the STZ-induced diabetic rats (Fazan *et al.*, 2006). Therefore, the functional and structural alterations of the baroreceptor afferent nerve

in type 1 diabetes still need to be further clarified in future study.

hairpin RNA (shRNA) transfection can solve this problem in future study.

**diabetes** 

**3.1.2 Role of aortic baroreceptor neurons in the arterial baroreflex in the type 1** 

Many studies have used the responses of blood pressure and heart rate to electrical stimulation of baroreceptor-containing nerve (aortic depressor nerve) for the evaluation of the baroreflex sensitivity in rats (Fan & Andresen, 1998; Salgado *et al.*, 2007; Tang &

By light, electron, and confocal microscopies, some researchers have identified the aortic baroreceptor terminals in the adventitia of the aortic arch from dogs, rabbits, cats, rats, and mice (Aumonier, 1972; Cheng *et al.*, 1997; Krauhs, 1979; Li *et al.*, 2010). More importantly, Li et al have demonstrated that diabetes induces morphological atrophy of the aortic baroreceptor terminals in type 1 diabetic mice (Li *et al.*, 2010). However, there is no report on the functional role of the aortic baroreceptor terminals in sham and type 1 diabetic animals because it is difficult to separate aortic baroreceptor terminals to other tissues (such as smooth muscle and endothelium) in the aortic arch. It is possible that using gene and short Dworkin, 2007). The aortic depressor nerves (the peripheral process of the aortic baroreceptor neuron) are composed of both afferent A-type (myelinated) axons (about 25%) and C-type (unmyelinated) axons (about 75%) (Yamasaki *et al.*, 2004). There are very different dynamic sensory discharge characteristics between A-type and C-type baroreceptor afferents. C-type afferents are activated mainly at very high pressure and have lower firing frequencies, irregular discharge patterns (Thoren *et al.*, 1999), and appear to be the primary regulators of tonic baseline levels of arterial blood pressure besides regulating the baroreflex sensitivity (Seagard *et al.*, 1993). A-type afferents have lower pressure thresholds with very stable, proportional firing patterns (Thoren *et al.*, 1999), which are thought to regulate the baroreflex sensitivity but not baseline levels of arterial blood pressure (Seagard *et al.*, 1993). Electrical Stimulation of the rat aortic depressor nerve has several advantages to examine the baroreflex function. First, the rat aortic depressor nerve contains only baroreceptor afferent fibers and no chemoreceptor afferent fibers to transmit the chemoreceptor information (Fan *et al.*, 1996; Kobayashi *et al.*, 1999; Sapru & Krieger, 1977; Sapru *et al.*, 1981). Second, the baroreflex induced by stimulating rat aortic depressor nerve is measured without the aortic baroreceptor terminals in the reflex arc, which allows us to specifically examine the role of electrical excitability of aortic baroreceptor in the baroreflex function (second process mentioned above). Third, by varying the frequency of stimulus, one can differentially activate A- and C- afferent fibers, and thus evaluate the relative contribution of each to the altered aortic baroreceptor excitability and baroreflex function in STZ-induced diabetes. In our preliminary study, the baroreflex responses of blood pressure and heart rate to the electrical stimulation of the aortic baroreceptor nerve are significantly depressed in STZ-induced diabetic rats (Fig. 1). In addition, our study also found that microinjection of angiotensin II type 1 (AT1) receptor antagonist (20 µM L158,809) into the nodose ganglia significantly improved the baroreflex sensitivity induced by aortic depressor nerve stimulation in STZ-induced diabetic rats (Fig. 1). Simultaneously, AT1 receptor antagonist also normalized the depressed cell excitability in the aortic baroreceptor neurons of STZ-induced diabetic rats (Li & Zheng, 2011). The fact is that nodose neurons are found to influence the conduction and frequency of the electrical impulses in the baroreceptor central axons projecting to the central nervous system when electrical signals in the baroreceptor peripheral axons reach the nodose neurons (Ducreux *et al.*, 1993). One review paper has concluded that the excitability of vagal afferent neurons has dramatic consequences for the regulation and modulation of vago-vagal reflex (Browning, 2003). Furthermore, Devor (Devor, 1999) has reported that electrical excitability of the soma in the dorsal root ganglia may be required to insure the reliable afferent electrical impulses transmitted to the spinal cord. These results, taken together, demonstrate that the reduced cell excitability of the aortic baroreceptor neurons contributes to the blunted baroreflex sensitivity in STZ-induced diabetic rats.

However, results from reflex experiments evoked by the electrical stimulation need to be tempered because the electrical stimulation technique does not represent a physiological substrate for baroreceptor activation. Thus, arterial baroreflex evoked by changes in arterial blood pressure should be done to further address the role of the aortic baroreceptor neurons in the arterial baroreflex in the type 1 diabetes. Of course, in this approach (blood pressuremediated baroreflex sensitivity), possible alterations in the mechanotransduction process at the baro-sensory nerve terminal may also play a role in the suppressed baroreceptor function in response to pressure changes.

Cardiovascular Autonomic Dysfunction in

Diabetes as a Complication: Cellular and Molecular Mechanisms 175

HCN channels have been found in various types of cells including cardiac and neuronal cells (DiFrancesco, 1985; DiFrancesco, 1993; Pape, 1996). In these spontaneously exciting cells, HCN channels normally associate to the cardiac pacemaker activity and the neuronal oscillatory behavior (Brown *et al.*, 1979; DiFrancesco, 1993; Kaupp & Seifert, 2001; Notomi & Shigemoto, 2004; Pape, 1996; Robinson & Siegelbaum, 2003). However, HCN channels may have a different role in the nodose ganglion neurons (the non-oscillatory and non-automatic exciting cells) because the nodose neurons are inactive except in response to a depolarizing stimulus generated by their peripheral sensory terminals (Doan & Kunze, 1999; Li *et al.*, 2008a). In the nodose neurons, the resting membrane potential is about -50 to -65 mV, in which voltage-dependent sodium, calcium, and potassium channels are almost inactivated (Robinson & Siegelbaum, 2003). The inactivation of these voltage-dependent channels can be recovered to the activation state during the hyperpolarization of the resting membrane potential, which means the number of available voltage-dependent channels for activation is increased if the nodose neurons receive the depolarizing stimulus (Doan & Kunze, 1999). Inhibition of HCN channels has been shown to hyperpolarize the nodose neurons (increasing the resting membrane potential) and to reduce action potential threshold in response to a depolarizing current stimulation, which suggests that HCN channels are involved in the cell excitability of the nodose neurons (Doan *et al.*, 2004; Li *et al.*, 2008a). Results from our recent studies (Li *et al.*, 2008a; Li & Zheng, 2011; Tu *et al.*, 2010) confirm that the HCN current density in A- and C-type aortic baroreceptor neurons from STZinduced diabetic rats is larger than that from the sham rats (Fig. 3). In addition, the resting membrane potential is depolarized and the current threshold induced the action potentials was elevated in the A-/C-type aortic baroreceptor neurons from STZ-induced diabetic rats, compared with that in sham rats (Li *et al.*, 2008a; Li & Zheng, 2011). Furthermore, HCN channel blockers (CsCl and ZD-7288) lowered the HCN current density, hyperpolarized the resting membrane potential, and raised the cell membrane excitability in A-/C-type aortic baroreceptor neurons from sham and STZ-induced diabetic rats (Li *et al.*, 2008a; Zhang *et al.*, 2010). These results clearly indicate that the HCN channels are involved in the regulation of aortic baroreceptor neuron excitability. The enhancement of HCN currents can contribute to the blunted aortic baroreceptor neuron excitability, and subsequently attenuate the arterial baroreflex sensitivity in STZ-induced diabetic rats. This is true because microinjection of HCN channel blocker (5 mM CsCl) improves the arterial baroreflex sensitivity induced by

the electrical stimulation of the aortic depressor nerve (Fig. 1) (Li *et al.*, 2008b).

Four mammalian genes encoding HCN channel isoforms (HCN1, HCN2, HCN3, and HCN4) have been identified (Doan *et al.*, 2004; Ishii *et al.*, 1999; Ludwig *et al.*, 1998; Santoro *et al.*, 1998; Vaccari *et al.*, 1999). In cell lines transfected HCN isoform cDNA, electrophysiological studies have shown that each channel isoform is activated by membrane hyperpolarization with distinct activation kinetics (Ludwig *et al.*, 1999; Moosmang *et al.*, 2001; Qu *et al.*, 2002; Santoro *et al.*, 1998). Activation of the HCN channels is also directly modulated by cAMP, which is dependent on the HCN channel isoform (Stieber *et al.*, 2003; Wainger *et al.*, 2001; Wang *et al.*, 2002). HCN channels are activated with the different activation rates in this order: HCN1>HCN2>HCN3>HCN4 (Accili *et al.*, 2002; Altomare *et al.*, 2001; Moosmang *et al.*, 2001; Stieber *et al.*, 2003; Stieber *et al.*, 2005). HCN1 and HCN3 are only weakly affected by cAMP whereas HCN2 and HCN4 are very sensitive to cAMP (Accili *et al.*, 2002; Stieber *et al.*, 2005; Wahl-Schott & Biel, 2009; Wang *et al.*, 2001). Our studies (Li *et al.*, 2008a; Tu *et al.*, 2010) have found that a fast-activated and cAMP-

Fig. 1. Reflex ΔMAP and ΔHR in response to different frequencies of ADN stimulation in anesthetized sham and STZ-induced diabetic rats (n=8 in each group). L158,809: AT1 receptor antagonist; CsCl: HCN channel blocker. MBP, mean blood pressure; HR, heart rate; ADN, aortic depressor nerve.

#### **3.1.3 Contribution of the HCN channel to the cell excitability of aortic baroreceptor neuron in the type 1 diabetes**

Until now it is controversial whether either severe degenerative changes or neuronal cell loss in sensory and autonomic nervous tissues are found in STZ-induced diabetic animals (Yagihashi, 1997). Apoptotic cell death was reported in the sensory neurons, satellite cells, and Schwann cells from dorsal root ganglia (DRG) of STZ-induced diabetic rats (Russell *et al.*, 1999; Srinivasan *et al.*, 2000). Kogawa et al. also found that apoptotic cell death of DRG neurons and impaired sensory nerve regeneration were induced by sciatic nerve crush in STZ-induced diabetic rats (Kogawa *et al.*, 2000). On the other hand, the findings from Sango et al (Sango *et al.*, 1991; Sango *et al.*, 1995; Sango *et al.*, 1997) indicated no difference in the dissociated neurons from DRG between sham and STZ-induced diabetic mice. Furthermore, some studies have demonstrated that there are no morphological changes of the peripheral nerves (Sharma & Thomas, 1987) and cell death of the nodose afferent neurons (Sango *et al.*, 2002) in STZ-induced diabetic animals. Our recent study (Tu *et al.*, 2010) also suggests that STZ-induced diabetes does not change the total cell number of the nodose afferent neurons and the ratio of A-/C-type neurons (Fig. 2). These results provide an important piece of information that the parasympathetic reflex dysfunction (Li *et al.*, 2008b; Thomas & Tomlinson, 1993; Ziegler, 1994) in STZ-induced diabetes might be not due to the structural changes in the nodose afferent neurons but most likely due to the functional changes at the cellular and molecular levels.

As everyone knows, many ion channels (such as sodium channels, calcium channels, potassium channels, etc) are responsible for the cell excitation in the excitable cells such as cardiac/skeletal myocytes and neurons including aortic baroreceptor neurons. However, much evidence has indicates that Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play an important role in the cell excitability of the aortic baroreceptor neurons from sham and STZ-induced diabetic rats.

Fig. 1. Reflex ΔMAP and ΔHR in response to different frequencies of ADN stimulation in anesthetized sham and STZ-induced diabetic rats (n=8 in each group). L158,809: AT1

**3.1.3 Contribution of the HCN channel to the cell excitability of aortic baroreceptor** 

Until now it is controversial whether either severe degenerative changes or neuronal cell loss in sensory and autonomic nervous tissues are found in STZ-induced diabetic animals (Yagihashi, 1997). Apoptotic cell death was reported in the sensory neurons, satellite cells, and Schwann cells from dorsal root ganglia (DRG) of STZ-induced diabetic rats (Russell *et al.*, 1999; Srinivasan *et al.*, 2000). Kogawa et al. also found that apoptotic cell death of DRG neurons and impaired sensory nerve regeneration were induced by sciatic nerve crush in STZ-induced diabetic rats (Kogawa *et al.*, 2000). On the other hand, the findings from Sango et al (Sango *et al.*, 1991; Sango *et al.*, 1995; Sango *et al.*, 1997) indicated no difference in the dissociated neurons from DRG between sham and STZ-induced diabetic mice. Furthermore, some studies have demonstrated that there are no morphological changes of the peripheral nerves (Sharma & Thomas, 1987) and cell death of the nodose afferent neurons (Sango *et al.*, 2002) in STZ-induced diabetic animals. Our recent study (Tu *et al.*, 2010) also suggests that STZ-induced diabetes does not change the total cell number of the nodose afferent neurons and the ratio of A-/C-type neurons (Fig. 2). These results provide an important piece of information that the parasympathetic reflex dysfunction (Li *et al.*, 2008b; Thomas & Tomlinson, 1993; Ziegler, 1994) in STZ-induced diabetes might be not due to the structural changes in the nodose afferent neurons but most likely due to the functional changes at the

As everyone knows, many ion channels (such as sodium channels, calcium channels, potassium channels, etc) are responsible for the cell excitation in the excitable cells such as cardiac/skeletal myocytes and neurons including aortic baroreceptor neurons. However, much evidence has indicates that Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play an important role in the cell excitability of the aortic baroreceptor

ADN, aortic depressor nerve.

**neuron in the type 1 diabetes** 

cellular and molecular levels.

neurons from sham and STZ-induced diabetic rats.

receptor antagonist; CsCl: HCN channel blocker. MBP, mean blood pressure; HR, heart rate;

HCN channels have been found in various types of cells including cardiac and neuronal cells (DiFrancesco, 1985; DiFrancesco, 1993; Pape, 1996). In these spontaneously exciting cells, HCN channels normally associate to the cardiac pacemaker activity and the neuronal oscillatory behavior (Brown *et al.*, 1979; DiFrancesco, 1993; Kaupp & Seifert, 2001; Notomi & Shigemoto, 2004; Pape, 1996; Robinson & Siegelbaum, 2003). However, HCN channels may have a different role in the nodose ganglion neurons (the non-oscillatory and non-automatic exciting cells) because the nodose neurons are inactive except in response to a depolarizing stimulus generated by their peripheral sensory terminals (Doan & Kunze, 1999; Li *et al.*, 2008a). In the nodose neurons, the resting membrane potential is about -50 to -65 mV, in which voltage-dependent sodium, calcium, and potassium channels are almost inactivated (Robinson & Siegelbaum, 2003). The inactivation of these voltage-dependent channels can be recovered to the activation state during the hyperpolarization of the resting membrane potential, which means the number of available voltage-dependent channels for activation is increased if the nodose neurons receive the depolarizing stimulus (Doan & Kunze, 1999). Inhibition of HCN channels has been shown to hyperpolarize the nodose neurons (increasing the resting membrane potential) and to reduce action potential threshold in response to a depolarizing current stimulation, which suggests that HCN channels are involved in the cell excitability of the nodose neurons (Doan *et al.*, 2004; Li *et al.*, 2008a). Results from our recent studies (Li *et al.*, 2008a; Li & Zheng, 2011; Tu *et al.*, 2010) confirm that the HCN current density in A- and C-type aortic baroreceptor neurons from STZinduced diabetic rats is larger than that from the sham rats (Fig. 3). In addition, the resting membrane potential is depolarized and the current threshold induced the action potentials was elevated in the A-/C-type aortic baroreceptor neurons from STZ-induced diabetic rats, compared with that in sham rats (Li *et al.*, 2008a; Li & Zheng, 2011). Furthermore, HCN channel blockers (CsCl and ZD-7288) lowered the HCN current density, hyperpolarized the resting membrane potential, and raised the cell membrane excitability in A-/C-type aortic baroreceptor neurons from sham and STZ-induced diabetic rats (Li *et al.*, 2008a; Zhang *et al.*, 2010). These results clearly indicate that the HCN channels are involved in the regulation of aortic baroreceptor neuron excitability. The enhancement of HCN currents can contribute to the blunted aortic baroreceptor neuron excitability, and subsequently attenuate the arterial baroreflex sensitivity in STZ-induced diabetic rats. This is true because microinjection of HCN channel blocker (5 mM CsCl) improves the arterial baroreflex sensitivity induced by the electrical stimulation of the aortic depressor nerve (Fig. 1) (Li *et al.*, 2008b).

Four mammalian genes encoding HCN channel isoforms (HCN1, HCN2, HCN3, and HCN4) have been identified (Doan *et al.*, 2004; Ishii *et al.*, 1999; Ludwig *et al.*, 1998; Santoro *et al.*, 1998; Vaccari *et al.*, 1999). In cell lines transfected HCN isoform cDNA, electrophysiological studies have shown that each channel isoform is activated by membrane hyperpolarization with distinct activation kinetics (Ludwig *et al.*, 1999; Moosmang *et al.*, 2001; Qu *et al.*, 2002; Santoro *et al.*, 1998). Activation of the HCN channels is also directly modulated by cAMP, which is dependent on the HCN channel isoform (Stieber *et al.*, 2003; Wainger *et al.*, 2001; Wang *et al.*, 2002). HCN channels are activated with the different activation rates in this order: HCN1>HCN2>HCN3>HCN4 (Accili *et al.*, 2002; Altomare *et al.*, 2001; Moosmang *et al.*, 2001; Stieber *et al.*, 2003; Stieber *et al.*, 2005). HCN1 and HCN3 are only weakly affected by cAMP whereas HCN2 and HCN4 are very sensitive to cAMP (Accili *et al.*, 2002; Stieber *et al.*, 2005; Wahl-Schott & Biel, 2009; Wang *et al.*, 2001). Our studies (Li *et al.*, 2008a; Tu *et al.*, 2010) have found that a fast-activated and cAMP-

Cardiovascular Autonomic Dysfunction in

Diabetes as a Complication: Cellular and Molecular Mechanisms 177

Fig. 3. Original HCN current recording and current density-voltage curves in A- and C-type neurons from sham and STZ-induced diabetic rats. \*P<0.05 vs. sham rats (Reprinted from

insensitive HCN current is induced in sham A-type aortic baroreceptor neurons whereas a slow-activated and cAMP-sensitive HCN current is induced in sham C-type aortic baroreceptor neurons. From these electrophysiological results, we can imagine that there is a differential distribution of the HCN channel isoforms in the A- and C-type aortic baroreceptor neurons. Data from immunofluorescent double staining also show that HCN1, HCN3, and HCN4 are expressed in sham A-type nodose neurons, whereas HCN2, HCN3, and HCN4 are expressed in sham C-type nodose neurons (Li *et al.*, 2008a; Tu *et al.*, 2010). Based on these results, it is reasonable to assume that there are marked different activation kinetics and cAMP sensitivity of HCN channels between A-fiber neurons and C-fiber neurons, which might be due to the neuron cell-specific expression of HCN channel

Tu *et al*., 2010, page 48, with permission from Elsevier).

isoforms.

Fig. 2. Ratio of A-type/C-type neurons (A and B) and total neuron number (C) in nodose ganglia from sham and STZ-induced diabetic rats. Calibration bar: 100 µm. RT97, A-type neuron marker; IB4, C-type neuron marker; DAPI, cell nucleus marker. Yellow arrows indicate nodose neurons in DAPI staining (Adapted and reprinted from Tu *et al*., 2010, page 42, with permission from Elsevier)

Fig. 2. Ratio of A-type/C-type neurons (A and B) and total neuron number (C) in nodose ganglia from sham and STZ-induced diabetic rats. Calibration bar: 100 µm. RT97, A-type neuron marker; IB4, C-type neuron marker; DAPI, cell nucleus marker. Yellow arrows indicate nodose neurons in DAPI staining (Adapted and reprinted from Tu *et al*., 2010, page

42, with permission from Elsevier)

Fig. 3. Original HCN current recording and current density-voltage curves in A- and C-type neurons from sham and STZ-induced diabetic rats. \*P<0.05 vs. sham rats (Reprinted from Tu *et al*., 2010, page 48, with permission from Elsevier).

insensitive HCN current is induced in sham A-type aortic baroreceptor neurons whereas a slow-activated and cAMP-sensitive HCN current is induced in sham C-type aortic baroreceptor neurons. From these electrophysiological results, we can imagine that there is a differential distribution of the HCN channel isoforms in the A- and C-type aortic baroreceptor neurons. Data from immunofluorescent double staining also show that HCN1, HCN3, and HCN4 are expressed in sham A-type nodose neurons, whereas HCN2, HCN3, and HCN4 are expressed in sham C-type nodose neurons (Li *et al.*, 2008a; Tu *et al.*, 2010). Based on these results, it is reasonable to assume that there are marked different activation kinetics and cAMP sensitivity of HCN channels between A-fiber neurons and C-fiber neurons, which might be due to the neuron cell-specific expression of HCN channel isoforms.

Cardiovascular Autonomic Dysfunction in

ganglia. This issue needs to be confirmed by further study.

sensitivity, etc) are responsible for this discrepancy.

Diabetes as a Complication: Cellular and Molecular Mechanisms 179

Above results suggest that elevation of local tissue angiotensin II plays an important role on the enhanced HCN channel activity and the blunted cell excitability in the AB neurons in diabetes. However, it is unclear how angiotensin II and its antagonist within an isolated aortic baroreceptor neuron from diabetic rat interact with AT1 receptor to affect the HCN channel activity and cell excitability. Classical viewpoint about the effects of angiotensin II binding with AT1 receptor is that angiotensin II binds with AT1 receptor at the cell membrane, and following the phosphorylation of the AT1 receptor, angiotensin II induces intracellular responses via activating intracellular downstream signal transduction. However, Zhuo, et al. (Zhuo *et al.*, 2002) have found that there is substantial intracellular accumulation of angiotensin II in renal cortical endosomes during angiotensin II-dependent hypertension via an AT1 receptor-mediated process. Recent studies have shown that intracellular administration of angiotensin II increases the peak inward calcium current density and decreases the junctional conductance via intracellular angiotensin II receptors in cardiac myocytes (De Mello, 2003; De Mello & Monterrubio, 2004). Intracellular treatment of losartan (a selective AT1 receptor antagonist) abolishes the effect of intracellular angiotensin II (Allen *et al.*, 1988; Bacal & Kunze, 1994). Based on these studies, we reason that diabetesinduced elevation of intracellular angiotensin II concentration in the nodose neurons contributes to the enhanced HCN channel activity and the blunted cell excitability in the AB neurons in diabetes. This viewpoint is confirmed by our observation that intracellular administration of losartan (added to the recording pipette solution) decreased the HCN current density and increased the cell excitability in the AB neurons from diabetic rats (Li & Zheng, 2011). Therefore, it is possible there is an intracellular angiotensin II production system in the nodose ganglion tissue. Of course, it would be optimal to measure intracellular angiotensin II concentration in the aortic baroreceptor neurons, but there is no appropriate measurement for it so far due to insufficient cellular material of tiny nodose

Growing evidence has shown that the AT1 and AT2 receptors are defined on the basis of their opposite pharmacological and biochemical effects (Levy, 2004). Activation of AT1 receptors mainly results in vasoconstriction, augmentation of cardiac contractility, cell proliferation, vascular and cardiac hypertrophy, oxidative stress, and inhibition of the neuronal potassium currents (Gelband *et al.*, 1999; Levy, 2004; Sumners *et al.*, 1996). On the other hand, stimulation of AT2 receptors induces vasodilation, anti-growth, antihypertrophy, and enhancement of the neuronal potassium currents (Horiuchi *et al.*, 1999; Kang *et al.*, 1995; Martens *et al.*, 1996; Matsubara, 1998; Siragy, 2000). Although AT2 receptors are expressed in the rat nodose neurons, activation of AT2 receptors does not affect the activation of HCN channels because AT2 receptor antagonist (PD123,319) does not alter the effect of angiotensin II on the HCN currents (Zhang *et al.*, 2010). Until now there is no study to explain this result, but it is possible that many factors (such as species, tissue, channel

Now the question is how angiotensin II regulates the activation of HCN channels and what is the downstream of angiotensin II-AT1 receptor. NADPH oxidase has been considered as a main source of intracellular superoxide in many tissues (Cifuentes *et al.*, 2000; Franco *et al.*, 2003; Gao *et al.*, 2004; Griendling *et al.*, 2000; Li *et al.*, 2007; Schieffer *et al.*, 2000). NADPH oxidase is a multicomponent enzyme composed of three cytosolic subunits (p40phox, p47phox, and p67phox), two membrane-associated subunits (gp91phox and p22phox), and the small Gproteins (Rac and Rap1a) (Kim & Iwao, 2000; Lassegue & Clempus, 2003). Angiotensin II significantly activates NADPH oxidase via AT1 receptors, resulting in the superoxide

Our studies further demonstrate that diabetes enhances the HCN currents and the expression of HCN1, HCN2, and HCH 3 channel proteins in A-type aortic baroreceptor neurons (Li *et al.*, 2008a; Tu *et al.*, 2010). Overexpression of HCN1, HCN2, and HCN3 but not HCN4 channel isoforms can link to the enhanced HCN currents, the slow-activated HCN channel kinetics, and the increased cAMP-sensitivity of HCN channels in diabetic A-type aortic baroreceptor neurons (Li *et al.*, 2008a; Tu *et al.*, 2010). Although diabetes also increases the HCN currents and the expression of HCN2 and HCN3 channel proteins in C-type aortic baroreceptor neurons, diabetes does not change the activation kinetics and the cAMP sensitivity of the HCN channels in C-type aortic baroreceptor neurons due to no expression of HCN1 channel in diabetic C-type aortic baroreceptor neurons (Li *et al.*, 2008a; Tu *et al.*, 2010). From these results, we propose that HCN currents are markedly enhanced via increasing the numbers of HCN channels and sensitivity of HCN channels to cAMP in the aortic baroreceptor neurons. The enhanced HCN currents can contribute to the depressed neuron excitability in diabetic aortic baroreceptor neurons. However, we do realize that these data cannot explain why diabetes induces the different changes of HCN channel protein expression and cannot identify the contribution of the various HCN channel isoforms to the enhanced HCN currents in diabetic A- and C-type aortic baroreceptor neurons.

#### **3.1.4 Regulation of the angiotensin II-superoxide signaling on the HCN channel in the type 1 diabetes**

Angiotensin II, an endogenous peptide, has been thought to be a prime candidate in the regulation of the HCN channel function and cell excitability in the diabetic state. It is known that circulating and tissue angiotensin II concentrations are elevated in human and animals with diabetes (Frustaci *et al.*, 2000; Sechi *et al.*, 1994; Shimoni & Liu, 2004). Previous autoradiographic study has identified a high density of angiotensin II receptor binding sites over the nodose neurons (Allen *et al.*, 1988). Widdop, et al. provided evidence for the direct neuronal effects of angiotensin II on the vagal afferent neurons (Widdop *et al.*, 1992). Indeed, our research data not only confirm AT1 and AT2 receptors exist in nodose neuronal cells, but also indicate that exogenous angiotensin II enhances the HCN currents and subsequently reduces cell excitability in the aortic baroreceptor neurons from normal rats (Zhang *et al.*, 2010). This is via NADPH oxidase-derived superoxide because a specific HCN channel blocker blunts the inhibitory effect of the exogenous angiotensin II on action potentials (Zhang *et al.*, 2010). More importantly, angiotensin II concentration and protein expression of AT1 receptors are increased in the nodose neuronal cells from STZ-induced diabetic rats (Li & Zheng, 2011). At the same time, mRNA expression of AT1 receptors measured by single cell real-time PCR technique is enhanced in the aortic baroreceptor neuron cells from the STZ-induced diabetic rats (Li & Zheng, 2011). In addition, AT1 receptor antagonist (losartan) significantly normalizes the enhanced HCN currents and the attenuated cell excitability (including depolarization of the resting membrane potential, fall in the input resistance, and decrease in the action potential number) in the aortic baroreceptor neurons induced by diabetes (Li & Zheng, 2011) or exogenous angiotensin II (Zhang *et al.*, 2010). Furthermore, angiotensin II-AT1 receptor is also involved in the attenuated arterial baroreflex sensitivity in STZ-induced diabetic rats (Fig. 1) (Li *et al.*, 2008b). Based on these results, it is reasonable to assume that elevation of local angiotensin II level can blunt the membrane excitability of the aortic baroreceptor neurons via enhancement of the HCN currents, and consequently attenuate the aortic baroreflex function in the type 1 diabetes.

Our studies further demonstrate that diabetes enhances the HCN currents and the expression of HCN1, HCN2, and HCH 3 channel proteins in A-type aortic baroreceptor neurons (Li *et al.*, 2008a; Tu *et al.*, 2010). Overexpression of HCN1, HCN2, and HCN3 but not HCN4 channel isoforms can link to the enhanced HCN currents, the slow-activated HCN channel kinetics, and the increased cAMP-sensitivity of HCN channels in diabetic A-type aortic baroreceptor neurons (Li *et al.*, 2008a; Tu *et al.*, 2010). Although diabetes also increases the HCN currents and the expression of HCN2 and HCN3 channel proteins in C-type aortic baroreceptor neurons, diabetes does not change the activation kinetics and the cAMP sensitivity of the HCN channels in C-type aortic baroreceptor neurons due to no expression of HCN1 channel in diabetic C-type aortic baroreceptor neurons (Li *et al.*, 2008a; Tu *et al.*, 2010). From these results, we propose that HCN currents are markedly enhanced via increasing the numbers of HCN channels and sensitivity of HCN channels to cAMP in the aortic baroreceptor neurons. The enhanced HCN currents can contribute to the depressed neuron excitability in diabetic aortic baroreceptor neurons. However, we do realize that these data cannot explain why diabetes induces the different changes of HCN channel protein expression and cannot identify the contribution of the various HCN channel isoforms to the enhanced HCN currents in diabetic A- and C-type aortic baroreceptor

**3.1.4 Regulation of the angiotensin II-superoxide signaling on the HCN channel in the** 

Angiotensin II, an endogenous peptide, has been thought to be a prime candidate in the regulation of the HCN channel function and cell excitability in the diabetic state. It is known that circulating and tissue angiotensin II concentrations are elevated in human and animals with diabetes (Frustaci *et al.*, 2000; Sechi *et al.*, 1994; Shimoni & Liu, 2004). Previous autoradiographic study has identified a high density of angiotensin II receptor binding sites over the nodose neurons (Allen *et al.*, 1988). Widdop, et al. provided evidence for the direct neuronal effects of angiotensin II on the vagal afferent neurons (Widdop *et al.*, 1992). Indeed, our research data not only confirm AT1 and AT2 receptors exist in nodose neuronal cells, but also indicate that exogenous angiotensin II enhances the HCN currents and subsequently reduces cell excitability in the aortic baroreceptor neurons from normal rats (Zhang *et al.*, 2010). This is via NADPH oxidase-derived superoxide because a specific HCN channel blocker blunts the inhibitory effect of the exogenous angiotensin II on action potentials (Zhang *et al.*, 2010). More importantly, angiotensin II concentration and protein expression of AT1 receptors are increased in the nodose neuronal cells from STZ-induced diabetic rats (Li & Zheng, 2011). At the same time, mRNA expression of AT1 receptors measured by single cell real-time PCR technique is enhanced in the aortic baroreceptor neuron cells from the STZ-induced diabetic rats (Li & Zheng, 2011). In addition, AT1 receptor antagonist (losartan) significantly normalizes the enhanced HCN currents and the attenuated cell excitability (including depolarization of the resting membrane potential, fall in the input resistance, and decrease in the action potential number) in the aortic baroreceptor neurons induced by diabetes (Li & Zheng, 2011) or exogenous angiotensin II (Zhang *et al.*, 2010). Furthermore, angiotensin II-AT1 receptor is also involved in the attenuated arterial baroreflex sensitivity in STZ-induced diabetic rats (Fig. 1) (Li *et al.*, 2008b). Based on these results, it is reasonable to assume that elevation of local angiotensin II level can blunt the membrane excitability of the aortic baroreceptor neurons via enhancement of the HCN currents, and consequently attenuate the aortic baroreflex function in the type 1 diabetes.

neurons.

**type 1 diabetes** 

Above results suggest that elevation of local tissue angiotensin II plays an important role on the enhanced HCN channel activity and the blunted cell excitability in the AB neurons in diabetes. However, it is unclear how angiotensin II and its antagonist within an isolated aortic baroreceptor neuron from diabetic rat interact with AT1 receptor to affect the HCN channel activity and cell excitability. Classical viewpoint about the effects of angiotensin II binding with AT1 receptor is that angiotensin II binds with AT1 receptor at the cell membrane, and following the phosphorylation of the AT1 receptor, angiotensin II induces intracellular responses via activating intracellular downstream signal transduction. However, Zhuo, et al. (Zhuo *et al.*, 2002) have found that there is substantial intracellular accumulation of angiotensin II in renal cortical endosomes during angiotensin II-dependent hypertension via an AT1 receptor-mediated process. Recent studies have shown that intracellular administration of angiotensin II increases the peak inward calcium current density and decreases the junctional conductance via intracellular angiotensin II receptors in cardiac myocytes (De Mello, 2003; De Mello & Monterrubio, 2004). Intracellular treatment of losartan (a selective AT1 receptor antagonist) abolishes the effect of intracellular angiotensin II (Allen *et al.*, 1988; Bacal & Kunze, 1994). Based on these studies, we reason that diabetesinduced elevation of intracellular angiotensin II concentration in the nodose neurons contributes to the enhanced HCN channel activity and the blunted cell excitability in the AB neurons in diabetes. This viewpoint is confirmed by our observation that intracellular administration of losartan (added to the recording pipette solution) decreased the HCN current density and increased the cell excitability in the AB neurons from diabetic rats (Li & Zheng, 2011). Therefore, it is possible there is an intracellular angiotensin II production system in the nodose ganglion tissue. Of course, it would be optimal to measure intracellular angiotensin II concentration in the aortic baroreceptor neurons, but there is no appropriate measurement for it so far due to insufficient cellular material of tiny nodose ganglia. This issue needs to be confirmed by further study.

Growing evidence has shown that the AT1 and AT2 receptors are defined on the basis of their opposite pharmacological and biochemical effects (Levy, 2004). Activation of AT1 receptors mainly results in vasoconstriction, augmentation of cardiac contractility, cell proliferation, vascular and cardiac hypertrophy, oxidative stress, and inhibition of the neuronal potassium currents (Gelband *et al.*, 1999; Levy, 2004; Sumners *et al.*, 1996). On the other hand, stimulation of AT2 receptors induces vasodilation, anti-growth, antihypertrophy, and enhancement of the neuronal potassium currents (Horiuchi *et al.*, 1999; Kang *et al.*, 1995; Martens *et al.*, 1996; Matsubara, 1998; Siragy, 2000). Although AT2 receptors are expressed in the rat nodose neurons, activation of AT2 receptors does not affect the activation of HCN channels because AT2 receptor antagonist (PD123,319) does not alter the effect of angiotensin II on the HCN currents (Zhang *et al.*, 2010). Until now there is no study to explain this result, but it is possible that many factors (such as species, tissue, channel sensitivity, etc) are responsible for this discrepancy.

Now the question is how angiotensin II regulates the activation of HCN channels and what is the downstream of angiotensin II-AT1 receptor. NADPH oxidase has been considered as a main source of intracellular superoxide in many tissues (Cifuentes *et al.*, 2000; Franco *et al.*, 2003; Gao *et al.*, 2004; Griendling *et al.*, 2000; Li *et al.*, 2007; Schieffer *et al.*, 2000). NADPH oxidase is a multicomponent enzyme composed of three cytosolic subunits (p40phox, p47phox, and p67phox), two membrane-associated subunits (gp91phox and p22phox), and the small Gproteins (Rac and Rap1a) (Kim & Iwao, 2000; Lassegue & Clempus, 2003). Angiotensin II significantly activates NADPH oxidase via AT1 receptors, resulting in the superoxide

Cardiovascular Autonomic Dysfunction in

arterial baroreflex.

central integration.

**4. Conclusion** 

diabetic patients.

**baroreflex in type 1 diabetes** 

Diabetes as a Complication: Cellular and Molecular Mechanisms 181

rats (do Carmo *et al.*, 2008), which indirectly suggests that impairment of the central neural system is associated with the arterial baroreflex dysfunction in type 1 diabetes. These findings allow us to assume the involvement of the impaired central neural integration in the blunted arterial baroreflex in type 1 diabetes even though there is no report focusing on the mechanisms responsible for the impairment of the central neural component of the

The autonomic neuroeffector component of the arterial baroreflex includes intracardiac ganglia, parasympathetic efferents, and sympathetic efferents. Morphological studies have shown that there is a remarkable structural remodeling of the intracardiac ganglia (such as cellular contraction, cytoplasmic condensation, degenerated axons, reduced cell size and number) in STZ-induced diabetic rats (Kamal *et al.*, 1991; Lund *et al.*, 1992), mice (Lin *et al.*, 2010), and diabetic patients (Tsujimura *et al.*, 1986). Biochemical studies also found a decrease in acetylcholine (a neurotransmitter in both the central and parasympathetic nervous system) concentration in alloxan-induced diabetic rats (Kuntscherova & Vlk, 1970) and a reduced choline acetyltransferase activity (an enzyme producing acetylcholine) in the hearts of the STZ-induced diabetic rats (Lund *et al.*, 1992). In addition, the function of the parasympathetic (vagal) efferent is reduced in STZ-induced diabetic rats (Maeda *et al.*, 1995; Yagihashi, 1995). However, functional studies have reported normal, reduced, or enhanced heart rate response to vagal efferent nerve stimulation in diabetic animal models (Dall'Ago *et al.*, 2007; de *et al.*, 2002; Lin *et al.*, 2010; Maeda *et al.*, 1995; McDowell *et al.*, 1994a). This discrepancy might be due to different animal species, experimental diabetic animal models, and time course of development of diabetes. Therefore, further studies are needed to explore whether the altered efferent component of the arterial baroreflex is responsible for the arterial baroreflex dysfunction in type 1 diabetes besides the arterial baroreceptor and

As a homeostatic mechanism, the arterial baroreflex normally alters heart rate and blood pressure in response to changes in arterial wall tension detected by the baroreceptors in the carotid sinus and aortic arch. As illustrated by the above evidence, arterial baroreflex impairment, a characteristic of the autonomic cardiovascular dysfunction is a frequent complication in type 1 diabetic patients and animal models. The arterial baroreflex dysfunction not only is an independent predictor for mortality of the type 1 diabetic patients, but also is associated with a poor prognosis and bad quality of life in the type 1

Although the mechanisms responsible for attenuated arterial baroreflex function in the type 1 diabetes are not yet fully understood, any part of the arterial baroreflex arc including an afferent limb, a central neural component, and an autonomic neuroeffector component can contribute to the arterial baroreflex dysfunction in the type 1 diabetic state. Especially at the level of the afferent limb, recent studies have revealed that aortic depressor nerve discharge and excitability of aortic baroreceptor neurons are blunted in the type 1 diabetic animals. HCN channels are significantly suppressed in the aortic baroreceptor neurons and are involved in the blunted baroreceptor neuron excitability in the type 1 diabetes. Angiotensin

**3.3 Participation of autonomic neuroeffector component in the blunted arterial** 

production (Touyz & Berry, 2002). In the nodose ganglia from STZ-induced diabetic rats, the protein expression of the NADPH oxidase components (gp91phox, p22phox, p40phox, p47phox, and p67phox) is elevated, compared to sham rats (Li & Zheng, 2011). In addition, NADPH oxidase inhibitor or superoxide scavenger significantly improves the superoxide overproduction, the enhanced HCN currents, and the lowered membrane excitability induced by exogenous angiotensin II (Zhang *et al.*, 2010) or diabetes (Li & Zheng, 2011). These results strongly indicate that NADPH-derived superoxide can mediate the effect of endogenous angiotensin II on the HCN channels and membrane excitability in diabetic rat aortic baroreceptor neurons.

#### **3.1.5 Role of other channels in the aortic baroreceptor neuron in the type 1 diabetes**

Using patch-clamp technique, all major voltage-gated ion channels including channels subunits are recorded in the nodose neurons, such as sodium channels (tetrodotoxinsensitive and tetrodotoxin-resistant sodium channels), calcium channels (N-type, L-type, T- /R-type, and other type calcium channels), and potassium channels (4-aminopyridinesensitive, tetraethylammonium-sensitive, and calcium-activated potassium channels) (Lancaster *et al.*, 2002; Li *et al.*, 2005; Li & Schild, 2006; Schild & Li, 2001). These channels all are involved in the initiation and formation of the action potential and affect the nodose neuron excitability. Angiotensin II is known to modulate the calcium channel kinetics in the nodose neurons (Bacal & Kunze, 1994; Moreira *et al.*, 2005). However, until now we did not obtain any information about the changes of these channels in the aortic baroreceptor neurons in the type 1 diabetes. Therefore, the role of these channels in the diabetic baroreceptor neurons remains to be revealed.

#### **3.2 Involvement of the central neural component in the blunted arterial baroreflex in type 1 diabetes**

Central neural integration of the input signals from the baroreceptors usually occurs at the level of nucleus tractus solitarii and rostral ventrolateral medulla (Spyer *et al.*, 1997). Although many studies have shown that diabetes causes a variety of functional and morphological disorders in the central nervous system (including hippocampus, cortex, and cerebellum) (Biessels *et al.*, 1999; Selvarajah & Tesfaye, 2006; Mooradian, 1997a; Mooradian, 1997b; Guven *et al.*, 2009), the role of the central neural component in the blunted arterial baroreflex in type 1 diabetes is less well documented. One recent study from Gu, et al (Gu *et al.*, 2008) suggests that a deficit of the central neural component contributes to the attenuation of arterial baroreflex in OVE26 type 1 diabetic mice. This is because they found that stimulation of the aortic depressor nerve induced a lesser magnitude of bradycardia in OVE26 type 1 diabetic mice as compared to sham mice, but the bradycardic response to vagal efferent stimulation was enhanced (Gu *et al.*, 2008). Immunoreactive study has shown that reduced c-Fos expression (an indicator of early cellular response to many extracellular signals) in the nucleus tractus solitarii links to the attenuated arterial baroreflex sensitivity in STZ-induced diabetic rats (Gouty *et al.*, 2001). In addition, Chen, et al (Chen *et al.*, 2008) have reported that neural firing activity of the nucleus tractus solitarii in STZ-induced diabetes is reduced, which is involved in the impaired arterial baroreflex function in STZ-induced diabetic rats. Furthermore, a chronic intracerebroventricular infusion of leptin (a hormone produced by fat cells and improving glucose utilization, Minokoshi *et al.*, 1999; Wang *et al.*, 1999) totally normalizes the impaired arterial baroreflex sensitivity in STZ-induced diabetic rats (do Carmo *et al.*, 2008), which indirectly suggests that impairment of the central neural system is associated with the arterial baroreflex dysfunction in type 1 diabetes. These findings allow us to assume the involvement of the impaired central neural integration in the blunted arterial baroreflex in type 1 diabetes even though there is no report focusing on the mechanisms responsible for the impairment of the central neural component of the arterial baroreflex.

#### **3.3 Participation of autonomic neuroeffector component in the blunted arterial baroreflex in type 1 diabetes**

The autonomic neuroeffector component of the arterial baroreflex includes intracardiac ganglia, parasympathetic efferents, and sympathetic efferents. Morphological studies have shown that there is a remarkable structural remodeling of the intracardiac ganglia (such as cellular contraction, cytoplasmic condensation, degenerated axons, reduced cell size and number) in STZ-induced diabetic rats (Kamal *et al.*, 1991; Lund *et al.*, 1992), mice (Lin *et al.*, 2010), and diabetic patients (Tsujimura *et al.*, 1986). Biochemical studies also found a decrease in acetylcholine (a neurotransmitter in both the central and parasympathetic nervous system) concentration in alloxan-induced diabetic rats (Kuntscherova & Vlk, 1970) and a reduced choline acetyltransferase activity (an enzyme producing acetylcholine) in the hearts of the STZ-induced diabetic rats (Lund *et al.*, 1992). In addition, the function of the parasympathetic (vagal) efferent is reduced in STZ-induced diabetic rats (Maeda *et al.*, 1995; Yagihashi, 1995). However, functional studies have reported normal, reduced, or enhanced heart rate response to vagal efferent nerve stimulation in diabetic animal models (Dall'Ago *et al.*, 2007; de *et al.*, 2002; Lin *et al.*, 2010; Maeda *et al.*, 1995; McDowell *et al.*, 1994a). This discrepancy might be due to different animal species, experimental diabetic animal models, and time course of development of diabetes. Therefore, further studies are needed to explore whether the altered efferent component of the arterial baroreflex is responsible for the arterial baroreflex dysfunction in type 1 diabetes besides the arterial baroreceptor and central integration.

#### **4. Conclusion**

180 Type 1 Diabetes Complications

production (Touyz & Berry, 2002). In the nodose ganglia from STZ-induced diabetic rats, the protein expression of the NADPH oxidase components (gp91phox, p22phox, p40phox, p47phox, and p67phox) is elevated, compared to sham rats (Li & Zheng, 2011). In addition, NADPH oxidase inhibitor or superoxide scavenger significantly improves the superoxide overproduction, the enhanced HCN currents, and the lowered membrane excitability induced by exogenous angiotensin II (Zhang *et al.*, 2010) or diabetes (Li & Zheng, 2011). These results strongly indicate that NADPH-derived superoxide can mediate the effect of endogenous angiotensin II on the HCN channels and membrane excitability in diabetic rat aortic

**3.1.5 Role of other channels in the aortic baroreceptor neuron in the type 1 diabetes**  Using patch-clamp technique, all major voltage-gated ion channels including channels subunits are recorded in the nodose neurons, such as sodium channels (tetrodotoxinsensitive and tetrodotoxin-resistant sodium channels), calcium channels (N-type, L-type, T- /R-type, and other type calcium channels), and potassium channels (4-aminopyridinesensitive, tetraethylammonium-sensitive, and calcium-activated potassium channels) (Lancaster *et al.*, 2002; Li *et al.*, 2005; Li & Schild, 2006; Schild & Li, 2001). These channels all are involved in the initiation and formation of the action potential and affect the nodose neuron excitability. Angiotensin II is known to modulate the calcium channel kinetics in the nodose neurons (Bacal & Kunze, 1994; Moreira *et al.*, 2005). However, until now we did not obtain any information about the changes of these channels in the aortic baroreceptor neurons in the type 1 diabetes. Therefore, the role of these channels in the diabetic

**3.2 Involvement of the central neural component in the blunted arterial baroreflex in** 

Central neural integration of the input signals from the baroreceptors usually occurs at the level of nucleus tractus solitarii and rostral ventrolateral medulla (Spyer *et al.*, 1997). Although many studies have shown that diabetes causes a variety of functional and morphological disorders in the central nervous system (including hippocampus, cortex, and cerebellum) (Biessels *et al.*, 1999; Selvarajah & Tesfaye, 2006; Mooradian, 1997a; Mooradian, 1997b; Guven *et al.*, 2009), the role of the central neural component in the blunted arterial baroreflex in type 1 diabetes is less well documented. One recent study from Gu, et al (Gu *et al.*, 2008) suggests that a deficit of the central neural component contributes to the attenuation of arterial baroreflex in OVE26 type 1 diabetic mice. This is because they found that stimulation of the aortic depressor nerve induced a lesser magnitude of bradycardia in OVE26 type 1 diabetic mice as compared to sham mice, but the bradycardic response to vagal efferent stimulation was enhanced (Gu *et al.*, 2008). Immunoreactive study has shown that reduced c-Fos expression (an indicator of early cellular response to many extracellular signals) in the nucleus tractus solitarii links to the attenuated arterial baroreflex sensitivity in STZ-induced diabetic rats (Gouty *et al.*, 2001). In addition, Chen, et al (Chen *et al.*, 2008) have reported that neural firing activity of the nucleus tractus solitarii in STZ-induced diabetes is reduced, which is involved in the impaired arterial baroreflex function in STZ-induced diabetic rats. Furthermore, a chronic intracerebroventricular infusion of leptin (a hormone produced by fat cells and improving glucose utilization, Minokoshi *et al.*, 1999; Wang *et al.*, 1999) totally normalizes the impaired arterial baroreflex sensitivity in STZ-induced diabetic

baroreceptor neurons.

**type 1 diabetes** 

baroreceptor neurons remains to be revealed.

As a homeostatic mechanism, the arterial baroreflex normally alters heart rate and blood pressure in response to changes in arterial wall tension detected by the baroreceptors in the carotid sinus and aortic arch. As illustrated by the above evidence, arterial baroreflex impairment, a characteristic of the autonomic cardiovascular dysfunction is a frequent complication in type 1 diabetic patients and animal models. The arterial baroreflex dysfunction not only is an independent predictor for mortality of the type 1 diabetic patients, but also is associated with a poor prognosis and bad quality of life in the type 1 diabetic patients.

Although the mechanisms responsible for attenuated arterial baroreflex function in the type 1 diabetes are not yet fully understood, any part of the arterial baroreflex arc including an afferent limb, a central neural component, and an autonomic neuroeffector component can contribute to the arterial baroreflex dysfunction in the type 1 diabetic state. Especially at the level of the afferent limb, recent studies have revealed that aortic depressor nerve discharge and excitability of aortic baroreceptor neurons are blunted in the type 1 diabetic animals. HCN channels are significantly suppressed in the aortic baroreceptor neurons and are involved in the blunted baroreceptor neuron excitability in the type 1 diabetes. Angiotensin

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#### **5. Acknowledgments**

This work was supported by a Scientist Development Grant from American Heart Association National Center (award no. 0730108N) to Yu-Long Li.

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II/AT1 receptor-NADPH oxidase-superoxide signaling regulates this alteration of the HCN channels in the aortic baroreceptor neurons and consequently decreases the arterial baroreflex function. In addition, we also consider that angiotensin II/AT1 receptor-NADPH oxidase-superoxide signaling affects the changes in the central neural and autonomic neuroeffector components beyond the afferent limb of the arterial baroreflex arc. These studies provide new information on the mechanisms underlying the impaired arterial baroreflex in the type 1 diabetes and unveil important pharmacological and genomic targets for improving the arterial baroreflex function and reducing the mortality in the type 1

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Leutenegger, M. & Attali, J.R. (2001). Predictive value of cardiac autonomic neuropathy in diabetic patients with or without silent myocardial ischemia.


**9** 

*Italy* 

**Type 1 Diabetes** 

**Microvascular and Macrovascular** 

Francesco Chiarelli and M. Loredana Marcovecchio

*Department of Paediatrics, University of Chieti,* 

**Complications in Children and Adolescents with** 

Diabetes mellitus is a serious chronic disorder of childhood and represents a major public health problem. Type 1 diabetes is the predominant form of diabetes during childhood and adolescence, accounting for about 90% of cases, although the growing epidemic of obesity has been associated with an increasing number of cases of childhood-onset type 2 diabetes

The global incidence of type 1 diabetes is increasing worldwide, at an annual rate of 3-5%, particularly in children under the age of 5 years, and this trend leads to a significant health burden (Patterson et al., 2009). Recent studies have shown that in European countries childhood-onset type 1 diabetes is associated with three to four fold increased mortality when compared with the general population (Asao et al., 2003; Skrivarhaug et al., 2006). Similar data emerged from a long-term study of a young cohort with type 1 diabetes in the USA, where mortality was 7 times higher than in the non-diabetic population (Secrest et al., 2010a). The high mortality reported for individuals with type 1 diabetes is mainly due to diabetes-related acute and chronic complications. As recently emerged from a large population-based cohort with long-standing childhood-onset type 1 diabetes, during the first decade of diabetes acute complications, such as diabetic ketoacidosis and hypoglycemia, are the main causes of death, being responsible for about 73% of cases, whereas during subsequent decades cardiovascular (CVD) and renal diseases become the

Diabetes vascular complications are divided in micro- and macrovascular disease. Microvascular complications include nephropathy (DN), retinopathy (DR) and neuropathy, whereas macrovascualar complications refer to cardiovascular, cerebrovascular and peripheral vascular disease (Marshall and Flyvbjerg, 2006). As a result of vascular complications, diabetes is the leading cause of blindness in working age people, is responsible for up to 40% cases of renal failure and is a major determinant of cardiovascular

Diabetic microvascular complications result from damage to the microvasculature of the

kidney, retina and neurons and they generally progress throughout different stages**.** 

**1. Introduction** 

(Craig et al., 2009; Patterson et al., 2009).

main determinants of mortality (Secrest et al., 2010b).

morbidity and mortality (Marshall and Flyvbjerg, 2006).

**2. Microvascular complications of type 1 diabetes** 


### **Microvascular and Macrovascular Complications in Children and Adolescents with Type 1 Diabetes**

Francesco Chiarelli and M. Loredana Marcovecchio *Department of Paediatrics, University of Chieti, Italy* 

#### **1. Introduction**

194 Type 1 Diabetes Complications

Yagihashi, S. (1995). Pathology and pathogenetic mechanisms of diabetic neuropathy.

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Yamasaki, M.; Shimizu, T.; Katahira, K.; Waki, H.; Nagayama, T.; Ishi, H.; Katsuda, S.;

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Ziegler D, Laude D, Akila F, & Elghozi JL (2001). Time- and frequency-domain estimation of

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4221

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911X

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treatment. *Diabetes/Metabolism Reviews*, Vol. 10, No. 4, (December, 1994), pp. 339-

early diabetic cardiovascular autonomic neuropathy. *Clinical Autonomic Research*,

Diabetes mellitus is a serious chronic disorder of childhood and represents a major public health problem. Type 1 diabetes is the predominant form of diabetes during childhood and adolescence, accounting for about 90% of cases, although the growing epidemic of obesity has been associated with an increasing number of cases of childhood-onset type 2 diabetes (Craig et al., 2009; Patterson et al., 2009).

The global incidence of type 1 diabetes is increasing worldwide, at an annual rate of 3-5%, particularly in children under the age of 5 years, and this trend leads to a significant health burden (Patterson et al., 2009). Recent studies have shown that in European countries childhood-onset type 1 diabetes is associated with three to four fold increased mortality when compared with the general population (Asao et al., 2003; Skrivarhaug et al., 2006). Similar data emerged from a long-term study of a young cohort with type 1 diabetes in the USA, where mortality was 7 times higher than in the non-diabetic population (Secrest et al., 2010a). The high mortality reported for individuals with type 1 diabetes is mainly due to diabetes-related acute and chronic complications. As recently emerged from a large population-based cohort with long-standing childhood-onset type 1 diabetes, during the first decade of diabetes acute complications, such as diabetic ketoacidosis and hypoglycemia, are the main causes of death, being responsible for about 73% of cases, whereas during subsequent decades cardiovascular (CVD) and renal diseases become the main determinants of mortality (Secrest et al., 2010b).

Diabetes vascular complications are divided in micro- and macrovascular disease. Microvascular complications include nephropathy (DN), retinopathy (DR) and neuropathy, whereas macrovascualar complications refer to cardiovascular, cerebrovascular and peripheral vascular disease (Marshall and Flyvbjerg, 2006). As a result of vascular complications, diabetes is the leading cause of blindness in working age people, is responsible for up to 40% cases of renal failure and is a major determinant of cardiovascular morbidity and mortality (Marshall and Flyvbjerg, 2006).

#### **2. Microvascular complications of type 1 diabetes**

Diabetic microvascular complications result from damage to the microvasculature of the kidney, retina and neurons and they generally progress throughout different stages**.** 

Microvascular and Macrovascular

as nephrin (Fioretto and Mauer, 2007).

**2.1.2 Epidemiology of diabetic nepropathy** 

DN (Mauer and Najafian, 2006).

progression in earlier studies.

the last decades.

2010).

Complications in Children and Adolescents with Type 1 Diabetes 197

Changes in the podocyte is another key feature of DN (Mauer and Najafian, 2006). Podocytes are highly specialised epithelial cells, interconnected by foot processes, which delimit the slit diaphragm, the main size-selective barrier in the glomerulus. The first detectable alteration in podocytes in the context of DN is a broadening and effacement of their foot processes (Kriz et al., 1998), which leads to a decrease in their density and number and a detachment from the glomerular basement membrane. The last phenomena are directly correlated to levels of albumin-creatinine ratio (ACR) and to the decline in GFR (Fioretto and Mauer, 2007). Podocytes can also undergo hypertrophy, apoptosis and increased synthesis of collagen IV, whereas there is a decreased synthesis of proteins, such

Another characteristic of DN is the hyalinosis of the afferent and efferent arterioles in the glomerulus, due to the accumulation of complement components, fibrinogen, immunoglobulins, albumin and other plasma proteins (Fioretto and Mauer, 2007). Hypertrophy and sclerosis of the iuxtaglomerular apparatus is another frequent finding in

DN affects about one third of people with type 1 diabetes after a disease duration of 20 years and represents an important determinant of mortality (Jones et al., 2005). This has been recently confirmed in patients with both childhood-onset and adult-onset type 1 diabetes (Groop et al., 2009; Orchard et al., 2010). In a large cohort of adults with type 1 diabetes the presence of microalbuminuria, macroalbuminuria and ESRD was associated with 2.8, 9.2, and 18.3 times higher standardized mortality ratio, respectively (Groop et al., 2009). Similarly, Orchard *et al*. reported a standardized mortality ratio of 6.4, 12.5 and 29.8 for individuals with microalbuminuria, proteinuria and ESRD, respectively, in a cohort with childhood-onset type 1 diabetes followed longitudinally for 30 years (Orchard et al.,

The natural history of DN has changed over the last decades and, whereas earlier landmark studies indicated that patients with microalbuminuria had a 60-85% risk of progressing to overt proteinuria within 6-14 years (Mogensen and Christensen, 1984; Parving et al., 1982; Viberti et al., 1982), more recent studies have reported a rate of progression of 30% over 10 years, and an increasing number of cases of regression to normoalbuminuria (31 to 58%) (de Boer et al., 2011; Hovind et al., 2004; Perkins et al., 2003). Regression to normoalbuminuria has been associated with a better metabolic control, a better lipid and blood pressure profile as well as with non-modifiable risk factors, such as younger age and shorter duration of microalbuminuria (Hovind et al., 2004; Perkins et al., 2003). These data are encouraging and highlight the positive effect of improvements in treatment, particularly glycemic (1993) and blood pressure control (Lewis et al., 1993), in influencing the natural history of DN. However, this changing trend may be also related to an overestimation of the rate of

With regards to young people with childhood onset type 1 diabetes, recent studies from Sweden (Nordwall et al., 2004) and Australia (Mohsin et al., 2005) have shown a decreasing trend in DN. However, these positive results have not been consistently reported. In fact, data from Iceland (Tryggvason et al., 2005) and the UK (Amin et al., 2009) indicated an unchanged trend in the incidence of microalbuminuria and DN over

#### **2.1 Diabetic nephropathy**

#### **2.1.1 Classification of diabetic nephropathy and structural kidney changes**

The changing occurring in the kidney in patients with type 1 diabetes are generally classified in five stages (Mogensen, 1999). The first stage is characterized by increases in glomerular filtration rate (GFR) and kidney hypertrophy. During the second phase subtle morphological changes occur together with progressive increases in urinary albumin excretion within the normal range. The third stage, also called incipient nephropathy, is characterized by the development of microalbuminuria, defined as an albumin excretion rate (AER) between 30-300 mg/24h or 20-200 µg/min, and by more profound structural changes. During the fourth phase there is a further increase in AER leading to macroalbuminuria (AER >200 µg/min or >300 mg/24h) and a consistent fall in GFR. Without any treatment this phase leads to the final stage of end stage renal disease (ESRD).

Typical morphological changes occurring in the diabetic kidney are represented by diffuse glomerular basement membrane thickening, mesangial expansion, hyalinosis of the mesangium and arteriolar walls, broadening and effacement of podocyte foot processes, reduction in podocyte number, glomerulosclerosis and tubule-interstitial fibrosis (Osterby, 1992). These morphological changes develop years before the clinical appearance of DN and this is an alarming aspect, given that when the disease is clinically evident some of the structural damage is already irreversible.

Thickening of the basement membrane is a common bioptic finding related to DN and is associated with loss of glycosaminoglycans and associated negative charges, with consequent increased loss of anionic albumin (Fioretto and Mauer, 2007; Fioretto et al., 1994; Mauer and Najafian, 2006). A subsequent increase in the size of membrane pores leads to the development of non-selective proteinuria. An imbalance between the production and the degradation of mesangial matrix proteins, together with an increase in mesangial cells number, is responsible for mesangial expansion in DN (Fioretto and Mauer, 2007; Mauer and Najafian, 2006).

Fig. 1. Stages of diabetic nephropathy

The changing occurring in the kidney in patients with type 1 diabetes are generally classified in five stages (Mogensen, 1999). The first stage is characterized by increases in glomerular filtration rate (GFR) and kidney hypertrophy. During the second phase subtle morphological changes occur together with progressive increases in urinary albumin excretion within the normal range. The third stage, also called incipient nephropathy, is characterized by the development of microalbuminuria, defined as an albumin excretion rate (AER) between 30-300 mg/24h or 20-200 µg/min, and by more profound structural changes. During the fourth phase there is a further increase in AER leading to macroalbuminuria (AER >200 µg/min or >300 mg/24h) and a consistent fall in GFR. Without any treatment this phase leads to the final stage of end stage renal disease (ESRD). Typical morphological changes occurring in the diabetic kidney are represented by diffuse glomerular basement membrane thickening, mesangial expansion, hyalinosis of the mesangium and arteriolar walls, broadening and effacement of podocyte foot processes, reduction in podocyte number, glomerulosclerosis and tubule-interstitial fibrosis (Osterby, 1992). These morphological changes develop years before the clinical appearance of DN and this is an alarming aspect, given that when the disease is clinically evident some of the

Thickening of the basement membrane is a common bioptic finding related to DN and is associated with loss of glycosaminoglycans and associated negative charges, with consequent increased loss of anionic albumin (Fioretto and Mauer, 2007; Fioretto et al., 1994; Mauer and Najafian, 2006). A subsequent increase in the size of membrane pores leads to the development of non-selective proteinuria. An imbalance between the production and the degradation of mesangial matrix proteins, together with an increase in mesangial cells number, is responsible

• AER: May be increased, but it becomes normal after starting insulin treatment

• AER: Normal with transient increases related to poor glycemic control or exercise

for mesangial expansion in DN (Fioretto and Mauer, 2007; Mauer and Najafian, 2006).

• Hyperfiltration/hyperperfusion phase (often at diagnosis)

• Normoalbuminuria/Silent phase (2-5 years after diagnosis)

• Microalbuminuria or incipient nephropathy phase (after 6-15 years) • Further basement membrane thickening and mesangial expansion

• Overt diabetic nephropathy/Macroalbuminuria phase (after 15-25 years) • Marked renal abnormalities, reduced renal function, increased blood pressure

• Advanced glomerulopathy; marked decreased renal function, hypertension • AER: Macroalbuminuria/ Often decreased due to glomerular occlusion

**2.1.1 Classification of diabetic nephropathy and structural kidney changes** 

**2.1 Diabetic nephropathy** 

structural damage is already irreversible.

• Increased renal size

• Basement membrane thickening

• AER: 20-200µg/min or 30-300 mg/24h

• AER: >200 µg/min or >300 mg/24h

• End stage renal disease phase (after 25-30 years)

Stage 1

Stage 2

Stage 3

Stage 4

Stage 5

Fig. 1. Stages of diabetic nephropathy

Changes in the podocyte is another key feature of DN (Mauer and Najafian, 2006). Podocytes are highly specialised epithelial cells, interconnected by foot processes, which delimit the slit diaphragm, the main size-selective barrier in the glomerulus. The first detectable alteration in podocytes in the context of DN is a broadening and effacement of their foot processes (Kriz et al., 1998), which leads to a decrease in their density and number and a detachment from the glomerular basement membrane. The last phenomena are directly correlated to levels of albumin-creatinine ratio (ACR) and to the decline in GFR (Fioretto and Mauer, 2007). Podocytes can also undergo hypertrophy, apoptosis and increased synthesis of collagen IV, whereas there is a decreased synthesis of proteins, such as nephrin (Fioretto and Mauer, 2007).

Another characteristic of DN is the hyalinosis of the afferent and efferent arterioles in the glomerulus, due to the accumulation of complement components, fibrinogen, immunoglobulins, albumin and other plasma proteins (Fioretto and Mauer, 2007). Hypertrophy and sclerosis of the iuxtaglomerular apparatus is another frequent finding in DN (Mauer and Najafian, 2006).

#### **2.1.2 Epidemiology of diabetic nepropathy**

DN affects about one third of people with type 1 diabetes after a disease duration of 20 years and represents an important determinant of mortality (Jones et al., 2005). This has been recently confirmed in patients with both childhood-onset and adult-onset type 1 diabetes (Groop et al., 2009; Orchard et al., 2010). In a large cohort of adults with type 1 diabetes the presence of microalbuminuria, macroalbuminuria and ESRD was associated with 2.8, 9.2, and 18.3 times higher standardized mortality ratio, respectively (Groop et al., 2009). Similarly, Orchard *et al*. reported a standardized mortality ratio of 6.4, 12.5 and 29.8 for individuals with microalbuminuria, proteinuria and ESRD, respectively, in a cohort with childhood-onset type 1 diabetes followed longitudinally for 30 years (Orchard et al., 2010).

The natural history of DN has changed over the last decades and, whereas earlier landmark studies indicated that patients with microalbuminuria had a 60-85% risk of progressing to overt proteinuria within 6-14 years (Mogensen and Christensen, 1984; Parving et al., 1982; Viberti et al., 1982), more recent studies have reported a rate of progression of 30% over 10 years, and an increasing number of cases of regression to normoalbuminuria (31 to 58%) (de Boer et al., 2011; Hovind et al., 2004; Perkins et al., 2003). Regression to normoalbuminuria has been associated with a better metabolic control, a better lipid and blood pressure profile as well as with non-modifiable risk factors, such as younger age and shorter duration of microalbuminuria (Hovind et al., 2004; Perkins et al., 2003). These data are encouraging and highlight the positive effect of improvements in treatment, particularly glycemic (1993) and blood pressure control (Lewis et al., 1993), in influencing the natural history of DN. However, this changing trend may be also related to an overestimation of the rate of progression in earlier studies.

With regards to young people with childhood onset type 1 diabetes, recent studies from Sweden (Nordwall et al., 2004) and Australia (Mohsin et al., 2005) have shown a decreasing trend in DN. However, these positive results have not been consistently reported. In fact, data from Iceland (Tryggvason et al., 2005) and the UK (Amin et al., 2009) indicated an unchanged trend in the incidence of microalbuminuria and DN over the last decades.

Microvascular and Macrovascular

Severe nonproliferative DR

Proliferative DR

1995; Scherrer et al., 1994).

Complications in Children and Adolescents with Type 1 Diabetes 199

hemorrhages, exudates and the development of vascular obstruction, intraretinal microvascular abnormalities, and infarction of the retinal nerve fibers causing cotton wool spots (Aiello et al., 1998; D'Amico, 1994; Williams and Pickup, 2004). Although this stage is not sight-threatening, it is highly predictive of progression to more advanced stages of retinopathy. Proliferative retinopathy is characterized by the development of new vessels, secondary to ischemia, on the surface of the retina and/or the optic disc (Aiello et al., 1998; D'Amico, 1994; Williams and Pickup, 2004). These new vessels can bleed into the vitroretinal space, and cause visual loss. In addition, the subsequent formation of fibrous tissue can cause tractional retinal detachment (Williams and Pickup, 2004). This stage is associated with a high

Diabetic macular oedema can complicate both non-proliferative and proliferative retinopathy and is a serious cause of vision loss in patients with diabetes (Ciulla et al., 2003). It is characterized by increased microvascular permeability and deposition of hard retinal exudates (Ciulla et al., 2003). This stage involves the breakdown of the blood-retinal barrier, with leakage of plasma from small blood vessels in the macula. Swelling of the macula is then followed by deposition of hard retinal exudates as a consequence of deposition of

**Proposed disease severity level Dilated ophthalmoscopy findings** 

Moderate nonproliferative DR More than just microaneurysms, but less than

Retinal damage in diabetes is mainly due to leakage of retinal blood vessels and inadequate retinal perfusion (Aiello et al., 1998; D'Amico, 1994; Williams and Pickup, 2004). It has been suggested that one of the initial alterations in the retinal hemodynamic is represented by retinal vasodilatation and hyperperfusion, due to hypoxia and increased release of nitric oxide (Joussen et al., 2004). This is followed by an impairment of the retinal vascular autoregulation with increased tension in the epithelial wall and increased vascular permeability, which, in turn, cause vascular leakage and aneurysm formation (Kohner et al.,

following:

quadrants

severe nonproliferative DR

each of four quadrants

One or more of the following: - Neovascularization


No signs of proliferative DR, with any of the




risk for visual impairment related to hemorrhages and retinal detachment.

lipids and lipoproteins following plasma re-absorption (Ciulla et al., 2003).

No apparent retinopathy No abnormalities

Table 1. International clinical DR disease severity scale

Mild nonproliferative DR Microaneurysms only

#### **2.1.3 Early manifestations of diabetic nephropathy in youth with type 1 diabetes**

Microalbuminuria is the most common abnormal finding in children and adolescents with type 1 diabetes, whereas overt proteinuria is found in less than 1–1.5% of them (Jones et al., 1998; Schultz et al., 1999b). Microalbuminuria often develops during puberty, whereas its prevalence during prepubertal years is rare (Janner et al., 1994; Lawson et al., 1996; Norgaard et al., 1989; Rudberg et al., 1993). The overall prevalence of microalbuminuria in youth with type 1 diabetes ranges between 4 and 26% (Bojestig et al., 1996; Cook and Daneman, 1990; Dahlquist and Rudberg, 1987; Joner et al., 1992; Jones et al., 1998; Mathiesen et al., 1986; Moore and Shield, 2000; Norgaard et al., 1989; Olsen et al., 2000; Rudberg et al., 1993; Schultz et al., 1999b). This large variation in the prevalence of microalbuminuria is due to differences in study design, duration of diabetes, age range, and glycemic control. Most studies investigating microalbuminuria have been cross-sectional and clinic based, with only a few being longitudinal, but mainly with a retrospective design. Jones *et al.* (Jones et al., 1998) reported a prevalence of 14.5% during 8.5 years of diabetes duration, whereas Rudberg *et al.* (Rudberg et al., 1993) reported a prevalence of 24% after 15 years diabetes duration. The most recent data from the Oxford Regional Prospective Study (ORPS), a population-based inception cohort of children with type 1 diabetes, has shown a cumulative prevalence of microalbuminuria of 25.7% after 10 years and of 50.7% after 19 years of diabetes duration (Amin et al., 2008).

After puberty, rates of rise in albumin excretion tend to decline and, longitudinal studies suggest that microalbuminuria is persistent in only 50% of adolescents; whereas in the other 40-50% urinary albumin excretion returns into the normal range after 3-10 years from the onset of microalbuminuria (Amin et al., 2008; Gorman et al., 1999). However, with longer follow-up cases of transient or intermittent microalbuminuria may become persistent. In addition, although urinary albumin excretion may return into the normal range, the renal morphological changes associated with microalbuminuria can persist and increase the risk of its recurrence and progression (Steinke and Mauer, 2008). Few data are available on the rate of progression of microalbuminuria to macroalbuminuria in young people with type 1 diabetes. The ORPS has shown a progression rate of around 13% after 3.2 years from the onset of microalbuminuria, a rate similar to that reported in adults (Amin et al., 2008).

Another renal abnormality often detected in youth with type 1 diabetes is increased GFR (Amin et al., 2005; Rudberg et al., 1992). Hyperfiltration, which has been associated with increased renal size often precedes the onset of microalbuminuria (Amin et al., 2005; Rudberg et al., 1992). In some, although not all studies, increased GFR has emerged as an independent predictor of microalbuminuria, and a recent meta-analysis has reported a 2.7 increased risk of developing microalbuminuria associated with hyperfiltration (Magee et al., 2009).

#### **2.2 Diabetic retinopathy**

#### **2.2.1 Classification and structural changes**

DR begins with the appearance of non-proliferative retinal abnormalities, which then progress to sight-threatening proliferative lesions (Aiello et al., 1998; Williams and Pickup, 2004) . The early stage of DR is characterised by the development of capillary microaneuryms, which consist in small blind outgrowths of retinal capillaries developing in areas where the wall is weakened (Aiello et al., 1998; D'Amico, 1994; Williams and Pickup, 2004). As the retinal damage progresses, there is the appearance of non-proliferative abnormalities, including

After puberty, rates of rise in albumin excretion tend to decline and, longitudinal studies suggest that microalbuminuria is persistent in only 50% of adolescents; whereas in the other 40-50% urinary albumin excretion returns into the normal range after 3-10 years from the onset of microalbuminuria (Amin et al., 2008; Gorman et al., 1999). However, with longer follow-up cases of transient or intermittent microalbuminuria may become persistent. In addition, although urinary albumin excretion may return into the normal range, the renal morphological changes associated with microalbuminuria can persist and increase the risk of its recurrence and progression (Steinke and Mauer, 2008). Few data are available on the rate of progression of microalbuminuria to macroalbuminuria in young people with type 1 diabetes. The ORPS has shown a progression rate of around 13% after 3.2 years from the onset of microalbuminuria, a rate similar to that reported in adults (Amin et al., 2008). Another renal abnormality often detected in youth with type 1 diabetes is increased GFR (Amin et al., 2005; Rudberg et al., 1992). Hyperfiltration, which has been associated with increased renal size often precedes the onset of microalbuminuria (Amin et al., 2005; Rudberg et al., 1992). In some, although not all studies, increased GFR has emerged as an independent predictor of microalbuminuria, and a recent meta-analysis has reported a 2.7 increased risk of developing microalbuminuria associated with hyperfiltration (Magee et al.,

DR begins with the appearance of non-proliferative retinal abnormalities, which then progress to sight-threatening proliferative lesions (Aiello et al., 1998; Williams and Pickup, 2004) . The early stage of DR is characterised by the development of capillary microaneuryms, which consist in small blind outgrowths of retinal capillaries developing in areas where the wall is weakened (Aiello et al., 1998; D'Amico, 1994; Williams and Pickup, 2004). As the retinal damage progresses, there is the appearance of non-proliferative abnormalities, including

**2.1.3 Early manifestations of diabetic nephropathy in youth with type 1 diabetes**  Microalbuminuria is the most common abnormal finding in children and adolescents with type 1 diabetes, whereas overt proteinuria is found in less than 1–1.5% of them (Jones et al., 1998; Schultz et al., 1999b). Microalbuminuria often develops during puberty, whereas its prevalence during prepubertal years is rare (Janner et al., 1994; Lawson et al., 1996; Norgaard et al., 1989; Rudberg et al., 1993). The overall prevalence of microalbuminuria in youth with type 1 diabetes ranges between 4 and 26% (Bojestig et al., 1996; Cook and Daneman, 1990; Dahlquist and Rudberg, 1987; Joner et al., 1992; Jones et al., 1998; Mathiesen et al., 1986; Moore and Shield, 2000; Norgaard et al., 1989; Olsen et al., 2000; Rudberg et al., 1993; Schultz et al., 1999b). This large variation in the prevalence of microalbuminuria is due to differences in study design, duration of diabetes, age range, and glycemic control. Most studies investigating microalbuminuria have been cross-sectional and clinic based, with only a few being longitudinal, but mainly with a retrospective design. Jones *et al.* (Jones et al., 1998) reported a prevalence of 14.5% during 8.5 years of diabetes duration, whereas Rudberg *et al.* (Rudberg et al., 1993) reported a prevalence of 24% after 15 years diabetes duration. The most recent data from the Oxford Regional Prospective Study (ORPS), a population-based inception cohort of children with type 1 diabetes, has shown a cumulative prevalence of microalbuminuria of 25.7% after 10 years and of 50.7% after 19 years of

diabetes duration (Amin et al., 2008).

2009).

**2.2 Diabetic retinopathy** 

**2.2.1 Classification and structural changes** 

hemorrhages, exudates and the development of vascular obstruction, intraretinal microvascular abnormalities, and infarction of the retinal nerve fibers causing cotton wool spots (Aiello et al., 1998; D'Amico, 1994; Williams and Pickup, 2004). Although this stage is not sight-threatening, it is highly predictive of progression to more advanced stages of retinopathy. Proliferative retinopathy is characterized by the development of new vessels, secondary to ischemia, on the surface of the retina and/or the optic disc (Aiello et al., 1998; D'Amico, 1994; Williams and Pickup, 2004). These new vessels can bleed into the vitroretinal space, and cause visual loss. In addition, the subsequent formation of fibrous tissue can cause tractional retinal detachment (Williams and Pickup, 2004). This stage is associated with a high risk for visual impairment related to hemorrhages and retinal detachment.

Diabetic macular oedema can complicate both non-proliferative and proliferative retinopathy and is a serious cause of vision loss in patients with diabetes (Ciulla et al., 2003). It is characterized by increased microvascular permeability and deposition of hard retinal exudates (Ciulla et al., 2003). This stage involves the breakdown of the blood-retinal barrier, with leakage of plasma from small blood vessels in the macula. Swelling of the macula is then followed by deposition of hard retinal exudates as a consequence of deposition of lipids and lipoproteins following plasma re-absorption (Ciulla et al., 2003).


Table 1. International clinical DR disease severity scale

Retinal damage in diabetes is mainly due to leakage of retinal blood vessels and inadequate retinal perfusion (Aiello et al., 1998; D'Amico, 1994; Williams and Pickup, 2004). It has been suggested that one of the initial alterations in the retinal hemodynamic is represented by retinal vasodilatation and hyperperfusion, due to hypoxia and increased release of nitric oxide (Joussen et al., 2004). This is followed by an impairment of the retinal vascular autoregulation with increased tension in the epithelial wall and increased vascular permeability, which, in turn, cause vascular leakage and aneurysm formation (Kohner et al., 1995; Scherrer et al., 1994).

Microvascular and Macrovascular

**Peripheral neuropathies** 


*Focal and multifocal neuropathies* 

Table 2. Neuropathies in diabetes




*Generalized symmetric polyneuropathies* 

**2.3 Diabetic neuropathy** 

Complications in Children and Adolescents with Type 1 Diabetes 201

Diabetic neuropathy is the most common neuropathy in industrialized countries, and it is associated with a wide range of clinical manifestations (Tesfaye et al., 2010). Diabetic neuropathy is defined as a clinical or subclinical disorder, without any additional causes other than diabetes, and can be either somatic or autonomic (Boulton et al., 2005). Early diagnosis and treatment of diabetic neuropathy are important given that peripheral neuropathy is associated with a high risk of feet injury in patients with type 1 diabetes (Boulton et al., 2005). In addition, diabetic autonomic neuropathy can lead to a significantly increased morbidity and

Chronic distal symmetric polyneuropathy (DPN) is the most common form of diabetic neuropathy and affects 30-50% of patients with type 1 diabetes and can be asymptomatic in up to 50% of them (Tesfaye et al., 2010). DPN implies symmetric damage of peripheral small sensory and large motor nerve fibres. It generally starts from the most distal end of the feet and then extend proximally over time and can lead to foot ulceration and amputation of lower limbs (Boulton et al., 2005). Dysfunction of peripheral small nerve fibres is characterised by parasthesiae, burning, and deep aching pain. If larger nerve fibres are affected, vibration, light touch and joint position senses are impaired, and tendon reflexes are absent (Boulton et al., 2005). Less common forms of diabetic somatic neuropathy include focal or multifocal neuropathies, which are characterized by entrapment of a peripheral

Diabetic autonomic neuropathy is a disorder of the autonomic nervous system in the context of diabetes and can affect the cardiovascular, gastrointestinal, urogenital systems and the sudomotor function (Tesfaye et al., 2010). Autonomic neuropathy can be observed in around 20% of asymptomatic patients with diabetes (Tesfaye et al., 2010). Clinical symptoms of autonomic neuropathy do not generally occur until long after the onset of diabetes. Subclinical autonomic dysfunction can, however, occur within two years of diagnosis in patients with type 1 diabetes (Boulton et al., 2005). Dysautonomic features may reflect the involvement of different systems and manifest as postural hypotension and orthostatic lightheadedness, gastroparesis, gastric fullness, early satiety, sexual dysfunction, bladder dysfunction, gustatory

**Autonomic neuropathy** 

inconinence

Erectile dysfunction - *Gastrointestinal:* Gastric paresis, constipation/diarrhea, fecal





Anhidrosis, Heat intolerance, Dry skin - *Metabolic:* hypoglycemia unawareness

mortality, mainly when involves the cardiovascular system (Boulton et al., 2005).

**2.3.1 Classification and epidemiological data of diabetic neuropathy** 

nerve commonly the median, ulnar or peroneal nerve (Boulton et al., 2005).

sweating or anhidrosis and pupillomotor dysfunction (Boulton et al., 2005).

Early structural abnormalities in the retinal microvasculature are characterised by thickening of the basement membrane, loss of endothelial cells and pericytes and increased capillary permeability (Kohner et al., 1995). An important characteristic of retinopathy is the loss of pericytes from the retinal capillaries (D'Amico, 1994; Feng et al., 2007; Frank, 2004). Pericytes are contractile cells which have an important role in the regulation of the capillary blood flow and their loss has been associated with the development of retinal microaneurysms (Feng et al., 2007; Kuwabara and Cogan, 1963). The loss of pericytes, associated with that of capillary endothelial cells, contributes to the disruption of the integrity of the blood–retinal barrier. Thickening of the capillary basement membrane and deposition of extracellular matrix components are additional mechanisms contributing to the alterations in the retinal blood flow (Feng et al., 2007).

Retinal leukostasis has been also associated with DR and, in particular, with capillary occlusion and the consequent appearance of retinal areas of non perfusion (Aiello et al., 1998; D'Amico, 1994; Williams and Pickup, 2004). These alterations contribute, in turn, to retinal ischemia, which is a potent stimulus for vascular neoformation (Miyamoto and Ogura, 1999).

#### **2.2.2 Epidemiology of retinopathy**

DR is the leading cause of blindness in people of working age in Western countries (Ciulla et al., 2003). The prevalence of DR increases with age and its was approximately 17.7 per 100 people with diabetes in the year 2005 (Deshpande et al., 2008). DR can be diagnosed already after 5 years from the onset of diabetes, and almost all patients will show variable degrees of DR after 20 years diabetes duration.

Recent data from the Wisconsin Epidemiologic Study of DR have shown that the 25-year cumulative incidence of visual impairment in adults with type 1 diabetes was 13% and that of severe visual impairment was 3%. Patients with onset of diabetes during more recent years have a lower prevalence of visual impairment when compared with those diagnosed in the past, independently of duration of diabetes (Klein et al., 2010). This is in agreement with some other recent studies reporting a declining incidence of retinopathy and other microvascular complications (Nathan et al., 2009; Nordwall et al., 2004).

#### **2.2.3 Early manifestations of retinopathy in youth with type 1 diabetes**

Early stages of DR can be detected in young people with type 1 diabetes, as shown by a population based study from Australia, where early background retinopathy was detected in 24% of the study population after 6-year diabetes duration (Donaghue et al., 2005). Similarly, in a Swedish study retinopathy was detected in 27% of young patients after 13 years of duration (Nordwall et al., 2006). Children with type 1 diabetes under the age of 10 years are at minimal risk of DR, but the prevalence rate increases after 5 years from diagnosis in post-pubertal patients (Klein et al., 1984). In an incident cohort, early retinopathy was detected in 12% of prepubertal children compared to 29% of adolescents, after 6 year type 1 diabetes duration (Donaghue et al., 2005). Interestingly, adolescents with type 1 diabetes have a higher risk of progression to sight-threatening DR when compared to adults and the progression may be particularly rapid when glycemic control is poor (Maguire et al., 2005). As for DN, cases of regression have also been reported for DR (Maguire et al., 2005).

#### **2.3 Diabetic neuropathy**

200 Type 1 Diabetes Complications

Early structural abnormalities in the retinal microvasculature are characterised by thickening of the basement membrane, loss of endothelial cells and pericytes and increased capillary permeability (Kohner et al., 1995). An important characteristic of retinopathy is the loss of pericytes from the retinal capillaries (D'Amico, 1994; Feng et al., 2007; Frank, 2004). Pericytes are contractile cells which have an important role in the regulation of the capillary blood flow and their loss has been associated with the development of retinal microaneurysms (Feng et al., 2007; Kuwabara and Cogan, 1963). The loss of pericytes, associated with that of capillary endothelial cells, contributes to the disruption of the integrity of the blood–retinal barrier. Thickening of the capillary basement membrane and deposition of extracellular matrix components are additional mechanisms contributing to

Retinal leukostasis has been also associated with DR and, in particular, with capillary occlusion and the consequent appearance of retinal areas of non perfusion (Aiello et al., 1998; D'Amico, 1994; Williams and Pickup, 2004). These alterations contribute, in turn, to retinal ischemia, which is a potent stimulus for vascular neoformation (Miyamoto and

DR is the leading cause of blindness in people of working age in Western countries (Ciulla et al., 2003). The prevalence of DR increases with age and its was approximately 17.7 per 100 people with diabetes in the year 2005 (Deshpande et al., 2008). DR can be diagnosed already after 5 years from the onset of diabetes, and almost all patients will show variable degrees of

Recent data from the Wisconsin Epidemiologic Study of DR have shown that the 25-year cumulative incidence of visual impairment in adults with type 1 diabetes was 13% and that of severe visual impairment was 3%. Patients with onset of diabetes during more recent years have a lower prevalence of visual impairment when compared with those diagnosed in the past, independently of duration of diabetes (Klein et al., 2010). This is in agreement with some other recent studies reporting a declining incidence of retinopathy and other

Early stages of DR can be detected in young people with type 1 diabetes, as shown by a population based study from Australia, where early background retinopathy was detected in 24% of the study population after 6-year diabetes duration (Donaghue et al., 2005). Similarly, in a Swedish study retinopathy was detected in 27% of young patients after 13 years of duration (Nordwall et al., 2006). Children with type 1 diabetes under the age of 10 years are at minimal risk of DR, but the prevalence rate increases after 5 years from diagnosis in post-pubertal patients (Klein et al., 1984). In an incident cohort, early retinopathy was detected in 12% of prepubertal children compared to 29% of adolescents, after 6 year type 1 diabetes duration (Donaghue et al., 2005). Interestingly, adolescents with type 1 diabetes have a higher risk of progression to sight-threatening DR when compared to adults and the progression may be particularly rapid when glycemic control is poor (Maguire et al., 2005). As for DN, cases of regression have also been reported for DR

microvascular complications (Nathan et al., 2009; Nordwall et al., 2004).

**2.2.3 Early manifestations of retinopathy in youth with type 1 diabetes** 

the alterations in the retinal blood flow (Feng et al., 2007).

Ogura, 1999).

**2.2.2 Epidemiology of retinopathy** 

DR after 20 years diabetes duration.

(Maguire et al., 2005).

Diabetic neuropathy is the most common neuropathy in industrialized countries, and it is associated with a wide range of clinical manifestations (Tesfaye et al., 2010). Diabetic neuropathy is defined as a clinical or subclinical disorder, without any additional causes other than diabetes, and can be either somatic or autonomic (Boulton et al., 2005). Early diagnosis and treatment of diabetic neuropathy are important given that peripheral neuropathy is associated with a high risk of feet injury in patients with type 1 diabetes (Boulton et al., 2005). In addition, diabetic autonomic neuropathy can lead to a significantly increased morbidity and mortality, mainly when involves the cardiovascular system (Boulton et al., 2005).

#### **2.3.1 Classification and epidemiological data of diabetic neuropathy**

Chronic distal symmetric polyneuropathy (DPN) is the most common form of diabetic neuropathy and affects 30-50% of patients with type 1 diabetes and can be asymptomatic in up to 50% of them (Tesfaye et al., 2010). DPN implies symmetric damage of peripheral small sensory and large motor nerve fibres. It generally starts from the most distal end of the feet and then extend proximally over time and can lead to foot ulceration and amputation of lower limbs (Boulton et al., 2005). Dysfunction of peripheral small nerve fibres is characterised by parasthesiae, burning, and deep aching pain. If larger nerve fibres are affected, vibration, light touch and joint position senses are impaired, and tendon reflexes are absent (Boulton et al., 2005). Less common forms of diabetic somatic neuropathy include focal or multifocal neuropathies, which are characterized by entrapment of a peripheral nerve commonly the median, ulnar or peroneal nerve (Boulton et al., 2005).

Diabetic autonomic neuropathy is a disorder of the autonomic nervous system in the context of diabetes and can affect the cardiovascular, gastrointestinal, urogenital systems and the sudomotor function (Tesfaye et al., 2010). Autonomic neuropathy can be observed in around 20% of asymptomatic patients with diabetes (Tesfaye et al., 2010). Clinical symptoms of autonomic neuropathy do not generally occur until long after the onset of diabetes. Subclinical autonomic dysfunction can, however, occur within two years of diagnosis in patients with type 1 diabetes (Boulton et al., 2005). Dysautonomic features may reflect the involvement of different systems and manifest as postural hypotension and orthostatic lightheadedness, gastroparesis, gastric fullness, early satiety, sexual dysfunction, bladder dysfunction, gustatory sweating or anhidrosis and pupillomotor dysfunction (Boulton et al., 2005).


Table 2. Neuropathies in diabetes

Microvascular and Macrovascular

Jorgensen et al., 2005).

2001).

**4.1 Glycemic control** 

inflammatory factors (Brownlee, 2001).

also of early signs of atherosclerosis (McGill et al., 1995).

Complications in Children and Adolescents with Type 1 Diabetes 203

Several studies have consistently shown that children and adolescents with type 1 diabetes present signs of endothelial dysfunction, as measured by flow-mediated dilation in the brachial artery (Jarvisalo et al., 2004; Singh et al., 2003). In addition, increased aortic and carotid intima-media thickness has been reported in children with type 1 diabetes (Harrington et al., 2010; Jarvisalo et al., 2001). Besides, markers of inflammation and oxidative stress are significantly increased in youth with type 1 diabetes when compared with age-matched

Progression of CVD in youth with type 1 diabetes can be more aggressive than in adults with diabetes, therefore highlighting the importance of early preventive strategies (Dahl-

**Early manifestations of CVD in youth with type 1 diabetes**

Vascular complications in the context of diabetes are the result of an interplay between hemodynamic and metabolic factors and the consequent activation of common intermediate pathways, associated with increased synthesis and release of growth factors, cytokines, chemokines and oxidant species, which are all final mediators of vascular damage (Cooper,

A strong association exists between the presence of micro- and macrovascular complications in people with type 1 diabetes (Girach and Vignati, 2006). In particular, it is well known that DN is a key risk factor for cardiovascular complications and many patients with renal impairment die of CVD-related causes even before developing ESRD (Groop et al., 2009). In addition, there is growing evidence suggesting that increases in albumin excretion, even within the normal range, in the general population as well as in people with type 1 diabetes, represents a determinant of CVD (Klausen et al., 2004). The link between micro- and macrovascular disease could be represented by endothelial dysfunction, as an underlining feature of both processes, as well as by a certain degree of inflammation, which has been associated to the presence of

Chronic hyperglycemia is known to activate several deleterious pathways implicated in the damage of vessels: protein glycation, increased glucose flux through alternative polyol and hexosamine pathways, increased oxidative stress, which then stimulate secondary intracellular signaling pathways leading to production of growth factors, cytokines and

Several epidemiological studies have shown a direct and strong association between longterm glycemic control, as expressed by HbA1c levels, and the risk of developing nephropathy, retinopathy and neuropathy (Amin et al., 2008; Danne et al., 1998; Gallego et al., 2008; Nordwall et al., 2009). In addition, HbA1c has been shown to be a key determinant

both micro- and macrovascular complications (Schalkwijk and Stehouwer, 2005).

controls and they can mediate vascular damage (Snell-Bergeon et al., 2010).

 Increased inflammatory markers Increased oxidative stress

 Increased intima-media thickness Decreased flow-mediated dilatation Increased pulse wave velocity

Table 3. Early manifestations of CVD in youth with type 1 diabetes

**4. Pathogenesis of micro- and macrovascular complications** 

Diabetic neuropathy is characterized by a reduction in the number of fibers, degeneration of the myelin sheath as well as changes affecting the endoneurial connective tissue, vessels and perineurium (Greene et al., 1992). The process of nerve demyelination may progress to Wallerian degeneration, in which the nerve axon is also injured and the distal part of the axon dies (Greene et al., 1992).

#### **2.3.2 Early signs of diabetic neuropathy in youth with type 1 diabetes**

In the most comprehensive epidemiological studies involving both adult and pediatric patients, DPN was detected in 9 to 58% of the study populations (Boulton et al., 2005). Data on autonomic neuropathy indicate a prevalence ranging from 14% to 75%, with a high number of youth with type 1 diabetes presenting suclinical signs of autonomic dysfunction, even after a short duration of T1D. This variability across different studies is mainly related to differences in the characteristics of the study cohorts as well as to the use of different testing modalities and different criteria and cut off values (Trotta et al., 2004; Verrotti et al., 2009).

#### **3. Macrovascular complications of type 1 diabetes**

Patients with type 1 diabetes have an increased risk of developing cardiovascular disease (CVD) relative to the nondiabetic population, and premature atherosclerosis represents the main cause of morbidity and mortality in type 1 diabetes populations (Laing et al., 2003). There is extensive evidence in support of the concept that atherosclerosis begins early in life (Ross, 1993) and therefore identification of CVD risk factors and preventive strategies should be started during childhood and adolescence (Dahl-Jorgensen et al., 2005). Children and adolescents with type 1 diabetes represent a high risk population with regards to CVD, given that cardiovascular risk factors are common among them (Dahl-Jorgensen et al., 2005; Margeirsdottir et al., 2008; van Vliet et al.) and they can contribute to their poor long-term prognosis (Skrivarhaug et al., 2006). A recent study has shown that as many as 86% of youth with type 1 diabetes has at least one, 45% at least two and 15% at least three CVD risk factors, including high HbA1c, high blood pressure, dyslipidemia, smoking and family history of CVD events (Margeirsdottir et al., 2008). These data are alarming given that it is well known that CVD risk factors can persist or track over time (Berenson, 2002) and therefore contribute to the overall burden associated with type 1 diabetes.

#### **3.1 Early signs of macrovascular disease in youth with type 1 diabetes**

The early stages of the atherosclerotic process are silent, but autopsy studies have detected early structural alterations in the arteries of youth, where they have been associated with the same risk factors than in adults (Berenson et al., 1998). The earliest recognizable pathologic intimal lesions are the fatty streaks, which make their appearance in the aorta of children even before 3 years of age (McGill et al., 2000; Williams et al., 2002). Fatty streaks represent an early manifestation of lipid accumulation in the vessel wall and they have been associated with increased cholesterol levels as well as with hyperglycemia (McGill et al., 2000).

Technology progresses over the last years have made possible to look for early surrogate markers of atherosclerotic vascular disease. These markers are represented by structural alterations such as increased intima-media thickness as well as functional changes represented by decreased flow-mediated dilatation and increased arterial stiffness, as detected by pulse wave velocity (Dahl-Jorgensen et al., 2005).

Diabetic neuropathy is characterized by a reduction in the number of fibers, degeneration of the myelin sheath as well as changes affecting the endoneurial connective tissue, vessels and perineurium (Greene et al., 1992). The process of nerve demyelination may progress to Wallerian degeneration, in which the nerve axon is also injured and the distal part of the

In the most comprehensive epidemiological studies involving both adult and pediatric patients, DPN was detected in 9 to 58% of the study populations (Boulton et al., 2005). Data on autonomic neuropathy indicate a prevalence ranging from 14% to 75%, with a high number of youth with type 1 diabetes presenting suclinical signs of autonomic dysfunction, even after a short duration of T1D. This variability across different studies is mainly related to differences in the characteristics of the study cohorts as well as to the use of different testing modalities

Patients with type 1 diabetes have an increased risk of developing cardiovascular disease (CVD) relative to the nondiabetic population, and premature atherosclerosis represents the main cause of morbidity and mortality in type 1 diabetes populations (Laing et al., 2003). There is extensive evidence in support of the concept that atherosclerosis begins early in life (Ross, 1993) and therefore identification of CVD risk factors and preventive strategies should be started during childhood and adolescence (Dahl-Jorgensen et al., 2005). Children and adolescents with type 1 diabetes represent a high risk population with regards to CVD, given that cardiovascular risk factors are common among them (Dahl-Jorgensen et al., 2005; Margeirsdottir et al., 2008; van Vliet et al.) and they can contribute to their poor long-term prognosis (Skrivarhaug et al., 2006). A recent study has shown that as many as 86% of youth with type 1 diabetes has at least one, 45% at least two and 15% at least three CVD risk factors, including high HbA1c, high blood pressure, dyslipidemia, smoking and family history of CVD events (Margeirsdottir et al., 2008). These data are alarming given that it is well known that CVD risk factors can persist or track over time (Berenson, 2002) and

**2.3.2 Early signs of diabetic neuropathy in youth with type 1 diabetes** 

and different criteria and cut off values (Trotta et al., 2004; Verrotti et al., 2009).

therefore contribute to the overall burden associated with type 1 diabetes.

**3.1 Early signs of macrovascular disease in youth with type 1 diabetes** 

detected by pulse wave velocity (Dahl-Jorgensen et al., 2005).

The early stages of the atherosclerotic process are silent, but autopsy studies have detected early structural alterations in the arteries of youth, where they have been associated with the same risk factors than in adults (Berenson et al., 1998). The earliest recognizable pathologic intimal lesions are the fatty streaks, which make their appearance in the aorta of children even before 3 years of age (McGill et al., 2000; Williams et al., 2002). Fatty streaks represent an early manifestation of lipid accumulation in the vessel wall and they have been associated with increased cholesterol levels as well as with hyperglycemia (McGill et al.,

Technology progresses over the last years have made possible to look for early surrogate markers of atherosclerotic vascular disease. These markers are represented by structural alterations such as increased intima-media thickness as well as functional changes represented by decreased flow-mediated dilatation and increased arterial stiffness, as

**3. Macrovascular complications of type 1 diabetes** 

axon dies (Greene et al., 1992).

2000).

Several studies have consistently shown that children and adolescents with type 1 diabetes present signs of endothelial dysfunction, as measured by flow-mediated dilation in the brachial artery (Jarvisalo et al., 2004; Singh et al., 2003). In addition, increased aortic and carotid intima-media thickness has been reported in children with type 1 diabetes (Harrington et al., 2010; Jarvisalo et al., 2001). Besides, markers of inflammation and oxidative stress are significantly increased in youth with type 1 diabetes when compared with age-matched controls and they can mediate vascular damage (Snell-Bergeon et al., 2010).

Progression of CVD in youth with type 1 diabetes can be more aggressive than in adults with diabetes, therefore highlighting the importance of early preventive strategies (Dahl-Jorgensen et al., 2005).

#### **Early manifestations of CVD in youth with type 1 diabetes**


Table 3. Early manifestations of CVD in youth with type 1 diabetes

#### **4. Pathogenesis of micro- and macrovascular complications**

Vascular complications in the context of diabetes are the result of an interplay between hemodynamic and metabolic factors and the consequent activation of common intermediate pathways, associated with increased synthesis and release of growth factors, cytokines, chemokines and oxidant species, which are all final mediators of vascular damage (Cooper, 2001).

A strong association exists between the presence of micro- and macrovascular complications in people with type 1 diabetes (Girach and Vignati, 2006). In particular, it is well known that DN is a key risk factor for cardiovascular complications and many patients with renal impairment die of CVD-related causes even before developing ESRD (Groop et al., 2009). In addition, there is growing evidence suggesting that increases in albumin excretion, even within the normal range, in the general population as well as in people with type 1 diabetes, represents a determinant of CVD (Klausen et al., 2004). The link between micro- and macrovascular disease could be represented by endothelial dysfunction, as an underlining feature of both processes, as well as by a certain degree of inflammation, which has been associated to the presence of both micro- and macrovascular complications (Schalkwijk and Stehouwer, 2005).

#### **4.1 Glycemic control**

Chronic hyperglycemia is known to activate several deleterious pathways implicated in the damage of vessels: protein glycation, increased glucose flux through alternative polyol and hexosamine pathways, increased oxidative stress, which then stimulate secondary intracellular signaling pathways leading to production of growth factors, cytokines and inflammatory factors (Brownlee, 2001).

Several epidemiological studies have shown a direct and strong association between longterm glycemic control, as expressed by HbA1c levels, and the risk of developing nephropathy, retinopathy and neuropathy (Amin et al., 2008; Danne et al., 1998; Gallego et al., 2008; Nordwall et al., 2009). In addition, HbA1c has been shown to be a key determinant also of early signs of atherosclerosis (McGill et al., 1995).

Microvascular and Macrovascular

**4.4 Puberty** 

**4.5 Gender** 

in girls (Schultz et al., 1999a).

**4.2 Blood pressure and plasma lipids** 

Kordonouri et al., 1996; Marcovecchio et al., 2009a).

**4.3 Duration and age at onset of type 1 diabetes** 

Complications in Children and Adolescents with Type 1 Diabetes 205

Elevated blood pressure and alterations in its circadian rhythm are common findings in people with type 1 diabetes and have been associated with the risk of developing vascular complications (Gallego et al., 2008; Marcovecchio et al., 2009b; Tesfaye et al., 1996). Increases in blood pressure have been found to precede or occur concomitant with the appearance of microalbuminuria in adolescents with type 1 diabetes (Marcovecchio et al., 2009b; Schultz et al., 2001). Similarly, higher than normal systolic and diastolic blood pressure independently contribute to the development of DR (Gallego et al., 2008). Blood pressure has been also

Lipid abnormalities have also been linked to the development and progression of microand macrovascular complications in adolescents with diabetes (Dahl-Jorgensen et al., 2005;

Duration of diabetes is another major determinant of complication risk. Although vascular complications rarely appear before puberty, prepubertal duration of diabetes is an important determinant for their development (Coonrod et al., 1993; Orchard et al., 1990). Patients with type 1 diabetes from early childhood and especially those diagnosed under five years of age seem to have slightly delayed onset of persistent microalbuminuria during the first 10 to 15 years duration compared with patients diagnosed later in childhood or during puberty. However, this initial protective effect of a younger age at diagnosis disappears over time (Amin et al., 2008; Donaghue et al., 2003). After 15 years of diabetes duration the risk of developing microalbuminuria is similar between subjects diagnosed with diabetes before 5 years of age when compared with those diagnosed between 5-11 years of age or after the age of 11 years, suggesting that age at the onset of diabetes does not influence the overall risk for microalbuminuria (Amin et al., 2008). A recent study has highlighted that also for DR, although patients diagnosed at a young age have a longer time free of proliferative retinopathy, this advantage then gradually disappears and youth diagnosed before the age of 15 years have a higher risk of proliferative retinopathy when

associated with early markers of atherosclerosis (Dahl-Jorgensen et al., 2005).

compared with those diagnosed when aged 15-40 years (Hietala et al., 2010).

Puberty is an important factor implicated in the development and progression of vascular complications. Poor glycemic control is a common finding among adolescents with type 1 diabetes (1994; Holl et al., 2003). In addition, puberty is associated with a decrease in insulin sensitivity, and adolescents with type 1 diabetes are more insulin resistant when compared with healthy controls (Dunger, 1992). Rapid growth, hormonal and metabolic changes

A gender dimorphism has been reported for vascular complications. In particular, during adolescence the risk for microalbuminuria is higher in female than in male subjects with comparable glycemic control (Amin et al., 2008; Jones et al., 1998), whereas among adults with type 1 diabetes the risk is higher for males (Hovind et al., 2004). These differences have been related to variations in the hormonal milieu and to a higher degree of insulin resistance

characterise this period of life and can influence complications risk (Dunger, 1992).

Fig. 2. Risk factors for the development of vascular complications in adolescents with type 1 diabetes

The Diabetes Control and Complications Trial (DCCT) and the Epidemiology of Diabetes Interventions and Complications (EDIC) studies have clearly shown the beneficial effect of strict glycemic control in reducing the risk of microvascular and macrovascular complications in subjects with type 1 diabetes (1993; 2003). In the adolescent cohort of the DCCT, a positive effect of improved glycemic control on complication risk was obtained. Intensive insulin therapy reduced the risk for the development and progression of DR by 76% and 54%; the occurrence of microalbuminuria and proteinuria by 39% and 54% and that of clinical neuropathy by 60% when compared to the conventional treated group (1994). Data from the DCCT/EDIC study also showed that intensive insulin therapy reduced the risk for any cardiovascular disease by 42% and of nonfatal myocardial infarction, stroke or death from CVD cause by 57% (Nathan et al., 2005). The beneficial effect of intensive insulin treatment in patients with childhood-onset type 1 diabetes also emerged from the Oslo study, where longterm glycemic control predicted coronary atherosclerosis (Larsen et al., 2002).

In addition, the EDIC study raised the important concept of 'metabolic memory'; in other words although after the end of the DCCT HbA1c levels became comparable between the intensively and conventionally treated groups, patients belonging to the first group still kept an advantage from prior better HbA1c levels (2003). Therefore, the EDIC data highlighted the need of implementing intensive management as soon as type 1 diabetes is diagnosed. This was further supported by recent data related to DR, showing that, although at year 10 of the EDIC there was still a decrease in DR progression risk in the intensively treated adult group, in the adolescent cohort retinopathy progression at year 10 of the EDIC did not differ between the previous DCCT intensively and conventionally treated groups, thus indicating loss of the metabolic memory (White et al., 2010). Interestingly, 79% of the difference in the metabolic effect between adults and adolescents after 10 years from the end of the DCCT was due to the difference in mean HbA1c levels during the DCCT between the two cohorts. This 1% difference, which did not seem to play a major role during the DCCT and early EDIC years with regards to the outcomes, seems to be a major player in the long run (White et al., 2010).

#### **4.2 Blood pressure and plasma lipids**

204 Type 1 Diabetes Complications

**Long-term glycemic control**

> **Familial/Genetic factors**

•CVD genes • Family history of hypertension, dyslipidemia, T2D, CVD

**Environmental/ biological factors**

•Blood pressure • Lipids • Puberty • Gender • Disease duration

• Sedentary lifestyle

• BMI • Smoking • Diet

diabetes

et al., 2010).

Microvascular & macrovascular complications

Fig. 2. Risk factors for the development of vascular complications in adolescents with type 1

The Diabetes Control and Complications Trial (DCCT) and the Epidemiology of Diabetes Interventions and Complications (EDIC) studies have clearly shown the beneficial effect of strict glycemic control in reducing the risk of microvascular and macrovascular complications in subjects with type 1 diabetes (1993; 2003). In the adolescent cohort of the DCCT, a positive effect of improved glycemic control on complication risk was obtained. Intensive insulin therapy reduced the risk for the development and progression of DR by 76% and 54%; the occurrence of microalbuminuria and proteinuria by 39% and 54% and that of clinical neuropathy by 60% when compared to the conventional treated group (1994). Data from the DCCT/EDIC study also showed that intensive insulin therapy reduced the risk for any cardiovascular disease by 42% and of nonfatal myocardial infarction, stroke or death from CVD cause by 57% (Nathan et al., 2005). The beneficial effect of intensive insulin treatment in patients with childhood-onset type 1 diabetes also emerged from the Oslo study, where long-

In addition, the EDIC study raised the important concept of 'metabolic memory'; in other words although after the end of the DCCT HbA1c levels became comparable between the intensively and conventionally treated groups, patients belonging to the first group still kept an advantage from prior better HbA1c levels (2003). Therefore, the EDIC data highlighted the need of implementing intensive management as soon as type 1 diabetes is diagnosed. This was further supported by recent data related to DR, showing that, although at year 10 of the EDIC there was still a decrease in DR progression risk in the intensively treated adult group, in the adolescent cohort retinopathy progression at year 10 of the EDIC did not differ between the previous DCCT intensively and conventionally treated groups, thus indicating loss of the metabolic memory (White et al., 2010). Interestingly, 79% of the difference in the metabolic effect between adults and adolescents after 10 years from the end of the DCCT was due to the difference in mean HbA1c levels during the DCCT between the two cohorts. This 1% difference, which did not seem to play a major role during the DCCT and early EDIC years with regards to the outcomes, seems to be a major player in the long run (White

term glycemic control predicted coronary atherosclerosis (Larsen et al., 2002).

Elevated blood pressure and alterations in its circadian rhythm are common findings in people with type 1 diabetes and have been associated with the risk of developing vascular complications (Gallego et al., 2008; Marcovecchio et al., 2009b; Tesfaye et al., 1996). Increases in blood pressure have been found to precede or occur concomitant with the appearance of microalbuminuria in adolescents with type 1 diabetes (Marcovecchio et al., 2009b; Schultz et al., 2001). Similarly, higher than normal systolic and diastolic blood pressure independently contribute to the development of DR (Gallego et al., 2008). Blood pressure has been also associated with early markers of atherosclerosis (Dahl-Jorgensen et al., 2005).

Lipid abnormalities have also been linked to the development and progression of microand macrovascular complications in adolescents with diabetes (Dahl-Jorgensen et al., 2005; Kordonouri et al., 1996; Marcovecchio et al., 2009a).

#### **4.3 Duration and age at onset of type 1 diabetes**

Duration of diabetes is another major determinant of complication risk. Although vascular complications rarely appear before puberty, prepubertal duration of diabetes is an important determinant for their development (Coonrod et al., 1993; Orchard et al., 1990). Patients with type 1 diabetes from early childhood and especially those diagnosed under five years of age seem to have slightly delayed onset of persistent microalbuminuria during the first 10 to 15 years duration compared with patients diagnosed later in childhood or during puberty. However, this initial protective effect of a younger age at diagnosis disappears over time (Amin et al., 2008; Donaghue et al., 2003). After 15 years of diabetes duration the risk of developing microalbuminuria is similar between subjects diagnosed with diabetes before 5 years of age when compared with those diagnosed between 5-11 years of age or after the age of 11 years, suggesting that age at the onset of diabetes does not influence the overall risk for microalbuminuria (Amin et al., 2008). A recent study has highlighted that also for DR, although patients diagnosed at a young age have a longer time free of proliferative retinopathy, this advantage then gradually disappears and youth diagnosed before the age of 15 years have a higher risk of proliferative retinopathy when compared with those diagnosed when aged 15-40 years (Hietala et al., 2010).

#### **4.4 Puberty**

Puberty is an important factor implicated in the development and progression of vascular complications. Poor glycemic control is a common finding among adolescents with type 1 diabetes (1994; Holl et al., 2003). In addition, puberty is associated with a decrease in insulin sensitivity, and adolescents with type 1 diabetes are more insulin resistant when compared with healthy controls (Dunger, 1992). Rapid growth, hormonal and metabolic changes characterise this period of life and can influence complications risk (Dunger, 1992).

#### **4.5 Gender**

A gender dimorphism has been reported for vascular complications. In particular, during adolescence the risk for microalbuminuria is higher in female than in male subjects with comparable glycemic control (Amin et al., 2008; Jones et al., 1998), whereas among adults with type 1 diabetes the risk is higher for males (Hovind et al., 2004). These differences have been related to variations in the hormonal milieu and to a higher degree of insulin resistance in girls (Schultz et al., 1999a).

Microvascular and Macrovascular

**When to start** 

**Screening Method:**  *1) Diabetic nephropathy* 

*2) Diabetic retinopathy* 

*3) Diabetic neuropathy* 

*4) Macrovascular disease* 

**5.2 Interventions** 

monitoring.

Complications in Children and Adolescents with Type 1 Diabetes 207

rest, QT interval, postural changes in BP and pupil responses to light and dark adaptation (Donaghue et al., 2007). These tests need to be carefully standardized and therefore they are

Additional screening concerns risk factors for CVD, such as dyslipidemia and hypertension. Blood lipids should be checked soon after diagnosis and if normal then repeated after 5 years (Donaghue et al., 2007). Office blood pressure should be assessed annually and in case of abnormal values, hypertension needs to be confirmed by ambulatory blood pressure

> Microvascular complications: at age 11 with 2 year duration of type 1 diabetes or from age 9 with 5 year duration (for

> Annual albumin-creatinine ratio in a spot urine sample or first

Annual dilated fundus ophthalmoscopy or fundal photography

Table 4. Screening for micro- and macrovascular complications in youth with type 1 diabetes

Improving glycemic control is the cornerstone of treatment strategies aiming at reducing the development and progression of microvascular and macrovascular complications. The DCCT and EDIC study clearly showed the importance of a strict glycemic control both in adults and in adolescents with type 1 diabetes (1994). The importance of keeping HbA1c within targets has also been highlighted by other studies, but similarly to the DCCT, subsequent studies have confirmed the difficulties encountered when dealing with young people with type 1 diabetes (Holl et al., 2003; Petitti et al., 2009). Tight glycemic control in the DCCT and subsequent studies was associated with a higher risk of complications, such as hypoglycemia and weight gain (1994). In addition, poor compliance is an important issue to be taken into account in adolescents with type 1 diabetes. Furthermore, other factors besides insulin therapy are relevant for metabolic control, such as dietary habits, education, family interactions, cultural and psychological aspects (Holl et al., 2003), and therefore they

Treatment with angiotensin converting enzyme inhibitors (ACEIs) is recommended when hypertension is confirmed (Donaghue et al., 2007). ACEIs are treatment of choice in adults with microalbuminuria, based on evidence that their use decrease the rate of progression and even increase rates of regression of microalbuminuria (Lewis et al., 1993). A beneficial

nephropathy and retinopathy; unclear for neuropathy)

largely used as screening methods for complications at a population level.

Macrovascular complications: after 12 years

morning albumin concentration

 History and physical examination Nerve conduction and autonomic tests

 Annual assessments of blood pressure Assessment of lipid levels every 5 years

need to be taken into account in order to define a successful treatment plan.

#### **4.6 Other factors**

Genetic factors represent another important contributing factor for the development of vascular complications, as suggested by their familial clustering, and by the observation that only a subset of patients with poor glycemic control develop severe long-term complications (1997). Family history of cardiovascular risk factors is associated with increased risk of microvascular complications in the offspring (Monti et al., 2007; Seaquist et al., 1989).

A high body mass index (BMI) represents another potential risk factor for microvascular complications in youth with type 1 diabetes (Stone et al., 2006). In addition, obesity is a wellknown risk factor for cardiovascular disease (Dahl-Jorgensen et al., 2005).

Environmental factors, including diet and lifestyle, can also contribute to the risk of developing vascular complications. Smoking in people with type 1 diabetes has been associated with an increased risk of developing vascular complications (Chase et al., 1991; Mohamed et al., 2007).

#### **5. Management of microvascular and macrovascular complications**

#### **5.1 Screening**

Diabetic microvascular and macrovascular complications are often asymptomatic during their early stages, and once symptoms develop, they may be difficult to reverse. Therefore, longitudinal repeated screening for vascular complications initiated during early adolescence is currently recommended

The American Diabetes Association (ADA) recommends to start screening in patients with type 1 diabetes aged 10 years or older after a disease duration of 3-5 years, with yearly follow-up (Silverstein et al., 2005). The International Society for Pediatric and Adolescent Diabetes (ISPAD) recommends screening from the age of 11 years in those with 2 year diabetes duration and from 9 years in those with a 5 year duration for both nephropathy and retinopathy (Donaghue et al., 2007). Measurement of urinary albumin excretion is the basis for early detection of microalbuminuria and can be achieved with: 1) 24-hour urine collection; 2) overnight timed urine collections; 3) albumin-creatinine ratio (ACR) or albumin concentration on a early morning spot urinary sample (Donaghue et al., 2007). 24 hour or timed urine collections are often difficult to collect in children and adolescents. Assessing ACR in early morning urines is the easiest method to carry out in an office setting and it generally provides accurate information (Donaghue et al., 2007).

With regards to DR, several techniques can be used, including direct and indirect ophthalmoscopy, stereoscopic digital and color film based fundus photography, mydriatic or nonmydriatic digital color or monochromatic single-field photography. The best technique to identify and grade retinopathy is represented by retinal photography, through dilated pupils, but dilated indirect ophtalmoscopy associated with biomicroscopy is an acceptable alternative (Ciulla et al., 2003).

In contrast to well-established criteria for when starting screening for DN and DR, it is unclear when to commence screening for neuropathy. History and physical examination are generally the recommended methods of screening (Donaghue et al., 2007). Clinical examination, including history of pain, paresthesia, numbness and physical examination of ankle reflexes and vibration and light touch sensation, is a fundamental part of screening, although not being as sensitive or specific as nerve conduction studies (Donaghue et al., 2007). Autonomic neuropathy can be assessed with specific autonomic nerve tests, such as heart rate response to deep breathing, standing from a lying position, Valsalva maneuver, heart rate variations at rest, QT interval, postural changes in BP and pupil responses to light and dark adaptation (Donaghue et al., 2007). These tests need to be carefully standardized and therefore they are largely used as screening methods for complications at a population level.

Additional screening concerns risk factors for CVD, such as dyslipidemia and hypertension. Blood lipids should be checked soon after diagnosis and if normal then repeated after 5 years (Donaghue et al., 2007). Office blood pressure should be assessed annually and in case of abnormal values, hypertension needs to be confirmed by ambulatory blood pressure monitoring.


Table 4. Screening for micro- and macrovascular complications in youth with type 1 diabetes

#### **5.2 Interventions**

206 Type 1 Diabetes Complications

Genetic factors represent another important contributing factor for the development of vascular complications, as suggested by their familial clustering, and by the observation that only a subset of patients with poor glycemic control develop severe long-term complications (1997). Family history of cardiovascular risk factors is associated with increased risk of

A high body mass index (BMI) represents another potential risk factor for microvascular complications in youth with type 1 diabetes (Stone et al., 2006). In addition, obesity is a well-

Environmental factors, including diet and lifestyle, can also contribute to the risk of developing vascular complications. Smoking in people with type 1 diabetes has been associated with an increased risk of developing vascular complications (Chase et al., 1991;

Diabetic microvascular and macrovascular complications are often asymptomatic during their early stages, and once symptoms develop, they may be difficult to reverse. Therefore, longitudinal repeated screening for vascular complications initiated during early

The American Diabetes Association (ADA) recommends to start screening in patients with type 1 diabetes aged 10 years or older after a disease duration of 3-5 years, with yearly follow-up (Silverstein et al., 2005). The International Society for Pediatric and Adolescent Diabetes (ISPAD) recommends screening from the age of 11 years in those with 2 year diabetes duration and from 9 years in those with a 5 year duration for both nephropathy and retinopathy (Donaghue et al., 2007). Measurement of urinary albumin excretion is the basis for early detection of microalbuminuria and can be achieved with: 1) 24-hour urine collection; 2) overnight timed urine collections; 3) albumin-creatinine ratio (ACR) or albumin concentration on a early morning spot urinary sample (Donaghue et al., 2007). 24 hour or timed urine collections are often difficult to collect in children and adolescents. Assessing ACR in early morning urines is the easiest method to carry out in an office setting

With regards to DR, several techniques can be used, including direct and indirect ophthalmoscopy, stereoscopic digital and color film based fundus photography, mydriatic or nonmydriatic digital color or monochromatic single-field photography. The best technique to identify and grade retinopathy is represented by retinal photography, through dilated pupils, but dilated indirect ophtalmoscopy associated with biomicroscopy is an

In contrast to well-established criteria for when starting screening for DN and DR, it is unclear when to commence screening for neuropathy. History and physical examination are generally the recommended methods of screening (Donaghue et al., 2007). Clinical examination, including history of pain, paresthesia, numbness and physical examination of ankle reflexes and vibration and light touch sensation, is a fundamental part of screening, although not being as sensitive or specific as nerve conduction studies (Donaghue et al., 2007). Autonomic neuropathy can be assessed with specific autonomic nerve tests, such as heart rate response to deep breathing, standing from a lying position, Valsalva maneuver, heart rate variations at

microvascular complications in the offspring (Monti et al., 2007; Seaquist et al., 1989).

known risk factor for cardiovascular disease (Dahl-Jorgensen et al., 2005).

**5. Management of microvascular and macrovascular complications** 

and it generally provides accurate information (Donaghue et al., 2007).

**4.6 Other factors** 

Mohamed et al., 2007).

adolescence is currently recommended

acceptable alternative (Ciulla et al., 2003).

**5.1 Screening** 

Improving glycemic control is the cornerstone of treatment strategies aiming at reducing the development and progression of microvascular and macrovascular complications. The DCCT and EDIC study clearly showed the importance of a strict glycemic control both in adults and in adolescents with type 1 diabetes (1994). The importance of keeping HbA1c within targets has also been highlighted by other studies, but similarly to the DCCT, subsequent studies have confirmed the difficulties encountered when dealing with young people with type 1 diabetes (Holl et al., 2003; Petitti et al., 2009). Tight glycemic control in the DCCT and subsequent studies was associated with a higher risk of complications, such as hypoglycemia and weight gain (1994). In addition, poor compliance is an important issue to be taken into account in adolescents with type 1 diabetes. Furthermore, other factors besides insulin therapy are relevant for metabolic control, such as dietary habits, education, family interactions, cultural and psychological aspects (Holl et al., 2003), and therefore they need to be taken into account in order to define a successful treatment plan.

Treatment with angiotensin converting enzyme inhibitors (ACEIs) is recommended when hypertension is confirmed (Donaghue et al., 2007). ACEIs are treatment of choice in adults with microalbuminuria, based on evidence that their use decrease the rate of progression and even increase rates of regression of microalbuminuria (Lewis et al., 1993). A beneficial

Microvascular and Macrovascular

**6. Conclusions** 

**7. References** 

prognosis.

Complications in Children and Adolescents with Type 1 Diabetes 209

The risk for micro- and macrovascular complications is high in young patients with childhood-onset type 1 diabetes and their development negatively influence their long-term

Early identification of risk factors and prevention of diabetes complications are of paramount importance in children and adolescents. Diabetic vascular complications are often asymptomatic during their early stages, and once symptoms develop, it can be difficult to reverse them. Therefore, screening for vascular complications started during early adolescence, as currently recommended, is essential. Identification of risk factors and subclinical signs of complications is of paramount importance for the early implementation of preventive and therapeutic strategies, which could change the course of vascular complications and improve the prognosis of youth with diabetes. Efforts should be made for optimizing glycemic control, to keep blood pressure and lipid levels within target levels, to avoid smoking, promote exercise and a healthy diet. Future studies are required to test the efficacy and safety of new therapies, which could target specific metabolic or hemodynamic

Further advances in genomics, proteomics and in other 'omics' and the integration of the findings of these different sciences will hopefully allow a better understanding of the pathogenesis of diabetic vascular complications in the near future and will potentially lead

(1993). The effect of intensive treatment of diabetes on the development and progression of

(1997). Clustering of long-term complications in families with diabetes in the diabetes

(2002). MRC/BHF Heart Protection Study of cholesterol lowering with simvastatin in 20,536 high-risk individuals: a randomised placebo-controlled trial. Lancet 360, 7-22. (2003). Sustained effect of intensive treatment of type 1 diabetes mellitus on development

Interventions and Complications (EDIC) study. Jama 290, 2159-2167. Aiello, L.P., Gardner, T.W., King, G.L., Blankenship, G., Cavallerano, J.D., Ferris, F.L., 3rd, and Klein, R. (1998). Diabetic retinopathy. Diabetes Care 21, 143-156. Amin, R., Turner, C., van Aken, S., Bahu, T.K., Watts, A., Lindsell, D.R., Dalton, R.N., and

long-term complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research Group. N Engl J Med 329, 977-986. (1994). Effect of intensive diabetes treatment on the development and progression of long-

term complications in adolescents with insulin-dependent diabetes mellitus: Diabetes Control and Complications Trial. Diabetes Control and Complications

control and complications trial. The Diabetes Control and Complications Trial

and progression of diabetic nephropathy: the Epidemiology of Diabetes

Dunger, D.B. (2005). The relationship between microalbuminuria and glomerular filtration rate in young type 1 diabetic subjects: The Oxford Regional Prospective

pathways implicated in the pathogenesis of diabetic complications.

to a personalized medicine for young patients with diabetes

Trial Research Group. J Pediatr 125, 177-188.

Research Group. Diabetes 46, 1829-1839.

Study. Kidney Int 68, 1740-1749.

effect of anti-hypertensive treatment, and in particular of treatment with ACEIs, has also been demonstrated for DR (Chaturvedi et al., 1998). The EURODIAB Controlled Trial of Lisinopril in type 1 diabetes showed a significant effect of lisinopril in reducing by around 50% the progression of retinopathy in normotensive and normo- or microalbuminuric patients (Chaturvedi et al., 1998). However, there is no universal recommendation for the use of ACEIs in children and adolescents with microalbuminuria. The ADA recommends to start treatment with ACEIs in presence of persistent microalbuminuria (Silverstein et al., 2005). Similarly, the recent ISPAD guidelines suggest to use ACEIs or angiotensin receptor blockers in presence of persistent microalbuminuria, in order to prevent progression to proteinuria (Donaghue et al., 2007), even though the lack of evidence in this context is acknowledged.

Dyslipidemia should be managed firstly with improvements in glycemic control and dietary changes and, in case of persistence of high cholesterol levels, treatment with statins should be considered, although there is no enough evidence of their use in children apart from those with familial hypercholesterolemia (Donaghue et al., 2007). However, in adults statins have been shown to be effective in the primary and secondary prevention of major cardiovascular events (2002).

Adolescents with type 1 diabetes need to be persuaded to avoid smoking. In addition, it is of paramount importance also to avoid increases in BMI, given that adiposity is a well known risk factor for cardiovascular complications, and in some studies it has also been associated with microvascular complications of type 1 diabetes (Donaghue et al., 2007).



Table 5. Recommendations to reduce diabetic vascular complications (ISPAD guideline 2006-2007)

New potential therapeutic possibilities for the treatment of vascular complications are emerging and they include drugs targeting specific pathways implicated in their pathogenesis. These include inhibitors of aldose reductase, inhibitors of protein kinase C, antagonists of advanced glycation end-products, glycosaminoglycans, inhibitors of growth factors and anti-oxidants (Soro-Paavonen and Forbes, 2006). Up to now, there are no definitive data to recommend the use of these new potential therapies but the overall objective of targeting specific metabolic and hemodynamic pathways implicated in the pathogenesis of diabetic vascular complications could lead to validation of these classes of drugs and discovery of novel pharmaceuticals.

#### **6. Conclusions**

208 Type 1 Diabetes Complications

effect of anti-hypertensive treatment, and in particular of treatment with ACEIs, has also been demonstrated for DR (Chaturvedi et al., 1998). The EURODIAB Controlled Trial of Lisinopril in type 1 diabetes showed a significant effect of lisinopril in reducing by around 50% the progression of retinopathy in normotensive and normo- or microalbuminuric patients (Chaturvedi et al., 1998). However, there is no universal recommendation for the use of ACEIs in children and adolescents with microalbuminuria. The ADA recommends to start treatment with ACEIs in presence of persistent microalbuminuria (Silverstein et al., 2005). Similarly, the recent ISPAD guidelines suggest to use ACEIs or angiotensin receptor blockers in presence of persistent microalbuminuria, in order to prevent progression to proteinuria (Donaghue et al., 2007), even though the lack of evidence in this context is

Dyslipidemia should be managed firstly with improvements in glycemic control and dietary changes and, in case of persistence of high cholesterol levels, treatment with statins should be considered, although there is no enough evidence of their use in children apart from those with familial hypercholesterolemia (Donaghue et al., 2007). However, in adults statins have been shown to be effective in the primary and secondary prevention of major

Adolescents with type 1 diabetes need to be persuaded to avoid smoking. In addition, it is of paramount importance also to avoid increases in BMI, given that adiposity is a well known risk factor for cardiovascular complications, and in some studies it has also been associated

**Targets to reduce diabetic vascular complications** 

with microvascular complications of type 1 diabetes (Donaghue et al., 2007).

HbA1c: ≤7.5% (without severe hypoglycemia)

Blood pressure: <90th percentile for age, sex and height

Table 5. Recommendations to reduce diabetic vascular complications (ISPAD guideline

New potential therapeutic possibilities for the treatment of vascular complications are emerging and they include drugs targeting specific pathways implicated in their pathogenesis. These include inhibitors of aldose reductase, inhibitors of protein kinase C, antagonists of advanced glycation end-products, glycosaminoglycans, inhibitors of growth factors and anti-oxidants (Soro-Paavonen and Forbes, 2006). Up to now, there are no definitive data to recommend the use of these new potential therapies but the overall objective of targeting specific metabolic and hemodynamic pathways implicated in the pathogenesis of diabetic vascular complications could lead to validation of these classes of

 LDL cholesterol: <2.6mmol/l HDL cholesterol: >1.1mmol/l Triglycerides: <1.7mmol/l

Physical activity: moderate: >1hr/day

Sedentary activities: <2hr/day

 BMI <95th centile Avoid smoking

Healthy diet

drugs and discovery of novel pharmaceuticals.

2006-2007)

acknowledged.

cardiovascular events (2002).

The risk for micro- and macrovascular complications is high in young patients with childhood-onset type 1 diabetes and their development negatively influence their long-term prognosis.

Early identification of risk factors and prevention of diabetes complications are of paramount importance in children and adolescents. Diabetic vascular complications are often asymptomatic during their early stages, and once symptoms develop, it can be difficult to reverse them. Therefore, screening for vascular complications started during early adolescence, as currently recommended, is essential. Identification of risk factors and subclinical signs of complications is of paramount importance for the early implementation of preventive and therapeutic strategies, which could change the course of vascular complications and improve the prognosis of youth with diabetes. Efforts should be made for optimizing glycemic control, to keep blood pressure and lipid levels within target levels, to avoid smoking, promote exercise and a healthy diet. Future studies are required to test the efficacy and safety of new therapies, which could target specific metabolic or hemodynamic pathways implicated in the pathogenesis of diabetic complications.

Further advances in genomics, proteomics and in other 'omics' and the integration of the findings of these different sciences will hopefully allow a better understanding of the pathogenesis of diabetic vascular complications in the near future and will potentially lead to a personalized medicine for young patients with diabetes

#### **7. References**


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**10** 

*Lebanon* 

**Type 1 Diabetes Mellitus: Redefining the Future** 

Diabetes Mellitus (DM) is a disease that was identified centuries ago, in around 1500 BC (King and Rubin, 2003). The word 'Diabetes' means "running through" (Holt, 2004) which is used to describe the excessive output of urine in this disease. "Mellitus" meaning "sweet" identifies the nature of the urine in patients suffering from DM (Widmaier *et al*., 2004). Thomas Willis was the first to differentiate DM from other polyurias in 1674 (Eknoyan and Nagy, 2005). In 1776, Matthew Dobson demonstrated that the sugar present in the urine was also present in the blood and was associated with its rise (Holt, 2004). In 1848, Claude Bernard's experiments on the liver showed that glycogen stored in the liver produced sugar and he hypothesized that glycogenolysis caused the disease. Although his discovery that "sugar production is a normal function of the animal" was revolutionary, it did not quite explain the etiology of the disease. In 1889, Oskar Minkowski confirmed that the ablation of the pancreas in dogs resulted in DM (Farmer, 1952). Frederick Banting and Charles Best were the medical scientists that discovered insulin, later in 1921, the lack of which, it was made clear, caused DM (Voet and Voet, 2004). They used the extract of a dog's fresh pancreas and demonstrated that upon administration of 10mL of extract to blood, blood glucose level is decreased from 0.3% to 0.17% (Rosenfeld, 2002). Some 30 years later, in 1953, Frederick Sanger was able to determine the complete amino

acid sequence of the protein for the first time in history (Boron and Boulpaep, 2009).

people by the year 2030 (Wild *et al.,* 2004).

**1.1 Type 1 diabetes mellitus** 

Today, DM is defined as a carbohydrate disorder characterized by impaired insulin secretion and/or peripheral insulin resistance leading to hyperglycemia (Beers and Berkow. 1999). It is considered to be the third leading cause of death after heart disease and cancer in the United States (Voet and Voet, 2004) and its incidence is expected to rise to 366 million

Diabetic patients have symptoms such as, thirst, polyuria, blurring of vision and weight loss. In extreme cases, ketoacidosis may develop leading to coma and ultimately death (Alberti and

Although both type 1 Diabetes Mellitus (type 1 DM) and type 2 Diabetes Mellitus (type 2 DM) are characterized clinically by hyperglycemia, they have their differences. Type 1 DM

Zimmet, 1998). Diabetes Mellitus is classified according to etiology to two major types:

**1. Introduction** 

**of Cardiovascular Complications** 

Anwar B. Bikhazi, Nadine S. Zwainy, Sawsan M. Al Lafi,

**with Novel Treatments** 

*American University of Beirut,* 

Shushan B. Artinian and Suzan S. Boutary


### **Type 1 Diabetes Mellitus: Redefining the Future of Cardiovascular Complications with Novel Treatments**

Anwar B. Bikhazi, Nadine S. Zwainy, Sawsan M. Al Lafi, Shushan B. Artinian and Suzan S. Boutary *American University of Beirut, Lebanon* 

#### **1. Introduction**

218 Type 1 Diabetes Complications

Viberti, G.C., Jarrett, R.J., and Keen, H. (1982). Microalbuminuria as prediction of

White, N.H., Sun, W., Cleary, P.A., Tamborlane, W.V., Danis, R.P., Hainsworth, D.P., and

Williams, C.L., Hayman, L.L., Daniels, S.R., Robinson, T.N., Steinberger, J., Paridon, S., and

Davis, M.D. (2010). Effect of prior intensive therapy in type 1 diabetes on 10-year progression of retinopathy in the DCCT/EDIC: comparison of adults and

Bazzarre, T. (2002). Cardiovascular health in childhood: A statement for health professionals from the Committee on Atherosclerosis, Hypertension, and Obesity in the Young (AHOY) of the Council on Cardiovascular Disease in the Young,

nephropathy in diabetics. Lancet 2, 611.

adolescents. Diabetes 59, 1244-1253.

American Heart Association. Circulation 106, 143-160.

Williams, G., and Pickup, J.C. (2004). Diabetic eye disease. (Blackwell Publishing).

Diabetes Mellitus (DM) is a disease that was identified centuries ago, in around 1500 BC (King and Rubin, 2003). The word 'Diabetes' means "running through" (Holt, 2004) which is used to describe the excessive output of urine in this disease. "Mellitus" meaning "sweet" identifies the nature of the urine in patients suffering from DM (Widmaier *et al*., 2004). Thomas Willis was the first to differentiate DM from other polyurias in 1674 (Eknoyan and Nagy, 2005). In 1776, Matthew Dobson demonstrated that the sugar present in the urine was also present in the blood and was associated with its rise (Holt, 2004). In 1848, Claude Bernard's experiments on the liver showed that glycogen stored in the liver produced sugar and he hypothesized that glycogenolysis caused the disease. Although his discovery that "sugar production is a normal function of the animal" was revolutionary, it did not quite explain the etiology of the disease. In 1889, Oskar Minkowski confirmed that the ablation of the pancreas in dogs resulted in DM (Farmer, 1952). Frederick Banting and Charles Best were the medical scientists that discovered insulin, later in 1921, the lack of which, it was made clear, caused DM (Voet and Voet, 2004). They used the extract of a dog's fresh pancreas and demonstrated that upon administration of 10mL of extract to blood, blood glucose level is decreased from 0.3% to 0.17% (Rosenfeld, 2002). Some 30 years later, in 1953, Frederick Sanger was able to determine the complete amino acid sequence of the protein for the first time in history (Boron and Boulpaep, 2009).

Today, DM is defined as a carbohydrate disorder characterized by impaired insulin secretion and/or peripheral insulin resistance leading to hyperglycemia (Beers and Berkow. 1999). It is considered to be the third leading cause of death after heart disease and cancer in the United States (Voet and Voet, 2004) and its incidence is expected to rise to 366 million people by the year 2030 (Wild *et al.,* 2004).

Diabetic patients have symptoms such as, thirst, polyuria, blurring of vision and weight loss. In extreme cases, ketoacidosis may develop leading to coma and ultimately death (Alberti and Zimmet, 1998). Diabetes Mellitus is classified according to etiology to two major types:

#### **1.1 Type 1 diabetes mellitus**

Although both type 1 Diabetes Mellitus (type 1 DM) and type 2 Diabetes Mellitus (type 2 DM) are characterized clinically by hyperglycemia, they have their differences. Type 1 DM

Type 1 Diabetes Mellitus: Redefining the Future

early intensive therapy for its management.

**2.1 Microvascular complications** 

**2.2 Macrovascular complications** 

**3. Renin angiotensin system** 

diagnosis (King *et al*., 2005).

of Cardiovascular Complications with Novel Treatments 221

2008; Rhoades and Bell, 2009). These injuries cause on the long run acceleration in atherosclerotic formation (Seifter *et al.*, 2005). In type 1 diabetic patients, hyperglycemia has been correlated with a variety of events in cardiovascular pathology, initiated with endothelial dysfunction and progressed to develop arterial stiffness (Tabit *et al*., 2010). Thus, a relation has been clearly established to link glycemia with cardiovascular events in type 1 DM, based on the fact that glycemia itself is the only factor mediating a risk factor for cardiovascular risk in the absence of other risk factors (Retenakaran and Zinman, 2008). One way of reducing cardiovascular risk and renal outcomes in type 1 DM is to initiate an

Microvascular complications include nephropathy, retinopathy and neuropathy (Coccheri, 2007; Fowler, 2008; Seifter *et al.*, 2005).There is a direct clinical practice to link these complications with DM type 1 rather than type 2 (Retenakaran and Zinman, 2008). Of note, type 2 DM may manifest development of microvascular complication 7 years preceding

These define the pathophysiology of cardiac disease in type 1 DM which includes atherosclerosis, coronary artery diseases, stroke and peripheral arterial disease (Retenakaran and Zinman, 2008; Seifter *et al*., 2005). These cardiovascular complications are mostly focused in clinical practice to type 2 DM (Fowler, 2008), and increased in rate as well in type 1 DM (Mcphee *et al*., 2008); however, the mortality impact of cardiovascular diseases in both DM types 1 and 2 is similar (Juutilainen *et al.,* 2008) contributing to 80 % of the mortality and morbidity (Coccheri, 2007). More alarmingly, development of macrovascular complication might not manifest in type 1 DM until 10 years proceeding diagnosis (King *et al.,* 2005). Endothelial and smooth muscle dysfunction has been associated with type 1 diabetic patients, which in turn might cause hypertension, a major risk factor for developing cardiovascular diseases (Retenakaran and Zinman, 2008). Also lipid abnormalities in type 1 DM, decrease in HDL and increase in LDL compositions, are being incidents of causing cardiovascular diseases (Retenakaran and Zinman, 2008). High levels of ketone bodies are produced from the body as an alternate fuel in the absence of glucose as a main source of energy in severe starving conditions, mainly hyperglycemia (Seifter *et al.*, 2005), or an acute multiplication of type 1 but not type 2 DM (Rhoades and Bell, 2009). Three types of ketones can be generated in the body these are acetone whose odor can be detected overtly *via* breath, acetacetic acid and βhydroxybutyric acid whose levels may elevate in the blood and be excreted in urine, which is

followed by cations and fluid loss ultimately leading to coma (Rhoades and Bell, 2009).

With the discovery of renin in 1898, Robert Tigerstedt and Per Bergman were able to fill a gap present in the understanding of fluid balance, hypertension and cardiovascular disease (Basso and Terragno, 2001**;** Phillips and Schmidt-Ott, 1999). The Renin Angiotensin System (RAS) plays an important role in maintaining normal blood volume and blood pressure. When salt and water intake is reduced, the role of RAS becomes critical (Rhoades and Bell, 2008). Upon low plasma volume, intrarenal baroreceptors found in the afferent arteriole

occurs commonly during childhood or adolescence therefore also named juvenile onset DM and may develop diabetic ketoacidosis (Beers and Berkow, 1999). Of all diabetes cases, 10% are type 1 DM (Holt, 2004). It includes all autoimmune and idiopathic causes of insulinsecreting β cell destruction resulting in absolute insulin deficiency (Alberti and Zimmet, 1998). The patient is usually genetically predisposed to DM, however, an environmental insult, such as a virus is needed to trigger the pathological process of the disease (Wilson *et al.* 1991). It is evident that CD4+ and CD8+ T lymphocytes are activated in the pancreatic lymph node and later infiltrate the pancreas causing inflammation (insulitis) (Yang and Santamaria, 2003). During this time, the body develops an immune response that sees pancreatic islet cells as 'nonself' and starts destroying its β cells (Wilson *et al.,* 1991). Specific causes of β cell destruction, such as cystic fibrosis or mitochondrial defects, are excluded from this classification (Alberti and Zimmet, 1998). Recently, type 1 DM was subdivided into type 1 A and type 1 B. Type 1A diabetes mellitus is described as a disease with immune mediated, selective destruction of insulin producing β cells, with the presence of anti-islet autoantibodies. Whereas type 1B diabetes mellitus, exhibits inflammation of the pancreas, but lack of anti-islet autoantibodies (Rhoades and Bell, 2009). Patients with type 1 DM require daily subcutaneous insulin administration as a treatment (Seifter *et al.,* 2005). Administration of exogenous insulin cannot be compared to the fine control of minute to minute insulin secretion that the pancreas provides (Hakim, 2002); for that reason, human islet cell transplantation has been accepted and applied as an alternative treatment for patients with type 1 DM (George, 2009).

#### **1.2 Type 2 diabetes mellitus**

Type 2 DM is also known "Adult-onset Diabetes" (Beers *et al.,* 2006), and represents 90-95% of diabetic patients (Creager *et al*., 2003; Seifter *et al.*, 2005). This percentage is expected to increase in the year of 2025 to reach 300 million diabetic patients (Rhoades and Bell, 2009) due to sedentary lifestyle and increase in obesity in addition to age progression (Boron and Boulpaep*,* 2009; King *et al.,* 2005). As the intake of glucose increases insulin secretion is elevated. After a while, insulin secretion becomes inadequate due to peripheral resistance of insulin receptors (Beers *et al*., 2006; King *et al.,* 2005). Insulin resistance decreases glucosemediated insulin transport and metabolism (Rhoades and Bell, 2009), resulting in a defect of the compensatory insulin secretion (Mcphee *et al.*, 2008). It is worth noting that in type 2 DM reactive oxygen species are generated resulting in endothelial dysfunction, cardiovascular complications and renal disease (Hayashi *et al.*, 2010; Seifter *et al*., 2005). Insulin resistance develops when insulin signaling pathway is interfered in adipose, skeletal and hepatic cells (Seifter *et al.,* 2005). As a result of insulin resistance, glucose transport

### **2. Complications of diabetes mellitus**

from the liver is impaired. (Rhoades and Bell, 2009)

Chronic hyperglycemia causes blood vessels injury (Seifter *et al.*, 2005) which is divided into two types depending on the size of vessels injured. Small vessels injury in diabetes is a microvascular complication whereas injury of large blood vessels determines a macrovascular complication (Hayashi *et al.*, 2010, Kalani, 2008). Microvascular and macrovascular complications occur in DM both types 1 and 2 (Retenakaran and Zinman,

inside adipocytes and skeletal muscle is reduced and the suppression of glucose output

2008; Rhoades and Bell, 2009). These injuries cause on the long run acceleration in atherosclerotic formation (Seifter *et al.*, 2005). In type 1 diabetic patients, hyperglycemia has been correlated with a variety of events in cardiovascular pathology, initiated with endothelial dysfunction and progressed to develop arterial stiffness (Tabit *et al*., 2010). Thus, a relation has been clearly established to link glycemia with cardiovascular events in type 1 DM, based on the fact that glycemia itself is the only factor mediating a risk factor for cardiovascular risk in the absence of other risk factors (Retenakaran and Zinman, 2008). One way of reducing cardiovascular risk and renal outcomes in type 1 DM is to initiate an early intensive therapy for its management.

#### **2.1 Microvascular complications**

220 Type 1 Diabetes Complications

occurs commonly during childhood or adolescence therefore also named juvenile onset DM and may develop diabetic ketoacidosis (Beers and Berkow, 1999). Of all diabetes cases, 10% are type 1 DM (Holt, 2004). It includes all autoimmune and idiopathic causes of insulinsecreting β cell destruction resulting in absolute insulin deficiency (Alberti and Zimmet, 1998). The patient is usually genetically predisposed to DM, however, an environmental insult, such as a virus is needed to trigger the pathological process of the disease (Wilson *et al.* 1991). It is evident that CD4+ and CD8+ T lymphocytes are activated in the pancreatic lymph node and later infiltrate the pancreas causing inflammation (insulitis) (Yang and Santamaria, 2003). During this time, the body develops an immune response that sees pancreatic islet cells as 'nonself' and starts destroying its β cells (Wilson *et al.,* 1991). Specific causes of β cell destruction, such as cystic fibrosis or mitochondrial defects, are excluded from this classification (Alberti and Zimmet, 1998). Recently, type 1 DM was subdivided into type 1 A and type 1 B. Type 1A diabetes mellitus is described as a disease with immune mediated, selective destruction of insulin producing β cells, with the presence of anti-islet autoantibodies. Whereas type 1B diabetes mellitus, exhibits inflammation of the pancreas, but lack of anti-islet autoantibodies (Rhoades and Bell, 2009). Patients with type 1 DM require daily subcutaneous insulin administration as a treatment (Seifter *et al.,* 2005). Administration of exogenous insulin cannot be compared to the fine control of minute to minute insulin secretion that the pancreas provides (Hakim, 2002); for that reason, human islet cell transplantation has been accepted and applied as an alternative treatment for

Type 2 DM is also known "Adult-onset Diabetes" (Beers *et al.,* 2006), and represents 90-95% of diabetic patients (Creager *et al*., 2003; Seifter *et al.*, 2005). This percentage is expected to increase in the year of 2025 to reach 300 million diabetic patients (Rhoades and Bell, 2009) due to sedentary lifestyle and increase in obesity in addition to age progression (Boron and Boulpaep*,* 2009; King *et al.,* 2005). As the intake of glucose increases insulin secretion is elevated. After a while, insulin secretion becomes inadequate due to peripheral resistance of insulin receptors (Beers *et al*., 2006; King *et al.,* 2005). Insulin resistance decreases glucosemediated insulin transport and metabolism (Rhoades and Bell, 2009), resulting in a defect of the compensatory insulin secretion (Mcphee *et al.*, 2008). It is worth noting that in type 2 DM reactive oxygen species are generated resulting in endothelial dysfunction, cardiovascular complications and renal disease (Hayashi *et al.*, 2010; Seifter *et al*., 2005). Insulin resistance develops when insulin signaling pathway is interfered in adipose, skeletal and hepatic cells (Seifter *et al.,* 2005). As a result of insulin resistance, glucose transport inside adipocytes and skeletal muscle is reduced and the suppression of glucose output

Chronic hyperglycemia causes blood vessels injury (Seifter *et al.*, 2005) which is divided into two types depending on the size of vessels injured. Small vessels injury in diabetes is a microvascular complication whereas injury of large blood vessels determines a macrovascular complication (Hayashi *et al.*, 2010, Kalani, 2008). Microvascular and macrovascular complications occur in DM both types 1 and 2 (Retenakaran and Zinman,

patients with type 1 DM (George, 2009).

from the liver is impaired. (Rhoades and Bell, 2009)

**2. Complications of diabetes mellitus** 

**1.2 Type 2 diabetes mellitus** 

Microvascular complications include nephropathy, retinopathy and neuropathy (Coccheri, 2007; Fowler, 2008; Seifter *et al.*, 2005).There is a direct clinical practice to link these complications with DM type 1 rather than type 2 (Retenakaran and Zinman, 2008). Of note, type 2 DM may manifest development of microvascular complication 7 years preceding diagnosis (King *et al*., 2005).

#### **2.2 Macrovascular complications**

These define the pathophysiology of cardiac disease in type 1 DM which includes atherosclerosis, coronary artery diseases, stroke and peripheral arterial disease (Retenakaran and Zinman, 2008; Seifter *et al*., 2005). These cardiovascular complications are mostly focused in clinical practice to type 2 DM (Fowler, 2008), and increased in rate as well in type 1 DM (Mcphee *et al*., 2008); however, the mortality impact of cardiovascular diseases in both DM types 1 and 2 is similar (Juutilainen *et al.,* 2008) contributing to 80 % of the mortality and morbidity (Coccheri, 2007). More alarmingly, development of macrovascular complication might not manifest in type 1 DM until 10 years proceeding diagnosis (King *et al.,* 2005).

Endothelial and smooth muscle dysfunction has been associated with type 1 diabetic patients, which in turn might cause hypertension, a major risk factor for developing cardiovascular diseases (Retenakaran and Zinman, 2008). Also lipid abnormalities in type 1 DM, decrease in HDL and increase in LDL compositions, are being incidents of causing cardiovascular diseases (Retenakaran and Zinman, 2008). High levels of ketone bodies are produced from the body as an alternate fuel in the absence of glucose as a main source of energy in severe starving conditions, mainly hyperglycemia (Seifter *et al.*, 2005), or an acute multiplication of type 1 but not type 2 DM (Rhoades and Bell, 2009). Three types of ketones can be generated in the body these are acetone whose odor can be detected overtly *via* breath, acetacetic acid and βhydroxybutyric acid whose levels may elevate in the blood and be excreted in urine, which is followed by cations and fluid loss ultimately leading to coma (Rhoades and Bell, 2009).

#### **3. Renin angiotensin system**

With the discovery of renin in 1898, Robert Tigerstedt and Per Bergman were able to fill a gap present in the understanding of fluid balance, hypertension and cardiovascular disease (Basso and Terragno, 2001**;** Phillips and Schmidt-Ott, 1999). The Renin Angiotensin System (RAS) plays an important role in maintaining normal blood volume and blood pressure. When salt and water intake is reduced, the role of RAS becomes critical (Rhoades and Bell, 2008). Upon low plasma volume, intrarenal baroreceptors found in the afferent arteriole

Type 1 Diabetes Mellitus: Redefining the Future

(EMEA) in August 2007 (Pool, 2007; Azizi, 2008).

**4. Endothelin system** 

(Penna *et al*., 2006; Prasad *et al.*, 2009).

**4.1 Endothelin-1** 

of Cardiovascular Complications with Novel Treatments 223

The idea of blocking RAS at its point of origin was initiated around 50 years ago (Pool, 2007). The origin of these direct renin inhibitors (DRIs) was either analogues of the prosegment peptide of renin or of the AGT's N-terminal amino acid sequence (Gradman and Kad, 2008). Many peptidomimetic synthetic drugs used for renin inhibition have been launched including pepsatitn (the first renin inhibtor) and other oral drugs, including remikiren, enalkiren and zankiren (Pimenta and Oparil, 2009), but their low efficiency related to large molecular size, first-pass metabolism, incomplete intestinal absorption, hydrophobicity and short half-lives attributed their poor oral activity and bioavailability, besides the high production cost (Pool, 2007; Waldmeier *et al.*, 2007). The drug Tekturna® (United States) or Rasilez ® (Europe) , known with the generic name of Aliskiren, ascribed to Dr. Alice Huxley (Gradman and Kad, 2008), manufactured by Novartis Pharmaceuticals, was the first of the nonpeptide DRIs to be approved by the Food and Drug Administration (FDA) (Azizi, 2008). Thereby becoming the commercially available renin blocker in markets, prescribed through United States and Canada for an effective essential hypertension treatment in March 2007 (Azizi, 2008), and approved by European Medicine Agency

In 1985, Hickey *et al.* discovered a potent vasoconstricting substance from cultured endothelial cells and named it endotensin or endothelial contracting factor. Later on, Yanagisawa *et al*. isolated the same substance from cultured porcine aortic and endothelial cells and renamed it endothelin. Endothelin (ET) is a naturally occurring polypeptide (Prasad *et al.,* 2009) produced by the endothelium (Shah et al.,2009; Wikes et al., 2003; Ying wu, 2003) and consists of 21 amino acids. It is present in three isoforms ET-1, ET-2 and ET-3 (Agapitov *et al.,* 2002; Prasad *et al.*, 2009). These three isoforms are encoded by different genes but have similar structure and function (Wilkes *et al.,* 2003). They have conserved sequences of amino acids mainly at the 6 C terminus and 4 cysteine residues that form 2 disulfide bridges between residues 1 to 15 and 3 to 11 and the main difference is at the Nterminus (Prasad *et al.*, 2009). They are vasoconstrictors synthesized by vascular, right atrial and left ventricular endothelial cells, vascular smooth muscle cells and fibromyocytes. In addition, they are synthesized in extra vascular tissues such as the lungs, spleen, pancreas and nervous system (Penna *et al*., 2006). All studies done on endothelial systems focused on ET-1, because represents the majority of the circulating endothelins (Prasad *et al.,* 2009; Schneider *et al*., 2002) and has important role in the regulation of the cardiovascular system

Endothelin-1 is the most common isoform correlated with the cardiovascular system (Kalani, 2008). It was identified in the late 1980s by Dr. Yanagisawa and his colleagues and found to be a very potent vasoconstrictor (Prasad *et al.,* 2009; Steiner and Preston, 2008; Thorin and Webb, 2009). It has ionotropic, chemotactic and mitogenic activities. It influences salt and water retention through its effect on RAS, vasopressin release and stimulation of sympathetic nervous system. So the ultimate role of ET-1 is to increase blood pressure (Agapitov *et al*., 2002) and to maintain vascular tone (Thorin and Webb, 2009). It is produced mostly from endothelial cells in addition to fibroblasts, cardiacmyocytes (Agapitov *et al.,*

walls sense a decrease in stretch and neighboring granular cells increase renin synthesis and release (Boron and Boulpaep, 2009). The granular cells of the juxtaglomerular apparatus of the kidney produce renin (Widmaier *et al.,* 2004). At first, preprorenin is synthesized in the granular cells; its 23 amino acid signal peptide is then cleaved to form prorenin (Sepehrdad *et al*., 2007). This proenzyme undergoes further modification- clipping the 43 amino acid segment from the N-terminal – to produce renin (Pool, 2007). Despite the conversion of prorenin to renin, prorenin remains the predominant form in the systemic circulation and represents 90% of the total plasma renin in humans (Pimenta and Oparil, 2009). Renin belongs to the family of aspartyl proteases, including pepsin, cathepsin D and chymosin (Verdeccia *et al*., 2008). Unlike other aspartyl proteases however, renin is able to work at neutral pH. Moreover, renin is specific for one substrate only- angiotensinogen (Sepehrdad *et al*., 2007). Made up of two lobes with a cleft in between renin accommodates the liverderived angiotensinogen in its active site, where a peptide bond of angiotensinogen is hydrolyzed and the decapeptide Angiotensin I (Ang I) is released (Verdeccia *et al*., 2008). Furthermore, this conversion is catalyzed 4-fold upon the binding of renin to its receptor (Pool, 2007). Angiotensin converting enzyme (ACE) is the principal enzyme that converts Ang I to the octapeptide Ang II (60% conversion); some other enzymes such as chymases, cathepsin G and other serine proteases account for the rest of the conversion (Weir, 2007).

#### **3.1 Cross talk between RAS and litus**

Angiotensin II was always considered in close proximity with diseases and vascular complications. This is implied from data linking high levels of Ang II to DM and vasoconstriction (causing hypertension) (Karam *et al*., 2005), in addition to glomerular damaging ending up with nephropathy (Coccheri, 2007). Other than its direct effect as a potent vasoconstrictor, it was denoted to alter Endothelin-1 (ET-1) production (Karam *et al*., 2005), remodel cardiovascular structure (Parmar and Jadav, 2007), modulate heart and vessels (Nuwayri-salti *et al.*, 2007), augment transforming growth factor–beta (TGF-β) and boost proliferative and inflammatory events (Wiggins and Kelly, 2009). In conjunction; modulation of insulin significance is obtained (Karam *et al*., 2005), either by directly halting signaling mechanism of insulin per se or damaging structure and function of β-cells *via* local pancreatic RAS (Coccheri, 2007). Angiotensin II has 2 receptors: Ang II Type 1 receptor (AT1R) and Ang II Type 2 receptor (AT2R). But until now AT1R rather than AT2R is assessed in the pathophysiology (Karam *et al.*, 2005; Rao, 2010) directing therapeutic agents either to target Ang II formation or block its binding to AT1R *via* Angiotensin converting enzyme inhibitors (ACEIs) and Ang II Receptor Blocker(ARB) respectively (Karam *et al.,* 2005). AT1R blockers improved vascular smooth muscle cell vasoconstriction and declined the hypertrophy of cardiomyocytes (CM) counteracting Ang II deleterious effect (Al Jaroudi *et al*., 2005) once bound to the G-coupled protein receptor (AT1 R) (Parmar and Jadav, 2007; Wiggins and Kelly, 2009). Quite interestingly, ACEIs ameliorated insulin sensitivity and mitigated new onset of DM type 2 (Hadi and Suwaidi, 2007; Scheen, 2004). Pooled together, these G-coupled transmembrane proteins mimic insulin receptor action (Nuwayri-Salti *et al*., 2007). Among several evidences, AT2R is more related to apoptotic actions thereby antagonizing insulin's growth effect, resulting in one way or another to boost cardiac hypertrophy mainly in type 1 DM (Al Jaroudi *et al.*, 2005). To complete this picture, a balance should be assured between both Ang II receptors to modulate a normal cardiac status in type 1 DM along the endothelial cells and cardiomyocytes (Al Jaroudi *et al*., 2005).

The idea of blocking RAS at its point of origin was initiated around 50 years ago (Pool, 2007). The origin of these direct renin inhibitors (DRIs) was either analogues of the prosegment peptide of renin or of the AGT's N-terminal amino acid sequence (Gradman and Kad, 2008). Many peptidomimetic synthetic drugs used for renin inhibition have been launched including pepsatitn (the first renin inhibtor) and other oral drugs, including remikiren, enalkiren and zankiren (Pimenta and Oparil, 2009), but their low efficiency related to large molecular size, first-pass metabolism, incomplete intestinal absorption, hydrophobicity and short half-lives attributed their poor oral activity and bioavailability, besides the high production cost (Pool, 2007; Waldmeier *et al.*, 2007). The drug Tekturna® (United States) or Rasilez ® (Europe) , known with the generic name of Aliskiren, ascribed to Dr. Alice Huxley (Gradman and Kad, 2008), manufactured by Novartis Pharmaceuticals, was the first of the nonpeptide DRIs to be approved by the Food and Drug Administration (FDA) (Azizi, 2008). Thereby becoming the commercially available renin blocker in markets, prescribed through United States and Canada for an effective essential hypertension treatment in March 2007 (Azizi, 2008), and approved by European Medicine Agency (EMEA) in August 2007 (Pool, 2007; Azizi, 2008).

#### **4. Endothelin system**

222 Type 1 Diabetes Complications

walls sense a decrease in stretch and neighboring granular cells increase renin synthesis and release (Boron and Boulpaep, 2009). The granular cells of the juxtaglomerular apparatus of the kidney produce renin (Widmaier *et al.,* 2004). At first, preprorenin is synthesized in the granular cells; its 23 amino acid signal peptide is then cleaved to form prorenin (Sepehrdad *et al*., 2007). This proenzyme undergoes further modification- clipping the 43 amino acid segment from the N-terminal – to produce renin (Pool, 2007). Despite the conversion of prorenin to renin, prorenin remains the predominant form in the systemic circulation and represents 90% of the total plasma renin in humans (Pimenta and Oparil, 2009). Renin belongs to the family of aspartyl proteases, including pepsin, cathepsin D and chymosin (Verdeccia *et al*., 2008). Unlike other aspartyl proteases however, renin is able to work at neutral pH. Moreover, renin is specific for one substrate only- angiotensinogen (Sepehrdad *et al*., 2007). Made up of two lobes with a cleft in between renin accommodates the liverderived angiotensinogen in its active site, where a peptide bond of angiotensinogen is hydrolyzed and the decapeptide Angiotensin I (Ang I) is released (Verdeccia *et al*., 2008). Furthermore, this conversion is catalyzed 4-fold upon the binding of renin to its receptor (Pool, 2007). Angiotensin converting enzyme (ACE) is the principal enzyme that converts Ang I to the octapeptide Ang II (60% conversion); some other enzymes such as chymases, cathepsin G and other serine proteases account for the rest of the conversion (Weir, 2007).

Angiotensin II was always considered in close proximity with diseases and vascular complications. This is implied from data linking high levels of Ang II to DM and vasoconstriction (causing hypertension) (Karam *et al*., 2005), in addition to glomerular damaging ending up with nephropathy (Coccheri, 2007). Other than its direct effect as a potent vasoconstrictor, it was denoted to alter Endothelin-1 (ET-1) production (Karam *et al*., 2005), remodel cardiovascular structure (Parmar and Jadav, 2007), modulate heart and vessels (Nuwayri-salti *et al.*, 2007), augment transforming growth factor–beta (TGF-β) and boost proliferative and inflammatory events (Wiggins and Kelly, 2009). In conjunction; modulation of insulin significance is obtained (Karam *et al*., 2005), either by directly halting signaling mechanism of insulin per se or damaging structure and function of β-cells *via* local pancreatic RAS (Coccheri, 2007). Angiotensin II has 2 receptors: Ang II Type 1 receptor (AT1R) and Ang II Type 2 receptor (AT2R). But until now AT1R rather than AT2R is assessed in the pathophysiology (Karam *et al.*, 2005; Rao, 2010) directing therapeutic agents either to target Ang II formation or block its binding to AT1R *via* Angiotensin converting enzyme inhibitors (ACEIs) and Ang II Receptor Blocker(ARB) respectively (Karam *et al.,* 2005). AT1R blockers improved vascular smooth muscle cell vasoconstriction and declined the hypertrophy of cardiomyocytes (CM) counteracting Ang II deleterious effect (Al Jaroudi *et al*., 2005) once bound to the G-coupled protein receptor (AT1 R) (Parmar and Jadav, 2007; Wiggins and Kelly, 2009). Quite interestingly, ACEIs ameliorated insulin sensitivity and mitigated new onset of DM type 2 (Hadi and Suwaidi, 2007; Scheen, 2004). Pooled together, these G-coupled transmembrane proteins mimic insulin receptor action (Nuwayri-Salti *et al*., 2007). Among several evidences, AT2R is more related to apoptotic actions thereby antagonizing insulin's growth effect, resulting in one way or another to boost cardiac hypertrophy mainly in type 1 DM (Al Jaroudi *et al.*, 2005). To complete this picture, a balance should be assured between both Ang II receptors to modulate a normal cardiac status in type 1 DM along the endothelial cells and cardiomyocytes (Al Jaroudi *et al*., 2005).

**3.1 Cross talk between RAS and litus** 

In 1985, Hickey *et al.* discovered a potent vasoconstricting substance from cultured endothelial cells and named it endotensin or endothelial contracting factor. Later on, Yanagisawa *et al*. isolated the same substance from cultured porcine aortic and endothelial cells and renamed it endothelin. Endothelin (ET) is a naturally occurring polypeptide (Prasad *et al.,* 2009) produced by the endothelium (Shah et al.,2009; Wikes et al., 2003; Ying wu, 2003) and consists of 21 amino acids. It is present in three isoforms ET-1, ET-2 and ET-3 (Agapitov *et al.,* 2002; Prasad *et al.*, 2009). These three isoforms are encoded by different genes but have similar structure and function (Wilkes *et al.,* 2003). They have conserved sequences of amino acids mainly at the 6 C terminus and 4 cysteine residues that form 2 disulfide bridges between residues 1 to 15 and 3 to 11 and the main difference is at the Nterminus (Prasad *et al.*, 2009). They are vasoconstrictors synthesized by vascular, right atrial and left ventricular endothelial cells, vascular smooth muscle cells and fibromyocytes. In addition, they are synthesized in extra vascular tissues such as the lungs, spleen, pancreas and nervous system (Penna *et al*., 2006). All studies done on endothelial systems focused on ET-1, because represents the majority of the circulating endothelins (Prasad *et al.,* 2009; Schneider *et al*., 2002) and has important role in the regulation of the cardiovascular system (Penna *et al*., 2006; Prasad *et al.*, 2009).

#### **4.1 Endothelin-1**

Endothelin-1 is the most common isoform correlated with the cardiovascular system (Kalani, 2008). It was identified in the late 1980s by Dr. Yanagisawa and his colleagues and found to be a very potent vasoconstrictor (Prasad *et al.,* 2009; Steiner and Preston, 2008; Thorin and Webb, 2009). It has ionotropic, chemotactic and mitogenic activities. It influences salt and water retention through its effect on RAS, vasopressin release and stimulation of sympathetic nervous system. So the ultimate role of ET-1 is to increase blood pressure (Agapitov *et al*., 2002) and to maintain vascular tone (Thorin and Webb, 2009). It is produced mostly from endothelial cells in addition to fibroblasts, cardiacmyocytes (Agapitov *et al.,*

Type 1 Diabetes Mellitus: Redefining the Future

of Cardiovascular Complications with Novel Treatments 225

a. Normal (N) b. Normal + Losartan (NL)

c. Diabetic (D) d. Diabetic + Losartan (DL)

Fig. 1. Indirect immunofluorescence of CM and vascular endothelium from the different rat groups depicting insulin receptors. Noted is the increase of fluorescence in the NL group (Fig. b) when compared to the normal (N) (Fig. a) at the myocytes (arrow). There is also increase fluorescence of the myocytes of the (DL) group as compared to the (D) group, and

f. Diabetic insulin +Losartan

of the (DIL) group as compared to the (DI) group (arrow).

e. Diabetic + insulin (DI)

2002; Schorlemmer *et al*., 2008), leukocytes, macrophages (Thorin and Webb, 2009), kidney, central nervous system and posterior pituitary (Agapitov *et al*., 2002).

Endothelin-1 exerts a paracrine/autocrine effect. In the circulation, the levels of ET-1 are in picomolars lower than that needed to cause vasoconstriction (Prasad *et al.,* 2009). Also endothelial cells secrete more ET-1 toward the vicinity of vascular smooth muscle cells than into the lumen of the blood vessels (Agapitov *et al*., 2002).

#### **4.2 Endothelin-1 synthesis and clearance**

Endothelin-1 synthesis begins with the cleavage of a 200 amino acid peptide called preproendothelin-1 (preproET-1) to form preendothelin-1 (preET-1) (Agapitov *et al.,* 2002; Penna *et al*., 2006). PreET-1 is then cleaved by furin endopeptidase to form big endothelin-1 (big ET-1) of 38-39 amino acids that is further metabolized by Endothelin Converting Enzyme-1 (ECE-1) to generate ET-1 of 21 amino acids (Agapitov *et al*., 2002; Penna *et al*., 2006). Moreover, chymase enzyme was found to produce ET(1-31) by breaking down big ET-1 at Tyr31-Gly32 bond (Agapitov *et al*., 2002).

Furthermore, the regulation of ET-1 synthesis is at the gene level which means at the level of ET-1 messenger RNA (mRNA). The ET-1 mRNA is up-regulated by several factors including: interleukins, insulin, Ang II, tumor necrosis factor alpha and growth factor while it is down regulated by hypoxia, shear stress and nitric oxide (Thorin and Webb, 2009). The clearance of ET-1 from the plasma involves different mechanisms including: (1) endocytosis in the lungs, (2) enzymatic degradation in the liver and the kidney and (3) ET-1 binding to ETB receptor thus forming ETB Receptor-Legand complex that is broken down by endocytosis (Kalani, 2008).

#### **4.3 Cross talk between endothelin system and renin angiotensin system**

The Renin Angiotensin and the Endothelin Systems are the most potent identified vasoconstrictory systems and has been suggested to be correlated with each other (Rossi *et al*., 1999; McEwan *et al*., 2000). This was demonstrated by several cell culture studies in cardiac fibroblasts. These studies showed that AngII increases preproET-1 mRNA expression, ET-1 levels and thus causes cardiomyocyte hypertrophy through regulation of ETAR. Also increased levels of ETBR via Ang II have also been identified in cultured CM (McEwan *et al.,* 2000).

The potential sites at which RAS increases ET-1 levels may be at the preproET-1 gene that possesses a *jun* sequence which is defined as the regulatory region of preproET-1. It is the site at which transcriptional regulation takes place by factors acting through G-proteinphospholipase and C-protein kinase C pathway. Another site for interaction of RAS and ET system is at the chymase enzyme which is abundant in the myocardium where it was found to produce ET (1-31) from big ETs**,** bearing in mind that the main function of chymase enzyme in the heart is to produce Ang II from Ang I (Rossi *et al*., 1999). Therefore, elevated levels of ET-1 can be reduced by blocking the RAS system. Studies on patients suffering from hypertension or diabetes showed decrease in the level of ET-1 when ACEI was administered (Schneider *et al*., 2002).

Therapies targeting these complications include the use of ARBs which proved their value as antihypertensive drugs. Losartan, an ARB, was found to decrease the destructive changes caused by Ang II on cardiac muscle beside its (Berk, 1999; Fiordaliso *et al*., 2000) capacity to reduce blood pressure. Recently, Al Jaroudi *et al*. proved that losartan in diabetic normotensive rats supplemented with insulin was able to prevent nearly totally myocardial degeneration caused by diabetes (Al Jaroudi *et al.*, 2005) (Fig. 1).

2002; Schorlemmer *et al*., 2008), leukocytes, macrophages (Thorin and Webb, 2009), kidney,

Endothelin-1 exerts a paracrine/autocrine effect. In the circulation, the levels of ET-1 are in picomolars lower than that needed to cause vasoconstriction (Prasad *et al.,* 2009). Also endothelial cells secrete more ET-1 toward the vicinity of vascular smooth muscle cells than

Endothelin-1 synthesis begins with the cleavage of a 200 amino acid peptide called preproendothelin-1 (preproET-1) to form preendothelin-1 (preET-1) (Agapitov *et al.,* 2002; Penna *et al*., 2006). PreET-1 is then cleaved by furin endopeptidase to form big endothelin-1 (big ET-1) of 38-39 amino acids that is further metabolized by Endothelin Converting Enzyme-1 (ECE-1) to generate ET-1 of 21 amino acids (Agapitov *et al*., 2002; Penna *et al*., 2006). Moreover, chymase enzyme was found to produce ET(1-31) by breaking down big

Furthermore, the regulation of ET-1 synthesis is at the gene level which means at the level of ET-1 messenger RNA (mRNA). The ET-1 mRNA is up-regulated by several factors including: interleukins, insulin, Ang II, tumor necrosis factor alpha and growth factor while it is down regulated by hypoxia, shear stress and nitric oxide (Thorin and Webb, 2009). The clearance of ET-1 from the plasma involves different mechanisms including: (1) endocytosis in the lungs, (2) enzymatic degradation in the liver and the kidney and (3) ET-1 binding to ETB receptor thus forming ETB Receptor-Legand complex that is broken down by

The Renin Angiotensin and the Endothelin Systems are the most potent identified vasoconstrictory systems and has been suggested to be correlated with each other (Rossi *et al*., 1999; McEwan *et al*., 2000). This was demonstrated by several cell culture studies in cardiac fibroblasts. These studies showed that AngII increases preproET-1 mRNA expression, ET-1 levels and thus causes cardiomyocyte hypertrophy through regulation of ETAR. Also increased levels of ETBR via Ang II have also been identified in cultured CM (McEwan *et al.,* 2000). The potential sites at which RAS increases ET-1 levels may be at the preproET-1 gene that possesses a *jun* sequence which is defined as the regulatory region of preproET-1. It is the site at which transcriptional regulation takes place by factors acting through G-proteinphospholipase and C-protein kinase C pathway. Another site for interaction of RAS and ET system is at the chymase enzyme which is abundant in the myocardium where it was found to produce ET (1-31) from big ETs**,** bearing in mind that the main function of chymase enzyme in the heart is to produce Ang II from Ang I (Rossi *et al*., 1999). Therefore, elevated levels of ET-1 can be reduced by blocking the RAS system. Studies on patients suffering from hypertension or diabetes showed decrease in the level of ET-1 when ACEI was

Therapies targeting these complications include the use of ARBs which proved their value as antihypertensive drugs. Losartan, an ARB, was found to decrease the destructive changes caused by Ang II on cardiac muscle beside its (Berk, 1999; Fiordaliso *et al*., 2000) capacity to reduce blood pressure. Recently, Al Jaroudi *et al*. proved that losartan in diabetic normotensive rats supplemented with insulin was able to prevent nearly totally myocardial

**4.3 Cross talk between endothelin system and renin angiotensin system** 

central nervous system and posterior pituitary (Agapitov *et al*., 2002).

into the lumen of the blood vessels (Agapitov *et al*., 2002).

**4.2 Endothelin-1 synthesis and clearance** 

ET-1 at Tyr31-Gly32 bond (Agapitov *et al*., 2002).

endocytosis (Kalani, 2008).

administered (Schneider *et al*., 2002).

degeneration caused by diabetes (Al Jaroudi *et al.*, 2005) (Fig. 1).

Fig. 1. Indirect immunofluorescence of CM and vascular endothelium from the different rat groups depicting insulin receptors. Noted is the increase of fluorescence in the NL group (Fig. b) when compared to the normal (N) (Fig. a) at the myocytes (arrow). There is also increase fluorescence of the myocytes of the (DL) group as compared to the (D) group, and of the (DIL) group as compared to the (DI) group (arrow).

Type 1 Diabetes Mellitus: Redefining the Future

the incretins, exemplified by GLP-1.

**6. Definition of incretins** 

Effect' (Rhoades and Bell, 2008).

**5.1 Insulin receptor** 

of Cardiovascular Complications with Novel Treatments 227

Insulin receptor is a member of the receptor tyrosine kinase family (De Meyts and Whittaker, 2002; Kanzaki, 2006; Klarlund *et al*., 2003; Lawrence *et al*., 2007; Stern, 1995; Ward *et al*., 2008). It is a large cell-surface multi-domain glycoprotein that consists of two extracellular α-subunits (MW~125 kDa) and two transmembrane β-subunits (MW~95 kDa) (Kanzaki, 2006; Klarlund *et al*., 2003; Lawrence *et al*., 2007; Stern, 1995; Ward *et al*., 2008) linked by disulfide bonds (Kanzaki, 2006; Lawrence *et al*., 2007) into an α2β2 heterotetrameric complex (Kanzaki, 2006). Extracellular α-subunits contain the insulin-binding site (Klarlund *et al*., 2003) while intracellularly transmembrane β-subunits contain the insulin-regulated tyrosine kinase catalytic domain (De Meyts and Whittaker, 2002; Klarlund *et al*., 2003; Ward *et al*., 2008). Two nearly identical isoforms (A and B) of the insulin receptor exist due to tissue specific alternative splicing of the receptor m-RNA at exon 11. Isoforms A and B differ by the presence or absence of a 12 amino acid sequence at the carboxyl-terminus of the αsubunit (Anderson *et al*. 1993; De Meyts and Whittaker. 2002; Klarlund *et al*., 2003; Lawrence *et al*., 2007). Despite the known biochemical and physiologic differences of these isoforms, changes in their expression levels have not been consistently found in patients with DM (Klarlund *et al*., 2003). Once insulin binds to the α-subunit of the insulin receptor, it induces a conformational change in the receptor that subsequently leads to the stimulation of the intrinsic tyrosine kinase activity in the β-subunits (Kanzaki, 2006; Klarlund *et al*., 2003; Stern, 1995). This results in the transfer of phosphate groups from ATP to several tyrosine residues on the insulin receptor itself as well as phosphorylation of cellular proteins such as insulin receptor substrate-1 (IRS-1) and Shc (Klarlund *et al*., 2003; Stern, 1995). The stimulation of the receptor tyrosine kinase allows transmission of the insulin signal to metabolic pathways such as glucose uptake upon translocation of GLUT-4 glucose transporters to the plasma membrane, glycogen synthesis, protein synthesis, and lipid metabolism within the cell. The various biological responses generated upon insulin receptor activation through insulin binding granted insulin a role in DM treatment due to its glucose lowering effects. Nevertheless, daily insulin injections continue to be a treatment for diabetic patients since 1922 despite the increasing worldwide prevalence of this disease (Sparre *et al*., 2005). Furthermore, insulin treatment dramatically prolongs survival, but does not cure diabetes (Myers and Zonszein, 2002). Insulin treatment does not seem to be beneficial for many patients and is associated with weight gain, hypoglycemia, and failure of their glycemic control (Halimi, 2008). Therefore, new treatment modalities are intensively studied mainly

The intravenous infusion of glucose at a constant rate results in a biphasic insulin secretory response, in which the first peak rises rapidly followed by a slower second peak. In contrast, an oral administration of glucose followed by its gastrointestinal absorption, triggers a hormonal pathway that eventually leads to a far greater response of insulin secretion which can last as long as glucose is administered. This phenomenon is termed as the 'Incretin

Incretins are hormones that are secreted by the gut upon feeding; their release alerts the pancreatic islets that nutrients will come through the gastrointestinal tract and islets start priming by vagal stimulation (Boron and Boulpaep, 2009). Glucagon-Like Peptide-1 (GLP-1) and Glucose Dependent Insulinotropic Peptide (GIP) are the major incretin hormones

Later, Karam *et al*. reported that the supplementation with insulin and administration of pharmacologic doses of losartan could improve cardiac contraction as well as coronary blood flow in the same normotensive rat model for type 1 DM as the one used by Jaroudi *et al.* through the modification of the affinity of ET type 1 receptor subtypes ETAR and ETBR to ET-1, a potent vasoconstrictor largely stimulated by Ang II (Karam *et al*., 2005). In fact, Kakoki *et al.* have demonstrated that the ETBRs are down-regulated by Ang II in the endothelial cells of the renal artery of diabetic rats through stimulation of increased ET-1 production (Kakoki *et al.*, 1999). On the other hand, these effects of Ang II are prevented by the ACE inhibitor Imidapril (Kakoki *et al.*, 1999). ACE inhibitors reduced also the plasma ET-1 levels in type 1 DM (Schneider *et al.*, 2002). Administration of Losartan to Ang IItreated rats restored the vasoconstrictive effect of ET-1 and decreased its tissue levels (D'Uscio *et al.*, 1997). Mc Ewan *et al.* showed that the blockade of the AT1Rs by Losartan, in the presence of a high plasma Ang II levels, is accompanied by an increase in ET-1 production by the CM of Ang II-treated rats.

In addition to this, an increase in the ETBRs mRNA but a decrease in the ETARs mRNA was also noted (Mc Ewan *et al.*, 2000). On the other hand, treatment with the selective ETAR blocker LU135252 normalized the increased ET-1 level in the aorta, femoral artery and kidney, and the ECE activity in isolated aorta and femoral artery of rats treated with Ang II (Barton *et al.*, 1997). In cultured heart endothelial cells, Ang II stimulates ppET-1 mRNA and ET-1 production via Protein Kinase C (PKC) - dependent pathway. Calphostin, a PKC inhibitor, blocks Ang II effects (Chua *et al.*, 1993). Taken together, these results show that the ET and the RA systems are related to each other. They also demonstrate the beneficial role of ARBs and ACE inhibitors in the amelioration of the blood flow which can be altered in some disease states due to the ET-1 and Ang II constrictor effects.

#### **5. Insulin**

The discovery of insulin in 1921 was one of the most dramatic events in the history of diabetes therapy. Insulin is a small globular protein comprising of two polypeptide chains A (21 amino acid residues) and B (30 amino acid residues) held together by two disulfide bonds (Docherty and Steiner. 2003; Joshi *et al*. 2007). It is produced in the β-cells of the islets of Langerhans in the form of preproinsulin which is rapidly cleaved by the action of proteolytic enzymes into proinsulin. Further catalysis of proinsulin results in insulin and a 31 amino acid connecting peptide, C-peptide, both of which are stored for secretion in secretory vesicles in β-cells (Docherty and Steiner. 2003). The biosynthesis and secretion of insulin by β-cells primarily occurs in response to increased circulating glucose levels. During feeding, elevated glucose concentrations in the blood increase the plasma insulin level, which facilitates glucose uptake through GLUT-4 into muscle tissues for utilization as a source of energy and into adipose tissues for synthesis of glycerol. Insulin also exerts its action on liver cells as well thus promoting glycogen formation. Consequently, glucose utilization by these different tissues contribute to the decrease in the concentration of glucose in blood. On top of its profound effect in carbohydrate metabolism, insulin has a fatsparing effect. Not only does it promote the synthesis of fatty acids in the liver, it also inhibits the breakdown of fats in adipose tissue thereby inducing fat accumulation. Hence, insulin is the major determinant of carbohydrate and lipid metabolism and has significant effects on protein metabolism. Insulin acts by binding to a receptor molecule embedded in the plasma membrane of its target tissues (Docherty and Steiner. 2003).

#### **5.1 Insulin receptor**

226 Type 1 Diabetes Complications

Later, Karam *et al*. reported that the supplementation with insulin and administration of pharmacologic doses of losartan could improve cardiac contraction as well as coronary blood flow in the same normotensive rat model for type 1 DM as the one used by Jaroudi *et al.* through the modification of the affinity of ET type 1 receptor subtypes ETAR and ETBR to ET-1, a potent vasoconstrictor largely stimulated by Ang II (Karam *et al*., 2005). In fact, Kakoki *et al.* have demonstrated that the ETBRs are down-regulated by Ang II in the endothelial cells of the renal artery of diabetic rats through stimulation of increased ET-1 production (Kakoki *et al.*, 1999). On the other hand, these effects of Ang II are prevented by the ACE inhibitor Imidapril (Kakoki *et al.*, 1999). ACE inhibitors reduced also the plasma ET-1 levels in type 1 DM (Schneider *et al.*, 2002). Administration of Losartan to Ang IItreated rats restored the vasoconstrictive effect of ET-1 and decreased its tissue levels (D'Uscio *et al.*, 1997). Mc Ewan *et al.* showed that the blockade of the AT1Rs by Losartan, in the presence of a high plasma Ang II levels, is accompanied by an increase in ET-1

In addition to this, an increase in the ETBRs mRNA but a decrease in the ETARs mRNA was also noted (Mc Ewan *et al.*, 2000). On the other hand, treatment with the selective ETAR blocker LU135252 normalized the increased ET-1 level in the aorta, femoral artery and kidney, and the ECE activity in isolated aorta and femoral artery of rats treated with Ang II (Barton *et al.*, 1997). In cultured heart endothelial cells, Ang II stimulates ppET-1 mRNA and ET-1 production via Protein Kinase C (PKC) - dependent pathway. Calphostin, a PKC inhibitor, blocks Ang II effects (Chua *et al.*, 1993). Taken together, these results show that the ET and the RA systems are related to each other. They also demonstrate the beneficial role of ARBs and ACE inhibitors in the amelioration of the blood flow which can be altered in some

The discovery of insulin in 1921 was one of the most dramatic events in the history of diabetes therapy. Insulin is a small globular protein comprising of two polypeptide chains A (21 amino acid residues) and B (30 amino acid residues) held together by two disulfide bonds (Docherty and Steiner. 2003; Joshi *et al*. 2007). It is produced in the β-cells of the islets of Langerhans in the form of preproinsulin which is rapidly cleaved by the action of proteolytic enzymes into proinsulin. Further catalysis of proinsulin results in insulin and a 31 amino acid connecting peptide, C-peptide, both of which are stored for secretion in secretory vesicles in β-cells (Docherty and Steiner. 2003). The biosynthesis and secretion of insulin by β-cells primarily occurs in response to increased circulating glucose levels. During feeding, elevated glucose concentrations in the blood increase the plasma insulin level, which facilitates glucose uptake through GLUT-4 into muscle tissues for utilization as a source of energy and into adipose tissues for synthesis of glycerol. Insulin also exerts its action on liver cells as well thus promoting glycogen formation. Consequently, glucose utilization by these different tissues contribute to the decrease in the concentration of glucose in blood. On top of its profound effect in carbohydrate metabolism, insulin has a fatsparing effect. Not only does it promote the synthesis of fatty acids in the liver, it also inhibits the breakdown of fats in adipose tissue thereby inducing fat accumulation. Hence, insulin is the major determinant of carbohydrate and lipid metabolism and has significant effects on protein metabolism. Insulin acts by binding to a receptor molecule embedded in

production by the CM of Ang II-treated rats.

**5. Insulin** 

disease states due to the ET-1 and Ang II constrictor effects.

the plasma membrane of its target tissues (Docherty and Steiner. 2003).

Insulin receptor is a member of the receptor tyrosine kinase family (De Meyts and Whittaker, 2002; Kanzaki, 2006; Klarlund *et al*., 2003; Lawrence *et al*., 2007; Stern, 1995; Ward *et al*., 2008). It is a large cell-surface multi-domain glycoprotein that consists of two extracellular α-subunits (MW~125 kDa) and two transmembrane β-subunits (MW~95 kDa) (Kanzaki, 2006; Klarlund *et al*., 2003; Lawrence *et al*., 2007; Stern, 1995; Ward *et al*., 2008) linked by disulfide bonds (Kanzaki, 2006; Lawrence *et al*., 2007) into an α2β2 heterotetrameric complex (Kanzaki, 2006). Extracellular α-subunits contain the insulin-binding site (Klarlund *et al*., 2003) while intracellularly transmembrane β-subunits contain the insulin-regulated tyrosine kinase catalytic domain (De Meyts and Whittaker, 2002; Klarlund *et al*., 2003; Ward *et al*., 2008). Two nearly identical isoforms (A and B) of the insulin receptor exist due to tissue specific alternative splicing of the receptor m-RNA at exon 11. Isoforms A and B differ by the presence or absence of a 12 amino acid sequence at the carboxyl-terminus of the αsubunit (Anderson *et al*. 1993; De Meyts and Whittaker. 2002; Klarlund *et al*., 2003; Lawrence *et al*., 2007). Despite the known biochemical and physiologic differences of these isoforms, changes in their expression levels have not been consistently found in patients with DM (Klarlund *et al*., 2003). Once insulin binds to the α-subunit of the insulin receptor, it induces a conformational change in the receptor that subsequently leads to the stimulation of the intrinsic tyrosine kinase activity in the β-subunits (Kanzaki, 2006; Klarlund *et al*., 2003; Stern, 1995). This results in the transfer of phosphate groups from ATP to several tyrosine residues on the insulin receptor itself as well as phosphorylation of cellular proteins such as insulin receptor substrate-1 (IRS-1) and Shc (Klarlund *et al*., 2003; Stern, 1995). The stimulation of the receptor tyrosine kinase allows transmission of the insulin signal to metabolic pathways such as glucose uptake upon translocation of GLUT-4 glucose transporters to the plasma membrane, glycogen synthesis, protein synthesis, and lipid metabolism within the cell. The various biological responses generated upon insulin receptor activation through insulin binding granted insulin a role in DM treatment due to its glucose lowering effects. Nevertheless, daily insulin injections continue to be a treatment for diabetic patients since 1922 despite the increasing worldwide prevalence of this disease (Sparre *et al*., 2005). Furthermore, insulin treatment dramatically prolongs survival, but does not cure diabetes (Myers and Zonszein, 2002). Insulin treatment does not seem to be beneficial for many patients and is associated with weight gain, hypoglycemia, and failure of their glycemic control (Halimi, 2008). Therefore, new treatment modalities are intensively studied mainly the incretins, exemplified by GLP-1.

#### **6. Definition of incretins**

The intravenous infusion of glucose at a constant rate results in a biphasic insulin secretory response, in which the first peak rises rapidly followed by a slower second peak. In contrast, an oral administration of glucose followed by its gastrointestinal absorption, triggers a hormonal pathway that eventually leads to a far greater response of insulin secretion which can last as long as glucose is administered. This phenomenon is termed as the 'Incretin Effect' (Rhoades and Bell, 2008).

Incretins are hormones that are secreted by the gut upon feeding; their release alerts the pancreatic islets that nutrients will come through the gastrointestinal tract and islets start priming by vagal stimulation (Boron and Boulpaep, 2009). Glucagon-Like Peptide-1 (GLP-1) and Glucose Dependent Insulinotropic Peptide (GIP) are the major incretin hormones

Type 1 Diabetes Mellitus: Redefining the Future

Rotella, 2008; Drucker, 2007).

**6.3 GLP-1 receptor** 

Nauk, 2006).

of Cardiovascular Complications with Novel Treatments 229

GLP-1 also exerts effects on the central nervous system: it promotes satiety and weight loss, inhibits food and water intake and improves memory and neuronal survival (Mannucci and

The GLP-1R belongs to the Guanine Nucleotide-Binding Protein (G-protein) coupled receptor family (GPCR) (Drucker, 2007). This seven transmembrane receptor protein is 90% identical to the rat GLP-1R and shows a 95% homology in amino acid. Its gene is found on the long arm of chromosome 6p21 in humans (Doyle and Egan, 2007). GLP-1 receptor is made up of 463 amino acid residues and has a molecular weight of approximately 65 kDa (Hui *et al*., 2005). GLP-1 receptors are expressed in many tissues including, the central and peripheral nervous systems, heart, kidney, lungs and the gastrointestinal tract (Drucker and

However, GLP-1 receptor expression in the pancreas is controversial. Studies have confirmed the detection of GLP-1 receptors in α, β and δ cells of the islets of Langerhans, whereas other studies indicate their expression exclusively in β cells (Doyle and Egan, 2007). In the heart, GLP-1 receptor expression was shown to exist in CM, microvascular endothelium, coronary smooth muscle cells and the highest in endocardium. In cardiac fibroblasts, however, there was no evidence in GLP-1R expression (Ban *et al.,* 2009). Low detection of GLP-1 receptor gene expression proves the existence of GLP-1 receptor in liver and muscles (MacDonald *et al*., 2002). Abundant GLP-1R specific transcripts were found in lungs and detectable amounts in the heart (Ban *et al.,* 2009). When fragments of the Nterminus are denatured, GLP-1R loses its affinity for GLP-1. This asserts that GLP-1 receptor's N-terminus plays an important role in recognizing and binding of the GLP-1 (Doyle and Egan. 2007). Upon binding of the GLP-1 to its receptor numerous signaling messengers are activated; first, GLP-1 receptor can couple to G proteins, including Gαs, Gαq, Gαi or Gαo (Baggio and Drucker, 2007). Adenylate cyclase uses ATP to form cAMP via the stimulatory G protein. Following the increase of cAMP is a chain of events is triggered: ATP-sensitive K+ channels shut and L-type voltage gated Ca2+ channels open; together with the efflux of Ca2+ molecules from intracellular Ca2+ stores, these ion channel alterations result in a subsequent intracellular rise in Ca2+ ions (Gomez *et al*., 2002) and the ultimate exocytosis of granules that contain insulin. A sustained receptor signaling results in the activation of Protein Kinase A (PKA), gene transcription, insulin biosynthesis and β-cell proliferation (Drucker and Nauk, 2006). Additionally, the binding of GLP-1 to its receptor activates protein kinase B, which is linked to glucose transporting in muscles, glycogen

A recent study showed that GLP-1 receptor expression is downregulated in β-cells exposed to high glucose concentrations in vitro and hyperglycemia in vivo (Xu *et al.,* 2007). Diabetic individuals' β-cells exhibit attenuated sensitivity to GLP-1. In both type 1 and type 2 DM, there is a marked reduction in the incretin effect (Knop *et al*., 2007). On the other hand, a study reported that GLP-1 levels are not decreased in type 2 diabetic patients (Lee *et al*., 2010). The European GLP-1 Club held a meeting recently and debated this controversial issue and came to a conclusion that more data is required to determine the exact effect of DM on GLP-1 secretion from L-cells (Burcelin, 2008). GLP-1 has been suggested to

synthesis and lipolysis in various tissues (Peyot *et al*., 2009).

**6.4 GLP-1 in diabetes mellitus and the heart** 

produced by the L cells of the ileum and colon, and K cells of the duodenum and jejunum, respectively (Inzucchi and McGuire, 2008).

#### **6.1 Synthesis of glucagon-like peptide-1**

The proglucagon gene, located on chromosome 2 in humans, has the coding sequence of GLP-1. Pancreatic α-cells, intestinal L-cells and neural cells in the caudal brainstem and hypothalamus, express the proglucagon gene (Baggio and Drucker, 2007) and the mRNAs produced in the pancreas and intestine are identical. Its post-translational processing, however, differs in the two tissues. The post translational processing of proglucagon in pancreatic α- cells results in Glucagon, Glicentin-Related Pancreatic Polypeptide (GRPP), Intervening peptide-1 (IP-1) and the major proglucagon fragment (MPGF) (Holst, 2007). The latter codes for the production of GLP-1 (1-36 amide) and GLP-1 (1-37) and GLP-2 in the pancreas (Hui *et al*., 2005). Whereas the intestinal L and brain cells produce GLP-1 (7-36 amide), GLP-1 (7-37), GLP-2, IP-2, and glicentin that if further cleaved produces GRPP and oxyntomodulin (Holst, 2007). The enzyme prohormone convertase (PC) 1/3 cleaves proglucagon to generate GLP-1 (Shin *et al*., 2008). GLP-1 is an incretin hormone produced primarily by the L-cells in the mucosa of the ileum and the colon (Mannucci and Rotella, 2008). Small neurons found in the Nucleus Tractus Solitarius (NTS), caudal brainstem, also produce GLP-1 that plays the role of a neuromodulator (Berthoud, 2009). The central nucleus of the amygdala (CeA) and the paraventricular nucleus of the hypothalamus (PVN) are also sites of GLP-1 production (Kinzig *et al.,* 2002).

Recently, GLP-1 production was shown to exist in taste bud cells (Shin *et al.,* 2008; Berthoud, 2009) and in particular in α-gustducin and the sweet taste receptor subunit T1R3, that play an important role in mediating the glucose dependent secretion of GLP-1 (Shin *et al*., 2008**)**. GLP-1 is usually present in the circulation minutes after meal intake- far before food reaches the L-cells in the gut. Hence, its stimulation is believed to be controlled by both endocrine and neural signals (Drucker, 2007). The two active forms of GLP-1 are the 30 amino acid GLP-1 (7-36) amide and the 31 amino acid glycine extended GLP-1 (7-37) (Ban *et al.,* 2009; Mannucci and Rotella, 2008). GLP-1 (7-36) amide is far more abundant than GLP-1 (7-37) in the circulation (Manucci and Rotella, 2008), but both have short half-lives (1.5-2 minutes) (Hui *et al*., 2002). They are rapidly degraded into their inactive forms, GLP-1(9-36) amide and GLP-1 (9-37), respectively, by the enzyme Dipeptidyl-Peptidase-IV (DPP-IV) (Mannucci and Rotella, 2008) and eliminated through renal clearance (Hui *et al.,* 2002).

#### **6.2 Effects of GLP-1**

GLP-1 has numerous effects on glucose homeostasis. Upon interaction with its receptor on β-cells, GLP-1 increases the intracellular levels of cAMP and calcium, thereby releasing insulin (Drucker, 2007). Furthermore, sustained GLP-1 receptor (GLP-1R) signaling leads to enhanced gene transcription, insulin biosynthesis and β-cell proliferation (Elahi *et al.,* 2008; Drucker, 2007). GLP-1 was shown to increase the expression of glucose transporter 2 (GLUT2) (Elahi *et al.,* 2008), a hepatic glucose transporter that facilitates glucose transport in or out of the liver regulated by insulin (Eisenberg *et al*., 2005) and glucokinase (Elahi *et al*., 2008) the enzyme that phosphorylates glucose to glucose-6-phosphate (G6P) (Voet and Voet, 2004). GLP-1 has also inhibitory effects on Glucagon secretion from the α-cells, gastric emptying and food ingestion (Drucker, 2007). The role of GLP-1 in relaxing the proximal stomach and increasing gastric capacity has been demonstrated (D'Alessio and Vahl, 2004). GLP-1 also exerts effects on the central nervous system: it promotes satiety and weight loss, inhibits food and water intake and improves memory and neuronal survival (Mannucci and Rotella, 2008; Drucker, 2007).

#### **6.3 GLP-1 receptor**

228 Type 1 Diabetes Complications

produced by the L cells of the ileum and colon, and K cells of the duodenum and jejunum,

The proglucagon gene, located on chromosome 2 in humans, has the coding sequence of GLP-1. Pancreatic α-cells, intestinal L-cells and neural cells in the caudal brainstem and hypothalamus, express the proglucagon gene (Baggio and Drucker, 2007) and the mRNAs produced in the pancreas and intestine are identical. Its post-translational processing, however, differs in the two tissues. The post translational processing of proglucagon in pancreatic α- cells results in Glucagon, Glicentin-Related Pancreatic Polypeptide (GRPP), Intervening peptide-1 (IP-1) and the major proglucagon fragment (MPGF) (Holst, 2007). The latter codes for the production of GLP-1 (1-36 amide) and GLP-1 (1-37) and GLP-2 in the pancreas (Hui *et al*., 2005). Whereas the intestinal L and brain cells produce GLP-1 (7-36 amide), GLP-1 (7-37), GLP-2, IP-2, and glicentin that if further cleaved produces GRPP and oxyntomodulin (Holst, 2007). The enzyme prohormone convertase (PC) 1/3 cleaves proglucagon to generate GLP-1 (Shin *et al*., 2008). GLP-1 is an incretin hormone produced primarily by the L-cells in the mucosa of the ileum and the colon (Mannucci and Rotella, 2008). Small neurons found in the Nucleus Tractus Solitarius (NTS), caudal brainstem, also produce GLP-1 that plays the role of a neuromodulator (Berthoud, 2009). The central nucleus of the amygdala (CeA) and the paraventricular nucleus of the hypothalamus (PVN)

Recently, GLP-1 production was shown to exist in taste bud cells (Shin *et al.,* 2008; Berthoud, 2009) and in particular in α-gustducin and the sweet taste receptor subunit T1R3, that play an important role in mediating the glucose dependent secretion of GLP-1 (Shin *et al*., 2008**)**. GLP-1 is usually present in the circulation minutes after meal intake- far before food reaches the L-cells in the gut. Hence, its stimulation is believed to be controlled by both endocrine and neural signals (Drucker, 2007). The two active forms of GLP-1 are the 30 amino acid GLP-1 (7-36) amide and the 31 amino acid glycine extended GLP-1 (7-37) (Ban *et al.,* 2009; Mannucci and Rotella, 2008). GLP-1 (7-36) amide is far more abundant than GLP-1 (7-37) in the circulation (Manucci and Rotella, 2008), but both have short half-lives (1.5-2 minutes) (Hui *et al*., 2002). They are rapidly degraded into their inactive forms, GLP-1(9-36) amide and GLP-1 (9-37), respectively, by the enzyme Dipeptidyl-Peptidase-IV (DPP-IV) (Mannucci

GLP-1 has numerous effects on glucose homeostasis. Upon interaction with its receptor on β-cells, GLP-1 increases the intracellular levels of cAMP and calcium, thereby releasing insulin (Drucker, 2007). Furthermore, sustained GLP-1 receptor (GLP-1R) signaling leads to enhanced gene transcription, insulin biosynthesis and β-cell proliferation (Elahi *et al.,* 2008; Drucker, 2007). GLP-1 was shown to increase the expression of glucose transporter 2 (GLUT2) (Elahi *et al.,* 2008), a hepatic glucose transporter that facilitates glucose transport in or out of the liver regulated by insulin (Eisenberg *et al*., 2005) and glucokinase (Elahi *et al*., 2008) the enzyme that phosphorylates glucose to glucose-6-phosphate (G6P) (Voet and Voet, 2004). GLP-1 has also inhibitory effects on Glucagon secretion from the α-cells, gastric emptying and food ingestion (Drucker, 2007). The role of GLP-1 in relaxing the proximal stomach and increasing gastric capacity has been demonstrated (D'Alessio and Vahl, 2004).

and Rotella, 2008) and eliminated through renal clearance (Hui *et al.,* 2002).

respectively (Inzucchi and McGuire, 2008).

**6.1 Synthesis of glucagon-like peptide-1** 

are also sites of GLP-1 production (Kinzig *et al.,* 2002).

**6.2 Effects of GLP-1** 

The GLP-1R belongs to the Guanine Nucleotide-Binding Protein (G-protein) coupled receptor family (GPCR) (Drucker, 2007). This seven transmembrane receptor protein is 90% identical to the rat GLP-1R and shows a 95% homology in amino acid. Its gene is found on the long arm of chromosome 6p21 in humans (Doyle and Egan, 2007). GLP-1 receptor is made up of 463 amino acid residues and has a molecular weight of approximately 65 kDa (Hui *et al*., 2005). GLP-1 receptors are expressed in many tissues including, the central and peripheral nervous systems, heart, kidney, lungs and the gastrointestinal tract (Drucker and Nauk, 2006).

However, GLP-1 receptor expression in the pancreas is controversial. Studies have confirmed the detection of GLP-1 receptors in α, β and δ cells of the islets of Langerhans, whereas other studies indicate their expression exclusively in β cells (Doyle and Egan, 2007). In the heart, GLP-1 receptor expression was shown to exist in CM, microvascular endothelium, coronary smooth muscle cells and the highest in endocardium. In cardiac fibroblasts, however, there was no evidence in GLP-1R expression (Ban *et al.,* 2009). Low detection of GLP-1 receptor gene expression proves the existence of GLP-1 receptor in liver and muscles (MacDonald *et al*., 2002). Abundant GLP-1R specific transcripts were found in lungs and detectable amounts in the heart (Ban *et al.,* 2009). When fragments of the Nterminus are denatured, GLP-1R loses its affinity for GLP-1. This asserts that GLP-1 receptor's N-terminus plays an important role in recognizing and binding of the GLP-1 (Doyle and Egan. 2007). Upon binding of the GLP-1 to its receptor numerous signaling messengers are activated; first, GLP-1 receptor can couple to G proteins, including Gαs, Gαq, Gαi or Gαo (Baggio and Drucker, 2007). Adenylate cyclase uses ATP to form cAMP via the stimulatory G protein. Following the increase of cAMP is a chain of events is triggered: ATP-sensitive K+ channels shut and L-type voltage gated Ca2+ channels open; together with the efflux of Ca2+ molecules from intracellular Ca2+ stores, these ion channel alterations result in a subsequent intracellular rise in Ca2+ ions (Gomez *et al*., 2002) and the ultimate exocytosis of granules that contain insulin. A sustained receptor signaling results in the activation of Protein Kinase A (PKA), gene transcription, insulin biosynthesis and β-cell proliferation (Drucker and Nauk, 2006). Additionally, the binding of GLP-1 to its receptor activates protein kinase B, which is linked to glucose transporting in muscles, glycogen synthesis and lipolysis in various tissues (Peyot *et al*., 2009).

#### **6.4 GLP-1 in diabetes mellitus and the heart**

A recent study showed that GLP-1 receptor expression is downregulated in β-cells exposed to high glucose concentrations in vitro and hyperglycemia in vivo (Xu *et al.,* 2007). Diabetic individuals' β-cells exhibit attenuated sensitivity to GLP-1. In both type 1 and type 2 DM, there is a marked reduction in the incretin effect (Knop *et al*., 2007). On the other hand, a study reported that GLP-1 levels are not decreased in type 2 diabetic patients (Lee *et al*., 2010). The European GLP-1 Club held a meeting recently and debated this controversial issue and came to a conclusion that more data is required to determine the exact effect of DM on GLP-1 secretion from L-cells (Burcelin, 2008). GLP-1 has been suggested to

Type 1 Diabetes Mellitus: Redefining the Future

**6.7 GLP-1 receptor agonists and GLP-1 analogues** 

(Halimi, 2008).

hormones (Laugero *et al*., 2009).

**6.7.1 GLP-1 analogue, exendin-4** 

**6.7.2 Mode of action of exendin-4** 

of Cardiovascular Complications with Novel Treatments 231

*al*., 2008) and enhance pancreatic islet cell function in animal models of type 2 diabetes and in diabetic patients (Lambeir *et al*., 2003). In contrast to GLP-1 mimetics, there are no data indicating inhibition of gastric emptying or appetite or weight reduction due to a treatment with DPP-IV inhibitors (Inzucchi and McGuire, 2008). Thus, chronic treatment with orally administered DPP-IV inhibitors has neutral effects on body weight and food consumption

In April 2005, the FDA approved the subcutaneous injectable exenatide with the brand name Byetta® to be launched as a GLP-1 analogue (Inzucchi and McGuire, 2008; Rhoades and Bell, 2009) with longer half life to maintain its glycemic control as diabetic medication (Behme *et al*., 2003). Exenatide exhibits a half-life of 60-90 minutes and a single injection of exenatide makes its concentration in the blood last for 4-6 hours (Drucker and Nauk, 2006). Exenatide exhibits antidiabetic effects similar to those of GLP-1 and is shown to improve β cell functioning (Baggio and Drucker, 2007). It also reduces the glycated Hemoglobin-HbA1c- levels that reflect mean blood glucose levels of the last 6-8 weeks. In addition, exenatide affects gastric emptying, appetite and consequently causes weight loss (Inzucchi and McGuire, 2008). GLP-1 agonists have exhibited vasodilating and diuretic effects. Recently, Laugero *et al.* showed that Exenatide may display antihypertensive effects mediated by pathways independent from glucose control, but possibly by altering steroid

The short half-life of GLP-1 limits itself from performing as a good therapeutic agent, as it is rapidly degraded by the serine protease DPP-IV (Hirata *et al.,* 2009). The search for biologically active peptides in lizard venom, led to the discovery of Exendin-4, a naturally occurring GLP-1 analogue (Drucker and Nauk, 2006). Exendin-4 is a peptide naturally found in the saliva of heloderma suspectum - the Gila monster (Laugero *et al*., 2009; Zhou *et al.,* 2008). Exendin-4 is a peptide made up of 39 amino acid residues that shares a 53% structural homology to GLP-1 (Zhou *et al.,* 2008).This long acting GLP-1 receptor agonist has an Nterminus almost identical to that of the GLP-1, except that the second amino acid residue is glycine in exendin-4 while alanine in GLP-1 (Hui *et al.,* 2005). This one amino acid difference makes it resistant to degradation by the enzyme DPP-IV, hence explaining its long half-life in vivo (Lovshin and Drucker, 2009). Studies have shown that GLP-1 receptor agonists like exendin-4, exhibit cardioprotective effects such as modifications in

Endogenous GLP-1 action is inhibited upon degradation by DPP-IV (De Koning *et al*., 2008; Mann *et al*., 2007), an enzyme expressed in many organs mainly kidney, small intestine, liver, and lung (Inzucchi and McGuire, 2008). More apt, exendin-4 is synthetic, thus not recognized by DPP-IV enzyme, thereby not degraded (Rhoades and Bell, 2009). This resistance allows it to stay in the circulation longer mimicking GLP-1 role in bolstering insulin secretion but with 5 to 10 times more powerful insulinotrpic effect (Hantouche *et al.*, 2010; Xu *et al.*, 1999). Moreover, it binds to GLP-1R with high affinity even after truncation of the first 8 amino acids at the N-terminal of the peptide unlike the endogenous GLP-1 (Mann

contractility, cardiac output and blood pressure (Ban *et al.,* 2009).

ameliorate left ventricular function, because of its antiapoptotic and insulin-like properties (Inzucchi and McGuire, 2008). In fact, one study confirmed that GLP-1 enhances the regulation of phosphatydil inositol 3 kinase (PI3K), that plays a key role in activating the antiapoptotic pathway, thus promoting cardioprotection in the ischemic rat hearts. Therefore, a direct effect of GLP-1 against apoptosis in cardiac cells is possible (Bose *et al.,* 2005).

#### **6.5 Dipeptidyl-peptidase IV**

DPP-IV grasped a great deal of the interest of the scientific, pharmaceutical, and medical community (Lambeir *et al*., 2003). Every year, an increasing number of publications tend to elucidate the diverse compelling questions concerning the various properties of DPP-IV and its multiple functions in the different fields of Biology (Lambeir *et al*., 2003). DPP-IV is a 766 amino acid serine protease that belongs to the prolyloligopeptidase family. It is a widely distributed cell surface peptidase expressed to different degrees in a variety of tissues such as the kidney, intestine, liver, placenta, uterus, prostate, skin, and capillary endothelium. Another soluble form of DPP-IV (sDPP-IV) also exists in the plasma and other body fluids (Drucker, 2007; Idris and Donnelly, 2007; Lambeir *et al*., 2003). This proteolytic enzyme acts by specifically cleaving the N-terminal dipeptide of peptide hormones containing proline or alanine in the second position (Drucker, 2007; Idris and Donnelly, 2007; Lambeir *et al*., 2003). Hence, DPP-IV truncates several biologically active peptides of medical importance. Furthermore, it has been implicated in glucose homeostasis through proteolytic degradation of the incretins (Drucker, 2007). In the case of GLP-1(7-36), proline and alanine are key determinants in incretin receptor activation therefore DPP-IV-mediated proteolysis results in the biologically inactive truncated molecules GLP-1(9-36) (Drucker, 2007; Idris and Donnelly, 2007). DPP-IV is a critical determinant of incretin inactivation (Drucker, 2007). Thus chemical inhibition of DPP-IV activity results in increased level of biologically active GLP-1 (Drucker, 2007). Therefore, extensive research studies were carried to create highly selective DPP-IV inhibitors.

#### **6.6 DPP-IV inhibitors**

DPP-IV inhibitors can be used as a potential treatment for diabetes due to their capability to potentiate the levels of active incretins (Lambeir *et al*., 2003; Halimi, 2008; Inzucchi and McGuire, 2008) by reducing the enzymatic activity of DPP-IV by more than 80% for duration up to 24 hours (Inzucchi and McGuire, 2008). Oral inhibitors of DPP-IV reduce glycosylated hemoglobin (HbA1C) (Drucker, 2007; Halimi, 2008; Moritoh *et al*., 2008). Various studies reported the multiple metabolic effects of DPP-IV inhibitors including enhancement of glucose-dependent stimulation of pancreatic insulin release as well as attenuation of glucagon secretion (Drucker, 2007; Halimi, 2008; Lambeir *et al*., 2003; Moritoh *et al.,* 2008). Furthermore, DPP-IV inhibitors demonstrated modestly effective glucose-lowering actions without being associated with hypoglycemia (Halimi, 2008; Lambeir *et al*., 2003; Moritoh *et al*., 2008) due to the fact that they are remarkably able to specifically end the insulinsecreting effect and glucagon inhibition once glycemic levels are normalized (Halimi, 2008). Moreover, loss of DPP-IV activity is associated with improved glucose tolerance, reduced glycemic excursion following oral glucose challenge, and increased pancreatic insulin content (Drucker, 2007). A cardinal role of DPP-IV inhibitors is their potential to significantly augment β-cell mass in streptozotocin (STZ)-injected diabetic rats (Moritoh *et* 

ameliorate left ventricular function, because of its antiapoptotic and insulin-like properties (Inzucchi and McGuire, 2008). In fact, one study confirmed that GLP-1 enhances the regulation of phosphatydil inositol 3 kinase (PI3K), that plays a key role in activating the antiapoptotic pathway, thus promoting cardioprotection in the ischemic rat hearts. Therefore, a direct effect of GLP-1 against apoptosis in cardiac cells is possible (Bose *et al.,*

DPP-IV grasped a great deal of the interest of the scientific, pharmaceutical, and medical community (Lambeir *et al*., 2003). Every year, an increasing number of publications tend to elucidate the diverse compelling questions concerning the various properties of DPP-IV and its multiple functions in the different fields of Biology (Lambeir *et al*., 2003). DPP-IV is a 766 amino acid serine protease that belongs to the prolyloligopeptidase family. It is a widely distributed cell surface peptidase expressed to different degrees in a variety of tissues such as the kidney, intestine, liver, placenta, uterus, prostate, skin, and capillary endothelium. Another soluble form of DPP-IV (sDPP-IV) also exists in the plasma and other body fluids (Drucker, 2007; Idris and Donnelly, 2007; Lambeir *et al*., 2003). This proteolytic enzyme acts by specifically cleaving the N-terminal dipeptide of peptide hormones containing proline or alanine in the second position (Drucker, 2007; Idris and Donnelly, 2007; Lambeir *et al*., 2003). Hence, DPP-IV truncates several biologically active peptides of medical importance. Furthermore, it has been implicated in glucose homeostasis through proteolytic degradation of the incretins (Drucker, 2007). In the case of GLP-1(7-36), proline and alanine are key determinants in incretin receptor activation therefore DPP-IV-mediated proteolysis results in the biologically inactive truncated molecules GLP-1(9-36) (Drucker, 2007; Idris and Donnelly, 2007). DPP-IV is a critical determinant of incretin inactivation (Drucker, 2007). Thus chemical inhibition of DPP-IV activity results in increased level of biologically active GLP-1 (Drucker, 2007). Therefore, extensive research studies were carried to create highly

DPP-IV inhibitors can be used as a potential treatment for diabetes due to their capability to potentiate the levels of active incretins (Lambeir *et al*., 2003; Halimi, 2008; Inzucchi and McGuire, 2008) by reducing the enzymatic activity of DPP-IV by more than 80% for duration up to 24 hours (Inzucchi and McGuire, 2008). Oral inhibitors of DPP-IV reduce glycosylated hemoglobin (HbA1C) (Drucker, 2007; Halimi, 2008; Moritoh *et al*., 2008). Various studies reported the multiple metabolic effects of DPP-IV inhibitors including enhancement of glucose-dependent stimulation of pancreatic insulin release as well as attenuation of glucagon secretion (Drucker, 2007; Halimi, 2008; Lambeir *et al*., 2003; Moritoh *et al.,* 2008). Furthermore, DPP-IV inhibitors demonstrated modestly effective glucose-lowering actions without being associated with hypoglycemia (Halimi, 2008; Lambeir *et al*., 2003; Moritoh *et al*., 2008) due to the fact that they are remarkably able to specifically end the insulinsecreting effect and glucagon inhibition once glycemic levels are normalized (Halimi, 2008). Moreover, loss of DPP-IV activity is associated with improved glucose tolerance, reduced glycemic excursion following oral glucose challenge, and increased pancreatic insulin content (Drucker, 2007). A cardinal role of DPP-IV inhibitors is their potential to significantly augment β-cell mass in streptozotocin (STZ)-injected diabetic rats (Moritoh *et* 

2005).

**6.5 Dipeptidyl-peptidase IV** 

selective DPP-IV inhibitors.

**6.6 DPP-IV inhibitors** 

*al*., 2008) and enhance pancreatic islet cell function in animal models of type 2 diabetes and in diabetic patients (Lambeir *et al*., 2003). In contrast to GLP-1 mimetics, there are no data indicating inhibition of gastric emptying or appetite or weight reduction due to a treatment with DPP-IV inhibitors (Inzucchi and McGuire, 2008). Thus, chronic treatment with orally administered DPP-IV inhibitors has neutral effects on body weight and food consumption (Halimi, 2008).

#### **6.7 GLP-1 receptor agonists and GLP-1 analogues**

In April 2005, the FDA approved the subcutaneous injectable exenatide with the brand name Byetta® to be launched as a GLP-1 analogue (Inzucchi and McGuire, 2008; Rhoades and Bell, 2009) with longer half life to maintain its glycemic control as diabetic medication (Behme *et al*., 2003). Exenatide exhibits a half-life of 60-90 minutes and a single injection of exenatide makes its concentration in the blood last for 4-6 hours (Drucker and Nauk, 2006). Exenatide exhibits antidiabetic effects similar to those of GLP-1 and is shown to improve β cell functioning (Baggio and Drucker, 2007). It also reduces the glycated Hemoglobin-HbA1c- levels that reflect mean blood glucose levels of the last 6-8 weeks. In addition, exenatide affects gastric emptying, appetite and consequently causes weight loss (Inzucchi and McGuire, 2008). GLP-1 agonists have exhibited vasodilating and diuretic effects. Recently, Laugero *et al.* showed that Exenatide may display antihypertensive effects mediated by pathways independent from glucose control, but possibly by altering steroid hormones (Laugero *et al*., 2009).

#### **6.7.1 GLP-1 analogue, exendin-4**

The short half-life of GLP-1 limits itself from performing as a good therapeutic agent, as it is rapidly degraded by the serine protease DPP-IV (Hirata *et al.,* 2009). The search for biologically active peptides in lizard venom, led to the discovery of Exendin-4, a naturally occurring GLP-1 analogue (Drucker and Nauk, 2006). Exendin-4 is a peptide naturally found in the saliva of heloderma suspectum - the Gila monster (Laugero *et al*., 2009; Zhou *et al.,* 2008). Exendin-4 is a peptide made up of 39 amino acid residues that shares a 53% structural homology to GLP-1 (Zhou *et al.,* 2008).This long acting GLP-1 receptor agonist has an Nterminus almost identical to that of the GLP-1, except that the second amino acid residue is glycine in exendin-4 while alanine in GLP-1 (Hui *et al.,* 2005). This one amino acid difference makes it resistant to degradation by the enzyme DPP-IV, hence explaining its long half-life in vivo (Lovshin and Drucker, 2009). Studies have shown that GLP-1 receptor agonists like exendin-4, exhibit cardioprotective effects such as modifications in contractility, cardiac output and blood pressure (Ban *et al.,* 2009).

#### **6.7.2 Mode of action of exendin-4**

Endogenous GLP-1 action is inhibited upon degradation by DPP-IV (De Koning *et al*., 2008; Mann *et al*., 2007), an enzyme expressed in many organs mainly kidney, small intestine, liver, and lung (Inzucchi and McGuire, 2008). More apt, exendin-4 is synthetic, thus not recognized by DPP-IV enzyme, thereby not degraded (Rhoades and Bell, 2009). This resistance allows it to stay in the circulation longer mimicking GLP-1 role in bolstering insulin secretion but with 5 to 10 times more powerful insulinotrpic effect (Hantouche *et al.*, 2010; Xu *et al.*, 1999). Moreover, it binds to GLP-1R with high affinity even after truncation of the first 8 amino acids at the N-terminal of the peptide unlike the endogenous GLP-1 (Mann

Type 1 Diabetes Mellitus: Redefining the Future

untreated) in normal and diabetic rats

in normal and diabetic rats.

of Cardiovascular Complications with Novel Treatments 233

**Rat Group τ (min) ± SEM**  Normal (N) 0.279 ± 0.004 a' Normal + Exendin-4 (NE) 0.370 ± 0.007 b' Normal + DPP-IV inhibitor (N-Dp) 0.311 ± 0.005 c' Diabetic (D) 0.252 ± 0.003 d' Diabetic + Insulin (DI) 0.292 ± 0.004 e' Diabetic + Exendin-4 (DE) 0.327 ± 0.005 f' Diabetic + Insulin + Exendin-4 (DIE) 0.295 ± 0.004 g' Diabetic + DPP-IV inhibitor (D-Dp) 0.255 ± 0.003 h' Diabetic + Insulin + DPP-IV inhibitor (DI-Dp) 0.321 ± 0.005 i'

Table 1. The calculated affinity constant (τ) of insulin to its receptor at CE (CHAPS-

**Rat Group τ (min) ± SEM**  Normal (N) 0.337 ± 0.006 a' Normal + Exendin-4 (NE) 0.424 ± 0.009 b' Normal + DPP-IV inhibitor (N-Dp) 0.234 ± 0.003 c' Diabetic (D) 0.368 ± 0.007 d' Diabetic + Insulin (DI) 0.331± 0.006 e' Diabetic + Exendin-4 (DE) 0.328 ± 0.005 f' Diabetic + Insulin + Exendin-4 (DIE) 0.331 ± 0.006 g' Diabetic + DPP-IV inhibitor (D-Dp) 0.376 ± 0.007 h' Diabetic + Insulin + DPP-IV inhibitor (DI-Dp) 0.382 ± 0.007 i'

Table 2. The calculated affinity constant (τ) of insulin to its receptor at CM (CHAPS-treated)

Therefore, in diabetes type 1, exendin-4 increases the insulin receptor affinity only by improving insulin sensitivity. As a conclusion, exendin-4 seems to have supplementary effects which might explain the increase in τ value at the level of the CE. First, it augments insulin release from pancreatic β-cells when present in the normal state. This is its insulinotropic effect. Second, it improves insulin sensitivity possibly by inducing a conformational change in the insulin receptor (Ebinger *et al*., 2000). On the other hand, DPP-IV inhibitor (KR-62436, Sigma Chemical Company) treatment solely does not seem to be implicated in modulating insulin receptor affinity at the endothelial site in diabetic rats treated with DPP-IV inhibitor as compared to diabetics. Yet the effect of DPP-IV inhibitor at the CE becomes obvious in the presence of insulin, as seen in the diabetic group co-treated with insulin and DPP-IV inhibitor (DI-Dp) compared to diabetics (D) and in the normal group treated with DPP-IV inhibitor (N-Dp) with respect to normal. This could be due to some kind of a cross-talk between insulin and DPP-IV inhibitor which results in an increase of τ value in both N-Dp and DI-Dp. Moreover, insulin receptor affinity is not altered at the level of the CM in diabetics treated with DPP-IV inhibitor (D-Dp) and/or insulin (DI-Dp) when compared to diabetic rats (D) (Table 2). The major difference between the CE and CM

*et al*., 2007). GLP-1R is a G-coupled transmembrane receptor (GPCR) that is found in many organs, most importantly the pancreatic β-cells and ducts, heart, lung, kidneys (Ban *et al.*, 2008), brain and stomach (Hantouche *et al.*, 2010). Conceptual understanding of this receptor delineates its advantages in cardiac management (Ban *et al*., 2008). On the initiation of treatment, slight adverse effects might arise as nausea and vomiting (Inzucchi and McGuire, 2008), which are dose-dependent and will vanish with time (Mcphee *et al.*, 2008). It can be used efficiently in type 1 DM (Behme *et al.*, 2003) in conjunction with insulin irrespective of hypoglycemic effects attributed to its glucose-dependent action; in spite of insulin low efficiency with type 2 DM (Inzucchi and McGuire, 2008).

#### **6.8 Exendin-4 in the treatment of diabetes mellitus correlated with cardiovascular risk**

In conjunction to insulinotropic action of GLP-1 role, exendin-4 also mimics the action of insulin itself, through reinforcing the heart to uptake glucose irrespective of insulin level in both diabetic and non-diabetic cases (Hantouche *et al*., 2010) STZ-induced diabetic rats acquire cardiomyopathic damage on day 1 starting with apoptosis and ending with hypertrophied hearts (Al Jaroudi *et al*., 2005), particularly myocardial atrophy (Poornima *et al*., 2006). Thereby, associated with its glucose lowering effect, certain data emphasized a potential benefit of exendin-4 in recovery of heart failure and function of left ventricle (Inzucchi and McGuire, 2008). Just similar to that of GLP-1 in improving ventricular contractions (Hantouche *et al.*, 2010) upon activation of pancreatic GLP-1R to boost insulin synthesis and secretion (De Koning *et al.,* 2008). Treatment with exendin-4 was concomitant with no cardiovascular complications, inexorable renal progression, or escalating plasma lipid (De Fronzo *et al*., 2005). In type 2 DM exendin-4 has completed a novel fruition in the arena of organ rejuvenation especially at the level of both and β cells mass, implementing a new born anti-hyperglycemic drug for solving the diabetes conundrum (Xu *et al*., 1999). This increase in pancreatic mass happens as a post hoc to β-cells neogenesis from either existing duct cells and/or replication of the already present β-cells (hyperplasia) and not from hypertrophy (escalating cell size) (De Koning *et al.*, 2008). Pooled together, this regimen was found to be concomitant with cardioprotection, judicious vascular and cardiac actions based on the fact that endogenous GLP-1 has the same sequence in humans, rats and mice (Ban *et al*., 2008). Recently, it was found that exendin-4 ameliorates the sensitivity of insulin receptors towards insulin at both the CM and coronary endothelium (CE) level (Hantouche *et al*., 2010). This effect is achieved by either enhancing the sensitivity of insulin towards insulin or augmenting β-cells to release more of the hypoglycemic hormone (De Koning *et al*., 2008). Valuable efficacy of treatment with both exendin-4 and insulin alleviates cardiomypathic regressions associated in type 1 DM at the level of receptor affinity enhancement and insulin secretion bolstering (Hantouche *et al.*, 2010) (Fig. 1 and 2).

Insulin affinity (τ) to its receptor was shown to be decreased in the diabetic state at the level of the CE as compared to normal. Exendin-4 treatment increases insulin receptor affinity at the CE in both diabetic and normal rat groups; whereas insulin tends to normalize the affinity irrespective of exendin-4 absence or presence (Table 1). Exendin-4 treatment probably augments insulin receptor affinity both in normal and diabetic state through exerting its insulinotropic actions (Nikolaidis *et al*., 2005) and/or by improving insulin sensitivity (Ebinger *et al*., 2000). The dramatic increase in the affinity of insulin to its receptor in the normal group treated with exendin-4 might be attributed to both of the previously mentioned causes. Yet the rise in insulin receptor affinity in exendin-4-treated diabetic group is not as remarkable as in NE which may be due to the lack of β-cells in diabetic type 1 state thus abolishing the insulinotropic effect of exendin-4.

*et al*., 2007). GLP-1R is a G-coupled transmembrane receptor (GPCR) that is found in many organs, most importantly the pancreatic β-cells and ducts, heart, lung, kidneys (Ban *et al.*, 2008), brain and stomach (Hantouche *et al.*, 2010). Conceptual understanding of this receptor delineates its advantages in cardiac management (Ban *et al*., 2008). On the initiation of treatment, slight adverse effects might arise as nausea and vomiting (Inzucchi and McGuire, 2008), which are dose-dependent and will vanish with time (Mcphee *et al.*, 2008). It can be used efficiently in type 1 DM (Behme *et al.*, 2003) in conjunction with insulin irrespective of hypoglycemic effects attributed to its glucose-dependent action; in spite of insulin low

**6.8 Exendin-4 in the treatment of diabetes mellitus correlated with cardiovascular risk**  In conjunction to insulinotropic action of GLP-1 role, exendin-4 also mimics the action of insulin itself, through reinforcing the heart to uptake glucose irrespective of insulin level in both diabetic and non-diabetic cases (Hantouche *et al*., 2010) STZ-induced diabetic rats acquire cardiomyopathic damage on day 1 starting with apoptosis and ending with hypertrophied hearts (Al Jaroudi *et al*., 2005), particularly myocardial atrophy (Poornima *et al*., 2006). Thereby, associated with its glucose lowering effect, certain data emphasized a potential benefit of exendin-4 in recovery of heart failure and function of left ventricle (Inzucchi and McGuire, 2008). Just similar to that of GLP-1 in improving ventricular contractions (Hantouche *et al.*, 2010) upon activation of pancreatic GLP-1R to boost insulin synthesis and secretion (De Koning *et al.,* 2008). Treatment with exendin-4 was concomitant with no cardiovascular complications, inexorable renal progression, or escalating plasma lipid (De Fronzo *et al*., 2005). In type 2 DM exendin-4 has completed a novel fruition in the arena of organ rejuvenation especially at the level of both and β cells mass, implementing a new born anti-hyperglycemic drug for solving the diabetes conundrum (Xu *et al*., 1999). This increase in pancreatic mass happens as a post hoc to β-cells neogenesis from either existing duct cells and/or replication of the already present β-cells (hyperplasia) and not from hypertrophy (escalating cell size) (De Koning *et al.*, 2008). Pooled together, this regimen was found to be concomitant with cardioprotection, judicious vascular and cardiac actions based on the fact that endogenous GLP-1 has the same sequence in humans, rats and mice (Ban *et al*., 2008). Recently, it was found that exendin-4 ameliorates the sensitivity of insulin receptors towards insulin at both the CM and coronary endothelium (CE) level (Hantouche *et al*., 2010). This effect is achieved by either enhancing the sensitivity of insulin towards insulin or augmenting β-cells to release more of the hypoglycemic hormone (De Koning *et al*., 2008). Valuable efficacy of treatment with both exendin-4 and insulin alleviates cardiomypathic regressions associated in type 1 DM at the level of receptor affinity

enhancement and insulin secretion bolstering (Hantouche *et al.*, 2010) (Fig. 1 and 2).

1 state thus abolishing the insulinotropic effect of exendin-4.

Insulin affinity (τ) to its receptor was shown to be decreased in the diabetic state at the level of the CE as compared to normal. Exendin-4 treatment increases insulin receptor affinity at the CE in both diabetic and normal rat groups; whereas insulin tends to normalize the affinity irrespective of exendin-4 absence or presence (Table 1). Exendin-4 treatment probably augments insulin receptor affinity both in normal and diabetic state through exerting its insulinotropic actions (Nikolaidis *et al*., 2005) and/or by improving insulin sensitivity (Ebinger *et al*., 2000). The dramatic increase in the affinity of insulin to its receptor in the normal group treated with exendin-4 might be attributed to both of the previously mentioned causes. Yet the rise in insulin receptor affinity in exendin-4-treated diabetic group is not as remarkable as in NE which may be due to the lack of β-cells in diabetic type

efficiency with type 2 DM (Inzucchi and McGuire, 2008).


Table 1. The calculated affinity constant (τ) of insulin to its receptor at CE (CHAPSuntreated) in normal and diabetic rats


Table 2. The calculated affinity constant (τ) of insulin to its receptor at CM (CHAPS-treated) in normal and diabetic rats.

Therefore, in diabetes type 1, exendin-4 increases the insulin receptor affinity only by improving insulin sensitivity. As a conclusion, exendin-4 seems to have supplementary effects which might explain the increase in τ value at the level of the CE. First, it augments insulin release from pancreatic β-cells when present in the normal state. This is its insulinotropic effect. Second, it improves insulin sensitivity possibly by inducing a conformational change in the insulin receptor (Ebinger *et al*., 2000). On the other hand, DPP-IV inhibitor (KR-62436, Sigma Chemical Company) treatment solely does not seem to be implicated in modulating insulin receptor affinity at the endothelial site in diabetic rats treated with DPP-IV inhibitor as compared to diabetics. Yet the effect of DPP-IV inhibitor at the CE becomes obvious in the presence of insulin, as seen in the diabetic group co-treated with insulin and DPP-IV inhibitor (DI-Dp) compared to diabetics (D) and in the normal group treated with DPP-IV inhibitor (N-Dp) with respect to normal. This could be due to some kind of a cross-talk between insulin and DPP-IV inhibitor which results in an increase of τ value in both N-Dp and DI-Dp. Moreover, insulin receptor affinity is not altered at the level of the CM in diabetics treated with DPP-IV inhibitor (D-Dp) and/or insulin (DI-Dp) when compared to diabetic rats (D) (Table 2). The major difference between the CE and CM

Type 1 Diabetes Mellitus: Redefining the Future

groups.

state.

**7. Conclusion** 

realms, warrant future investigation.

Medical Practice Plan of the American University of Beirut.

**8. Acknowledgement** 

of Cardiovascular Complications with Novel Treatments 235

Fig. 3. Insulin receptor β-subunit (MW~95 kDa) density in the heart of the different rat

There is no further attenuation in the level of insulin receptor upon insulin administration in combination with exendin-4 in diabetic rat groups. Thus, it is conceivable that GLP-1 is a key player in insulin receptor regulation in the diabetic state. GLP-1 also induces its insulinomimetic effects in the normal state thereby resulting in a slight decrease of insulin receptor density in NE. DPP-IV inhibitor induces similar effects as exendin-4 on IR-α and IR-β levels and this could be attributed as well to the insulinomimetic effects of GLP-1 (Fig. 2 and 3). There is a similar attenuation in receptor density in non-diabetic DPP-IV inhibitor treated rats (N-Dp). This reflects the actions of DPP-IV inhibitor treatment on the modulation of insulin receptor density not only in the diabetic but also in the normal

In conclusion, insulin, Losartan, exendin-4 and DPP-IV inhibitor have demonstrated their ability as promising candidates for treating type 1 diabetic patients. Moreover, exendin-4 and DPP-IV inhibitor appear to be promising in their efficacy and prolonged antidiabetic properties. Their actions on cardiovascular function, in both the preclinical and clinical

The authors would like to acknowledge funding from the University Research Board and

upon DPP-IV inhibitor treatment could be attributed to the fact that the already limited increase of GLP-1 by DPP-IV inhibitor is primarily imposed on endothelial cells which are the first site of encounter with systemic GLP-1. The difference in affinities observed between DPP-IV inhibitor and exendin-4 treatment might be due to the fact that DPP-IV inhibitor increases the systemically available GLP-1 levels; whereas exendin-4, besides its quantitative systemic effect, has a higher physiologic quality in term of being 5- to 10- fold more potent than GLP-1 (Saraceni and Broderick, 2007).

Fig. 2. Insulin receptor α-subunit (MW~125 kDa) density in the heart of the different rat groups.

Western blotting was performed on protein extracts from rat heart homogenates in order to assess the variation in the insulin receptor subunits density among the different treated and untreated normal and diabetic rat groups. Both insulin receptor α-subunit (IR-α) (Fig. 2) and insulin receptor β-subunit (IR-β) (Fig. 3) levels are augmented in diabetic state indicating that there is an increase in the level of cardiac insulin receptor protein in diabetics (D) compared with normal controls (N). These results indicate that cardiac insulin receptors are up-regulated in the heart of diabetic rats as a feedback mechanism probably due to the lack of insulin. This is consistent with the results demonstrated by Al Jaroudi *et al*. 2005 in the study done on insulin receptor regulation in the diabetic heart. Our results indicate that insulin administration to diabetic rats reduces the number of IR-α and IR-β to near normal values. Interestingly, exendin-4 treatment normalizes insulin receptor subunits density in diabetic rat hearts (Fig. 2 and 3). The regression in insulin receptor density with exendin-4 treatment is suggested to be attributable to the insulinomimetic effects of exendin-4 (Sokos *et al*., 2006) which result in a cross-talk between GLP-1 and insulin signaling pathways (Ebinger *et al*., 2000).

upon DPP-IV inhibitor treatment could be attributed to the fact that the already limited increase of GLP-1 by DPP-IV inhibitor is primarily imposed on endothelial cells which are the first site of encounter with systemic GLP-1. The difference in affinities observed between DPP-IV inhibitor and exendin-4 treatment might be due to the fact that DPP-IV inhibitor increases the systemically available GLP-1 levels; whereas exendin-4, besides its quantitative systemic effect, has a higher physiologic quality in term of being 5- to 10- fold more potent

Fig. 2. Insulin receptor α-subunit (MW~125 kDa) density in the heart of the different rat

Western blotting was performed on protein extracts from rat heart homogenates in order to assess the variation in the insulin receptor subunits density among the different treated and untreated normal and diabetic rat groups. Both insulin receptor α-subunit (IR-α) (Fig. 2) and insulin receptor β-subunit (IR-β) (Fig. 3) levels are augmented in diabetic state indicating that there is an increase in the level of cardiac insulin receptor protein in diabetics (D) compared with normal controls (N). These results indicate that cardiac insulin receptors are up-regulated in the heart of diabetic rats as a feedback mechanism probably due to the lack of insulin. This is consistent with the results demonstrated by Al Jaroudi *et al*. 2005 in the study done on insulin receptor regulation in the diabetic heart. Our results indicate that insulin administration to diabetic rats reduces the number of IR-α and IR-β to near normal values. Interestingly, exendin-4 treatment normalizes insulin receptor subunits density in diabetic rat hearts (Fig. 2 and 3). The regression in insulin receptor density with exendin-4 treatment is suggested to be attributable to the insulinomimetic effects of exendin-4 (Sokos *et al*., 2006) which result in a cross-talk between GLP-1 and insulin signaling pathways

than GLP-1 (Saraceni and Broderick, 2007).

groups.

(Ebinger *et al*., 2000).

Fig. 3. Insulin receptor β-subunit (MW~95 kDa) density in the heart of the different rat groups.

There is no further attenuation in the level of insulin receptor upon insulin administration in combination with exendin-4 in diabetic rat groups. Thus, it is conceivable that GLP-1 is a key player in insulin receptor regulation in the diabetic state. GLP-1 also induces its insulinomimetic effects in the normal state thereby resulting in a slight decrease of insulin receptor density in NE. DPP-IV inhibitor induces similar effects as exendin-4 on IR-α and IR-β levels and this could be attributed as well to the insulinomimetic effects of GLP-1 (Fig. 2 and 3). There is a similar attenuation in receptor density in non-diabetic DPP-IV inhibitor treated rats (N-Dp). This reflects the actions of DPP-IV inhibitor treatment on the modulation of insulin receptor density not only in the diabetic but also in the normal state.

#### **7. Conclusion**

In conclusion, insulin, Losartan, exendin-4 and DPP-IV inhibitor have demonstrated their ability as promising candidates for treating type 1 diabetic patients. Moreover, exendin-4 and DPP-IV inhibitor appear to be promising in their efficacy and prolonged antidiabetic properties. Their actions on cardiovascular function, in both the preclinical and clinical realms, warrant future investigation.

#### **8. Acknowledgement**

The authors would like to acknowledge funding from the University Research Board and Medical Practice Plan of the American University of Beirut.

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Unraveling the pathogenesis of type 1 diabetes with proteomics: present and future


**11** 

*Serbia* 

**Diabetic Nephrophaty in Children** 

Aleksandar N. Jovanovic2 and Radojica V. Stolic2 *1Pediatric clinic, Medical Faculty Prishtina, Kosovska Mitrovica 2Internal clinic, Medical Faculty Prishtina, Kosovska Mitrovica* 

Diabetic nephropathy (DN) is one of the consequences of a long-term diabetes, typically defined by macroalbuminuria—that is, a urinary albumin excretion of more than 300 mg in a 24-hour collection—or macroalbuminuria and abnormal renal function as represented by an abnormality in serum creatinine, calculated creatinine clearance, or glomerular filtration

Long-term microvascular and neurologic complications cause major morbidity and mortality in patients with insulin-dependent diabetes mellitus (IDDM) ². The incidence of overt nephropathy rapidly grows 10-15 years after the onset of type 1 diabetes mellitus; the incidence of nephropathy declines after that period and the occurence of nephropathy after 35 years of duration of type 1 diabetes is uncommon. Diabetic nephropathy rarely develops before 10 years' duration of IDDM. The peak incidence (3% per year) is usually found in persons who have had diabetes for 10-20 years. The increased mortality risk in long-term

Epidemiologic data derived from one of the studies – EDC – showed that in group of patients aged <18 and with the duration of diabetes over 5 years the prevalence of microalbuminia reached as much as 14%. Long-term diabetes further increased the prevalence of albuminiria, thus 80% of male and 50% of female patients having diabetes up

Microalbuminuria is a marker for more serious proteinuria followed by azotemia in type 1 diabetic nephropathy5 . Microalbuminuria develops in 40-60% type 1 diabetic patients and over 50% of patients with microalbuminuria evolve to macroalbuminuric stage e.g.

Reported risk factors for the development of diabetic renal disease include a longer duration of IDDM, an earlier age at the time of diagnosis, onset of puberty, poorer glycemic control

T1DM may be due to nephropathy, which may account for about 50% of deaths.

to 30 years of duration had proteinuria in micro- or macroalbuminuric range. (³-4)

**1. Introduction** 

rate (GFR). ¹

**2. Prevalence** 

**3. What is microalbuminuria** 

advanced phase of overt diabetic nephropathy.

**4. Risk factor for diabetes nephropathy** 

Snezana Markovic-Jovanovic1,


## **Diabetic Nephrophaty in Children**

Snezana Markovic-Jovanovic1,

Aleksandar N. Jovanovic2 and Radojica V. Stolic2 *1Pediatric clinic, Medical Faculty Prishtina, Kosovska Mitrovica 2Internal clinic, Medical Faculty Prishtina, Kosovska Mitrovica Serbia* 

#### **1. Introduction**

244 Type 1 Diabetes Complications

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and bioactivity of exendin-4, a peptide analogue of GLP-1, expressed in Pichia

Diabetic nephropathy (DN) is one of the consequences of a long-term diabetes, typically defined by macroalbuminuria—that is, a urinary albumin excretion of more than 300 mg in a 24-hour collection—or macroalbuminuria and abnormal renal function as represented by an abnormality in serum creatinine, calculated creatinine clearance, or glomerular filtration rate (GFR). ¹

#### **2. Prevalence**

Long-term microvascular and neurologic complications cause major morbidity and mortality in patients with insulin-dependent diabetes mellitus (IDDM) ². The incidence of overt nephropathy rapidly grows 10-15 years after the onset of type 1 diabetes mellitus; the incidence of nephropathy declines after that period and the occurence of nephropathy after 35 years of duration of type 1 diabetes is uncommon. Diabetic nephropathy rarely develops before 10 years' duration of IDDM. The peak incidence (3% per year) is usually found in persons who have had diabetes for 10-20 years. The increased mortality risk in long-term T1DM may be due to nephropathy, which may account for about 50% of deaths.

Epidemiologic data derived from one of the studies – EDC – showed that in group of patients aged <18 and with the duration of diabetes over 5 years the prevalence of microalbuminia reached as much as 14%. Long-term diabetes further increased the prevalence of albuminiria, thus 80% of male and 50% of female patients having diabetes up to 30 years of duration had proteinuria in micro- or macroalbuminuric range. (³-4)

#### **3. What is microalbuminuria**

Microalbuminuria is a marker for more serious proteinuria followed by azotemia in type 1 diabetic nephropathy5 . Microalbuminuria develops in 40-60% type 1 diabetic patients and over 50% of patients with microalbuminuria evolve to macroalbuminuric stage e.g. advanced phase of overt diabetic nephropathy.

#### **4. Risk factor for diabetes nephropathy**

Reported risk factors for the development of diabetic renal disease include a longer duration of IDDM, an earlier age at the time of diagnosis, onset of puberty, poorer glycemic control

Diabetic Nephrophaty in Children 247

Hyperglycemia increases the expression of transforming growth factor-beta (TGF-beta) in the glomeruli and of matrix proteins specifically stimulated by this cytokine. TGF-beta may contribute to the cellular hypertrophy and enhanced collagen synthesis observed in persons with diabetic nephropathy. 10 High glucosae levels may also may activate protein kinase C, which further contribute to renal disease and other vascular complications of diabetes.

Fig. 1. Nodular glomerularsclerosis – *Nodular glomerulosclerosis in the kidney of a patient with diabetic nephropathy. US Federal Government public domain image. Source: CDC. (This image was* 

Tissue hypoxia, augmented capillary permeability and capillary blood flow are the factors which cause increased production of angiotensin II in 45% of diabetics. Angiotensin II might generate precapillary and capillary hypertension. The presence of risk factors for hypertension is particularlly important in patients with relatively poor glucose control (hemoglobin A1 concentration above 12 percent). These patients are at increased risk of developing overt nephropathy within 20 years. 11. Furthermore, the risk for developing diabetic nephropathy in adolescents with type 1 diabetes whose parents suffered hypertension is threefold increased. 12 The effects of prostaglandin PGI2 produced by hyperertrophic mesangial cells and atrial natriuretic polypeptide may be important in development of microvascular complications

In our previous study13, performed among the 55 pediatric patients with type 1 diabetes, who had urinary albumin excretion in the range of microalbuminuria was found in almost half (17 of 35 e.g. 48.6%) of children with diabetic ketoacidosis. Furthermore, among the 20

patients with normoalbuminuric UAE only 3 (15.0%) had ketoacidosis.

*copied from wikipediaen)* 

including diabetic nepropathy.

during the first five years of diabetes, smoking, and a family history of diabetic nephropathy.6-7

Some of the factors leading to occurrence of diabetic nephropathy are hereditary predisposition (ACE genotype), poor glycemic control, diabetes - induced hyperfiltration, tissue hypoxia owing to reduction in capillary permeability, and increased postcapillary resistance . The capillary changes are caused by accumulation of glycated proteins and interaction of these proteins with cellular elements in the capillary walls, that effectuate the decrement in capillary ability for dilatation and ultimately, lead to capillary obstruction. In diabetes, morphological changes in type 4 collagen, heparin – sulphat proteoglycane, fibronectin and enectin molecules leads to structural and functional changes in basal membrane of the renal capillary bed . 8

The theory of a reduction in nephron number at birth indicates that individuals born with a reduced number of glomeruli may be predisposed to subsequent renal injury and progressive nephropathy. This has been shown in animal studies in which the mother was exposed to hyperglycemia at the time of pregnancy. If this linkage is true in humans, that would have important implications concerning the role of maternal factors in the eventual development of kidney disease. 1

Certain ethnic groups, particularly African Americans, persons of Hispanic origin, and American Indians, may be particularly disposed to renal disease as a complication of diabetes.

#### **5. Pathology and patohistologic changes in diabetic nephropathy**

The earliest morphologic abnormality in diabetic nephropathy is the thickening of the glomerular basement membrane (GBM) and expansion of the mesangium due to accumulation of extracellular matrix. Light microscopy findings in diffuse diabetic nephropathy show an increase in the solid spaces of the tuft, most frequently observed (by the positive periodic-acid Schiff reaction) as coarse branching of solid material. Large acellular accumulations also may be observed within these areas. These are circular on section and are known as the Kimmelstiel-Wilson lesions/nodules. Three major histologic changes occur in the glomeruli of persons with diabetic nephropathy. First, mesangial expansion which may be directly induced by hyperglycemia, perhaps via increased matrix production or glycation of matrix proteins. Second, a glomerulal basement membrane thickening may occur. Third, glomerular sclerosis being caused by intraglomerular hypertension (induced by renal vasodilatation or from ischemic injury induced by hyaline narrowing of the vessels supplying the glomeruli). These different histological patterns appear to have similar prognostic significance.

Hyperglycemia induces diverse metabolic changes which may give birth to variety of microvascular lesions. The glycation (also called non-enzymatic glycosilation) of proteins is a process induced by the incubation of soluble proteins in the solution with a high glucose concentrations. Glucation lead to three-dimensional structural and/or functional alterations of the proteins involved: thus, the glycation of the erythrocyte membrane proteins leads to its increased adherence ability; likewise, the glycation of heparin – sulphate proteoglycans leads to proliferation of mesangial cells.

High glucose level induces activation of polyol metabolic pathway resulting in increased sobritol production. Sorbitol, in turn, reduces intracellular myoinositol levels leading to increased capillary permeability and structural basal membrane changes. 9

during the first five years of diabetes, smoking, and a family history of diabetic

Some of the factors leading to occurrence of diabetic nephropathy are hereditary predisposition (ACE genotype), poor glycemic control, diabetes - induced hyperfiltration, tissue hypoxia owing to reduction in capillary permeability, and increased postcapillary resistance . The capillary changes are caused by accumulation of glycated proteins and interaction of these proteins with cellular elements in the capillary walls, that effectuate the decrement in capillary ability for dilatation and ultimately, lead to capillary obstruction. In diabetes, morphological changes in type 4 collagen, heparin – sulphat proteoglycane, fibronectin and enectin molecules leads to structural and functional changes in basal

The theory of a reduction in nephron number at birth indicates that individuals born with a reduced number of glomeruli may be predisposed to subsequent renal injury and progressive nephropathy. This has been shown in animal studies in which the mother was exposed to hyperglycemia at the time of pregnancy. If this linkage is true in humans, that would have important implications concerning the role of maternal factors in the eventual

Certain ethnic groups, particularly African Americans, persons of Hispanic origin, and American Indians, may be particularly disposed to renal disease as a complication of

The earliest morphologic abnormality in diabetic nephropathy is the thickening of the glomerular basement membrane (GBM) and expansion of the mesangium due to accumulation of extracellular matrix. Light microscopy findings in diffuse diabetic nephropathy show an increase in the solid spaces of the tuft, most frequently observed (by the positive periodic-acid Schiff reaction) as coarse branching of solid material. Large acellular accumulations also may be observed within these areas. These are circular on section and are known as the Kimmelstiel-Wilson lesions/nodules. Three major histologic changes occur in the glomeruli of persons with diabetic nephropathy. First, mesangial expansion which may be directly induced by hyperglycemia, perhaps via increased matrix production or glycation of matrix proteins. Second, a glomerulal basement membrane thickening may occur. Third, glomerular sclerosis being caused by intraglomerular hypertension (induced by renal vasodilatation or from ischemic injury induced by hyaline narrowing of the vessels supplying the glomeruli). These different histological patterns

Hyperglycemia induces diverse metabolic changes which may give birth to variety of microvascular lesions. The glycation (also called non-enzymatic glycosilation) of proteins is a process induced by the incubation of soluble proteins in the solution with a high glucose concentrations. Glucation lead to three-dimensional structural and/or functional alterations of the proteins involved: thus, the glycation of the erythrocyte membrane proteins leads to its increased adherence ability; likewise, the glycation of heparin – sulphate proteoglycans

High glucose level induces activation of polyol metabolic pathway resulting in increased sobritol production. Sorbitol, in turn, reduces intracellular myoinositol levels leading to

increased capillary permeability and structural basal membrane changes. 9

**5. Pathology and patohistologic changes in diabetic nephropathy** 

nephropathy.6-7

diabetes.

membrane of the renal capillary bed . 8

development of kidney disease. 1

appear to have similar prognostic significance.

leads to proliferation of mesangial cells.

Hyperglycemia increases the expression of transforming growth factor-beta (TGF-beta) in the glomeruli and of matrix proteins specifically stimulated by this cytokine. TGF-beta may contribute to the cellular hypertrophy and enhanced collagen synthesis observed in persons with diabetic nephropathy. 10 High glucosae levels may also may activate protein kinase C, which further contribute to renal disease and other vascular complications of diabetes.

Fig. 1. Nodular glomerularsclerosis – *Nodular glomerulosclerosis in the kidney of a patient with diabetic nephropathy. US Federal Government public domain image. Source: CDC. (This image was copied from wikipediaen)* 

Tissue hypoxia, augmented capillary permeability and capillary blood flow are the factors which cause increased production of angiotensin II in 45% of diabetics. Angiotensin II might generate precapillary and capillary hypertension. The presence of risk factors for hypertension is particularlly important in patients with relatively poor glucose control (hemoglobin A1 concentration above 12 percent). These patients are at increased risk of developing overt nephropathy within 20 years. 11. Furthermore, the risk for developing diabetic nephropathy in adolescents with type 1 diabetes whose parents suffered hypertension is threefold increased. 12 The effects of prostaglandin PGI2 produced by hyperertrophic mesangial cells and atrial natriuretic polypeptide may be important in development of microvascular complications including diabetic nepropathy.

In our previous study13, performed among the 55 pediatric patients with type 1 diabetes, who had urinary albumin excretion in the range of microalbuminuria was found in almost half (17 of 35 e.g. 48.6%) of children with diabetic ketoacidosis. Furthermore, among the 20 patients with normoalbuminuric UAE only 3 (15.0%) had ketoacidosis.

Diabetic Nephrophaty in Children 249

The results of the study13 revealed a quite significant correlation between microalbuminuria and plasma pH and bicarbonates; moreover, the level of plasma bicarbonate was a very good predictor of UAE values, depicting the aggravation of urinary albumin excretion in a

There are 5 clinical stages of diabetic nephropathy. In the first stage, there is the substantial increase of glomerular filtration rate. Hyperglycemia and the lack of insulin along with several other hormones and factors undoubtedly contribute to development of hyperfiltration. The increase in glomerular filtration rate correlates with the enlargement of total filtration surface owing to glomerular hypertrophy; the latter reaches as much as 80%

In the second ("silent") phase hypefunctional periods alternate the periods of normal function, and vice-versa. The periodicity of these alterations depend on quality of metabolic control. Glomerular filtration rate is, therefore, variable, arterial blood pressure is mainly within the normal range, although a mild hypertension might be present; UAE excretion rate is normal. The third stage (the incipient nephropathy) is characterized by UAE within microalbuminuric range (30-3oo mg/24 h ), there is progressive increment of arterial blood pressure, usually 5-15mmHg above the normal values, while the glomerular filtration rate

In our previous study, the total of 40% microalbuminuric children had systolic prehypertension and systolic hypertension (17.1% and 22.9%, respectively) and even 60% had diastolic blood pressure disorders: diastolic prehypertension was found in 35% and diastolic hypertension in 25% patients with microalbuminuria. While the microalbuminuric patients had significantly higher blood pressure comparing to normoalbuminuric group, its noteworthy that the percentage (15% with systolic and 20% with diastolic disturbances) of prehypertensive and hypertensive patients among the type 1 diabetic children with normal UAE was also relatively high (Fig.1 and 2). The level of blood pressure correlated significantly with UAE, but was not

The fourth stage, also known as manifest nephropathy, a clinically manifest proteinuria ensues, with UAE excretion rate exceeding 3oo mg/24h; albuminuria tends to be progressive, possibly leading to the clinical manifestations of nephritic syndrome. As nephropathy evolves to early overt stage with proteinuria (UAE >300 mg/24 hr, or >200 μg/min), it is accompanied by hypertension. The arterial blood pressure is generally raised by 7% per year, followed by the variable reduction in glomerular filtration rate. In the majority of patients, in the midst of this phase a manifest renal insufficiency characterized by overt azotemia occurs. The fourth and fifth stages almost never occur in children and are

The fifth phase (renal insufficiency) is characterized by the general glomerular collapse,

At the beginning, proteinuria is mild and intermittent and may remain that way during 5-10 years following the first discovery. However, an increase of the amount of excreted protein and of frequency of proteinuric episodes might be expected afterwards. The beginning of retinopathy may precede or follow the occurrence of nephropathy, but in the later stages

proved to be a predictor of microalbuminuria in children with type 1 diabetes.

quite rare in adolescents, mostly being related to the adult population.

these two diabetic complications usually have roughly parallel course.

followed by overt azotemia, manifest proteinuria and grave hypertension. Microalbuminuria is also a well-established marker of increased CVD risk 15-16

response to the development of diabetic ketoacidosis

of the values existing prior to diabetes.

may be normal, increased or reduced.

**6. Alterations in renal function in diabetic nephropathy** 

Fig. 2. Showing the correlations between the level of UAE and main parameters of acid-base staus.

Fig. 3. The pathogenesis of diabetic nephropathy, Soman SS, Soman AS>Diabetic Nephropathy eMedicine Specialities, Endocrinology, Diabetes Mellitus, 2009.

Fig. 2. Showing the correlations between the level of UAE and main parameters of acid-base

Fig. 3. The pathogenesis of diabetic nephropathy, Soman SS, Soman AS>Diabetic Nephropathy eMedicine Specialities, Endocrinology, Diabetes Mellitus, 2009.

staus.

The results of the study13 revealed a quite significant correlation between microalbuminuria and plasma pH and bicarbonates; moreover, the level of plasma bicarbonate was a very good predictor of UAE values, depicting the aggravation of urinary albumin excretion in a response to the development of diabetic ketoacidosis

#### **6. Alterations in renal function in diabetic nephropathy**

There are 5 clinical stages of diabetic nephropathy. In the first stage, there is the substantial increase of glomerular filtration rate. Hyperglycemia and the lack of insulin along with several other hormones and factors undoubtedly contribute to development of hyperfiltration. The increase in glomerular filtration rate correlates with the enlargement of total filtration surface owing to glomerular hypertrophy; the latter reaches as much as 80% of the values existing prior to diabetes.

In the second ("silent") phase hypefunctional periods alternate the periods of normal function, and vice-versa. The periodicity of these alterations depend on quality of metabolic control. Glomerular filtration rate is, therefore, variable, arterial blood pressure is mainly within the normal range, although a mild hypertension might be present; UAE excretion rate is normal.

The third stage (the incipient nephropathy) is characterized by UAE within microalbuminuric range (30-3oo mg/24 h ), there is progressive increment of arterial blood pressure, usually 5-15mmHg above the normal values, while the glomerular filtration rate may be normal, increased or reduced.

In our previous study, the total of 40% microalbuminuric children had systolic prehypertension and systolic hypertension (17.1% and 22.9%, respectively) and even 60% had diastolic blood pressure disorders: diastolic prehypertension was found in 35% and diastolic hypertension in 25% patients with microalbuminuria. While the microalbuminuric patients had significantly higher blood pressure comparing to normoalbuminuric group, its noteworthy that the percentage (15% with systolic and 20% with diastolic disturbances) of prehypertensive and hypertensive patients among the type 1 diabetic children with normal UAE was also relatively high (Fig.1 and 2). The level of blood pressure correlated significantly with UAE, but was not proved to be a predictor of microalbuminuria in children with type 1 diabetes.

The fourth stage, also known as manifest nephropathy, a clinically manifest proteinuria ensues, with UAE excretion rate exceeding 3oo mg/24h; albuminuria tends to be progressive, possibly leading to the clinical manifestations of nephritic syndrome. As nephropathy evolves to early overt stage with proteinuria (UAE >300 mg/24 hr, or >200 μg/min), it is accompanied by hypertension. The arterial blood pressure is generally raised by 7% per year, followed by the variable reduction in glomerular filtration rate. In the majority of patients, in the midst of this phase a manifest renal insufficiency characterized by overt azotemia occurs. The fourth and fifth stages almost never occur in children and are quite rare in adolescents, mostly being related to the adult population.

The fifth phase (renal insufficiency) is characterized by the general glomerular collapse, followed by overt azotemia, manifest proteinuria and grave hypertension.

Microalbuminuria is also a well-established marker of increased CVD risk 15-16

At the beginning, proteinuria is mild and intermittent and may remain that way during 5-10 years following the first discovery. However, an increase of the amount of excreted protein and of frequency of proteinuric episodes might be expected afterwards. The beginning of retinopathy may precede or follow the occurrence of nephropathy, but in the later stages these two diabetic complications usually have roughly parallel course.

Diabetic Nephrophaty in Children 251

Phase of constant and massive proteinuria may follow the stage of intermittent proteinuria. During the period, serum creatinine remains normal or slightly increased. The duration of this stage may be variable. Its course may accelerate leading to terminal renal insufficiency.

Advanced stage nephropathy is defined by a progressive decline in renal function (declining glomerular filtration rate and elevation of serum blood urea and creatinine), progressive proteinuria, and hypertension. Progression to end-stage renal disease (ESRD) is recognized

Fig. 6. Soman SS, Soman AS: Diabetic Nephropathy eMedicine Specialities, Endocrinology,

The first system manifestation of diabetic nephropathy is the occurrence of **peripheral oedema** usually on ankles. Contrary to the widely accepted conviction that only hypoalbuminemia gives rise to ankle oedema, the direct cause of this symptom is usually not found. It seems that the pathogenesis of this symptom is quite complex. In most of the patients, the frail capillary walls are detected very often and, thus, the oedema might be due

Regardless of cause, the peripheral oedema always indicate advanced stage of diabetic nephropathy and the occurrence and extent of the swelling is dependent on the duration of clinically manifest proteinuria. Also, the complaints related to lower leg muscle cramps are

**Hypertension**: Incipient to mild structural glomerular lesions do not correlate with the raise in arterial blood pressure. However, in the advanced stage of diabetic nephropathy the arterial hypertension is almost always present and relates with the duration of clinically

Some of our previous results13 showed a very good correlation between the level of urinary albumin excretion and the values of systolic and diastolic arterial blood pressure. Still, the

**7. The clinical features and laboratory findings in diabetic nephropathy** 

The first sign of this acceleration is the rise in serum creatinine level.

by the appearance of uremia, the nephritic syndrome.

Diabetes Mellitus, 2009.

not unusual.

manifest proteinuria.

to increased capillary permeability.

Fig. 4 and 5. Showing the percentage of blood pressure disturbances in microalbuminuric and normoalbuminuric group of children with type 1 diabetes .

Fig. 4 and 5. Showing the percentage of blood pressure disturbances in microalbuminuric

and normoalbuminuric group of children with type 1 diabetes .

Phase of constant and massive proteinuria may follow the stage of intermittent proteinuria. During the period, serum creatinine remains normal or slightly increased. The duration of this stage may be variable. Its course may accelerate leading to terminal renal insufficiency. The first sign of this acceleration is the rise in serum creatinine level.

Advanced stage nephropathy is defined by a progressive decline in renal function (declining glomerular filtration rate and elevation of serum blood urea and creatinine), progressive proteinuria, and hypertension. Progression to end-stage renal disease (ESRD) is recognized by the appearance of uremia, the nephritic syndrome.


Fig. 6. Soman SS, Soman AS: Diabetic Nephropathy eMedicine Specialities, Endocrinology, Diabetes Mellitus, 2009.

#### **7. The clinical features and laboratory findings in diabetic nephropathy**

The first system manifestation of diabetic nephropathy is the occurrence of **peripheral oedema** usually on ankles. Contrary to the widely accepted conviction that only hypoalbuminemia gives rise to ankle oedema, the direct cause of this symptom is usually not found. It seems that the pathogenesis of this symptom is quite complex. In most of the patients, the frail capillary walls are detected very often and, thus, the oedema might be due to increased capillary permeability.

Regardless of cause, the peripheral oedema always indicate advanced stage of diabetic nephropathy and the occurrence and extent of the swelling is dependent on the duration of clinically manifest proteinuria. Also, the complaints related to lower leg muscle cramps are not unusual.

**Hypertension**: Incipient to mild structural glomerular lesions do not correlate with the raise in arterial blood pressure. However, in the advanced stage of diabetic nephropathy the arterial hypertension is almost always present and relates with the duration of clinically manifest proteinuria.

Some of our previous results13 showed a very good correlation between the level of urinary albumin excretion and the values of systolic and diastolic arterial blood pressure. Still, the

Diabetic Nephrophaty in Children 253

 Minimal proteinuria may indicate nondiabetic kidney disease. Other specific diagnostic tests for other systemic disorders associated with nondiabetic kidney disease

 The characteristic test results that differ from DN are: the presence of paraproteinemia/paraproteinuria;

immunoglobulin; and lytic bone lesions. Bataille R, Harousseau JL. Multiple myeloma. N Engl J Med.

 Urinalysis with sulfosalicylic acid (SSA) was classically utilized to evaluate for discrepancy between albumin and total protein, as standard urinalysis dipstick detects albumin only. SSA causes precipitation of all of the urinary proteins, including paraproteins

hypercalcemia; impaired production of normal

1997;336:1657-1664

(Bence Jones proteins). Serum protein electrophoresis (SPEP), urine protein electrophoresis (UPEP): paraprotein spike.

light chain in serum.

lesions.

Proliferation

obstruction. Kidney ultrasound: hydronephrosis, stones. Prostate ultrasound: hypertrophy,

cancer.

 Serum and urine free light chains: increased concentrations of free

Skull x-rays, CT or MRI bone: lytic

Bone marrow biopsy: plasma cell

 Passage of Foley catheter will result in flow of urine and relief of

may be positive.

Disease Differentiating Signs/Symptoms Differentiating Tests

(although onset is insidious in type 2, and DN may occasionally be the presenting manifestation of type 2

 Multiple myeloma (MM) patients also may present with renal failure and

 Symptoms of bone pain and anemia are the most common presenting features, affecting 80% of patients

Can be caused by stones, cancer,

hypertrophy/cancer, neurogenic bladder, or pelviureteric junction

 Obstruction to urine flow can result in postrenal failure. Symptoms

fibrosis, prostate

obstruction.

DM).

proteinuria.

with MM.

 Since both diabetes mellitus and chronic kidney disease (CKD) are common disorders, patients with both conditions may or may not have DN. A diagnosis other than DN should be considered if: there is a rapid progression of renal failure, evidence of another systemic disease, or short duration of diabetes

Non diabetic kidney disease

Multiple myeloma (MM)

Renal tract obstruction

arterial blood pressure level was not predictive for microalbuminuria, judging by the multiple regression analysis, as already stated above.

Fig. 7. Showing the relation between the arterial blood pressure level and UAE values

Paradoxally, however, there is no good correlation between the reduction in glomerular filtration rate and the rise in arterial blood pressure 17-18*.*Yet, most certainly, the therapy aimed at lowering high blood pressure leads to slowing down in reduction of glomerular filtration.

**Macroproteinuria**: The amount of proteins daily excreted in patients with diabetic nephropathy ranges from the minimal concentrations of 30 mg/day to more than 20g/day. The average excretion is about 2,5g/24h. In about 20% of patients with nephropathy the amount is less than 1g/day and in 15% it exceeds 5g/day (nephritic syndrome). Therefore, although proteinuria is considered a marker of of chronic diabetic nephropathy, yet it relatively rarely reach the nephritic range.

Other symptoms of kidney disease include loss of sleep, poor appetite, upset stomach, weakness, and difficulty concentrating. Characteristic signs are:


#### **8. Differential diagnosis**

Other pathological states of potential significance in differential diagnosis are:


arterial blood pressure level was not predictive for microalbuminuria, judging by the

Microalbumi nuria

Ageadjusted systolic blood pressure Ageadjused diastolic blood pressure

multiple regression analysis, as already stated above.

**ρ = 0.559, p < 0.01**

weakness, and difficulty concentrating. Characteristic signs are:

Other pathological states of potential significance in differential diagnosis are:

Fig. 7. Showing the relation between the arterial blood pressure level and UAE values

Paradoxally, however, there is no good correlation between the reduction in glomerular filtration rate and the rise in arterial blood pressure 17-18*.*Yet, most certainly, the therapy aimed at lowering high blood pressure leads to slowing down in reduction of glomerular filtration. **Macroproteinuria**: The amount of proteins daily excreted in patients with diabetic nephropathy ranges from the minimal concentrations of 30 mg/day to more than 20g/day. The average excretion is about 2,5g/24h. In about 20% of patients with nephropathy the amount is less than 1g/day and in 15% it exceeds 5g/day (nephritic syndrome). Therefore, although proteinuria is considered a marker of of chronic diabetic nephropathy, yet it

Other symptoms of kidney disease include loss of sleep, poor appetite, upset stomach,

**-1**

relatively rarely reach the nephritic range.

 Going to the bathroom more often at night Less need for insulin or antidiabetic medications

Morning sickness, nausea, and vomiting

Weakness, paleness, and anemia

**8. Differential diagnosis** 

 Cholesterol embolization Chronic obstruction Interstitial nephritis Amyloidosis

Itching

**0**

**1**

**2**

**rank**

**3**

**4**

**ρ = 0.465, p < 0.02**


Diabetic Nephrophaty in Children 255

Urinary albumin for creatinine ratio microalbuminuria: between 30 and 300 mg/g;

Serum creatinine with GFR estimation Glomerular filtration rate (GFR) may be raised

Table 2. Clinical tests in diagnosis of diabetic nephropathyClinical tests in diagnostics of

24 hour urine to collection microalbuminuria: albumin 30 to 300 mg/24

CT-abdomen hydronephrosis; wedge-shaped areas of low

Kidney biopsy mesangial expansion, fibrosis, Kimmelstiel-

High blood pressure often goes along with diabetic nephropathy. You may have high blood

Annual screening for microalbuminuria, with a random spot urine sample for microalbumin-to-creatinine ratio, should be initiated once the child is 10 years of age and

Confirmed, persistently elevated microalbumin levels on two additional urine specimens should be treated with an ACE inhibitor titrated to normalization of microalbumin excretion

Once albuminuria is diagnosed, a number of factors attenuate the effect of hyperfiltration on

1. Control of hyperglycemia- The Diabetes Control and Complications Trial (DCCT) 21 and the United Kingdom Prospective Diabetes Study (UKPDS) 21 have definitively shown that intensive diabetes therapy can significantly reduce the risk of the development of

microalbuminuria and overt nephropathy in people with diabetes.

Kidney ultrasound normal-to-large kidneys with increased

macroalbuminuria: >300 mg/g

reduced in CKD stages 3 to 5

superimposed

mg/24 hours

Wilson nodules

in CKD stage 1, normal in CKD stage 2, and

echogenicity; may show hydronephrosis if vesiculopathy and/or obstruction is

hours; macroalbuminuria: albumin >300

stranding; cysts; masses; stones

attenuation; loss of the ability to distinguish the corticomedullary border; perinephric

Urinanalysis proteinuria

Test Results

pressure that develops rapidly or is difficult to control.

Magnet resonance angiography renal artery stenosis

Doppler ultrasound may show renal artery stenosis

diabetic nephropathy:

The source: epocrates online com, 2010 Table 3. Other tests to consider

**9. Treatment and therapy** 

has had diabetes for 5 years.

if possible

kidneys:

Blood biochemistry elevated creatinine


Table 1. BMJ Group: Diabetic nephropathy, diferential diagnosis , epocrates online, 2010

#### **8.1 The diagnosis of diabetic nephropathy <sup>19</sup>**

Patients with diabetes should be screened annually for DKD. Initial screening should commence:


An elevated ACR should be confirmed in the absence of urinary tract infection with 2 additional first-void specimens collected during the next 3 to 6 months.


Using the CKD staging likelihood of DN can be determined as follows:


 CT abdomen: hydronephrosis, stones, mass, congenital abnormalities, fibrosis. PSA: elevated in BPH, prostate

 MRI: not routine but may show hydronephrosis, stones, mass, congenital abnormalities, fibrosis

Urinalysis: hematuria, proteinuria, RBC casts, dysmorphic red cells.

Positive serology (e.g., ANA, ANCA,

Complement: decreased in immune glomerulonephritis (e.g., lupus). Kidney biopsy: glomerulonephritis.

Ultrasound, CT scan, MRI: shrunken kidney, decreased flow through the

Magnetic resonance angiography (MRA): renal artery stenosis. Renal angiogram: renal artery

cancer.

Albuminuria.

renal artery.

stenosis.

hepatitis serology).

Disease Differentiating Signs/Symptoms Differentiating Tests include trouble passing urine, anuria, oliguria, hematuria, pain (with kidney stones), and urinary leakage/incontinence. Physical examination findings include enlarged prostate on rectal examination, costovertebral angle tenderness, suprapubic tenderness,

and bladder fullness.

involvement.

2001;344:431-442

5 years after the diagnosis of type 1 diabetes; or

 Measurements of urinary ACR in a spot urine sample; Measurement of serum creatinine and estimation of GFR.

Macroalbuminuria is defined as an ACR > 300 mg/g.

additional first-void specimens collected during the next 3 to 6 months. Microalbuminuria is defined as an ACR between 30-300 mg/g.

Using the CKD staging likelihood of DN can be determined as follows: - Normoalbuminuria in CKD stages 3 to 5 (GFR <60) is unlikely to be DN. - Microalbuminuria in CKD stages 1 to 3 (GFR >30) is possible DN. - Microalbuminuria in CKD stages 4 to 5 (GFR <30) is unlikely to be DN. - Macroalbuminuria at all stages of CKD is highly likely to be DN.

**8.1 The diagnosis of diabetic nephropathy <sup>19</sup>**

From diagnosis of type 2 diabetes.

Screening should include:

confirm classification.

Glomerulonephritis, such as lupus nephritis and cryoglobulinemia, is in the differential for DN. Patient presentation and physical examination may be similar to that of DN. However, there may be symptoms and signs of other systemic disease, such as rashes or joint

Renal artery stenosis presents either as hypertension refractory to multiple maximized antihypertensives or as renal failure shortly after the initiation

Table 1. BMJ Group: Diabetic nephropathy, diferential diagnosis , epocrates online, 2010

Patients with diabetes should be screened annually for DKD. Initial screening should

An elevated ACR should be confirmed in the absence of urinary tract infection with 2

2 of 3 samples should fall within the microalbuminuric or macroalbuminuric range to

of an ACE inhibitor. Physical examination is significant for an abdominal bruit. Safian RD, Textor SC. Renal-artery stenosis. N Engl J Med.

Glomerulonep

Renal artery stenosis

commence:

hritis


Table 2. Clinical tests in diagnosis of diabetic nephropathyClinical tests in diagnostics of diabetic nephropathy:


The source: epocrates online com, 2010

Table 3. Other tests to consider

High blood pressure often goes along with diabetic nephropathy. You may have high blood pressure that develops rapidly or is difficult to control.

#### **9. Treatment and therapy**

Annual screening for microalbuminuria, with a random spot urine sample for microalbumin-to-creatinine ratio, should be initiated once the child is 10 years of age and has had diabetes for 5 years.

Confirmed, persistently elevated microalbumin levels on two additional urine specimens should be treated with an ACE inhibitor titrated to normalization of microalbumin excretion if possible

Once albuminuria is diagnosed, a number of factors attenuate the effect of hyperfiltration on kidneys:

1. Control of hyperglycemia- The Diabetes Control and Complications Trial (DCCT) 21 and the United Kingdom Prospective Diabetes Study (UKPDS) 21 have definitively shown that intensive diabetes therapy can significantly reduce the risk of the development of microalbuminuria and overt nephropathy in people with diabetes.

Diabetic Nephrophaty in Children 257

Treatment of hi gh-normal blood pressure (systolic or diastolic blood pressure consistently above the 90th percentile for age, sex, and height) should include dietary intervention and exercise aimed at weight control and increased physical activity, if appropriate. If target blood pressure is not reached with 3–6 months of lifestyle intervention, pharmacologic

Pharmacologic treatment of hypertension (systolic or diastolic blood pressure consistently above the 95th percentile for age, sex, and height or consistently >130/80 mmHg, if 95%

The principal agents for lowering high blood pressure are angiotensin convertase inhibitors (ACE). The beneficial effect of ACE inhibition on preventing progression from microalbuminuria to overt diabetic nephropathy is long-lasting (8 y) and is associated with the preservation of a normal GFR. ACE-I reduces the risk of progression of overt type 1 diabetic nephropathy to ESRD and in type 1 patients with microalbuminuria to overt nephropathy. 25 The results of multicentric studies showed that blood pressure in patients with chronic renal insufficiency should be lowered below normal range (as recommended

A meta-analysis of several small studies has shown that protein restriction may be of benefit in some patients whose nephropathy seems to be progressing despite optimal glucose and

Hypertensive people with diabetes and CKD stages 1-4 should be treated with an ACE inhibitor or an ARB (Angiotensin Receptor Blocer), usually in combination with a

If needed to achieve blood pressure targets, a thiazide diuretic should be added to those with an estimated glomerular filtration rate (GFR) (see below) ≥50 ml/min per 1.73 m2 and

Normotensive people with diabetes and macroalbuminuria should be treated with an ACE inhibitor or an ARB.In type 1 diabetes with macroalbuminuria, ACE inhibitors decrease albuminuria and reduce the risk of clinical outcomes regardless of the presence or absence of hypertension. A randomized controlled trial in people with type 1 diabetes and macroalbuminuria found that ACE inhibitors reduced the risk of the combined outcome of doubling of serum creatinine level, CKD stage 5, and death. 28 A quarter of the participants were normotensive. There was no significant difference in the treatment effect between the

Future agents: Wenzel et al. 29 *.* have examined the role of avosentan (endothelin antagonist) on progression of microalbuminuria. Avosentan have demonstrated antifibrotic, antiinflammatory, and antiproteinuric effects in experimental studies. Wenzel et al conducted a randomized, placebo-controlled, double-blind, parallel-design, dosage-range study on the effect of the endothelin-A antagonist avosentan (SPP301) on urinary albumin excretion rate in 286 patients with diabetic nephropathy, macroalbuminuria, and a blood pressure of <180/110 mm Hg. All dosages of avosentan, administered in addition to standard ACE inhibitor/ARB treatment, were found to reduce the mean relative urinary albumin excretion

3. Selective control of arteriolar dilation by use of angiotensin-converting enzyme (ACE) inhibitors (thus decreasing transglomerular capillary pressure) This is particularly significant when lowering of systemic blood pressure is accompanied with concomitant lessening of glomerular capillary pressure. however the optimal lower limit for systolic

exceeds that value) should be initiated as soon as the diagnosis is confirmed.

by WHO) in order to achieve beneficial effect on progression of the disease.

a loop diuretic for those with an estimated GFR <50 ml/min per 1.73 m2. (E)

rate (-16.3% to -29.9%, relative to baseline) in the study's patients.

treatment should be initiated.

blood pressure control 26

normotensive and hypertensive individuals.

blood pressure is unclear. 30

diuretic27

Fig. 8. Showing the relation of the parameters of metabolic regulation and the UAE level in children with type 1 diabetes

Accordingly the earlier study13 revealed that hemoglobin A1/c and, particularly, blood glucose level were significantly related to urinary albumin excretion rate. Furthermore, blood glucose was the major predictor of microalbuminuria in children with type 1 diabetes. Lowering A1C to an average of ~7% has been shown to reduce microvascular and neuropathic complications of diabetes and, possibly, macrovascular disease. Preliminary results of the multicenter A1C-Derived Average Glucose (ADAG) Trial, presented at the European Association for the Study of Diabetes meeting in September 2007, confirmed a close correlation of A1C with mean glucose in patients with type 1, type 2, or no diabetes. Final results of this study, not available at the time this statement was completed, should allow more accurate reporting of the estimated average glucose (eAG) and improve patients' understanding of this measure of glycemia22

Target HbA1c for people with diabetes should be < 7.0%, irrespective of the presence or absence of CKD. 23

Considering the high incidence of overnight hypoglycemias, in children and adolescents during the optimal regulation of blood glucosae levels, the A1C level achieved in the "intensive" adolescent cohort of the DCCT group was >1% higher than that achieved by adult DCCT subjects and above current ADA recommendations for patients in general. Standards of Medical Care in Diabetes—2008 American Diabetes Association Tight glycemic control will delay the progression of microalbuminuria and slow the progression of diabetic nephropathy.

2. Aggressive control of systemic blood pressure- In patients with type 1 diabetes, hypertension is usually caused by underlying diabetic nephropathy and typically becomes manifest about the time that patients develop microalbuminuria . Hypertension in childhood is defined as an average systolic or diastolic blood pressure ≥95th percentile for age, sex, and height percentile measured on at least three separate days. "High-normal" blood pressure is defined as an average systolic or diastolic blood pressure ≥90th but <95th percentile for age, sex, and height percentile measured on at least three separate days.

microalbumi nuria

glycaemia

 Hemoglobin A1-c

**ρ = 0.0.697, p < 0.001**

**0**

children with type 1 diabetes

absence of CKD. 23

patients' understanding of this measure of glycemia22

**2**

**4**

**rank**

**6**

**8**

**ρ = 0.382, p < 0.05**

Fig. 8. Showing the relation of the parameters of metabolic regulation and the UAE level in

Accordingly the earlier study13 revealed that hemoglobin A1/c and, particularly, blood glucose level were significantly related to urinary albumin excretion rate. Furthermore, blood glucose was the major predictor of microalbuminuria in children with type 1 diabetes. Lowering A1C to an average of ~7% has been shown to reduce microvascular and neuropathic complications of diabetes and, possibly, macrovascular disease. Preliminary results of the multicenter A1C-Derived Average Glucose (ADAG) Trial, presented at the European Association for the Study of Diabetes meeting in September 2007, confirmed a close correlation of A1C with mean glucose in patients with type 1, type 2, or no diabetes. Final results of this study, not available at the time this statement was completed, should allow more accurate reporting of the estimated average glucose (eAG) and improve

Target HbA1c for people with diabetes should be < 7.0%, irrespective of the presence or

Considering the high incidence of overnight hypoglycemias, in children and adolescents during the optimal regulation of blood glucosae levels, the A1C level achieved in the "intensive" adolescent cohort of the DCCT group was >1% higher than that achieved by adult DCCT subjects and above current ADA recommendations for patients in general. Standards of Medical Care in Diabetes—2008 American Diabetes Association Tight glycemic control will delay the progression of microalbuminuria and slow the progression of diabetic nephropathy. 2. Aggressive control of systemic blood pressure- In patients with type 1 diabetes, hypertension is usually caused by underlying diabetic nephropathy and typically becomes manifest about the time that patients develop microalbuminuria . Hypertension in childhood is defined as an average systolic or diastolic blood pressure ≥95th percentile for age, sex, and height percentile measured on at least three separate days. "High-normal" blood pressure is defined as an average systolic or diastolic blood pressure ≥90th but <95th percentile for age, sex, and height percentile measured on at least three separate days.

Treatment of hi gh-normal blood pressure (systolic or diastolic blood pressure consistently above the 90th percentile for age, sex, and height) should include dietary intervention and exercise aimed at weight control and increased physical activity, if appropriate. If target blood pressure is not reached with 3–6 months of lifestyle intervention, pharmacologic treatment should be initiated.

Pharmacologic treatment of hypertension (systolic or diastolic blood pressure consistently above the 95th percentile for age, sex, and height or consistently >130/80 mmHg, if 95% exceeds that value) should be initiated as soon as the diagnosis is confirmed.

The principal agents for lowering high blood pressure are angiotensin convertase inhibitors (ACE). The beneficial effect of ACE inhibition on preventing progression from microalbuminuria to overt diabetic nephropathy is long-lasting (8 y) and is associated with the preservation of a normal GFR. ACE-I reduces the risk of progression of overt type 1 diabetic nephropathy to ESRD and in type 1 patients with microalbuminuria to overt nephropathy. 25 The results of multicentric studies showed that blood pressure in patients with chronic renal insufficiency should be lowered below normal range (as recommended by WHO) in order to achieve beneficial effect on progression of the disease.

A meta-analysis of several small studies has shown that protein restriction may be of benefit in some patients whose nephropathy seems to be progressing despite optimal glucose and blood pressure control 26

Hypertensive people with diabetes and CKD stages 1-4 should be treated with an ACE inhibitor or an ARB (Angiotensin Receptor Blocer), usually in combination with a diuretic27

If needed to achieve blood pressure targets, a thiazide diuretic should be added to those with an estimated glomerular filtration rate (GFR) (see below) ≥50 ml/min per 1.73 m2 and a loop diuretic for those with an estimated GFR <50 ml/min per 1.73 m2. (E)

Normotensive people with diabetes and macroalbuminuria should be treated with an ACE inhibitor or an ARB.In type 1 diabetes with macroalbuminuria, ACE inhibitors decrease albuminuria and reduce the risk of clinical outcomes regardless of the presence or absence of hypertension. A randomized controlled trial in people with type 1 diabetes and macroalbuminuria found that ACE inhibitors reduced the risk of the combined outcome of doubling of serum creatinine level, CKD stage 5, and death. 28 A quarter of the participants were normotensive. There was no significant difference in the treatment effect between the normotensive and hypertensive individuals.

Future agents: Wenzel et al. 29 *.* have examined the role of avosentan (endothelin antagonist) on progression of microalbuminuria. Avosentan have demonstrated antifibrotic, antiinflammatory, and antiproteinuric effects in experimental studies. Wenzel et al conducted a randomized, placebo-controlled, double-blind, parallel-design, dosage-range study on the effect of the endothelin-A antagonist avosentan (SPP301) on urinary albumin excretion rate in 286 patients with diabetic nephropathy, macroalbuminuria, and a blood pressure of <180/110 mm Hg. All dosages of avosentan, administered in addition to standard ACE inhibitor/ARB treatment, were found to reduce the mean relative urinary albumin excretion rate (-16.3% to -29.9%, relative to baseline) in the study's patients.

3. Selective control of arteriolar dilation by use of angiotensin-converting enzyme (ACE) inhibitors (thus decreasing transglomerular capillary pressure) This is particularly significant when lowering of systemic blood pressure is accompanied with concomitant lessening of glomerular capillary pressure. however the optimal lower limit for systolic blood pressure is unclear. 30

Diabetic Nephrophaty in Children 259

[9] Williamson JR , Kilo C,Basement membrane physiology and patohystology in (eds):

[11] Kostadaras A. Risk Factors for Diabetic Nephropathy. Astoria Hypertension

[12] Zdravkovic S. Dragan: Klinička Pedijatrijska Endokrinologija, 2001, Hronične

[13] Markovic-Jovanovic Snezana; Peric Vladan; Jovanovic Aleksandar; Stolic Radojica The

[15] Garg JP, Bakris GL: Microalbuminuria: marker of vascular dysfunction, risk factor for

[16] Klausen K, Borch-Johnsen K, Feldt-Rasmussen B, Jensen G, Clausen P, Scharling H,

[17] Parving HH, Smidt UM, Frusberg B et al.: A prospective study of glomerular filtration

[18] Viberti GC i sar. 1983). Viberti GC i sar.: Long term correction of hyperglycemia and

[19] The National Kidney Foundation Kidney Disease Outcomes Quality Initiative (KDOQI)

[20] DCCT DCCT Research Group. Are continued studies of metabolic controll and

[21] United Kingdom Prospective Diabetes Study (UKPDS), American Diabetes Assotiation,

[22] Rohlfing CL, Wiedmeyer HM, Little RR, England JD, Tennill A, Goldstein DE: Defining

[23] KDOQI Clinical Practice Guidelines and Clinical Practice Recommendations for Diabetes and Chronic Kidney Disease, 2007 National Kidney Foundation [24] Standards of Medical Care for Patients with Diabetes, Diabetes Care 27 (Suppl. 1), 2004:

[25] Laight DW. Therapeutic inhibition of the renin angiotensin aldosterone system Expert

Influence of Acid-Base Disturbances on Development of Microalbuminuria in Children With Type 1 Diabetes. Endocrinologist. 18(4):182-186, July/August 2008. [14] Viberti GC, Wiseman MJ, The kidney in diabetes: significance of the early

Appleyard M, Jensen JS: Very low levels of microalbuminuria are associated with increased risk of coronary heart disease and death independently of renal function,

rate and arterial blood pressure in insulin-dependent diabetics with diabetic

progression of renal failure in insulin -dipendent diabetes. Br J Med 1983, 2 86: 598-

National Kidney Foundation. KDOQI clinical practice guidelines and clinical practice recommendations for diabetes and chronic kidney disease. Am J Kidney

microvascular complications in insulin-dependent diabetes mellitus justified?

Diabetes Care Implication of the United kingdom Prospective Diabetes Study,

the relationship between plasma glucose and HbA(1c): analysis of glucose profiles and HbA(1c) in the Diabetes Control and Complications Trial. Diabetes Care

RA, Keen H and Zimmet P, 1992. John Wiley and Sons LTD, p 1245-65 [10] Chiarelli F, Gaspari S, Marcovecchio ML. Role of growth factors in diabetic kidney

disease. Horm Metab Res. May 18 2009

komplikacije dijabetes melitusa: s.446-459

abnormalities.Clin.Endocrinol.Metab.,151986;p.753-82

hypertension, and diabetes. Circulation 110:32–35, 2004

cardiovascular disease. Vasc Med 7:35–43, 2002

nephropathy. Diabetologia, 20, 1981; p.457-61

Dis. 2007;49(suppl 2):s1-s179

January 2002, vol 25

25:275–278, 2002

N.Engl.J.Med. 318, 1988, p.246-9

S15-S35. 3 American Diabetes Association

Opin Ther Pat. May 21 2009

602

Clinic. Available at Accessed 7/2/09

International Textbook of Diabetes Mellitus. Edited by Alberty KGMM,Defronzo

4. Dietary protein restriction (because high protein intake increases renal perfusion rate).

A meta-analysis examining the effects of dietary protein restriction (0.5-0.85 g/kg/d) in diabetic patients suggested a beneficial effect on the GFR, creatinine clearance, and albuminuria. However, a large, long-term prospective study is needed to establish the safety, efficacy, and compliance with protein restriction in diabetic patients with nephropathy. Considering the importance and role of dietary proteins in the process of growth and development, a sharp restriction (<15% of total daily amount of nutrients) of proteins in adolescents with diabetic nephropathy is not justified.

Other standard modalities for the treatment of progressive renal disease and its complications (e.g., osteodystrophy) must also be used when indicated, such as sodium and phosphate restriction and use of phosphate binders. When the GFR begins to decline substantially, referral to a physician experienced in the care of such patients is indicated. Radiocontrast media are particularly nephrotoxic in patients with diabetic nephropathy, and azotemic patients should be carefully hydrated before receiving any procedures requiring contrast that cannot be avoided. 31

As for any other patient with ESRD, diabetic patients with ESRD can be offered hemodialysis, peritoneal dialysis, kidney transplantation, or combined kidney-pancreas transplantation.

#### **10. References**


4. Dietary protein restriction (because high protein intake increases renal perfusion rate). A meta-analysis examining the effects of dietary protein restriction (0.5-0.85 g/kg/d) in diabetic patients suggested a beneficial effect on the GFR, creatinine clearance, and albuminuria. However, a large, long-term prospective study is needed to establish the safety, efficacy, and compliance with protein restriction in diabetic patients with nephropathy. Considering the importance and role of dietary proteins in the process of growth and development, a sharp restriction (<15% of total daily amount of nutrients) of

Other standard modalities for the treatment of progressive renal disease and its complications (e.g., osteodystrophy) must also be used when indicated, such as sodium and phosphate restriction and use of phosphate binders. When the GFR begins to decline substantially, referral to a physician experienced in the care of such patients is indicated. Radiocontrast media are particularly nephrotoxic in patients with diabetic nephropathy, and azotemic patients should be carefully hydrated before receiving any procedures requiring

As for any other patient with ESRD, diabetic patients with ESRD can be offered hemodialysis, peritoneal dialysis, kidney transplantation, or combined kidney-pancreas

[1] National kidney Fondation:Prevention of progression in diabetic nephropathy. Diabetes

[2] The Diabetes Control and Complications Trial Research Group, (1993). Effect of

[3] EURODIAB ACE study Group, variationand trends in incidence of childhood diabetes in

[4] Orchard TJ, DormanJS, Maser R, et al.Prevalence of comlicationes in IDDM, by sex and

[5] Bernardo, J.F., Ellis, D., Orchard, T. (1997). Predictors of microalbuminuria in individuals

[6] Andersen, A.R., Christiansen, J.S., Andersen, J.K., Kreiner, S., Deckert, T. (1983) Diabetic

[7] Rudberg, S., Ullman, E., Dahlquist, G. (1993). Relationship between early metabolic

[8] Shimomura, H., Spiro, R. (1987). Studies on macromolecular components of human

heparin-sulfate proteoglycane and laminin. Diabetes 36:374-81.

Intensive Treatment of Diabetes on the Development and Progression of Long-Term Complications in Insulin-Dependent Diabetes Mellitus. The New England

duration. Pittsburg Epidemiology of Diabetes Complicationes StadyII. Diabetes 39:

with insulin dependent diabetes mellitus (IDDM). An update, J Am Soc Nephrol

nephropathy in type I (insulin-dependent) diabetes: an epidemiological study.

control and the development of microalbuminuria – a longitudinal study in children with type-1 (insulin-dependent) diabetes mellitus. Diabetologia 36:1309-

glomerular basement membrane and alterations in diabetes: decreased levels of

proteins in adolescents with diabetic nephropathy is not justified.

contrast that cannot be avoided. 31

Spectrum. 19: 2006; 18-24.

Journal of Medicine 14(329):977-986.

Europe. Lancet 355,873,2000.

Diabetologia 25:496-501

1116, 1990

8:109A.

1314.

transplantation.

**10. References** 


**12** 

*Brazil* 

**Type 1 Diabetes** 

and Giovanna A. Balarini Lima3 *1Federal University of Rio de Janeiro (UFRJ), 2State University of Rio de Janeiro (UERJ)* 

*3Fluminense Federal University of Rio de Janeiro (UFF)* 

**Understanding Pancreatic Secretion in** 

Mirella Hansen De Almeida1, Alessandra Saldanha De Mattos Matheus2

Type 1 Diabetes Mellitus (T1DM) is a chronic disease characterized by the immune-mediated destruction of β cells of pancreatic islets. Despite the increase of its incidence observed in last decades, it has not been fully elucidated the immunogenetic and environmental factors

The pathogenic process in T1DM begins with insulitis, which progresses and expands so as to be accompanied by cell necrosis. The rate of progression of the lesion is variable and may be related to age at onset of the disease, being faster in cases diagnosed in children (3). When approximately 80-90% of insulin-secreting cells have been destroyed, T1DM is

T1DM patients may have some residual insulin secretion at diagnosis (5-8). Several studies indicate that the loss of functional capacity usually occurs within three to five years. Most patients with long duration of disease do not present clinical evidence of preservation of β cells (9-11). However, the conservation of some residual secretion, even insufficient to cure or prevent T1DM from using insulin, has been associated with a better prognosis regarding glycemic control, lower rates of hypoglycemia, diabetic retinopathy

One of the most frequently used method to assess β cell function in patients with T1DM is the determination of C-peptide (CP), a molecule secreted by β cells in equimolar concentrations with insulin and without significant liver metabolism (13-15). Taking into consideration the potential benefits of preserving a residual insulin secretion in patients with T1DM, experimental treatments have been proposed in order to maintain detectable

Developments in research aiming for the prevention and cure of T1DM, increased the need to elucidate the contributing factors for the maintenance of residual insulin secretion in patients affected by the disease. This is because individuals who still exhibit residual insulin secretion could be ideal candidates for new curative treatments. Furthermore, most studies of pancreatic function in T1DM have been performed in Caucasians and Asians. We do not know whether

the results obtained so far can be extrapolated to other ethnic populations (16).

associated with the initiation and perpetuation of the pancreatic injury(1,2).

**1. Introduction** 

clinically overt (4).

and nephropathy (12).

levels of CP in these individuals.


### **Understanding Pancreatic Secretion in Type 1 Diabetes**

Mirella Hansen De Almeida1, Alessandra Saldanha De Mattos Matheus2 and Giovanna A. Balarini Lima3

*1Federal University of Rio de Janeiro (UFRJ), 2State University of Rio de Janeiro (UERJ) 3Fluminense Federal University of Rio de Janeiro (UFF) Brazil* 

#### **1. Introduction**

260 Type 1 Diabetes Complications

[26] Anderson S, Tarnow L, Rossing P, Hansen BV, Parving HH: Renoprotective effects of

[27] KDOQI Clinical Practice Guidelines and Clinical Practice Recommendations for

[28] Lewis EJ, Hunsicker LG, Bain RP, Rohde RD: The effect of angiotensin-converting-

[29] Wenzel rene et. Al. Avosentan Reduces Albumin Excretion in Diabetes with

[31] Nephropathy in Diabetes , American Diabetes Association Diabetes Care January 2004

[30] Diabetes Guidelines. Royal Free Hampstead NHS Trust. Accessed 7/2/09.

nephropathy. Kidney Int 57:601–606, 2000

diabetes and chronic kidney disease,2007

Engl J Med 329:1456-1462, 1993

vol. 27 no. suppl 1 s79-s83

10

angiotensin II receptor blockade in type 1 diabetic patients with diabetic

Diabetes and Chronic Kidney Disease, Guideline 3: Management of hypertension in

enzyme inhibition on diabetic nephropathy. The Collaborative Study Group. N

Macroalbuminuria; jurnal of the American Society of Nephrology, January, 2009, 1-

Type 1 Diabetes Mellitus (T1DM) is a chronic disease characterized by the immune-mediated destruction of β cells of pancreatic islets. Despite the increase of its incidence observed in last decades, it has not been fully elucidated the immunogenetic and environmental factors associated with the initiation and perpetuation of the pancreatic injury(1,2).

The pathogenic process in T1DM begins with insulitis, which progresses and expands so as to be accompanied by cell necrosis. The rate of progression of the lesion is variable and may be related to age at onset of the disease, being faster in cases diagnosed in children (3). When approximately 80-90% of insulin-secreting cells have been destroyed, T1DM is clinically overt (4).

T1DM patients may have some residual insulin secretion at diagnosis (5-8). Several studies indicate that the loss of functional capacity usually occurs within three to five years. Most patients with long duration of disease do not present clinical evidence of preservation of β cells (9-11). However, the conservation of some residual secretion, even insufficient to cure or prevent T1DM from using insulin, has been associated with a better prognosis regarding glycemic control, lower rates of hypoglycemia, diabetic retinopathy and nephropathy (12).

One of the most frequently used method to assess β cell function in patients with T1DM is the determination of C-peptide (CP), a molecule secreted by β cells in equimolar concentrations with insulin and without significant liver metabolism (13-15). Taking into consideration the potential benefits of preserving a residual insulin secretion in patients with T1DM, experimental treatments have been proposed in order to maintain detectable levels of CP in these individuals.

Developments in research aiming for the prevention and cure of T1DM, increased the need to elucidate the contributing factors for the maintenance of residual insulin secretion in patients affected by the disease. This is because individuals who still exhibit residual insulin secretion could be ideal candidates for new curative treatments. Furthermore, most studies of pancreatic function in T1DM have been performed in Caucasians and Asians. We do not know whether the results obtained so far can be extrapolated to other ethnic populations (16).

Understanding Pancreatic Secretion in Type 1 Diabetes 263

In the pathogenesis of T1DM, the activation of the immune system mediated by T cells plays a central role. This process leads to an inflammatory reaction (insulitis) characterized by infiltration of pancreatic islets by mononuclear cells such as dendritic cells, macrophages, B lymphocytes and CD4 and CD8 (3,17,32). With the progression of inflammatory injury, there is a development of cellular necrosis and cellular immunity seems to be primarily

The pathogenic process that culminates with the onset of T1DM begins with the loss of selftolerance of T lymphocytes. Self-tolerance is defined as the process in which T cells or autoreactive B are eliminated from the body. This can be divided into central and peripheral. The central tolerance is the deletion of autoreactive T cells in the thymus and requires the presence of autoantigens in thymic environment. Peripheral tolerance mechanisms are responsible for destroying or inhibiting the function of self-reactive cells that crossed the thymic deletion process, through mechanisms such as immunological

Some of the risk genes for the development of T1DM are responsible for the concentration of insulin within the thymus. Thus, these genes are linked to the process of central tolerance. Moreover, changes in peripheral tolerance may also be related to the etiopathogenesis and the release of super-antigens after viral infections. Typically, the peripheral tolerance would be responsible for the sequestration of these super-antigens mediated by the immune system, which would not lead to lymphocyte activation (34,35). Another mechanism of pancreatic injury is associated with infection as a possible molecular mimicry between viral antigens and autoantigens aggravating the process of insulitis. This molecular similarity could activate autoreactive T lymphocytes and direct them to attack pancreatic cells (34). Overt T1DM occurs when approximately 80 to 90% of the β cells have been destroyed (4). The rate of progression to classical T1DM symptoms may be related to age of onset of

The presence of one or more autoantibodies associated with T1DM may precede the onset of

T1DM diagnosed in childhood may have autoantibodies to the major antigens of the pancreatic islets detectable in the first two years of life (38). This seems to be a reflection of cellular injury and its cause is not specific (33). The role of B lymphocyte cells in the pathogenesis of T1DM must also be emphasized. Several diseases mediated by T lymphocytes present B lymphocytes in the process of antigen presentation (39). A recent study revealed that patients with newly diagnosed T1DM treated with Rituximab (monoclonal anti-CD20) showed improvement in clinical and metabolic parameters after selective depletion of B lymphocytes. The explanation for this fact lies on the reduction of antigen presentation mediated by B lymphocytes, or even the reduction of cytokine production in pancreatic or peri-pancreatic lymph nodes. This suggests that B lymphocytes

may have a greater role in the pathogenesis of T1DM than previously thought (40).

Although there are many hypotheses about the onset and progression to autoimmunity of

Production of pancreatic autoantibodies does not appear to be the primary mechanism of destruction of pancreatic islets (41,42). The release of antigen caused by β cell destruction leads to its detection by the immune system and immune activation, subsequently causing

**2.3 Etiopathogenesis** 

responsible for this process (33).

ignorance, deletion, anergy or immune inhibition (34).

symptoms, being faster in cases diagnosed at young age (3).

T1DM, the definitive mechanism is not fully understood.

**2.4 Autoantibodies** 

clinical disease by months or even years (28,36,37).

### **2. Type 1 diabetes mellitus (T1DM)**

#### **2.1 Definition**

T1DM is a chronic disease characterized by the destruction of pancreatic β cells, which leads to absolute deficiency in insulin production.

There are two subgroups of T1DM: type 1A, mediated by autoimmune destruction of β cells and type 1B, without an identifiable cause and more common among Asian and Afro-American people (17).

#### **2.2 Epidemiology**

The incidence of T1DM is rising in recent decades (18-20). This has been observed mainly in developed countries, mainly among children younger than four years-old (18,20-22). It is estimated that 15 to 30 million people in the world population have the disease with a growth rate of approximately 3-5% per year (19,20,23,24).

Epidemiological data suggest that 30 to 50% of T1DM cases may occur after the age of 20 and 50-60% of these patients were younger than 16-18 years at diagnosis. The incidence declines throughout the adult life (25-28).

The incidence among men and women is equal. However, there is a predominance of females in populations at low risk for T1DM, while the opposite occurs in high-risk populations (29).

There is also variation in relation to different countries. There is a high incidence in Finland and Sardinia, Italy (36.5 and 36.8 individuals per 100,000, respectively) and low in Germany and Pakistan. In countries like Brazil and Portugal, the incidence of T1DM is intermediate, as shown in Table 1. In Brazil, there is about 8.0 new cases per 100,000 inhabitants (30).

Regarding ethnicity, individuals of Caucasian origin have a higher incidence of T1DM than Hispanic, African, Asian or Indian descendents (31).


Adapted from The WHO Diamond Project Group, 2000 (30).

Table 1. Incidence of T1DM in population under 15 years-old, in different countries.

#### **2.3 Etiopathogenesis**

262 Type 1 Diabetes Complications

T1DM is a chronic disease characterized by the destruction of pancreatic β cells, which leads

There are two subgroups of T1DM: type 1A, mediated by autoimmune destruction of β cells and type 1B, without an identifiable cause and more common among Asian and Afro-

The incidence of T1DM is rising in recent decades (18-20). This has been observed mainly in developed countries, mainly among children younger than four years-old (18,20-22). It is estimated that 15 to 30 million people in the world population have the disease with a

Epidemiological data suggest that 30 to 50% of T1DM cases may occur after the age of 20 and 50-60% of these patients were younger than 16-18 years at diagnosis. The incidence

The incidence among men and women is equal. However, there is a predominance of females in populations at low risk for T1DM, while the opposite occurs in high-risk populations (29). There is also variation in relation to different countries. There is a high incidence in Finland and Sardinia, Italy (36.5 and 36.8 individuals per 100,000, respectively) and low in Germany and Pakistan. In countries like Brazil and Portugal, the incidence of T1DM is intermediate, as shown in Table 1. In Brazil, there is about 8.0 new cases per 100,000 inhabitants (30). Regarding ethnicity, individuals of Caucasian origin have a higher incidence of T1DM than

**Country Term Total** Germany 1990 – 1994 1.0 Brazil 1990 – 1992 8.0 Canada 1990 – 1994 24.0 Denmark 1990 – 1994 15.5 Spain 1990 – 1994 12.5

1990 – 1994

1990 – 1994

Table 1. Incidence of T1DM in population under 15 years-old, in different countries.

Finland 1987 – 1989 36.5

Mexico 1990 – 1993 1.5 Norway 1990 – 1994 21.2 Pakistan 1990 0.7 Portugal 1990 – 1994 13.2 Sweden 1978 – 1987 24.2

Incidence per 100,000 people

16.4 13.3

36.8 11.7 7.2

**2. Type 1 diabetes mellitus (T1DM)** 

to absolute deficiency in insulin production.

declines throughout the adult life (25-28).

growth rate of approximately 3-5% per year (19,20,23,24).

Hispanic, African, Asian or Indian descendents (31).

Adapted from The WHO Diamond Project Group, 2000 (30).

**2.1 Definition** 

American people (17).

**2.2 Epidemiology** 

USA

Italy

White Non-white

Sardinia Sicily Lombardy In the pathogenesis of T1DM, the activation of the immune system mediated by T cells plays a central role. This process leads to an inflammatory reaction (insulitis) characterized by infiltration of pancreatic islets by mononuclear cells such as dendritic cells, macrophages, B lymphocytes and CD4 and CD8 (3,17,32). With the progression of inflammatory injury, there is a development of cellular necrosis and cellular immunity seems to be primarily responsible for this process (33).

The pathogenic process that culminates with the onset of T1DM begins with the loss of selftolerance of T lymphocytes. Self-tolerance is defined as the process in which T cells or autoreactive B are eliminated from the body. This can be divided into central and peripheral. The central tolerance is the deletion of autoreactive T cells in the thymus and requires the presence of autoantigens in thymic environment. Peripheral tolerance mechanisms are responsible for destroying or inhibiting the function of self-reactive cells that crossed the thymic deletion process, through mechanisms such as immunological ignorance, deletion, anergy or immune inhibition (34).

Some of the risk genes for the development of T1DM are responsible for the concentration of insulin within the thymus. Thus, these genes are linked to the process of central tolerance. Moreover, changes in peripheral tolerance may also be related to the etiopathogenesis and the release of super-antigens after viral infections. Typically, the peripheral tolerance would be responsible for the sequestration of these super-antigens mediated by the immune system, which would not lead to lymphocyte activation (34,35). Another mechanism of pancreatic injury is associated with infection as a possible molecular mimicry between viral antigens and autoantigens aggravating the process of insulitis. This molecular similarity could activate autoreactive T lymphocytes and direct them to attack pancreatic cells (34).

Overt T1DM occurs when approximately 80 to 90% of the β cells have been destroyed (4). The rate of progression to classical T1DM symptoms may be related to age of onset of symptoms, being faster in cases diagnosed at young age (3).

The presence of one or more autoantibodies associated with T1DM may precede the onset of clinical disease by months or even years (28,36,37).

T1DM diagnosed in childhood may have autoantibodies to the major antigens of the pancreatic islets detectable in the first two years of life (38). This seems to be a reflection of cellular injury and its cause is not specific (33). The role of B lymphocyte cells in the pathogenesis of T1DM must also be emphasized. Several diseases mediated by T lymphocytes present B lymphocytes in the process of antigen presentation (39). A recent study revealed that patients with newly diagnosed T1DM treated with Rituximab (monoclonal anti-CD20) showed improvement in clinical and metabolic parameters after selective depletion of B lymphocytes. The explanation for this fact lies on the reduction of antigen presentation mediated by B lymphocytes, or even the reduction of cytokine production in pancreatic or peri-pancreatic lymph nodes. This suggests that B lymphocytes may have a greater role in the pathogenesis of T1DM than previously thought (40).

Although there are many hypotheses about the onset and progression to autoimmunity of T1DM, the definitive mechanism is not fully understood.

#### **2.4 Autoantibodies**

Production of pancreatic autoantibodies does not appear to be the primary mechanism of destruction of pancreatic islets (41,42). The release of antigen caused by β cell destruction leads to its detection by the immune system and immune activation, subsequently causing

Understanding Pancreatic Secretion in Type 1 Diabetes 265

Genes from the histocompatibility system HLA *(Human Leukocyte Antigen Complex), IDDM* 1 - chromosome 6q21.31, are polymorphic and have different amino acid sequences between individuals. They are divided into HLA class I (HLA-A, B and C) and HLA class II (DP, DQ

HLA class II polymorphisms are associated with an increased risk of T1DM. The main risk alleles are HLA DQB1 \* 2002 / \* 0302 and HLA DR03/04. The DQB1 \* 0602 allele is

Other genes have been associated with the pathogenesis of T1DM, as shown in Table 2,

mellitus Genetic Product Cromossomic

IDDM1 HLA 6p21.31

IDDM2 Insulin 11p15.5

IDDM3 - 15q.26

IDDM4 - 11q13

IDDM5 - 6q25

IDDM6 - 18q21

IDDM7 - 2q31-31

IDDM8 - 6q27

IDDM9 - 3q21

IDDM10 - 10p11-q11

IDDM11 - 14q4.3-14q31

IDDM12 CTLA-4 2q33

IDDM13 - 2q34

IDDM14 - 6q21

IDDM15 - 10q25.1

IDDM17 - CR10

PTPN22 -

Adapted from Kelly et al. 2003 (3) and Eisenbarth, 2005 (32). Table 2. Genes involved in the Pathogenesis of T1DM location

**2.5 Genetic factors associated with T1DM** 

\* IDDM - insulin dependent diabetes

considered protective (5).

and DR) and both are related to immune response (52).

among which are the insulin gene PTPN22 and CTL4 (17,27,53-55).

the production of antibodies against pancreatic components. Thus, antibodies appear to be predominantly markers of immune activation and β cell destruction, and not the cause itself (31,33).

The first autoantibody isolated was the antibody against the cytoplasm of the pancreatic islet *(islet cell cytoplasmic antibody* - ICA*)* and three major autoantibodies contribute to their positivity: the antiglutamic acid decarboxylase (GADA65), antibody against tyrosine phosphatase (anti-IA2, also known as ICA512) and antibodies against glycolipids (43)

The glutamic acid decarboxylase (GADA) is an enzyme involved in the synthesis of γamino-butiric acid (GABA) in the central nervous system (CNS) and in pancreatic islets. GADA is expressed in all cell types of pancreatic islets, not only in β cells. Two isoforms of GADA (GADA64 and GADA67) synthesized in other tissues have also been identified. GADA, used in current assays, detects pancreatic isoform of this enzyme of 65 kd and is found in 70-80% of newly diagnosed Caucasian. A lower incidence is observed in children younger than 10 years of age (43,44).

After GADA discovery, anti-IA-2 (40 kd) or ICA512, and anti-IA-2β (37 kd), known as *phorin* (45,46), were identified. Most patients with anti-IA 2β also exhibit anti-IA-2. However, about 10% of T1DM with anti-IA-2 autoantibodies are not the type-2 anti-IA. Until now, anti-IA-2 appears to be the most specific immunological marker of T1DM and is present in 32 to 75% of the new cases (47,48). Once detected, most individuals concomitantly present positive GADA and anti-insulin (IAA) (28). Another antibody has been identified as IAA. In children diagnosed under 10 years of age, sensitivity of IAA is of 50 to 60%, while in patients between 10 and 30 years sensitivity is of 10% (47,48). There is cross-reactivity between antibodies produced against endogenous and exogenous insulin and most patients develop IAA after initiation of insulin therapy, even with the use of recombinant analogues. Thus, the measurement of this antibody in the blood is recommended only before or within 5 to 7 days after initiation of insulin therapy (32).

It has been recently described another auto-antigen associated with T1DM - the zinc transporter 8 *(zinc transporter* 8 - Znt8). Present in β cells, the Znt8 is involved in regulating the insulin secretion pathway. It regulates the entry of zinc in the lumen of the granules, where this cation binds to hexamers of insulin. Nearly 60 to 80% of individuals with newly diagnosed T1DM have antibodies against this antigen (49,50).

Most pancreatic autoantibody titers decline after the diagnosis of the disease, but GADA may remain positive for many years after diagnosis (51). This feature makes it ideal for antibody studies in patients with T1DM of long duration.

Some patients may not have pancreatic autoantibodies detectable at diagnosis. This can be explained by some reasons (5):


At diagnosis, traditionally it has been estimated that 90% of children had one or more pancreatic antibody positive. However, with the availability of anti-Znt8, measuring the combination of the four main pancreatic autoantibodies (GADA, IAA, anti-IA2 and anti-Znt8) in patients with newly diagnosed T1DM, showed a detection rate of autoimmunity against β cells of 98% (49,50).

#### **2.5 Genetic factors associated with T1DM**

264 Type 1 Diabetes Complications

the production of antibodies against pancreatic components. Thus, antibodies appear to be predominantly markers of immune activation and β cell destruction, and not the cause itself

The first autoantibody isolated was the antibody against the cytoplasm of the pancreatic islet *(islet cell cytoplasmic antibody* - ICA*)* and three major autoantibodies contribute to their positivity: the antiglutamic acid decarboxylase (GADA65), antibody against tyrosine phosphatase (anti-IA2, also known as ICA512) and antibodies against glycolipids (43) The glutamic acid decarboxylase (GADA) is an enzyme involved in the synthesis of γamino-butiric acid (GABA) in the central nervous system (CNS) and in pancreatic islets. GADA is expressed in all cell types of pancreatic islets, not only in β cells. Two isoforms of GADA (GADA64 and GADA67) synthesized in other tissues have also been identified. GADA, used in current assays, detects pancreatic isoform of this enzyme of 65 kd and is found in 70-80% of newly diagnosed Caucasian. A lower incidence is observed in children

After GADA discovery, anti-IA-2 (40 kd) or ICA512, and anti-IA-2β (37 kd), known as *phorin* (45,46), were identified. Most patients with anti-IA 2β also exhibit anti-IA-2. However, about 10% of T1DM with anti-IA-2 autoantibodies are not the type-2 anti-IA. Until now, anti-IA-2 appears to be the most specific immunological marker of T1DM and is present in 32 to 75% of the new cases (47,48). Once detected, most individuals concomitantly present positive GADA and anti-insulin (IAA) (28). Another antibody has been identified as IAA. In children diagnosed under 10 years of age, sensitivity of IAA is of 50 to 60%, while in patients between 10 and 30 years sensitivity is of 10% (47,48). There is cross-reactivity between antibodies produced against endogenous and exogenous insulin and most patients develop IAA after initiation of insulin therapy, even with the use of recombinant analogues. Thus, the measurement of this antibody in the blood is recommended only before or within 5 to 7

It has been recently described another auto-antigen associated with T1DM - the zinc transporter 8 *(zinc transporter* 8 - Znt8). Present in β cells, the Znt8 is involved in regulating the insulin secretion pathway. It regulates the entry of zinc in the lumen of the granules, where this cation binds to hexamers of insulin. Nearly 60 to 80% of individuals with newly

Most pancreatic autoantibody titers decline after the diagnosis of the disease, but GADA may remain positive for many years after diagnosis (51). This feature makes it ideal for

Some patients may not have pancreatic autoantibodies detectable at diagnosis. This can be

1. These patients may have detectable titers in the pre clinical and became negative before

2. Tests available for determination of these autoantibodies were not sensitive enough to

4. This is possibly a case of idiopathic T1DM (type 1B) without autoimmune etiology. At diagnosis, traditionally it has been estimated that 90% of children had one or more pancreatic antibody positive. However, with the availability of anti-Znt8, measuring the combination of the four main pancreatic autoantibodies (GADA, IAA, anti-IA2 and anti-Znt8) in patients with newly diagnosed T1DM, showed a detection rate of autoimmunity

3. There may be yet unidentified antibodies in research conducted so far.

(31,33).

younger than 10 years of age (43,44).

days after initiation of insulin therapy (32).

explained by some reasons (5):

diagnosis.

detect low titers.

against β cells of 98% (49,50).

diagnosed T1DM have antibodies against this antigen (49,50).

antibody studies in patients with T1DM of long duration.

Genes from the histocompatibility system HLA *(Human Leukocyte Antigen Complex), IDDM* 1 - chromosome 6q21.31, are polymorphic and have different amino acid sequences between individuals. They are divided into HLA class I (HLA-A, B and C) and HLA class II (DP, DQ and DR) and both are related to immune response (52).

HLA class II polymorphisms are associated with an increased risk of T1DM. The main risk alleles are HLA DQB1 \* 2002 / \* 0302 and HLA DR03/04. The DQB1 \* 0602 allele is considered protective (5).

Other genes have been associated with the pathogenesis of T1DM, as shown in Table 2, among which are the insulin gene PTPN22 and CTL4 (17,27,53-55).


Adapted from Kelly et al. 2003 (3) and Eisenbarth, 2005 (32).

Table 2. Genes involved in the Pathogenesis of T1DM

Understanding Pancreatic Secretion in Type 1 Diabetes 267

and the use of exogenous insulin. Another limiting factor is the presence of anti-insulin

Thus, other means to measure β cell function have been developed, and among them the

Pancreatic β cells secrete, in addition to insulin, proinsulin, conversion intermediates of

The pro-insulin is cleaved in the Golgi apparatus of islet cells. This reaction leads to the formation of insulin, CP and two pairs of basic amino acids. Insulin and the CP are released into circulation at a ratio of 1:1, as well as small amounts of proinsulin and intermediates. The proinsulin sum 20% of molecules with insulin-immunoreactivity *simile,* seems to have no metabolic effect, undergoes extra-hepatic metabolism and is excreted exclusively by the

The CP is a connection between the peptide chains A and B of proinsulin and facilitates the processing of biologically active insulin in secretory granules of pancreatic islets. After the cleavage of proinsulin, the intact CP is stored with insulin in these granules and is cosecreted with insulin. For this reason, the CP can be considered an independent marker of insulin secretion (16). However, in some situations, such as renal failure, the serum concentration of CP is not proportional to the rate of insulin secretion. About 85% of CP is metabolized by the kidneys and the remainder excreted intact in urine. A decrease in renal

The CP plasma half-life is of thirty minutes, greater than that of insulin, which is only of

Under standard conditions of measurement, the CP has been widely accepted as a rough measure of insulin secretion, since it is secreted into the portal circulation in equimolar, concentrations, does not undergo hepatic metabolism, its half-life is longer (30 minutes) and

In adverse conditions, such as hyper- or hypoglycemia, CP concentrations are not proportional to the rate of insulin secretion, and its *clearance* may vary between different

Therefore, at the moment, the most appropriate, accepted and clinically validated method to measure the ability of β cell secretion under ideal conditions is the measurement of baseline and/or stimulated CP (76). This stimulation can be done with glucose or insulin secretagogues such as glucagon, standard mixed meal or oral glucose tolerance test with 75g

The standard mixed meal test consists of oral administration of a liquid diet (Sustacal ® / Boost) of approximately 500 kcal containing 50% carbohydrate, 30% fat and 20% protein. Blood samples for measurement of blood glucose and CP are collected in fasting and 30, 60 and/or 90 minutes after the meal (76,80). This test shows the typical postprandial response of the cell β and its interaction with the various hormones secreted during oral feeding. It is

Oral glucose tolerance test (OGTT) is the determination of glucose, insulin and CP after 10 hours of fasting and 30, 60, 90, 120 minutes after ingestion of 75g anhydrous glucose orally administered. It is used to measure glucose tolerance and the residual function of the β cell in patients at risk, but has not yet been validated for use in T1DM patients. It is useful to

antibodies (IAA) to interfere in the measurement of serum insulin (12,72,73).

function leads to reduced metabolism of CP and elevated serum levels (74)

has low cross reactivity with proinsulin and insulin antibodies (12, 76,77).

four minutes (13-15,75). The normal value of the CP varies from 1.1 to 5.0 ng / mL.

measurement of baseline and/or stimulated CP.

insulin (proinsulin *split)* and the connecting peptide (CP) (15).

**3.1 C-peptide** 

kidneys.

individuals (78,79).

of anhydrous glucose (OGTT) (12,80-82).

the most physiological test among the above cited (76,83).

#### **2.6 Environmental factors associated with T1DM**

Besides genetic susceptibility, exposure to environmental factors is also important for the development of T1DM. Studies suggest that these factors would be responsible for triggering the immune process that leads to β cell destruction. Specific viral infectious diseases have been included among the causes (56,57). Some of the viruses suggested as associated with T1DM are Enteroviruses, Coxsackie virus, congenital Rubella. Toxins such as nitrosamines and protein foods such as cow's milk, cereals and gluten are also considered as potential immunological triggers (58-60). Moreover, the presence of multiple infections in early life is associated with a reduced risk for the disease (1).

Recently, there has been a significant increase in diagnosed cases aged younger than four years (61,62). This shift may be explained by increased exposure to environmental factors or the increased prevalence of obesity (63,64).

One of the hypotheses to explain the development of T1DM is the controversial theory of acceleration suggested by Wilkin. It argues that T1DM and type 2 diabetes (T2DM) constitute a single disease and not two distinct comorbidities. The rate of loss of β cell mass, associated with three main factors accelerators, would define the disease. The first factor would be the intrinsic potential for high speed apoptosis of β cells, essential, but insufficient for the development of DM. The second accelerator would be insulin resistance, a result of obesity and physical inactivity, central link between the two entities. Insulin resistance overtaxes the β cell mass already at risk for accelerated apoptosis, contributing to the clinical expression of DM. The third accelerator was present only in individuals with genetic susceptibility to autoimmunity. Individuals with metabolically more active β cells, insulin resistance and genetic susceptibility would be more prone to rapid deterioration of functional and clinical expression of T1DM. In the absence of autoimmune accelerator, apoptosis rate would be slower and thus there would be progression to T2DM (65).

#### **3. Assessment of pancreatic β cell function**

A major limitation of studies of T1DM in humans is the inability to measure the mass of β cells *in vivo,* since pancreatic biopsies are associated with high morbidity and mortality. Therefore, indirect methods have been developed. The assessment of pancreatic function was shown to have a rough correlation with the mass of β cells used in islet transplantation in patients with DM (25).

Methods of imaging and nuclear medicine are being studied to assist the measurement of the mass of pancreatic islets and its correlation with insulin production, but with conflicting results (66-68).

The measurement of β cell mass does not always correlate with functional capacity. In prediabetes, there can be no proliferation or maintenance of cell mass (69,70). Marchetti et al suggested that patients with T1DM can have β cell secretory dysfunction and not just cell destruction (71).

In an attempt to understand the β cell function *in vivo,* it was initially developed a radioimmunoassay for measurement of serum insulin. For years, this was the gold standard for assessing the secretory activity. However, there are several factors limiting the use of serum insulin for the evaluation of pancreatic β cell function. The first is that 50 to 60% of the insulin produced by the pancreas undergoes hepatic metabolism and does not reach the systemic circulation. In addition, the peripheral *clearance* of insulin is variable and the tests available for their determination do not differentiate insulin, proinsulin, its intermediates and the use of exogenous insulin. Another limiting factor is the presence of anti-insulin antibodies (IAA) to interfere in the measurement of serum insulin (12,72,73).

Thus, other means to measure β cell function have been developed, and among them the measurement of baseline and/or stimulated CP.

#### **3.1 C-peptide**

266 Type 1 Diabetes Complications

Besides genetic susceptibility, exposure to environmental factors is also important for the development of T1DM. Studies suggest that these factors would be responsible for triggering the immune process that leads to β cell destruction. Specific viral infectious diseases have been included among the causes (56,57). Some of the viruses suggested as associated with T1DM are Enteroviruses, Coxsackie virus, congenital Rubella. Toxins such as nitrosamines and protein foods such as cow's milk, cereals and gluten are also considered as potential immunological triggers (58-60). Moreover, the presence of multiple infections

Recently, there has been a significant increase in diagnosed cases aged younger than four years (61,62). This shift may be explained by increased exposure to environmental factors or

One of the hypotheses to explain the development of T1DM is the controversial theory of acceleration suggested by Wilkin. It argues that T1DM and type 2 diabetes (T2DM) constitute a single disease and not two distinct comorbidities. The rate of loss of β cell mass, associated with three main factors accelerators, would define the disease. The first factor would be the intrinsic potential for high speed apoptosis of β cells, essential, but insufficient for the development of DM. The second accelerator would be insulin resistance, a result of obesity and physical inactivity, central link between the two entities. Insulin resistance overtaxes the β cell mass already at risk for accelerated apoptosis, contributing to the clinical expression of DM. The third accelerator was present only in individuals with genetic susceptibility to autoimmunity. Individuals with metabolically more active β cells, insulin resistance and genetic susceptibility would be more prone to rapid deterioration of functional and clinical expression of T1DM. In the absence of autoimmune accelerator,

apoptosis rate would be slower and thus there would be progression to T2DM (65).

A major limitation of studies of T1DM in humans is the inability to measure the mass of β cells *in vivo,* since pancreatic biopsies are associated with high morbidity and mortality. Therefore, indirect methods have been developed. The assessment of pancreatic function was shown to have a rough correlation with the mass of β cells used in islet transplantation

Methods of imaging and nuclear medicine are being studied to assist the measurement of the mass of pancreatic islets and its correlation with insulin production, but with conflicting

The measurement of β cell mass does not always correlate with functional capacity. In prediabetes, there can be no proliferation or maintenance of cell mass (69,70). Marchetti et al suggested that patients with T1DM can have β cell secretory dysfunction and not just cell

In an attempt to understand the β cell function *in vivo,* it was initially developed a radioimmunoassay for measurement of serum insulin. For years, this was the gold standard for assessing the secretory activity. However, there are several factors limiting the use of serum insulin for the evaluation of pancreatic β cell function. The first is that 50 to 60% of the insulin produced by the pancreas undergoes hepatic metabolism and does not reach the systemic circulation. In addition, the peripheral *clearance* of insulin is variable and the tests available for their determination do not differentiate insulin, proinsulin, its intermediates

**2.6 Environmental factors associated with T1DM** 

the increased prevalence of obesity (63,64).

**3. Assessment of pancreatic β cell function** 

in patients with DM (25).

results (66-68).

destruction (71).

in early life is associated with a reduced risk for the disease (1).

Pancreatic β cells secrete, in addition to insulin, proinsulin, conversion intermediates of insulin (proinsulin *split)* and the connecting peptide (CP) (15).

The pro-insulin is cleaved in the Golgi apparatus of islet cells. This reaction leads to the formation of insulin, CP and two pairs of basic amino acids. Insulin and the CP are released into circulation at a ratio of 1:1, as well as small amounts of proinsulin and intermediates. The proinsulin sum 20% of molecules with insulin-immunoreactivity *simile,* seems to have no metabolic effect, undergoes extra-hepatic metabolism and is excreted exclusively by the kidneys.

The CP is a connection between the peptide chains A and B of proinsulin and facilitates the processing of biologically active insulin in secretory granules of pancreatic islets. After the cleavage of proinsulin, the intact CP is stored with insulin in these granules and is cosecreted with insulin. For this reason, the CP can be considered an independent marker of insulin secretion (16). However, in some situations, such as renal failure, the serum concentration of CP is not proportional to the rate of insulin secretion. About 85% of CP is metabolized by the kidneys and the remainder excreted intact in urine. A decrease in renal function leads to reduced metabolism of CP and elevated serum levels (74)

The CP plasma half-life is of thirty minutes, greater than that of insulin, which is only of four minutes (13-15,75). The normal value of the CP varies from 1.1 to 5.0 ng / mL.

Under standard conditions of measurement, the CP has been widely accepted as a rough measure of insulin secretion, since it is secreted into the portal circulation in equimolar, concentrations, does not undergo hepatic metabolism, its half-life is longer (30 minutes) and has low cross reactivity with proinsulin and insulin antibodies (12, 76,77).

In adverse conditions, such as hyper- or hypoglycemia, CP concentrations are not proportional to the rate of insulin secretion, and its *clearance* may vary between different individuals (78,79).

Therefore, at the moment, the most appropriate, accepted and clinically validated method to measure the ability of β cell secretion under ideal conditions is the measurement of baseline and/or stimulated CP (76). This stimulation can be done with glucose or insulin secretagogues such as glucagon, standard mixed meal or oral glucose tolerance test with 75g of anhydrous glucose (OGTT) (12,80-82).

The standard mixed meal test consists of oral administration of a liquid diet (Sustacal ® / Boost) of approximately 500 kcal containing 50% carbohydrate, 30% fat and 20% protein. Blood samples for measurement of blood glucose and CP are collected in fasting and 30, 60 and/or 90 minutes after the meal (76,80). This test shows the typical postprandial response of the cell β and its interaction with the various hormones secreted during oral feeding. It is the most physiological test among the above cited (76,83).

Oral glucose tolerance test (OGTT) is the determination of glucose, insulin and CP after 10 hours of fasting and 30, 60, 90, 120 minutes after ingestion of 75g anhydrous glucose orally administered. It is used to measure glucose tolerance and the residual function of the β cell in patients at risk, but has not yet been validated for use in T1DM patients. It is useful to

Understanding Pancreatic Secretion in Type 1 Diabetes 269

some residual insulin secretion at diagnosis especially in cases diagnosed in adulthood (5-8,

The loss of this residual secretion usually occurs within three to five years after the diagnosis (86). Patients with long duration of illness tend not to have CP secretory reserves, demonstrating the exhaustion of pancreatic secretion (9-11). However, Meier et al demonstrated the preservation of β cells secreting insulin in most patients with T1DM evaluated in histopathological studies (90). In addition, Keenan and colleagues demonstrated detectable levels of CP in 18% of individuals with T1DM for over 50 years and

The reason why some β cells are maintained for years after diagnosis of T1DM remains unclear. It is possible that some cells are not equally susceptible to destruction or even that the destructive process is attenuated over the years. Another possibility to explain this persistence would be a recovery in β cell by replication, which seems unlikely. Another explanation is that some cells could be inactive and not destroyed and recover their function

The rate of decline of pancreatic function in T1DM is heterogeneous, ranging from 13 to 58% in the first year after diagnosis (84). Some factors such as age at diagnosis and sex seem to influence this fall. Association between residual pancreatic function and female sex has also been found (93). Karjalainen et al reported that T1DM that begins in adulthood (20 to 55.8 years old) is characterized by a longer asymptomatic period before diagnosis and better preservation of residual β cell function than T1DM beginning in

Some studies have shown that baseline serum CP levels in patients diagnosed in adulthood and post-pubertal period are higher than in those diagnosed in the pre-puberty (7,8,12). This fact indicates a greater destruction of β cells in younger people. However, there could be a change in CP levels according to age. According to Palmer JP et al, the serum CP levels in adulthood are around 0.6 to 1.3 nmol / L during puberty between 0.3 and 0.9 nmol / L and

In the *Diabetes Control and Complications Trial* (DCCT), patients with short disease duration (≤ 5 years) had a CP after the stimulus with mixed meal detectable (greater than 0.2 nmol / L) in 33% of those with <18 years of age and 48% in adults (10). Stimulated CP > 0.2 pmol / mL was found in 3% of children and 8% of adults with long duration of illness (> 5 years). Basal

The presence of antibodies is another predictor of reduction in β cell function. High levels of ICA were associated with a faster decrease in the secretion of CP (81). Aimed to modulate the immune system and prevent the destruction of β cells, studies using vaccination against GADA showed some preservation of the CP, although it did not change the needs of insulin

There is evidence that intensive glucose control can reduce, at least temporarily, the failure of insulin secretion in T1DM. However, it is possible that the maintenance of some residual secretion facilitates the achievement of adequate metabolic control (20). The mechanisms by which intensive insulin prolongs the β-cell function in T1DM can be due to reduced

and stimulated CP were negatively correlated with disease duration (80).

glucotoxicity or by direct action in the autoimmune destruction (97).

absence of chronic complications of the disease (91).

**3.2.1 Factors influencing the residual insulin secretion and CP** 

89).

over time (92).

childhood (5,16,94).

(95,96).

in pre-pubertal <0.2 nmol / L (12).

predict early changes in glucose metabolism in relatives of T1DM or individuals with positive autoantibodies (76).

The glucagon stimulation test is done by measurement of CP at baseline, after 8 hours fasting, and 6 minutes after intravenous administration of 1 mg of glucagon, while its maximum concentration is observed (76,79). The most commonly observed side effects are facial flushing and nausea due to decreased gastrointestinal motility (76). The advantages of the glucagon test in relation to the standard meal test are:


To avoid inaccuracy in CP measurement, caution is necessary during blood collection and processing. As the CP is a small molecule, linear and prone to degradation by proteolytic enzymes blood samples should be cleared by centrifugation for a short period of time (not more than a few hours). Palmer et al suggest that this is done within one month after collection because immunoreactivity falls with prolonged blood storage generating falsely lower results (84). This time, however, it is not well defined (12). Until the proper measurement, the serum should be stored -80 ° C.

Changes in blood glucose are factors that acutely interfere with the measurement of serum CP and can underestimate the ability of secretion. The Immunology of Diabetes Society has established that the glucagon test must be conducted during fasting and with glucose levels between 70 and 200mg/dL. The optimal level of blood glucose in these patients is around 126mg/dL. Hypoglycemia (<70 mg / dL) inhibits the insulin response while acute hyperglycemia (> 200mg/dL) may potentiate the secretory response or inhibit it. On the other hand, chronic hyperglycemia can reduce β cell function due to the phenomenon of glucotoxicity (85-86). Moreover, high glucose concentrations have been shown to damage β cells *in vitro* and *in vivo,* compromising insulin secretion (87).

The characteristics of the methods used to quantitate the CP must be well defined. The presence of cross-reactivity with proinsulin and its intermediate results in falsely elevated CP concentrations. In general, it is expected that the rate of cross-reactivity of a method is less than 10% (12).

#### **3.2 Role of the pancreatic β cell in T1DM**

In T1DM, there is a progressive loss of the ability of insulin secretion. During the process of destruction of pancreatic β cells, the first abnormality observed in the preclinical phase is the loss of first phase insulin secretion - *FPIR, First phase insulin release* (31,88). The FPIR is the sum of the plasma insulin at 1 and 3 minutes after the glucose load during an intravenous glucose tolerance test (76). Moreover, impairment in glucose tolerance test has been correlated with an increased risk of progressing from preclinical to clinical diabetes (4).

One of the first authors who studied β cell function in diabetic patients and healthy controls was OK Faber et al in 1977. He compared measurements of the CP after the standard meal and after glucagon (1 mg intravenous) and noticed that the CP, at baseline and after fasting and stimulation with glucagon, was higher in healthy controls when compared to patients with T1DM (79). Subsequently, it was identified that individuals with T1DM may have

predict early changes in glucose metabolism in relatives of T1DM or individuals with

The glucagon stimulation test is done by measurement of CP at baseline, after 8 hours fasting, and 6 minutes after intravenous administration of 1 mg of glucagon, while its maximum concentration is observed (76,79). The most commonly observed side effects are facial flushing and nausea due to decreased gastrointestinal motility (76). The advantages of

1. Faster action, since glucagon is a potent supra-physiological stimulus for insulin

2. Minor influence of glucotoxicity in patients with high glycated hemoglobin (HbA1C)

To avoid inaccuracy in CP measurement, caution is necessary during blood collection and processing. As the CP is a small molecule, linear and prone to degradation by proteolytic enzymes blood samples should be cleared by centrifugation for a short period of time (not more than a few hours). Palmer et al suggest that this is done within one month after collection because immunoreactivity falls with prolonged blood storage generating falsely lower results (84). This time, however, it is not well defined (12). Until the proper

Changes in blood glucose are factors that acutely interfere with the measurement of serum CP and can underestimate the ability of secretion. The Immunology of Diabetes Society has established that the glucagon test must be conducted during fasting and with glucose levels between 70 and 200mg/dL. The optimal level of blood glucose in these patients is around 126mg/dL. Hypoglycemia (<70 mg / dL) inhibits the insulin response while acute hyperglycemia (> 200mg/dL) may potentiate the secretory response or inhibit it. On the other hand, chronic hyperglycemia can reduce β cell function due to the phenomenon of glucotoxicity (85-86). Moreover, high glucose concentrations have been shown to damage β

The characteristics of the methods used to quantitate the CP must be well defined. The presence of cross-reactivity with proinsulin and its intermediate results in falsely elevated CP concentrations. In general, it is expected that the rate of cross-reactivity of a method is

In T1DM, there is a progressive loss of the ability of insulin secretion. During the process of destruction of pancreatic β cells, the first abnormality observed in the preclinical phase is the loss of first phase insulin secretion - *FPIR, First phase insulin release* (31,88). The FPIR is the sum of the plasma insulin at 1 and 3 minutes after the glucose load during an intravenous glucose tolerance test (76). Moreover, impairment in glucose tolerance test has been correlated with an increased risk of progressing from preclinical to clinical diabetes (4). One of the first authors who studied β cell function in diabetic patients and healthy controls was OK Faber et al in 1977. He compared measurements of the CP after the standard meal and after glucagon (1 mg intravenous) and noticed that the CP, at baseline and after fasting and stimulation with glucagon, was higher in healthy controls when compared to patients with T1DM (79). Subsequently, it was identified that individuals with T1DM may have

secretion, directly and indirectly, also influenced by hyperglycemia (76).

positive autoantibodies (76).

3. Simple technical achievement (76).

(76,80).

less than 10% (12).

the glucagon test in relation to the standard meal test are:

4. Good reproducibility between individuals (76).

measurement, the serum should be stored -80 ° C.

**3.2 Role of the pancreatic β cell in T1DM** 

cells *in vitro* and *in vivo,* compromising insulin secretion (87).

some residual insulin secretion at diagnosis especially in cases diagnosed in adulthood (5-8, 89).

The loss of this residual secretion usually occurs within three to five years after the diagnosis (86). Patients with long duration of illness tend not to have CP secretory reserves, demonstrating the exhaustion of pancreatic secretion (9-11). However, Meier et al demonstrated the preservation of β cells secreting insulin in most patients with T1DM evaluated in histopathological studies (90). In addition, Keenan and colleagues demonstrated detectable levels of CP in 18% of individuals with T1DM for over 50 years and absence of chronic complications of the disease (91).

The reason why some β cells are maintained for years after diagnosis of T1DM remains unclear. It is possible that some cells are not equally susceptible to destruction or even that the destructive process is attenuated over the years. Another possibility to explain this persistence would be a recovery in β cell by replication, which seems unlikely. Another explanation is that some cells could be inactive and not destroyed and recover their function over time (92).

#### **3.2.1 Factors influencing the residual insulin secretion and CP**

The rate of decline of pancreatic function in T1DM is heterogeneous, ranging from 13 to 58% in the first year after diagnosis (84). Some factors such as age at diagnosis and sex seem to influence this fall. Association between residual pancreatic function and female sex has also been found (93). Karjalainen et al reported that T1DM that begins in adulthood (20 to 55.8 years old) is characterized by a longer asymptomatic period before diagnosis and better preservation of residual β cell function than T1DM beginning in childhood (5,16,94).

Some studies have shown that baseline serum CP levels in patients diagnosed in adulthood and post-pubertal period are higher than in those diagnosed in the pre-puberty (7,8,12). This fact indicates a greater destruction of β cells in younger people. However, there could be a change in CP levels according to age. According to Palmer JP et al, the serum CP levels in adulthood are around 0.6 to 1.3 nmol / L during puberty between 0.3 and 0.9 nmol / L and in pre-pubertal <0.2 nmol / L (12).

In the *Diabetes Control and Complications Trial* (DCCT), patients with short disease duration (≤ 5 years) had a CP after the stimulus with mixed meal detectable (greater than 0.2 nmol / L) in 33% of those with <18 years of age and 48% in adults (10). Stimulated CP > 0.2 pmol / mL was found in 3% of children and 8% of adults with long duration of illness (> 5 years). Basal and stimulated CP were negatively correlated with disease duration (80).

The presence of antibodies is another predictor of reduction in β cell function. High levels of ICA were associated with a faster decrease in the secretion of CP (81). Aimed to modulate the immune system and prevent the destruction of β cells, studies using vaccination against GADA showed some preservation of the CP, although it did not change the needs of insulin (95,96).

There is evidence that intensive glucose control can reduce, at least temporarily, the failure of insulin secretion in T1DM. However, it is possible that the maintenance of some residual secretion facilitates the achievement of adequate metabolic control (20). The mechanisms by which intensive insulin prolongs the β-cell function in T1DM can be due to reduced glucotoxicity or by direct action in the autoimmune destruction (97).

Understanding Pancreatic Secretion in Type 1 Diabetes 271

The need for insulin and/or progression of pancreatic β cell damage can be avoided by preserving the ability of insulin secretion. The maintenance of some residual function, even if insufficient to avoid insulin therapy may have important advantages in better metabolic

The DCCT showed that intensive insulin therapy can reduce the progression of β cell damage, with positive effects for up to 6 years after diagnosis (8). Brown et al in their study also confirmed this benefit (103). Intensive insulin therapy may promote survival of β cells,

Immunosuppressive therapy, such as cyclosporine, azathioprine, prednisone, and antithymocyte globulin, aiming for depletion and inactivation of β cells, were used in newly diagnosed patients, but with limited efficacy and temporary effects due to its toxicity (104). Immunomodulators, such as anti-CD3 monoclonal antibodies, used in newly diagnosed T1DM also allowed the maintenance of the secretion of CP for one to two years with low toxicity, and benefits in glycemic control (105-107). After 48 months of follow up, patients who received the monoclonal antibodies anti-CD3 had lower daily insulin requirements, with improved A1C and glycemic variations smaller than the control. The best results have been found in individuals under the age of 27 years and with higher CP at baseline (108). The induction of immunological tolerance to self antigens such as GAD, insulin and oral *heat shock protein* 60 (HSP60) has been tried with controversial results in preservation of islet

Another measure that has been tested for secondary prevention of T1DM is the autologous non-myeloablative hematopoietic stem cell transplant. Voltarelli and colleagues conducted this transplant in 15 newly diagnosed patients with T1DM. Five patients have remained insulin free for up to 21 months and seven remained for more than six months without use of exogenous insulin (111). Despite the low rate of complications reported, there is a potential risk for more serious effects related to immunosuppression. (107,110). Mesenchymal stem cell therapies and combination of multiple immunomodulatory drugs

Several studies aiming at the preservation of β cell mass are being conducted, with a main goal: search for the cure of T1DM. The attempt to preserve β cell function even if insufficient to cure the disease, can be useful in the prevention of microvascular complications, improves glycemic control and reduced the frequency of hypoglycemic events (7,8,12,98). In summary, we tried to emphasize some aspects of the natural history of T1DM. At first, a large mass of beta cell function is lost between the period from the surveillance diagnosis to the period of overt T1DM. Then, residual beta cell function results in better glycemic control and less microvascular complications. The rate of progression of beta cell failure may be due to several factors such as underlying genetic predisposition, age of the patient and metabolic control. Finally, CP has its role on diagnosis reserve of beta cell mass and higher levels are associated to a better glycemic control and preservation of pancreatic function. Moreover, interventions that are being held nowadays are clinically important to quality of life,

[1] Atkinson MA et al. Type 1 Diabetes: New Perspectives On Disease Pathogenesis And

control and lower risk of chronic complications as previously described (8,84).

**3.2.3 Methods of preserving pancreatic function** 

reducing the metabolic demand and glucotoxicity.

function (104,109,110).

are currently under study.

**4. References** 

mortality and morbidity of patients with T1DM.

Treatment. Lancet 2001; 358: 221-229.

#### **3.2.2 Impact of maintenance of residual insulin secretion and PC**

The persistence of detectable CP serum levels, especially in patients with long duration of disease, may have clinical importance. Some studies have shown that this is a prognostic factor for improved glycemic control, lower frequency of hypoglycemia, retinopathy and diabetic nephropathy (8,80,98). So far, the main information about the importance of preservation of some residual secretion in the development of chronic complications, glycemic control and incidence of hypoglycemia T1DM were obtained from the DCCT (8).

In the DCCT, the intensive glucose control significantly reduced the loss of β cell function in relation to conventional treatment. Furthermore, patients with CP ≥ 0.2 nmol / L in the intensive treatment group had lower HbA1C at baseline and through the four year followup period (8).

The presence of a residual capacity for insulin secretion has also been associated with a reduced risk of hypoglycemia. Data from the DCCT showed that patients with stimulated CP≥ 0.2 nmol / L for at least one year had the prevalence of hypoglycemia reduced in 30%. Among patients in intensive control group, the risk of hypoglycemia was three times lower in those who remained detectable CP than in others. In the conventional group, this difference was not observed (8).

In relation to chronic complications, the DCCT showed that patients with T1DM and undetectable levels of CP (<0.04 nmol / L) had 4.6 times greater chance of progression to diabetic retinopathy and 4.4 times greater chance of developing microalbuminuria in relation to others (8).

Some authors found no association between the frequency of chronic complications and residual β cell function. Klein and colleagues studied the relationship between serum levels of CP and severity of diabetic retinopathy in different types of diabetes in the *Wisconsin Epidemiologic Study of Diabetic Retinopathy.* Young subjects with T1DM using insulin did not present any association between CP levels and the frequency or severity of diabetic retinopathy (99). Winocour et al, on the other hand, found an association between the presence of residual secretion of CP and reduced risk of proliferative diabetic retinopathy in T1DM but no correlation to peripheral neuropathy or autonomic, hypertension, nephropathy or coronary heart disease (100).

Other studies also found no influence of CP stimulated with the development of retinopathy, neuropathy and/or microalbuminuria in T1DM. However, these studies included a small number of patients, with short time for monitoring and/or few chronic complications (100-102).

In the pathological study of Meier et al, the number of β cells found in patients with T1DM was not associated with the disease duration, but rather with glycemic control, being higher at lower blood glucose levels (90).

Today, the role of the CP only as a marker of insulin secretion is questionable. Some suggests that it may also have a direct action on target organs of chronic complications of T1DM (84). Potential actions of the CP include: improvement in nerve conduction velocity, improving the sensory and autonomic nerve function, improvement in cardiac function, decreased microalbuminuria; stimulation of the activity of nitric oxide synthase *(eNOS)* inhibition of smooth muscle cells proliferation; and decreased signal transduction of NF-kβ *(nuclear factor kappa-light-chain-enhancer of activated B cells)* with reduced inflammation. These findings have been observed *in vitro* and in clinical studies in animals and humans with T1DM (84).

The persistence of detectable CP serum levels, especially in patients with long duration of disease, may have clinical importance. Some studies have shown that this is a prognostic factor for improved glycemic control, lower frequency of hypoglycemia, retinopathy and diabetic nephropathy (8,80,98). So far, the main information about the importance of preservation of some residual secretion in the development of chronic complications, glycemic control and incidence of hypoglycemia T1DM were obtained from the DCCT (8). In the DCCT, the intensive glucose control significantly reduced the loss of β cell function in relation to conventional treatment. Furthermore, patients with CP ≥ 0.2 nmol / L in the intensive treatment group had lower HbA1C at baseline and through the four year follow-

The presence of a residual capacity for insulin secretion has also been associated with a reduced risk of hypoglycemia. Data from the DCCT showed that patients with stimulated CP≥ 0.2 nmol / L for at least one year had the prevalence of hypoglycemia reduced in 30%. Among patients in intensive control group, the risk of hypoglycemia was three times lower in those who remained detectable CP than in others. In the conventional group, this

In relation to chronic complications, the DCCT showed that patients with T1DM and undetectable levels of CP (<0.04 nmol / L) had 4.6 times greater chance of progression to diabetic retinopathy and 4.4 times greater chance of developing microalbuminuria in

Some authors found no association between the frequency of chronic complications and residual β cell function. Klein and colleagues studied the relationship between serum levels of CP and severity of diabetic retinopathy in different types of diabetes in the *Wisconsin Epidemiologic Study of Diabetic Retinopathy.* Young subjects with T1DM using insulin did not present any association between CP levels and the frequency or severity of diabetic retinopathy (99). Winocour et al, on the other hand, found an association between the presence of residual secretion of CP and reduced risk of proliferative diabetic retinopathy in T1DM but no correlation to peripheral neuropathy or autonomic, hypertension,

Other studies also found no influence of CP stimulated with the development of retinopathy, neuropathy and/or microalbuminuria in T1DM. However, these studies included a small number of patients, with short time for monitoring and/or few chronic

In the pathological study of Meier et al, the number of β cells found in patients with T1DM was not associated with the disease duration, but rather with glycemic control, being higher

Today, the role of the CP only as a marker of insulin secretion is questionable. Some suggests that it may also have a direct action on target organs of chronic complications of T1DM (84). Potential actions of the CP include: improvement in nerve conduction velocity, improving the sensory and autonomic nerve function, improvement in cardiac function, decreased microalbuminuria; stimulation of the activity of nitric oxide synthase *(eNOS)* inhibition of smooth muscle cells proliferation; and decreased signal transduction of NF-kβ *(nuclear factor kappa-light-chain-enhancer of activated B cells)* with reduced inflammation. These findings have been observed *in vitro* and in clinical studies in animals and humans with

**3.2.2 Impact of maintenance of residual insulin secretion and PC** 

up period (8).

difference was not observed (8).

nephropathy or coronary heart disease (100).

relation to others (8).

complications (100-102).

T1DM (84).

at lower blood glucose levels (90).

#### **3.2.3 Methods of preserving pancreatic function**

The need for insulin and/or progression of pancreatic β cell damage can be avoided by preserving the ability of insulin secretion. The maintenance of some residual function, even if insufficient to avoid insulin therapy may have important advantages in better metabolic control and lower risk of chronic complications as previously described (8,84).

The DCCT showed that intensive insulin therapy can reduce the progression of β cell damage, with positive effects for up to 6 years after diagnosis (8). Brown et al in their study also confirmed this benefit (103). Intensive insulin therapy may promote survival of β cells, reducing the metabolic demand and glucotoxicity.

Immunosuppressive therapy, such as cyclosporine, azathioprine, prednisone, and antithymocyte globulin, aiming for depletion and inactivation of β cells, were used in newly diagnosed patients, but with limited efficacy and temporary effects due to its toxicity (104).

Immunomodulators, such as anti-CD3 monoclonal antibodies, used in newly diagnosed T1DM also allowed the maintenance of the secretion of CP for one to two years with low toxicity, and benefits in glycemic control (105-107). After 48 months of follow up, patients who received the monoclonal antibodies anti-CD3 had lower daily insulin requirements, with improved A1C and glycemic variations smaller than the control. The best results have been found in individuals under the age of 27 years and with higher CP at baseline (108).

The induction of immunological tolerance to self antigens such as GAD, insulin and oral *heat shock protein* 60 (HSP60) has been tried with controversial results in preservation of islet function (104,109,110).

Another measure that has been tested for secondary prevention of T1DM is the autologous non-myeloablative hematopoietic stem cell transplant. Voltarelli and colleagues conducted this transplant in 15 newly diagnosed patients with T1DM. Five patients have remained insulin free for up to 21 months and seven remained for more than six months without use of exogenous insulin (111). Despite the low rate of complications reported, there is a potential risk for more serious effects related to immunosuppression. (107,110). Mesenchymal stem cell therapies and combination of multiple immunomodulatory drugs are currently under study.

Several studies aiming at the preservation of β cell mass are being conducted, with a main goal: search for the cure of T1DM. The attempt to preserve β cell function even if insufficient to cure the disease, can be useful in the prevention of microvascular complications, improves glycemic control and reduced the frequency of hypoglycemic events (7,8,12,98).

In summary, we tried to emphasize some aspects of the natural history of T1DM. At first, a large mass of beta cell function is lost between the period from the surveillance diagnosis to the period of overt T1DM. Then, residual beta cell function results in better glycemic control and less microvascular complications. The rate of progression of beta cell failure may be due to several factors such as underlying genetic predisposition, age of the patient and metabolic control. Finally, CP has its role on diagnosis reserve of beta cell mass and higher levels are associated to a better glycemic control and preservation of pancreatic function. Moreover, interventions that are being held nowadays are clinically important to quality of life, mortality and morbidity of patients with T1DM.

#### **4. References**

[1] Atkinson MA et al. Type 1 Diabetes: New Perspectives On Disease Pathogenesis And Treatment. Lancet 2001; 358: 221-229.

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**Part 3** 

**Retinopathy** 


**Part 3** 

**Retinopathy** 

278 Type 1 Diabetes Complications

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Hematopoietic Stem Cell Transplantation In Newly Diagnosed Type 1 Diabetes

**13** 

*Spain* 

**Review of the Relationship Between Renal** 

**and Retinal Microangiopathy in Type 1** 

Pedro Romero-Aroca1 , Juan Fernández-Ballart2, Nuria Soler1,

*1Department of Ophthalmology, University Hospital Sant Joan, Institut de Investigació* 

*2Epidemiology, Department of Basic Sciences, University Rovira i Virgili (Tarragona),* 

Diabetes mellitus is a group of metabolic disorders of carbohydrate metabolism in which glucose is underutilized, producing hyperglycemia. The disease is classified into several categories. The revised classification, published in 1997 (ADA, 1997; The Expert Comitee on the Diagnosis and Classification of Diabetes mellitus, 2000), defines Type 1 diabetes mellitus (formerly known as the insulin-dependent diabetes mellitus or juvenile-onset diabetes mellitus) as a disorder caused by autoimmune destruction of pancreatic h-cells, rendering the pancreas unable to synthesize and secrete insulin. In 85–90% of cases, antibodies appear against pancreatic h-cells (ICA), acting as anti-insulin (IAA), or others such as GAD, IA-2

The latter complications of diabetes mellitus include both microvascular complications (predominantly retinopathy, nephropathy and neuropathy) and macrovascular complications, particularly stroke and coronary artery disease. Together, these make diabetes the seventh most common cause of death in the developed world (Geiss et al 1997). The major microvascular complications, retinopathy and nephropathy, are the more important causes of blindness and end-stage renal disease in Europe. There are few similarities in the coexistence of DR and DN being both as microvascular disease and microscopically both have capillary basement membrane thickening. However, capillary closure is apparent in the retina and kidney after sufficient exposure to disease with duration. The pathophysiology of DN and DR are more or less similar, which commence with increase in vascular permeability. The selective increase in permeability to albumin in early DN is caused by loss of polarity across the glomerular basement membrane (Myers et al, 1982) and the disease mechanism in the eye is probably a breakdown of tight junctions between cells. The onset of proteinuria and proliferative retinopathy are both related to

The detection of retinopathy is easy (by the use of fundus periodical retinographies), but the diagnosis of the early stages of nephropathy needs microalbuminuria to be determined in

previous poor glycemic control, duration of diabetes and hypertension.

**1. Introduction** 

and IA-2h (Geiss et al 1997).

**Diabetes Mellitus Patients** 

*Sanitaria Pere Virgili (IISPV), Reus,* 

Marc Baget-Bernaldiz1 and Isabel Mendez-Marin1

### **Review of the Relationship Between Renal and Retinal Microangiopathy in Type 1 Diabetes Mellitus Patients**

Pedro Romero-Aroca1 , Juan Fernández-Ballart2, Nuria Soler1, Marc Baget-Bernaldiz1 and Isabel Mendez-Marin1 *1Department of Ophthalmology, University Hospital Sant Joan, Institut de Investigació Sanitaria Pere Virgili (IISPV), Reus, 2Epidemiology, Department of Basic Sciences, University Rovira i Virgili (Tarragona), Spain* 

#### **1. Introduction**

Diabetes mellitus is a group of metabolic disorders of carbohydrate metabolism in which glucose is underutilized, producing hyperglycemia. The disease is classified into several categories. The revised classification, published in 1997 (ADA, 1997; The Expert Comitee on the Diagnosis and Classification of Diabetes mellitus, 2000), defines Type 1 diabetes mellitus (formerly known as the insulin-dependent diabetes mellitus or juvenile-onset diabetes mellitus) as a disorder caused by autoimmune destruction of pancreatic h-cells, rendering the pancreas unable to synthesize and secrete insulin. In 85–90% of cases, antibodies appear against pancreatic h-cells (ICA), acting as anti-insulin (IAA), or others such as GAD, IA-2 and IA-2h (Geiss et al 1997).

The latter complications of diabetes mellitus include both microvascular complications (predominantly retinopathy, nephropathy and neuropathy) and macrovascular complications, particularly stroke and coronary artery disease. Together, these make diabetes the seventh most common cause of death in the developed world (Geiss et al 1997).

The major microvascular complications, retinopathy and nephropathy, are the more important causes of blindness and end-stage renal disease in Europe. There are few similarities in the coexistence of DR and DN being both as microvascular disease and microscopically both have capillary basement membrane thickening. However, capillary closure is apparent in the retina and kidney after sufficient exposure to disease with duration. The pathophysiology of DN and DR are more or less similar, which commence with increase in vascular permeability. The selective increase in permeability to albumin in early DN is caused by loss of polarity across the glomerular basement membrane (Myers et al, 1982) and the disease mechanism in the eye is probably a breakdown of tight junctions between cells. The onset of proteinuria and proliferative retinopathy are both related to previous poor glycemic control, duration of diabetes and hypertension.

The detection of retinopathy is easy (by the use of fundus periodical retinographies), but the diagnosis of the early stages of nephropathy needs microalbuminuria to be determined in

Review of the Relationship Between

measures taken at one month intervals).

**2.3 Diagnostic methods** 

(Wilkinson et al 2003): Mild non proliferative Moderate non proliferative Severe non proliferative

disappeared)

Proliferative

**2.2 Design** 

Renal and Retinal Microangiopathy in Type 1 Diabetes Mellitus Patients 283

The present study is prospective and was initiated in 1990 with 126 patients recruited with type I diabetes mellitus. The initial conditions included the absence of retinopathy and nephropathy (determined by the absence of microalbuminuria in three consecutive

Two previous results were obtained at 5 and 10 years of the study (Romero-Aroca et al, 2000; Romero-Aroca et al, 2003). At the end of the study in 2007 only 112 patients were still being controlled (14 patients had dropped out during the follow up). At the end of the study (seventeen years of follow-up) the authors have determined the incidence of diabetic macular oedema and their risk factors, related to the appearance of renal overt nephropathy.

Diabetic retinopathy was evaluated by retinal photographs through dilated pupils, of two 50º fields of each eye centred firstly at the temporal to the macula and secondly at the nasal to the papilla (Aldington et al, 1995 ). The results were then classified into four groups

Macular edema was diagnosed under stereoscopic viewing of the macula with a slit lamp

 hard exudates at or within 500 μ m of the centre of the macula, if associated with thickening of adjacent retina (but no hard exudates remaining after retinal thickening

a zone or zones of retinal thickening, one disc area or larger in size, any part of which is

The clinical classification used was the international clinical diabetic retinopathy disease severity scale, proposed by the American Academy of Ophthalmology in 2002 (Wilkinson et al 2003). In all patients with diabetic macular oedema, a fluorescein angiography was obtained, centred on macular region to determine the leakage in that area. The fluorescein

focal leakage type, which was predominantly well-defined focal areas of leakage from

diffuse leakage type, predominantly widespread and ill-defined leakage involving the

cystoid leakage type, predominantly diffuse leakage but with pooling of dye in the

Since 2000, all patients with diabetic macular oedema have been given an optical coherence tomography OCT), repeated every 4 months as a control test. Optical coherence tomography was performed with a OCT model TOPCON TRC NW 7SF. The retinal map algorithm uses measurements along 6 radial lines, 6 mm in length, to produce a circular plot in which the foveal zone is the central circular zone of 1.00 mm in diameter. Macular edema measured by OCT was defined as a retinal thickening of more than 216 microns, and was classified as follows, using the Otani et al patterns amplified by the two tractional forms

and Goldmann fundus contact lens, and was considered present if we found: retinal thickening involving or within 500 μ m of the centre of the macula

within 1 disc diameter of the centre of the macula.

angiographic findings were categorized into three types:

microaneurysm or localized dilated capillaries;

cystic spaces of the macula in the late phase.

whole circumference of the fovea;

urinalysis. Microalbuminuria has prognostic significance; thus, in 80% of people with Type 1 diabetes mellitus and microalbuminuria, urinary albumin excretion increases at a rate of 10–20% per year, with the development of clinical proteinuria within 10– 15 years. After the development of clinical grade proteinuria (>80%), patients go on to develop decreased glomerular filtration rate and, given enough time, end-stage renal disease (Geiss et al 1997). Several factors appear to influence susceptibility to the microvascular complications of diabetes mellitus, but our knowledge of the role and the importance of these genetic and environmental factors are still incomplete. The most powerful risk factor for microvascular complications was the duration of diabetes, but frequency of both retinopathy and nephropathy was impressively related to the level of plasma glucose at the time of examination.

From the recent studies, it is evident that the presence of retinopathy itself may reveal patients at risk for nephropathy (Estacio et al, 1998; El Asrar et al, 2002; Rossing et al, 2002; Villar et al, 1999). In a cross sectional study, patients with DR were 5.68, 13.39 and 3.51 times as likely to have DN among type1 and type2 diabetic patients (El-Asrar et al, 2002).

However, there is lack of evidence that determine the association of retinal-renal complications using the gold standard methods. The DR is characterized by microvascular abnormalities, proliferation of retinal vessels and increased retinal vascular permeability leading to the development of non-proliferative and proliferative DR, and macular edema (Williams et al 2004). The DN is a life threatening complication which predisposes to excess morbidity and mortality resulting from renal failure and cardiovascular disease (Ritz et al, 1999; Adler at al, 2003).

Our hypothesis was that the severity of DR correlates with the presence and severity of DN in people with type 1 diabetes. Studies in other populations documented a wellknown association between advanced DR stages and overt nephropathy in type 1 diabetic patient (Looker et al, 2003; Gall et al, 1997). Similarly, our results provide further support to the close relationship between presence of DR and severity of DN in type1 diabetic patients.

It was reported that at least one fifth of the diabetic individuals are affected by multiple complications and the frequency increases with increasing age and duration of diabetes.

In the study of WESDR, there was a strong correlation between DN and severity of DR in all age groups (Klein et al, 1984; Klein et al 1984).

In the present study we determine the epidemiological risk factors that influence the appearance of diabetic retinopathy, and overt nephropathy, in a seventeen -year follow-up of a population sample of 112 patients who did not have diabetic retinopathy or microalbuminuria at the beginning of the study.

#### **2. Methods**

#### **2.1 Sample size and study population**

Since 1987 a register has been kept of any new cases of type I diabetes mellitus in Catalonia (Spain). The incidence of new cases over that period has been 11.4 cases per 100000 inhabitants (13.2 cases in men and 9.6 cases in women) (Castell et al, 1999).

Since 1990, there has been an ongoing registration of all diabetic patients (type I and 2) at St Joan Hospital, which is the only surgical ophthalmology centre in Reus (Spain), and having a dependent population of around 207,500 inhabitants. In 1999, there were 1495 patients with diabetes mellitus type I (Castell et al, 1999).

#### **2.2 Design**

282 Type 1 Diabetes Complications

urinalysis. Microalbuminuria has prognostic significance; thus, in 80% of people with Type 1 diabetes mellitus and microalbuminuria, urinary albumin excretion increases at a rate of 10–20% per year, with the development of clinical proteinuria within 10– 15 years. After the development of clinical grade proteinuria (>80%), patients go on to develop decreased glomerular filtration rate and, given enough time, end-stage renal disease (Geiss et al 1997). Several factors appear to influence susceptibility to the microvascular complications of diabetes mellitus, but our knowledge of the role and the importance of these genetic and environmental factors are still incomplete. The most powerful risk factor for microvascular complications was the duration of diabetes, but frequency of both retinopathy and nephropathy was impressively related to the level of plasma glucose at the time of

From the recent studies, it is evident that the presence of retinopathy itself may reveal patients at risk for nephropathy (Estacio et al, 1998; El Asrar et al, 2002; Rossing et al, 2002; Villar et al, 1999). In a cross sectional study, patients with DR were 5.68, 13.39 and 3.51 times

However, there is lack of evidence that determine the association of retinal-renal complications using the gold standard methods. The DR is characterized by microvascular abnormalities, proliferation of retinal vessels and increased retinal vascular permeability leading to the development of non-proliferative and proliferative DR, and macular edema (Williams et al 2004). The DN is a life threatening complication which predisposes to excess morbidity and mortality resulting from renal failure and cardiovascular disease (Ritz et al,

Our hypothesis was that the severity of DR correlates with the presence and severity of DN in people with type 1 diabetes. Studies in other populations documented a wellknown association between advanced DR stages and overt nephropathy in type 1 diabetic patient (Looker et al, 2003; Gall et al, 1997). Similarly, our results provide further support to the close relationship between presence of DR and severity of DN in type1 diabetic

It was reported that at least one fifth of the diabetic individuals are affected by multiple complications and the frequency increases with increasing age and duration of diabetes. In the study of WESDR, there was a strong correlation between DN and severity of DR in all

In the present study we determine the epidemiological risk factors that influence the appearance of diabetic retinopathy, and overt nephropathy, in a seventeen -year follow-up of a population sample of 112 patients who did not have diabetic retinopathy or

Since 1987 a register has been kept of any new cases of type I diabetes mellitus in Catalonia (Spain). The incidence of new cases over that period has been 11.4 cases per 100000

Since 1990, there has been an ongoing registration of all diabetic patients (type I and 2) at St Joan Hospital, which is the only surgical ophthalmology centre in Reus (Spain), and having a dependent population of around 207,500 inhabitants. In 1999, there were 1495 patients

inhabitants (13.2 cases in men and 9.6 cases in women) (Castell et al, 1999).

as likely to have DN among type1 and type2 diabetic patients (El-Asrar et al, 2002).

examination.

1999; Adler at al, 2003).

age groups (Klein et al, 1984; Klein et al 1984).

microalbuminuria at the beginning of the study.

with diabetes mellitus type I (Castell et al, 1999).

**2.1 Sample size and study population** 

patients.

**2. Methods** 

The present study is prospective and was initiated in 1990 with 126 patients recruited with type I diabetes mellitus. The initial conditions included the absence of retinopathy and nephropathy (determined by the absence of microalbuminuria in three consecutive measures taken at one month intervals).

Two previous results were obtained at 5 and 10 years of the study (Romero-Aroca et al, 2000; Romero-Aroca et al, 2003). At the end of the study in 2007 only 112 patients were still being controlled (14 patients had dropped out during the follow up). At the end of the study (seventeen years of follow-up) the authors have determined the incidence of diabetic macular oedema and their risk factors, related to the appearance of renal overt nephropathy.

#### **2.3 Diagnostic methods**

Diabetic retinopathy was evaluated by retinal photographs through dilated pupils, of two 50º fields of each eye centred firstly at the temporal to the macula and secondly at the nasal to the papilla (Aldington et al, 1995 ). The results were then classified into four groups (Wilkinson et al 2003):


Macular edema was diagnosed under stereoscopic viewing of the macula with a slit lamp and Goldmann fundus contact lens, and was considered present if we found:


The clinical classification used was the international clinical diabetic retinopathy disease severity scale, proposed by the American Academy of Ophthalmology in 2002 (Wilkinson et al 2003). In all patients with diabetic macular oedema, a fluorescein angiography was obtained, centred on macular region to determine the leakage in that area. The fluorescein angiographic findings were categorized into three types:


Since 2000, all patients with diabetic macular oedema have been given an optical coherence tomography OCT), repeated every 4 months as a control test. Optical coherence tomography was performed with a OCT model TOPCON TRC NW 7SF. The retinal map algorithm uses measurements along 6 radial lines, 6 mm in length, to produce a circular plot in which the foveal zone is the central circular zone of 1.00 mm in diameter. Macular edema measured by OCT was defined as a retinal thickening of more than 216 microns, and was classified as follows, using the Otani et al patterns amplified by the two tractional forms

Review of the Relationship Between

collection (Geiss et al, 1997).

ischemic attack, stroke.

**2.7 Statistical methods** 

acuity an OCT findings.

*Demographic variables of the patients* 

and age a priori.

in 44 patients (39.3%).

**3. Results** 

indicate statistical significance.

Blood Cholesterol in Adults, 2001).

Renal and Retinal Microangiopathy in Type 1 Diabetes Mellitus Patients 285

 Presence of diabetic nephropathy, defined as clinical albuminuria or overt nephropathy by the American Diabetes Association, corresponding to protein excretion >300 mg/24h (>200 µg/min or >300 µg/mg of albumin: creatinine ratio). Measurement of creatinine clearance as an index of glomerular filtration rate was performed on the same urine

 Patients were classified as having macro vascular disease if one or more of the following were present: symptoms of angina pectoris, history of myocardial infarction, coronary artery by pass grafting, percutaneous tranluminal coronary angioplasty, symptoms of or operation for intermittent claudication, history of amputation, transient

 Levels of triglycerides and fractions of cholesterol (HDL-cholesterol and LDLchorlesterol). In the statistical analysis, we classified the patient into normal or higher values, according to the ADA categories as patients with high risk if LDL-cholesterol 3.35 mmol/L (130 mg/dl), HDL-cholesterol 0.90 mmol/L for men and >1.15 mmol/L for women (35 mg/dL for men and 45 mg/dL for women), and triglycerides 1.5 mmol/dL (400 mg/dL), (Expert Panel on Detection, Evaluation, And Treatment of high

All statistical analyses were carried out using the SPSS software package (version 18.0), results are expressed as mean±standard error, a P-value of less than 0.05 was considered to

Differences between those included in analyses were examined using the two sample Student T-tests or one-way ANOVA, for continuous or quantitative data, as visual acuity or current age. For the qualitative or categorical data we used the Chi-square test in the

The Kruskal-Wallis test and the least significant difference test using ranks for multiple comparisons were carried out to evaluate the correlation between best-corrected visual

In the multivariate phase of analysis the relationship of diabetic retinopathy, microalbuminuria and overt nephropathy, to various demographic and other risk factors were examined using logistic regression analysis; the full model was built including gender

The mean of age was 39.94 10.53 years old (24 – 61 years), the mean of diabetes mellitus type I duration was 23.42 7.57 years (12 – 45 years). The arterial hypertension was present

univariate phase of study, with determination of Odds ratio for each variable.

Gender: 54 patients were men (48.2%) and 58 were women (51.8%)

Glycosylated haemoglobin A1c: 7.69% 1.24 (4.50% – 11.40%)

The means of the different quantitative data were:

LDL-Cholesterol: 3.50 0,57 mmol/l (3.00 – 4.00)

repeated testing was made within a period of 3-4 months.

1997). The test was performed annually. After microalbuminuria was diagnosed,

described later (Otani et al, 1999 ): Sponge-like retinal thickness, defined as increased retinal thickness with reduced intra retinal reflectivity and expanded areas of lower reflectivity; cystoid macular oedema, characterized by the intra retinal cystoid spaces at the macular area; serous retinal detachment was thought to be present if the posterior surface of the retina was elevated above the outer border of the highly reflective band, regarded as the signal generated mainly by the retinal pigment epithelium. Only patients with a visible separation between the layer of photoreceptors and the pigment epithelium, was classified as serous detachment; if we observed the photoreceptor layer adjacent to pigment epithelium, we classified the case as cystoid macular oedema.

#### **2.4 Inclusion criteria**

Patients with type I diabetes mellitus (insulin dependent or young-onset diabetes mellitus)

#### **2.5 Exclusion criteria**

Presence of diabetic retinopathy at the beginning of study, presence of diabetic nephropathy at the beginning of study, presence of microalbuminuria at the beginning of study, patients with LADA diabetes (latent autoimmune adult diabetes), patients with type 2 diabetes mellitus (not insulin-dependent or older-onset diabetes mellitus), patients with type 2 diabetes mellitus appeared before 30 years of age (MODY diabetes mellitus)

#### **2.6 Definition of variables**

Visual acuity in each eye was measured on the Snellen chart and recorded as a decimal value, with best refraction for distance. All data manipulations were performed on visual acuities expressed in log MAR form. The legal blind subject was defined as corrected visual acuity less than or equal to 0.1 in the better eye; reduced visual acuity as less than or equal to 0.4 and greater than 0.1 in the better eye.

The epidemiological risk factors included in the study were:


1997). The test was performed annually. After microalbuminuria was diagnosed, repeated testing was made within a period of 3-4 months.


#### **2.7 Statistical methods**

284 Type 1 Diabetes Complications

described later (Otani et al, 1999 ): Sponge-like retinal thickness, defined as increased retinal thickness with reduced intra retinal reflectivity and expanded areas of lower reflectivity; cystoid macular oedema, characterized by the intra retinal cystoid spaces at the macular area; serous retinal detachment was thought to be present if the posterior surface of the retina was elevated above the outer border of the highly reflective band, regarded as the signal generated mainly by the retinal pigment epithelium. Only patients with a visible separation between the layer of photoreceptors and the pigment epithelium, was classified as serous detachment; if we observed the photoreceptor layer adjacent to pigment

Patients with type I diabetes mellitus (insulin dependent or young-onset diabetes mellitus)

Presence of diabetic retinopathy at the beginning of study, presence of diabetic nephropathy at the beginning of study, presence of microalbuminuria at the beginning of study, patients with LADA diabetes (latent autoimmune adult diabetes), patients with type 2 diabetes mellitus (not insulin-dependent or older-onset diabetes mellitus), patients with type 2

Visual acuity in each eye was measured on the Snellen chart and recorded as a decimal value, with best refraction for distance. All data manipulations were performed on visual acuities expressed in log MAR form. The legal blind subject was defined as corrected visual acuity less than or equal to 0.1 in the better eye; reduced visual acuity as less than or equal

Duration of diabetes mellitus, classified in the statistical study in two groups: below 20

 Type of diabetic retinopathy classified into two groups: first with a diabetic retinopathy lower than severe pattern, and the second with patients with severe or proliferative pattern, patients who need scattered photocoagulation were classified in tis second

 Arterial hypertension, which indicates a systolic measurement above or equal to 140 mm Hg and the diastolic measurement above or equal to 90 mm Hg, or when the

 Levels of glycated haemoglobin (HbA1c) as recommended i by the American Diabetes Association (ADA, 1997) as the major component of HbA1c (accounting for 80% of HbA1c), was measured every 3 months. The control of glycaemia was considered in concordance with the European Diabetes Policy Group, into two groups of patients i.e. over or under 7.0% (European Diabetes Policy Group, 1999). The value included in the

 Presence of microalbuminuria, defined as increased albumin excretion (30-300 mg of albumin/24 h or 20-200 µg/min of creatinine) on two of three tests repeated at intervals of 3-6 months as well as exclusion of conditions that invalidate the test (Geiss et al,

statistical analysis was the mean of all values obtained over the 15 years.

diabetes mellitus appeared before 30 years of age (MODY diabetes mellitus)

epithelium, we classified the case as cystoid macular oedema.

**2.4 Inclusion criteria** 

**2.5 Exclusion criteria** 

**2.6 Definition of variables** 

Gender and current age.

group.

to 0.4 and greater than 0.1 in the better eye.

The epidemiological risk factors included in the study were:

patient is taking anti-hypertensive medications.

years of duration and equal to or more than 20 years duration.

All statistical analyses were carried out using the SPSS software package (version 18.0), results are expressed as mean±standard error, a P-value of less than 0.05 was considered to indicate statistical significance.

Differences between those included in analyses were examined using the two sample Student T-tests or one-way ANOVA, for continuous or quantitative data, as visual acuity or current age. For the qualitative or categorical data we used the Chi-square test in the univariate phase of study, with determination of Odds ratio for each variable.

The Kruskal-Wallis test and the least significant difference test using ranks for multiple comparisons were carried out to evaluate the correlation between best-corrected visual acuity an OCT findings.

In the multivariate phase of analysis the relationship of diabetic retinopathy, microalbuminuria and overt nephropathy, to various demographic and other risk factors were examined using logistic regression analysis; the full model was built including gender and age a priori.

#### **3. Results**

*Demographic variables of the patients* 

Gender: 54 patients were men (48.2%) and 58 were women (51.8%)

The mean of age was 39.94 10.53 years old (24 – 61 years), the mean of diabetes mellitus type I duration was 23.42 7.57 years (12 – 45 years). The arterial hypertension was present in 44 patients (39.3%).

The means of the different quantitative data were:


Review of the Relationship Between

*Logistic regression of diabetic nephropathy (Table 2).* 

Risk factor Chi square Logistic

Nephropathy 0.001 6.097 0.021 3.498

Odds ratio

Signifi cance (p)

chi squared test had a result of p<0.001

Significance (p)

Glycated haemoglobin (HbA1c) >8%)

Arterial

Duration of diabetes mellitus (20 years)

HDL-

LDL-

Overt

Proliferative diabetic retinopathy

microalbuminuria.

Renal and Retinal Microangiopathy in Type 1 Diabetes Mellitus Patients 287

The significant factors were: the presence of arterial hypertension p<0.001, and high levels of HbA1c p<0.001, high levels of LDL-Cholesterol p=0.002, high levels of triglycerides p=0.009,. Also the presence of diabetic retinopathy was significant p=0,021. When we introduced the presence of proliferative diabetic retinopathy against the presence of any retinopathy, the

Diabetic retinopathy Overt Nephropathy

<0.001 11.011 <0.001 5.575 <0.001 55.687 <0.001 38.360

<0.001 33.623 <0001 12.096 0.913 0.012 0.170 0.394

Significance (p)

Odds ratio

Gender 0.237 1.397 0.829 0.881 0.743 0.107 0.870 0.910

hypertension <0.001 28.193 0.007 6.579 <0.001 12.777 0.023 10.271

Cholesterol 0.828 0.047 0.813 0.837 0.019 5.201 0.061 0.195

Cholesterol <0001 18984 0.002 10.304 0.010 2.715 0.002 3.555

Triglycerides 0.003 8.442 0.004 1.528 0.003 3.513 0.009 2.912

Retinopathy 0.021 6.097 0.021 2.153

Table 1. Chi squared and logistic regression analysis for diabetic retinopathy and

<0.001 14.814 <0.001 4.306

regression Chi square Logistic regression

Odds ratio

Significance (p)

Odds ratio


#### Visual acuity study

Mean visual acuity after 17 years was 0.77 0.34 ( 0,02 – 1) in the Snellen chart test; and +0.37 +0.72 (+ 1.7 - + 0) in the Log MAR test.

Low vision (defined as vision in the best eye >0,1 and < 0.4 in the Snellen chart) was detected in 13 patients (11.6%) and blindness (AV < 0.1 in Snellen chart) in 14 patients (12.5%).

#### *Incidence of diabetic retinopathy (Table 1)*

After 17 years there were 62 patients (55.4%) with different types of diabetic retinopathy. The Rate of progression was 8.30 person-year.


There were 23 patients (20.5%) with diabetic macular edema after 17 years, the Rate of progression was 3.08 person-year. The mild or moderate form of diabetic macular edema was present in 13 patients (11.6%) and the severe form of macular edema in 10 patients (8.9%).

In the 62 patients with diabetic retinopathy 17 (27.42%) developed overt nephropathy, with a rate of progression 4.11 person-year. In patients with proliferative form of diabetic retinopathy (23 patients) 11 developed microalbuminuria (47.82%), the rate of progression was 7.17 person-year.

#### *Statistical study of diabetic retinopathy*

Univariate study with the application of chi squared test (Table 1).

The factors significant in the appearance of diabetic retinopathy were as follows: duration of diabetic retinopathy p<0.001, presence of arterial hypertension p<0.001, levels of glycated haemoglobin (HbA1c) > 7.5% p<0.001, high levels of LDL-cholesterol p<0.001, high levels of tryglicerides p=0.003 and presence of overt nephropathy p= 0.001.

Logistic regression of diabetic retinopathy (Table 2).

The followings factors studied were significant in the appearance of diabetic retinopathy: Duration of diabetes mellitus more than 20 years p< 0.001, presence of arterial hypertension p<0.001, high levels of HbA1c p< 0.001, high levels of tryglicerides p=0.004, high levels of LDL-Cholesterol p= 0.002, and overt nephropathy p=0.021.

#### *Statistical study of diabetic nephropathy*

Univariate analysis with the application of chi squared test(Table 1).

The factors significant in the apparition of overt nephropathy were: presence of arterial hypertension p<0.001, high levels of HbA1c p<0.001, high levels of LDL-Cholesterol p=0.010, high levels of triglycerides p=0.003, and presence of diabetic retinopathy p=0.021. When we introduced the presence of proliferative diabetic retinopathy against the presence of any retinopathy, the chi squared test had a result of p<0.001, and for proliferative diabetic retinopathy p< 0.001.

#### *Logistic regression of diabetic nephropathy (Table 2).*

286 Type 1 Diabetes Complications

Mean visual acuity after 17 years was 0.77 0.34 ( 0,02 – 1) in the Snellen chart test; and

Low vision (defined as vision in the best eye >0,1 and < 0.4 in the Snellen chart) was detected in 13 patients (11.6%) and blindness (AV < 0.1 in Snellen chart) in 14 patients

After 17 years there were 62 patients (55.4%) with different types of diabetic retinopathy.

There were 23 patients (20.5%) with diabetic macular edema after 17 years, the Rate of progression was 3.08 person-year. The mild or moderate form of diabetic macular edema was present in 13 patients (11.6%) and the severe form of macular edema in 10 patients

In the 62 patients with diabetic retinopathy 17 (27.42%) developed overt nephropathy, with a rate of progression 4.11 person-year. In patients with proliferative form of diabetic retinopathy (23 patients) 11 developed microalbuminuria (47.82%), the rate of progression

The factors significant in the appearance of diabetic retinopathy were as follows: duration of diabetic retinopathy p<0.001, presence of arterial hypertension p<0.001, levels of glycated haemoglobin (HbA1c) > 7.5% p<0.001, high levels of LDL-cholesterol p<0.001, high levels of

The followings factors studied were significant in the appearance of diabetic retinopathy: Duration of diabetes mellitus more than 20 years p< 0.001, presence of arterial hypertension p<0.001, high levels of HbA1c p< 0.001, high levels of tryglicerides p=0.004, high levels of

The factors significant in the apparition of overt nephropathy were: presence of arterial hypertension p<0.001, high levels of HbA1c p<0.001, high levels of LDL-Cholesterol p=0.010, high levels of triglycerides p=0.003, and presence of diabetic retinopathy p=0.021. When we introduced the presence of proliferative diabetic retinopathy against the presence of any retinopathy, the chi squared test had a result of p<0.001, and for proliferative diabetic

 HDL-Cholesterol: 1.12 0,42 mmol/l (0.70 – 2.02) Triglycerides: 1.47 0.76 mmol/l (0.90 – 3.00)

+0.37 +0.72 (+ 1.7 - + 0) in the Log MAR test.

The Rate of progression was 8.30 person-year. Mild diabetic retinopathy in31 patients (27.7%) Moderate diabetic retinopathy in 7 patients (6.3%) Severe diabetic retinopathy in 5 patients (4.5%)

Proliferative diabetic retinopathy in 18 patients (16.1%)

Univariate study with the application of chi squared test (Table 1).

tryglicerides p=0.003 and presence of overt nephropathy p= 0.001.

Univariate analysis with the application of chi squared test(Table 1).

Logistic regression of diabetic retinopathy (Table 2).

LDL-Cholesterol p= 0.002, and overt nephropathy p=0.021.

*Incidence of diabetic retinopathy (Table 1)* 

Visual acuity study

(12.5%).

(8.9%).

was 7.17 person-year.

*Statistical study of diabetic retinopathy* 

*Statistical study of diabetic nephropathy* 

retinopathy p< 0.001.

The significant factors were: the presence of arterial hypertension p<0.001, and high levels of HbA1c p<0.001, high levels of LDL-Cholesterol p=0.002, high levels of triglycerides p=0.009,. Also the presence of diabetic retinopathy was significant p=0,021. When we introduced the presence of proliferative diabetic retinopathy against the presence of any retinopathy, the chi squared test had a result of p<0.001


Table 1. Chi squared and logistic regression analysis for diabetic retinopathy and microalbuminuria.

Review of the Relationship Between

proteinuria (1%-2% annually).

that influence any of these groups.

were more important than the duration of diabetes mellitus.

diabetic retinopathy and overt nephropathy.

high levels of HbA1c.

Renal and Retinal Microangiopathy in Type 1 Diabetes Mellitus Patients 289

increases the prevalence, since this point to a levels upper 80% after 20 years diabetes duration, without a decreases after these 20 years of duration, as we observed in our study. The incidence of DR was 55.4%, and was lower than in other studies such as Klein et al 1998 (Klein et al, 1998), but this may be because the sample studied did not present diabetic retinopathy at the beginning and patient controls were stricter than in the rest of the patients with diabetes mellitus type I (controls every 3 months), we could concluded that the mean level of HbA1c was 7.69% 1.24 (4.50% – 11.40%) is better than the achieved by WESDR (Klein et al, 1998). The diabetic macular edema appeared in 20.5% of patients, which was more similar to the findings in the Klein study at 14 years (26%). In the present study the incidence of diabetic macular edema is higher than the proliferative form of diabetic

With regard to nephropathy diabetes accountings for more than 19.6% of all cases of DN. The DN presents initially as intermittent microalbuminuria that progresses to persistent microalbuminuria, and is accompanied by a decline in the glomerular filtration rate. These dates were in agreement with other studies published in our country on renal failure in diabetes mellitus type I patients Smatjes et al (Esmatjes et al, 1998) found an incidence of

The relationship between DN and DR was well described, thus the Wisconsin Epidemiologic Study of Diabetic Retinopathy (Klein et al, 1993) associated the presence of gross-proteinuria at baseline examination with a 96% increase in the risk of progression to proliferative retinopathy. Also in the Steno study (Kofoed-Enevoldsen et al, 1987), people with type I diabetes mellitus and gross-proteinuria at baseline had an increase risk of progression to proliferative retinopathy (12% annually) compared to those without

At the end of our study we can see that four groups of patients had formed: those without overt nephropathy or diabetic retinopathy (45 patients), patients with only overt nephropathy (5 patients), those with only diabetic retinopathy (45 patients), and those with overt nephropathy and diabetic retinopathy (17 patients). The statistical test used for examining these data was a discriminate test, which allowed us to identify the risk factors

In the group of patients with only DR the duration of diabetes mellitus was the more important risk factor and for the group with Dr and DN the most important risk factor is the

We may assume then, that for the development of only retinal lesions in diabetes mellitus, the duration of the disease is the most important followed by and in a second level of importance the levels of HbA1c and arterial hypertension; and for the development of renal and retinal lesion simultaneously poor control of glycaemia measured by levels of HbA1c

Two broader groups of patients can be assumed to have been formed in this study, the first being those patients who developed only diabetic retinopathy, and the second those with both diabetic retinopathy and renal lesion (overt nephropathy). This conclusion is consistent with previous studies as that of Lövestam-Adrian in 1998 (Lövestam-Adrian, et al, 1998), in which after a 10-year follow-up of a population of 24 patients, with proliferative diabetic retinopathy at the beginning of the study, only two developed microalbuminuria, That study concluded that there are, at least partly, different pathogenic mechanisms behind

retinopathy in type I diabetes mellitus patients, as was observed in other studies.

44.5% with some form of renal failure at 20 years in type I diabetes mellitus.


\* = Function of classification coefficients.

Table 2. Fisher's classification coefficient.

*Statistical application of discriminate analysis* 

At the end of the study we may observe that four groups of patients had been formed:


In this case we needed to apply a discriminate analysis to evaluate the risk factors for the different groups.

Applying Fisher's coefficient indicated that (Table 2):


#### **4. Discussion**

The diabetic retinopathy (DR) and diabetic nephropathy (DN) are the two major complications of diabetes mellitus. The proliferative diabetic retinopathy and proteinuria secondary to DN are both late complications of diabetic overt nephropathy, these usually occur 10 to 15 years after the onset of type 1 DM, and are strongly associated with each other. The epidemiology of diabetic overt nephropathy and retinopathy in type 1 DM, are different, thus for diabetic overt nephropathy increases its prevalence since 10% at ten year duration of diabetes mellitus, achieving the highest value after 40 years with a 40% of diabetic patients with nephropathy, since this point the curve levels off, and only a minority of patients develop clinically significant renal abnormalities, and patients who survive 35 years of type 1 DM without developing DN are at extremely low risk of doing so in the future. Against this curve the diabetic retinopathy succeed in a different form in type 1 diabetic patients, thus the diabetic retinopathy is rare before 10 years DM duration, and

Patients with retinopathy and overt nephropathy

High levels of triglycerides (2.810)\*

Duration of diabetes mellitus (2.269)\*

High levels of LDL-cholesterol (1.232)\*

(7.250)\* High levels of HbA1c (6.471)\*

mellitus (1.029)\* Arterial hypertension (2.657)\*

Patients only with overt nephropathy

High levels of HbA1c

High levels of triglycerides (1.713)\*

Duration of diabetes

Arterial hypertension (0.742)\*

High levels of LDLcholesterol (-1.360)\*

At the end of the study we may observe that four groups of patients had been formed: those without any form of microangiopathy (overt nephropathy or retinopathy) 45

In this case we needed to apply a discriminate analysis to evaluate the risk factors for the

for group B the risk factors were: duration of diabetes mellitus (4.679), high levels of

 for the group D the significant factors were: high levels of HbA1c (6,471), presence of arterial hypertension (2.657), high levels of triglycerides (2.810) and duration of diabetes

The diabetic retinopathy (DR) and diabetic nephropathy (DN) are the two major complications of diabetes mellitus. The proliferative diabetic retinopathy and proteinuria secondary to DN are both late complications of diabetic overt nephropathy, these usually occur 10 to 15 years after the onset of type 1 DM, and are strongly associated with each other. The epidemiology of diabetic overt nephropathy and retinopathy in type 1 DM, are different, thus for diabetic overt nephropathy increases its prevalence since 10% at ten year duration of diabetes mellitus, achieving the highest value after 40 years with a 40% of diabetic patients with nephropathy, since this point the curve levels off, and only a minority of patients develop clinically significant renal abnormalities, and patients who survive 35 years of type 1 DM without developing DN are at extremely low risk of doing so in the future. Against this curve the diabetic retinopathy succeed in a different form in type 1 diabetic patients, thus the diabetic retinopathy is rare before 10 years DM duration, and

those with both overt nephropathy and retinopathy (17 patients) (group D).

for the group C only the high levels of HbA1c (7,250) were highly correlated

Patients only with retinopathy

Duration of diabetes mellitus (4.679)\*

High levels of HbA1c (2.250)\*

Arterial hypertension (2.668)\*

High levels of LDLcholesterol (1.277)\*

High levels of triglycerides (1.254)\*

patients (group A).

different groups.

mellitus (2.269)

**4. Discussion** 

\* = Function of classification coefficients. Table 2. Fisher's classification coefficient. *Statistical application of discriminate analysis* 

 those with only retinopathy (45 patients) (group B). those with only overt nephropathy (5 patients) (group C).

Applying Fisher's coefficient indicated that (Table 2):

HbA1c (2.250) and high levels of LDL-Cholesterol (2.268)

increases the prevalence, since this point to a levels upper 80% after 20 years diabetes duration, without a decreases after these 20 years of duration, as we observed in our study.

The incidence of DR was 55.4%, and was lower than in other studies such as Klein et al 1998 (Klein et al, 1998), but this may be because the sample studied did not present diabetic retinopathy at the beginning and patient controls were stricter than in the rest of the patients with diabetes mellitus type I (controls every 3 months), we could concluded that the mean level of HbA1c was 7.69% 1.24 (4.50% – 11.40%) is better than the achieved by WESDR (Klein et al, 1998). The diabetic macular edema appeared in 20.5% of patients, which was more similar to the findings in the Klein study at 14 years (26%). In the present study the incidence of diabetic macular edema is higher than the proliferative form of diabetic retinopathy in type I diabetes mellitus patients, as was observed in other studies.

With regard to nephropathy diabetes accountings for more than 19.6% of all cases of DN. The DN presents initially as intermittent microalbuminuria that progresses to persistent microalbuminuria, and is accompanied by a decline in the glomerular filtration rate. These dates were in agreement with other studies published in our country on renal failure in diabetes mellitus type I patients Smatjes et al (Esmatjes et al, 1998) found an incidence of 44.5% with some form of renal failure at 20 years in type I diabetes mellitus.

The relationship between DN and DR was well described, thus the Wisconsin Epidemiologic Study of Diabetic Retinopathy (Klein et al, 1993) associated the presence of gross-proteinuria at baseline examination with a 96% increase in the risk of progression to proliferative retinopathy. Also in the Steno study (Kofoed-Enevoldsen et al, 1987), people with type I diabetes mellitus and gross-proteinuria at baseline had an increase risk of progression to proliferative retinopathy (12% annually) compared to those without proteinuria (1%-2% annually).

At the end of our study we can see that four groups of patients had formed: those without overt nephropathy or diabetic retinopathy (45 patients), patients with only overt nephropathy (5 patients), those with only diabetic retinopathy (45 patients), and those with overt nephropathy and diabetic retinopathy (17 patients). The statistical test used for examining these data was a discriminate test, which allowed us to identify the risk factors that influence any of these groups.

In the group of patients with only DR the duration of diabetes mellitus was the more important risk factor and for the group with Dr and DN the most important risk factor is the high levels of HbA1c.

We may assume then, that for the development of only retinal lesions in diabetes mellitus, the duration of the disease is the most important followed by and in a second level of importance the levels of HbA1c and arterial hypertension; and for the development of renal and retinal lesion simultaneously poor control of glycaemia measured by levels of HbA1c were more important than the duration of diabetes mellitus.

Two broader groups of patients can be assumed to have been formed in this study, the first being those patients who developed only diabetic retinopathy, and the second those with both diabetic retinopathy and renal lesion (overt nephropathy). This conclusion is consistent with previous studies as that of Lövestam-Adrian in 1998 (Lövestam-Adrian, et al, 1998), in which after a 10-year follow-up of a population of 24 patients, with proliferative diabetic retinopathy at the beginning of the study, only two developed microalbuminuria, That study concluded that there are, at least partly, different pathogenic mechanisms behind diabetic retinopathy and overt nephropathy.

Review of the Relationship Between

Atlanta GA.

0326

0326

ISSN 1523-1755

341:1127-33. ISSN 1533-4406

684. ISSN 0181-5512

1801-1815. ISSN 0161-6420

Renal and Retinal Microangiopathy in Type 1 Diabetes Mellitus Patients 291

Geiss L, Engelgau M, Fraizer E & Tierney E (1997). Diabetes surveillance, 1997. Centers for

Klein R, Klein BEK, Moss SE & Cruickshanks KJ (1998). The 14-year incidence and

Klein R, Klein BEK, Moss SE, Davis MD & DeMets DL (1984). The Wisconsin Epidemiologic

Klein R, Klein BEK, Moss SE, Davis MD & DeMets DL (1984). The Wisconsin Epidemiologic

Klein R, Moss SE & Klein BEK (1993). Is gross proteinuria a risk factor for the incidence of proliferative diabetic retinopathy?. Ophthalmology, 100: 1140-1146 ISSN0161-6420 Kofoed-Enevoldsen A, Jensen T, Borch-Johnsen K & Deckert T (1987). Incidence of

Looker HC, Krakoff J, Knowler WC, Bennett PH, Klein R & Hanson RL (2003). Longitudinal

Myers BD, Winetz JA, Chui F, Michaels AS. Mechanism of proteinuria in diabetic

Otani T, Kishi S & Maruyana Y (1999). Patterns of diabetic macular edema with optical coherence tomography. Am J Ophthalmol , 127, 688-693. ISSN 0002- 939 Ritz E, Orth SR. Nephropathy in patients with type2 diabetes mellitus. N Eng J Med 1999;

Romero-Aroca P, Espeso-Sentis O, Sarda-Aure P & del Castillo-Dejardin D (2000).

Romero-Aroca P, Salvat-Serra M, Mendez-Marin I & Martinez-Salcedo I (2003). Is

Rossing P, Hougaard P & Parving HH (2002). Risk factors for the development of incipient

observational study. Diabetes Care 2002;25:859-864. ISSN 0149-5992 The Expert Comitee on the Diagnosis and Classification of Diabetes mellitus (2000). Report

photography in Pima Indians. Diabetes Care;26:320-326. ISSN 0149-5992 Lövestam-Adrian M, Agardh E & Agardh CD (1998). The incidence of nephropathy in type

nephropathy. J Diabet Complications,1, 96-99. ISSN 1056-8727

Diabetes Res Clin Pract.;39(1):11-17. ISSN 0168-8227

mellitus. Rev Clin Esp. 200,351-354. ISSN 0014-2565

Diabetes Care; 23: S4-S19 ISSN 0149-5992

Disease Control and Prevention. U.S. Departement of Health and Human Services

progression of diabetic retinopathy an associate risk factors in type I diabetes. The Wisconsin Epidemiologic Study of Diabetic Retinopathy XVII. Ophthalmology, 105,

Study of Diabetic Retinopathy II: prevalence and risk of diabetic retinopathy when age at diagnosis is less than 30 years. Arch Ophthalmology;102:520-526. ISSN 0093-

Study of Diabetic Retinopathy II: prevalence and risk of diabetic retinopathy when age at diagnosis is 30 or more years. Arch Ophthalmology;102:527-32. ISSN 0093-

retinopathy in type I (insulin-dependent) diabetes: association with clinical

studies of incidence and progression of diabetic retinopathy assessed by retinal

1 diabetic patients with prol iferative retinopathy: a 10-year follow-up study.

nephropathy: A study of glomerular barrier functions. Kidney Int 1982;21:633-41

Relationship between microalbuminuria and diabetic retinopathy in type I diabetes

microalbuminuria a risk factor for diabetic retinopathy?. J Fr Ophtalmol, 26, 7, 680-

and overt diabetic nephropathy in type 1 diabetic patients: A 10 year prospective

of the Expert Committee on the Diagnosis and classification of Diabetes Mellitus.

#### **5. Conclusion**

Despite there being a poor relationship between overt nephropathy and diabetic retinopathy (p=0.021 in the present study), the presence of overt nephropathy correlated well with severe forms of diabetic retinopathy (as proliferative Dr p<0.001 in the present study); and in addition, at the end of study two broad group of patients had been configured, the first those who developed only diabetic retinopathy, and the second with diabetic retinopathy and renal lesion (overt nephropathy). For the first group with only DR , duration of diabetes mellitus is the most important risk factor, and for the second group (patients with DR and DN) the levels of HbA1c and blood pressure are the most important.

#### **6. References**


Despite there being a poor relationship between overt nephropathy and diabetic retinopathy (p=0.021 in the present study), the presence of overt nephropathy correlated well with severe forms of diabetic retinopathy (as proliferative Dr p<0.001 in the present study); and in addition, at the end of study two broad group of patients had been configured, the first those who developed only diabetic retinopathy, and the second with diabetic retinopathy and renal lesion (overt nephropathy). For the first group with only DR , duration of diabetes mellitus is the most important risk factor, and for the second group (patients with

Adler AI, Stevens RJ, Manley SE, Bilous RW, Cull CA & Holman RR (2003). UKPDS Group.

Aldington SJ, Kohner EM, Meuer S, Klein R & Sjolie AK (1995). Methodology for retinal

American Diabetes Association (1997). Report of the Expert Committee on the diagnosis and classification of diabetes mellitus. Diabetes Care; 20:1183-1201. ISSN 0149-5992 Castell C, Tresserras R, Lloveras G, Goday A, Serra J & Salleras L (1999). Prevalence of

El-Asrar AM, Al Rubeaan KA, Al-Amor SA, Moharram OA & Kangave D (2000).

Esmatjes E, Castell C, Goday A, Montanya E, Pou JM & cols (1998). Prevalence of

Estacio RO, McFarling E, Biggerstaff S, Jeffers BW, Johnson D & Schrier RW (1998). Overt

European Diabetes Policy Group (1999). A Desktop guide to type I (insulin-dependent)

Expert Panel on Detection, Evaluation, And Treatment of high Blood Cholesterol in Adults

Gall MA, Hougaard P, Borch-Johnsen K & Parving HH (1997). Risk factors for development

complications study. Diabetologia, 38 , 437-444 ISSN 0012-186X

Development and progression of nephropathy in type2 diabetes. The United Kingdom Prospective Diabetes Study (UKPDS 64). Kidney Int; 63:225-232. ISSN

photography and assessment of diabetic retinopathy , The EURODIAB IDDM

diabetes in Catalonia, an OGTT-based population study. Diab Res Clin Prac, 43, 33-

Retinopathy as a predictor of other diabetic complications. International

nephropathy in type I diabetes Med Clin (Barc). 1998 17; 110(1):6-10. ISSN 0025-

albuminuria predicts diabetic retinopathy in Hispanics with NIDDM. Am J of

diabetes mellitus. 1998-199. Guidelines for Diabetes care. Diabetic Med, 16, 253-266.

(Adult Treatment Panel III) (2001). Executive summary of the third report of the National Cholesterol Education Program (NCEP). JAMA, 285, 2486-2497 ISSN

of incipient and overt diabetic nephropathy in patients with non-insulin dependent diabetes mellitus: prospective, observational study. BMJ;314:783-789. ISSN 0959-

DR and DN) the levels of HbA1c and blood pressure are the most important.

**5. Conclusion** 

**6. References** 

0085-2538

40 ISSN 0168-8227

ISSN 1464-5491

0098-7484

8138

7753

Ophthalmology;24:1-11. ISSN 0161-6420

Kidney Disease; 31:947-953. ISSN 0272-6386


**14** 

 *Israel* 

**Ocular Complications of Type 1 Diabetes** 

*Ophthalmology Department, Kaplan Medical Center, POB 1, Rehovot,* 

Daniel Rappoport, Yoel Greenwald, Ayala Pollack and Guy Kleinmann

Type 1 diabetes is a complex metabolic disease involving multiple organ systems which may cause severe visual impairment. Almost all parts of the eye may be affected including:

Diabetes is the leading cause of blindness between the ages of 20 and 74 in many developed countries (Cheung et al., 2010; Powers, 2008). Individuals with diabetes are 25 times more likely to become legally blind than individuals without diabetes. Blindness is primarily the result of diabetic retinopathy that accounts for ¼ of blind registrations in the western world

Prevention of severe visual impairment in type 1 diabetes includes: optimal glycemic control, the treatment of ancillary risk factors such as hypertension, regular ophthalmic

In the following chapter we will describe the ocular complications of diabetes and the

Diabetics may present with a sudden onset of diplopia (double vision). This is usually caused by the partial or complete paralysis of one of the extra-ocular muscles due to microvascular damage to the third, fourth or sixth cranial nerve (Thomas & Graham, 2008;

When a third cranial nerve palsy occurs, it is important to differentiate between a diabetic nerve palsy and paresis due to compression of the nerve from an aneurysm at the junction of the posterior communicating and internal carotid arteries. In diabetic nerve palsy the pupil is often spared meaning that it continues to react to light appropriately despite damage to the motor capabilities of the third cranial nerve. Typically in cranial nerve palsy due to a space occupying lesion such as an aneurysm, the pupil in the affected eye is dilated. In 20% of patients with diabetic nerve palsy there may be pupil involvement, however this is usually a mild efferent defect (Kline et al. 2010). Aneurysms are rare in children but may be present in adolescents, so ruling it out, by neuroimaging, is crucial. Pain may also be present in diabetic third nerve palsy. When there is paresis of the fourth or sixth cranial nerve, referral to a neuro-ophthalmolgist is also recommended for follow-up and to exclude other causes, such as myasthenia gravis

the extra-ocular muscles, intra-ocular lens, the optic nerve, and retina.

screening, and early diagnosis and treatment of ocular complications.

**1. Introduction** 

(Cheung et al., 2010; Powers, 2008).

treatments for these conditions.

**2. Extra-ocular muscles** 

Kline et al. 2010).

or brain lesions.


### **Ocular Complications of Type 1 Diabetes**

Daniel Rappoport, Yoel Greenwald, Ayala Pollack and Guy Kleinmann *Ophthalmology Department, Kaplan Medical Center, POB 1, Rehovot, Israel* 

#### **1. Introduction**

292 Type 1 Diabetes Complications

Villar G, Gracia Y, Goicolea I & Barquees J (1999). Determinants of development of

Wilkinson CP, Ferris FL, Klein RE, Lee PP, Agardh CD, Davis M, Dills D, Kampic A,

Williams R, Airey M, Baxter H, Forrester J, Kennedy-Martin T & Giarach A (2004).

Diabetes and Metabolism; 25:246-254. ISSN 1520-7560

ISSN 0161-6420

Eye; 18:963-983. ISSN 0950-222X

microalbuminuria in normotensive patients with type 1 and type 2 diabetes.

Pararajasegaram R & Verdaguer JT, representing the Global Diabetic Retinopathy Project Group (2003). Proposed international clinical diabetic retinopathy and diabetic macular edema disease severity scales. Ophthalmology, 110, 1677-1682

Epidemiology of diabetic retinopathy and macular edema: a systematic review.

Type 1 diabetes is a complex metabolic disease involving multiple organ systems which may cause severe visual impairment. Almost all parts of the eye may be affected including: the extra-ocular muscles, intra-ocular lens, the optic nerve, and retina.

Diabetes is the leading cause of blindness between the ages of 20 and 74 in many developed countries (Cheung et al., 2010; Powers, 2008). Individuals with diabetes are 25 times more likely to become legally blind than individuals without diabetes. Blindness is primarily the result of diabetic retinopathy that accounts for ¼ of blind registrations in the western world (Cheung et al., 2010; Powers, 2008).

Prevention of severe visual impairment in type 1 diabetes includes: optimal glycemic control, the treatment of ancillary risk factors such as hypertension, regular ophthalmic screening, and early diagnosis and treatment of ocular complications.

In the following chapter we will describe the ocular complications of diabetes and the treatments for these conditions.

#### **2. Extra-ocular muscles**

Diabetics may present with a sudden onset of diplopia (double vision). This is usually caused by the partial or complete paralysis of one of the extra-ocular muscles due to microvascular damage to the third, fourth or sixth cranial nerve (Thomas & Graham, 2008; Kline et al. 2010).

When a third cranial nerve palsy occurs, it is important to differentiate between a diabetic nerve palsy and paresis due to compression of the nerve from an aneurysm at the junction of the posterior communicating and internal carotid arteries. In diabetic nerve palsy the pupil is often spared meaning that it continues to react to light appropriately despite damage to the motor capabilities of the third cranial nerve. Typically in cranial nerve palsy due to a space occupying lesion such as an aneurysm, the pupil in the affected eye is dilated. In 20% of patients with diabetic nerve palsy there may be pupil involvement, however this is usually a mild efferent defect (Kline et al. 2010). Aneurysms are rare in children but may be present in adolescents, so ruling it out, by neuroimaging, is crucial. Pain may also be present in diabetic third nerve palsy. When there is paresis of the fourth or sixth cranial nerve, referral to a neuro-ophthalmolgist is also recommended for follow-up and to exclude other causes, such as myasthenia gravis or brain lesions.

Ocular Complications of Type 1 Diabetes 295

oxidative stress to the lens (Obrosova et al., 2010). This causes diabetic patients to develop age related lens changes similar to non-diabetic age related cataracts, except at a younger age than non-diabetics (Bobrow et al. 2010). Several studies have analyzed the effect of vitamin and anti-oxidant supplements, such as vitamin C, E and beta carotene and zinc on preventing or slowing progression of age related cataracts in diabetes without showing any statistically significant benefit with their use (AREDS report no. 9, 2001 as cited in Obrosova

A rarer form of cataract in diabetics that is seldom encountered in clinical practice today is called the 'true diabetic cataract'. This is typically seen in young patients with uncontrolled diabetes. Any rapidly maturing (i.e whitening) cataract in a child or a young adult should

Cataract surgery is indicated when visual function is significantly impaired by the cataract. Surgery is also indicated if the cataract obscures the view of the retina and makes the diagnosis and treatment of diabetic retinopathy difficult. Cataract surgery is safe in diabetic patients and there is a 95% success rate in terms of improved visual acuity (Obrosova et al., 2010). Good glycemic control, fluid and electrolyte balance should be maintained perioperatively and the patient's primary care physician and anesthesiologist should be involved. It is recommended that the surgery be scheduled in the morning to minimize changes in the patient's usual schedule (Purdy et al., 2010). Some controversy exists regarding a potential association between cataract surgery and a subsequent worsening of diabetic retinopathy. Patients should be made aware of this risk pre-operatively. Cataract surgery and its effect on diabetic retinopathy will be discussed in more detail the diabetic

Structural changes to the corneal basement membrane in diabetes decrease the adhesion of corneal epithelial cells to the deeper stromal tissue. This increases the risk of recurrent corneal erosions (Reidy et al., 2010). In addition, accumulation of sorbitol in the cornea during periods of hyperglycemia leads to hypoesthesia (a loss of corneal sensation). Both hypoesthesia and epithelial adhesion dysfunction occur more frequently with increased severity and duration of diabetes. In these patients, any epithelial injury, either from trauma, during ocular surgery or from routine contact lens use, may result in prolonged healing times. This increases the risk of severe complications such as bacterial infiltration and

Bilateral tonic pupils may be seen in diabetic patients (Kline et al., 2010). This manifests with sluggish, segmented pupillary reactions to light and better response to near effort, followed by slow redilation of the pupil. Tonic pupils are caused by microvascular damage to postganglionic parasympathetic pupillomotor nerve fibers. Diminished pupillary response is also seen due to glycogen infiltration of the pigment epithelium and sphincter and dilator

Rubeosis iridis, neovascularization in the iris, is a serious complication of diabetes which occurs in patients with severe diabetic retinopathy (Thomas & Graham, 2008). Growth factors released from the ischemic retina induce the development of intertwining blood

et al., 2010).

raise awareness to the possibility of diabetes.

retinopathy section (7.6.2).

**4. Cornea** 

ulceration.

**5. Iris and pupil** 

muscles (Thomas & Graham, 2008).

When the oculomotor defect is due to microvascular complications of diabetes the prognosis is good.

Recovery of ocular motor function generally begins within three months of onset and recovery is usually complete. Although the diplopia can be debilitating, due to the generally limited course of these complaints, patients can usually be effectively managed conservatively with eye patching. Surgery is rarely indicated.

#### **3. Lens: Cataract and refractive changes**

Hyperglycemia can reduce lens clarity. Hyperglycemia also induces changes in the refractive index and accommodative amplitude of the lens, both of which also act to reduce visual function (Flynn & Smiddy, 2000).

#### **3.1 Refractive and accommodative changes**

One of the most frequently encountered ocular manifestations of diabetes is abrupt changes in the refractive power of the lens. When the blood glucose level is high, the glucose concentration in the aqueous humor, the fluid surrounding the lens, increases as well. This causes the glucose concentration in the lens to increase by diffusion (Flynn & Smiddy, 2000). Under normal conditions glucose is metabolized inside the lens by glycolysis. However, when the glucose level in the lens is very high, glycolysis enzymes are overridden and some glucose is reduced by the enzyme aldose reductase and converted to sorbitol (Stirban et al., 2008). Sorbitol is metabolized slowly by the lens cells and accumulates, increasing the osmotic pressure inside the lens. This increased osmotic pressure leads to an influx of water from the aqueous humor and the lens swells. This larger lens is more convex and therefore more powerful at bending incoming light which alters the focal point of the eye, causing acute nearsightedness (myopia) and blurred distance vision.

These refractive changes may be up to three to four diopters and may last for several weeks. Thus, patients with poorly controlled blood glucose levels experience transient refractive changes due to fluctuating levels of glucose in the blood. Acute blurring of vision may be the first symptom of undiagnosed or poorly controlled diabetes.

Accommodation, the ability to adjust focus for near tasks such as reading, is also affected in patients with diabetes. Studies have shown that diabetics have decreased amplitude of accommodation compared to age matched controls, and require spectacle correction for near work at a younger age than non-diabetics (Flynn & Smiddy, 2000).

#### **3.2 Cataract**

Cataract is a common cause of visual impairment in patients with diabetes. Epidemiological studies have revealed an up to five-fold increased prevalence of cataracts in diabetic patients. Individuals with type 1 diabetes manifested a greater prevalence of cataracts between the ages of 18 to 44 than age-matched controls (Obrosova et al., 2010). Duration of diabetes and quality of glycemic control are the major risk factors for early cataract development.

Potential mechanisms of diabetic cataract formation include accumulation of lenticular sorbitol, as described in the previous section (3.1). This reduces lens clarity leading to early cataract formation. It has also been postulated that recurrent high levels of glucose in the lens lead to the glycolation of lens proteins from increased non-enzymatic glycation and

When the oculomotor defect is due to microvascular complications of diabetes the prognosis

Recovery of ocular motor function generally begins within three months of onset and recovery is usually complete. Although the diplopia can be debilitating, due to the generally limited course of these complaints, patients can usually be effectively managed

Hyperglycemia can reduce lens clarity. Hyperglycemia also induces changes in the refractive index and accommodative amplitude of the lens, both of which also act to reduce

One of the most frequently encountered ocular manifestations of diabetes is abrupt changes in the refractive power of the lens. When the blood glucose level is high, the glucose concentration in the aqueous humor, the fluid surrounding the lens, increases as well. This causes the glucose concentration in the lens to increase by diffusion (Flynn & Smiddy, 2000). Under normal conditions glucose is metabolized inside the lens by glycolysis. However, when the glucose level in the lens is very high, glycolysis enzymes are overridden and some glucose is reduced by the enzyme aldose reductase and converted to sorbitol (Stirban et al., 2008). Sorbitol is metabolized slowly by the lens cells and accumulates, increasing the osmotic pressure inside the lens. This increased osmotic pressure leads to an influx of water from the aqueous humor and the lens swells. This larger lens is more convex and therefore more powerful at bending incoming light which alters the focal point of the eye, causing

These refractive changes may be up to three to four diopters and may last for several weeks. Thus, patients with poorly controlled blood glucose levels experience transient refractive changes due to fluctuating levels of glucose in the blood. Acute blurring of vision may be

Accommodation, the ability to adjust focus for near tasks such as reading, is also affected in patients with diabetes. Studies have shown that diabetics have decreased amplitude of accommodation compared to age matched controls, and require spectacle correction for near

Cataract is a common cause of visual impairment in patients with diabetes. Epidemiological studies have revealed an up to five-fold increased prevalence of cataracts in diabetic patients. Individuals with type 1 diabetes manifested a greater prevalence of cataracts between the ages of 18 to 44 than age-matched controls (Obrosova et al., 2010). Duration of diabetes and quality of glycemic control are the major risk factors for early cataract

Potential mechanisms of diabetic cataract formation include accumulation of lenticular sorbitol, as described in the previous section (3.1). This reduces lens clarity leading to early cataract formation. It has also been postulated that recurrent high levels of glucose in the lens lead to the glycolation of lens proteins from increased non-enzymatic glycation and

conservatively with eye patching. Surgery is rarely indicated.

acute nearsightedness (myopia) and blurred distance vision.

the first symptom of undiagnosed or poorly controlled diabetes.

work at a younger age than non-diabetics (Flynn & Smiddy, 2000).

**3. Lens: Cataract and refractive changes** 

**3.1 Refractive and accommodative changes** 

visual function (Flynn & Smiddy, 2000).

is good.

**3.2 Cataract** 

development.

oxidative stress to the lens (Obrosova et al., 2010). This causes diabetic patients to develop age related lens changes similar to non-diabetic age related cataracts, except at a younger age than non-diabetics (Bobrow et al. 2010). Several studies have analyzed the effect of vitamin and anti-oxidant supplements, such as vitamin C, E and beta carotene and zinc on preventing or slowing progression of age related cataracts in diabetes without showing any statistically significant benefit with their use (AREDS report no. 9, 2001 as cited in Obrosova et al., 2010).

A rarer form of cataract in diabetics that is seldom encountered in clinical practice today is called the 'true diabetic cataract'. This is typically seen in young patients with uncontrolled diabetes. Any rapidly maturing (i.e whitening) cataract in a child or a young adult should raise awareness to the possibility of diabetes.

Cataract surgery is indicated when visual function is significantly impaired by the cataract. Surgery is also indicated if the cataract obscures the view of the retina and makes the diagnosis and treatment of diabetic retinopathy difficult. Cataract surgery is safe in diabetic patients and there is a 95% success rate in terms of improved visual acuity (Obrosova et al., 2010). Good glycemic control, fluid and electrolyte balance should be maintained perioperatively and the patient's primary care physician and anesthesiologist should be involved. It is recommended that the surgery be scheduled in the morning to minimize changes in the patient's usual schedule (Purdy et al., 2010). Some controversy exists regarding a potential association between cataract surgery and a subsequent worsening of diabetic retinopathy. Patients should be made aware of this risk pre-operatively. Cataract surgery and its effect on diabetic retinopathy will be discussed in more detail the diabetic retinopathy section (7.6.2).

#### **4. Cornea**

Structural changes to the corneal basement membrane in diabetes decrease the adhesion of corneal epithelial cells to the deeper stromal tissue. This increases the risk of recurrent corneal erosions (Reidy et al., 2010). In addition, accumulation of sorbitol in the cornea during periods of hyperglycemia leads to hypoesthesia (a loss of corneal sensation). Both hypoesthesia and epithelial adhesion dysfunction occur more frequently with increased severity and duration of diabetes. In these patients, any epithelial injury, either from trauma, during ocular surgery or from routine contact lens use, may result in prolonged healing times. This increases the risk of severe complications such as bacterial infiltration and ulceration.

#### **5. Iris and pupil**

Bilateral tonic pupils may be seen in diabetic patients (Kline et al., 2010). This manifests with sluggish, segmented pupillary reactions to light and better response to near effort, followed by slow redilation of the pupil. Tonic pupils are caused by microvascular damage to postganglionic parasympathetic pupillomotor nerve fibers. Diminished pupillary response is also seen due to glycogen infiltration of the pigment epithelium and sphincter and dilator muscles (Thomas & Graham, 2008).

Rubeosis iridis, neovascularization in the iris, is a serious complication of diabetes which occurs in patients with severe diabetic retinopathy (Thomas & Graham, 2008). Growth factors released from the ischemic retina induce the development of intertwining blood

Ocular Complications of Type 1 Diabetes 297

Fig. 2. Diabetic Papillopathy. A chronic swelling of the optic disc often associated with mild visual impairment. This color fundus photograph shows blurred disc margins (white arrows), flame shaped hemorrhages (black arrowhead). The dilated blood vessels on the optic disc (black arrow) may be confused with neovscularization but are radially dilated, do not enter the vitreous cavity and do not leak on fluorescein angigraphy.[ marked by the

When diabetic papillopathy is suspected, it is important to perform fluorescein angiography. In diabetic papillopathy, dye leakage is limited to the disc and peripapillary retina as opposed to the intravitreal leakage seen in the case of neovascular lesions. Diabetic retinopathy is usually present at diagnosis, but in 20% of reported cases there was no clinical evidence of any diabetic retinopathy (Kline et al., 2010). If the optic disc edema is bilateral, the initial evaluation should include brain imaging and lumbar puncture to rule out intracranial space occupying lesions and elevated intracranial

The optic disc edema resolves in many cases without treatment, usually within two to ten months. Minimal optic atrophy is seen in 20% of cases. In rare cases, especially in poorly controlled patients, diabetic papillopathy may progress to non-arteritic anterior ischemic optic neuropathy (NAION) with significant optic atrophy and arcuate visual field defects. In most cases, long term visual acuity depends on the associated diabetic retinopathy. There is

Non-arteritic anterior ischemic optic neuropathy (NAION) occurs as a consequence of the interruption of blood flow to the optic nerve at the level of the optic disc. NAION is characterized by diffuse or segmental, hyperemic or pale optic disc edema. It is usually

white arrowhead the pointer aiding the patient's fixation during photography].

pressure.

no proven treatment for diabetic papillopathy.

**6.2 Non-arteritic anterior ischemic optic neuropathy (NAION)** 

vessels on the anterior surface of the iris (figure 1). These vessels can block the normal drainage of fluid from the anterior chamber, leading to a sharp and persistent rise in intraocular pressure. This complication is known as neovascular glaucoma. This type of glaucoma is often refractory to treatment and can be associated with pain from very high ocular pressure. Topical medical therapy used commonly in other forms of glaucoma is often less effective. Treatment should include aggressive control of the underlying retinal disease with peripheral laser ablation to reduce ischemia. The treatment of proliferative diabetic retinopathy will be discussed in more detail in section 7.6.2.3.

Fig. 1. Neovascularization of the Iris: These pathologic blood vessels on the iris (white arrows) are associated with retinal ischemia in proliferative diabetic retinopathy. The normal iris has no visible surface blood vessels.

#### **6. Optic nerve**

#### **6.1 Diabetic papillopathy**

In diabetic papillopathy, chronic swelling of the optic disc often associated with mild visual impairment. The suspected cause is mild reversible ischemia of the optic nerve head (Ostri et al., 2010; Kline et al., 2010). Risk factors include pronounced recent decrease in hemoglobin A1C and a small cup to disc ratio of the optic nerve head. Patients often present with no visual complaints or with a mild nonspecific visual disturbance such as mild distortion or blurring. There is no pain and visual acuity is usually normal but may be slightly diminished. There is no afferent pupillary defect. An enlarged blind spot is seen on visual fields. Clinical examination reveals unilateral or bilateral hyperemic edema of the optic disc, accompanied by dilation of inner disc surface vessels, vascular leakage and axonal swelling (cotton wool spots). These enlarged vessels may be confused with neovascularization of the disc but these radially dilated vessels do not extend into the vitreous (Figure 2).

vessels on the anterior surface of the iris (figure 1). These vessels can block the normal drainage of fluid from the anterior chamber, leading to a sharp and persistent rise in intraocular pressure. This complication is known as neovascular glaucoma. This type of glaucoma is often refractory to treatment and can be associated with pain from very high ocular pressure. Topical medical therapy used commonly in other forms of glaucoma is often less effective. Treatment should include aggressive control of the underlying retinal disease with peripheral laser ablation to reduce ischemia. The treatment of proliferative

Fig. 1. Neovascularization of the Iris: These pathologic blood vessels on the iris (white arrows) are associated with retinal ischemia in proliferative diabetic retinopathy. The

In diabetic papillopathy, chronic swelling of the optic disc often associated with mild visual impairment. The suspected cause is mild reversible ischemia of the optic nerve head (Ostri et al., 2010; Kline et al., 2010). Risk factors include pronounced recent decrease in hemoglobin A1C and a small cup to disc ratio of the optic nerve head. Patients often present with no visual complaints or with a mild nonspecific visual disturbance such as mild distortion or blurring. There is no pain and visual acuity is usually normal but may be slightly diminished. There is no afferent pupillary defect. An enlarged blind spot is seen on visual fields. Clinical examination reveals unilateral or bilateral hyperemic edema of the optic disc, accompanied by dilation of inner disc surface vessels, vascular leakage and axonal swelling (cotton wool spots). These enlarged vessels may be confused with neovascularization of the disc but these radially dilated vessels do not extend into the

normal iris has no visible surface blood vessels.

**6. Optic nerve** 

vitreous (Figure 2).

**6.1 Diabetic papillopathy** 

diabetic retinopathy will be discussed in more detail in section 7.6.2.3.

Fig. 2. Diabetic Papillopathy. A chronic swelling of the optic disc often associated with mild visual impairment. This color fundus photograph shows blurred disc margins (white arrows), flame shaped hemorrhages (black arrowhead). The dilated blood vessels on the optic disc (black arrow) may be confused with neovscularization but are radially dilated, do not enter the vitreous cavity and do not leak on fluorescein angigraphy.[ marked by the white arrowhead the pointer aiding the patient's fixation during photography].

When diabetic papillopathy is suspected, it is important to perform fluorescein angiography. In diabetic papillopathy, dye leakage is limited to the disc and peripapillary retina as opposed to the intravitreal leakage seen in the case of neovascular lesions. Diabetic retinopathy is usually present at diagnosis, but in 20% of reported cases there was no clinical evidence of any diabetic retinopathy (Kline et al., 2010). If the optic disc edema is bilateral, the initial evaluation should include brain imaging and lumbar puncture to rule out intracranial space occupying lesions and elevated intracranial pressure.

The optic disc edema resolves in many cases without treatment, usually within two to ten months. Minimal optic atrophy is seen in 20% of cases. In rare cases, especially in poorly controlled patients, diabetic papillopathy may progress to non-arteritic anterior ischemic optic neuropathy (NAION) with significant optic atrophy and arcuate visual field defects. In most cases, long term visual acuity depends on the associated diabetic retinopathy. There is no proven treatment for diabetic papillopathy.

#### **6.2 Non-arteritic anterior ischemic optic neuropathy (NAION)**

Non-arteritic anterior ischemic optic neuropathy (NAION) occurs as a consequence of the interruption of blood flow to the optic nerve at the level of the optic disc. NAION is characterized by diffuse or segmental, hyperemic or pale optic disc edema. It is usually

Ocular Complications of Type 1 Diabetes 299

3. Hypertension: Good blood pressure control is important in reducing the risk of retinopathy. Every 10 mmHg reduction in systolic blood pressure leads to a reduction of 35% in the risk of retinopathy progression and a reduction of 50% in the risk of visual

4. Hyperlipidemia: High cholesterol may also be a risk factor for diabetic retinopathy

5. Genetic factors: The Diabetes Control and Complications Trial (DCCT group, 1997) showed a heritable tendency for developing diabetic retinopathy, regardless of other

6. Ethnicity: Diabetic retinopathy in America is more prevalent among African Americans, Hispanic and south Asian groups than in Caucasians with otherwise similar risk

7. Pregnancy: Pregnancy is associated with worsening of diabetic retinopathy (DCCT group, 2000). All pregnant women need to be closely monitored throughout pregnancy.

The normal retina has a blood–retinal barrier (BRB) which consists of cells that are tightly joined together to prevent certain substances from entering the retinal tissue. An important part of the BRB is the non-fenestrated capillaries of the retinal circulation. In diabetic retinopathy, damage to retinal blood vessels leads to a breakdown of the BRB and the

Diabetic retinopathy is induced when hyperglycemia and other causal risk factors trigger a cascade of biochemical changes leading to microvascular damage in the retina. Hyperglycemia leads to rise of sorbitol concentrations via the action of aldose reductase. This process increases oxidative stress by reducing intracellular levels of reduced glutathione, an important antioxidant (Stirban et al., 2008). Intracellular hyperglycemia also increases synthesis of diacylglycerol an activating cofactor for protein kinase C (PKC). Activated PKC decreases the production of anti-artherosclerotic factors and increases production of pro-artherogenic factors, pro-adhesive and pro-inflammatory factors (Stirban et al., 2008). Hyperglycemia also leads to accumulation of advanced glycated end products which are pro-inflammatory and activate PKC (Stirban et al., 2008). Intracellular hyperglycaemia increases intracellular N-acetylglucosamine levels. This byproduct reacts with serine and threonine residues in transcription factors, resulting in pathologic changes in gene expression (Stirban et al., 2008). The final consequence of these pathological processes is increased inflammation and increased oxidative stress which cause endothelial

Endothelial cell dysfunction induces retinal arteriolar dilatation which increases capillary bed pressure. This results in microaneurysm formation, vessel leakage and rupture (Cheung et al., 2010). Vascular permeability is also increased from loss of pericytes and increased endothelial proliferation in retinal capillaries. The breakdown of the bloodretinal barrier allows fluid to accumulate in the deep retinal layers where it damages photoreceptors and other neural tissues. This is the mechanism by which macular edema

Pregnancy in type 1 diabetes is discussed in further detail in section 7.6.1.

reduction in the risk of blindness (Cheung et al. 2010, DCCT group, 1995).

loss (Cheung et al. 2010).

risk factors.

**7.3 Pathophysiology** 

progression (Cheung et al., 2010).

profiles (Cheung et al., 2010).

cell dysfunction in retinal blood vessels.

reduces visual acuity.

leakage of fluid, blood and protein into the retinal tissue.

reduction in the risk of progression to vision threatening retinopathy and a 15%

unilateral, and in contrast to diabetic papillopathy, damage to the optic nerve ganglions causes decreased visual acuity and/or visual field loss. Vision is usually not worse than 20/200 and the typical visual field defect is altitudinal. Diabetes is a risk factor for NAION and the condition may present at a younger age in diabetic patients (Kline et al., 2010).

After an episode of NAION visual acuity may be stable but can also decline slowly over weeks to months until eventual stabilization. The initial finding upon examination is optic disc swelling, but this resolves over time and is replaced by optic disc atrophy within 4-8 weeks. There is no proven treatment for this condition.

### **7. Diabetic retinopathy**

Damage to the retinal capillaries and other small vessels is the hallmark of diabetic eye disease and is known as diabetic retinopathy. This condition is the major cause of blindness and visual disability in patients with type 1 diabetes.

#### **7.1 Epidemiology**

Diabetic retinopathy is one of the most frequent causes of blindness in working aged adults (20-74 years) (Regillo et al., 2010; Cheung et al., 2010). In the USA an estimated 86% of patients with type 1 diabetes have some degree of diabetic retinopathy. Data from the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR) showed that within 5 years of diagnosis of type 1 diabetes, 14% of patients developed retinopathy, with the incidence rising to 74% by 10 years (Klein et al., 2008; Varma, 2008). In people with retinopathy at the WESDR baseline examination, 64% had their retinopathy worsen, 17% progressed to proliferative diabetic retinopathy (PDR) and about 20% developed diabetic macular edema during 10 years of follow-up.

The WESDR data in type 1 diabetics showed that 25 years after diagnosis, 97% of patients developed retinopathy, 43% progressed to PDR, 29% developed diabetic macular edema and 3.6% of patients younger than 30 at diagnosis were legally blind (Klein et al., 2008). Fortunately, recent advances in glycemic control, ophthalmic treatment and patient education seem to be working. The WESDR results also showed a reduction in the yearly incidence and progression of diabetic retinopathy during the past 15 years (Varma 2008).

The course of diabetic retinal disease in children is fairly benign. Severe complications such as proliferative diabetic retinopathy are uncommon in children before puberty (Raab et al., 2010).

#### **7.2 Risk factors**

Several risk factors influence the development and progression of diabetic retinopathy. The following list contains most of the important risk factors known today.


unilateral, and in contrast to diabetic papillopathy, damage to the optic nerve ganglions causes decreased visual acuity and/or visual field loss. Vision is usually not worse than 20/200 and the typical visual field defect is altitudinal. Diabetes is a risk factor for NAION and the condition may present at a younger age in diabetic patients (Kline et al.,

After an episode of NAION visual acuity may be stable but can also decline slowly over weeks to months until eventual stabilization. The initial finding upon examination is optic disc swelling, but this resolves over time and is replaced by optic disc atrophy within 4-8

Damage to the retinal capillaries and other small vessels is the hallmark of diabetic eye disease and is known as diabetic retinopathy. This condition is the major cause of blindness

Diabetic retinopathy is one of the most frequent causes of blindness in working aged adults (20-74 years) (Regillo et al., 2010; Cheung et al., 2010). In the USA an estimated 86% of patients with type 1 diabetes have some degree of diabetic retinopathy. Data from the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR) showed that within 5 years of diagnosis of type 1 diabetes, 14% of patients developed retinopathy, with the incidence rising to 74% by 10 years (Klein et al., 2008; Varma, 2008). In people with retinopathy at the WESDR baseline examination, 64% had their retinopathy worsen, 17% progressed to proliferative diabetic retinopathy (PDR) and about 20% developed diabetic

The WESDR data in type 1 diabetics showed that 25 years after diagnosis, 97% of patients developed retinopathy, 43% progressed to PDR, 29% developed diabetic macular edema and 3.6% of patients younger than 30 at diagnosis were legally blind (Klein et al., 2008). Fortunately, recent advances in glycemic control, ophthalmic treatment and patient education seem to be working. The WESDR results also showed a reduction in the yearly incidence and progression of diabetic retinopathy during the past

The course of diabetic retinal disease in children is fairly benign. Severe complications such as proliferative diabetic retinopathy are uncommon in children before puberty (Raab et al.,

Several risk factors influence the development and progression of diabetic retinopathy. The

1. Diabetes duration: The longer the duration of diabetes, the higher the risk of developing diabetic retinopathy and of having a severe manifestation of the disease. (Simon et al.,

2. Hyperglycemia: Good glycemic control has been shown to significantly prevent the development and progression of diabetic retinopathy. Every 1% decrease in hemoglobin A1C leads to a 40% reduction in the risk of developing retinopathy, a 25%

following list contains most of the important risk factors known today.

weeks. There is no proven treatment for this condition.

and visual disability in patients with type 1 diabetes.

macular edema during 10 years of follow-up.

2010).

**7. Diabetic retinopathy** 

**7.1 Epidemiology** 

15 years (Varma 2008).

2010; Cheung et al., 2010).

**7.2 Risk factors** 

2010).

reduction in the risk of progression to vision threatening retinopathy and a 15% reduction in the risk of blindness (Cheung et al. 2010, DCCT group, 1995).


#### **7.3 Pathophysiology**

The normal retina has a blood–retinal barrier (BRB) which consists of cells that are tightly joined together to prevent certain substances from entering the retinal tissue. An important part of the BRB is the non-fenestrated capillaries of the retinal circulation. In diabetic retinopathy, damage to retinal blood vessels leads to a breakdown of the BRB and the leakage of fluid, blood and protein into the retinal tissue.

Diabetic retinopathy is induced when hyperglycemia and other causal risk factors trigger a cascade of biochemical changes leading to microvascular damage in the retina. Hyperglycemia leads to rise of sorbitol concentrations via the action of aldose reductase. This process increases oxidative stress by reducing intracellular levels of reduced glutathione, an important antioxidant (Stirban et al., 2008). Intracellular hyperglycemia also increases synthesis of diacylglycerol an activating cofactor for protein kinase C (PKC). Activated PKC decreases the production of anti-artherosclerotic factors and increases production of pro-artherogenic factors, pro-adhesive and pro-inflammatory factors (Stirban et al., 2008). Hyperglycemia also leads to accumulation of advanced glycated end products which are pro-inflammatory and activate PKC (Stirban et al., 2008). Intracellular hyperglycaemia increases intracellular N-acetylglucosamine levels. This byproduct reacts with serine and threonine residues in transcription factors, resulting in pathologic changes in gene expression (Stirban et al., 2008). The final consequence of these pathological processes is increased inflammation and increased oxidative stress which cause endothelial cell dysfunction in retinal blood vessels.

Endothelial cell dysfunction induces retinal arteriolar dilatation which increases capillary bed pressure. This results in microaneurysm formation, vessel leakage and rupture (Cheung et al., 2010). Vascular permeability is also increased from loss of pericytes and increased endothelial proliferation in retinal capillaries. The breakdown of the bloodretinal barrier allows fluid to accumulate in the deep retinal layers where it damages photoreceptors and other neural tissues. This is the mechanism by which macular edema reduces visual acuity.

Ocular Complications of Type 1 Diabetes 301

Fig. 3. A: Color photograph of normal fundus (left eye) normal optic disc (white arrow) and macula (black arrow). B: Color photograph (right eye) showing non-proliferative diabetic retinopathy and macular edema. Findings include; dot and blot hemorrhages (black

arrows), flame shaped hemorrhages (white arrow), and cotton wool spot (white arrowhead) which represent nerve fiber layer infarcts. Hard exudates (black arrowheads) are lipid filled macrophages and in this photograph are radially distributed around the central macula.

Fig. 4. Color photograph of fundus with moderate non-proliferative diabetic retinopathy

with macular edema. Multiple dot and blot retinal hemorrhages are seen (white

arrowheads) and hard exudates (black arrows).

In some capillaries there is endothelial cell apoptosis. Vessels become acellular leading to vascular occlusion and non-perfusion of local retinal tissue (Stirban et al., 2008). The resultant retinal ischemia promotes the release of inflammatory growth factors, such as vascular endothelial growth factor (VEGF), growth hormone- insulin growth factor and erythropoietin (Cheung et al., 2010). These factors influence neovascularization, the proliferation of new capillaries, which is the hallmark of proliferative diabetic retinopathy.

#### **7.4 Classification and clinical features**

Diabetic retinopathy is classified into two stages: non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). In NPDR the vascular changes occur within the retina and do not cross the retinal surface. The more advanced stage of PDR is marked by neovascularization wherein new blood vessels grow out from the retinal surface towards the vitreous cavity.

A major cause of vision loss in diabetic retinopathy is diabetic macular edema (DME). DME occurs when leaky capillary beds allow fluid to accumulate in the part of the retina responsible for central vision. This edema can occur in patients with any stage of underlying retinopathy from mild NPDR to severe PDR.

Visual impairment is usually related to the state of macular disease and the consequences of neovascularisation such as vitreous hemorrhage and retinal detachment. As such, the level of retinal disease does not always correlate with visual function and severe diabetic retinopathy can be present initially without significant visual loss.

#### **7.4.1 Non-proliferative diabetic retinopathy (NPDR)**

In NPDR the retinal microvascular changes occur within the retina and do not extend beyond the surface of the retina. The patient with NPDR is usually asymptomatic and visual acuity is preserved unless the macula is affected.

Clinical findings include microaneurysms (saccular enlargements of weakened capillaries), intra-retinal hemorrhages, hard exudates (lipid filled macrophages), cotton wool spots (nerve fiber layer infarcts)(figures 3-4), venous beading (focal venous dilatations and constrictions) and intra-retinal microvascular abnormalities (IRMA's, dilated pre-existing capillaries) (Regillo et al., 2010, Cheung et al., 2008).

Fluorescein angiography (FA) is an essential tool for evaluating the retinal circulation and retinopathy stage. Sodium fluorescein is injected into the systemic circulation, and an angiogram is obtained by photographing the fluorescence emitted after illumination of the retina. In NPDR, the FA shows microaneurysms as dye filled outpouchings. Hemorrhages appear as black dots as the blood obscures the fluorescence from the retina and choroid below (figure 5).

NPDR is classified as mild, moderate or severe, reflecting the risk of progression to PDR (Table 1) as determined by the Early Treatment in Diabetic Retinopathy Study (ETDRS) (ETDRS group, 1995). The diagnosis of severe NPDR is made when one of three findings is present: diffuse intra-retinal hemorrhages and microaneurysms in all 4 retinal quadrants, venous beading in 2 quadrants or one intra-retinal microvascular abnormalities anywhere. Fifteen persent of patients with severe NPDR will progress to high-risk PDR within 1 year. When any two features of severe NPDR are present, the patient is said to suffer from very severe NPDR and the one year risk of progression to high risk PDR increases to 45%.

In some capillaries there is endothelial cell apoptosis. Vessels become acellular leading to vascular occlusion and non-perfusion of local retinal tissue (Stirban et al., 2008). The resultant retinal ischemia promotes the release of inflammatory growth factors, such as vascular endothelial growth factor (VEGF), growth hormone- insulin growth factor and erythropoietin (Cheung et al., 2010). These factors influence neovascularization, the proliferation of new capillaries, which is the hallmark of proliferative diabetic retinopathy.

Diabetic retinopathy is classified into two stages: non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). In NPDR the vascular changes occur within the retina and do not cross the retinal surface. The more advanced stage of PDR is marked by neovascularization wherein new blood vessels grow out from the retinal surface

A major cause of vision loss in diabetic retinopathy is diabetic macular edema (DME). DME occurs when leaky capillary beds allow fluid to accumulate in the part of the retina responsible for central vision. This edema can occur in patients with any stage of underlying

Visual impairment is usually related to the state of macular disease and the consequences of neovascularisation such as vitreous hemorrhage and retinal detachment. As such, the level of retinal disease does not always correlate with visual function and severe diabetic

In NPDR the retinal microvascular changes occur within the retina and do not extend beyond the surface of the retina. The patient with NPDR is usually asymptomatic and visual

Clinical findings include microaneurysms (saccular enlargements of weakened capillaries), intra-retinal hemorrhages, hard exudates (lipid filled macrophages), cotton wool spots (nerve fiber layer infarcts)(figures 3-4), venous beading (focal venous dilatations and constrictions) and intra-retinal microvascular abnormalities (IRMA's, dilated pre-existing

Fluorescein angiography (FA) is an essential tool for evaluating the retinal circulation and retinopathy stage. Sodium fluorescein is injected into the systemic circulation, and an angiogram is obtained by photographing the fluorescence emitted after illumination of the retina. In NPDR, the FA shows microaneurysms as dye filled outpouchings. Hemorrhages appear as black dots as the blood obscures the fluorescence from the retina and choroid

NPDR is classified as mild, moderate or severe, reflecting the risk of progression to PDR (Table 1) as determined by the Early Treatment in Diabetic Retinopathy Study (ETDRS) (ETDRS group, 1995). The diagnosis of severe NPDR is made when one of three findings is present: diffuse intra-retinal hemorrhages and microaneurysms in all 4 retinal quadrants, venous beading in 2 quadrants or one intra-retinal microvascular abnormalities anywhere. Fifteen persent of patients with severe NPDR will progress to high-risk PDR within 1 year. When any two features of severe NPDR are present, the patient is said to suffer from very severe NPDR and the one year risk of progression to

**7.4 Classification and clinical features** 

retinopathy from mild NPDR to severe PDR.

retinopathy can be present initially without significant visual loss.

**7.4.1 Non-proliferative diabetic retinopathy (NPDR)** 

acuity is preserved unless the macula is affected.

capillaries) (Regillo et al., 2010, Cheung et al., 2008).

towards the vitreous cavity.

below (figure 5).

high risk PDR increases to 45%.

Fig. 3. A: Color photograph of normal fundus (left eye) normal optic disc (white arrow) and macula (black arrow). B: Color photograph (right eye) showing non-proliferative diabetic retinopathy and macular edema. Findings include; dot and blot hemorrhages (black arrows), flame shaped hemorrhages (white arrow), and cotton wool spot (white arrowhead) which represent nerve fiber layer infarcts. Hard exudates (black arrowheads) are lipid filled macrophages and in this photograph are radially distributed around the central macula.

Fig. 4. Color photograph of fundus with moderate non-proliferative diabetic retinopathy with macular edema. Multiple dot and blot retinal hemorrhages are seen (white arrowheads) and hard exudates (black arrows).

Ocular Complications of Type 1 Diabetes 303

neovascular lesions as the new vessels are porous and leak fluorescent dye into the vitreous

The new vessels in PDR evolve in three stages. Initially, the fine new vessels grow with minimal fibrous tissue. Then the new vessels increase in gauge and length with an increased fibrous component. Finally, the vessels regress and the residual fibrovascular tissue along

Retinal neovascularizations (NV) are divided into two subtypes based on their relative risk of causing severe visual loss as demonstrated by the Diabetic Retinopathy Study (DRS). Vascular proliferations on or near the optic disc are termed NV-disc (NVD) and proliferations elsewhere are termed NV-elsewhere (NVE) (figures 6 - 8). The presence of NVD carries the higher risk of severe visual loss and requires more urgent treatment (DRS

 PDR is graded from early to high risk based on the risk of severe visual loss as determined by the extent of the neovascular proliferations. The DRS (DRS research group, 1979, 1981) defined high risk PDR as the presence of either: NVD with a vitreous hemorrhage, NVD larger than a quarter disc area without vitreous hemorrhage or NVE larger than half disc area with vitreous hemorrhage. Without treatment, patients with early PDR have 50% risk of developing high risk PDR in 1 year and those with high risk PDR have a 25% risk of severe visual loss within 2 years. Treatment of PDR involves extensive peripheral laser

Fig. 6. Proliferative Diabetic Retinopathy with Neovascularization of the Optic Disc (NVD). Vascular proliferations on or near the optic disc are termed NV-disc (NVD).This Color photograph shows fine jumbled vessels (black arrows) typical of NVD. Multiple dot and

the posterior surface of the vitreous body contracts.

ablation of the retina and is discussed in section 7.5.2.3.

blot retinal hemorrhages are seen (white arrows).

research group, 1979, 1981).

cavity.

Fig. 5. Fluorescein angiography of non proliferative diabetic retinopathyof the right eye. Sodium fluorescein is injected into the systemic circulation, and an angiogram is obtained by photographing the fluorescence emitted after illumination of the retina. In NPDR the FA shows microaneurysms filled with dye (small white arrows). Hemorrhages appear as black dots because the transmission of fluorescence from below the hemorrhage is blocked (white arrowheads) . The optic disc marked with long white arrow.


Table 1. Clinical classification of non-proliferative diabetic retinopathy

#### **7.4.2 Proliferative diabetic retinopathy (PDR)**

Diabetic retinopathy advances to the proliferative stage when new vessels (neovascularizations) are formed which grow up from the retina towards the vitreous cavity. The development of these pathological blood vessels is induced by pro-angiogenic factors released as a result of the severe retinal ischemia caused by the progression of diabetic retinal microvascular disease. Neovascularizations can be identified clinically as a jumble of disorganized, fine vessels emanating from the organized retinal vessel architecture (figure 6). Fluorescein angiography is also very effective at identifying

Fig. 5. Fluorescein angiography of non proliferative diabetic retinopathyof the right eye. Sodium fluorescein is injected into the systemic circulation, and an angiogram is obtained by photographing the fluorescence emitted after illumination of the retina. In NPDR the FA shows microaneurysms filled with dye (small white arrows). Hemorrhages appear as black dots because the transmission of fluorescence from below the hemorrhage is blocked (white

Mild NPDR Few microaneurysms 5% progress to PDR within

 Extensive intraretinal hemorrhages and microaneurysms in all four

Venous beading in two or

Diabetic retinopathy advances to the proliferative stage when new vessels (neovascularizations) are formed which grow up from the retina towards the vitreous cavity. The development of these pathological blood vessels is induced by pro-angiogenic factors released as a result of the severe retinal ischemia caused by the progression of diabetic retinal microvascular disease. Neovascularizations can be identified clinically as a jumble of disorganized, fine vessels emanating from the organized retinal vessel architecture (figure 6). Fluorescein angiography is also very effective at identifying

1 year

within 1 year

within 1 year

PDR within 1 year

PDR within 1 year

12-16% progress to PDR

52% progress to PDR

15% progress to high risk

45% progress to high risk

**Stage of NPDR Clinical Features Progression Risk** 

microvascular lesions

more quadrants

quadrants

One IRMA

severe NPDR

Table 1. Clinical classification of non-proliferative diabetic retinopathy

arrowheads) . The optic disc marked with long white arrow.

Moderate NPDR Microaneurysms and other

Very severe NPDR Any two of the features of

**7.4.2 Proliferative diabetic retinopathy (PDR)** 

Severe NPDR (Meets 1 of 3 criteria) neovascular lesions as the new vessels are porous and leak fluorescent dye into the vitreous cavity.

The new vessels in PDR evolve in three stages. Initially, the fine new vessels grow with minimal fibrous tissue. Then the new vessels increase in gauge and length with an increased fibrous component. Finally, the vessels regress and the residual fibrovascular tissue along the posterior surface of the vitreous body contracts.

Retinal neovascularizations (NV) are divided into two subtypes based on their relative risk of causing severe visual loss as demonstrated by the Diabetic Retinopathy Study (DRS). Vascular proliferations on or near the optic disc are termed NV-disc (NVD) and proliferations elsewhere are termed NV-elsewhere (NVE) (figures 6 - 8). The presence of NVD carries the higher risk of severe visual loss and requires more urgent treatment (DRS research group, 1979, 1981).

 PDR is graded from early to high risk based on the risk of severe visual loss as determined by the extent of the neovascular proliferations. The DRS (DRS research group, 1979, 1981) defined high risk PDR as the presence of either: NVD with a vitreous hemorrhage, NVD larger than a quarter disc area without vitreous hemorrhage or NVE larger than half disc area with vitreous hemorrhage. Without treatment, patients with early PDR have 50% risk of developing high risk PDR in 1 year and those with high risk PDR have a 25% risk of severe visual loss within 2 years. Treatment of PDR involves extensive peripheral laser ablation of the retina and is discussed in section 7.5.2.3.

Fig. 6. Proliferative Diabetic Retinopathy with Neovascularization of the Optic Disc (NVD). Vascular proliferations on or near the optic disc are termed NV-disc (NVD).This Color photograph shows fine jumbled vessels (black arrows) typical of NVD. Multiple dot and blot retinal hemorrhages are seen (white arrows).

Ocular Complications of Type 1 Diabetes 305

The most frequent complication of PDR is vitreous hemorrhage (figure 9) caused by rupture in the fragile neovascular vessels. The initial complaint is often of black dots partially obscuring vision and can evolve to severe visual loss over a period of hours to days as the

Fig. 9. Vitreous Hemorrhage. Vitreous hemorrhage is the most frequent complication of PDR. It is caused by rupture of neovascular vessels. This color figure shows partial vitreous

photocoagulation as a treatment for PDR. Multiple white-yellow laser burns are seen (black arrows). This treatment is discussed in section 7.5.2.3. The optic disc is also seen (white

hemorrhage causing general haze. Dense blood in the vitreous cavity is seen (white arrowheads). This hemorrhage occurred in eye after the initiation of pan-retinal

eye fills with blood.

arrow).

Fig. 7. High Risk Proliferative Diabetic Retinopathy with Large NVD. PDR is graded from early to high risk based on the risk of severe visual loss as determined by the extent of the neovascular proliferation. A: Color photograph showing a large neovascularization of the optic disc (black arrows) this NVD is larger than a quarter disc area therefore consistent with high risk PDR. Retinal hemorrhages (white arrows) and hard exudates (white arrowheads) are also seen. B: Fluorescein angiography in the same patient demonstrating hyper-fluorescence due to dye leakage from the disc neovascularization (white arrows).

Fig. 8. High Risk Proliferative Diabetic Retinopathy with large NVE. This neovascular lesion located away from the optic disc is known as a Neovascularization-Elsewhere (NVE). This large NVE (black arrow) is associated with a small hemorrhage (white arrow).

Fig. 7. High Risk Proliferative Diabetic Retinopathy with Large NVD. PDR is graded from early to high risk based on the risk of severe visual loss as determined by the extent of the neovascular proliferation. A: Color photograph showing a large neovascularization of the optic disc (black arrows) this NVD is larger than a quarter disc area therefore consistent with high risk PDR. Retinal hemorrhages (white arrows) and hard exudates (white arrowheads) are also seen. B: Fluorescein angiography in the same patient demonstrating hyper-fluorescence due to dye leakage from the disc neovascularization (white arrows).

Fig. 8. High Risk Proliferative Diabetic Retinopathy with large NVE. This neovascular lesion located away from the optic disc is known as a Neovascularization-Elsewhere (NVE). This

large NVE (black arrow) is associated with a small hemorrhage (white arrow).

The most frequent complication of PDR is vitreous hemorrhage (figure 9) caused by rupture in the fragile neovascular vessels. The initial complaint is often of black dots partially obscuring vision and can evolve to severe visual loss over a period of hours to days as the eye fills with blood.

Fig. 9. Vitreous Hemorrhage. Vitreous hemorrhage is the most frequent complication of PDR. It is caused by rupture of neovascular vessels. This color figure shows partial vitreous hemorrhage causing general haze. Dense blood in the vitreous cavity is seen (white arrowheads). This hemorrhage occurred in eye after the initiation of pan-retinal photocoagulation as a treatment for PDR. Multiple white-yellow laser burns are seen (black arrows). This treatment is discussed in section 7.5.2.3. The optic disc is also seen (white arrow).

Ocular Complications of Type 1 Diabetes 307

living such as reading and driving. Diabetic macular edema is assessed separately from the stage of retinopathy (NPDR/PDR) and it can manifest along a different and

The edema evolves when damage to the macular capillary bed causes a breakdown of the blood-retina barrier. This results in increased retinal vascular permeability and to the accumulation of fluid in the macula. Macular edema may be 'focal' and emanate from a small cluster of leaky vessels, or 'diffuse' and involve the entire macula without a clear point of origin. Clinical examination can reveal rings of hard exudates (lipid filled macrophages) which delineate the area of focal leakage (figures 3 & 4). These subtypes are often differentiated by angiography which demonstrates areas of focal leakage from specific capillary lesions and microaneurysms in focal edema. In diffuse macular edema, angiography reveals widespread leakage with no definitive point of origin from extensive

Treatment decisions are based on the clinical examination in DME. Intervention is recommended only when the retinal edema involves or threatens the center of the macula.

Optical Coherence Tomography (OCT) is a useful ancillary imaging technique in DME. Recent technological advances in OCT technology have provided ophthalmologists with high-resolution images of the retina in cross-sectional slices. Aside from demonstrating areas of retinal thickening and intra-retinal fluid (figure 11), OCT obtains quantitative measurements of central retinal thickness that are important for close monitoring and follow-up of macular edema. Serial OCT examinations are often used as a non-invasive and accurate method analyzing treatment response in DME patients (Cheung et al.,

Fig. 11. Optical Coherence Tomography (OCT) of the Macula. A: OCT scan demonstrating the normal anatomic indentation in the central macula in a healthy eye (white arrow). B: Diabetic macular edema: There is loss of central macular indentation due to retinal cysts

Macular ischemia is a devastating complication of diabetic retinopathy. It is caused by extensive loss of retinal capillary perfusion in the macula. Clinical exam often reveals microaneurysms clustering at the margins of the non-perfused retina. Angiography can demonstrate the presence and extent of the area with capillary non-perfusion. This entity is

generally associated with significantly decreased vision (Regillo et al., 2010).

(white arrow). Hard exudates are also seen (white arrowhead).

**7.4.4 Diabetic macular ischemia** 

breakdown of the blood-retinal barrier (Regillo et al., 2010).

In all other cases, close follow-up alone is indicated (ETDRS group, 1995).

independent course.

2008).

Another cause of severe vision loss in PDR is traction retinal detachment. This detachment occurs when the neovascular tissue connecting the retinal surface to the vitreous contracts causing a separation between the surface and deep retinal layers (figure 10). If this occurs in the center of the macula severe vision loss can result.

Fig. 10. Traction Retinal Detachment. This detachment is one of the serious complications in PDR and may cause severe visual loss. It occurs when the neovascular tissue connecting the retinal surface to the vitreous contracts causing a separation between the surface and deep retinal layers. This color fundus photograph shows elevated and detached retina (black arrowheads) involving the optic disc area (white arrow). The retina at the center of the macula (black arrow) is not detached.

A third serious complication of PDR occurs when proliferative and pro-angiogenic factors released from the ischemic retina induce the propagation of new vessels on the iris (figure 1). These vessels can block the normal outflow of fluid from the eye causing often severely increased ocular pressure. This complication is known as neovascular glaucoma and is discussed further previously in section 5.

Visual acuity in the absence of macular disease is often good in PDR until a complication occurs, most commonly vitreous hemorrhage. This sudden transition from good vision to near blindness can be traumatic for patients who were unaware of the severity of their diabetic eye disease.

#### **7.4.3 Diabetic macular edema**

Diabetic macular edema (DME) is responsible for most of the moderate visual loss in retinopathy patients. The vision loss is often mild at first, but without effective treatment it can progress and patients can lose the ability to perform activities of daily

Another cause of severe vision loss in PDR is traction retinal detachment. This detachment occurs when the neovascular tissue connecting the retinal surface to the vitreous contracts causing a separation between the surface and deep retinal layers (figure 10). If this occurs in

Fig. 10. Traction Retinal Detachment. This detachment is one of the serious complications in PDR and may cause severe visual loss. It occurs when the neovascular tissue connecting the retinal surface to the vitreous contracts causing a separation between the surface and deep retinal layers. This color fundus photograph shows elevated and detached retina (black arrowheads) involving the optic disc area (white arrow). The retina at the center of the

A third serious complication of PDR occurs when proliferative and pro-angiogenic factors released from the ischemic retina induce the propagation of new vessels on the iris (figure 1). These vessels can block the normal outflow of fluid from the eye causing often severely increased ocular pressure. This complication is known as neovascular glaucoma and is

Visual acuity in the absence of macular disease is often good in PDR until a complication occurs, most commonly vitreous hemorrhage. This sudden transition from good vision to near blindness can be traumatic for patients who were unaware of the severity of their

Diabetic macular edema (DME) is responsible for most of the moderate visual loss in retinopathy patients. The vision loss is often mild at first, but without effective treatment it can progress and patients can lose the ability to perform activities of daily

the center of the macula severe vision loss can result.

macula (black arrow) is not detached.

discussed further previously in section 5.

diabetic eye disease.

**7.4.3 Diabetic macular edema** 

living such as reading and driving. Diabetic macular edema is assessed separately from the stage of retinopathy (NPDR/PDR) and it can manifest along a different and independent course.

The edema evolves when damage to the macular capillary bed causes a breakdown of the blood-retina barrier. This results in increased retinal vascular permeability and to the accumulation of fluid in the macula. Macular edema may be 'focal' and emanate from a small cluster of leaky vessels, or 'diffuse' and involve the entire macula without a clear point of origin. Clinical examination can reveal rings of hard exudates (lipid filled macrophages) which delineate the area of focal leakage (figures 3 & 4). These subtypes are often differentiated by angiography which demonstrates areas of focal leakage from specific capillary lesions and microaneurysms in focal edema. In diffuse macular edema, angiography reveals widespread leakage with no definitive point of origin from extensive breakdown of the blood-retinal barrier (Regillo et al., 2010).

Treatment decisions are based on the clinical examination in DME. Intervention is recommended only when the retinal edema involves or threatens the center of the macula. In all other cases, close follow-up alone is indicated (ETDRS group, 1995).

Optical Coherence Tomography (OCT) is a useful ancillary imaging technique in DME. Recent technological advances in OCT technology have provided ophthalmologists with high-resolution images of the retina in cross-sectional slices. Aside from demonstrating areas of retinal thickening and intra-retinal fluid (figure 11), OCT obtains quantitative measurements of central retinal thickness that are important for close monitoring and follow-up of macular edema. Serial OCT examinations are often used as a non-invasive and accurate method analyzing treatment response in DME patients (Cheung et al., 2008).

Fig. 11. Optical Coherence Tomography (OCT) of the Macula. A: OCT scan demonstrating the normal anatomic indentation in the central macula in a healthy eye (white arrow). B: Diabetic macular edema: There is loss of central macular indentation due to retinal cysts (white arrow). Hard exudates are also seen (white arrowhead).

#### **7.4.4 Diabetic macular ischemia**

Macular ischemia is a devastating complication of diabetic retinopathy. It is caused by extensive loss of retinal capillary perfusion in the macula. Clinical exam often reveals microaneurysms clustering at the margins of the non-perfused retina. Angiography can demonstrate the presence and extent of the area with capillary non-perfusion. This entity is generally associated with significantly decreased vision (Regillo et al., 2010).

Ocular Complications of Type 1 Diabetes 309

as clinically significant macular edema (CSME), benefited from focal laser treatment. Focal laser treatment reduced the risk of moderate visual loss (loss of three lines of vision) by 50% over two years, increased the chance of improved vision and reduced central macular

In eyes with macular edema that does not meet the criteria for CSME, laser treatment is not indicated. Close follow-up is recommended to determine the progression of the macular edema. Unfortunately, when the macular edema is associated with macular ischemia from a loss of macular capillary perfusion, the ETDRS showed a lesser beneficial effect for focal

Side effects of focal laser photocoagulation include: paracentral visual field loss, transiently increased macular edema with decreased visual acuity and choroidal neovascularization

Inflammatory factors play an important role in the development of diabetic retinopathy and macular edema (see section 7.3). For this reason it has long been thought that ocular steroid injections may be beneficial in DME treatment. Several trials have shown modestly improved visual acuity and central macular thickness after injection of intravitreal Triamcinolone (Grover at al., 2008; Yilmaz et al., 2009). A few recent trials on long acting steroid implants, such as Fluocinolone Acetate or Dexamethasone, have also reported short

The Diabetic Retinopathy Clinical Research Network (DRCR network, 2008) compared intravitreal injection of Triamcinolone to focal laser treatment in eyes with DME. There was no difference in visual acuity between the two groups after 1 year, and at 2 years eyes treated with laser had better vision. However, complications of intravitreal steroids, including elevated ocular pressure and increased cataract progression limit the usefulness of these drugs in DME. Intravitreal sterioid injections may be considered in patients who have previously undergone cataract surgery and in cases where the macular edema is refractory to focal laser. In these cases the injections may be given either alone or as an adjunct to laser

Vascular Endothelial Growth Factor (VEGF) is a major cause of the increased retinal vascular permeability which causes macular edema (Stirban et al., 2008). Several VEGF inhibitors have been investigated as treatments for DME with a beneficial effect on visual

Injection of intravitreal Pegaptanib, a pegylated aptamer that inhibits one isoform of VEGF, was found to be better than sham injections in improving in visual acuity and decreasing the need for focal laser treatment, in the Macugen Diabetic Retinopathy Study group

The injection of monoclonal antibodies that block all isoforms of VEGF has also been investigated as a treatment for DME. Several studies have shown a beneficial effect on visual acuity in eyes treated with Bevacizumab (trade name Avastin), a recombinant full length humanized antibody to VEGF (Nicholson & Shachat, 2010). Based on data from multiple studies (Nicholson & Shachat, 2010) repeated doses of Bevacizumab increase its average positive effect. The optimal timing for repeat dosing is unclear, but is probably between 3 to

12 weeks, with maximal effect with a 3 to 6 weeks interval between treatments.

thickness compared to no treatment.

term visual acuity improvements (Grover et al., 2009).

treatment (Gillies et al., 2006; Maia et al., 2009).

acuity and central macular thickness.

(Cunningham et al., 2005).

*7.5.2.2.3 Anti-vascular endothelial growth factor (VEGF) compounds* 

laser (ETDRS Group, 1995).

(Regillo et al, 2010). *7.5.2.2.2 Steroid injections* 

#### **7.5 Treatment and prevention of diabetic retinopathy**

The main goal of treatment of diabetic retinopathy is preventing complications which can lead to vision loss. Treatment should include both ocular therapy and systemic medical intervention.

#### **7.5.1 Medical treatment**

Hyperglycemia, hypertension and hyperlipidemia are known risk factors for the development and progression of diabetic retinopathy. Treating and controlling these factors is crucial to preventing and limiting disease progression.

The Diabetes Control and Complications Trial (DCCT group, 1995) showed that intensive glycemic control reduced both the risk of developing retinopathy and the rate of progression of existing retinopathy. Intensive glycemic control reduced the risk for progression to severe NPDR and PDR, the incidence of diabetic macular edema and the need for laser treatments. Every percent reduction in hemoglobin A1C lowers the risk of retinopathy development by 30-40%.

Several other systemic therapies have been investigated and found to reduce the risk of retinopathy progression including Angiotensin Converting Enzyme Inhibitors, Protein Kinase C Inhibitors and inhibitors of Advanced Glycosylation End-products formation (Cheung et al., 2008).

#### **7.5.2 Ocular treatment**

Ocular therapy in diabetic retinopathy includes panretinal or focal laser photocoagulation, intravitreal injections of either steroids or inhibitors of Vascular Endothelial Growth Factor (VEGF), surgery or a combination of the aforementioned treatments. The suitable treatment regimen must be tailored individually for each patient and is based on clinical status of the patient (ocular and systemic), previous treatments and data from several reports and ongoing studies.

#### **7.5.2.1 NPDR**

Visual acuity is not usually affected in NPDR unless there is damage to the macula in the form of macular edema or ischemia. Ocular treatment at this stage is definitively indicated only if there is evidence of macular disease. In patients with very severe NPDR who are at high risk for progression to PDR, laser treatment can be considered if the patient is not considered a suitable candidate for close follow-up. In such cases the recommended treatment is Pan- Retinal laser Photocoagulation (PRP) which will be discussed in section 7.5.2.3 (ETDRS group, 1995).

#### **7.5.2.2 Diabetic macular edema (DME)**

Treatment options for DME include focal laser photocoagulation, intravitreal injections of either steroids or anti-VEGF compounds and surgery.

#### *7.5.2.2.1 Focal laser*

The mainstay of DME treatment is focal laser photocoagulation. Focal laser treatment for DME involves the application of discrete laser burns to areas of leakage in the macula. The treatment is not painful and can be repeated up to every 4 months if edema persists. Treatment criteria are based on the ETDRS recommendations (ETDRS group, 1995) which showed that eyes with macular edema involving or threatening the central macula, defined

The main goal of treatment of diabetic retinopathy is preventing complications which can lead to vision loss. Treatment should include both ocular therapy and systemic medical

Hyperglycemia, hypertension and hyperlipidemia are known risk factors for the development and progression of diabetic retinopathy. Treating and controlling these factors

The Diabetes Control and Complications Trial (DCCT group, 1995) showed that intensive glycemic control reduced both the risk of developing retinopathy and the rate of progression of existing retinopathy. Intensive glycemic control reduced the risk for progression to severe NPDR and PDR, the incidence of diabetic macular edema and the need for laser treatments. Every percent reduction in hemoglobin A1C lowers the risk of

Several other systemic therapies have been investigated and found to reduce the risk of retinopathy progression including Angiotensin Converting Enzyme Inhibitors, Protein Kinase C Inhibitors and inhibitors of Advanced Glycosylation End-products formation

Ocular therapy in diabetic retinopathy includes panretinal or focal laser photocoagulation, intravitreal injections of either steroids or inhibitors of Vascular Endothelial Growth Factor (VEGF), surgery or a combination of the aforementioned treatments. The suitable treatment regimen must be tailored individually for each patient and is based on clinical status of the patient (ocular and systemic), previous treatments and data from several reports and

Visual acuity is not usually affected in NPDR unless there is damage to the macula in the form of macular edema or ischemia. Ocular treatment at this stage is definitively indicated only if there is evidence of macular disease. In patients with very severe NPDR who are at high risk for progression to PDR, laser treatment can be considered if the patient is not considered a suitable candidate for close follow-up. In such cases the recommended treatment is Pan- Retinal laser Photocoagulation (PRP) which will be discussed in section

Treatment options for DME include focal laser photocoagulation, intravitreal injections of

The mainstay of DME treatment is focal laser photocoagulation. Focal laser treatment for DME involves the application of discrete laser burns to areas of leakage in the macula. The treatment is not painful and can be repeated up to every 4 months if edema persists. Treatment criteria are based on the ETDRS recommendations (ETDRS group, 1995) which showed that eyes with macular edema involving or threatening the central macula, defined

**7.5 Treatment and prevention of diabetic retinopathy** 

is crucial to preventing and limiting disease progression.

intervention.

**7.5.1 Medical treatment** 

(Cheung et al., 2008).

ongoing studies. **7.5.2.1 NPDR** 

**7.5.2 Ocular treatment** 

7.5.2.3 (ETDRS group, 1995).

*7.5.2.2.1 Focal laser* 

**7.5.2.2 Diabetic macular edema (DME)** 

either steroids or anti-VEGF compounds and surgery.

retinopathy development by 30-40%.

as clinically significant macular edema (CSME), benefited from focal laser treatment. Focal laser treatment reduced the risk of moderate visual loss (loss of three lines of vision) by 50% over two years, increased the chance of improved vision and reduced central macular thickness compared to no treatment.

In eyes with macular edema that does not meet the criteria for CSME, laser treatment is not indicated. Close follow-up is recommended to determine the progression of the macular edema. Unfortunately, when the macular edema is associated with macular ischemia from a loss of macular capillary perfusion, the ETDRS showed a lesser beneficial effect for focal laser (ETDRS Group, 1995).

Side effects of focal laser photocoagulation include: paracentral visual field loss, transiently increased macular edema with decreased visual acuity and choroidal neovascularization (Regillo et al, 2010).

#### *7.5.2.2.2 Steroid injections*

Inflammatory factors play an important role in the development of diabetic retinopathy and macular edema (see section 7.3). For this reason it has long been thought that ocular steroid injections may be beneficial in DME treatment. Several trials have shown modestly improved visual acuity and central macular thickness after injection of intravitreal Triamcinolone (Grover at al., 2008; Yilmaz et al., 2009). A few recent trials on long acting steroid implants, such as Fluocinolone Acetate or Dexamethasone, have also reported short term visual acuity improvements (Grover et al., 2009).

The Diabetic Retinopathy Clinical Research Network (DRCR network, 2008) compared intravitreal injection of Triamcinolone to focal laser treatment in eyes with DME. There was no difference in visual acuity between the two groups after 1 year, and at 2 years eyes treated with laser had better vision. However, complications of intravitreal steroids, including elevated ocular pressure and increased cataract progression limit the usefulness of these drugs in DME. Intravitreal sterioid injections may be considered in patients who have previously undergone cataract surgery and in cases where the macular edema is refractory to focal laser. In these cases the injections may be given either alone or as an adjunct to laser treatment (Gillies et al., 2006; Maia et al., 2009).

#### *7.5.2.2.3 Anti-vascular endothelial growth factor (VEGF) compounds*

Vascular Endothelial Growth Factor (VEGF) is a major cause of the increased retinal vascular permeability which causes macular edema (Stirban et al., 2008). Several VEGF inhibitors have been investigated as treatments for DME with a beneficial effect on visual acuity and central macular thickness.

Injection of intravitreal Pegaptanib, a pegylated aptamer that inhibits one isoform of VEGF, was found to be better than sham injections in improving in visual acuity and decreasing the need for focal laser treatment, in the Macugen Diabetic Retinopathy Study group (Cunningham et al., 2005).

The injection of monoclonal antibodies that block all isoforms of VEGF has also been investigated as a treatment for DME. Several studies have shown a beneficial effect on visual acuity in eyes treated with Bevacizumab (trade name Avastin), a recombinant full length humanized antibody to VEGF (Nicholson & Shachat, 2010). Based on data from multiple studies (Nicholson & Shachat, 2010) repeated doses of Bevacizumab increase its average positive effect. The optimal timing for repeat dosing is unclear, but is probably between 3 to 12 weeks, with maximal effect with a 3 to 6 weeks interval between treatments.

Ocular Complications of Type 1 Diabetes 311

Fig. 12. Pan-Retinal Photocoagulation. The mainstay of treatment for PDR is laser ablation of the peripheral retina. In this treatment, known as panretinal photocoagulation (PRP), laser burns are placed over the entire retina (small black arrows), sparing only the central macula

Full PRP treatment as recommended by the DRS and the ETDRS includes as many as 4000 laser burns. PRP can be painful and is often performed over several sessions. After the initial treatment course, additional therapy can be applied if there is persistent neovascularisation. After treatment, proliferative retinal tissue may regress and contract causing a vitreous hemorrhage or a traction retinal detachment from contracture of fibrovascular tissue. Side effects of PRP treatment also include; decreased in night vision, decreased color vision and loss of peripheral vision. These side effects can be reduced by spreading out the treatment

When PDR presents with macular edema, PRP treatment may initially increase the amount of the edema (ETDRS Group, 1991). In such case it is recommended to treat the macular edema with either focal laser or an intravitreal injection before initiating PRP (Silva et al.,

In patients with new onset vitreous hemorrhages, laser PRP treatment is recommended if visualization of the retina is adequate. A severe, dense, non-clearing vitreous hemorrhage is an indication for vitrectomy surgery. The Diabetic Retinopathy Vitrectomy Study (DRVS Group, 1985) recommended surgery within 1 to 6 months of vitreous hemorrhage onset in type 1 diabetes patients with a non-clearing vitreous hemorrhage. Early vitrectomy improved visual acuity outcomes compared with waiting up to a year for spontaneous

(large black arrow) and the optic disc (white arrow).

2009, Mirshani et al., 2008). *7.5.2.3.2 Vitreous hemorrhage* 

resolution before resorting to surgery.

sessions and by using less energy at each session (Regillo et al. 2010).

The Bevacizumab Or Laser Treatment study (Michaelidis et al., BOLT study, 2010) compared 6-weekly bevacizumab injections to focal laser treatment in DME. One year postrandomization, eyes treated with bevacizumab had significantly better vision by over one line of acuity and less macular edema compared to eyes treated with focal laser. Bevacizumab treated eyes also had fivefold greater odds gaining at least 2 lines of vision.

Another promising drug that targets VEGF is Ranibizumab (trade name Lucentis). This a recombinant, humanized antibody fragment binds and inhibits all isoforms of VEGF. A recent study compared four treatment options; monthly injections of Ranibizumab combined with focal laser, monthly Ranibizumab alone with the option for rescue focal laser treatment, focal laser treatment alone and focal laser combined with intravitreal injections of Triamcinolone (DRCR network 2010a). After 1 year, eyes that received intravitreal injections of Ranibizumab, either combined with laser or with the option for rescue laser had better visual acuity compared with the other treatment groups. On average, eyes receiving Ranibizumab gained 1 line in visual acuity after 1 year. Half of Ranibizumab treated patients gained more than 2 lines in visual acuity, and 30% gained 3 lines or more. Two years results showed a similar positive treatment effect in DME with Ranibizumab injections.

The injection of anti-VEGF agents to the vitreous is both effective and safe. Adverse ocular effects include: cataract formation, retinal detachment, vitreous hemorrhage and infection. Potential systemic adverse effects include: hypertension, stroke, and myocardial infarction but these are very uncommon (Cheung et al., 2010; Nicholson & Shachat, 2010).

#### *7.5.2.2.4 Surgical intervention*

DME can also be treated surgically by performing a vitrectomy. This option is used sparingly because of the utility of both laser treatments and intravitreal injections in controlling the disease. Surgery is indicated in cases refractory to other treatments or when there is mechanical traction on the macula from vitreo-retinal adhesions. In such cases edema resolution can often not be obtained without resorting to intraocular surgery where the traction can be definitively released (Kaiser et al., 2001; DRCR network, 2010b).

#### **7.5.2.3 Proliferative diabetic retinopathy (PDR) treatment**

#### *7.5.2.3.1 Panretinal photocoagulation (PRP)*

The goal of treatment in PDR is to prevent complications and lower the risk of severe vision loss. The mainstay of treatment for PDR is laser ablation of the peripheral retina. In this treatment, known as panretinal photocoagulation (PRP), laser burns are placed over the entire retina, sparing only the central macula (figure 12). PRP promotes the regression and arrest of progression of retinal neovascularizations by destroying ischemic retinal tissue and reducing ischemia driven VEGF production (Cheung et al., 2010; Regillo et al., 2010).

The Diabetic Retinopathy Study (DRS) evaluated efficacy of PRP treatment in eyes with advanced NPDR or PDR (DRS Group, 1981). The DRS study recommended prompt treatment in eyes with high risk PDR (defined in section 7.4.2), because these eyes had the highest risk for severe visual loss. PRP treatment in these patients reduced the risk of severe visual loss by 50% over 5 years. The ETDRS study found that PRP treatment in eyes with early PDR reduced the risk of progression to high risk PDR by 50%, and significantly reduced the risk of severe visual loss (ETDRS Group, 1995). Based on these results, PRP treatment should be considered in eyes with any stage PDR especially if there is poor metabolic control, a non compliant patient or difficulty in maintaining close follow-up.

The Bevacizumab Or Laser Treatment study (Michaelidis et al., BOLT study, 2010) compared 6-weekly bevacizumab injections to focal laser treatment in DME. One year postrandomization, eyes treated with bevacizumab had significantly better vision by over one line of acuity and less macular edema compared to eyes treated with focal laser. Bevacizumab treated eyes also had fivefold greater odds gaining at least 2 lines of vision. Another promising drug that targets VEGF is Ranibizumab (trade name Lucentis). This a recombinant, humanized antibody fragment binds and inhibits all isoforms of VEGF. A recent study compared four treatment options; monthly injections of Ranibizumab combined with focal laser, monthly Ranibizumab alone with the option for rescue focal laser treatment, focal laser treatment alone and focal laser combined with intravitreal injections of Triamcinolone (DRCR network 2010a). After 1 year, eyes that received intravitreal injections of Ranibizumab, either combined with laser or with the option for rescue laser had better visual acuity compared with the other treatment groups. On average, eyes receiving Ranibizumab gained 1 line in visual acuity after 1 year. Half of Ranibizumab treated patients gained more than 2 lines in visual acuity, and 30% gained 3 lines or more. Two years results showed a similar positive

The injection of anti-VEGF agents to the vitreous is both effective and safe. Adverse ocular effects include: cataract formation, retinal detachment, vitreous hemorrhage and infection. Potential systemic adverse effects include: hypertension, stroke, and myocardial infarction

DME can also be treated surgically by performing a vitrectomy. This option is used sparingly because of the utility of both laser treatments and intravitreal injections in controlling the disease. Surgery is indicated in cases refractory to other treatments or when there is mechanical traction on the macula from vitreo-retinal adhesions. In such cases edema resolution can often not be obtained without resorting to intraocular surgery where

The goal of treatment in PDR is to prevent complications and lower the risk of severe vision loss. The mainstay of treatment for PDR is laser ablation of the peripheral retina. In this treatment, known as panretinal photocoagulation (PRP), laser burns are placed over the entire retina, sparing only the central macula (figure 12). PRP promotes the regression and arrest of progression of retinal neovascularizations by destroying ischemic retinal tissue and reducing ischemia driven VEGF production (Cheung et al., 2010; Regillo et al.,

The Diabetic Retinopathy Study (DRS) evaluated efficacy of PRP treatment in eyes with advanced NPDR or PDR (DRS Group, 1981). The DRS study recommended prompt treatment in eyes with high risk PDR (defined in section 7.4.2), because these eyes had the highest risk for severe visual loss. PRP treatment in these patients reduced the risk of severe visual loss by 50% over 5 years. The ETDRS study found that PRP treatment in eyes with early PDR reduced the risk of progression to high risk PDR by 50%, and significantly reduced the risk of severe visual loss (ETDRS Group, 1995). Based on these results, PRP treatment should be considered in eyes with any stage PDR especially if there is poor metabolic control, a non compliant patient or difficulty in maintaining close follow-up.

but these are very uncommon (Cheung et al., 2010; Nicholson & Shachat, 2010).

the traction can be definitively released (Kaiser et al., 2001; DRCR network, 2010b).

treatment effect in DME with Ranibizumab injections.

**7.5.2.3 Proliferative diabetic retinopathy (PDR) treatment** 

*7.5.2.3.1 Panretinal photocoagulation (PRP)* 

*7.5.2.2.4 Surgical intervention* 

2010).

Fig. 12. Pan-Retinal Photocoagulation. The mainstay of treatment for PDR is laser ablation of the peripheral retina. In this treatment, known as panretinal photocoagulation (PRP), laser burns are placed over the entire retina (small black arrows), sparing only the central macula (large black arrow) and the optic disc (white arrow).

Full PRP treatment as recommended by the DRS and the ETDRS includes as many as 4000 laser burns. PRP can be painful and is often performed over several sessions. After the initial treatment course, additional therapy can be applied if there is persistent neovascularisation. After treatment, proliferative retinal tissue may regress and contract causing a vitreous hemorrhage or a traction retinal detachment from contracture of fibrovascular tissue. Side effects of PRP treatment also include; decreased in night vision, decreased color vision and loss of peripheral vision. These side effects can be reduced by spreading out the treatment sessions and by using less energy at each session (Regillo et al. 2010).

When PDR presents with macular edema, PRP treatment may initially increase the amount of the edema (ETDRS Group, 1991). In such case it is recommended to treat the macular edema with either focal laser or an intravitreal injection before initiating PRP (Silva et al., 2009, Mirshani et al., 2008).

#### *7.5.2.3.2 Vitreous hemorrhage*

In patients with new onset vitreous hemorrhages, laser PRP treatment is recommended if visualization of the retina is adequate. A severe, dense, non-clearing vitreous hemorrhage is an indication for vitrectomy surgery. The Diabetic Retinopathy Vitrectomy Study (DRVS Group, 1985) recommended surgery within 1 to 6 months of vitreous hemorrhage onset in type 1 diabetes patients with a non-clearing vitreous hemorrhage. Early vitrectomy improved visual acuity outcomes compared with waiting up to a year for spontaneous resolution before resorting to surgery.

Ocular Complications of Type 1 Diabetes 313

In refractory cases Cyclodestructive procedures are required if medical therapy fails to provide symptomatic relief. With cyclocryotherapy, the IOP-lowering effect is achieved by destroying secretory ciliary epithelium and/or reducing blood flow to the ciliary body. It is

In women with preexisting diabetes, pregnancy is considered an independent risk factor for the development and progression of diabetic retinopathy (Shultz et al., 2005). Gestational diabetes, in absence of preexisting diabetes does not show a similar association with diabetic retinopathy. Most of the progression of diabetic retinopathy in pregnancy occurs by the end of the second trimester. Although regression of retinopathy usually occurs postpartum, there is still an increased risk for progression during the first year postpartum (Shultz et al., 2005). Risk factors for the development and progression of diabetic retinopathy in pregnancy include longer duration of diabetes before conception, rapid normalization of hemoglobin A1C at the beginning of pregnancy, poor glycemic control during pregnancy, diabetic nephropathy, high blood pressure and preeclampsia (Shultz et al., 2005; Vestgaard

Severity of diabetic retinopathy before or at beginning of pregnancy is also a strong predictor of progression of retinopathy during and after pregnancy. The Diabetes in Early Pregnancy Study (Chew et al., 1995) showed that 10.3% of women without diabetic retinopathy and 18.8% with mild NPDR experienced retinopathy progression during pregnancy, and 6.3% of women with mild NPDR progressed to PDR. In women with moderate NPDR, 54.8% suffered retinopathy progression and 29% developed PDR. Overall, progression to sight threatening diabetic retinopathy, including macular edema and PDR,

Progression of retinopathy during pregnancy is probably related to the hypervolemic and hyper-coagulable states in pregnancy, as well as elevated pro-inflammatory and angiogenic factor levels. This results in capillary occlusion and leakage aggravating diabetic retinopathy mechanisms (Shultz et al., 2005; Kastelan et al., 2010). Ideally, good glycemic control and full treatment of pre-existing diabetic retinopathy complications should be attained before

All diabetic women who plan pregnancy should be referred by their treating physician to an ophthalmologist. The recommended follow-up of pregnant women with type 1 diabetes includes an ophthalmologic exam at the beginning of pregnancy and during the first trimester. Subsequent follow-up depends on the stage of diabetic retinopathy found on the initial examinations. In women with no retinopathy or very mild NPDR, an ophthalmologic exam is indicated when there are visual complaints. In moderate NPDR an exam should be done at least once during the second trimester and every 4-6 weeks during the third trimester. In severe NPDR and PDR, close follow-up is needed, and an exam should be done

Treatment of diabetic retinopathy during pregnancy includes maximal control of both glucose levels and blood pressure (Vestgaard et al., 2010). Ocular therapy such as PRP should definitely be performed for PDR and be strongly considered in cases of severe NPDR. Disease progression can be very fast in pregnancy and waiting for PDR to clearly

indicated as a last resort only if relief of pain is the main goal.

occurs in 6% of pregnant diabetic women (Vestgaard et al., 2010).

every 4-6 weeks, from the beginning of the second trimester.

**7.6 Special considerations** 

et al., 2010).

conception.

**7.6.1 Diabetic retinopathy in pregnancy** 

Recent advances in vitreoretinal surgery, including smaller gauge instruments and the ability to perform laser ablation during surgery, have changed treatment recommendations. If a patient with a vitreous hemorrhage has not previously undergone PRP, vitrectomy is recommended when a dense vitreous hemorrhage persists beyond one to three months. Patients with vitreous hemorrhage that have preexisting complete PRP may undergo a longer observation period (Regillo et al., 2010).

Several studies have evaluated the efficacy of intravitreal anti-VEGF injections in patients with PDR (Nicholson & Shachat, 2010). Intravitreal Bevacizumab as adjunctive therapy with PRP was found to decrease leakage area from neovascularizations, improve visual acuity outcomes and reduce macular edema compared with PRP alone. In eyes with PDR and a dense vitreous hemorrhage preventing full PRP treatment, a Bevacizumab injection has been shown to aid significantly in clearing the hemorrhage (Moradian et al., 2008).

Bevacizumab has also been shown to enhance retinal surgery in patients with PDR. A single Bevacizumab injection given 1-2 weeks before vitrectomy for vitreous hemorrhage, results in decreased bleeding during surgery, decreased operating time and less post operative vitreous hemorrhage as compared to vitrectomy alone (Nicholson & Shachat, 2010; Ahmadieh et al.,2009).

#### *7.5.2.3.3 Traction retinal detachment*

Traction retinal detachment from the contraction of the neovascular tissue connecting the retinal surface to the vitreous is another serious complication of PDR. However, traction detachments which do not involve the macula can remain stable for years. Vitrectomy surgery is indicated only when the traction retinal detachment involves or threatens the central macula or if a retinal tear develops (Regillo et al. 2010).

#### *7.5.2.3.4 Neovascular glaucoma*

A third serious complication of PDR occurs when high levels of VEGF in the retina induce the development of new vessels on the iris. These vessels threaten to block the outflow of aqueous fluid from the eye and raise ocular pressure. The treatment of patients with neovascularisation of the iris involves both the minimization of retinal ischemia and the aggressive reduction of intraocular pressure if elevated. Retinal ischemia is treated with aggressive and extensive ablation of peripheral retinal tissue with PRP regardless of the stage of PDR. Injection of intravitreal anti-VEGF agents as adjunctive therapy to PRP can induce a rapid reduction or resolution of neovascularisation of the iris (Ahmadieh et al., 2009; Wasik et al., 2009). However, these injections should be seen as an adjunct to full PRP which remains the definitive treatment and not as a viable replacement.

Elevated ocular pressure in neovascular glaucoma is treated initially with topical medications. Often multiple drops are required to reduce pressure to below the target level of approximately 20 millimeters of mercury. In advanced cases, topical treatment alone may not be sufficient and systemic treatment with carbonic anhydrase inhibitors such as Acetazolamide may be considered. Common side effects of Acetazolamide include numbness and tingling in the fingers and toes, and taste alterations. Acetazolamide also increases the risk of dehydration and metabolic acidosis. Serial electrolyte and kidney function tests are recommended in all patients receiving this medication.

Recent advances in vitreoretinal surgery, including smaller gauge instruments and the ability to perform laser ablation during surgery, have changed treatment recommendations. If a patient with a vitreous hemorrhage has not previously undergone PRP, vitrectomy is recommended when a dense vitreous hemorrhage persists beyond one to three months. Patients with vitreous hemorrhage that have preexisting complete PRP may undergo a

Several studies have evaluated the efficacy of intravitreal anti-VEGF injections in patients with PDR (Nicholson & Shachat, 2010). Intravitreal Bevacizumab as adjunctive therapy with PRP was found to decrease leakage area from neovascularizations, improve visual acuity outcomes and reduce macular edema compared with PRP alone. In eyes with PDR and a dense vitreous hemorrhage preventing full PRP treatment, a Bevacizumab injection has been shown to aid significantly in clearing the hemorrhage

Bevacizumab has also been shown to enhance retinal surgery in patients with PDR. A single Bevacizumab injection given 1-2 weeks before vitrectomy for vitreous hemorrhage, results in decreased bleeding during surgery, decreased operating time and less post operative vitreous hemorrhage as compared to vitrectomy alone (Nicholson & Shachat, 2010;

Traction retinal detachment from the contraction of the neovascular tissue connecting the retinal surface to the vitreous is another serious complication of PDR. However, traction detachments which do not involve the macula can remain stable for years. Vitrectomy surgery is indicated only when the traction retinal detachment involves or threatens the

A third serious complication of PDR occurs when high levels of VEGF in the retina induce the development of new vessels on the iris. These vessels threaten to block the outflow of aqueous fluid from the eye and raise ocular pressure. The treatment of patients with neovascularisation of the iris involves both the minimization of retinal ischemia and the aggressive reduction of intraocular pressure if elevated. Retinal ischemia is treated with aggressive and extensive ablation of peripheral retinal tissue with PRP regardless of the stage of PDR. Injection of intravitreal anti-VEGF agents as adjunctive therapy to PRP can induce a rapid reduction or resolution of neovascularisation of the iris (Ahmadieh et al., 2009; Wasik et al., 2009). However, these injections should be seen as an adjunct to full PRP which remains the definitive

Elevated ocular pressure in neovascular glaucoma is treated initially with topical medications. Often multiple drops are required to reduce pressure to below the target level of approximately 20 millimeters of mercury. In advanced cases, topical treatment alone may not be sufficient and systemic treatment with carbonic anhydrase inhibitors such as Acetazolamide may be considered. Common side effects of Acetazolamide include numbness and tingling in the fingers and toes, and taste alterations. Acetazolamide also increases the risk of dehydration and metabolic acidosis. Serial electrolyte and kidney function tests are recommended in all patients receiving this

longer observation period (Regillo et al., 2010).

(Moradian et al., 2008).

Ahmadieh et al.,2009).

*7.5.2.3.3 Traction retinal detachment* 

*7.5.2.3.4 Neovascular glaucoma* 

medication.

treatment and not as a viable replacement.

central macula or if a retinal tear develops (Regillo et al. 2010).

In refractory cases Cyclodestructive procedures are required if medical therapy fails to provide symptomatic relief. With cyclocryotherapy, the IOP-lowering effect is achieved by destroying secretory ciliary epithelium and/or reducing blood flow to the ciliary body. It is indicated as a last resort only if relief of pain is the main goal.

#### **7.6 Special considerations**

#### **7.6.1 Diabetic retinopathy in pregnancy**

In women with preexisting diabetes, pregnancy is considered an independent risk factor for the development and progression of diabetic retinopathy (Shultz et al., 2005). Gestational diabetes, in absence of preexisting diabetes does not show a similar association with diabetic retinopathy. Most of the progression of diabetic retinopathy in pregnancy occurs by the end of the second trimester. Although regression of retinopathy usually occurs postpartum, there is still an increased risk for progression during the first year postpartum (Shultz et al., 2005). Risk factors for the development and progression of diabetic retinopathy in pregnancy include longer duration of diabetes before conception, rapid normalization of hemoglobin A1C at the beginning of pregnancy, poor glycemic control during pregnancy, diabetic nephropathy, high blood pressure and preeclampsia (Shultz et al., 2005; Vestgaard et al., 2010).

Severity of diabetic retinopathy before or at beginning of pregnancy is also a strong predictor of progression of retinopathy during and after pregnancy. The Diabetes in Early Pregnancy Study (Chew et al., 1995) showed that 10.3% of women without diabetic retinopathy and 18.8% with mild NPDR experienced retinopathy progression during pregnancy, and 6.3% of women with mild NPDR progressed to PDR. In women with moderate NPDR, 54.8% suffered retinopathy progression and 29% developed PDR. Overall, progression to sight threatening diabetic retinopathy, including macular edema and PDR, occurs in 6% of pregnant diabetic women (Vestgaard et al., 2010).

Progression of retinopathy during pregnancy is probably related to the hypervolemic and hyper-coagulable states in pregnancy, as well as elevated pro-inflammatory and angiogenic factor levels. This results in capillary occlusion and leakage aggravating diabetic retinopathy mechanisms (Shultz et al., 2005; Kastelan et al., 2010). Ideally, good glycemic control and full treatment of pre-existing diabetic retinopathy complications should be attained before conception.

All diabetic women who plan pregnancy should be referred by their treating physician to an ophthalmologist. The recommended follow-up of pregnant women with type 1 diabetes includes an ophthalmologic exam at the beginning of pregnancy and during the first trimester. Subsequent follow-up depends on the stage of diabetic retinopathy found on the initial examinations. In women with no retinopathy or very mild NPDR, an ophthalmologic exam is indicated when there are visual complaints. In moderate NPDR an exam should be done at least once during the second trimester and every 4-6 weeks during the third trimester. In severe NPDR and PDR, close follow-up is needed, and an exam should be done every 4-6 weeks, from the beginning of the second trimester.

Treatment of diabetic retinopathy during pregnancy includes maximal control of both glucose levels and blood pressure (Vestgaard et al., 2010). Ocular therapy such as PRP should definitely be performed for PDR and be strongly considered in cases of severe NPDR. Disease progression can be very fast in pregnancy and waiting for PDR to clearly

up.

**9. Summary** 

**10. References** 

0161-6420

Ocular Complications of Type 1 Diabetes 315

The timing and frequency of follow-up ocular examinations depends on individual patient's status. In high risk patients with long term diabetes and poor systemic risk factor control annual examinations should be performed even in the absence of retinopathy. In patients with known retinopathy, the examination schedule is based on the degree of retinopathy, and on the patient's compliance and adherence to regular follow-up. In more advances stages such as PDR and when macular edema is present, more frequent and careful followup is suggested. (Regillo et al., 2010). Table 2 shows the recommended schedule for follow–

**Retinopathy Stage Follow-up Schedule** 

Normal or rare microaneurysms Annually

Mild NPDR Every 9 months

Moderate NPDR Every 6 months

Severe NPDR Every 2-4 months

Clinically significant macular edema Every 2-4 months

Academy of Ophthalmology, 2003, as cited in Regillo et al., 2010).

threatening complications, significantly enhancing patient quality of life.

PDR Every 2-3 months (careful follow-up)

Table 2. Suggested time table for follow-up in diabetic retinopathy (modified from the Preferred Practice Patterns committee, retina panel, diabetic retinopathy, American

Management of type 1 diabetes involves close cooperation between the treating primary physician and the many specialists who help manage the complications of this disease. Recent advances, including intraocular anti-VEGF injections, have added important new tools which minimize vision loss in diabetic eye disease. Proactive, interdisciplinary coordination of treatment and timely referrals can aid in the minimization of visually

Ahmadieh H.; Shoeibi N.; Entezari M.; Monshizadeh R. (2009). Intravitreal Bevacizumab for

Bobrow JC.; Blecher MH.; Glasser D. et al. (Eds.) (2010). Section 11: Lens and cataract. *Basic* 

Americam Academy of Ophthalmology, ISBN 9781615251391

prevention of early postvitrectomy hemorrhage in diabetic patients: a randomized clinical trial. *Ophthalmology*, Vol. 116, No.10, (October 2010), pp. 1943-1948, ISSN

*and Clinical Science Course, 2010-2011, American Academy of Ophthalmology.*

develop may result in severe complications that necessitate invasive surgery. Ocular therapy for PDR and macular edema during pregnancy can include PRP, focal laser and intravitreal injections of Triamcinolone. Although there is not much data on the safety of intravitreal injections of anti-VEGF agents during pregnancy, the literature includes some reports on the safe and effective use of Bevacizumab (Tarantola et al., 2010).

#### **7.6.2 Cataract surgery in patients with diabetic retinopathy**

Cataract is a major factor which compromises vision in diabetic patients. While diabetics may benefit from cataract extraction, a controversy exists in the ophthalmic community as to whether cataract surgery potentiates diabetic retinopathy progression. Several studies have reported worsening of diabetic retinopathy and macular edema after surgery (Pollack et al., 1991; Hauser et al., 2004; Jaffe et al., 1992, Hayashi et al., 2009). Progression was seen during the first year after surgery and was highest in the first 3 months postoperatively. A review of several other studies, especially in the cataract surgery era using the smaller incision phacoemulsification technique, showed no significant progression of diabetic retinopathy and macular edema after surgery (Rashid & Young, 2010; Shah & Chen, 2010). Overall, diabetics with cataracts benefit from surgery, and improved visual acuity is reported in 92-94% of patients (Rashid & Young, 2010). The combined evidence suggests that in patients with low risk or absent diabetic retinopathy and no clinically significant macular edema at the time of surgery, there is little increased risk of retinopathy progression. However, in patients with severe NPDR, PDR or significant macular edema, cataract surgery carries an increased risk for retinopathy progression and a worse visual acuity outcome.

Recent studies have shown a potential benefit using intravitreal injection of Bevacizumab at the end of cataract surgery (Cheema et al., 2009; Chen et al., 2009; Nicholson & Shachat, 2010) especially in cases with poorly controlled or refractory macular edema and diabetic retinopathy before surgery. Patients who received intravitreal Bevacizumab enjoyed better outcomes in terms of visual acuity, macular thickness and retinopathy progression.

A thorough evaluation of patients with diabetes is warranted before cataract surgery. Patients who have severe NPDR or PDR should be considered for PRP treatment prior to cataract removal (Chew et al., 1999). Patients with clinically significant macular edema should undergo treatment, such as focal laser or intravitreal injection of anti- VEGF agents pre-operatively. Ideally, surgery should be delayed until stabilization of retinopathy and macular edema is achieved. In refractory cases, adjunctive therapy with an anti-VEGF agent at the end of cataract surgery should be considered. Close post-operative follow-up with an ophthalmologist is highly recommended in all patients with preexisting diabetic retinopathy.

#### **8. Schedule for ophthalmologic examinations**

Regular ocular examination can detect early ocular disease such as cataracts and glaucoma as well as retinopathy. Diabetic retinopathy in type 1 diabetes is rare during the first 5 years after diagnosis, so the baseline ophthalmologic examination could be extended to 5 years after diagnosis if blood glucose has been well controlled. In children with pre-pubertal diabetes, the baseline examination should be done at puberty (Raab et al. 2010).

develop may result in severe complications that necessitate invasive surgery. Ocular therapy for PDR and macular edema during pregnancy can include PRP, focal laser and intravitreal injections of Triamcinolone. Although there is not much data on the safety of intravitreal injections of anti-VEGF agents during pregnancy, the literature includes some reports on the

Cataract is a major factor which compromises vision in diabetic patients. While diabetics may benefit from cataract extraction, a controversy exists in the ophthalmic community as to whether cataract surgery potentiates diabetic retinopathy progression. Several studies have reported worsening of diabetic retinopathy and macular edema after surgery (Pollack et al., 1991; Hauser et al., 2004; Jaffe et al., 1992, Hayashi et al., 2009). Progression was seen during the first year after surgery and was highest in the first 3 months postoperatively. A review of several other studies, especially in the cataract surgery era using the smaller incision phacoemulsification technique, showed no significant progression of diabetic retinopathy and macular edema after surgery (Rashid & Young, 2010; Shah & Chen, 2010). Overall, diabetics with cataracts benefit from surgery, and improved visual acuity is reported in 92-94% of patients (Rashid & Young, 2010). The combined evidence suggests that in patients with low risk or absent diabetic retinopathy and no clinically significant macular edema at the time of surgery, there is little increased risk of retinopathy progression. However, in patients with severe NPDR, PDR or significant macular edema, cataract surgery carries an increased risk for retinopathy progression and

Recent studies have shown a potential benefit using intravitreal injection of Bevacizumab at the end of cataract surgery (Cheema et al., 2009; Chen et al., 2009; Nicholson & Shachat, 2010) especially in cases with poorly controlled or refractory macular edema and diabetic retinopathy before surgery. Patients who received intravitreal Bevacizumab enjoyed better outcomes in terms of visual acuity, macular thickness and retinopathy

A thorough evaluation of patients with diabetes is warranted before cataract surgery. Patients who have severe NPDR or PDR should be considered for PRP treatment prior to cataract removal (Chew et al., 1999). Patients with clinically significant macular edema should undergo treatment, such as focal laser or intravitreal injection of anti- VEGF agents pre-operatively. Ideally, surgery should be delayed until stabilization of retinopathy and macular edema is achieved. In refractory cases, adjunctive therapy with an anti-VEGF agent at the end of cataract surgery should be considered. Close post-operative follow-up with an ophthalmologist is highly recommended in all patients with preexisting diabetic

Regular ocular examination can detect early ocular disease such as cataracts and glaucoma as well as retinopathy. Diabetic retinopathy in type 1 diabetes is rare during the first 5 years after diagnosis, so the baseline ophthalmologic examination could be extended to 5 years after diagnosis if blood glucose has been well controlled. In children with pre-pubertal

diabetes, the baseline examination should be done at puberty (Raab et al. 2010).

safe and effective use of Bevacizumab (Tarantola et al., 2010).

**7.6.2 Cataract surgery in patients with diabetic retinopathy** 

a worse visual acuity outcome.

**8. Schedule for ophthalmologic examinations** 

progression.

retinopathy.

The timing and frequency of follow-up ocular examinations depends on individual patient's status. In high risk patients with long term diabetes and poor systemic risk factor control annual examinations should be performed even in the absence of retinopathy. In patients with known retinopathy, the examination schedule is based on the degree of retinopathy, and on the patient's compliance and adherence to regular follow-up. In more advances stages such as PDR and when macular edema is present, more frequent and careful followup is suggested. (Regillo et al., 2010). Table 2 shows the recommended schedule for follow– up.


Table 2. Suggested time table for follow-up in diabetic retinopathy (modified from the Preferred Practice Patterns committee, retina panel, diabetic retinopathy, American Academy of Ophthalmology, 2003, as cited in Regillo et al., 2010).

#### **9. Summary**

Management of type 1 diabetes involves close cooperation between the treating primary physician and the many specialists who help manage the complications of this disease. Recent advances, including intraocular anti-VEGF injections, have added important new tools which minimize vision loss in diabetic eye disease. Proactive, interdisciplinary coordination of treatment and timely referrals can aid in the minimization of visually threatening complications, significantly enhancing patient quality of life.

#### **10. References**


Ocular Complications of Type 1 Diabetes 317

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**Part 4** 

**Treatment** 


## **Part 4**

## **Treatment**

320 Type 1 Diabetes Complications

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**15** 

*Italy* 

*University of Torino,* 

**Perspectives of Cell Therapy in Type 1 Diabetes** 

Type 1 diabetes is an autoimmune disease leading to the destruction of pancreatic cells. The reduction of cell mass results in insulin deficiency that leads to a failure of glucose homeostasis with increased levels of glucose in blood. Hyperglycemia which *per se* is detrimental for the organism and may be life-threatening, may in the long term associate with chronic complications involving blood vessels and nerves. The gold standard treatment for diabetes patients aimed to reach a tight control of glycemia, relies on intensive insulin therapy based on multiple daily injections or continuous subcutaneous infusion of insulin. A tight control of glycemia reached with such regimens was shown to significantly reduce the incidence of microvascular complications in respect to the conventional insulin therapy. Nevertheless, to reach an optimal control of blood levels may prove to be difficult when compared to the physiological condition where this is guaranteed by the pancreatic cells (Suckale, 2008). Therefore, preservation of cell mass could be an important therapeutic target to reduce microvascular complications and to improve the glycemia control (Gonez & Knight, 2010). It is generally accepted that the endocrine pancreas has some regenerative capabilities, although it is still debated which cells are involved in cell turnover. In rodents the capability of adult pancreas to increase cell mass has been documented in physiological conditions and after injury. The understanding of mechanisms involved in cell turnover may therefore be relevant to design new therapeutic strategies aimed to maintain a cell mass or to favour regeneration of cells. These strategies however, should take to account the problem of recurrent autoimmunity that in type 1 diabetes not only impairs the original cell mass, but may also limit the regenerative process. Indeed, autoimmune T lymphocytes may kill the cells newly formed in response to injury (Fan &

**2. cell regeneration: Contribution of stem/progenitor cells or replication of** 

The physiological turnover of the long-lasting endocrine cells of pancreas requires the

generation of new cells even with a very slow kinetic.

**1. Introduction** 

Rudert, 2009).

 **cell?** 

Maria M. Zanone, Vincenzo Cantaluppi, Enrica Favaro,

Elisa Camussi, Maria Chiara Deregibus and Giovanni Camussi

*Renal and Vascular Physiopathology Laboratory, Department of Internal Medicine, Molecular Biotechnology Centre and Research Centre for Molecular Medicine,* 

### **Perspectives of Cell Therapy in Type 1 Diabetes**

Maria M. Zanone, Vincenzo Cantaluppi, Enrica Favaro, Elisa Camussi, Maria Chiara Deregibus and Giovanni Camussi *Renal and Vascular Physiopathology Laboratory, Department of Internal Medicine, Molecular Biotechnology Centre and Research Centre for Molecular Medicine, University of Torino, Italy* 

**1. Introduction** 

Type 1 diabetes is an autoimmune disease leading to the destruction of pancreatic cells. The reduction of cell mass results in insulin deficiency that leads to a failure of glucose homeostasis with increased levels of glucose in blood. Hyperglycemia which *per se* is detrimental for the organism and may be life-threatening, may in the long term associate with chronic complications involving blood vessels and nerves. The gold standard treatment for diabetes patients aimed to reach a tight control of glycemia, relies on intensive insulin therapy based on multiple daily injections or continuous subcutaneous infusion of insulin. A tight control of glycemia reached with such regimens was shown to significantly reduce the incidence of microvascular complications in respect to the conventional insulin therapy. Nevertheless, to reach an optimal control of blood levels may prove to be difficult when compared to the physiological condition where this is guaranteed by the pancreatic cells (Suckale, 2008). Therefore, preservation of cell mass could be an important therapeutic target to reduce microvascular complications and to improve the glycemia control (Gonez & Knight, 2010). It is generally accepted that the endocrine pancreas has some regenerative capabilities, although it is still debated which cells are involved in cell turnover. In rodents the capability of adult pancreas to increase cell mass has been documented in physiological conditions and after injury. The understanding of mechanisms involved in cell turnover may therefore be relevant to design new therapeutic strategies aimed to maintain a cell mass or to favour regeneration of cells. These strategies however, should take to account the problem of recurrent autoimmunity that in type 1 diabetes not only impairs the original cell mass, but may also limit the regenerative process. Indeed, autoimmune T lymphocytes may kill the cells newly formed in response to injury (Fan & Rudert, 2009).

#### **2. cell regeneration: Contribution of stem/progenitor cells or replication of cell?**

The physiological turnover of the long-lasting endocrine cells of pancreas requires the generation of new cells even with a very slow kinetic.

Perspectives of Cell Therapy in Type 1 Diabetes 325

A problem in the identification of tissue resident stem cells is that we do not know specific markers allowing tracing of pluripotent stem cells in various tissues. Therefore, resident stem cells are mainly defined by functional *in vitro* assays using cultured cells, and their *in vivo* exact localization and function remains elusive. Several studies suggest that the adult tissue resident stem cells belong to the MSC lineage (da Silva Meirelles et al., 2006). The minimal criteria to define human MSC established by *Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy* (da Silva Meirelles et al., 2008; Dominici et al., 2006), include cell positivity for CD105, CD73, and CD90 and negativity for CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR as well as osteo-, chondro-, and

A perivascular location for MSC has been suggested, correlating these cells with pericytes providing an explanation for the presence of MSC virtually in all vascularized tissues (da Silva Meirelles et al., 2006). The perivascular zone may act as a MSC niche *in vivo*, where microenvironment factors may modulate their phenotype with transition to progenitor and mature cells. For many years the concept of niche has been associated with a hierarchical nature of stem cells that undergoing asymmetric division insure self-renewal and generation of a progeny with progressive loss of proliferative potential and gain of differentiated characteristics (Till et al., 1964). More recently, a continuum model of stem cell biology has been proposed (Colvin et al., 2004; Quesenberry et al., 2005). It has been postulated that the phenotype of stem cells is labile, it varies with position in the cell cycle and that it is reversible (Colvin et al., 2007). This cell-cycle reversibility is at the basis of the continuum model of stem cell biology, in which the phenotype of stem cells is reversibly changing during the cell cycle transit awaiting for a terminal-differentiating stimulus at a cyclesusceptible time. In this model the status of the cell cycle and the exposure to environmental factors play critical roles in the acquirement of different phenotypes by the same cell in different functional states (Quesenberry et al., 2007). Recently, Quesenberry and Aliotta proposed that the so-called niche consists in areas of influence which are continually adjusting to individual circumstances (Quesenberry & Aliotta, 2008). Based on these considerations, the refined regenerative system in mammalians does not need to position in each organ different stem cell types but it would be sufficient to maintain few undifferentiated cells with self-renewal capability that depending on the circumstances may vary their phenotype to replace the loss of differentiated cells. On the other hand, differentiated cells may re-acquire an undifferentiated phenotype and re-entry in to cell cycle first to restore the cell mass and subsequently to re-differentiate and restore functional integrity. In this context, the exchange of genetic information among cells by microvesicles in a defined environment plays a critical role in modulating plasticity of stem cells as well as

the response of differentiated cells to injury (Deregibus et al., 2010).

capacity to replicate.

Studies on cell proliferation in humans are limited, but there is evidence that this process occurs at relatively high levels in the first 2 years of life declining thereafter with the possibility, at least in animals, of re-induction under conditions of insulin-resistance, such as pregnancy or obesity. (Meier et al., 2008). This suggests that cells may retain an intrinsic

In the adult, endocrine pancreas cells are considered to have a very low turnover. However, albeit quite slowly, cells undergo senescence and should be continuously replaced by newly formed cells. By combining abdominal CT scans and morphometric analysis of human pancreatic tissue, Meier et al reported that the cell mass expands by

adipogenic-differentiation capabilities.

The organ growth after birth requires a coordinated increase in the number of constitutive cells. Moreover, in the adult body most of the tissues and organs have the ability to replace the cells that die for physiological senescence or following limited injury. The source of newly formed cells may derive from resident stem/progenitor cells or from the ability of differentiated cells to re-entry into cell cycle and replicate themselves. The prevalence of these two mechanisms varies in different organs and tissues; however, they may result in the restoration of the original tissue conformation and function. We therefore should expect that also the endocrine pancreas retains the ability of regeneration in appropriate physiological conditions. The nature and the location of the cells involved in such processes, as well as the mechanisms involved in the activation of the regenerative processes, still remain largely unknown.

The concept of "stem cell" implies the ability of unlimited self renewal and of high multilineage differentiation potential into different types of mature cells. Therefore, stem cells play fundamental roles in organogenesis during embryonic development, and in the adult are responsible for the growth, homeostasis and repair of many tissues.

In the haematopoietic system, the intestine and the skin, tissues that require a high cell turnover, the stem cells are critical for maintaining their homeostasis. However, adult stem/progenitor cells are present in the majority of tissues and organs of mammalian organisms, including the central nervous system (Reynolds & Weiss, 1992), retina (Tropepe et al., 2000), skeletal muscle (Jackson et al., 1999), liver (Herrera et al., 2006) and kidney (Bussolati et al., 2005).

In tissues with a low rate of cell turnover, such as the kidney, the lung, the skeletal muscle and the liver, the resident stem cells may activate after injury and participate in tissue repair. Tissue resident stem cells preferentially generate differentiated cells of the tissue of origin, suggesting a relevant role in the postnatal growth of organs, in physiological turnover and in tissue repair. Tissue-resident adult stem cells are thought to co-localize with supporting cells within specific regions or specialized microenvironments in each tissue/organ, called stem cell niche (Jones & Wagers, 2008; L. Li et al., 2005; Moore & Lemischka, 2006). In bone marrow the haematopoietic stem cells (HSC) are located in the endosteal niche, associated with the osteoblasts of the inner surface of the cavities of trabecular bone that could provide factors able to regulate number and function of HSC (Mitsiadis et al., 2007) and in the perivascular area of sinusoids that could ensure homeostatic blood cell production and prompt responses to haematological stresses (Kiel et al., 2005). The other stem cells present in bone marrow are the mesenchymal stem cells (MSC), undifferentiated adult stem cells of mesodermal origin, which localize in perivascular areas in the bone marrow in close association with HSC (Shi & Gronthos, 2003) and that have the capacity to differentiate into cells of connective tissue lineages, including bone, fat, cartilage and muscle (Y. Jiang et al., 2002).

Other stem cell niches detected in mammals (da Silva Meirelles et al., 2008; L. Li & Xie, 2005) are the epithelial stem cell niche in skin that resides in the bulge area of the hair follicle beneath the sebaceous gland (Cotsarelis et al., 1990; Niemann & Watt, 2002; Sun et al., 1991), the intestinal stem cell niche located at the fourth or fifth position above the Paneth cells from the crypt bottom (Booth & Potten, 2000; He et al., 2004; Sancho et al., 2004) and the neural stem cell niche at the subventricular zone and the subgranular zone of the hippocampus where neural stem cells could reside and support neurogenesis in the adult brain (Doetsch et al., 1999; Temple, 2001).

The organ growth after birth requires a coordinated increase in the number of constitutive cells. Moreover, in the adult body most of the tissues and organs have the ability to replace the cells that die for physiological senescence or following limited injury. The source of newly formed cells may derive from resident stem/progenitor cells or from the ability of differentiated cells to re-entry into cell cycle and replicate themselves. The prevalence of these two mechanisms varies in different organs and tissues; however, they may result in the restoration of the original tissue conformation and function. We therefore should expect that also the endocrine pancreas retains the ability of regeneration in appropriate physiological conditions. The nature and the location of the cells involved in such processes, as well as the mechanisms involved in the activation of the regenerative processes, still

The concept of "stem cell" implies the ability of unlimited self renewal and of high multilineage differentiation potential into different types of mature cells. Therefore, stem cells play fundamental roles in organogenesis during embryonic development, and in the

In the haematopoietic system, the intestine and the skin, tissues that require a high cell turnover, the stem cells are critical for maintaining their homeostasis. However, adult stem/progenitor cells are present in the majority of tissues and organs of mammalian organisms, including the central nervous system (Reynolds & Weiss, 1992), retina (Tropepe et al., 2000), skeletal muscle (Jackson et al., 1999), liver (Herrera et al., 2006) and kidney

In tissues with a low rate of cell turnover, such as the kidney, the lung, the skeletal muscle and the liver, the resident stem cells may activate after injury and participate in tissue repair. Tissue resident stem cells preferentially generate differentiated cells of the tissue of origin, suggesting a relevant role in the postnatal growth of organs, in physiological turnover and in tissue repair. Tissue-resident adult stem cells are thought to co-localize with supporting cells within specific regions or specialized microenvironments in each tissue/organ, called stem cell niche (Jones & Wagers, 2008; L. Li et al., 2005; Moore & Lemischka, 2006). In bone marrow the haematopoietic stem cells (HSC) are located in the endosteal niche, associated with the osteoblasts of the inner surface of the cavities of trabecular bone that could provide factors able to regulate number and function of HSC (Mitsiadis et al., 2007) and in the perivascular area of sinusoids that could ensure homeostatic blood cell production and prompt responses to haematological stresses (Kiel et al., 2005). The other stem cells present in bone marrow are the mesenchymal stem cells (MSC), undifferentiated adult stem cells of mesodermal origin, which localize in perivascular areas in the bone marrow in close association with HSC (Shi & Gronthos, 2003) and that have the capacity to differentiate into cells of connective tissue lineages, including

Other stem cell niches detected in mammals (da Silva Meirelles et al., 2008; L. Li & Xie, 2005) are the epithelial stem cell niche in skin that resides in the bulge area of the hair follicle beneath the sebaceous gland (Cotsarelis et al., 1990; Niemann & Watt, 2002; Sun et al., 1991), the intestinal stem cell niche located at the fourth or fifth position above the Paneth cells from the crypt bottom (Booth & Potten, 2000; He et al., 2004; Sancho et al., 2004) and the neural stem cell niche at the subventricular zone and the subgranular zone of the hippocampus where neural stem cells could reside and support neurogenesis in the adult

adult are responsible for the growth, homeostasis and repair of many tissues.

remain largely unknown.

(Bussolati et al., 2005).

bone, fat, cartilage and muscle (Y. Jiang et al., 2002).

brain (Doetsch et al., 1999; Temple, 2001).

A problem in the identification of tissue resident stem cells is that we do not know specific markers allowing tracing of pluripotent stem cells in various tissues. Therefore, resident stem cells are mainly defined by functional *in vitro* assays using cultured cells, and their *in vivo* exact localization and function remains elusive. Several studies suggest that the adult tissue resident stem cells belong to the MSC lineage (da Silva Meirelles et al., 2006). The minimal criteria to define human MSC established by *Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy* (da Silva Meirelles et al., 2008; Dominici et al., 2006), include cell positivity for CD105, CD73, and CD90 and negativity for CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR as well as osteo-, chondro-, and adipogenic-differentiation capabilities.

A perivascular location for MSC has been suggested, correlating these cells with pericytes providing an explanation for the presence of MSC virtually in all vascularized tissues (da Silva Meirelles et al., 2006). The perivascular zone may act as a MSC niche *in vivo*, where microenvironment factors may modulate their phenotype with transition to progenitor and mature cells. For many years the concept of niche has been associated with a hierarchical nature of stem cells that undergoing asymmetric division insure self-renewal and generation of a progeny with progressive loss of proliferative potential and gain of differentiated characteristics (Till et al., 1964). More recently, a continuum model of stem cell biology has been proposed (Colvin et al., 2004; Quesenberry et al., 2005). It has been postulated that the phenotype of stem cells is labile, it varies with position in the cell cycle and that it is reversible (Colvin et al., 2007). This cell-cycle reversibility is at the basis of the continuum model of stem cell biology, in which the phenotype of stem cells is reversibly changing during the cell cycle transit awaiting for a terminal-differentiating stimulus at a cyclesusceptible time. In this model the status of the cell cycle and the exposure to environmental factors play critical roles in the acquirement of different phenotypes by the same cell in different functional states (Quesenberry et al., 2007). Recently, Quesenberry and Aliotta proposed that the so-called niche consists in areas of influence which are continually adjusting to individual circumstances (Quesenberry & Aliotta, 2008). Based on these considerations, the refined regenerative system in mammalians does not need to position in each organ different stem cell types but it would be sufficient to maintain few undifferentiated cells with self-renewal capability that depending on the circumstances may vary their phenotype to replace the loss of differentiated cells. On the other hand, differentiated cells may re-acquire an undifferentiated phenotype and re-entry in to cell cycle first to restore the cell mass and subsequently to re-differentiate and restore functional integrity. In this context, the exchange of genetic information among cells by microvesicles in a defined environment plays a critical role in modulating plasticity of stem cells as well as the response of differentiated cells to injury (Deregibus et al., 2010).

Studies on cell proliferation in humans are limited, but there is evidence that this process occurs at relatively high levels in the first 2 years of life declining thereafter with the possibility, at least in animals, of re-induction under conditions of insulin-resistance, such as pregnancy or obesity. (Meier et al., 2008). This suggests that cells may retain an intrinsic capacity to replicate.

In the adult, endocrine pancreas cells are considered to have a very low turnover. However, albeit quite slowly, cells undergo senescence and should be continuously replaced by newly formed cells. By combining abdominal CT scans and morphometric analysis of human pancreatic tissue, Meier et al reported that the cell mass expands by

Perspectives of Cell Therapy in Type 1 Diabetes 327

bone marrow derived stem cells of both haemopoietic (BM-derived HSC) and mesenchymal (BM-derived MSC) origin. These cells act by a paracrine mechanism releasing factors that favour tissue repair. Moreover, transdifferentiation of ductal and acinal cells may generate

Fig. 1. Mechanisms potentially involved in cell neogenesis.

The discrepancy in identification of cells that in the adult sustain cell neogenesis, may result from the differential contribution in condition of physiological turnover and in condition of injury where the regenerative processes are accelerated and cells other than insulin producing cells are involved to insure a reparative process. The study of Thorel et al showing the conversion of adult pancreatic cells into after extreme cell loss stands in this line of interpretation (Thorel et al., 2010). In this study the Authors used *in vivo* genetic approaches to obtain near total cell loss without autoimmunity combined with cell lineage tracing. For this purpose, they created a model of inducible, rapid β cell removal (>99%) by administration of diphtheria toxin (DT) in transgenic mice in which the DT receptor was expressed only in cells (Thorel et al., 2010). In transgenic mice the systemic administration of DT permitted a specific cell ablation by apoptosis (Saito et al., 2001). In this model, β cell regeneration was monitored in combination with cell lineage tracing to investigate the

insulin secreting cells.

several fold from birth to adulthood as result of an expansion in size of islets rather than an increase in number of islets (Meier et al., 2008). The increase in cell number per islet mainly occurs in young children in coincidence with the growth of the organ size. Cnop et al provided evidence for a long lifespan and low turnover of human islet cells estimated by mathematical modelling of lipofuscin accumulation (Cnop et al., 2010). Human cells, unlike those of young rodents, are long-lived and in the adult human cell population is mainly established in the first 20 years of life. Dor et al using a method for genetic lineage tracing to determine the contribution of stem cells to pancreatic cell neogenesis showed that pre-existing cells, rather than pluripotent stem cells, are the major source of new cells during adult life and after pancreatectomy in mice (Dor et al., 2004). These results suggest that terminally differentiated cells retain, at least in mice, a significant proliferative capacity *in vivo*. Nir et al used a transgenic mouse model to study the dynamics of cell regeneration from a transiently induced diabetic state (Nir et al., 2007). Lineage tracing analysis in this model indicated that enhanced proliferation of surviving cells played the major role in regeneration. These studies provided evidence that adult pancreatic cells are formed by self duplication rather than stem cell differentiation (Dor et al., 2004; Nir et al., 2007). On the other hand there are studies suggesting that regenerated cells derive from precursors located within pancreatic ducts in the proximity of islets (Juhl et al., 2010). The origin from these precursors has been demonstrated in rodent models of pancreatic damage (Bonner-Weir et al., 2004; Xu et al., 2008). Monitoring the expression of Neurogenin 3 (Ngn3), the earliest islet cell-specific transcription factor in embryonic development, Xu et al showed activation of cell progenitors located in the ductal lining in injured adult mouse pancreas (Xu et al., 2008). They found that differentiation of the adult progenitors is Ngn3 dependent and generates all islet cell types, including glucose responsive cells that proliferate, both *in situ* and when cultured in embryonic pancreas explants. This study suggests that multipotent progenitor cells present in the pancreas of adult mice can increase the functional cell mass by differentiation and proliferation rather than by self-duplication of pre-existing cells only. Li et al investigated whether after partial pancreatectomy in adult rats, pancreatic-duct cells serve as a source of regeneration by undergoing a dedifferentiation and redifferentiation (W.C. Li et al., 2010). The Authors detected after pancreatectomy an early loss by the mature ducts of the ductal differentiation marker Hnf6, followed by the transient formation of areas composed of proliferating ductules, called foci of regeneration. These ductules expressed markers of the embryonic pancreatic epithelium Pdx1, Tcf2 and Sox9 (W.C. Li et al., 2010). Since foci subsequently form new pancreatic lobes, it was suggested that these cells act as progenitors of the regenerating pancreas. Islets in foci initially resemble embryonic islets as they transiently expressed the endocrine-lineage-specific transcription factor Ngn3 and lacked of MafA expression and contained low percentage of cells. The numbers of MafA(+) insulin(+) cells progressively increased with the maturation of foci (W.C. Li et al., 2010). Based on these observations, it was suggested that adult pancreatic duct cells may recapitulate aspects of embryonic pancreas in response to injury (W.C. Li et al., 2010). This mechanism of regeneration implicates the plasticity of the differentiated cells within the pancreas.

As schematized in figure 1, after injury cells may be replaced by replication of cells or from differentiation of stem cells (SC) localized within the islets or in exocrine pancreatic tissues (ductal and acinal cells). After extreme loss of cell mass, glucagon producing cells may transdifferentiate in cells. A possible contribution to cell neogenesis comes from

several fold from birth to adulthood as result of an expansion in size of islets rather than an increase in number of islets (Meier et al., 2008). The increase in cell number per islet mainly occurs in young children in coincidence with the growth of the organ size. Cnop et al provided evidence for a long lifespan and low turnover of human islet cells estimated by mathematical modelling of lipofuscin accumulation (Cnop et al., 2010). Human cells, unlike those of young rodents, are long-lived and in the adult human cell population is mainly established in the first 20 years of life. Dor et al using a method for genetic lineage tracing to determine the contribution of stem cells to pancreatic cell neogenesis showed that pre-existing cells, rather than pluripotent stem cells, are the major source of new cells during adult life and after pancreatectomy in mice (Dor et al., 2004). These results suggest that terminally differentiated cells retain, at least in mice, a significant proliferative capacity *in vivo*. Nir et al used a transgenic mouse model to study the dynamics of cell regeneration from a transiently induced diabetic state (Nir et al., 2007). Lineage tracing analysis in this model indicated that enhanced proliferation of surviving cells played the major role in regeneration. These studies provided evidence that adult pancreatic cells are formed by self duplication rather than stem cell differentiation (Dor et al., 2004; Nir et al., 2007). On the other hand there are studies suggesting that regenerated cells derive from precursors located within pancreatic ducts in the proximity of islets (Juhl et al., 2010). The origin from these precursors has been demonstrated in rodent models of pancreatic damage (Bonner-Weir et al., 2004; Xu et al., 2008). Monitoring the expression of Neurogenin 3 (Ngn3), the earliest islet cell-specific transcription factor in embryonic development, Xu et al showed activation of cell progenitors located in the ductal lining in injured adult mouse pancreas (Xu et al., 2008). They found that differentiation of the adult progenitors is Ngn3 dependent and generates all islet cell types, including glucose responsive cells that proliferate, both *in situ* and when cultured in embryonic pancreas explants. This study suggests that multipotent progenitor cells present in the pancreas of adult mice can increase the functional cell mass by differentiation and proliferation rather than by self-duplication of pre-existing cells only. Li et al investigated whether after partial pancreatectomy in adult rats, pancreatic-duct cells serve as a source of regeneration by undergoing a dedifferentiation and redifferentiation (W.C. Li et al., 2010). The Authors detected after pancreatectomy an early loss by the mature ducts of the ductal differentiation marker Hnf6, followed by the transient formation of areas composed of proliferating ductules, called foci of regeneration. These ductules expressed markers of the embryonic pancreatic epithelium Pdx1, Tcf2 and Sox9 (W.C. Li et al., 2010). Since foci subsequently form new pancreatic lobes, it was suggested that these cells act as progenitors of the regenerating pancreas. Islets in foci initially resemble embryonic islets as they transiently expressed the endocrine-lineage-specific transcription factor Ngn3 and lacked of MafA expression and contained low percentage of cells. The numbers of MafA(+) insulin(+) cells progressively increased with the maturation of foci (W.C. Li et al., 2010). Based on these observations, it was suggested that adult pancreatic duct cells may recapitulate aspects of embryonic pancreas in response to injury (W.C. Li et al., 2010). This mechanism of

regeneration implicates the plasticity of the differentiated cells within the pancreas.

As schematized in figure 1, after injury cells may be replaced by replication of cells or from differentiation of stem cells (SC) localized within the islets or in exocrine pancreatic tissues (ductal and acinal cells). After extreme loss of cell mass, glucagon producing cells may transdifferentiate in cells. A possible contribution to cell neogenesis comes from bone marrow derived stem cells of both haemopoietic (BM-derived HSC) and mesenchymal (BM-derived MSC) origin. These cells act by a paracrine mechanism releasing factors that favour tissue repair. Moreover, transdifferentiation of ductal and acinal cells may generate insulin secreting cells.

Fig. 1. Mechanisms potentially involved in cell neogenesis.

The discrepancy in identification of cells that in the adult sustain cell neogenesis, may result from the differential contribution in condition of physiological turnover and in condition of injury where the regenerative processes are accelerated and cells other than insulin producing cells are involved to insure a reparative process. The study of Thorel et al showing the conversion of adult pancreatic cells into after extreme cell loss stands in this line of interpretation (Thorel et al., 2010). In this study the Authors used *in vivo* genetic approaches to obtain near total cell loss without autoimmunity combined with cell lineage tracing. For this purpose, they created a model of inducible, rapid β cell removal (>99%) by administration of diphtheria toxin (DT) in transgenic mice in which the DT receptor was expressed only in cells (Thorel et al., 2010). In transgenic mice the systemic administration of DT permitted a specific cell ablation by apoptosis (Saito et al., 2001). In this model, β cell regeneration was monitored in combination with cell lineage tracing to investigate the

Perspectives of Cell Therapy in Type 1 Diabetes 329

a significant reduction in cell mass is clearly established in Type 2 diabetes (Donath & Halban, 2004; Weir & Bonner-Weir, 2004). The same mechanisms of glucotoxicity reported are involved, together with dyslipidaemia. In murine models of type 2 diabetes, short-term hyperglycemia has been shown to increase islet capillary blood pressure and perfusion, in a glucose dependent and reversible pathway, possibly mediated by a NOS–dependent mechanism (Bonner-Weir, 2004; Carlsson et al., 1998). However, with age, persistent hyperglycemia induces islet hypoperfusion. Inducible NOS (iNOS) increases the islet blood perfusion also in prediabetic low dose streptozotocin-treated mice, a model of type 1 diabetes (Carlsson et al., 2000). Increased islet capillary flow and pressure could, over time, contribute to the damage of the islet endothelium and thickening of the capillary wall, thus decreasing the islet perfusion. The hyperglycemia-induced NO production by the endothelium could also result cytotoxic to the islets and directly impair insulin release. In Zucker diabetic fatty rats, a model of type 2 diabetes, it has been shown that changes in the islet vasculature play a key pathogenetic role in the development of diabetes (X. Li et al., 2006). In a biphasic pattern, an early vascular hyperplasia was followed by endothelial cell

Allogenic pancreatic islet transplantation has become a suitable therapeutic option for the treatment of patients with unstable type I diabetes after the introduction of the Edmonton Protocol based on the optimization of islet isolation techniques and the development of a rapamycin-based glucocorticoid-free immunosuppressive regimen (Ricordi, 2003; Shapiro et al., 2000, 2003, 2006). However, after 5 years only 10% of the recipients were insulin independent (Ryan et al., 2005; Shapiro, 2006). In addition, although a sufficient islet mass can be obtained from good quality pancreata, to achieve insulin independence usually are needed islet preparations derived from multiple donors (Biancone & Ricordi, 2002). Therefore, this procedure is limited by the supply of cadaveric donors. Moreover, several factors may be responsible of the progressive dysfunction of transplanted islets. After an initial islet mass loss following the intraportal infusion, due to an inflammatory reaction, engraftment requires efficient islet revascularization by a chimeric vascular tree formed by host and recipient endothelial cells (Brissova et al., 2004). A poor vascular engraftment is one of the main causes of islet loss. Other factors that concur to islet loss include the exposure of islets to increased lipid levels, a side effect of immunosuppressive therapy based on mTOR inhibitors (Hafiz et al., 2005; Pileggi et al., 2006). Exposure to high-dose of calcineurin inhibitors (CNI) is recognized to induce direct β cell toxicity and functional impairment. The antiproliferative effects of mTOR inhibitors and CNI inhibit tissue remodelling and reduce β cell self-renewal (Nir et al., 2007). Moreover, the anti-angiogenic activity of rapamycin is a potential limitation of the current immunosuppressive protocols, that may be particularly detrimental in the early engraftment phase (Cantaluppi et al., 2006). Therefore, to overcome these problems it is necessary to improve the recovery and quality of islet cells from a single-donor pancreas and to develop strategies allowing the inhibition of inflammatory reactions, the improvement of islet vascularization and of islet engraftment using safer and less cytotoxic immunomodulatory approaches (Mineo et al., 2009; Pileggi et al., 2006; Ricordi, 2003). Recently, increased islet yields have been obtained by improving techniques of pancreas recovery and preservation and of islet isolation and purification (Pileggi et al., 2006;Ricordi, 2003). On the other hand, peritransplant interventions based on

loss and vasculature disruption, in parallel to progressive islet failure.

**4. Islet transplantation as strategy for cell replacement** 

origin of newly formed β cells. The results obtained showed that the adult pancreas can generate new β cells after their near total loss, mainly by the spontaneous reprogramming of glucagon-secreting cells (Saito et al., 2001). Therefore, cell replication may account for maintaining homeostasis, whereas transdifferentiation of other cell types could be required after injury to replace the lost cells.

Summarizing these studies, one can envisage a scenario in which cells may replicate themselves to maintain homeostasis (Dor et al., 2004), tissue specific precursors localized within pancreatic ductal cells generate and cells after pancreatic injury and glucagon secreting cells can transdifferentiate into cells to repair an extremely severe selective cell loss. Translation of these experimental studies in humans may be difficult without the help of lineage tracing or of specific cellular markers.

#### **3. cell mass in type 1 and type 2 diabetes**

Despite the evidence of some turnover in adult humans and the apparent capacity of endocrine pancreatic cells to regenerate throughout life, patients with type 1 as well as type 2 diabetes have substantial deficit in cell mass, approximately 99% in long-standing type 1 diabetes and 65% in long-standing type 2 diabetes. This loss of cells implies that restoration of endogenous insulin secretion might be accomplished through replacement or regeneration of islet cells.

The cell deficit in type 1 diabetes is related to the established autoimmune destruction of the target cells. However, several lines of research indicate that some cell regeneration may occur in recent onset type 1 diabetic patients (Willcox et al., 2010) and even in patients with long-standing type 1 diabetes (Meier et al., 2005). The failure of cell regeneration in type 1 diabetes may be related to the increased vulnerability of the newly forming cells to apoptosis induced by inflammatory cytokines (Meier et al., 2006).

Other mechanisms, involved in all forms of diabetes, include the hyperglycemic induction of the nitric oxide synthase (NOS)-dependent mechanisms in islet microendothelium, with production of the vasoactive mediator nitric oxide (NO) (Suschek et al., 1994), endothelial cell loss and vasculature disruption (Zanone et al., 2008). NO is in fact increased in hyperglycemic conditions, and has an established direct islet cytotoxicity and potentially impairs insulin release (Steiner et al, 1997; Suschek et al., 1994; Kröncke et al., 1993). Islet microendothelial cells are also source of the proinflammatory cytokine IL-1 under hypeglycemic conditions, independently of any viral or immune-mediated process. IL-1 impairs insulin release in human islet, induces Fas expression enabling Fas-mediated apoptosis and IL-1 is thus implicated as a mediator of glucotoxicity (Maedler et al., 2001; Loweth et al., 1998). The metabolic mechanisms by which hyperglycemia initiates apoptosis in vascular endothelium are incompletely understood. These mechanisms include oxidative stress, increased intracellular Ca++, mitocondrial dysfunction, changes in intracellular fattyacid metabolism, and impaired phosphorylation of the protein kinase Akt (Favaro et al., 2008). Akt signaling pathway plays a pivotal role in preventing apoptosis in a variety of settings (Datta et al., 1999), and, in particular, Akt activation is crucial for the ability of factors such as insulin, IGF-I and VEGF to inhibit apoptosis in cultured endothelium (Jung et al., 2000). Recent data highlight the Akt role also in insulin-mediated glucose transport and pancreatic cell mass and function (Bernal-Mizrachi et al., 2004; Elghazi et al., 2006). As for type 2 diabetes, the progressive increase in glucose levels that characterizes its natural history has been claimed to be due to gradual reduction of function and mass of cells, and

origin of newly formed β cells. The results obtained showed that the adult pancreas can generate new β cells after their near total loss, mainly by the spontaneous reprogramming of glucagon-secreting cells (Saito et al., 2001). Therefore, cell replication may account for maintaining homeostasis, whereas transdifferentiation of other cell types could be required

Summarizing these studies, one can envisage a scenario in which cells may replicate themselves to maintain homeostasis (Dor et al., 2004), tissue specific precursors localized within pancreatic ductal cells generate and cells after pancreatic injury and glucagon secreting cells can transdifferentiate into cells to repair an extremely severe selective cell loss. Translation of these experimental studies in humans may be difficult without the

Despite the evidence of some turnover in adult humans and the apparent capacity of endocrine pancreatic cells to regenerate throughout life, patients with type 1 as well as type 2 diabetes have substantial deficit in cell mass, approximately 99% in long-standing type 1 diabetes and 65% in long-standing type 2 diabetes. This loss of cells implies that restoration of endogenous insulin secretion might be accomplished through replacement or

The cell deficit in type 1 diabetes is related to the established autoimmune destruction of the target cells. However, several lines of research indicate that some cell regeneration may occur in recent onset type 1 diabetic patients (Willcox et al., 2010) and even in patients with long-standing type 1 diabetes (Meier et al., 2005). The failure of cell regeneration in type 1 diabetes may be related to the increased vulnerability of the newly forming cells to

Other mechanisms, involved in all forms of diabetes, include the hyperglycemic induction of the nitric oxide synthase (NOS)-dependent mechanisms in islet microendothelium, with production of the vasoactive mediator nitric oxide (NO) (Suschek et al., 1994), endothelial cell loss and vasculature disruption (Zanone et al., 2008). NO is in fact increased in hyperglycemic conditions, and has an established direct islet cytotoxicity and potentially impairs insulin release (Steiner et al, 1997; Suschek et al., 1994; Kröncke et al., 1993). Islet microendothelial cells are also source of the proinflammatory cytokine IL-1 under hypeglycemic conditions, independently of any viral or immune-mediated process. IL-1 impairs insulin release in human islet, induces Fas expression enabling Fas-mediated apoptosis and IL-1 is thus implicated as a mediator of glucotoxicity (Maedler et al., 2001; Loweth et al., 1998). The metabolic mechanisms by which hyperglycemia initiates apoptosis in vascular endothelium are incompletely understood. These mechanisms include oxidative stress, increased intracellular Ca++, mitocondrial dysfunction, changes in intracellular fattyacid metabolism, and impaired phosphorylation of the protein kinase Akt (Favaro et al., 2008). Akt signaling pathway plays a pivotal role in preventing apoptosis in a variety of settings (Datta et al., 1999), and, in particular, Akt activation is crucial for the ability of factors such as insulin, IGF-I and VEGF to inhibit apoptosis in cultured endothelium (Jung et al., 2000). Recent data highlight the Akt role also in insulin-mediated glucose transport and pancreatic cell mass and function (Bernal-Mizrachi et al., 2004; Elghazi et al., 2006). As for type 2 diabetes, the progressive increase in glucose levels that characterizes its natural history has been claimed to be due to gradual reduction of function and mass of cells, and

after injury to replace the lost cells.

regeneration of islet cells.

help of lineage tracing or of specific cellular markers.

**3. cell mass in type 1 and type 2 diabetes** 

apoptosis induced by inflammatory cytokines (Meier et al., 2006).

a significant reduction in cell mass is clearly established in Type 2 diabetes (Donath & Halban, 2004; Weir & Bonner-Weir, 2004). The same mechanisms of glucotoxicity reported are involved, together with dyslipidaemia. In murine models of type 2 diabetes, short-term hyperglycemia has been shown to increase islet capillary blood pressure and perfusion, in a glucose dependent and reversible pathway, possibly mediated by a NOS–dependent mechanism (Bonner-Weir, 2004; Carlsson et al., 1998). However, with age, persistent hyperglycemia induces islet hypoperfusion. Inducible NOS (iNOS) increases the islet blood perfusion also in prediabetic low dose streptozotocin-treated mice, a model of type 1 diabetes (Carlsson et al., 2000). Increased islet capillary flow and pressure could, over time, contribute to the damage of the islet endothelium and thickening of the capillary wall, thus decreasing the islet perfusion. The hyperglycemia-induced NO production by the endothelium could also result cytotoxic to the islets and directly impair insulin release.

In Zucker diabetic fatty rats, a model of type 2 diabetes, it has been shown that changes in the islet vasculature play a key pathogenetic role in the development of diabetes (X. Li et al., 2006). In a biphasic pattern, an early vascular hyperplasia was followed by endothelial cell loss and vasculature disruption, in parallel to progressive islet failure.

#### **4. Islet transplantation as strategy for cell replacement**

Allogenic pancreatic islet transplantation has become a suitable therapeutic option for the treatment of patients with unstable type I diabetes after the introduction of the Edmonton Protocol based on the optimization of islet isolation techniques and the development of a rapamycin-based glucocorticoid-free immunosuppressive regimen (Ricordi, 2003; Shapiro et al., 2000, 2003, 2006). However, after 5 years only 10% of the recipients were insulin independent (Ryan et al., 2005; Shapiro, 2006). In addition, although a sufficient islet mass can be obtained from good quality pancreata, to achieve insulin independence usually are needed islet preparations derived from multiple donors (Biancone & Ricordi, 2002). Therefore, this procedure is limited by the supply of cadaveric donors. Moreover, several factors may be responsible of the progressive dysfunction of transplanted islets. After an initial islet mass loss following the intraportal infusion, due to an inflammatory reaction, engraftment requires efficient islet revascularization by a chimeric vascular tree formed by host and recipient endothelial cells (Brissova et al., 2004). A poor vascular engraftment is one of the main causes of islet loss. Other factors that concur to islet loss include the exposure of islets to increased lipid levels, a side effect of immunosuppressive therapy based on mTOR inhibitors (Hafiz et al., 2005; Pileggi et al., 2006). Exposure to high-dose of calcineurin inhibitors (CNI) is recognized to induce direct β cell toxicity and functional impairment. The antiproliferative effects of mTOR inhibitors and CNI inhibit tissue remodelling and reduce β cell self-renewal (Nir et al., 2007). Moreover, the anti-angiogenic activity of rapamycin is a potential limitation of the current immunosuppressive protocols, that may be particularly detrimental in the early engraftment phase (Cantaluppi et al., 2006). Therefore, to overcome these problems it is necessary to improve the recovery and quality of islet cells from a single-donor pancreas and to develop strategies allowing the inhibition of inflammatory reactions, the improvement of islet vascularization and of islet engraftment using safer and less cytotoxic immunomodulatory approaches (Mineo et al., 2009; Pileggi et al., 2006; Ricordi, 2003). Recently, increased islet yields have been obtained by improving techniques of pancreas recovery and preservation and of islet isolation and purification (Pileggi et al., 2006;Ricordi, 2003). On the other hand, peritransplant interventions based on

Perspectives of Cell Therapy in Type 1 Diabetes 331

implanted islets, although soluble mediators may still reach cells and induce cell death. On the other hand, this strategy might allow full maturation of embryonic stem cells into glucose sensitive insulin secreting cells. Indeed, human embryonic stem cells differentiated into pancreatic endoderm could be encapsulated in a protective device before transplantation (D'Amour et al., 2005; Kroon et al., 2008). Experiments in NOD mice demonstrated that encapsulated human cell precursors may generate functional insulin producing cells after implantation improving diabetes (S.H. Lee et al., 2009). As an alternative, ongoing studies are evaluating the use of encapsulated porcine islets to correct type 1 diabetes (Elliott et al., 2007). Preliminary studies in humans showed long term survival of xenotransplantation of porcine neonatal islets and Sertoli cells using a technology to provide an immune protective environment (Valdes-Gonzales et al., 2005). A vascularized chamber model allowing tissue growth in threedimension under influence of hypoxia that triggers angiogenesis has been developped in mouse (Cronin et al., 2004) and rat (Mian et al., 2000; Tanaka et al., 2000). Vascularized chambers containing syngenic islets were used to improve glucose control in diabetic mice (Hussey et al., 2009). In this study islets were transplanted into prevascularized chambers implanted on the epigastric pedicle in the groin of diabetic mice resulting in a significant reduction in blood glucose levels and improvement of glycemic control. This study suggests that islet survival and function are enhanced by prevascularization of tissue engineering chambers before islet transplantation (Hussey et al., 2009). Opara et al. reported a new design of a bioartificial pancreas comprising islets co-encapsulated with angiogenic protein in permselective multilayer alginate-poly-L-ornithine-alginate microcapsules and transplanted in an omentum pouch in diabetic experimental animals (Opara et al., 2010). A great effort is invested to improve the encapsulation techniques to avoid clogging of the device that may reduce the influx of nutrients and glucose and impair the insulin efflux. Teramura & Iwata recently reviewed the obstacles and the new techniques to overcome these problems, such as conformal coating

In consideration of the shortage of pancreata for islet transplantation and of the side effects of current immunosuppressive protocols, the research has been focused on the possible development of alternative sources of functional β cells. The ideal approach to overcome the current inadequate supply of human pancreatic islet cells for transplantation is the availability of an unlimited source of transplantable insulin-producing cells. The potential of adult and embryonic stem cells to generate islet cells as well as development of appropriate conditions for expansion and differentiation to β cells of pancreatic islet cell precursors or of cells that share common embryonic origin such as liver cells is under intense investigation (Mineo et al., 2009). Finally, xenogeneic islet transplantation remains a viable therapeutic

The consideration that stem cells play a crucial role to self-renewal in physiologic and pathologic conditions in the endocrine pancreas (Bouvens, 2006; Sarvetnick et al., 2007a, 2007b)

In the last years, a variety of approaches have been employed to induce β cell neogenesis using pluripotent stem cells (embryonic stem cells or induced pluripotent stem cells), multipotent adult stem cells such as the hepatic oval cells or terminally differentiated cells such as the exocrine pancreatic cells (Yechoor & Chan, 2010). Brolén et al investigated the potential of

prompted researchers to develop SC-based therapy to stimulate β cell regeneration.

and islet enclosure with cells (Teramura & Iwata, 2010).

**5. In search for alternative sources of cells** 

option for the future (Ricordi, 2003).

combination therapies have been proposed to inhibit allo- and auto-immune response minimizing the side effects and favoring development of T regulatory cells (Treg) to maintain long term tolerance and to favor cell regeneration. Targeting the costimulatory molecules involved in T-cell activation and/or adhesion molecules by immunomodulatory agents now available for clinical applications could be an option to reduce the side effects of immunosuppression and the islet toxicity and to achieve specific immune tolerance (Ricordi & Strom, 2004). Several experimental studies suggest that a combined islet transplant and cell therapy with bone marrow–derived cells, mesenchymal cells, Treg, and tolerogenic dendritic cells may modulate recipient immune response and increase the engraftment and long term survival of islets (Mineo et al., 2008). This possibility is supported by recent clinical trials demonstrating stable mixed haematopoietic chimerism and/or improved tolerance in kidney allograft recipients using nonmyeloablative conditioning and donor haematopoietic stem cell infusion (Sykes, 2009).

Another factor limiting successful islet engraftment is the inflammatory reaction that takes place in the liver after portal vein infusion of islets. This observation led to experiments of co-transplantation of islets with bone marrow-derived mesenchymal stem cells to take advantage of the anti-inflammatory action of these cells (Ito et al., 2010).

To improve islet vascularization is also a must for a better engraftment of islets. It has been shown that bone marrow-derived endothelial progenitor cells (EPC) isolated from peripheral blood specifically localize within sites of endothelial injury inducing a regenerative program. EPC are able to chimerize with donor vessels in transplanted organs, suggesting a putative role of these cells in graft revascularization (Schuh et al., 2008¸ Koopmans et al., 2006). EPC are recruited to the pancreas in response to islet injury and EPC-mediated pancreas neovascularization may facilitate the recovery of injured cells improving islet allograft function (Mathews et al., 2004). In a murine model of islet transplantation, the increase of EPC in the peripheral circulation obtained by mobilization with granulocyte-macrophage colony-stimulating factor has been associated with higher vascular density and engraftment (Contreras et al., 2003). Therefore, the identification of factors able to enhance neoangiogenesis may increase the success of islet transplantation.

Another goal of combination therapies is to preserve islet function after detection of graft dysfunction (Froud et al., 2008). For this purpose it has been suggested, for instance, the use of exenatide, glucagon-like peptide synthetic analog, anti-tumor necrosis factor agents or immunomodulatory therapy (Faradji et al., 2008; Froud et al., 2008). However, the recently released results of two trials addressing strategies of combination therapies are disappointing. The phase three trial based on combination of anti-CD25 mAb (Daclizumab) that blocks IL-2 signalling pathway in activated T cells without interferring with Treg, in combination with mycophenolate mofetil that bloks the *de novo* purine synthesis in T and B lymphocytes, reported no improvement of cell preservation (Gottlieb et al., 2010). The anti-CD25 mAb was tested in another trial in association with exenatide. Also in this trial the improvement of cell function was not observed (Rother et al., 2009). Several other phase II-III National Institutes of Health (NIH)–sponsored randomized trials in islet transplantation alone and in islet-after-kidney transplantation are currently under evaluation by the Clinical Islet Transplantation Consortium (http://www.citisletstudy.org/). An alternative to allow long term survival of islets after transplantation is the development of efficient encapsulation techniques aimed to guarantee immune-isolation, an adequate exchange of nutrients to islet cells and release of insulin (Calafiore et al., 2006). This kind of approach may protect from cell-mediated rejection of

combination therapies have been proposed to inhibit allo- and auto-immune response minimizing the side effects and favoring development of T regulatory cells (Treg) to maintain long term tolerance and to favor cell regeneration. Targeting the costimulatory molecules involved in T-cell activation and/or adhesion molecules by immunomodulatory agents now available for clinical applications could be an option to reduce the side effects of immunosuppression and the islet toxicity and to achieve specific immune tolerance (Ricordi & Strom, 2004). Several experimental studies suggest that a combined islet transplant and cell therapy with bone marrow–derived cells, mesenchymal cells, Treg, and tolerogenic dendritic cells may modulate recipient immune response and increase the engraftment and long term survival of islets (Mineo et al., 2008). This possibility is supported by recent clinical trials demonstrating stable mixed haematopoietic chimerism and/or improved tolerance in kidney allograft recipients using nonmyeloablative conditioning and donor

Another factor limiting successful islet engraftment is the inflammatory reaction that takes place in the liver after portal vein infusion of islets. This observation led to experiments of co-transplantation of islets with bone marrow-derived mesenchymal stem cells to take

To improve islet vascularization is also a must for a better engraftment of islets. It has been shown that bone marrow-derived endothelial progenitor cells (EPC) isolated from peripheral blood specifically localize within sites of endothelial injury inducing a regenerative program. EPC are able to chimerize with donor vessels in transplanted organs, suggesting a putative role of these cells in graft revascularization (Schuh et al., 2008¸ Koopmans et al., 2006). EPC are recruited to the pancreas in response to islet injury and EPC-mediated pancreas neovascularization may facilitate the recovery of injured cells improving islet allograft function (Mathews et al., 2004). In a murine model of islet transplantation, the increase of EPC in the peripheral circulation obtained by mobilization with granulocyte-macrophage colony-stimulating factor has been associated with higher vascular density and engraftment (Contreras et al., 2003). Therefore, the identification of factors able to enhance neoangiogenesis may increase the success of islet transplantation. Another goal of combination therapies is to preserve islet function after detection of graft dysfunction (Froud et al., 2008). For this purpose it has been suggested, for instance, the use of exenatide, glucagon-like peptide synthetic analog, anti-tumor necrosis factor agents or immunomodulatory therapy (Faradji et al., 2008; Froud et al., 2008). However, the recently released results of two trials addressing strategies of combination therapies are disappointing. The phase three trial based on combination of anti-CD25 mAb (Daclizumab) that blocks IL-2 signalling pathway in activated T cells without interferring with Treg, in combination with mycophenolate mofetil that bloks the *de novo* purine synthesis in T and B lymphocytes, reported no improvement of cell preservation (Gottlieb et al., 2010). The anti-CD25 mAb was tested in another trial in association with exenatide. Also in this trial the improvement of cell function was not observed (Rother et al., 2009). Several other phase II-III National Institutes of Health (NIH)–sponsored randomized trials in islet transplantation alone and in islet-after-kidney transplantation are currently under evaluation by the Clinical Islet Transplantation Consortium (http://www.citisletstudy.org/). An alternative to allow long term survival of islets after transplantation is the development of efficient encapsulation techniques aimed to guarantee immune-isolation, an adequate exchange of nutrients to islet cells and release of insulin (Calafiore et al., 2006). This kind of approach may protect from cell-mediated rejection of

advantage of the anti-inflammatory action of these cells (Ito et al., 2010).

haematopoietic stem cell infusion (Sykes, 2009).

implanted islets, although soluble mediators may still reach cells and induce cell death. On the other hand, this strategy might allow full maturation of embryonic stem cells into glucose sensitive insulin secreting cells. Indeed, human embryonic stem cells differentiated into pancreatic endoderm could be encapsulated in a protective device before transplantation (D'Amour et al., 2005; Kroon et al., 2008). Experiments in NOD mice demonstrated that encapsulated human cell precursors may generate functional insulin producing cells after implantation improving diabetes (S.H. Lee et al., 2009). As an alternative, ongoing studies are evaluating the use of encapsulated porcine islets to correct type 1 diabetes (Elliott et al., 2007). Preliminary studies in humans showed long term survival of xenotransplantation of porcine neonatal islets and Sertoli cells using a technology to provide an immune protective environment (Valdes-Gonzales et al., 2005). A vascularized chamber model allowing tissue growth in threedimension under influence of hypoxia that triggers angiogenesis has been developped in mouse (Cronin et al., 2004) and rat (Mian et al., 2000; Tanaka et al., 2000). Vascularized chambers containing syngenic islets were used to improve glucose control in diabetic mice (Hussey et al., 2009). In this study islets were transplanted into prevascularized chambers implanted on the epigastric pedicle in the groin of diabetic mice resulting in a significant reduction in blood glucose levels and improvement of glycemic control. This study suggests that islet survival and function are enhanced by prevascularization of tissue engineering chambers before islet transplantation (Hussey et al., 2009). Opara et al. reported a new design of a bioartificial pancreas comprising islets co-encapsulated with angiogenic protein in permselective multilayer alginate-poly-L-ornithine-alginate microcapsules and transplanted in an omentum pouch in diabetic experimental animals (Opara et al., 2010). A great effort is invested to improve the encapsulation techniques to avoid clogging of the device that may reduce the influx of nutrients and glucose and impair the insulin efflux. Teramura & Iwata recently reviewed the obstacles and the new techniques to overcome these problems, such as conformal coating and islet enclosure with cells (Teramura & Iwata, 2010).

In consideration of the shortage of pancreata for islet transplantation and of the side effects of current immunosuppressive protocols, the research has been focused on the possible development of alternative sources of functional β cells. The ideal approach to overcome the current inadequate supply of human pancreatic islet cells for transplantation is the availability of an unlimited source of transplantable insulin-producing cells. The potential of adult and embryonic stem cells to generate islet cells as well as development of appropriate conditions for expansion and differentiation to β cells of pancreatic islet cell precursors or of cells that share common embryonic origin such as liver cells is under intense investigation (Mineo et al., 2009). Finally, xenogeneic islet transplantation remains a viable therapeutic option for the future (Ricordi, 2003).

#### **5. In search for alternative sources of cells**

The consideration that stem cells play a crucial role to self-renewal in physiologic and pathologic conditions in the endocrine pancreas (Bouvens, 2006; Sarvetnick et al., 2007a, 2007b) prompted researchers to develop SC-based therapy to stimulate β cell regeneration.

In the last years, a variety of approaches have been employed to induce β cell neogenesis using pluripotent stem cells (embryonic stem cells or induced pluripotent stem cells), multipotent adult stem cells such as the hepatic oval cells or terminally differentiated cells such as the exocrine pancreatic cells (Yechoor & Chan, 2010). Brolén et al investigated the potential of

Perspectives of Cell Therapy in Type 1 Diabetes 333

human albumin, suggesting a partial hepatocyte commitment. At variance with oval cells, the HLSC isolated from normal adult liver, did not express the CD34, c-kit and CK19 markers, and were pluripotent. Moreover, the HLSC were able to differentiate in appropriate conditions into pancreatic insulin producing cells (Herrera et al., 2006). When cultured in DMEM with high glucose content (23mM) for one month followed by 5-7 days of culture in the presence of 10 mM nicotinamide, HLSC formed small spheroid cell clusters positive for human insulin and Glut2 which is a glucose transporter that has been suggested to function as a glucose sensor in pancreatic cells (L. Yang et al., 2002). Moreover, HLSC under this differentiating condition, were positively stained with the Zn-chelating agent dithizone, that is specific for the insulin containing granules (Shiroi et al., 2002). It has been recently shown that adult human pancreas contains rare multipotent stem cells that express

Several studies demonstrate that the cell phenotype can be induced both *in vitro* and *in vivo* by transfection of pancreatic transcription factors into the liver that develops from the same embryological origin of pancreas (Kojima et al., 2003; Lemaigre & Zaret, 2004; Nagaya

The potential of bone marrow derived MSC has also been explored. Genetically modified MSC by recombinant PDX-1 adenovirus or by non-virus gene transfection, were able to express insulin sufficient to reduce blood glucose in the streptozotocin mouse model of diabetes (Y. Li et al., 2007). The differentiated PDX-1+ human MSC expressed multiple isletcell genes such as neurogenin3 (Ngn3), insulin, GK, Glut2, and glucagon, produced and released insulin/C-peptide. After transplatation into STZ-induced diabetic mice the differentiated PDX-1+ human MSC induced within 2 weeks euglycemia and maintained it for at least 42 days. These findings suggest that appropriately modified MSC may allow enrichment of human cells and represent a possible source for cell replacement therapy in

Human umbilical cord blood contains several types of stem/precursor cells. Recent studies indicate the possibility to obtain multipotent stem cells from umbilical cord blood able to confer protection to cells and to stimulate cell neogenesis (Zhao & Mazzone, 2010). Isletlike cell clusters were obtained from MSC derived from humbilical cord vein. However, they secreted very low amounts of insulin in vitro (Chao, 2008). Denner et al showed that directed engineering of human cord blood stem cells may produce C-peptide and insulin

A better understanding of the mechanisms of β cell regeneration may provide further insight for development of cell based therapeutic approaches. Besides the potential contribution of circulating and resident stem cells, the transdifferentiation of existing pancreatic cells such as pancreatic ductal/acinar cells into insulin secreting cells could be potentially exploited (Baeyens et al., 2005; Cantaluppi et al., 2008; Minami et al., 2005;

The successful differentiation of stem cells into cells may prove not to be easy. The secretion of insulin in physiological concentration in response to glucose concentration is the goal to be achieved. One of the more difficult tasks is to maintain an intact coupling of stimulus-secretion in order to obtain a regulated insulin secretion to keep physiological

Another pre-requisite for cell replacement therapy is to obtain a non limiting source of cells possibly derived from the patient himself to avoid the allo-immune response (Wagner et al., 2010). The possibility of reprogramming patient's own cells could be an

insulin (Smukler et al., 2011).

diabetes (Y. Li et al., 2007).

(Denner et al., 2007).

Rosenberg, 1995).

glucose concentration.

et al., 2009).

human embryonic stem cells to differentiate into cells (Brolen et al., 2005). They found that signals from the embryonic mouse pancreas induce differentiation of human embryonic stem cells into insulin-producing cell-like cells. Human embryonic stem cells (hESC) under twodimensional growth conditions spontaneously differentiated in Pdx1(+)/Foxa2(+) pancreatic progenitors and Pdx1(+)/Isl1(+) endocrine progenitors but not in insulin-producing cells. The differentiation of cell-like cell clusters required co-transplantation with the dorsal pancreas from mouse embryos. hESC-derived insulin(+) cell clusters exhibited several features of normal cells, such as synthesis (proinsulin) and processing (C-peptide) of insulin and nuclear localization of key cell transcription factors, including Foxa2, Pdx1, and Isl1 (Brolén et al., 2005). Insulin-producing islet-like cells were also generated from human embryonic stem cells under feeder-free conditions (Jiang et al., 2007). Cell aggregates formed in the presence of epidermal growth factor, basic fibroblast growth factor, and noggin were finally matured in the presence of insulin-like growth factor II and nicotinamide. The temporal kinetic of pancreas-specific gene expression was considerably similar to that of *in vivo* pancreas development. The final population contained cells representative of the ductal, exocrine, and endocrine pancreas. Ricordi et al reported a diabetes reversal in mice by embryonic-derived stem cells (Ricordi & Edlund, 2008). They showed that endodermal-derived embryonic stem cells were able to differentiate into cells that expressed typical pancreatic endodermal markers and, once transplanted in diabetic mice, these cells showed a diffuse staining for insulin, other hormones and markers of fully differentiated β cells (Ricordi & Edlund, 2008). The therapy with embryonic stem cells represents an exciting approach towards cell regeneration and function. So far, the most promising data are from a group of NovoCell who demonstrated the generation of glucose-responsive insulin-secreting cells *in vivo* by pancreatic endoderm derived from human embryonic stem cells into immune-deficient mice (Kroon et al., 2008). However, several problems remain to be solved, such as the reproducibility of the advance, the protection of the engrafted cells against tumor formation and immune-protection that could be achieved by encapsulation devices. Recently, Matveyenko et al tried to reproduce and extend the studies of NovoCell by the implantation of human embryonic stem cell-derived pancreatic endoderm into athimic nude rats, analysing the metabolic parameters of insulin sensitivity and glucose-stimulated insulin secretion to verify the development of viable glucose-responsive insulin secreting cells. The implantation was assessed into the epididymal fat pads of the athymic nude rats or subcutaneously into Theracyte encapsulation devices for 20 weeks (Matveyenko et al., 2010). The data resulting from this study not completely confirmed the development of islet-like structures from human embryonic stem cells differentiated to pancreatic endoderm, since the extent of endocrine cell formation and secretory function was insufficient to be clinically relevant given that human C-peptide and insulin were detectable at very low levels, no increase in human C-peptide/insulin levels after glucose challenge and no development of viable pancreatic tissue or efficient insulin secretion by implantation in the encapsulation devices were present (Matveyenko et al., 2010).

Nevertheless, because the use of embryonic stem cells is burdened with the limited access to embryonic tissues and with the complex ethical concerns involved with the use of embryonic cells, more attention has been focused on adult stem cells. We found that stem cells derived from normal adult human liver (HLSC) with multiple differentiating capabilities distinct from those of oval stem cells may generate islet-like structures. HLSC expressed several MSC markers such as CD73, CD90, CD29, CD44 and the stem cell marker nestin (Herrera et al., 2006). At variance of MSC, HLSC did not express -SMA and expressed liver tissue-specific proteins such as AFP, a marker of hepatocyte precursors, and

human embryonic stem cells to differentiate into cells (Brolen et al., 2005). They found that signals from the embryonic mouse pancreas induce differentiation of human embryonic stem cells into insulin-producing cell-like cells. Human embryonic stem cells (hESC) under twodimensional growth conditions spontaneously differentiated in Pdx1(+)/Foxa2(+) pancreatic progenitors and Pdx1(+)/Isl1(+) endocrine progenitors but not in insulin-producing cells. The differentiation of cell-like cell clusters required co-transplantation with the dorsal pancreas from mouse embryos. hESC-derived insulin(+) cell clusters exhibited several features of normal cells, such as synthesis (proinsulin) and processing (C-peptide) of insulin and nuclear localization of key cell transcription factors, including Foxa2, Pdx1, and Isl1 (Brolén et al., 2005). Insulin-producing islet-like cells were also generated from human embryonic stem cells under feeder-free conditions (Jiang et al., 2007). Cell aggregates formed in the presence of epidermal growth factor, basic fibroblast growth factor, and noggin were finally matured in the presence of insulin-like growth factor II and nicotinamide. The temporal kinetic of pancreas-specific gene expression was considerably similar to that of *in vivo* pancreas development. The final population contained cells representative of the ductal, exocrine, and endocrine pancreas. Ricordi et al reported a diabetes reversal in mice by embryonic-derived stem cells (Ricordi & Edlund, 2008). They showed that endodermal-derived embryonic stem cells were able to differentiate into cells that expressed typical pancreatic endodermal markers and, once transplanted in diabetic mice, these cells showed a diffuse staining for insulin, other hormones and markers of fully differentiated β cells (Ricordi & Edlund, 2008). The therapy with embryonic stem cells represents an exciting approach towards cell regeneration and function. So far, the most promising data are from a group of NovoCell who demonstrated the generation of glucose-responsive insulin-secreting cells *in vivo* by pancreatic endoderm derived from human embryonic stem cells into immune-deficient mice (Kroon et al., 2008). However, several problems remain to be solved, such as the reproducibility of the advance, the protection of the engrafted cells against tumor formation and immune-protection that could be achieved by encapsulation devices. Recently, Matveyenko et al tried to reproduce and extend the studies of NovoCell by the implantation of human embryonic stem cell-derived pancreatic endoderm into athimic nude rats, analysing the metabolic parameters of insulin sensitivity and glucose-stimulated insulin secretion to verify the development of viable glucose-responsive insulin secreting cells. The implantation was assessed into the epididymal fat pads of the athymic nude rats or subcutaneously into Theracyte encapsulation devices for 20 weeks (Matveyenko et al., 2010). The data resulting from this study not completely confirmed the development of islet-like structures from human embryonic stem cells differentiated to pancreatic endoderm, since the extent of endocrine cell formation and secretory function was insufficient to be clinically relevant given that human C-peptide and insulin were detectable at very low levels, no increase in human C-peptide/insulin levels after glucose challenge and no development of viable pancreatic tissue or efficient insulin secretion

by implantation in the encapsulation devices were present (Matveyenko et al., 2010).

Nevertheless, because the use of embryonic stem cells is burdened with the limited access to embryonic tissues and with the complex ethical concerns involved with the use of embryonic cells, more attention has been focused on adult stem cells. We found that stem cells derived from normal adult human liver (HLSC) with multiple differentiating capabilities distinct from those of oval stem cells may generate islet-like structures. HLSC expressed several MSC markers such as CD73, CD90, CD29, CD44 and the stem cell marker nestin (Herrera et al., 2006). At variance of MSC, HLSC did not express -SMA and expressed liver tissue-specific proteins such as AFP, a marker of hepatocyte precursors, and human albumin, suggesting a partial hepatocyte commitment. At variance with oval cells, the HLSC isolated from normal adult liver, did not express the CD34, c-kit and CK19 markers, and were pluripotent. Moreover, the HLSC were able to differentiate in appropriate conditions into pancreatic insulin producing cells (Herrera et al., 2006). When cultured in DMEM with high glucose content (23mM) for one month followed by 5-7 days of culture in the presence of 10 mM nicotinamide, HLSC formed small spheroid cell clusters positive for human insulin and Glut2 which is a glucose transporter that has been suggested to function as a glucose sensor in pancreatic cells (L. Yang et al., 2002). Moreover, HLSC under this differentiating condition, were positively stained with the Zn-chelating agent dithizone, that is specific for the insulin containing granules (Shiroi et al., 2002). It has been recently shown that adult human pancreas contains rare multipotent stem cells that express insulin (Smukler et al., 2011).

Several studies demonstrate that the cell phenotype can be induced both *in vitro* and *in vivo* by transfection of pancreatic transcription factors into the liver that develops from the same embryological origin of pancreas (Kojima et al., 2003; Lemaigre & Zaret, 2004; Nagaya et al., 2009).

The potential of bone marrow derived MSC has also been explored. Genetically modified MSC by recombinant PDX-1 adenovirus or by non-virus gene transfection, were able to express insulin sufficient to reduce blood glucose in the streptozotocin mouse model of diabetes (Y. Li et al., 2007). The differentiated PDX-1+ human MSC expressed multiple isletcell genes such as neurogenin3 (Ngn3), insulin, GK, Glut2, and glucagon, produced and released insulin/C-peptide. After transplatation into STZ-induced diabetic mice the differentiated PDX-1+ human MSC induced within 2 weeks euglycemia and maintained it for at least 42 days. These findings suggest that appropriately modified MSC may allow enrichment of human cells and represent a possible source for cell replacement therapy in diabetes (Y. Li et al., 2007).

Human umbilical cord blood contains several types of stem/precursor cells. Recent studies indicate the possibility to obtain multipotent stem cells from umbilical cord blood able to confer protection to cells and to stimulate cell neogenesis (Zhao & Mazzone, 2010). Isletlike cell clusters were obtained from MSC derived from humbilical cord vein. However, they secreted very low amounts of insulin in vitro (Chao, 2008). Denner et al showed that directed engineering of human cord blood stem cells may produce C-peptide and insulin (Denner et al., 2007).

A better understanding of the mechanisms of β cell regeneration may provide further insight for development of cell based therapeutic approaches. Besides the potential contribution of circulating and resident stem cells, the transdifferentiation of existing pancreatic cells such as pancreatic ductal/acinar cells into insulin secreting cells could be potentially exploited (Baeyens et al., 2005; Cantaluppi et al., 2008; Minami et al., 2005; Rosenberg, 1995).

The successful differentiation of stem cells into cells may prove not to be easy. The secretion of insulin in physiological concentration in response to glucose concentration is the goal to be achieved. One of the more difficult tasks is to maintain an intact coupling of stimulus-secretion in order to obtain a regulated insulin secretion to keep physiological glucose concentration.

Another pre-requisite for cell replacement therapy is to obtain a non limiting source of cells possibly derived from the patient himself to avoid the allo-immune response (Wagner et al., 2010). The possibility of reprogramming patient's own cells could be an

Perspectives of Cell Therapy in Type 1 Diabetes 335

Cell-based therapeutic strategies for immunemodulation that answer to the requirement of safety and effectiveness are under development. The intravenous use of humanized antibodies against CD3 (part of the T-cell receptor complex) soon after the initial onset of diabetic clinical symptoms decreased the insulin requirement compared to controls without this monoclonal antibody therapy. However, this therapy is burdened by several adverse

Other approaches of immunemodulation included subcutaneous administration of heatshock protein (Raz et al., 2001) or intravenous injection of rabbit polyclonal anti T-cell globulin (Saudek et al., 2004). In many of these studies a short term effective preservation of cell function was observed; however, only few patients no longer required insulin treatment. Using human recombinant glutamic acid decarboxylase (GAD65) as therapeutic vaccine it was observed a preservation of C-peptide levels and a decrease in insulin dose requirement only in patients with very recent-onset of diabetes (Ludvigsson et al., 2008; Uibo and Lernmark 2008). Compelling evidence indicates the relevant role of Treg in the inhibition of autoimmune response. A decreased number and function of Treg have been reported in patients with type 1 diabetes (Roncarolo & Battaglia, 2007). Therefore, intervention to restore Treg function in diabetic patients is an attractive therapeutic strategy. Nevertheless, only few studies have shown re-establishment of Treg function in diabetes. Zhao et al demonstrated the possibility to correct functional defects of CD4+ CD62L Treg using human cord blood stem cells. This strategy allowed reversal of autoimmune diabetes in NOD mice (Y. Zhao et al., 2009) providing proof of principle that cord blood stem cells

Treg may act by producing IL-10 and in particular transforming growth factor-1 (TGF1), cytokines that are known to contribute to the induction of immunetolerance. It has been suggested that protection of newly generated cells achieved by treatment with Treg depends on the formation of TGF1 cell ring surrounding pancreatic islands and conferring protection against autoimmune T lymphocytes inducing their apoptosis (Y. Zhao et al., 2009). In a pilot study in Type 1 diabetic children based on administration of autologous umbilical cord blood shortly after disease onset a lower level of HbA1c and a reduced insulin requirement was reported. However, none of the patients achieved insulin-treatment

Recent studies raised great interest on the immunemodulatory potential of mesenchymal stem cells (Le Blanc & Pittenger, 2005). MSC can modulate several T cell functions exerting an immunesuppressive effect. MSC that lack MHC class II molecules and do not express key costimulatory molecules B7-1, B7-2, CD40 and CD40L, were shown to reduce the expression of several lymphocyte activation markers (Aggarwal & Pittenger, 2005). The mechanism has been related to the induction of an anti-inflammatory phenotype in dendritic cells, naive and activated T cells and NK cells, and to an increase of the regulatory T cell population. This results not only in the inhibition of T cell proliferation to polyclonal stimuli, but also in inhibition of naive and memory antigen-specific T cells response to their cognate peptide (Krampera et al., 2003). The MSC-induced suppression has been ascribed to several soluble factors, including hepatocyte growth factor (HGF), TGF-1 and prostaglandin E2 (PGE2) (Aggarwal & Pittenger, 2005). Moreover, MSC have been shown to induce mature dendritic cells type 2 (DC2) to increase IL-10 secretion thus promoting anti-infammatory DC2 signaling. Several studies suggest that MSC improve the outcome of allogenic transplantation and hamper graft-versus-host disease (Le Blanc et al., 2004; Ringden et al.,

effects (Keymeulen et al., 2005; Herold et al., 2002; McDevitt & Unanue, 2008).

can correct function of Treg.

independency (Haller et al., 2007).

approach to these requirements. This was rendered possible by the discovery that induced pluripotent stem cells (iPS) can be generated from adult human somatic cells such as fibroblasts by transfection with selected genes encoding for transcription factors able to confer the characteristics of pluripotency to cells (Takahashi & Yamanaka, 2006; Yamanaka et al., 2007). iPS share the same differentiation potential of embryonic stem cells and they can differentiate into cells of different lineages including insulin producing cells. Tateishi et al tried to generate human iPS cells by retroviral expression of human Oct4, Sox2, Klf4 and c-Myc in the human foreskin fibroblast cells and tested the differentiation potential of human iPS cells into insulin secreting islet-like clusters, demonstrating that iPS cells derived from human skin cells can be differentiated into pancreatic islet-like cluster cells. These cluster cells were shown to contain C-peptidepositive and glucagon-positive cells and, more importantly, to release the C-peptide upon glucose stimulation (Tateishi et al., 2008). iPS cells were also generated from patients with type 1 diabetes by reprogramming their adult fibroblasts with three transcription factors (*OCT4, SOX2, KLF4*). The iPS generated from patients were shown to differentiate into insulin-producing cells (Maehr et al., 2009). A proof of principle for potential clinical applications of reprogrammed somatic cells in the treatment of diabetes type 1 or 2 was provided by Alipio et al who were able to reverse hyperglycemia in diabetic mouse models using iPS-derived pancreatic like cells (Alipio et al., 2010). iPS could overcome the ethical issues and the immunogenicity of embryonic stem cells. The possibility to use iPS individually tailored for each patient is certainly appealing. However, several problems of bio-safety must be solved before iPS enter in clinical use. These include an aberrant or incomplete differentiation and a tumorigenic potential. Reprogramming cells typically requires integration of genes such as *c-MYC, OCT4 AND KLF4* that are known to be oncogenic and may favour development of tumors (Miura et al., 2009). Although after reprogramming the transgenes undergo silencing, it is always possible a reactivation. Therefore, the research in this field is actively searching for alternative strategies to induce pluripotency.

#### **6. Stem cell therapy to counteract the autoimmune destruction of cells**

Assuming that we could generate pluripotent stem cell lines for each individual patient and that after infusion they would be able to restore the cell mass, we still have to face the problem of recurrent autoimmunity. The autoimmune destruction of islet cells by reactive T lymphocytes not only plays a role in the establishment of type 1 diabetes but also impairs an efficient regenerative process. Newly forming cells show increased vulnerability to apoptosis induced by inflammatory cytokines (Meier et al., 2006), explaining the failure of cell regeneration in type 1 diabetes. Therefore, newly generated cells are systematically killed as they acquire the mature phenotype, thus impairing the regenerative attempts in type 1 diabetic patients. Furthermore, therapeutic intervention studies in new onset type 1 diabetes involving broadly immunosuppressive agents, such as cyclosporin A, failed to produce lasting remission, demonstrating the inherent tendency of the autoimmune effector response in humans to recur. Therefore, it is critical that any immunemodulatory therapy induces tolerance to cell antigens, while minimizing detrimental effects on host defence. This is strengthened by recurrence of type 1 diabetes in syngenic pancreas transplantation (Sutherland et al., 1984).

approach to these requirements. This was rendered possible by the discovery that induced pluripotent stem cells (iPS) can be generated from adult human somatic cells such as fibroblasts by transfection with selected genes encoding for transcription factors able to confer the characteristics of pluripotency to cells (Takahashi & Yamanaka, 2006; Yamanaka et al., 2007). iPS share the same differentiation potential of embryonic stem cells and they can differentiate into cells of different lineages including insulin producing cells. Tateishi et al tried to generate human iPS cells by retroviral expression of human Oct4, Sox2, Klf4 and c-Myc in the human foreskin fibroblast cells and tested the differentiation potential of human iPS cells into insulin secreting islet-like clusters, demonstrating that iPS cells derived from human skin cells can be differentiated into pancreatic islet-like cluster cells. These cluster cells were shown to contain C-peptidepositive and glucagon-positive cells and, more importantly, to release the C-peptide upon glucose stimulation (Tateishi et al., 2008). iPS cells were also generated from patients with type 1 diabetes by reprogramming their adult fibroblasts with three transcription factors (*OCT4, SOX2, KLF4*). The iPS generated from patients were shown to differentiate into insulin-producing cells (Maehr et al., 2009). A proof of principle for potential clinical applications of reprogrammed somatic cells in the treatment of diabetes type 1 or 2 was provided by Alipio et al who were able to reverse hyperglycemia in diabetic mouse models using iPS-derived pancreatic like cells (Alipio et al., 2010). iPS could overcome the ethical issues and the immunogenicity of embryonic stem cells. The possibility to use iPS individually tailored for each patient is certainly appealing. However, several problems of bio-safety must be solved before iPS enter in clinical use. These include an aberrant or incomplete differentiation and a tumorigenic potential. Reprogramming cells typically requires integration of genes such as *c-MYC, OCT4 AND KLF4* that are known to be oncogenic and may favour development of tumors (Miura et al., 2009). Although after reprogramming the transgenes undergo silencing, it is always possible a reactivation. Therefore, the research in this field is actively searching for alternative strategies to

**6. Stem cell therapy to counteract the autoimmune destruction of cells** 

Assuming that we could generate pluripotent stem cell lines for each individual patient and that after infusion they would be able to restore the cell mass, we still have to face the problem of recurrent autoimmunity. The autoimmune destruction of islet cells by reactive T lymphocytes not only plays a role in the establishment of type 1 diabetes but also impairs an efficient regenerative process. Newly forming cells show increased vulnerability to apoptosis induced by inflammatory cytokines (Meier et al., 2006), explaining the failure of cell regeneration in type 1 diabetes. Therefore, newly generated cells are systematically killed as they acquire the mature phenotype, thus impairing the regenerative attempts in type 1 diabetic patients. Furthermore, therapeutic intervention studies in new onset type 1 diabetes involving broadly immunosuppressive agents, such as cyclosporin A, failed to produce lasting remission, demonstrating the inherent tendency of the autoimmune effector response in humans to recur. Therefore, it is critical that any immunemodulatory therapy induces tolerance to cell antigens, while minimizing detrimental effects on host defence. This is strengthened by recurrence of type 1 diabetes in syngenic pancreas transplantation

induce pluripotency.

(Sutherland et al., 1984).

Cell-based therapeutic strategies for immunemodulation that answer to the requirement of safety and effectiveness are under development. The intravenous use of humanized antibodies against CD3 (part of the T-cell receptor complex) soon after the initial onset of diabetic clinical symptoms decreased the insulin requirement compared to controls without this monoclonal antibody therapy. However, this therapy is burdened by several adverse effects (Keymeulen et al., 2005; Herold et al., 2002; McDevitt & Unanue, 2008).

Other approaches of immunemodulation included subcutaneous administration of heatshock protein (Raz et al., 2001) or intravenous injection of rabbit polyclonal anti T-cell globulin (Saudek et al., 2004). In many of these studies a short term effective preservation of cell function was observed; however, only few patients no longer required insulin treatment. Using human recombinant glutamic acid decarboxylase (GAD65) as therapeutic vaccine it was observed a preservation of C-peptide levels and a decrease in insulin dose requirement only in patients with very recent-onset of diabetes (Ludvigsson et al., 2008; Uibo and Lernmark 2008). Compelling evidence indicates the relevant role of Treg in the inhibition of autoimmune response. A decreased number and function of Treg have been reported in patients with type 1 diabetes (Roncarolo & Battaglia, 2007). Therefore, intervention to restore Treg function in diabetic patients is an attractive therapeutic strategy. Nevertheless, only few studies have shown re-establishment of Treg function in diabetes. Zhao et al demonstrated the possibility to correct functional defects of CD4+ CD62L Treg using human cord blood stem cells. This strategy allowed reversal of autoimmune diabetes in NOD mice (Y. Zhao et al., 2009) providing proof of principle that cord blood stem cells can correct function of Treg.

Treg may act by producing IL-10 and in particular transforming growth factor-1 (TGF1), cytokines that are known to contribute to the induction of immunetolerance. It has been suggested that protection of newly generated cells achieved by treatment with Treg depends on the formation of TGF1 cell ring surrounding pancreatic islands and conferring protection against autoimmune T lymphocytes inducing their apoptosis (Y. Zhao et al., 2009). In a pilot study in Type 1 diabetic children based on administration of autologous umbilical cord blood shortly after disease onset a lower level of HbA1c and a reduced insulin requirement was reported. However, none of the patients achieved insulin-treatment independency (Haller et al., 2007).

Recent studies raised great interest on the immunemodulatory potential of mesenchymal stem cells (Le Blanc & Pittenger, 2005). MSC can modulate several T cell functions exerting an immunesuppressive effect. MSC that lack MHC class II molecules and do not express key costimulatory molecules B7-1, B7-2, CD40 and CD40L, were shown to reduce the expression of several lymphocyte activation markers (Aggarwal & Pittenger, 2005). The mechanism has been related to the induction of an anti-inflammatory phenotype in dendritic cells, naive and activated T cells and NK cells, and to an increase of the regulatory T cell population. This results not only in the inhibition of T cell proliferation to polyclonal stimuli, but also in inhibition of naive and memory antigen-specific T cells response to their cognate peptide (Krampera et al., 2003). The MSC-induced suppression has been ascribed to several soluble factors, including hepatocyte growth factor (HGF), TGF-1 and prostaglandin E2 (PGE2) (Aggarwal & Pittenger, 2005). Moreover, MSC have been shown to induce mature dendritic cells type 2 (DC2) to increase IL-10 secretion thus promoting anti-infammatory DC2 signaling. Several studies suggest that MSC improve the outcome of allogenic transplantation and hamper graft-versus-host disease (Le Blanc et al., 2004; Ringden et al.,

Perspectives of Cell Therapy in Type 1 Diabetes 337

autoimmune diseases. In experimental autoimmune encephalomyelitis, animal model of multiple sclerosis mediated by autoreactive T cells, injected MSC home to lymphoid organs where they cluster around T cells, and ameliorate the disease onset (Gerdoni et al., 2007). In this setting, MSC induce peripheral tolerance, impairing both the cellular and humoral arm of the encephalitogenic immune response, without evidence of transdifferentiation into neural cells (Zappia et al., 2005; Gerdoni et al., 2007). Furthermore, in a murine model of rheumatoid arthritis MSC have been demonstrated to exert an immunemodulatory effect by educating antigen-specific regulatory T cells (Augello et al., 2007). MSCs are also able to inhibit autoreactive T and B cells in experimental models of systemic lupus erythematosus (Deng et al., 2005). Overall, studies on murine models of type 1 diabetes (Fiorina et al., 2009; Madec et al., 2009) and multiple sclerosis (Zappia et al., 2005) as well as non-autoimmune diseases (Herrera et al., 2007) indicate that a key feature of MSC is their ability to selectively migrate into sites of injury, where they are likely to interact with activated T cells. Diabetogenic T cells are generated in pancreatic lymph nodes where they are introduced to antigens by dendritic cells. The preferential homing of MSC to pancreatic lympho nodes (Fiorina et al., 2009; Madec et al., 2009), supports the hypothesis that these cells could directly suppress autoreactive T cells *in vivo* within the pancreatic environment. Further, the desired therapeutic effects could be achieved by modulation of chemokines/receptors to promote the homing of MSC to specific anatomical sites (Sackstein et al., 2008). The first report on transplantation of human allogenic MSC, into a patient with autoimmune systemic sclerosis, indicates its safety and, notably, striking efficacy by selective immunesuppression and regeneration of impaired endothelial progenitors (Christopeit et al., 2008). Thus, results on the use of MSC infusion for treatment of severe graft-versus-host disease or autoimmune diseases (Lazarus et al., 2005; Fouillard et al., 2003; Le Blanc et al., 2004; Ringden et al., 2006; Christopeit et al., 2008) suggest a potential use in patients at risk of type 1 diabetes or at disease onset, to preserve or reduce loss of ß cells (Abdi et al., 2008;

Staeva-Vieira et al., 2007).

of self renewal.

**7. MSC treatment of complications of diabetes mellitus** 

The ability of MSC to differentiate into tissue of mesodermal origin makes them attractive also as therapeutic agents for a number of complications of diabetes, including cardiomyopathy, nephropathy, polyneuropathy and diabetic wounds. MSC have been shown to differentiate into several cell types, including cardiomyocytes, endothelial cells, neurons, hepatocytes, epithelial cells and adipocytes, characteristics coupled with capacity

Chronic hyperglycemia is responsible for myocardial remodelling leading to ventricular dysfunction with hypertrophy and apoptosis of cardiomyocytes, microcirculatory defects, altered extracellular matrix and matrix metalloproteinasis (Jesmin et al., 2003). MSC can induce myogenesis and angiogenesis by different mitogenic, angiogenic and antiapoptotic factors, such as VEGF, IGF-1 and HGF (Zhang et al., 2008). In a diabetic rat model, intravenous MSC have been shown to improve cardiac function, potentially by differentiation into cardiomyocytes and improvement of myogenesis and angiogenesis. Metalloproteinase activity was also modulated, leading to increased arteriolar density and decreased collagen volume. Cardioprotection is probably more mediated by release of paracrine factors by MSC. These include VEGF, HGF, Bcl-2, Hsp20, activation of Akt

2006). Studies on the immunemodulatory potential of MSC in human type 1 diabetes are still lacking but it has been reported their regenerative potential in diabetic NOD/SCID mice leading to an increased number of pancreatic islets and cells (R.H. Lee et al., 2006). We recently demonstrated that human allogenic bone marrow derived MSC can abrogate *in vitro* a pro-inflammatory Th1 response to islet antigen GAD in new onset type 1 diabetes, by impairing the production of IFN- and by inducing anti-inflammatory IL-4 production (Zanone et al., 2010). These data stimulate further studies on MSC immunemodulation in diabetes and open a perspective for immune-intervention strategies. The mechanisms of MSC interaction with the immune system cells are still controversial (van Laar & Tyndall, 2006; Abdi et al., 2008), and include reduction of the expression of lymphocyte activation markers, change of the cytokine profile of dendritic cells, naive and activated T cells and NK cells to an anti-inflammatory phenotype, and increase of the regulatory T cell population (Aggarwal & Pittenger, 2005; Le Blanc et al., 2004). In the reported study in type 1 diabetes, MSC induced in peripheral blood mononuclear cells (PBMC) of responder patients IL-4 producing cells and IL-4 secretion, suggesting a possible switch to an anti-inflammatory Th2 signalling of T cells (Zanone et al., 2010). Increased IL-4 secretion has been shown in studies of MSC cocultured with subpopulations of PHA-stimulated immune cells (Aggarwal & Pittenger, 2005) but not in studies of T cells activated by encephalitogenic peptide (Zappia et al., 2005). Lymphocyte activation is extremely complex and it is likely that several mechanisms are involved in the MSC-mediated immunesuppression and that the specific factors may depend on the lymphocyte population tested, the stimulus used, the timing of analysis and the context of the immune disease.

Further, inhibition of PGE2 production abrogated the MSC-mediated IFN- suppression, indicating that PGE2 secretion plays a key role in MSC-mediated immune effects, and the contact between MSC and PBMC enhances the production of prostaglandin E2 (Zanone et al., 2010). This observation suggests the requirement of both soluble factors and cell-contact in line with the interpretation that the immunemodulatory effects of MSC might require an initial cell-contact phase (Krampera et al., 2003). Nevertheless, the requirement of cell contact for MSC to operate their inhibition is a controversial issue, and the results reported in the literature may depend on the species and the type of stimulus.

Other studies in murine diabetic models on the regenerative capabilities of MSC, showed that injection of MSC into immunodeficient diabetic NOD/SCID mice resulted in the selective homing of MSC to pancreatic islets and in an increased number of pancreatic islets and functioning cells (R.H. Lee et al., 2006). Further, MSC can be influenced to differentiate into cells with properties of the cell phenotype, becoming more efficient after transplantation in mice (M. Zhao et al., 2008). Indeed, genetically modified MSC by recombinant Pdx-1 adenovirus or by non-virus gene transfection, were able to express insulin sufficient to reduce blood glucose in the streptozocin mouse model of diabetes (Karnieli et al., 2007; M. Zhao et al., 2008). More recently, allogenic MSC obtained from diabetes-prone as well as -resistant mice and injected into NOD mice, have been shown to delay the onset of diabetes or to reverse hyperglycemia (Fiorina et al., 2009). This study indicates that the beneficial effects observed could also be ascribed to the immunemodulatory capacities of MSC, as for other studies focusing on MSC-induced repair of cell injury (Abdi et al., 2008; Morigi et al., 2004; Duffield et al., 2005).

Studies on *ex vivo* expanded MSC to improve the outcome of allogenic transplantation and of acute graft-versus-host disease paved the way for the clinical use of MSC also in

2006). Studies on the immunemodulatory potential of MSC in human type 1 diabetes are still lacking but it has been reported their regenerative potential in diabetic NOD/SCID mice leading to an increased number of pancreatic islets and cells (R.H. Lee et al., 2006). We recently demonstrated that human allogenic bone marrow derived MSC can abrogate *in vitro* a pro-inflammatory Th1 response to islet antigen GAD in new onset type 1 diabetes, by impairing the production of IFN- and by inducing anti-inflammatory IL-4 production (Zanone et al., 2010). These data stimulate further studies on MSC immunemodulation in diabetes and open a perspective for immune-intervention strategies. The mechanisms of MSC interaction with the immune system cells are still controversial (van Laar & Tyndall, 2006; Abdi et al., 2008), and include reduction of the expression of lymphocyte activation markers, change of the cytokine profile of dendritic cells, naive and activated T cells and NK cells to an anti-inflammatory phenotype, and increase of the regulatory T cell population (Aggarwal & Pittenger, 2005; Le Blanc et al., 2004). In the reported study in type 1 diabetes, MSC induced in peripheral blood mononuclear cells (PBMC) of responder patients IL-4 producing cells and IL-4 secretion, suggesting a possible switch to an anti-inflammatory Th2 signalling of T cells (Zanone et al., 2010). Increased IL-4 secretion has been shown in studies of MSC cocultured with subpopulations of PHA-stimulated immune cells (Aggarwal & Pittenger, 2005) but not in studies of T cells activated by encephalitogenic peptide (Zappia et al., 2005). Lymphocyte activation is extremely complex and it is likely that several mechanisms are involved in the MSC-mediated immunesuppression and that the specific factors may depend on the lymphocyte population tested, the stimulus used, the timing of

Further, inhibition of PGE2 production abrogated the MSC-mediated IFN- suppression, indicating that PGE2 secretion plays a key role in MSC-mediated immune effects, and the contact between MSC and PBMC enhances the production of prostaglandin E2 (Zanone et al., 2010). This observation suggests the requirement of both soluble factors and cell-contact in line with the interpretation that the immunemodulatory effects of MSC might require an initial cell-contact phase (Krampera et al., 2003). Nevertheless, the requirement of cell contact for MSC to operate their inhibition is a controversial issue, and the results reported

Other studies in murine diabetic models on the regenerative capabilities of MSC, showed that injection of MSC into immunodeficient diabetic NOD/SCID mice resulted in the selective homing of MSC to pancreatic islets and in an increased number of pancreatic islets and functioning cells (R.H. Lee et al., 2006). Further, MSC can be influenced to differentiate into cells with properties of the cell phenotype, becoming more efficient after transplantation in mice (M. Zhao et al., 2008). Indeed, genetically modified MSC by recombinant Pdx-1 adenovirus or by non-virus gene transfection, were able to express insulin sufficient to reduce blood glucose in the streptozocin mouse model of diabetes (Karnieli et al., 2007; M. Zhao et al., 2008). More recently, allogenic MSC obtained from diabetes-prone as well as -resistant mice and injected into NOD mice, have been shown to delay the onset of diabetes or to reverse hyperglycemia (Fiorina et al., 2009). This study indicates that the beneficial effects observed could also be ascribed to the immunemodulatory capacities of MSC, as for other studies focusing on MSC-induced repair

Studies on *ex vivo* expanded MSC to improve the outcome of allogenic transplantation and of acute graft-versus-host disease paved the way for the clinical use of MSC also in

analysis and the context of the immune disease.

in the literature may depend on the species and the type of stimulus.

of cell injury (Abdi et al., 2008; Morigi et al., 2004; Duffield et al., 2005).

autoimmune diseases. In experimental autoimmune encephalomyelitis, animal model of multiple sclerosis mediated by autoreactive T cells, injected MSC home to lymphoid organs where they cluster around T cells, and ameliorate the disease onset (Gerdoni et al., 2007). In this setting, MSC induce peripheral tolerance, impairing both the cellular and humoral arm of the encephalitogenic immune response, without evidence of transdifferentiation into neural cells (Zappia et al., 2005; Gerdoni et al., 2007). Furthermore, in a murine model of rheumatoid arthritis MSC have been demonstrated to exert an immunemodulatory effect by educating antigen-specific regulatory T cells (Augello et al., 2007). MSCs are also able to inhibit autoreactive T and B cells in experimental models of systemic lupus erythematosus (Deng et al., 2005). Overall, studies on murine models of type 1 diabetes (Fiorina et al., 2009; Madec et al., 2009) and multiple sclerosis (Zappia et al., 2005) as well as non-autoimmune diseases (Herrera et al., 2007) indicate that a key feature of MSC is their ability to selectively migrate into sites of injury, where they are likely to interact with activated T cells. Diabetogenic T cells are generated in pancreatic lymph nodes where they are introduced to antigens by dendritic cells. The preferential homing of MSC to pancreatic lympho nodes (Fiorina et al., 2009; Madec et al., 2009), supports the hypothesis that these cells could directly suppress autoreactive T cells *in vivo* within the pancreatic environment. Further, the desired therapeutic effects could be achieved by modulation of chemokines/receptors to promote the homing of MSC to specific anatomical sites (Sackstein et al., 2008). The first report on transplantation of human allogenic MSC, into a patient with autoimmune systemic sclerosis, indicates its safety and, notably, striking efficacy by selective immunesuppression and regeneration of impaired endothelial progenitors (Christopeit et al., 2008). Thus, results on the use of MSC infusion for treatment of severe graft-versus-host disease or autoimmune diseases (Lazarus et al., 2005; Fouillard et al., 2003; Le Blanc et al., 2004; Ringden et al., 2006; Christopeit et al., 2008) suggest a potential use in patients at risk of type 1 diabetes or at disease onset, to preserve or reduce loss of ß cells (Abdi et al., 2008; Staeva-Vieira et al., 2007).

#### **7. MSC treatment of complications of diabetes mellitus**

The ability of MSC to differentiate into tissue of mesodermal origin makes them attractive also as therapeutic agents for a number of complications of diabetes, including cardiomyopathy, nephropathy, polyneuropathy and diabetic wounds. MSC have been shown to differentiate into several cell types, including cardiomyocytes, endothelial cells, neurons, hepatocytes, epithelial cells and adipocytes, characteristics coupled with capacity of self renewal.

Chronic hyperglycemia is responsible for myocardial remodelling leading to ventricular dysfunction with hypertrophy and apoptosis of cardiomyocytes, microcirculatory defects, altered extracellular matrix and matrix metalloproteinasis (Jesmin et al., 2003). MSC can induce myogenesis and angiogenesis by different mitogenic, angiogenic and antiapoptotic factors, such as VEGF, IGF-1 and HGF (Zhang et al., 2008). In a diabetic rat model, intravenous MSC have been shown to improve cardiac function, potentially by differentiation into cardiomyocytes and improvement of myogenesis and angiogenesis. Metalloproteinase activity was also modulated, leading to increased arteriolar density and decreased collagen volume. Cardioprotection is probably more mediated by release of paracrine factors by MSC. These include VEGF, HGF, Bcl-2, Hsp20, activation of Akt

Perspectives of Cell Therapy in Type 1 Diabetes 339

Abdi, R., Fiorina, P., Adra, C.N., Atkinson, M. & Sayegh, M.H. (2008). Immunomodulation

Aggarwal, S. & Pittenger, M.F. (2005). Human mesenchymal stem cells modulate allogeneic

Alipio, Z., Liao, W., Roemer, E.J., Waner, M., Fink, L.M., Ward, D.C. & Ma, Y. (2010).

Augello, A., Tasso.,R., Negrini, S.M., Cancedda, R. & Pennesi, G. (2007). Cell therapy using

Baeyens, L., De Breuck, S., Lardon, J., Mfopou, J.K., Rooman, I. & Bouwens, L. (2005). In

Bonner-Weir, S., Toschi, E., Inada, A., Reitz, P., Fonseca, S.Y., Aye, T. & Sharma, A. (2004).

Booth, C. & Potten, C.S. (2000). Gut instincts: thoughts on intestinal epithelial stem cells. *J Clin Invest,* Vol. 105, No. 11, (June 2000), pp. 1493-1499, ISSN 0021-9738 Bouvens, L. (2006). Beta cell regeneration. *Curr Diabetes Rev,* Vol. 2, pp. 3-9 ISSN 1573-3998 Brissova, M., Fowler, M., Wiebe, P., Shostak, A., Shiota, M., Radhika, A., Lin, P.C., Gannon,

Brolén, G.K., Heins, N., Edsbagge, J. & Semb, H. (2005). Signals from the embryonic mouse

Bussolati, B., Bruno, S., Grange, C., Buttiglieri, S., Deregibus, M.C., Cantino, D. & Camussi,

Calafiore, R., Basta, G., Luca, G., Lemmi, A., Montanucci, M.P., Calabrese, G., Racanicchi, L.,

Vol. 166, No. 2, (February 2005), pp. 545-555, ISSN 0002-9440

Vol. 29, No. 1, (January 2006), pp. 137-138, ISSN 0149-5992

*Diabetologia*, Vol. 48, No. 1, (January 2005), pp. 49-57, ISSN 0012-186X Bernal-Mizrachi, E., Fatrai, S., Johnson, J.D., Ohsugi, M., Otani, K., Han, Z., Polonsky, K.S. &

*Diabetes*, Vol. 57, No. 7, (July 2008), pp. 1759-1767, ISSN 0012-1797.

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by mesenchymal stem cells: a potential therapeutic strategy for type 1 diabetes.

immune cell responses. *Blood,* Vol. 105, No. 4, (February 2005), pp. 1815-1822, ISSN

Reversal of hyperglycemia in diabetic Augello mouse models using inducedpluripotent stem (iPS)-derived pancreatic beta-like cells. *Proc Natl Acad Sci U S A*,

allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen-induced arthritis. *Arthritis Rheum*, Vol. 56, No. 4, (April 2007), pp. 1175-

vitro generation of insulin-producing beta cells from adult exocrine pancreatic cells.

Permutt, M.A. (2004). Defective insulin secretion and increased susceptibility to experimental diabetes are induced by reduced Akt activity in pancreatic islet beta cells. *J Clin Invest*, Vol. 114, No. 7, (October 2004), pp. 928-936, ISSN 0021-9738 Biancone, L. & Ricordi, C. (2002). Pancreatic islet transplantation: an update. *Cell Transplant,*

The pancreatic ductal epithelium serves as a potential pool of progenitor cells.

M. & Powers, A.C. (2004). Intraislet endothelial cells contribute to revascularization of transplanted pancreatic islets. *Diabetes*, Vol. 53, No. 5, (May 2004), pp. 1318-1325,

pancreas induce differentiation of human embryonic stem cells into insulinproducing beta-cell-like cells. *Diabetes*, Vol. 54, No. 10, (October 2005), pp. 2867-

G. (2005). Isolation of renal progenitor cells from adult human kidney. *Am J Pathol*,

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**10. References** 

0006-4971

1186, ISSN 00043591

ISSN 0012-1797

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(Wang et al., 2009), which can affect remodelling, repair and neovascularization. Intravenous autologous MSC in post-infarction patients have indeed been shown to reduce episodes of ventricular tachycardia and to increase ventricular ejection fraction (R.H. Lee et al., 2010).

Diabetic limb ischemia could also be improved by MSC-derived pro-angiogenic factors (Comerota et al., 2010).

MSC have also been used for treatment of diabetic nephropathy in NOD/SCID and streptozocin C57B1/6 mice (R.H. Lee et al., 2006). The injected cells engraft in damaged kidneys, potentially differentiate into renal cells and endothelial cells and can regulate the immune response. This resulted in improved kidney function and regeneration of glomerular structure. MSC, however, were unable to proliferate; therefore, it is conceivable that MSC contribute to the repair by releasing paracrine factors that promote neovasculatization and limit cytotoxic injury.

As for diabetic polyneuropathy, in diabetic rats, intramuscular injection of MSC led to increased ratio of capillaries to muscle fibers, improvement of hyperalgesia and function of neural fibers. MSC settled in the gap between muscle fibers at the transplanted site, and produced VEGF and basic bFGF, without differentiating into neural cells (Shibata et al., 2008).

By releasing paracrine factors and by differentiation into photoreceptor and glial-like cells in the retina, transplanted MSC have also been shown to improve the integrity of the bloodretinal barrier, ameliorating diabetic retinopathy in streptozocin diabetic rats (Z. Yang et al., 2010).

Prolonged and uncompleted wound healing can complicate the diabetic condition. Injection of MSC in animal models of diabetes improved wound healing, with increase of collagen levels and of wound-breaking strength, together with increased levels of TFG, KGF, EGF, PDGF, VEGF, all involved in repair (Wu et al., 2007). Besides these paracrine effects MSC were shown to differentiate and regenerate damaged epithelium.

Limitation of the potential therapeutic use of MSC also for diabetic chronic complications are, at present, mainly the poor engraftment and the limited differentiation under *in vivo* conditions, together with the potential differentiation into unwanted mesenchymal lineages.

#### **8. Conclusion**

A number of issues should be addressed before a cell based therapy may come to a clinical setting. The first challenge is to define which kind of cells are more suitable for cell substitution. This implies to develop efficient strategies of stem cell differentiation that lead to cells that produce and secrete insulin in physiological amounts under the control of glycemia. Moreover, safety of a cell based therapy remains a critical point, as any precursor or stem cell types might induce tumor formation. Whether the achievement of fully differentiated cells would reduce this risk remains to be proved. Another relevant point is to define the strategies that allow an immune modulation to avoid the recurrence of autoimmune destruction of newly formed cells.

#### **9. Acknowledgement**

Funded by Regione Piemonte, Piattaforme Biotecnologiche, Pi-Stem project.

#### **10. References**

338 Type 1 Diabetes Complications

(Wang et al., 2009), which can affect remodelling, repair and neovascularization. Intravenous autologous MSC in post-infarction patients have indeed been shown to reduce episodes of ventricular tachycardia and to increase ventricular ejection fraction

Diabetic limb ischemia could also be improved by MSC-derived pro-angiogenic factors

MSC have also been used for treatment of diabetic nephropathy in NOD/SCID and streptozocin C57B1/6 mice (R.H. Lee et al., 2006). The injected cells engraft in damaged kidneys, potentially differentiate into renal cells and endothelial cells and can regulate the immune response. This resulted in improved kidney function and regeneration of glomerular structure. MSC, however, were unable to proliferate; therefore, it is conceivable that MSC contribute to the repair by releasing paracrine factors that promote

As for diabetic polyneuropathy, in diabetic rats, intramuscular injection of MSC led to increased ratio of capillaries to muscle fibers, improvement of hyperalgesia and function of neural fibers. MSC settled in the gap between muscle fibers at the transplanted site, and produced VEGF and basic bFGF, without differentiating into neural cells (Shibata et al.,

By releasing paracrine factors and by differentiation into photoreceptor and glial-like cells in the retina, transplanted MSC have also been shown to improve the integrity of the bloodretinal barrier, ameliorating diabetic retinopathy in streptozocin diabetic rats (Z. Yang et al.,

Prolonged and uncompleted wound healing can complicate the diabetic condition. Injection of MSC in animal models of diabetes improved wound healing, with increase of collagen levels and of wound-breaking strength, together with increased levels of TFG, KGF, EGF, PDGF, VEGF, all involved in repair (Wu et al., 2007). Besides these paracrine effects MSC

Limitation of the potential therapeutic use of MSC also for diabetic chronic complications are, at present, mainly the poor engraftment and the limited differentiation under *in vivo* conditions, together with the potential differentiation into unwanted mesenchymal

A number of issues should be addressed before a cell based therapy may come to a clinical setting. The first challenge is to define which kind of cells are more suitable for cell substitution. This implies to develop efficient strategies of stem cell differentiation that lead to cells that produce and secrete insulin in physiological amounts under the control of glycemia. Moreover, safety of a cell based therapy remains a critical point, as any precursor or stem cell types might induce tumor formation. Whether the achievement of fully differentiated cells would reduce this risk remains to be proved. Another relevant point is to define the strategies that allow an immune modulation to avoid the recurrence of

Funded by Regione Piemonte, Piattaforme Biotecnologiche, Pi-Stem project.

were shown to differentiate and regenerate damaged epithelium.

autoimmune destruction of newly formed cells.

(R.H. Lee et al., 2010).

(Comerota et al., 2010).

2008).

2010).

lineages.

**8. Conclusion** 

**9. Acknowledgement** 

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**16** 

*Iran* 

Nepton Soltani

**Prevention of Diabetes Complications** 

*Molecular Medicine Research Center, Hormozgan University of Medical Science,* 

The constellation of abnormalities caused by insulin deficiency is called diabetes mellitus. The cause of clinical diabetes is always a deficiency of the effect of insulin at the tissue level. Type I diabetes or insulin- dependent diabetes mellitus (IDDM), is due to insulin deficiency caused by autoimmune destruction of the B cell in the pancreatic islets, and it accounts for 3- 5 % of cases and usually presents in children. Type 2 diabetes, or non-insulin-dependent diabetes mellitus (NIDDM), is characterized by the dysregulation of insulin release from the B cells, along with insulin resistance in peripheral tissues such as skeletal muscle, brain, and

The incidence of diabetes in the human population has reached epidemic proportions worldwide and it is increasing at the rapid rate.150 million people in 2000, which is

In animals, it can be produced by pancreatectomy; by administration of alloxan, streptozocin, or other toxins that in appropriate doses cause selective destruction of the beta cells of the pancreatic islets; by administration of drugs that inhibit insulin secretion; and by administration of anti-insulin anti-bodies. Strains of mice, rats, hamsters, guinea pigs, miniature swine, and monkeys that have a high incidence of spontaneous diabetes mellitus

Diabetes is characterized by polyuria, polydipisa, weight loss in spite of polyphagia, hyperglycemia, glycosuria, ketosis, acidosis, and coma. Widespread biochemical abnormalities are present, but the fundamental defects to which most of the abnormalities can be traced are 1) reduced entry of glucose into various peripheral tissues and 2) increased liberation of glucose into the circulation from the liver. Therefore there is an extracellular glucose excess and, in many cells, an intracellular glucose deficiency a situation that has been called starvation in the midst of plenty. Also, the entry of amino acids into muscle is

Diabetic complications are divided to two parts: 1) metabolic complication and 2) vascular

Obesity is increasing in incidence, and relates to the regulation of food intake and energy balance and overall nutrition. It is special relation to disordered carbohydrate metabolism

liver. Type 2 diabetes usually presents in overweight or obese adults.

predicted to rise to 220 million in 2010.

decreased and lipolysis is increased.

**2. Diabetes complication** 

**2.1 Metabolic complication** 

complication.

have also been described.

**1. Introduction** 


## **Prevention of Diabetes Complications**

#### Nepton Soltani

*Molecular Medicine Research Center, Hormozgan University of Medical Science, Iran* 

#### **1. Introduction**

352 Type 1 Diabetes Complications

Yang, Z., Li, K., Yan, X., Dong, F. & Zhao, C. (2010). Amelioration of diabetic retinopathy by

Zappia, E., Casazza, S., Pedemonte, E., Benvenuto, F., Bonanni, I., Gerdoni, E., Giunti, D.,

Zhang, N., Li, J., Luo, R., Jiang, J. & Wang, J.A. (2008). Bone marrow mesenchymal stem cells

Zhao, M., Amiel, S.A., Ajami, S., Jiang, J., Rela, M., Heaton, N. & Huang, G.C. (2008).

Zhao, Y., Lin, B., Darflinger, R., Zhang, Y., Holterman, M.J. & Skidgel, R.A. (2009). Human

Zhao, Y. & Mazzone, T. (2010). Human cord blood stem cells and the journey to a cure for

Zanone, M.M., Favaro, E. & Camussi, G. (2008). From endothelial to beta cells: insights into

Zanone, M.M., Favaro, E., Miceli, I., Grassi, G., Camussi, E., Caorsi, C., Amoroso, A.,

Vol. 4, No. 1, (January 2009), pp. e4226, ISSN 1932-6203

1415-1422, ISSN 0721-832X

ISSN 0006-4971

7349

1932-6203

1568-9972

pp. 1-9, ISSN 1573-3998

ISSN 0021-972X

engrafted human adipose-derived mesenchymal stem cells in streptozotocin diabetic rats. *Graefes Arch Clin Exp Ophthalmol*, Vol. 248, No. 10, (October 2010), pp.

Ceravolo, A., Cazzanti, F., Frassoni, F., Mancardi, G. & Uccelli, A. (2005). Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. *Blood*, Vol. 106, No. 5, (September 2005), pp. 1755-1761,

induce angiogenesis and attenuate the remodeling of diabetic cardiomyopathy. *Exp Clin Endocrinol Diabetes*, Vol. 116, No. 2, (February 2008), pp. 104-111, ISSN 0947-

Amelioration of streptozotocin-induced diabetes in mice with cells derived from human marrow stromal cells. *PLoS One*, Vol. 3, No. 7, (July 2008), pp. e2666, ISSN

cord blood stem cell-modulated regulatory T lymphocytes reverse the autoimmune-caused type 1 diabetes in nonobese diabetic (NOD) mice. *PLoS One*,

type 1 diabetes. *Autoimmun Rev*, Vol. 10, No. 2, (December 2010), pp. 103-107, ISSN

pancreatic islet microendothelium. *Curr Diabetes Rev*, Vol. 4, No. 1, (February 2008),

Giovarelli, M., Perin, P.C. & Camussi, G. (2010). Human mesenchymal stem cells modulate cellular immune response to islet antigen glutamic acid decarboxylase in type 1 diabetes. *J Clin Endocrinol Metab*, Vol. 95, No. 8, (August 2010), pp. 3788-97, The constellation of abnormalities caused by insulin deficiency is called diabetes mellitus. The cause of clinical diabetes is always a deficiency of the effect of insulin at the tissue level. Type I diabetes or insulin- dependent diabetes mellitus (IDDM), is due to insulin deficiency caused by autoimmune destruction of the B cell in the pancreatic islets, and it accounts for 3- 5 % of cases and usually presents in children. Type 2 diabetes, or non-insulin-dependent diabetes mellitus (NIDDM), is characterized by the dysregulation of insulin release from the B cells, along with insulin resistance in peripheral tissues such as skeletal muscle, brain, and liver. Type 2 diabetes usually presents in overweight or obese adults.

The incidence of diabetes in the human population has reached epidemic proportions worldwide and it is increasing at the rapid rate.150 million people in 2000, which is predicted to rise to 220 million in 2010.

In animals, it can be produced by pancreatectomy; by administration of alloxan, streptozocin, or other toxins that in appropriate doses cause selective destruction of the beta cells of the pancreatic islets; by administration of drugs that inhibit insulin secretion; and by administration of anti-insulin anti-bodies. Strains of mice, rats, hamsters, guinea pigs, miniature swine, and monkeys that have a high incidence of spontaneous diabetes mellitus have also been described.

Diabetes is characterized by polyuria, polydipisa, weight loss in spite of polyphagia, hyperglycemia, glycosuria, ketosis, acidosis, and coma. Widespread biochemical abnormalities are present, but the fundamental defects to which most of the abnormalities can be traced are 1) reduced entry of glucose into various peripheral tissues and 2) increased liberation of glucose into the circulation from the liver. Therefore there is an extracellular glucose excess and, in many cells, an intracellular glucose deficiency a situation that has been called starvation in the midst of plenty. Also, the entry of amino acids into muscle is decreased and lipolysis is increased.

#### **2. Diabetes complication**

Diabetic complications are divided to two parts: 1) metabolic complication and 2) vascular complication.

#### **2.1 Metabolic complication**

Obesity is increasing in incidence, and relates to the regulation of food intake and energy balance and overall nutrition. It is special relation to disordered carbohydrate metabolism

Prevention of Diabetes Complications 355

Vascular disease is one of the complicating features of diabetes mellitus. Several prospective studies have indicated that hypertension in diabetic patient's takes place at a rate more than twice compared to the normal population. The hypertension is also considered an independent risk factor for cardiovascular mortality in patients with diabetes. It has been suggested that alterations in the reactivity of blood vessels to neurotransmitters and circulating hormones are responsible for the cardiovascular complications of diabetes. Some studies showed that Ca/Mg ratio is a marker of vascular tone; its increase represents increased vascular reactivity and atherogenic risk. Atherogenic lesion is poorly correlated with serum cholesterol level and is highly dependent on plasma magnesium level and Ca/Mg ratio. Pervious studies showed that Ca/Mg ratio increase in diabetic case. Endothelial function is altered early in diabetes. It has been demonstrated that in diabetic subjects, the vasodilating response to stimuli is diminished and that this anomaly is related to glycemic control. In vivo studies have demonstrated that hyperglycemic spikes induce, in both diabetic and normal subjects, an endothelial dysfunction. This effect of hyperglycemia is probably linked with a reduced production/bioavailability of nitric oxide (NO), since hyperglycemia-induced endothelial dysfunction is counterbalanced by arginine. Furthermore, it is very interesting that a rapid decrease of flow-mediated vasodilation has been shown in the postprandial phase in type II diabetes patients and that the decrease

correlated inversely with the magnitude of postprandial hyperglycemia.

Type I diabetes usually develops before the age 40, patients with this disease are not obese and they have a high incidence of ketosis and acidosis. Various anti-B cell antibodies are present in plasma, but the current thinking is that type I diabetes is primarily a T lymphocyte-mediated disease. But type II diabetes is the most common type of diabetes and is usually associated with obesity. It usually develops after age 40 and is not associated with total loss of the ability to secrete insulin. It has an insidious onset, is rarely associated with ketosis, and is usually associated with normal beta-cells morphology and insulin content if the beta-cells have not become exhausted. So it seems we should look for different methods

Several lifestyle factors affect the incidence of type 2 diabetes. Obesity and weight gain dramatically increase the risk, and physical inactivity further elevates the risk, independently of obesity. Cigarette smoking is associated with a small increase and moderate alcohol consumption with a decrease in the risk of diabetes. In addition, a low-fiber diet with a high glycemic index has been associated with an increased risk of diabetes, and specific dietary

Excess body fat is the single most important determinant of type II diabetes. Weight control would be the most effective way to reduce the risk of type II diabetes, but current strategies have not been very successful on a population basis, and the prevalence of obesity continues to increase. The public generally does not recognize the connection between overweight or

Low-fat vegetarian and vegan diets are associated with reduced body weight, increased insulin sensitivity, and reductions in cardiovascular risk factors. The potential cardiovascular benefits of vegetarian and vegan diets may be especially important for

fatty acids may differentially affect insulin resistance and the risk of diabetes.

obesity and diabetes. Thus, greater efforts at education are needed.

**3. Prevention of diabetes complications** 

for prevention of type I and II diabetes.

**3.1 Diabetes diet** 

and diabetes. As body weight increase, insulin resistance increase, that is, there is a decreased ability of insulin to move glucose into fat and muscle and shut off glucose release from liver. The liver takes up glucose from the bloodstream and stores it as glycogen, but because the liver contains glucose 6- phosphates it also discharges glucose into the blood– stream. Insulin facilitates glycogen synthesis and inhibits hepatic glucose output. When the plasma glucose is high, insulin secretion is normally increased and hepatic glucogenesis is decreased. This response dose not occurs in type I and II diabetes.

When plasma glucose is episodically elevated over time, small amounts of hemoglobin A are nonenzymatically glycated to from HbA1c. Careful control of the quently HbA1c level is measured clinically as an integrated index of diabetic control for the 4 to 6 weeks period before the measurement. Many studies showed that the mean HbA1c value was a good predictor of ischemic heart disease. In particular, the multivariate analysis showed that per each 1% increment in HbA1c there was a 10% increase in the risk of coronary heart disease. Some studies believed that 1% reduction in HbA1c level led to a 16% reduction in the occurrence of myocardial infarction.

Peripheral neuropathy, often expressed as hypersensitivity to painful stimuli, is among the most common complications of diabetes that develops in up to 60% of patients. It occurs in both type I and II diabetes and its incidence is linked to duration of disease. Neuropathic pain is a chronic or persistent pain characterized by alterations in pain perception, enhanced sensitivity to noxious stimuli (hyperalgesia) and abnormal pain sensitivity to previously non-painful stimuli (allodynia). Though the pathophysiology of neuropathy in diabetes has not been fully elucidated, hyperglycemia induced by diabetes is though to contribute to its development and maintaining good glycemic control could restrict the onset and progression of diabetic neuropathy.

#### **2.2 Vascular complication**

Hyperglycemia has a direct, harmful effect on the cardiovascular system requires, at the very least, a link between acute hyperglycemia and one or more risk factors for cardiovascular disease (CVD). Associated with obesity there is hyperinsulinemia, high circulating triglyceride and low HDL, and accelerated development of atherosclerosis. In diabetes, the plasma cholesterol level is usually elevated and this plays a role in the accelerated development of the atherosclerotic vascular disease that is a major long-term complication of diabetes in humans.

As usual diabetes is characterized by a high incidence of CVD, and poor control of hyperglycemia appears to play a significant role in the development of CVD in diabetes. Recently, there has been increasing evidence that the postprandial state is an important contributing factor to the development of atherosclerosis. In diabetes the postprandial phase is characterized by a rapid and large increase in blood glucose levels, and the possibility that these postprandial hyperglycemic spikes may be relevant to the pathophysiology of the late diabetes complications.

Insulin resistance (IR) has profound, negative effects on the function of arteries and arterioles throughout the body. In addition to the obvious link between IR and the development of type 2 diabetes, IR-associated dysfunction of resistance vessels is associated with arterial hypertension and vascular occlusive diseases. IR affects arteries and arterioles at both the endothelium and smooth muscle levels. For example, IR causes reduced responsiveness of vascular smooth muscle to dilator agents; predominantly due to impaired potassium channel function.

and diabetes. As body weight increase, insulin resistance increase, that is, there is a decreased ability of insulin to move glucose into fat and muscle and shut off glucose release from liver. The liver takes up glucose from the bloodstream and stores it as glycogen, but because the liver contains glucose 6- phosphates it also discharges glucose into the blood– stream. Insulin facilitates glycogen synthesis and inhibits hepatic glucose output. When the plasma glucose is high, insulin secretion is normally increased and hepatic glucogenesis is

When plasma glucose is episodically elevated over time, small amounts of hemoglobin A are nonenzymatically glycated to from HbA1c. Careful control of the quently HbA1c level is measured clinically as an integrated index of diabetic control for the 4 to 6 weeks period before the measurement. Many studies showed that the mean HbA1c value was a good predictor of ischemic heart disease. In particular, the multivariate analysis showed that per each 1% increment in HbA1c there was a 10% increase in the risk of coronary heart disease. Some studies believed that 1% reduction in HbA1c level led to a 16% reduction in the

Peripheral neuropathy, often expressed as hypersensitivity to painful stimuli, is among the most common complications of diabetes that develops in up to 60% of patients. It occurs in both type I and II diabetes and its incidence is linked to duration of disease. Neuropathic pain is a chronic or persistent pain characterized by alterations in pain perception, enhanced sensitivity to noxious stimuli (hyperalgesia) and abnormal pain sensitivity to previously non-painful stimuli (allodynia). Though the pathophysiology of neuropathy in diabetes has not been fully elucidated, hyperglycemia induced by diabetes is though to contribute to its development and maintaining good glycemic control could restrict the onset and

Hyperglycemia has a direct, harmful effect on the cardiovascular system requires, at the very least, a link between acute hyperglycemia and one or more risk factors for cardiovascular disease (CVD). Associated with obesity there is hyperinsulinemia, high circulating triglyceride and low HDL, and accelerated development of atherosclerosis. In diabetes, the plasma cholesterol level is usually elevated and this plays a role in the accelerated development of the atherosclerotic vascular disease that is a major long-term

As usual diabetes is characterized by a high incidence of CVD, and poor control of hyperglycemia appears to play a significant role in the development of CVD in diabetes. Recently, there has been increasing evidence that the postprandial state is an important contributing factor to the development of atherosclerosis. In diabetes the postprandial phase is characterized by a rapid and large increase in blood glucose levels, and the possibility that these postprandial hyperglycemic spikes may be relevant to the pathophysiology of the late

Insulin resistance (IR) has profound, negative effects on the function of arteries and arterioles throughout the body. In addition to the obvious link between IR and the development of type 2 diabetes, IR-associated dysfunction of resistance vessels is associated with arterial hypertension and vascular occlusive diseases. IR affects arteries and arterioles at both the endothelium and smooth muscle levels. For example, IR causes reduced responsiveness of vascular smooth muscle to dilator agents; predominantly due to impaired

decreased. This response dose not occurs in type I and II diabetes.

occurrence of myocardial infarction.

progression of diabetic neuropathy.

complication of diabetes in humans.

**2.2 Vascular complication** 

diabetes complications.

potassium channel function.

Vascular disease is one of the complicating features of diabetes mellitus. Several prospective studies have indicated that hypertension in diabetic patient's takes place at a rate more than twice compared to the normal population. The hypertension is also considered an independent risk factor for cardiovascular mortality in patients with diabetes. It has been suggested that alterations in the reactivity of blood vessels to neurotransmitters and circulating hormones are responsible for the cardiovascular complications of diabetes. Some studies showed that Ca/Mg ratio is a marker of vascular tone; its increase represents increased vascular reactivity and atherogenic risk. Atherogenic lesion is poorly correlated with serum cholesterol level and is highly dependent on plasma magnesium level and Ca/Mg ratio. Pervious studies showed that Ca/Mg ratio increase in diabetic case. Endothelial function is altered early in diabetes. It has been demonstrated that in diabetic subjects, the vasodilating response to stimuli is diminished and that this anomaly is related to glycemic control. In vivo studies have demonstrated that hyperglycemic spikes induce, in both diabetic and normal subjects, an endothelial dysfunction. This effect of hyperglycemia is probably linked with a reduced production/bioavailability of nitric oxide (NO), since hyperglycemia-induced endothelial dysfunction is counterbalanced by arginine. Furthermore, it is very interesting that a rapid decrease of flow-mediated vasodilation has been shown in the postprandial phase in type II diabetes patients and that the decrease correlated inversely with the magnitude of postprandial hyperglycemia.

#### **3. Prevention of diabetes complications**

Type I diabetes usually develops before the age 40, patients with this disease are not obese and they have a high incidence of ketosis and acidosis. Various anti-B cell antibodies are present in plasma, but the current thinking is that type I diabetes is primarily a T lymphocyte-mediated disease. But type II diabetes is the most common type of diabetes and is usually associated with obesity. It usually develops after age 40 and is not associated with total loss of the ability to secrete insulin. It has an insidious onset, is rarely associated with ketosis, and is usually associated with normal beta-cells morphology and insulin content if the beta-cells have not become exhausted. So it seems we should look for different methods for prevention of type I and II diabetes.

#### **3.1 Diabetes diet**

Several lifestyle factors affect the incidence of type 2 diabetes. Obesity and weight gain dramatically increase the risk, and physical inactivity further elevates the risk, independently of obesity. Cigarette smoking is associated with a small increase and moderate alcohol consumption with a decrease in the risk of diabetes. In addition, a low-fiber diet with a high glycemic index has been associated with an increased risk of diabetes, and specific dietary fatty acids may differentially affect insulin resistance and the risk of diabetes.

Excess body fat is the single most important determinant of type II diabetes. Weight control would be the most effective way to reduce the risk of type II diabetes, but current strategies have not been very successful on a population basis, and the prevalence of obesity continues to increase. The public generally does not recognize the connection between overweight or obesity and diabetes. Thus, greater efforts at education are needed.

Low-fat vegetarian and vegan diets are associated with reduced body weight, increased insulin sensitivity, and reductions in cardiovascular risk factors. The potential cardiovascular benefits of vegetarian and vegan diets may be especially important for

Prevention of Diabetes Complications 357

in plasma endothelin I due to magnesium deficiency and a direct effect of magnesium deficiency on vascular smooth muscle are involved in the elevation of vascular tone in diabetic pateint. Elevated vascular tone can contribute to increased blood pressure. Some studies have observed that systolic and diastolic blood pressure and mean arterial blood pressure in Mg-treated chronic diabetic subject are lower than in chronic diabetic. The administration of magnesium can decrease vascular bed sensitivity to phenylephrine and decrease Ca/Mg ratio. Studies also showed that magnesium decreases collagen thickness, intima/media thickness and the lumen/ media ratio in aorta. This suggests that the administration of magnesium can decrease blood pressure and prevent vascular morphological changes and decrease in vascular sensitivity to neurotransmitter. Hemoglobin deficiency is observed in diabetic subjects. This can probably be explained by the inhibition of ð-aminolevulinate dehydratase (ð-ALA-D) in diabetes. Studies have found that this enzyme is inhibited by glycation of the active site lysine residue involved in Schiff's base formation with the first ð-ALA-D molecule. Magnesium administration reduces this glycation via blood glucose reduction and, thus, prevents hemoglobin deficiency. So it seems that magnesium administration may play in the management of diabetes and the

Type I diabetes is a complex disease that results from an autoimmune T-lymphocytedependent islet infiltration and destruction of islet beta- cells, with consequent insulin deficiency and dependence on exogenous insulin treatment. A strikingly decreased functional beta- cell mass owing to apoptosis constitutes the histopathological hallmark of the disease at diagnosis. Recently, strategies employing beta-cell growth factors to enhance functional beta-cell mass and restore insulin secretion have been proposed for the treatment and prevention of diabetes. One such promising beta-cell growth factor identified is glucagone-like peptide-1 (GLP-1). GLP-1 is an insulinotropic hormone that is secreted from intestinal L-cell in response to nutrient ingestion and promotes nutrient absorption via regulation of islet hormone secretion. GLP-1 receptor is expressed mainly by pancreatic beta-cells, and to some extent in other tissues like lung, kidney and brain. GLP-1 enhances pancreatic islet beta-cell proliferation and inhibits beta-cell apoptosis in a glucose-dependent fashion. Other actions of GLP-1 are to decrease glucagons secretion and gastric emptying. Together, all these actions tend to lower the plasma glucose concentration and to limit plasma glucose rises with meals. GLP-1 is another gastrointestinal hormone that is also expressed in the hypothalamus and brainstem. Their CNS actions are to decrease food intake, decrease water intake, and increase diuresis. However, native GLP-1 has a short circulating half-life (less than 2 min) that results mainly from rapid enzymatic inactivation by dipeptidyl-peptidase IV (DPP- IV), and/ or renal clearance. Therefore, continuous subcutaneous infusion by pump is necessary to maintain GLP-1 action. DPP- IV-resistant GLP-1 analogues and other formulations appear to be promising therapeutic drug candidates for the treatment and prevention of diabetes, but these peptides require once or twice-daily injections and/or combination therapies with oral diabetic medications. Scientifics recently developed a novel GLP-1 fusion peptide consisting of the active human GLP-1 molecule and the murine IgG1 constant heavy-chain (IgG-Fc). Plasmid-based, electroporation-enhanced intramuscular gene therapy with GLP-1/IgG-Fc improved insulin

prevention of its vascular complications in diabetic patients.

production and normalized glucose tolerance in type one or two diabetes.

**5. Glucagone-like peptide-1 (GLP-1)** 

individuals with diabetes, for whom cardiovascular disease is a main cause of premature mortality; the effects of such diets on cardiovascular risk factors appear to be similar in individuals with and without diabetes.

Prior studies have shown that near-vegetarian diets reduce the need for insulin and oral medications in individuals with type 2 diabetes. We previously reported that in individuals with type 2 diabetes, a low-fat, vegan diet was associated with improved glycemic control, weight loss, and improved plasma lipid control during a 22-wk study period. What is particularly critical in diabetes management is long-term improvement in clinical measures, particularly glycemia and cardiovascular risk factors. Well-planned low-fat vegan diets are nutritionally adequate and, in research studies, have shown acceptability comparable with that of other therapeutic diets, suggesting they are suitable for long-term use.

#### **3.2 Immunosupression drug**

If type one diabetic patient give immunosupression drugs like cyclosporine ameliorate early in the disease, before all beta cells are lost can be useful for prevention of disease. But chose the low fat and low carbohydrate diets could be useful for prevention of type 2 diabetes.

#### **4. Magnesium**

Some studies indicated that magnesium is a novel factor implicated in the pathogenesis of the complication of diabetes. Magnesium plays a fundamental role as a cofactor in various enzymatic reactions of energy metabolism. Magnesium is a cofactor in cell membrane glucose-transporting mechanisms, as well as in various enzymes in carbohydrate oxidation. It is also involved, at multiple levels, in insulin secretion, binding and activity. Magnesium deficit has been described in patients with type I diabetes. Hypomagnesemia can also be the cause or a result of diabetes complications. If it is followed by diabetes, osmotic diuresis may play a role in the mechanisms responsible for magnesium deficiency. Magnesium loss may be linked to the development of diabetes complications via a reduction in the rate of inositol transport and its subsequent intracellular depletion that might enhance the development of complications. Studies showed that the administration of magnesium corrected hyperglycemia and has brought blood glucose back to normal levels within 24 h of its administration. Moreover, magnesium appears to have some reparative effect on the pancrease of diabetic case. Accordingly, during long-term treatment, pancreatic repair may have an effective role in the control of plasma glucose levels. Magnesium also is a necessary cofactor for many enzymes which is involved in lipid metabolism. Mg-deficiency enhances catecholamine secretion which result in an increase in lipolysis and blood plasma magnesium has been shown to decrease when lipolysis is increased. Enhancement in lipolysis and subsequent elevation of plasma free fatty acids levels may lead to an increase in hepatic VLDL and triglycerides synthesis and secretion and elevated plasma triglyceride concentration. The hepato-biliary pathway is the main rout for removal of cholesterol from the body. Bile flow is significantly lower in Mg-deficient subject than in controls and the cholesterol concentration in bile is decreased. Magnesium administration could decrease triglycerides, cholesterol and LDL cholesterol and also increased HDL cholesterol. The decrease in serum triglycerides was associated with the change in serum total Mg concentration. Other supporting evidence is accumulating for the role of magnesium in the modulation of serum lipids and lipids uptake in macrophages. Studies showed that increase

individuals with diabetes, for whom cardiovascular disease is a main cause of premature mortality; the effects of such diets on cardiovascular risk factors appear to be similar in

Prior studies have shown that near-vegetarian diets reduce the need for insulin and oral medications in individuals with type 2 diabetes. We previously reported that in individuals with type 2 diabetes, a low-fat, vegan diet was associated with improved glycemic control, weight loss, and improved plasma lipid control during a 22-wk study period. What is particularly critical in diabetes management is long-term improvement in clinical measures, particularly glycemia and cardiovascular risk factors. Well-planned low-fat vegan diets are nutritionally adequate and, in research studies, have shown acceptability comparable with

If type one diabetic patient give immunosupression drugs like cyclosporine ameliorate early in the disease, before all beta cells are lost can be useful for prevention of disease. But chose the low fat and low carbohydrate diets could be useful for prevention of type 2

Some studies indicated that magnesium is a novel factor implicated in the pathogenesis of the complication of diabetes. Magnesium plays a fundamental role as a cofactor in various enzymatic reactions of energy metabolism. Magnesium is a cofactor in cell membrane glucose-transporting mechanisms, as well as in various enzymes in carbohydrate oxidation. It is also involved, at multiple levels, in insulin secretion, binding and activity. Magnesium deficit has been described in patients with type I diabetes. Hypomagnesemia can also be the cause or a result of diabetes complications. If it is followed by diabetes, osmotic diuresis may play a role in the mechanisms responsible for magnesium deficiency. Magnesium loss may be linked to the development of diabetes complications via a reduction in the rate of inositol transport and its subsequent intracellular depletion that might enhance the development of complications. Studies showed that the administration of magnesium corrected hyperglycemia and has brought blood glucose back to normal levels within 24 h of its administration. Moreover, magnesium appears to have some reparative effect on the pancrease of diabetic case. Accordingly, during long-term treatment, pancreatic repair may have an effective role in the control of plasma glucose levels. Magnesium also is a necessary cofactor for many enzymes which is involved in lipid metabolism. Mg-deficiency enhances catecholamine secretion which result in an increase in lipolysis and blood plasma magnesium has been shown to decrease when lipolysis is increased. Enhancement in lipolysis and subsequent elevation of plasma free fatty acids levels may lead to an increase in hepatic VLDL and triglycerides synthesis and secretion and elevated plasma triglyceride concentration. The hepato-biliary pathway is the main rout for removal of cholesterol from the body. Bile flow is significantly lower in Mg-deficient subject than in controls and the cholesterol concentration in bile is decreased. Magnesium administration could decrease triglycerides, cholesterol and LDL cholesterol and also increased HDL cholesterol. The decrease in serum triglycerides was associated with the change in serum total Mg concentration. Other supporting evidence is accumulating for the role of magnesium in the modulation of serum lipids and lipids uptake in macrophages. Studies showed that increase

that of other therapeutic diets, suggesting they are suitable for long-term use.

individuals with and without diabetes.

**3.2 Immunosupression drug** 

diabetes.

**4. Magnesium** 

in plasma endothelin I due to magnesium deficiency and a direct effect of magnesium deficiency on vascular smooth muscle are involved in the elevation of vascular tone in diabetic pateint. Elevated vascular tone can contribute to increased blood pressure. Some studies have observed that systolic and diastolic blood pressure and mean arterial blood pressure in Mg-treated chronic diabetic subject are lower than in chronic diabetic. The administration of magnesium can decrease vascular bed sensitivity to phenylephrine and decrease Ca/Mg ratio. Studies also showed that magnesium decreases collagen thickness, intima/media thickness and the lumen/ media ratio in aorta. This suggests that the administration of magnesium can decrease blood pressure and prevent vascular morphological changes and decrease in vascular sensitivity to neurotransmitter. Hemoglobin deficiency is observed in diabetic subjects. This can probably be explained by the inhibition of ð-aminolevulinate dehydratase (ð-ALA-D) in diabetes. Studies have found that this enzyme is inhibited by glycation of the active site lysine residue involved in Schiff's base formation with the first ð-ALA-D molecule. Magnesium administration reduces this glycation via blood glucose reduction and, thus, prevents hemoglobin deficiency. So it seems that magnesium administration may play in the management of diabetes and the prevention of its vascular complications in diabetic patients.

#### **5. Glucagone-like peptide-1 (GLP-1)**

Type I diabetes is a complex disease that results from an autoimmune T-lymphocytedependent islet infiltration and destruction of islet beta- cells, with consequent insulin deficiency and dependence on exogenous insulin treatment. A strikingly decreased functional beta- cell mass owing to apoptosis constitutes the histopathological hallmark of the disease at diagnosis. Recently, strategies employing beta-cell growth factors to enhance functional beta-cell mass and restore insulin secretion have been proposed for the treatment and prevention of diabetes. One such promising beta-cell growth factor identified is glucagone-like peptide-1 (GLP-1). GLP-1 is an insulinotropic hormone that is secreted from intestinal L-cell in response to nutrient ingestion and promotes nutrient absorption via regulation of islet hormone secretion. GLP-1 receptor is expressed mainly by pancreatic beta-cells, and to some extent in other tissues like lung, kidney and brain. GLP-1 enhances pancreatic islet beta-cell proliferation and inhibits beta-cell apoptosis in a glucose-dependent fashion. Other actions of GLP-1 are to decrease glucagons secretion and gastric emptying. Together, all these actions tend to lower the plasma glucose concentration and to limit plasma glucose rises with meals. GLP-1 is another gastrointestinal hormone that is also expressed in the hypothalamus and brainstem. Their CNS actions are to decrease food intake, decrease water intake, and increase diuresis. However, native GLP-1 has a short circulating half-life (less than 2 min) that results mainly from rapid enzymatic inactivation by dipeptidyl-peptidase IV (DPP- IV), and/ or renal clearance. Therefore, continuous subcutaneous infusion by pump is necessary to maintain GLP-1 action. DPP- IV-resistant GLP-1 analogues and other formulations appear to be promising therapeutic drug candidates for the treatment and prevention of diabetes, but these peptides require once or twice-daily injections and/or combination therapies with oral diabetic medications. Scientifics recently developed a novel GLP-1 fusion peptide consisting of the active human GLP-1 molecule and the murine IgG1 constant heavy-chain (IgG-Fc). Plasmid-based, electroporation-enhanced intramuscular gene therapy with GLP-1/IgG-Fc improved insulin production and normalized glucose tolerance in type one or two diabetes.

Prevention of Diabetes Complications 359

antiproliferative effects of nitric oxide. By increasing intracellular oxidative stress, AGEs activate the transcription factor NF-kB, thus promoting up-regulation of various NF-kB controlled target genes. NF-kB enhances production of nitric oxide, which is believed to be a

Considerable evidence also implicates activation of the sorbitol pathway by glucose as a component in the pathogenesis of diabetic complications, for example, in lens cataract formation or peripheral neuropathy. Efforts to understand cataract formation have provoked various hypotheses. In the aldose reductase osmotic hypothesis, accumulation of polyols initiates lenticular osmotic changes. In addition, oxidative stress is linked to decreased glutathione levels and depletion of NADPH levels. Alternatively, increased sorbitol dehydrogenase activity is associated with altered NAD+ levels, which results in

Mechanisms linking the changes in diabetic neuropathy and induced sorbitol pathway are not well delineated. One possible mechanism, metabolic imbalances in the neural tissues, has been implicated in impaired neurotrophism, neurotrsnsmission changes, Schwann cell

While on the one hand hyperglycemia engenders free radicals, on the other hand it also impairs the endogenous antioxidant defense system in many ways during diabetes. Antioxidant defense mechanism involves both enzymatic and nonenzymatic strategies. Common antioxidants include the vitamins A, C and E, antioxidant minerals (copper, zinc, manganese, and selenium), and the cofactors (folic acid, vitamins B1, B2, B6, B12). They work in synergy with each other and against different types of free radicals. Vitamin E suppresses the propagation of lipid peroxidation; vitamin C with vitamin E inhibits hydroperoxide formation; metal complexing agents, such as penicillamine, bind transition metal involved in some reactions in lipid peroxidation and inhibit Fenton and Haber-weiss-

Recently, the search for appropriate hypoglycemic agents has been focused on plants. Many herbal medicines have been recommended for the treatment of diabetes. Plant drugs are frequently considered to be less toxic and free from side effect than synthetic ones. The leaf of Psidium guava, Teucrium polium, Cinnamon and Garlic are used traditionally in many countries to manage, control and treat of diabetes. Some recent studies have shown that administration of Psidium guava or Teucrium polium leaves decrease blood glucose via

Psidium guajva Linn., commonly known as guava, is a native plant in tropical American and has long been naturalized in south east Asia and in south of Iran. Different parts of the plant are used in traditional medicine for the treatment of various human aliments such as wound, ulcers, bronchitis, cyesores and diarrhea. In folklore guava has been used for a long time as a medicinal herb to cure diabetes mellitus. Many people in some countries including Japan, Taiwan and Iran boil guava leaves in water and drink the exact as a folk medicine for diabetes and hypertension. Psidium guajava leaves have a beneficial effects on diabetes

Photochemical analysis of Psidium guajava leaves have revealed the presence of flavonoids, which include quercetin and its derivatives (guajaverin, isoquercitrin, hyperin, quercitrin, avicularin), morin and its derivatives (morin-3-O-α-L-lixopyranoside and morin-3-O-α-L-

protein modification by nonenzymatic glycosylation of lens proteins.

type reactions; vitamins A and E scavenge free radicals.

metabolic syndrome and vascular complications.

mediator of islet beta cell damage.

injury, and axonopathy.

**8. Herbal medicine** 

enhance insulin secretion.

#### **6. Gama amino butyric acid (GABA)**

Gama amino butyric acid (GABA) is an important neurotransmitter which was initially identified in the central nervous system and is also found in islet beta-cells. GABA has an important role in pathogenesis of diabetes. Excessive secretion of glucagon is a major contributor to the development of diabetic hyperglycemia. Secretion of glucagon is regulated by various nutrients, with glucose being a primary determinant of the rate of alpha-cell glucagon secretion. The intra-islet action of insulin is essential to exert of insulin, glucose is not able to suppress glucagons release in vivo. However, the precise mechanism by which insulin suppresses glucagon secretion from alpha-cells is unknown. Studies showed that insulin induces activation of GABAA Akt kinase-dependent pathway. This leads to membrane hyperpolarization in the alpha-cells and, ultimately, suppression of glucagon secretion. Researchers propose that defects in this pathway contribute to diabetic hyperglycemia. It is well known that the secretion of glucagon is abnormal in human type I diabetes patients. The patients do not secrete glucagon in response to hypoglycemia and they have an exaggerated response of glucagon to stimuli such as arginine infusion and a protein meal. In studies of patients with type I diabetes there are indications of an increase in alpha cell numbers. GABA decreases in diabetic patients. Some studies indicated that a reduction in cellular GABA level is more sensitive than insulin as a marker for the presence of dead beta-cells in isolated preparations. Pancreatic GABA content also rapidly decreased after diabetes induction and remained unaffected by 12 h of hyperglycemia. It seems that GABA therapy can has some beneficial effect to prevention or treatment type I diabetes.

#### **7. Antioxidants**

Increasing evidence in both experimental and clinical studies suggests that oxidative stress play a major role in the pathogenesis of both types of diabetes mellitus. Diabetes is usually accompanied by increased production of free radicals or impaired antioxidant defenses. Mechanisms by which increased oxidative stress is involved in the diabetic complication are partly known, including activation of transcription factors, advanced glycated end products (AGEs), and protein kinase C.

Excessively high levels of tree radicals cause damage to cellular proteins, membrane lipids and nucleic acids, and eventually cell death. Various mechanisms have been suggested to contribute to the formation of these reactive oxygen-free radicals. Glucose oxidation is believed to be the main source of free radicals. In its enediol form, glucose is oxidized in a transition-metal-dependent reaction to an enddiol radical anion that is converted into reactive ketoaldehydes and to superoxide anion radicals. The superoxide anion radicals undergo dismutation to hydrogen peroxide, which if not degraded by catalase or glutathione peroxidase, and in the presence of transition metals, can lead to production of extremely reactive hydroxyl radicals. Superoxide anion radicals can also react with nitric oxide to form reactive peroxynitrite radicals. Hyperglycemia is also found to promote lipid peroxidation of LDL by a superoxide- dependent pathway resulting in the generation of free radicals. Another important source of free radicals in diabetes is the interaction of glucose with proteins leading to the formation of Amadori product and then advanced glycation endproducts (AGEs). These AGEs, via their receptors (RAGEs), inactivate enzymes and alter their structures and functions, promote free radical formation, and quench and block

Gama amino butyric acid (GABA) is an important neurotransmitter which was initially identified in the central nervous system and is also found in islet beta-cells. GABA has an important role in pathogenesis of diabetes. Excessive secretion of glucagon is a major contributor to the development of diabetic hyperglycemia. Secretion of glucagon is regulated by various nutrients, with glucose being a primary determinant of the rate of alpha-cell glucagon secretion. The intra-islet action of insulin is essential to exert of insulin, glucose is not able to suppress glucagons release in vivo. However, the precise mechanism by which insulin suppresses glucagon secretion from alpha-cells is unknown. Studies showed that insulin induces activation of GABAA Akt kinase-dependent pathway. This leads to membrane hyperpolarization in the alpha-cells and, ultimately, suppression of glucagon secretion. Researchers propose that defects in this pathway contribute to diabetic hyperglycemia. It is well known that the secretion of glucagon is abnormal in human type I diabetes patients. The patients do not secrete glucagon in response to hypoglycemia and they have an exaggerated response of glucagon to stimuli such as arginine infusion and a protein meal. In studies of patients with type I diabetes there are indications of an increase in alpha cell numbers. GABA decreases in diabetic patients. Some studies indicated that a reduction in cellular GABA level is more sensitive than insulin as a marker for the presence of dead beta-cells in isolated preparations. Pancreatic GABA content also rapidly decreased after diabetes induction and remained unaffected by 12 h of hyperglycemia. It seems that GABA therapy can has some beneficial

Increasing evidence in both experimental and clinical studies suggests that oxidative stress play a major role in the pathogenesis of both types of diabetes mellitus. Diabetes is usually accompanied by increased production of free radicals or impaired antioxidant defenses. Mechanisms by which increased oxidative stress is involved in the diabetic complication are partly known, including activation of transcription factors, advanced glycated end products

Excessively high levels of tree radicals cause damage to cellular proteins, membrane lipids and nucleic acids, and eventually cell death. Various mechanisms have been suggested to contribute to the formation of these reactive oxygen-free radicals. Glucose oxidation is believed to be the main source of free radicals. In its enediol form, glucose is oxidized in a transition-metal-dependent reaction to an enddiol radical anion that is converted into reactive ketoaldehydes and to superoxide anion radicals. The superoxide anion radicals undergo dismutation to hydrogen peroxide, which if not degraded by catalase or glutathione peroxidase, and in the presence of transition metals, can lead to production of extremely reactive hydroxyl radicals. Superoxide anion radicals can also react with nitric oxide to form reactive peroxynitrite radicals. Hyperglycemia is also found to promote lipid peroxidation of LDL by a superoxide- dependent pathway resulting in the generation of free radicals. Another important source of free radicals in diabetes is the interaction of glucose with proteins leading to the formation of Amadori product and then advanced glycation endproducts (AGEs). These AGEs, via their receptors (RAGEs), inactivate enzymes and alter their structures and functions, promote free radical formation, and quench and block

**6. Gama amino butyric acid (GABA)** 

effect to prevention or treatment type I diabetes.

**7. Antioxidants** 

(AGEs), and protein kinase C.

antiproliferative effects of nitric oxide. By increasing intracellular oxidative stress, AGEs activate the transcription factor NF-kB, thus promoting up-regulation of various NF-kB controlled target genes. NF-kB enhances production of nitric oxide, which is believed to be a mediator of islet beta cell damage.

Considerable evidence also implicates activation of the sorbitol pathway by glucose as a component in the pathogenesis of diabetic complications, for example, in lens cataract formation or peripheral neuropathy. Efforts to understand cataract formation have provoked various hypotheses. In the aldose reductase osmotic hypothesis, accumulation of polyols initiates lenticular osmotic changes. In addition, oxidative stress is linked to decreased glutathione levels and depletion of NADPH levels. Alternatively, increased sorbitol dehydrogenase activity is associated with altered NAD+ levels, which results in protein modification by nonenzymatic glycosylation of lens proteins.

Mechanisms linking the changes in diabetic neuropathy and induced sorbitol pathway are not well delineated. One possible mechanism, metabolic imbalances in the neural tissues, has been implicated in impaired neurotrophism, neurotrsnsmission changes, Schwann cell injury, and axonopathy.

While on the one hand hyperglycemia engenders free radicals, on the other hand it also impairs the endogenous antioxidant defense system in many ways during diabetes. Antioxidant defense mechanism involves both enzymatic and nonenzymatic strategies. Common antioxidants include the vitamins A, C and E, antioxidant minerals (copper, zinc, manganese, and selenium), and the cofactors (folic acid, vitamins B1, B2, B6, B12). They work in synergy with each other and against different types of free radicals. Vitamin E suppresses the propagation of lipid peroxidation; vitamin C with vitamin E inhibits hydroperoxide formation; metal complexing agents, such as penicillamine, bind transition metal involved in some reactions in lipid peroxidation and inhibit Fenton and Haber-weisstype reactions; vitamins A and E scavenge free radicals.

#### **8. Herbal medicine**

Recently, the search for appropriate hypoglycemic agents has been focused on plants. Many herbal medicines have been recommended for the treatment of diabetes. Plant drugs are frequently considered to be less toxic and free from side effect than synthetic ones. The leaf of Psidium guava, Teucrium polium, Cinnamon and Garlic are used traditionally in many countries to manage, control and treat of diabetes. Some recent studies have shown that administration of Psidium guava or Teucrium polium leaves decrease blood glucose via enhance insulin secretion.

Psidium guajva Linn., commonly known as guava, is a native plant in tropical American and has long been naturalized in south east Asia and in south of Iran. Different parts of the plant are used in traditional medicine for the treatment of various human aliments such as wound, ulcers, bronchitis, cyesores and diarrhea. In folklore guava has been used for a long time as a medicinal herb to cure diabetes mellitus. Many people in some countries including Japan, Taiwan and Iran boil guava leaves in water and drink the exact as a folk medicine for diabetes and hypertension. Psidium guajava leaves have a beneficial effects on diabetes metabolic syndrome and vascular complications.

Photochemical analysis of Psidium guajava leaves have revealed the presence of flavonoids, which include quercetin and its derivatives (guajaverin, isoquercitrin, hyperin, quercitrin, avicularin), morin and its derivatives (morin-3-O-α-L-lixopyranoside and morin-3-O-α-L-

Prevention of Diabetes Complications 361

Abe A, Kawasoe C, Kondo Y, Sato K. 2003. Enhancement of norepinephrine-induced

Adeghate E, Ponery AS. 2002. GABA in the endocrine pancreas: cellular localization and

Afifi FU, Al-Khalidi B, Khalil E. 2005. Studies on the in vivo hypoglycemic activities of two

Altura BM, Altura BT. 1995. Magnesium and cardiovascular biology an important link

Altura BM, Altura BT. 1991. Cardiovascular risk factors and magnesium: relationships to

Anetor, JI, Senjobi A, Ajose OA, Agbedana, E.O. 2002. Decreased serum magnesium and

Anwar N, Mason DF. 1982. Two actions of gamma-aminobutyric acid on the responses of the isolated basilar artery from the rabbit. Br J Pharmacol. 75(1): 177-81. Ashraf R, Aamir K, Shaikh AR, Ahmed T. 2005. Effect of Garlic on dyslipidemia in patients with type 2 diabetes mellitus. J Ayub Med Coll Abbottabad. 17(3): 1-5 Baluchnejadmojarad T, Roghani M, Homayounfar H, Hosseini M. 2003. Benefical effect of

Begum S, Hassan SI, Ali SN, Siddiqui BS. 2004. Chemical constituents from the leaves of

Borboni P, Porzio O, Fusco A, Sesti G, Lauro R, Marlier LN. 1994. Molecular and cellular

Busija DW, Miller AW, Katakam P, Simandle S, Erdös B. 2004. Mechanisms of vascular dysfunctionin insulin resistance. Curr Opin Investig Drugs. 5(9): 929-35 Ceriello A. 2005. Postprandial hyperglycemia and diabetes complications. Is it time to treat?

Chiwororo DH, Ojewole J. 2008. Biphasic effect of psidium guajava Linn. (Myrtaceae) leaf

Corica F, Allegra A, Di BA, Giacobbe MS, Romano G, Cucinotta D, Buemi M, Ceruso D.

following intranasal administration. J. Ethnopharmacol. 100: 314-318 Ajay M, Achike FI, Mustafa AM, Mustafa MR. 2006. Effect of quercetin on altered vascular

function in normal and diabetic rats. Tissue Cell. 34(1):1-6.

transient contraction in aortic smooth muscle of diabetic mice. Acta. Med. Okayama

medicinal plants used in the treatment of diabetes in Jordanian traditional medicine

reactivity in aorta isolated from sterptozotocin-induced diabetic rats. Diabetes Res.

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arabopyranoside), rutin, myricetin, luteolin and kaempferol. The leaves of the plant have also been shown to contain essential oil, fixed oil, volatile oil, saponin, resin, tannin, triterpenoids, asiatic acid and ellagic acid.

The relaxant effect of Psidium guajva Linn., on endothelium-intact aortic rings were only partially inhibited by N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, suggesting that the vasorelaxant effect of Psidium guajva Linn., on aortic rings is probably mediated via both endothelium-derived relaxing factor (EDRF)-dependent and EDRF-independent mechanisms but it seems this mechanism is not follow in diabetic rat vessel.

Teucrium polium L. is one of 300 species of the genus Teucrium and found mainly in the Mediterranean and Western Irano-Turanian sphere. It is widely distributed in Iran, Jordan and Palestine. The leaves 1-3 cm long, are sessile, oblong or linear, the stems are ending in a shortly paniculate or corymbose infoflorescences, corolla is white or pale cream colored. Several researchers have evaluated Teucrium polium grown in different geographic origin and it has flavonoids and iridoids. Hypoglycemic activity has been reported - in addition to the flavonoids- also for the volatile oils. Traditionally, especially in the Mediterranean countries and in Iran, Teucrium polium, is used for its antispasmodic and hypoglycemic activities by the native inhabitants and recommended by the herbalists. Anti-inflammatory, anti-hypertensive, antinociceptive, anti-ulcer and anorexic effects are other activities reported. Some investigators have reported reduction in blood glucose concentrations of animal diabetic model after treatment with a single i.v., i.p. and oral dose of Teucrium polium aqueous decoction. Some Iranian researchers have observed significant decrease in blood glucose in animal diabetic model after six weeks of consecutive oral treatment with ethanol/ water extract.

Spices such as Cinnamon display insulin-enhancing activity in vitro. Cinnamon can improve glucose metabolism and the overall condition of individuals with diabetes not only by hypoglycemic effects but also by improving lipid metabolism, antioxidant status, and capillary function. Aqueous extracts from Cinnamon have also been shown to increase in vitro glucose uptake and glycogen synthesis and to increase phosphorylation of the insulin receptor; in addition, these Cinnamon extracts are likely to aid in triggering the insulin cascade system. Because insulin also plays a key role in lipid metabolism, consumption of Cinnamon would lead to improved glucose and blood lipids in vitro. The mechanism of the effect of Cinnamon on glucose and blood lipids is not completely understood but the researchers believe that extracts of Cinnamon activated glycogen synthase, increased glucose uptake, and inhibited glycogen synthase kinase-3β. Extracts of Cinnamon also activated insulin receptor kinse and inhibited dephosphorylation of the insulin receptor, leading to maximal phosphorylation of the insulin receptor. All of these effects would lead to increased insulin sensitivity. The extract of Cinnamon also has function as potent antioxidants, which would lead to additional health benefits of this substance.

Garlic was known to be effective in decreasing, cholesterol and can inhibit LDL-Oxidation. Many clinical trials have been conducted to determine the lipid-lowering effects of fresh garlic and garlic supplements. Garlic consumption also can decrease blood glucose in diabetic patients and has beneficial effect on diabetic vessel. It seems that daily Garlic consumption can be useful to prevent of diabetes.

#### **9. References**

360 Type 1 Diabetes Complications

arabopyranoside), rutin, myricetin, luteolin and kaempferol. The leaves of the plant have also been shown to contain essential oil, fixed oil, volatile oil, saponin, resin, tannin,

The relaxant effect of Psidium guajva Linn., on endothelium-intact aortic rings were only partially inhibited by N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, suggesting that the vasorelaxant effect of Psidium guajva Linn., on aortic rings is probably mediated via both endothelium-derived relaxing factor (EDRF)-dependent and EDRF-independent mechanisms but it seems this mechanism is not follow in diabetic rat

Teucrium polium L. is one of 300 species of the genus Teucrium and found mainly in the Mediterranean and Western Irano-Turanian sphere. It is widely distributed in Iran, Jordan and Palestine. The leaves 1-3 cm long, are sessile, oblong or linear, the stems are ending in a shortly paniculate or corymbose infoflorescences, corolla is white or pale cream colored. Several researchers have evaluated Teucrium polium grown in different geographic origin and it has flavonoids and iridoids. Hypoglycemic activity has been reported - in addition to the flavonoids- also for the volatile oils. Traditionally, especially in the Mediterranean countries and in Iran, Teucrium polium, is used for its antispasmodic and hypoglycemic activities by the native inhabitants and recommended by the herbalists. Anti-inflammatory, anti-hypertensive, antinociceptive, anti-ulcer and anorexic effects are other activities reported. Some investigators have reported reduction in blood glucose concentrations of animal diabetic model after treatment with a single i.v., i.p. and oral dose of Teucrium polium aqueous decoction. Some Iranian researchers have observed significant decrease in blood glucose in animal diabetic model after six weeks of consecutive oral treatment with

Spices such as Cinnamon display insulin-enhancing activity in vitro. Cinnamon can improve glucose metabolism and the overall condition of individuals with diabetes not only by hypoglycemic effects but also by improving lipid metabolism, antioxidant status, and capillary function. Aqueous extracts from Cinnamon have also been shown to increase in vitro glucose uptake and glycogen synthesis and to increase phosphorylation of the insulin receptor; in addition, these Cinnamon extracts are likely to aid in triggering the insulin cascade system. Because insulin also plays a key role in lipid metabolism, consumption of Cinnamon would lead to improved glucose and blood lipids in vitro. The mechanism of the effect of Cinnamon on glucose and blood lipids is not completely understood but the researchers believe that extracts of Cinnamon activated glycogen synthase, increased glucose uptake, and inhibited glycogen synthase kinase-3β. Extracts of Cinnamon also activated insulin receptor kinse and inhibited dephosphorylation of the insulin receptor, leading to maximal phosphorylation of the insulin receptor. All of these effects would lead to increased insulin sensitivity. The extract of Cinnamon also has function as potent antioxidants, which would lead to additional health benefits of this

Garlic was known to be effective in decreasing, cholesterol and can inhibit LDL-Oxidation. Many clinical trials have been conducted to determine the lipid-lowering effects of fresh garlic and garlic supplements. Garlic consumption also can decrease blood glucose in diabetic patients and has beneficial effect on diabetic vessel. It seems that daily Garlic

triterpenoids, asiatic acid and ellagic acid.

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ethanol/ water extract.

substance.

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Olatunji-Bello I, Odusanya AJ, Raji I, Ladipo CO. 2007. Contractile effect of aqueous extract of Psidium guajava leaves on aortic rings in rat. Fitoterapia 78: 241-243 Ozcelikay AT, Tay A, Guner S, Tasyaran V, Yildizoglu-Ar, N, Dincer UD, Altan

Ozdem SS, Sadan G. 1999. Impairment of GABA-mediated contractions of rat isolated ileum

Pari L, Umamaheswari J. 2000. Antihyperglycaemic activity of Musa Sapientum flowers: effect on lipid peroxidation in alloxan diabetic rats. Phytother. Res. 14: 1-3 Prudhomme GJ, Chang Y. 1999. Prevention of autoimmune diabetes by intramuscular gene

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**17** 

*University of Pittsburgh* 

*University of Pittsburgh* 

*1,3USA 2Italy* 

**The Enigma of -Cell Regeneration in the Adult** 

*1Division of Immunogenetics, Department of Pediatrics, Children's Hospital of Pittsburgh,* 

The pancreas is constituted by two distinctly different tissues: the exocrine component, i.e., pancreatic acinar cells that secrete digestive enzymes; and the endocrine component, the islets of Langerhans, constituted by hormone secreting cells. In the islet, the α-cells produce glucagon; the β-cells, insulin; the δ-cells, somatostatin; γ-cells, pancreatic polypeptide (Figure 1). Diabetes is caused either by an absolute (type 1) or relative (type 2) defect of insulin-producing β-cells in the pancreas. Therefore, regardless of the different pathogenesis, diabetes is the perfect candidate for cell replacement therapy. Currently the two available alternatives for β-cell replacement therapy are whole pancreas or isolated islet transplantation (Shapiro et al., 2000). However, these approaches are severely limited by a shortage of human organ donors and the need of lifelong immunosuppressive therapy. In the absence of other clearly suitable and renewable sources of surrogate β-cells, an alternative strategy to exogenous cell replacement therapy might be fostering endogenous β-cell regeneration. Therefore, knowledge of the mechanisms regulating β-cell plasticity in both embryonic and adult life, as well as in pathological conditions, is of particular interest. During pancreatic development, β-cells derive from a population of endocrine precursors arising from the pancreatic epithelium (Gittes, 2009). Activation of cell-specific transcription factors guides the initially multipotent progenitors and determines their differentiation into mature β-cells. The final size of the endocrine pancreas is limited by the size of the progenitor cell pool in the developing pancreatic bud (Stanger et al., 2007). After birth, most β-cells are considered quiescent, however, it has been shown that the β-cell mass can adaptively expand under some physiologic or pathologic circumstances, such as pregnancy

and obesity, both in mammals and rodents (Bernard-Kargar & Ktorza, 2001).

In mouse models there is also evidence that the pancreas preserves the ability to regenerate its β-cell mass in response to several non-physiological injuries, such as selective chemical destruction or surgical excision (Trucco, 2005; Thorel, 2010). Furthermore, in non-obese diabetic mice (NOD) it has been shown that recovery of sufficient endogenous insulin

**1. Introduction** 

**Pancreas: Self-Renewal Versus Neogenesis** 

A. Criscimanna1,2,3, S. Bertera1, F. Esni3, M. Trucco1 andR. Bottino1

*2Division of Endocrinology, DOSAC, Universita' degli Studi di Palermo* 

*3Department of Surgery, Children's Hospital of Pittsburgh* 


### **The Enigma of -Cell Regeneration in the Adult Pancreas: Self-Renewal Versus Neogenesis**

A. Criscimanna1,2,3, S. Bertera1, F. Esni3, M. Trucco1 andR. Bottino1 *1Division of Immunogenetics, Department of Pediatrics, Children's Hospital of Pittsburgh, University of Pittsburgh* 

#### **1. Introduction**

366 Type 1 Diabetes Complications

Yajnik CS. 2001. The insulin resistance epidemic in India: fetal origins, later lifestyle, or

Zizzo MG, Mulè F, Serio R. 2007. Functional evidence for GABA as modulator of the

GABA(C) receptors. Neuropharmacology. 52(8):1685-90.

contractility of the longitudinal muscle in mouse duodenum: role of GABA(A) and

both? Nutr. Rev. 59: 1-9

The pancreas is constituted by two distinctly different tissues: the exocrine component, i.e., pancreatic acinar cells that secrete digestive enzymes; and the endocrine component, the islets of Langerhans, constituted by hormone secreting cells. In the islet, the α-cells produce glucagon; the β-cells, insulin; the δ-cells, somatostatin; γ-cells, pancreatic polypeptide (Figure 1). Diabetes is caused either by an absolute (type 1) or relative (type 2) defect of insulin-producing β-cells in the pancreas. Therefore, regardless of the different pathogenesis, diabetes is the perfect candidate for cell replacement therapy. Currently the two available alternatives for β-cell replacement therapy are whole pancreas or isolated islet transplantation (Shapiro et al., 2000). However, these approaches are severely limited by a shortage of human organ donors and the need of lifelong immunosuppressive therapy. In the absence of other clearly suitable and renewable sources of surrogate β-cells, an alternative strategy to exogenous cell replacement therapy might be fostering endogenous β-cell regeneration. Therefore, knowledge of the mechanisms regulating β-cell plasticity in both embryonic and adult life, as well as in pathological conditions, is of particular interest.

During pancreatic development, β-cells derive from a population of endocrine precursors arising from the pancreatic epithelium (Gittes, 2009). Activation of cell-specific transcription factors guides the initially multipotent progenitors and determines their differentiation into mature β-cells. The final size of the endocrine pancreas is limited by the size of the progenitor cell pool in the developing pancreatic bud (Stanger et al., 2007). After birth, most β-cells are considered quiescent, however, it has been shown that the β-cell mass can adaptively expand under some physiologic or pathologic circumstances, such as pregnancy and obesity, both in mammals and rodents (Bernard-Kargar & Ktorza, 2001).

In mouse models there is also evidence that the pancreas preserves the ability to regenerate its β-cell mass in response to several non-physiological injuries, such as selective chemical destruction or surgical excision (Trucco, 2005; Thorel, 2010). Furthermore, in non-obese diabetic mice (NOD) it has been shown that recovery of sufficient endogenous insulin

*<sup>2</sup>Division of Endocrinology, DOSAC, Universita' degli Studi di Palermo 3Department of Surgery, Children's Hospital of Pittsburgh University of Pittsburgh 1,3USA 2Italy* 

The Enigma of β-Cell Regeneration in the Adult Pancreas: Self-Renewal Versus Neogenesis 369

pancreatic response. Most of these models allow exploring the plasticity of the pancreas in the absence of concurrent autoimmunity, which might prevent or block any attempt of β-cell

Partial pancreatectomy (>90% removal) was shown to induce limited re-growth of the remnant organ in rats (Pearson et al., 1977). In comparison to liver regeneration second to partial hepatectomy, subtotal pancreatectomy is followed only by a limited regenerative growth that is proportional to the size of the excision. In addition, regeneration is mostly related to the exocrine tissue, while the endocrine part was shown to rescue, at its best, only approximately 30% of its initial mass (De Leon et al., 2003). The extent of the surgical intervention seems to be important, and could explain discrepancies in some reports describing absent (Dor et al., 2004) or vigorous pancreatic regeneration [Bonner-Weir et al., 1993] after 70% and 90% organ resection, respectively. In addition, hyperglycemia, which is

Pancreatic duct ligation (PDL) was also used to determine obstruction and consequently local inflammation and stimulation of pancreatic regeneration. During the first week postligation an increase in the β-cell number and the presence of intermediate ductal/endocrine (Wang et al., 1995) or acinar/endocrine phenotypes (Bertelli & Bendayan, 1997; Inada et al., 2008) has been observed. Additional stimulation of the expansion of the β-cell mass

Wrapping the pancreas with cellophane has also been used to induce islet neogenesis from ducts, and it has been reported to reverse streptozotocin-induced diabetes in hamsters

Selective β-cell destruction can be obtained by chemical ablation with streptozotocin or alloxan, alone and in combination with pancreatectomy (Finegood et al., 1999; Wang et al., 1996; Rood et al., 2006). Streptozotocin (STZ) is a drug that leads to cell death by DNA alkylation, while alloxan is a generator of oxygen free radicals causing extensive DNA damage. Adult mice rendered diabetic with a high dose of STZ or alloxan are unable to recover endogenous β-cell function (Szkudelski, 2001). Interestingly, β-cell neogenesis can be stimulated in STZ-diabetic newborn rats by administration of the hormone glucagon-like peptide-1 (GLP-1), resulting in improved glucose homeostasis persisting at adult age (Tourrel et al., 2001). In another murine experimental model of alloxan-induced beta-cell destruction, treatment with gastrin and epidermal growth factor (EGF) was found to restore glycemic control and 30–40% of the normal beta-cell mass within 7 days (Rooman & Bouwens, 2004). Combination of the same growth factors proved to be effective also in facilitating islet β-cell neogeneisis in NOD mice with autoimmune diabetes (Suarez-Pinzon et al., 2005). In addition, rescue of endogenous islet function was shown in STZ-diabetic mice after removal of the kidney bearing syngeneic islets, which temporarily maintained mice normoglicemic (Yin et al., 2006), thus indicating that glucose control might be relevant

restoration.

**2.1 Partial pancreatectomy** 

**2.2 Pancreatic duct ligation** 

(Rosenberg et al., 1996).

**2.4 Selective β-cell destruction** 

present only in the latter case, could act as co-stimulator.

**2.3 Wrapping of the pancreas with cellophane** 

following PDL can be achieved by gastrin infusion (Rooman et al., 2002).

production is possible via combination of strategies involving reversal of the autoimmune attack (Zorina et al., 2003; Kodama et al., 2003; Suri et al., 2006; Chong et al., 2006; Nishio et al., 2006).

In humans it is still debated whether this recovery is possible and, if so, to what extent it is feasible. Spontaneous recovery of β-cell function has been reported in patients with recent onset type 1 diabetes, suggesting that β-cells can regenerate despite underlying autoimmunity (Karges et al., 2004, 2006; Meier et al., 2006a; Butler et al., 2007). Additionally, the observation that people with long-standing type 1 diabetes still possess β-cells despite their destruction by the enduring autoimmunity and glucotoxicity, suggests that new β-cell formation might occur throughout life [Meier et al., 2005]. However, it remains largely unclear through which molecular and cellular mechanisms it occurs.

Fig. 1. Cells of the pancreas

The pancreas houses two different tissues. Its bulk is comprised of exocrine tissue, which is made up of acinar cells. These cells secrete pancreatic enzymes delivered to the intestine to facilitate the digestion of food. Scattered throughout the exocrine tissue are many thousands of clusters of endocrine cells known as islets of Langerhans. Within the islet, α-cells produce glucagon; β-cells, insulin; δ-cells, somatostatin; and γ-cells, pancreatic polypeptide — all of which are delivered into the blood stream.

#### **2. Can the endocrine pancreas regenerate? Models of artificially induced diabetes**

To address the question whether the pancreas possesses the ability to regenerate, several models have been used to artificially reduce the β-cell mass in order to stimulate a pancreatic response. Most of these models allow exploring the plasticity of the pancreas in the absence of concurrent autoimmunity, which might prevent or block any attempt of β-cell restoration.

#### **2.1 Partial pancreatectomy**

368 Type 1 Diabetes Complications

production is possible via combination of strategies involving reversal of the autoimmune attack (Zorina et al., 2003; Kodama et al., 2003; Suri et al., 2006; Chong et al., 2006; Nishio et

In humans it is still debated whether this recovery is possible and, if so, to what extent it is feasible. Spontaneous recovery of β-cell function has been reported in patients with recent onset type 1 diabetes, suggesting that β-cells can regenerate despite underlying autoimmunity (Karges et al., 2004, 2006; Meier et al., 2006a; Butler et al., 2007). Additionally, the observation that people with long-standing type 1 diabetes still possess β-cells despite their destruction by the enduring autoimmunity and glucotoxicity, suggests that new β-cell formation might occur throughout life [Meier et al., 2005]. However, it remains largely

The pancreas houses two different tissues. Its bulk is comprised of exocrine tissue, which is made up of acinar cells. These cells secrete pancreatic enzymes delivered to the intestine to facilitate the digestion of food. Scattered throughout the exocrine tissue are many thousands of clusters of endocrine cells known as islets of Langerhans. Within the islet, α-cells produce glucagon; β-cells, insulin; δ-cells, somatostatin; and γ-cells, pancreatic polypeptide — all of

**2. Can the endocrine pancreas regenerate? Models of artificially induced** 

To address the question whether the pancreas possesses the ability to regenerate, several models have been used to artificially reduce the β-cell mass in order to stimulate a

unclear through which molecular and cellular mechanisms it occurs.

al., 2006).

Fig. 1. Cells of the pancreas

**diabetes** 

which are delivered into the blood stream.

Partial pancreatectomy (>90% removal) was shown to induce limited re-growth of the remnant organ in rats (Pearson et al., 1977). In comparison to liver regeneration second to partial hepatectomy, subtotal pancreatectomy is followed only by a limited regenerative growth that is proportional to the size of the excision. In addition, regeneration is mostly related to the exocrine tissue, while the endocrine part was shown to rescue, at its best, only approximately 30% of its initial mass (De Leon et al., 2003). The extent of the surgical intervention seems to be important, and could explain discrepancies in some reports describing absent (Dor et al., 2004) or vigorous pancreatic regeneration [Bonner-Weir et al., 1993] after 70% and 90% organ resection, respectively. In addition, hyperglycemia, which is present only in the latter case, could act as co-stimulator.

#### **2.2 Pancreatic duct ligation**

Pancreatic duct ligation (PDL) was also used to determine obstruction and consequently local inflammation and stimulation of pancreatic regeneration. During the first week postligation an increase in the β-cell number and the presence of intermediate ductal/endocrine (Wang et al., 1995) or acinar/endocrine phenotypes (Bertelli & Bendayan, 1997; Inada et al., 2008) has been observed. Additional stimulation of the expansion of the β-cell mass following PDL can be achieved by gastrin infusion (Rooman et al., 2002).

#### **2.3 Wrapping of the pancreas with cellophane**

Wrapping the pancreas with cellophane has also been used to induce islet neogenesis from ducts, and it has been reported to reverse streptozotocin-induced diabetes in hamsters (Rosenberg et al., 1996).

#### **2.4 Selective β-cell destruction**

Selective β-cell destruction can be obtained by chemical ablation with streptozotocin or alloxan, alone and in combination with pancreatectomy (Finegood et al., 1999; Wang et al., 1996; Rood et al., 2006). Streptozotocin (STZ) is a drug that leads to cell death by DNA alkylation, while alloxan is a generator of oxygen free radicals causing extensive DNA damage. Adult mice rendered diabetic with a high dose of STZ or alloxan are unable to recover endogenous β-cell function (Szkudelski, 2001). Interestingly, β-cell neogenesis can be stimulated in STZ-diabetic newborn rats by administration of the hormone glucagon-like peptide-1 (GLP-1), resulting in improved glucose homeostasis persisting at adult age (Tourrel et al., 2001). In another murine experimental model of alloxan-induced beta-cell destruction, treatment with gastrin and epidermal growth factor (EGF) was found to restore glycemic control and 30–40% of the normal beta-cell mass within 7 days (Rooman & Bouwens, 2004). Combination of the same growth factors proved to be effective also in facilitating islet β-cell neogeneisis in NOD mice with autoimmune diabetes (Suarez-Pinzon et al., 2005). In addition, rescue of endogenous islet function was shown in STZ-diabetic mice after removal of the kidney bearing syngeneic islets, which temporarily maintained mice normoglicemic (Yin et al., 2006), thus indicating that glucose control might be relevant

The Enigma of β-Cell Regeneration in the Adult Pancreas: Self-Renewal Versus Neogenesis 371

Cells can be irreversibly marked using the Cre/LoxP system, thus permitting detection of progeny cells that no longer express the gene of interest. This system uses two transgenic

The reporter line uses promoter 1 (Pro.1, black rectangle) to drive reporter gene expression (green rectangle). Upstream of the reporter gene coding region is a STOP cassette made of three repeats of a polyadenilation signal (red rectangle). Flanking the blocking signal are two LoxP sites (blue arrows). Promoter 1 can be tissue specific or ubiquitous. In the deletor line, another tissue specific promoter (Pro. 2, black rectangle) is used to drive the expression of *Cre* recombinase (yellow rectangle). When the two mouse lines are crossed, CRE is expressed in the cells in which promoter 2 is active, thus deleting the blocking signal. This results in the expression of the reporter gene in

The first transgenic mouse uses a promoter (promoter 1), which can be tissue specific or ubiquitous, to drive the expression of a reporter gene, such as LacZ or green fluorescent protein (GFP). The second mouse carries a transgene that uses a different tissue specific promoter (promoter 2) to drive the expression of Cre recombinase. In the absence of the Cre deletor transgene, the expression of the reporter protein is prevented by a STOP cassette (multiple repeats of a poly-adenylation signal) upstream of the reporter coding sequence. However, in the presence of Cre recombinase, two LoxP sites flanking the blocking sequence permit this block to be removed. Thus, in double transgenic animals, the reporter gene will be expressed in cells following the excision event, thereby labeling all progeny derived from

A fine-tuning of the Cre/LoxP system can be achieved with the CRE-ER™ recombinase, which is a fusion between the catalytic domain of the CRE recombinase and the ligandbinding domain of a modified estrogen receptor. The CRE-ER™ protein requires an artificial ligand, tamoxifen, to catalyze LoxP mediated recombination. Because tamoxifen is active within mouse embryos for less than 48 h, cells expressing Cre-ER™ at a specific developmental stage can be selectively labeled by administration of tamoxifen during that stage. After tamoxifen treatment, the conventional CRE recombinase activates the reporter transgene expression as soon as CRE protein is generated, and labeled cells accumulate in any lineage where Cre has been expressed. This type of recombinase can be used to follow selectively the progeny of cells born at defined developmental stages, including postnatal

**3.1 Cre/LoxP system** 

cells that also express promoter 1.

growth and during regeneration.

Fig. 2. Design plan for direct cell lineage analysis.

those precursors that express the deletor transgene.

mouse lines, the "reporter" and the "deletor" (Figure 2).

to facilitate the regenerative process. On the other hand, the possibility that recovery of the endogenous β-cell function may occur independently of glucose control exists, i.e., by the mediation of cytokines, which may activate residual β-cell proliferation or progenitor cell differentiation. To note, in the study by Yin et al., a facilitating role was exerted by the presence of the spleen, which probably plays an indirect role as modulator of the inflammatory process in the pancreas, thereby stimulating recovery of the STZ-damaged islets. STZ seems to trigger a pancreatic regenerative response also in non-human primate models, although it does not lead to a substantial endogenous β-cell recovery in absence of additional stimuli, like the failure of exogenous islet transplantation in the liver (Bottino et al., 2009).


CA-II: carbonic anhydrase II

Table 1. Summary of the models used to investigate pancreatic regeneration.

#### **3. Lineage tracing techniques**

Lineage tracing techniques have been widely used to investigate both the ontogeny of pancreatic cell fates during mouse embryogenesis as well as the identification of progenitor cells *in vivo* during regeneration (see Figure 2 and 3 for further explanations).

In lineage analysis, specific cells are labeled or marked so that their progeny can be identified later during development. In the pancreas, lineage analysis has been used to recognize not only the progenitor cells giving rise to mature endocrine and exocrine cells, but also the stage at which each set of progenitors is restricted to a particular cell fate. Lineage tracing is also useful to label and isolate marked cells in order to study their gene expression profile and *in vitro* differentiation.

In pancreatic lineage analysis, cells can be labeled using distinct approaches. A physical label - such as dye or a replication-incompetent retrovirus - can be directly injected into embryos to label cells within a tissue. The tissue is allowed to mature *in vivo* or in culture, and the cell types that become labeled reveal the lineage of the starting cells. However, since this method marks cells indiscriminately, in most tissues it cannot be used to label specific sub-populations. A more reliable approach is to genetically mark progenitor cells using endogenous gene expression patterns. This method selectively labels cells that express a particular gene, thus revealing the fate of their progeny. In most cases, a tissue specific promoter (for example Pdx-1) driving Cre recombinase is used to irreversibly tag cells. Other options include the use of a transgene driven by a specific promoter within different cell types, and lineage ablation, either using gene-inactivation mutants (knockout) or transgenic expression of cellular toxins. All these approaches have been used to follow pancreatic cell lineages (reviewed in Gu et al. 2003).

#### **3.1 Cre/LoxP system**

370 Type 1 Diabetes Complications

to facilitate the regenerative process. On the other hand, the possibility that recovery of the endogenous β-cell function may occur independently of glucose control exists, i.e., by the mediation of cytokines, which may activate residual β-cell proliferation or progenitor cell differentiation. To note, in the study by Yin et al., a facilitating role was exerted by the presence of the spleen, which probably plays an indirect role as modulator of the inflammatory process in the pancreas, thereby stimulating recovery of the STZ-damaged islets. STZ seems to trigger a pancreatic regenerative response also in non-human primate models, although it does not lead to a substantial endogenous β-cell recovery in absence of additional stimuli, like the failure of exogenous islet transplantation in the liver (Bottino et

> Replication of pre-existing β-cells Reactivation of embryonic program

Regeneration through Ngn3+ precursors

Regeneration through Ca-II and Sox-9+ progenitors

**Method Target Cells Potential Mechanism for Regeneration** 

**Streptozotocin** β-cell β-cell neogenesis from ductal cells **Alloxan** β-cell β-cell neogenesis from ductal cells

Table 1. Summary of the models used to investigate pancreatic regeneration.

cells *in vivo* during regeneration (see Figure 2 and 3 for further explanations).

used to follow pancreatic cell lineages (reviewed in Gu et al. 2003).

Lineage tracing techniques have been widely used to investigate both the ontogeny of pancreatic cell fates during mouse embryogenesis as well as the identification of progenitor

In lineage analysis, specific cells are labeled or marked so that their progeny can be identified later during development. In the pancreas, lineage analysis has been used to recognize not only the progenitor cells giving rise to mature endocrine and exocrine cells, but also the stage at which each set of progenitors is restricted to a particular cell fate. Lineage tracing is also useful to label and isolate marked cells in order to study their gene

In pancreatic lineage analysis, cells can be labeled using distinct approaches. A physical label - such as dye or a replication-incompetent retrovirus - can be directly injected into embryos to label cells within a tissue. The tissue is allowed to mature *in vivo* or in culture, and the cell types that become labeled reveal the lineage of the starting cells. However, since this method marks cells indiscriminately, in most tissues it cannot be used to label specific sub-populations. A more reliable approach is to genetically mark progenitor cells using endogenous gene expression patterns. This method selectively labels cells that express a particular gene, thus revealing the fate of their progeny. In most cases, a tissue specific promoter (for example Pdx-1) driving Cre recombinase is used to irreversibly tag cells. Other options include the use of a transgene driven by a specific promoter within different cell types, and lineage ablation, either using gene-inactivation mutants (knockout) or transgenic expression of cellular toxins. All these approaches have been

al., 2009).

**Pancreatic duct ligation** 

CA-II: carbonic anhydrase II

**3. Lineage tracing techniques** 

expression profile and *in vitro* differentiation.

**Pancreatectomy** Endocrine

Exocrine

Endocrine Exocrine

Cells can be irreversibly marked using the Cre/LoxP system, thus permitting detection of progeny cells that no longer express the gene of interest. This system uses two transgenic mouse lines, the "reporter" and the "deletor" (Figure 2).

The reporter line uses promoter 1 (Pro.1, black rectangle) to drive reporter gene expression (green rectangle). Upstream of the reporter gene coding region is a STOP cassette made of three repeats of a polyadenilation signal (red rectangle). Flanking the blocking signal are two LoxP sites (blue arrows). Promoter 1 can be tissue specific or ubiquitous. In the deletor line, another tissue specific promoter (Pro. 2, black rectangle) is used to drive the expression of *Cre* recombinase (yellow rectangle). When the two mouse lines are crossed, CRE is expressed in the cells in which promoter 2 is active, thus deleting the blocking signal. This results in the expression of the reporter gene in cells that also express promoter 1.

Fig. 2. Design plan for direct cell lineage analysis.

The first transgenic mouse uses a promoter (promoter 1), which can be tissue specific or ubiquitous, to drive the expression of a reporter gene, such as LacZ or green fluorescent protein (GFP). The second mouse carries a transgene that uses a different tissue specific promoter (promoter 2) to drive the expression of Cre recombinase. In the absence of the Cre deletor transgene, the expression of the reporter protein is prevented by a STOP cassette (multiple repeats of a poly-adenylation signal) upstream of the reporter coding sequence. However, in the presence of Cre recombinase, two LoxP sites flanking the blocking sequence permit this block to be removed. Thus, in double transgenic animals, the reporter gene will be expressed in cells following the excision event, thereby labeling all progeny derived from those precursors that express the deletor transgene.

A fine-tuning of the Cre/LoxP system can be achieved with the CRE-ER™ recombinase, which is a fusion between the catalytic domain of the CRE recombinase and the ligandbinding domain of a modified estrogen receptor. The CRE-ER™ protein requires an artificial ligand, tamoxifen, to catalyze LoxP mediated recombination. Because tamoxifen is active within mouse embryos for less than 48 h, cells expressing Cre-ER™ at a specific developmental stage can be selectively labeled by administration of tamoxifen during that stage. After tamoxifen treatment, the conventional CRE recombinase activates the reporter transgene expression as soon as CRE protein is generated, and labeled cells accumulate in any lineage where Cre has been expressed. This type of recombinase can be used to follow selectively the progeny of cells born at defined developmental stages, including postnatal growth and during regeneration.

The Enigma of β-Cell Regeneration in the Adult Pancreas: Self-Renewal Versus Neogenesis 373

During pancreatic organogenesis, stem cells within the duct pancreatic epithelium give rise to both the endocrine and acinar cells (Gittes, 2009). Therefore it seems reasonable to think that the regeneration process could start within the ductal compartment, recapitulating embryonic and fetal development. In addition, there are similarities between islet regeneration and embryonic pancreas development at the gene expression level. Evidence of adult duct cells harboring stem cells capable of differentiating into βcells was reported both *in vivo* and *in vitro* (Dudek et al., 1991; Ramiya et al., 2000; Bonner-Weir et al., 2000, 2008; Gao et al., 2003). In 2000 Ramiya et al. claimed that long-term cultivation of pancreatic ductal epithelial cells isolated from pre-diabetic, adult, non-obese diabetic mice contained nestin-positive stem cells able to differentiate into islets of Langerhans (Ramiya et al., 2000). These "surrogate" islets responded *in vitro* to glucose challenge, and reversed insulin-dependent diabetes after being implanted into diabetic NOD mice. Similar observations were reported using more defined culture conditions in which isolated human pancreatic duct preparations led to formation and propagation of human islet-like structures (Bonner-Weir et al., 2000, 2008; Gao et al., 2003). Ogata et al. also derived a similar subset of islet-like insulin secreting cells from pancreatic ducts of neonatal rats (Ogata et al., 2004). After incubation with activin A and betacellulin, cells showed tolbutamide- and glucose-responsive insulin secretion. Transplantation of these pseudo-islets in STZ-diabetic NOD mice improved blood glucose levels. Hao et al. confirmed the existence of endocrine stem or progenitor cells within the epithelial compartment of the adult human pancreas, by isolating stem cells from the non-endocrine fraction after islet separation of adult human pancreas digests (Hao et al., 2006). Following elimination of the contaminating mesenchymal cells, the highly purified population of non-endocrine pancreatic epithelial cells (NEPECs) was transplanted under the kidney capsule of immunodeficient (SCID) mice. Although NEPECs produced only low amounts of insulin, when co-transplanted with fetal pancreatic cells, they were capable of endocrine differentiation. No evidence of -cell replication or cell fusion was observed. To directly test whether ductal cells serve as pancreatic progenitors after birth and give rise to new islets, a transgenic mouse expressing human carbonic anhydrase II (CAII) promoter was generated. This study showed that CAII-expressing cells within the pancreas act as progenitors that give rise to both new islets and acini normally after birth

Additional evidence of the existence of endocrine precursor cells within the ductal compartment come from the detection of the nuclear transcription factor Neurogenin-3 (Ngn3) in the ducts during regeneration after STZ. Ngn3 is a basic helix–loop–helix transcription factor, which is able to commit pancreatic cells to an endocrine cell fate (Schwitzgebel et al., 2000). Lack of Ngn3 leads to an absence of islets (Gradwhol et al., 2000); its ectopic expression determines premature over-commission towards the endocrine lineage (Apelqvist et al., 1999). Presence of Ngn3 is very convincing evidence that pancreatic regeneration starts from pancreatic progenitors and mimics the same pathway followed during normal development (Figure 4). By using an inducible Cre-ERTM-LoxP system to

**4. Evidence of pancreatic endocrine progenitors/stem cells in the pancreas**  Several cells in the pancreas have been described as potential sources of β-cell renewal.

**4.1 Pancreatic ductal progenitor cells** 

and after injury (PDL) (Inada et al., 2008).

#### **3.2 Lineage analysis based on simple transgenes**

A simpler transgenic approach drives expression of a reporter gene, such as LacZ or green fluorescent protein (GFP), under the promoter of interest. The drawback of this approach is that any progeny of these cells, which cease expression of the chosen protein cannot be followed. In addition, since this method marks cells from the first time the promoter is activated, and as these cells accumulate during development, it becomes impossible to distinguish new members of the population. Thus, one cannot distinguish the progeny of cells born during embryogenesis from those born in adults.

#### **3.3 Lineage analysis based on cell ablation**

Another method to investigate lineage relationship is cell ablation. This can be accomplished by specific gene inactivation mutations (knockout), such as in the *Pdx1* knockout mouse, which has no mature pancreatic cells. Alternatively, a tissue specific promoter can be used to drive the expression of a cellular toxin, such as the Diphtheria Toxin A (DTA) subunit. In these transgenic animals, the DTA subunit will kill those cells whose progenitors express that specific transgene. A similar approach is represented by the Diphtheria Toxin Receptor (DTR)-mediated conditional cell ablation model. DTR is a membrane-anchored form of the heparin-binding EGF-like growth factor (HB-EGF precursor). The human and simian HB-EGF precursors bind DT and function as toxin receptors, whereas HB-EGF from mice and rats do not bind the toxin and therefore remain insensitive to DT. Thus, transgenic expression of the simian or human DTR in mice can render cells DT-sensitive. Recently, a mouse strain was generated (iDTR), in which the gene encoding DTR has been introduced into the *ROSA26* locus (R26*DTR*), but its expression is dependent on the Cre-mediated removal of a transcriptional STOP cassette. Therefore, only Cre-expressing cells and their progeny will undergo Cre-recombinase activity and subsequently will transcribe DTR. Although viable and normally functioning, these cells are rapidly killed upon DT administration (Figure 3) (Buch et al. 2003).

The STOP cassette, which prohibits DTR expression, is removed by crossing the iDTR strain to a tissuespecific Cre-expressing mouse strain. Consecutive expression of the DTR renders the respective tissues sensitive to cell death induced by injection of diphtheria toxin. Filled rectangles, *loxP* sites; arrows, transcriptional activity; open ovals, promoter.

Fig. 3. Design plan of the inducible DTR mouse strain (iDTR).

Several cells in the pancreas have been described as potential sources of β-cell renewal.

#### **4.1 Pancreatic ductal progenitor cells**

372 Type 1 Diabetes Complications

A simpler transgenic approach drives expression of a reporter gene, such as LacZ or green fluorescent protein (GFP), under the promoter of interest. The drawback of this approach is that any progeny of these cells, which cease expression of the chosen protein cannot be followed. In addition, since this method marks cells from the first time the promoter is activated, and as these cells accumulate during development, it becomes impossible to distinguish new members of the population. Thus, one cannot distinguish the progeny of

Another method to investigate lineage relationship is cell ablation. This can be accomplished by specific gene inactivation mutations (knockout), such as in the *Pdx1* knockout mouse, which has no mature pancreatic cells. Alternatively, a tissue specific promoter can be used to drive the expression of a cellular toxin, such as the Diphtheria Toxin A (DTA) subunit. In these transgenic animals, the DTA subunit will kill those cells whose progenitors express that specific transgene. A similar approach is represented by the Diphtheria Toxin Receptor (DTR)-mediated conditional cell ablation model. DTR is a membrane-anchored form of the heparin-binding EGF-like growth factor (HB-EGF precursor). The human and simian HB-EGF precursors bind DT and function as toxin receptors, whereas HB-EGF from mice and rats do not bind the toxin and therefore remain insensitive to DT. Thus, transgenic expression of the simian or human DTR in mice can render cells DT-sensitive. Recently, a mouse strain was generated (iDTR), in which the gene encoding DTR has been introduced into the *ROSA26* locus (R26*DTR*), but its expression is dependent on the Cre-mediated removal of a transcriptional STOP cassette. Therefore, only Cre-expressing cells and their progeny will undergo Cre-recombinase activity and subsequently will transcribe DTR. Although viable and normally functioning, these cells

The STOP cassette, which prohibits DTR expression, is removed by crossing the iDTR strain to a tissuespecific Cre-expressing mouse strain. Consecutive expression of the DTR renders the respective tissues sensitive to cell death induced by injection of diphtheria toxin. Filled rectangles, *loxP* sites; arrows,

**3.2 Lineage analysis based on simple transgenes** 

cells born during embryogenesis from those born in adults.

are rapidly killed upon DT administration (Figure 3) (Buch et al. 2003).

**3.3 Lineage analysis based on cell ablation** 

transcriptional activity; open ovals, promoter.

Fig. 3. Design plan of the inducible DTR mouse strain (iDTR).

During pancreatic organogenesis, stem cells within the duct pancreatic epithelium give rise to both the endocrine and acinar cells (Gittes, 2009). Therefore it seems reasonable to think that the regeneration process could start within the ductal compartment, recapitulating embryonic and fetal development. In addition, there are similarities between islet regeneration and embryonic pancreas development at the gene expression level. Evidence of adult duct cells harboring stem cells capable of differentiating into βcells was reported both *in vivo* and *in vitro* (Dudek et al., 1991; Ramiya et al., 2000; Bonner-Weir et al., 2000, 2008; Gao et al., 2003). In 2000 Ramiya et al. claimed that long-term cultivation of pancreatic ductal epithelial cells isolated from pre-diabetic, adult, non-obese diabetic mice contained nestin-positive stem cells able to differentiate into islets of Langerhans (Ramiya et al., 2000). These "surrogate" islets responded *in vitro* to glucose challenge, and reversed insulin-dependent diabetes after being implanted into diabetic NOD mice. Similar observations were reported using more defined culture conditions in which isolated human pancreatic duct preparations led to formation and propagation of human islet-like structures (Bonner-Weir et al., 2000, 2008; Gao et al., 2003). Ogata et al. also derived a similar subset of islet-like insulin secreting cells from pancreatic ducts of neonatal rats (Ogata et al., 2004). After incubation with activin A and betacellulin, cells showed tolbutamide- and glucose-responsive insulin secretion. Transplantation of these pseudo-islets in STZ-diabetic NOD mice improved blood glucose levels. Hao et al. confirmed the existence of endocrine stem or progenitor cells within the epithelial compartment of the adult human pancreas, by isolating stem cells from the non-endocrine fraction after islet separation of adult human pancreas digests (Hao et al., 2006). Following elimination of the contaminating mesenchymal cells, the highly purified population of non-endocrine pancreatic epithelial cells (NEPECs) was transplanted under the kidney capsule of immunodeficient (SCID) mice. Although NEPECs produced only low amounts of insulin, when co-transplanted with fetal pancreatic cells, they were capable of endocrine differentiation. No evidence of -cell replication or cell fusion was observed. To directly test whether ductal cells serve as pancreatic progenitors after birth and give rise to new islets, a transgenic mouse expressing human carbonic anhydrase II (CAII) promoter was generated. This study showed that CAII-expressing cells within the pancreas act as progenitors that give rise to both new islets and acini normally after birth and after injury (PDL) (Inada et al., 2008).

Additional evidence of the existence of endocrine precursor cells within the ductal compartment come from the detection of the nuclear transcription factor Neurogenin-3 (Ngn3) in the ducts during regeneration after STZ. Ngn3 is a basic helix–loop–helix transcription factor, which is able to commit pancreatic cells to an endocrine cell fate (Schwitzgebel et al., 2000). Lack of Ngn3 leads to an absence of islets (Gradwhol et al., 2000); its ectopic expression determines premature over-commission towards the endocrine lineage (Apelqvist et al., 1999). Presence of Ngn3 is very convincing evidence that pancreatic regeneration starts from pancreatic progenitors and mimics the same pathway followed during normal development (Figure 4). By using an inducible Cre-ERTM-LoxP system to

The Enigma of β-Cell Regeneration in the Adult Pancreas: Self-Renewal Versus Neogenesis 375

Ngn3+ cells presumably give rise to the adult duct system and account for the heterogeneity in developmental potential among 'duct-like structures'. Kodama et al. also suggested that in STZ-treated mice, regeneration occurs mainly from intra-islet Ngn3+ progenitor cells rather than from ductal precursors (Kodama et al., 2005). Recently, Xu et al. showed that PDL in the pancreatic tail resulted in some β-cell proliferation and, more strikingly, in a large upregulation of Ngn3 gene expression (Xu et al., 2008). Ngn3 positive cells were found to be closely associated with ducts, and possibly cells of the ductal lineage themselves. After isolation, these cells could give rise to all islet cell types, including glucose responsive β-cells, both *in situ* and when cultured in Ngn3−/<sup>−</sup> embryonic pancreas explants. In addition, White et al. utilized a system based on Ngn3– enhanced green fluorescent protein knock-in mouse model to isolate endocrine progenitor cells from embryonic pancreata to generate an ample gene expression profile of these progenitors and their immediate descendants (White et al., 2008). On the other hand, a recent publication reported low level expression of Ngn3 in adult endocrine cells, raising concerns about using Ngn3 expression as a marker of endocrine progenitors and neogenesis in the adult pancreas (Wang et al., 2009). Furthermore, another study indicates that although PDL leads to Ngn3 expression in a sub-population of cells within the ducts, it does not induce appropriate cues to allow for completion of the entire β-cell neogenesis

The potential for the pancreatic organ to recover the endocrine function after injury has been also investigated in non-human primates, where a ductal involvement was observed in STZdiabetic monkeys that recovered endogenous β-cell function following pig islet

Further evidence that hormone-positive cells arise from the ducts comes from the comparison of 16 donor pancreas specimens and pancreas biopsies from 8 simultaneous pancreas/kidney transplantations (Martin-Pagoda et al., 2008). While in the donor pancreas the frequency of insulin+ duct cells was low (0.45%), in five pancreatic transplants with recurrent autoimmunity, 57.5% of the duct cells expressed insulin protein. If new islets were generated from pre-existing ductal tissue, transient co-expression of hormones and residual duct markers could be expected. Indeed, this has been demonstrated in grafts of purified

Besides ductal progenitors, other groups proposed that pancreatic stem cells may also reside

Acinar cells, which represent the main portion of pancreatic tissue, have been shown to transdifferentiate into islet cells *in vivo* and *in vitro*, through the generation of duct cells as an intermediate step (Lardon et al., 2004; Baeyens et al., 2005; Rooman et al., 2000; Lipsett et al., 2007). Lineage tracing has been used *in vitro* to further strengthen the conclusion that endocrine cells can be generated from exocrine cells via transdifferentiation [Minami et al., 2005]. Earlier *in vivo* lineage tracing experiments in mice showed that acinar cells scarcely contribute to generate new β-cells and duct cells (Desai et al., 2007). However, more recent studies by Collombat et al. demonstrated that upon expression of Pax4, adult -cells can transdifferentiate to β-cells (Collombat et al., 2009). The ectopic expression of Pax4 forces

program (Kopp et al., 2011).

transplantation in the liver (Bottino et al., 2009).

human duct cells (Yatoh et al., 2007).

**4.2.1 Acinar cells** 

**4.2 Pancreatic non-ductal progenitor cells** 

within the islets or in the acinar compartment.

Fig. 4. Regulatory nuclear transcription factors controlling cell type lineages during embryonic pancreas development.

mark the progeny of cells expressing either Ngn3 or Pdx1 at different stages of development, Gu et al. showed that endocrine/exocrine and ductal lineages are separated before E12.5 (Gu et al., 2002). Authors demonstrate that, while cells expressing Pdx1 give rise to all three types of pancreatic tissue (exocrine, endocrine and duct), only the subset Pdx 1/Ngn3+ cells are islet progenitors. The duct cells that do not contain progeny of Ngn3+ cells presumably give rise to the adult duct system and account for the heterogeneity in developmental potential among 'duct-like structures'. Kodama et al. also suggested that in STZ-treated mice, regeneration occurs mainly from intra-islet Ngn3+ progenitor cells rather than from ductal precursors (Kodama et al., 2005). Recently, Xu et al. showed that PDL in the pancreatic tail resulted in some β-cell proliferation and, more strikingly, in a large upregulation of Ngn3 gene expression (Xu et al., 2008). Ngn3 positive cells were found to be closely associated with ducts, and possibly cells of the ductal lineage themselves. After isolation, these cells could give rise to all islet cell types, including glucose responsive β-cells, both *in situ* and when cultured in Ngn3−/<sup>−</sup> embryonic pancreas explants. In addition, White et al. utilized a system based on Ngn3– enhanced green fluorescent protein knock-in mouse model to isolate endocrine progenitor cells from embryonic pancreata to generate an ample gene expression profile of these progenitors and their immediate descendants (White et al., 2008). On the other hand, a recent publication reported low level expression of Ngn3 in adult endocrine cells, raising concerns about using Ngn3 expression as a marker of endocrine progenitors and neogenesis in the adult pancreas (Wang et al., 2009). Furthermore, another study indicates that although PDL leads to Ngn3 expression in a sub-population of cells within the ducts, it does not induce appropriate cues to allow for completion of the entire β-cell neogenesis program (Kopp et al., 2011).

The potential for the pancreatic organ to recover the endocrine function after injury has been also investigated in non-human primates, where a ductal involvement was observed in STZdiabetic monkeys that recovered endogenous β-cell function following pig islet transplantation in the liver (Bottino et al., 2009).

Further evidence that hormone-positive cells arise from the ducts comes from the comparison of 16 donor pancreas specimens and pancreas biopsies from 8 simultaneous pancreas/kidney transplantations (Martin-Pagoda et al., 2008). While in the donor pancreas the frequency of insulin+ duct cells was low (0.45%), in five pancreatic transplants with recurrent autoimmunity, 57.5% of the duct cells expressed insulin protein. If new islets were generated from pre-existing ductal tissue, transient co-expression of hormones and residual duct markers could be expected. Indeed, this has been demonstrated in grafts of purified human duct cells (Yatoh et al., 2007).

#### **4.2 Pancreatic non-ductal progenitor cells**

Besides ductal progenitors, other groups proposed that pancreatic stem cells may also reside within the islets or in the acinar compartment.

#### **4.2.1 Acinar cells**

374 Type 1 Diabetes Complications

Fig. 4. Regulatory nuclear transcription factors controlling cell type lineages during

mark the progeny of cells expressing either Ngn3 or Pdx1 at different stages of development, Gu et al. showed that endocrine/exocrine and ductal lineages are separated before E12.5 (Gu et al., 2002). Authors demonstrate that, while cells expressing Pdx1 give rise to all three types of pancreatic tissue (exocrine, endocrine and duct), only the subset Pdx 1/Ngn3+ cells are islet progenitors. The duct cells that do not contain progeny of

embryonic pancreas development.

Acinar cells, which represent the main portion of pancreatic tissue, have been shown to transdifferentiate into islet cells *in vivo* and *in vitro*, through the generation of duct cells as an intermediate step (Lardon et al., 2004; Baeyens et al., 2005; Rooman et al., 2000; Lipsett et al., 2007). Lineage tracing has been used *in vitro* to further strengthen the conclusion that endocrine cells can be generated from exocrine cells via transdifferentiation [Minami et al., 2005]. Earlier *in vivo* lineage tracing experiments in mice showed that acinar cells scarcely contribute to generate new β-cells and duct cells (Desai et al., 2007). However, more recent studies by Collombat et al. demonstrated that upon expression of Pax4, adult -cells can transdifferentiate to β-cells (Collombat et al., 2009). The ectopic expression of Pax4 forces

The Enigma of β-Cell Regeneration in the Adult Pancreas: Self-Renewal Versus Neogenesis 377

cells derived from human β-cells. Nevertheless, it appears that induction of significant

In addition to pancreatic progenitors, cells from other organs, such as liver, spleen, bone marrow, adipose tissue and limbus have been identified as either new sources of islets or

Pancreas and liver share the same origin from the embryonic endoderm (Zaret, 2000). It has been reported that transdifferentiation of pancreas into liver occurs both *in vitro* and *in vivo* in animal models after a number of experimental treatments (Rao et al., 1986, 1995; Dabeva et al., 1997; Kralowski et al., 1999; Shen et al., 2000). The opposite conversion of liver into pancreas is also possible (Horb et al., 2003). Zalzman et al. were able to immortalize a population of human fetal liver epithelial progenitor cells that, once transfected with the Pdx1 gene, generated a stable population of insulin-producing cells (Zalzman et al., 2003). Intraperitoneal transplantation of these cells into immunodeficient mice led to reversal of diabetes for 80 days. However, Yang et al. showed that expression of Pdx1 in hepatocytes does not result in the formation of functional endocrine pancreas in Pdx1 deficient mice, thus suggesting that Pdx1 is necessary but not sufficient to induce differentiation of the

The hypothesis that the spleen may harbor stem cells capable of differentiating into β-cells has also been investigated. Faustman and colleagues initially showed that splenocytes contributed to the reversal of autoimmunity in the NOD mouse model when injected with Freund's complete adjuvant (Ryu et al., 2001). Later they also suggested that splenocytes may directly contribute to islet regeneration by differentiation into β-cells (Kodama et al., 2003). However, these findings proved to be controversial. Indeed several other groups, confirming a partial recovery from the autoimmune attack following splenocyte injections, actually failed to display evidence of a direct contribution of donor cells to β-cell regeneration (Suri et al., 2006; Chong et al., 2006; Nishio et al., 2006). Nonetheless, Yin and colleagues supported a facilitating

There are numerous reports suggesting that the bone marrow not only harbors haemopoietic stem cells, which are committed to differentiate into blood cells, but also mesenchymal stem cells (MSCs), capable of differentiation into β-cells (Oh et al., 2004; Moriscot et al., 2005). MSCs were reported to differentiate *in vivo* into glucose-competent pancreatic endocrine cells when transplanted in NOD mice (Ianus et al., 2003). However, following studies resulted in controversial outcomes, suggesting that MSCs do not become *per se* insulin producing cells, rather they take part in islet vascularization, eventually promoting β-cell regeneration (Hess et al., 2003 Chamson-Reig et al., 2010). Transplantation of human MSCs was also shown to induce repair of pancreatic islets and renal glomeruli in immunodeficient mice (NOD/SCID) suffering from STZ-induced diabetes (Lee et al., 2006). The role exerted by bone marrow cells

role of the spleen in the regeneration of endogenous β-cell mass (Yin et al., 2006).

**5. Evidence of pancreatic progenitors/stem cells outside the pancreas** 

replication *in vitro* results in dedifferentiation. (Russ et al, 2008).

stimulators of islet regeneration.

pancreatic tissue (Yang et al., 2002).

**5.2 Splenic stem cells** 

**5.3 Mesenchymal stem cells** 

**5.1 Liver stem cells** 

endocrine precursor cells as well as mature -cells, to adopt a β-cell fate. In addition, since -cells were constantly recruited and converted to β-cells, the resulting glucagon deficiency provoked a compensatory and continuous glucagon+ cell neogenesis through Ngn3+ precursors. On the other hand, Arx misexpression in β-cells, using either an InsCre or in adult β-cells using an inducible Pdx1CreERT system reduced insulin-expressing cells and increased alpha and PP-positive cells (Collombat et al., 2007).

Alpha-to-Beta-cell transdifferentiation was also recently described in a transgenic model of diphtheria-toxin-induced acute selective near-total β-cell ablation (Thorel et al., 2010). Lineage-tracing to label the glucagon-producing -cells before β-cell ablation, tracked large fractions of regenerated β-cells as deriving from -cells, revealing a previously unknown flexibility in the functioning of the pancreas in relation to hormone secretion, with the potential for exploiting it to cure diabetes.

Generation of β-cells from α-cells has also been shown with a unique model that combines PDL with alloxan-mediated β-cells destruction (Chung et al., 2010). In this model, large numbers of β-cells were generated primarily from α-cells by two mechanisms: the first involved extensive α-cell proliferation, which provided a large pool of precursors that, in turn, would become β-cells via asymmetric division; the second demonstrated that β-cells could form directly from α-cells via transdifferentiation. This latter mechanism was put forward by the finding of intermediate cells co-expressing α- and β-cell-specific markers. Double-positive cells were detectable in the first week after injury, but their number gradually declined and by the second week some converted into mature β-cells, as shown by loss of glucagon and new expression of MafA, a β-cell-specific transcriptional activator.

#### **4.2.2 Nestin-positive cells**

Nestin-positive cells have been identified within adult rat islets as being capable of differentiating into insulin-positive cells *in vitro* (Zulewski et al., 2001). Nestin is an intermediate filament protein expressed by the neural lineage, which, according to some groups can be found in the pancreas (Edlund, 2002), in contrast to others that could not find its expression during development of the human pancreatic epithelium (Piper et al., 2002). More recent lineage-tracing experiments showed that nestin-positive cells contribute to the vasculature as well as acinar lineages but not to the endocrine lineage (Treutelaar et al., 2003; Esni et al., 2004; Delacour et al., 2004).

#### **4.2.3 Proliferative human islet precursor cells (hIPCs)**

Proliferative human islet precursor cells (hIPCs) were obtained *in vitro* from preparations of adult human islets after extensive *in vitro* proliferation (Gershengorn et al., 2004). Authors believed that these cells, showing a mesenchymal phenotype, derived from insulinexpressing cells undergoing epithelial-to-mesenchymal transition (EMT). hIPCs could be redifferentiated into insulin-expressing islet-like cell aggregates (ICAs) and secreted insulin when transplanted under the kidney capsule of immunodeficient mice**.** However, many criticisms were advanced from other groups, claiming that, at least in mouse pancreatic cultures, islet-derived fibroblast-like cells are not generated via EMT from pancreatic β-cells (Chase et al., 2007; Atouf et al., 2007). Later, Gershengorn et al. further confirmed the basic differences between human and mouse cultures, and claimed that hIPCs are a special kind of pancreatic mesenchymal stromal cells (Morton et al., 2007). More recently, using a lineage-tracing *in vitro* technique, Russ et al. found evidence for massive proliferation of cells derived from human β-cells. Nevertheless, it appears that induction of significant replication *in vitro* results in dedifferentiation. (Russ et al, 2008).

#### **5. Evidence of pancreatic progenitors/stem cells outside the pancreas**

In addition to pancreatic progenitors, cells from other organs, such as liver, spleen, bone marrow, adipose tissue and limbus have been identified as either new sources of islets or stimulators of islet regeneration.

#### **5.1 Liver stem cells**

376 Type 1 Diabetes Complications

endocrine precursor cells as well as mature -cells, to adopt a β-cell fate. In addition, since -cells were constantly recruited and converted to β-cells, the resulting glucagon deficiency provoked a compensatory and continuous glucagon+ cell neogenesis through Ngn3+ precursors. On the other hand, Arx misexpression in β-cells, using either an InsCre or in adult β-cells using an inducible Pdx1CreERT system reduced insulin-expressing cells and increased

Alpha-to-Beta-cell transdifferentiation was also recently described in a transgenic model of diphtheria-toxin-induced acute selective near-total β-cell ablation (Thorel et al., 2010). Lineage-tracing to label the glucagon-producing -cells before β-cell ablation, tracked large fractions of regenerated β-cells as deriving from -cells, revealing a previously unknown flexibility in the functioning of the pancreas in relation to hormone secretion, with the

Generation of β-cells from α-cells has also been shown with a unique model that combines PDL with alloxan-mediated β-cells destruction (Chung et al., 2010). In this model, large numbers of β-cells were generated primarily from α-cells by two mechanisms: the first involved extensive α-cell proliferation, which provided a large pool of precursors that, in turn, would become β-cells via asymmetric division; the second demonstrated that β-cells could form directly from α-cells via transdifferentiation. This latter mechanism was put forward by the finding of intermediate cells co-expressing α- and β-cell-specific markers. Double-positive cells were detectable in the first week after injury, but their number gradually declined and by the second week some converted into mature β-cells, as shown by loss of glucagon and new expression of MafA, a β-cell-specific transcriptional activator.

Nestin-positive cells have been identified within adult rat islets as being capable of differentiating into insulin-positive cells *in vitro* (Zulewski et al., 2001). Nestin is an intermediate filament protein expressed by the neural lineage, which, according to some groups can be found in the pancreas (Edlund, 2002), in contrast to others that could not find its expression during development of the human pancreatic epithelium (Piper et al., 2002). More recent lineage-tracing experiments showed that nestin-positive cells contribute to the vasculature as well as acinar lineages but not to the endocrine lineage (Treutelaar et al.,

Proliferative human islet precursor cells (hIPCs) were obtained *in vitro* from preparations of adult human islets after extensive *in vitro* proliferation (Gershengorn et al., 2004). Authors believed that these cells, showing a mesenchymal phenotype, derived from insulinexpressing cells undergoing epithelial-to-mesenchymal transition (EMT). hIPCs could be redifferentiated into insulin-expressing islet-like cell aggregates (ICAs) and secreted insulin when transplanted under the kidney capsule of immunodeficient mice**.** However, many criticisms were advanced from other groups, claiming that, at least in mouse pancreatic cultures, islet-derived fibroblast-like cells are not generated via EMT from pancreatic β-cells (Chase et al., 2007; Atouf et al., 2007). Later, Gershengorn et al. further confirmed the basic differences between human and mouse cultures, and claimed that hIPCs are a special kind of pancreatic mesenchymal stromal cells (Morton et al., 2007). More recently, using a lineage-tracing *in vitro* technique, Russ et al. found evidence for massive proliferation of

alpha and PP-positive cells (Collombat et al., 2007).

potential for exploiting it to cure diabetes.

**4.2.2 Nestin-positive cells** 

2003; Esni et al., 2004; Delacour et al., 2004).

**4.2.3 Proliferative human islet precursor cells (hIPCs)** 

Pancreas and liver share the same origin from the embryonic endoderm (Zaret, 2000). It has been reported that transdifferentiation of pancreas into liver occurs both *in vitro* and *in vivo* in animal models after a number of experimental treatments (Rao et al., 1986, 1995; Dabeva et al., 1997; Kralowski et al., 1999; Shen et al., 2000). The opposite conversion of liver into pancreas is also possible (Horb et al., 2003). Zalzman et al. were able to immortalize a population of human fetal liver epithelial progenitor cells that, once transfected with the Pdx1 gene, generated a stable population of insulin-producing cells (Zalzman et al., 2003). Intraperitoneal transplantation of these cells into immunodeficient mice led to reversal of diabetes for 80 days. However, Yang et al. showed that expression of Pdx1 in hepatocytes does not result in the formation of functional endocrine pancreas in Pdx1 deficient mice, thus suggesting that Pdx1 is necessary but not sufficient to induce differentiation of the pancreatic tissue (Yang et al., 2002).

#### **5.2 Splenic stem cells**

The hypothesis that the spleen may harbor stem cells capable of differentiating into β-cells has also been investigated. Faustman and colleagues initially showed that splenocytes contributed to the reversal of autoimmunity in the NOD mouse model when injected with Freund's complete adjuvant (Ryu et al., 2001). Later they also suggested that splenocytes may directly contribute to islet regeneration by differentiation into β-cells (Kodama et al., 2003). However, these findings proved to be controversial. Indeed several other groups, confirming a partial recovery from the autoimmune attack following splenocyte injections, actually failed to display evidence of a direct contribution of donor cells to β-cell regeneration (Suri et al., 2006; Chong et al., 2006; Nishio et al., 2006). Nonetheless, Yin and colleagues supported a facilitating role of the spleen in the regeneration of endogenous β-cell mass (Yin et al., 2006).

#### **5.3 Mesenchymal stem cells**

There are numerous reports suggesting that the bone marrow not only harbors haemopoietic stem cells, which are committed to differentiate into blood cells, but also mesenchymal stem cells (MSCs), capable of differentiation into β-cells (Oh et al., 2004; Moriscot et al., 2005). MSCs were reported to differentiate *in vivo* into glucose-competent pancreatic endocrine cells when transplanted in NOD mice (Ianus et al., 2003). However, following studies resulted in controversial outcomes, suggesting that MSCs do not become *per se* insulin producing cells, rather they take part in islet vascularization, eventually promoting β-cell regeneration (Hess et al., 2003 Chamson-Reig et al., 2010). Transplantation of human MSCs was also shown to induce repair of pancreatic islets and renal glomeruli in immunodeficient mice (NOD/SCID) suffering from STZ-induced diabetes (Lee et al., 2006). The role exerted by bone marrow cells

The Enigma of β-Cell Regeneration in the Adult Pancreas: Self-Renewal Versus Neogenesis 379

disguising the innate regenerative response, such as the persistence of circulating autoreactive T cells. To further sustain this hypothesis, it has been demonstrated how therapeutic protocols intended at blocking autoimmunity in NOD mice (Chatenoud et al., 1994) and in humans with type 1 diabetes (Herold et al., 2002, 2005, Bresson et al., 2006) resulted in partial remission from the disease. Whether this is due to a true regeneration

Fig. 5. Schematic illustration of potential cell sources for postnatal ß cell regeneration

β-cell pool homogeneously possesses replication ability.

Recently, Brennand et al. also demonstrated that in adult mice all β-cells, not just a subpopulation, equally contribute to islet growth and maintenance (Brennand et al., 2007). Two approaches were performed to address this issue. First, evaluation of the replicative potential of the entire β-cell mass was performed by monitoring the disappearance of a fluorescent marker accompanying cell division. Second, clonal analysis of dividing β-cells was completed. Because a uniform loss of label (cell division) across the entire cell population was observed, and all clones were of comparable size, authors conclude that the

In humans, increased β-cell replication has been documented adjacent to intrapancreatic gastrinomas, suggesting that adult human β-cells can be driven, under specific circumstances, into the cell cycle (Meier et al., 2006b). Support to this remark was also given by another study, which demonstrated that β-cell replication is the primary mechanism responsible for the postnatal expansion of the β-cell mass in a population of young nondiabetic individuals [Meier et al., 2008]. In particular, it was shown that β-cell mass is able to (1) expand by several folds from birth to adulthood, (2) this is accomplished by an increase in number of β-cells per islet with a concomitant expansion in islet size (3) the relative rate

process or just recovery of dysfunctional β-cells is still debated.

by quenching autoimmunity allowing therefore functional recovery of residual β-cell mass in the pancreas, has been proven by Zorina et al. (Zorina et al., 2003). In the NOD autoimmune diabetes mouse model" in place of, restoration of endogenous β-cell function to physiologically sufficient levels was achievable after allogeneic bone marrow transplantation. Abrogation of autoimmunity and consequent β-cell mass recovery interestingly occurred even when allogeneic bone marrow cell tranplantation was performed after the clinical onset of diabetes. A recent study has suggested that bone marrow cells might have a role in permitting survival of endogenous β-cells also in humans (Voltarelli et al., 2007). Authors reported insulin independence for up to a year in more than half the cases of a small number of patients with recent-onset type 1 diabetes. Patients were administered high-dose immunosuppressive therapy to kill autoreactive T cell clones followed by autologous non-myeloablative stem cell transplantation. Nonetheless, because autologous bone marrow cell transplantation could not change indefinitely the genetic susceptibility to develop autoimmune diabetes, autoimmunity recurred soon after full immunocompetence was re-established. Therefore, different approaches should be used to obtain durable abrogation of β-cell specific autoimmunity, and allow recovery of insulin production (Giannoukakis et al., 2008).

Collectively these series of reports suggest that bone marrow cells do not give rise directly to new insulin producing cells - however they can indirectly facilitate regeneration of the endocrine pancreas, perhaps by secreting appropriate regenerative factors that still need to be characterized.

Other mesenchymal stem cells have been taken into consideration as a potential source of cells. Human and rat multipotent adipose tissue-derived stem cells (ADSCs) have been reported to generate insulin-producing cells after transduction with Pdx1 gene. The surrogate -cells improved glucose sensitivity when transplanted under the renal capsule of STZinduced diabetic rats [Lin et al., 2009]. In addition, intraportal infusion of human ADSCs together with bone marrow stem cells could increase endogenous insulin levels reducing exogenous insulin requirements in patients affected by type 1 diabetes (Trivedi et al., 2008).

The human limbus has also been indicated as a source of stem cells. The limbus is a highly specialized region of the eye hosting a well-recognized population of epithelial stem cells, which continuously renew the corneal surface. Additionally, the limbal niche also hosts stromal fibroblast-like stem cells (f-LSCs), with multilineage transdifferentiation potential.

f-LSCs were able to generate functional pancreatic hormone-expressing cells *in vitro* recapitulating pancreatic organogenesis (Criscimanna et al., 2011).

#### **6. Evidence of β-cell regeneration via proliferation of pre-existing β-cells**

During the fetal stage, differentiation from precursor cells is the major mechanism by which cells are formed, while β-cells replication is enhanced during the perinatal and neonatal period. Lineage-tracing experiments in rodents provided convincing proof to the theory that adult β-cells predominantly arise from other β-cells without significant contributions from underlying stem or progenitor cell populations (Cano et al., 2008). Several studies conducted by the group of Melton and colleagues showed that, after pancreatic resection (Dor et al., 2004), or in a diabetic status induced by transgenic expression of diphtheria toxin (Nir et al., 2007), mouse β-cells possess significant capacity for spontaneous regeneration, sufficient to recover from overt diabetes. Authors claim that failure of β-cell regeneration in both autoimmune and pharmacological models of diabetes is due to confounding factors

by quenching autoimmunity allowing therefore functional recovery of residual β-cell mass in the pancreas, has been proven by Zorina et al. (Zorina et al., 2003). In the NOD autoimmune diabetes mouse model" in place of, restoration of endogenous β-cell function to physiologically sufficient levels was achievable after allogeneic bone marrow transplantation. Abrogation of autoimmunity and consequent β-cell mass recovery interestingly occurred even when allogeneic bone marrow cell tranplantation was performed after the clinical onset of diabetes. A recent study has suggested that bone marrow cells might have a role in permitting survival of endogenous β-cells also in humans (Voltarelli et al., 2007). Authors reported insulin independence for up to a year in more than half the cases of a small number of patients with recent-onset type 1 diabetes. Patients were administered high-dose immunosuppressive therapy to kill autoreactive T cell clones followed by autologous non-myeloablative stem cell transplantation. Nonetheless, because autologous bone marrow cell transplantation could not change indefinitely the genetic susceptibility to develop autoimmune diabetes, autoimmunity recurred soon after full immunocompetence was re-established. Therefore, different approaches should be used to obtain durable abrogation of β-cell specific autoimmunity, and

Collectively these series of reports suggest that bone marrow cells do not give rise directly to new insulin producing cells - however they can indirectly facilitate regeneration of the endocrine pancreas, perhaps by secreting appropriate regenerative factors that still need to

Other mesenchymal stem cells have been taken into consideration as a potential source of cells. Human and rat multipotent adipose tissue-derived stem cells (ADSCs) have been reported to generate insulin-producing cells after transduction with Pdx1 gene. The surrogate -cells improved glucose sensitivity when transplanted under the renal capsule of STZinduced diabetic rats [Lin et al., 2009]. In addition, intraportal infusion of human ADSCs together with bone marrow stem cells could increase endogenous insulin levels reducing exogenous insulin requirements in patients affected by type 1 diabetes (Trivedi et al., 2008). The human limbus has also been indicated as a source of stem cells. The limbus is a highly specialized region of the eye hosting a well-recognized population of epithelial stem cells, which continuously renew the corneal surface. Additionally, the limbal niche also hosts stromal fibroblast-like stem cells (f-LSCs), with multilineage transdifferentiation potential. f-LSCs were able to generate functional pancreatic hormone-expressing cells *in vitro*

**6. Evidence of β-cell regeneration via proliferation of pre-existing β-cells** 

During the fetal stage, differentiation from precursor cells is the major mechanism by which cells are formed, while β-cells replication is enhanced during the perinatal and neonatal period. Lineage-tracing experiments in rodents provided convincing proof to the theory that adult β-cells predominantly arise from other β-cells without significant contributions from underlying stem or progenitor cell populations (Cano et al., 2008). Several studies conducted by the group of Melton and colleagues showed that, after pancreatic resection (Dor et al., 2004), or in a diabetic status induced by transgenic expression of diphtheria toxin (Nir et al., 2007), mouse β-cells possess significant capacity for spontaneous regeneration, sufficient to recover from overt diabetes. Authors claim that failure of β-cell regeneration in both autoimmune and pharmacological models of diabetes is due to confounding factors

allow recovery of insulin production (Giannoukakis et al., 2008).

recapitulating pancreatic organogenesis (Criscimanna et al., 2011).

be characterized.

disguising the innate regenerative response, such as the persistence of circulating autoreactive T cells. To further sustain this hypothesis, it has been demonstrated how therapeutic protocols intended at blocking autoimmunity in NOD mice (Chatenoud et al., 1994) and in humans with type 1 diabetes (Herold et al., 2002, 2005, Bresson et al., 2006) resulted in partial remission from the disease. Whether this is due to a true regeneration process or just recovery of dysfunctional β-cells is still debated.

Fig. 5. Schematic illustration of potential cell sources for postnatal ß cell regeneration

Recently, Brennand et al. also demonstrated that in adult mice all β-cells, not just a subpopulation, equally contribute to islet growth and maintenance (Brennand et al., 2007). Two approaches were performed to address this issue. First, evaluation of the replicative potential of the entire β-cell mass was performed by monitoring the disappearance of a fluorescent marker accompanying cell division. Second, clonal analysis of dividing β-cells was completed. Because a uniform loss of label (cell division) across the entire cell population was observed, and all clones were of comparable size, authors conclude that the β-cell pool homogeneously possesses replication ability.

In humans, increased β-cell replication has been documented adjacent to intrapancreatic gastrinomas, suggesting that adult human β-cells can be driven, under specific circumstances, into the cell cycle (Meier et al., 2006b). Support to this remark was also given by another study, which demonstrated that β-cell replication is the primary mechanism responsible for the postnatal expansion of the β-cell mass in a population of young nondiabetic individuals [Meier et al., 2008]. In particular, it was shown that β-cell mass is able to (1) expand by several folds from birth to adulthood, (2) this is accomplished by an increase in number of β-cells per islet with a concomitant expansion in islet size (3) the relative rate

The Enigma of β-Cell Regeneration in the Adult Pancreas: Self-Renewal Versus Neogenesis 381

Co-expression of the epithelial marker CK19 (green) and Ngn3 (red), a transcription factor typically expressed in progenitor cells committed to the endocrine lineage in the pancreas of a patient with chronic pancreatitis (right panel). Control healthy pancreas (left panel). A number of questions can be asked regarding the process by which damage, and pancreatitis specifically, mobilize these cells. For example, what is the potential role of the immune response to a damage signal in the pancreas in the differentiation of ductal and exocrine cells towards the endocrine lineage? Do immune cells produce soluble factors that facilitate the process of differentiation? What are the characteristics of differentiating nonendocrine and endocrine cells in response to pancreatitis? Can endocrine progenitor cells be better characterized using pancreatitis as an inductive event? Would isolation of such progenitor cells offer a framework for *in vitro* genetic manipulation to further differentiate such cells into defined and fully functional endocrine cells? Would *in vivo* genetic manipulation of the microenvironment exhibiting differentiation processes in response to pancreatitis offer a framework to direct such cells into endocrine lineages? These questions are important in the context of identifying progenitor cells that can serve as the source of

In conclusion, caution is required in interpreting studies unraveling the mechanisms involved in the maintenance of the β-cell mass. The extrapolation of information from animal studies can be misleading and not necessarily indicative for regenerative therapy in diabetic patients because mechanisms of regeneration/maintenance of the β-cell mass might be different across species (Hanley et al., 2008). For example, in obese rodents, an increment in β-cell mass is achieved by a massive increase of islet size and β-cell number per islet, consistent with a predominant mechanism of β-cell replication. On the contrary, in obese humans, islets are only modestly increased in size, consistent with minor involvement of βcell replication (Butler et al., 2003). Also, a 90% pancreatectomy performed in young rodents leads to transient diabetes and regeneration of β-cell mass of ~50% within 2 weeks (Bonner-Weir et al., 1983, 1994), while only a 50% pancreatectomy is sufficient to determine diabetic status in adult humans who afterward became obese too. Certainly, one potential explanation for the reported differences between rodents and humans could be that rodents are more frequently studied at 1–3 months of age, when there is a high capacity for β-cell

Fig. 7. Peculiar cell phenotypes in chronic pancreatitis.

endocrine cells, especially insulin-producing β**-**cells.

**8. Conclusions** 

of β-cell growth is higher in infancy and gradually declines thereafter to adulthood with no secondary accelerated growth phase during adolescence, (4) β-cell mass (and presumably growth) is highly variable between individuals, (5) a high rate of β-cell replication is coincident with the major postnatal expansion of -cell mass. A summary of the theories on β-cell origin is presented in Figure 5.

#### **7. The chronic pancreatitis model**

Regenerative responses from the pancreatic tissue can be the result of a surgical or inflammatory injury. It has been described that pancreatic stellate cells, the star shaped cells representing approximately 4% of the resident pancreatic cell pool, are involved in fibrogenesis and pancreas regeneration. In chronic pancreatitis, these cells undergo transformation from quiescent to activated myofibroblast-like cells. Upon activation, which can be triggered by reactive oxygen intermediates, ethanol, Transforming Growth Factor (TGF) and - 1, they can disclose special features, including the capability to increase the synthesis of collagen, fibronectins, in addition to cytokines. Interestingly TGF- is expressed by ductal cells in chronic pancreatitis, with a role in development of fibrosis and glandular atrophy (Demois et al. 2002). Recently it has been shown in the adult human pancreas of patients with chronic pancreatitis increased numbers of insulin as well as glucagon -containing cells in the ducts, in addition to other cells containing endocrine and exocrine markers. Such findings were also associated to higher numbers of proliferating cells (Ki67 positive) and Pdx1+ cells, suggestive of a "metaplastic" status (Philips et al.,2007).

In our own experience as a reference center for the isolation of human islet cells, we observed peculiar morphological features in the pancreatic sections in patients with chronic pancreatitis. Not only did we find a higher density of islets, probably due to the destruction of the surrounding exocrine tissue (Figure 6), but, interestingly, we found that the intra-islet -cell area relative to the -cell area was significantly higher in chronic pancreatitis patients. Co-expression of the epithelial marker CK19, typical of ductal cells, with Ngn3, a transcription factor expressed in endocrine-committed cells during pancreatic development (Figure 7) was also identified.

Fig. 6. Histological pancreatic features in chronic pancreatitis.

Islets of Langerhans (immunostained for insulin in brown/red) of a healthy individual (left) in comparison with those of a patient affected by chronic pancreatitis (right).

Fig. 7. Peculiar cell phenotypes in chronic pancreatitis.

Co-expression of the epithelial marker CK19 (green) and Ngn3 (red), a transcription factor typically expressed in progenitor cells committed to the endocrine lineage in the pancreas of a patient with chronic pancreatitis (right panel). Control healthy pancreas (left panel).

A number of questions can be asked regarding the process by which damage, and pancreatitis specifically, mobilize these cells. For example, what is the potential role of the immune response to a damage signal in the pancreas in the differentiation of ductal and exocrine cells towards the endocrine lineage? Do immune cells produce soluble factors that facilitate the process of differentiation? What are the characteristics of differentiating nonendocrine and endocrine cells in response to pancreatitis? Can endocrine progenitor cells be better characterized using pancreatitis as an inductive event? Would isolation of such progenitor cells offer a framework for *in vitro* genetic manipulation to further differentiate such cells into defined and fully functional endocrine cells? Would *in vivo* genetic manipulation of the microenvironment exhibiting differentiation processes in response to pancreatitis offer a framework to direct such cells into endocrine lineages? These questions are important in the context of identifying progenitor cells that can serve as the source of endocrine cells, especially insulin-producing β**-**cells.

#### **8. Conclusions**

380 Type 1 Diabetes Complications

of β-cell growth is higher in infancy and gradually declines thereafter to adulthood with no secondary accelerated growth phase during adolescence, (4) β-cell mass (and presumably growth) is highly variable between individuals, (5) a high rate of β-cell replication is coincident with the major postnatal expansion of -cell mass. A summary of the theories on

Regenerative responses from the pancreatic tissue can be the result of a surgical or inflammatory injury. It has been described that pancreatic stellate cells, the star shaped cells representing approximately 4% of the resident pancreatic cell pool, are involved in fibrogenesis and pancreas regeneration. In chronic pancreatitis, these cells undergo transformation from quiescent to activated myofibroblast-like cells. Upon activation, which can be triggered by reactive oxygen intermediates, ethanol, Transforming Growth Factor (TGF) and - 1, they can disclose special features, including the capability to increase the synthesis of collagen, fibronectins, in addition to cytokines. Interestingly TGF- is expressed by ductal cells in chronic pancreatitis, with a role in development of fibrosis and glandular atrophy (Demois et al. 2002). Recently it has been shown in the adult human pancreas of patients with chronic pancreatitis increased numbers of insulin as well as glucagon -containing cells in the ducts, in addition to other cells containing endocrine and exocrine markers. Such findings were also associated to higher numbers of proliferating cells (Ki67 positive) and Pdx1+ cells, suggestive of a "metaplastic" status

In our own experience as a reference center for the isolation of human islet cells, we observed peculiar morphological features in the pancreatic sections in patients with chronic pancreatitis. Not only did we find a higher density of islets, probably due to the destruction of the surrounding exocrine tissue (Figure 6), but, interestingly, we found that the intra-islet -cell area relative to the -cell area was significantly higher in chronic pancreatitis patients. Co-expression of the epithelial marker CK19, typical of ductal cells, with Ngn3, a transcription factor expressed in endocrine-committed cells during pancreatic development

Islets of Langerhans (immunostained for insulin in brown/red) of a healthy individual (left)

in comparison with those of a patient affected by chronic pancreatitis (right).

β-cell origin is presented in Figure 5.

(Philips et al.,2007).

(Figure 7) was also identified.

Fig. 6. Histological pancreatic features in chronic pancreatitis.

**7. The chronic pancreatitis model** 

In conclusion, caution is required in interpreting studies unraveling the mechanisms involved in the maintenance of the β-cell mass. The extrapolation of information from animal studies can be misleading and not necessarily indicative for regenerative therapy in diabetic patients because mechanisms of regeneration/maintenance of the β-cell mass might be different across species (Hanley et al., 2008). For example, in obese rodents, an increment in β-cell mass is achieved by a massive increase of islet size and β-cell number per islet, consistent with a predominant mechanism of β-cell replication. On the contrary, in obese humans, islets are only modestly increased in size, consistent with minor involvement of βcell replication (Butler et al., 2003). Also, a 90% pancreatectomy performed in young rodents leads to transient diabetes and regeneration of β-cell mass of ~50% within 2 weeks (Bonner-Weir et al., 1983, 1994), while only a 50% pancreatectomy is sufficient to determine diabetic status in adult humans who afterward became obese too. Certainly, one potential explanation for the reported differences between rodents and humans could be that rodents are more frequently studied at 1–3 months of age, when there is a high capacity for β-cell

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In conclusion, the debate between the supporters of neogenesis and self-renewal for maintenance and replacement of β-cells is still open. It is perhaps acceptable to conclude that there is some truth in both proposed hypothesis, and that one does not exclude the other. Understanding the potential contribution of each mechanism will be crucial to find better curative approaches for diabetes.

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 Benhamou PY. Human bone marrow mesenchymal stem cells can express insulin and key transcription factors of the endocrine pancreas developmental pathway upon genetic and/or microenvironmental manipulation in vitro. Stem Cells 2005;

cells from mouse islets are not generated by epithelial-to-mesenchymal transition

of murine type 1 diabetes in the absence of any infused spleen cell contribution.

transplantation of pseudoislets consisting of beta cells derived from ductal cells .

marrow-derived cells trans-differentiating into insulin producing cells for the

 differentiation during human development does not rely on nestin-positive precursors: implications for stem cell-derived replacement therapy. Diabetologia

islet progenitor cells in their ducts, but reversal of overt diabetes in NOD mice by anti-CD3 shows no evidence for islet regeneration. Diabetes. 2007 Mar;56(3):634-40.

dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat

physiologic self-regeneration: a step beyond islet cell replacement. Pharm Res 2006;

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Means AL, Meszoely IM, Suzuki K, Miyamoto Y, Rustgi AK, Coffey RJ Jr, Wright CV,

Meier JJ, Bhushan A, Butler AE, Rizza RA, Butler PC. Sustained beta cell apoptosis in

Meier JJ, Lin JC, Butler AE, Galasso R, Martinez DS, Butler PC. Direct evidence of attempted

Meier JJ, Butler AE, Galasso R, Rizza RA, Butler PC. Increased islet beta cell replication

Meier JJ, Butler AE, Saisho Y, Monchamp T, Galasso R, Bhushan A, Rizza RA, Butler PC.

Minami K, Okuno M, Miyawaki K, Okumachi A, Ishizaki K, Oyama K, Kawaguchi M,

 hepatocytes and proliferation of duct cells. Am J Pathol 1999;154:683-91. Lardon J, Huyens N, Rooman I, Bouwens L. Exocrine cell transdifferentiation in

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Pancreatic expression of keratinocyte growth factor leads to differentiation of islet

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adipose tissue-derived stem cells expressing pancreatic duodenal homeobox 1.

model to study human acinar-to-duct-to-islet differentiation. Pancreas 2007; 34:452-

 Nepom GT, Ricordi C, Ruiz P, Sageshima J, Ciancio G, Burke GW, Pugliese A. Insulin protein and proliferation in ductal cells in the transplanted pancreas of patients with type 1 diabetes and recurrence of autoimmunity. Diabetologia. 2008

 Stoffers DA, Leach SD. Pancreatic epithelial plasticity mediated by acinar cell transdifferentiation and generation of nestin-positive intermediates. Development

patients with long-standing type 1 diabetes: indirect evidence for islet

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Beta-cell replication is the primary mechanism subserving the postnatal expansion

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**18** 

**Cell Replacement Therapy: The Rationale** 

Stephen J. M. Skinner, Paul L. J. Tan, Olga Garkavenko, Marija Muzina, Livia Escobar and Robert B. Elliott

*Living Cell Technologies (NZ) Ltd* 

*New Zealand*

**for Encapsulated Porcine Islet Transplantation** 

Among the problems posed by chronic diseases today, one of the most daunting is that posed by diabetes mellitus, which is a very significant public health problem resulting in substantial morbidity and mortality (American Diabetes Association, 2011). With the increasing life-expectancy of the world's population, increasing exposure to environmental trigger factors, the rising incidence of obesity, and lifestyle changes such as unhealthy diets and decreased physical activity, the prevalence of diabetes has risen dramatically over recent years and is now reaching epidemic proportions globally. This rapid increase is a significant cause for concern, with an additional 7 million diagnosed each year (International Diabetes Federation, 2011). Diabetes is now the fourth or fifth leading cause of death in most developed countries (International Diabetes Federation 2000). Globally, it is estimated that more than 200 million adults now have diabetes and this number is expected to increase alarmingly in the coming decades. By the year 2025, it is estimated that almost

333 million people will have the disease (International Diabetes Federation 2006).

Type 1 (insulin-dependent) diabetes accounts for 5% to 10% of all diagnosed cases. In 2003, approximately 4.9 million people (0.09% of the world's population) were estimated to have type 1 disease, with Europe having the highest number of sufferers (1.27 million) followed by North America (1.04 million) and Southeast Asia (0.91 million). The highest prevalence of type 1 diabetes was in North America (0.25%) followed by Europe (0.19%) (International Diabetes Federation 2006). In 2002, there were an estimated 0.9 to 1.2 million people with type 1 diabetes in the USA (American Diabetes Association 2006). The incidence in recent years may have accelerated alarmingly as shown in a recent study from Finland where the rate increased from 31.4 per 100,000 per year in 1980 to 64.2 per 100,000 per year in 2005

In New Zealand, the incidence of type 1 diabetes has doubled in the last 15 years, reflecting international trends. In 2003, the estimated prevalence of type 1 disease among the population aged <25 years was 0.18%, with the total number of sufferers in this age range numbering 2540. The majority, 85% (2158 people), were of European descent, while 9% were

Although the life-expectancy of patients with type 1 (insulin-dependent) diabetes mellitus has vastly improved since the introduction of insulin, the ability of insulin injections to

Maori, 2.9% were Pacific peoples, and 3.0% were Asian (Wu et al. 2005).

**1. Introduction** 

(Harjutsalo et al, 2008).

Zulewski H, Abraham EJ, Gerlach MJ, Daniel PB, Moritz W, Muller B, Vallejo M, Thomas MK, Habener JF. Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine and hepatic phenotypes. Diabetes 2001; 50:521-33.

### **Cell Replacement Therapy: The Rationale for Encapsulated Porcine Islet Transplantation**

Stephen J. M. Skinner, Paul L. J. Tan, Olga Garkavenko, Marija Muzina, Livia Escobar and Robert B. Elliott *Living Cell Technologies (NZ) Ltd New Zealand*

#### **1. Introduction**

390 Type 1 Diabetes Complications

Zulewski H, Abraham EJ, Gerlach MJ, Daniel PB, Moritz W, Muller B, Vallejo M, Thomas

hepatic phenotypes. Diabetes 2001; 50:521-33.

 MK, Habener JF. Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine and

> Among the problems posed by chronic diseases today, one of the most daunting is that posed by diabetes mellitus, which is a very significant public health problem resulting in substantial morbidity and mortality (American Diabetes Association, 2011). With the increasing life-expectancy of the world's population, increasing exposure to environmental trigger factors, the rising incidence of obesity, and lifestyle changes such as unhealthy diets and decreased physical activity, the prevalence of diabetes has risen dramatically over recent years and is now reaching epidemic proportions globally. This rapid increase is a significant cause for concern, with an additional 7 million diagnosed each year (International Diabetes Federation, 2011). Diabetes is now the fourth or fifth leading cause of death in most developed countries (International Diabetes Federation 2000). Globally, it is estimated that more than 200 million adults now have diabetes and this number is expected to increase alarmingly in the coming decades. By the year 2025, it is estimated that almost 333 million people will have the disease (International Diabetes Federation 2006).

> Type 1 (insulin-dependent) diabetes accounts for 5% to 10% of all diagnosed cases. In 2003, approximately 4.9 million people (0.09% of the world's population) were estimated to have type 1 disease, with Europe having the highest number of sufferers (1.27 million) followed by North America (1.04 million) and Southeast Asia (0.91 million). The highest prevalence of type 1 diabetes was in North America (0.25%) followed by Europe (0.19%) (International Diabetes Federation 2006). In 2002, there were an estimated 0.9 to 1.2 million people with type 1 diabetes in the USA (American Diabetes Association 2006). The incidence in recent years may have accelerated alarmingly as shown in a recent study from Finland where the rate increased from 31.4 per 100,000 per year in 1980 to 64.2 per 100,000 per year in 2005 (Harjutsalo et al, 2008).

> In New Zealand, the incidence of type 1 diabetes has doubled in the last 15 years, reflecting international trends. In 2003, the estimated prevalence of type 1 disease among the population aged <25 years was 0.18%, with the total number of sufferers in this age range numbering 2540. The majority, 85% (2158 people), were of European descent, while 9% were Maori, 2.9% were Pacific peoples, and 3.0% were Asian (Wu et al. 2005).

> Although the life-expectancy of patients with type 1 (insulin-dependent) diabetes mellitus has vastly improved since the introduction of insulin, the ability of insulin injections to

Cell Replacement Therapy: The Rationale for Encapsulated Porcine Islet Transplantation 393

increased by breeding more donor animals; (b) pig and human insulins have close structural and biological similarities; and (c) physiological glucose levels in pigs are similar to those in humans (Elliott, 2011). The rationale for this treatment approach (termed 'xenotransplantation') is that the implanted pig islets have the potential to mimic the normal physiological insulin response in type 1 diabetics, such that near-normal blood glucose levels may be achievable without insulin injections or with reduced requirements. As a consequence, long-term diabetes complications may be prevented and patients should experience less hypoglycaemia than they do with the currently recommended 'intensive' insulin regimens. Thus the need and rationale for improved diabetes control is clear but the effectiveness and practicality of islet transplants, whether from human, porcine or other sources, has yet to be firmly established. As with any transplanted tissue, organ or cells, whether from human or animal, the host immune system must be considered. Immunosuppression has been studied and used extensively in islet transplant patients. An increasingly successful alternative is immune-isolation, that is to isolate the transplanted cells in capsules or devices that exclude immune cells and antibodies, but allow the free diffusion of glucose, insulin, nutrients and dissolved oxygen and carbon dioxide.

Some of the **key issues** in implementing **porcine islet xenotransplantation** in the treatment

**1.1.1.1** Have 'proof of concept' pre-clinical experiments demonstrated sufficiently effective

**1.1.1.2** Have the risks to the patient's safety been sufficiently evaluated, including: (a) the risk of transmission of infectious diseases; and (b) risks to the individual from the transplant procedure itself, including allergic reactions and other immunological responses that might

**1.1.1.3** How rejection of the cells by the recipient's immune system can be effectively

**1.1.1.4** Whether porcine islet xenotransplants can restore, at least partially, the normal regulation of blood glucose (as reflected in decreased insulin requirements and decrease in

**1.1.1.5** The duration of effectiveness of the transplanted islets (i.e. whether they remain effective over a sufficiently prolonged period to justify the inconvenience and cost of the procedure), and the extent to which the patient's well-being is enhanced and long-term

**1.1.1.6** Ethical considerations, including cultural, ethical and spiritual dimensions, informed

**1.1.1.7** If clinical trials are judged successful by rigorous independent review, will resources to expand the availability of the treatment be provided by commercial and/or government

The main body of information from our laboratory on the efficacy and safety of current preparations of porcine islets is derived from studies with islets sourced from Auckland

**2. Pre-clinical studies of porcine islet xenotransplantation in non-human** 

The latter approach eliminates the risks associated with immunosuppression.

improvement in the control of Type 1 diabetes in experimental animals?

of patients with Type 1 diabetes are:

compromise the success of the procedure.

diabetes complications are prevented.

HbA1c), and the number of islets needed to achieve this.

consent issues, and measures to ensure animal welfare.

prevented.

investment?

**primates and rodents** 

reliably prevent wide fluctuations in blood glucose levels is often inadequate and many patients develop complications of the disease. These complications cause considerable disability and suffering, and their management has major morbidity and cost consequences. High blood glucose concentrations not only cause acute metabolic problems but also lasting and accumulative damage by chemical reaction (glycation, e.g. Haemoglobin A1c) with a host of physiologically critical proteins. Hence the long term damage to the cardiovascular system, eye, kidney, heart and nervous system. On the other hand, low blood glucose levels, known as hypoglycaemic episodes, are usually perceived by the patient and treated by ingesting glucose or food. But in a significant minority of patients, hypoglycaemic episodes are not perceived and may cause loss of consciousness. These can lead to fatal outcomes for the patient and sometimes for others, such as in situations where the patient is in control of a moving vehicle.

For these reasons, the investment in, and search for, newer treatments that can provide a 'cure' for the disease with normoglycaemic control, or that at least minimizes the damaging effects of extremely high and low blood glucose excursions with markedly better control of metabolic disturbances, has been energetically pursued. In terms of health economics, the benefits in preventing eye, heart and kidney diseases would more than justify a substantial investment in such research and development (Beckwith et al. 2010).

#### **1.1 Background and rationale for cell transplantation**

Insulin is essential for normal glucose metabolism. It is released by the beta-islet cells of the pancreas in response to rising blood glucose levels. The feedback mechanisms involved provide a precise, finely tuned response, keeping blood glucose at a concentration of around 4.5mM. Type 1 diabetes is an endocrine disorder caused by autoimmune destruction of the beta-islet cells leading to insulin deficiency. Treatment by injecting various commercially produced insulins subcutaneously, while life-sustaining, can not provide the control of blood glucose provided by a full complement of functional islets.

In order to optimize the control of blood sugar and thus prevent the acute and chronic damage, the most likely way to improve these outcomes is to replace the patient's pancreatic islets with a new pancreas or new islets. Transplanting a whole new pancreas is a very demanding procedure that requires many resources and is found to be less than practical.

#### **1.1.1 Porcine islet cell transplants**

Among the newer treatment strategies that have been proposed, transplantation of pancreatic islets, obtained either from other human or animal donors, has received considerable attention worldwide. This is because islet transplantation can restore not only the insulin-secreting unit, but also the precise insulin release in response to rising blood glucose and multiple signals arising within and beyond the islets.

Because human islet transplantation is limited by the shortage of human islet tissue, human embryonic stem cells or induced pluripotent stem (iPS) cells from the patient are being developed into transplantable insulin producing cells. Recent reports indicate that iPS cells may not be transplantable into mice of the same strain without immune rejection (Zhao et al, 2011). The US FDA guidelines highlight concerns that stem cell derived lines may undergo malignant transformation or develop into teratomas after transplantation (Fink et al 2009).

While stem cell line-derived insulin producing cells are still at an early stage of research, pig islets are viewed as a promising alternative since: (a) the supply of pig pancreatic cells can be

reliably prevent wide fluctuations in blood glucose levels is often inadequate and many patients develop complications of the disease. These complications cause considerable disability and suffering, and their management has major morbidity and cost consequences. High blood glucose concentrations not only cause acute metabolic problems but also lasting and accumulative damage by chemical reaction (glycation, e.g. Haemoglobin A1c) with a host of physiologically critical proteins. Hence the long term damage to the cardiovascular system, eye, kidney, heart and nervous system. On the other hand, low blood glucose levels, known as hypoglycaemic episodes, are usually perceived by the patient and treated by ingesting glucose or food. But in a significant minority of patients, hypoglycaemic episodes are not perceived and may cause loss of consciousness. These can lead to fatal outcomes for the patient and sometimes for others, such as in situations where the patient is in control of

For these reasons, the investment in, and search for, newer treatments that can provide a 'cure' for the disease with normoglycaemic control, or that at least minimizes the damaging effects of extremely high and low blood glucose excursions with markedly better control of metabolic disturbances, has been energetically pursued. In terms of health economics, the benefits in preventing eye, heart and kidney diseases would more than justify a substantial

Insulin is essential for normal glucose metabolism. It is released by the beta-islet cells of the pancreas in response to rising blood glucose levels. The feedback mechanisms involved provide a precise, finely tuned response, keeping blood glucose at a concentration of around 4.5mM. Type 1 diabetes is an endocrine disorder caused by autoimmune destruction of the beta-islet cells leading to insulin deficiency. Treatment by injecting various commercially produced insulins subcutaneously, while life-sustaining, can not provide the control of

In order to optimize the control of blood sugar and thus prevent the acute and chronic damage, the most likely way to improve these outcomes is to replace the patient's pancreatic islets with a new pancreas or new islets. Transplanting a whole new pancreas is a very demanding procedure that requires many resources and is found to be less than practical.

Among the newer treatment strategies that have been proposed, transplantation of pancreatic islets, obtained either from other human or animal donors, has received considerable attention worldwide. This is because islet transplantation can restore not only the insulin-secreting unit, but also the precise insulin release in response to rising blood

Because human islet transplantation is limited by the shortage of human islet tissue, human embryonic stem cells or induced pluripotent stem (iPS) cells from the patient are being developed into transplantable insulin producing cells. Recent reports indicate that iPS cells may not be transplantable into mice of the same strain without immune rejection (Zhao et al, 2011). The US FDA guidelines highlight concerns that stem cell derived lines may undergo malignant transformation or develop into teratomas after transplantation (Fink et al 2009). While stem cell line-derived insulin producing cells are still at an early stage of research, pig islets are viewed as a promising alternative since: (a) the supply of pig pancreatic cells can be

investment in such research and development (Beckwith et al. 2010).

blood glucose provided by a full complement of functional islets.

glucose and multiple signals arising within and beyond the islets.

**1.1 Background and rationale for cell transplantation** 

**1.1.1 Porcine islet cell transplants** 

a moving vehicle.

increased by breeding more donor animals; (b) pig and human insulins have close structural and biological similarities; and (c) physiological glucose levels in pigs are similar to those in humans (Elliott, 2011). The rationale for this treatment approach (termed 'xenotransplantation') is that the implanted pig islets have the potential to mimic the normal physiological insulin response in type 1 diabetics, such that near-normal blood glucose levels may be achievable without insulin injections or with reduced requirements. As a consequence, long-term diabetes complications may be prevented and patients should experience less hypoglycaemia than they do with the currently recommended 'intensive' insulin regimens. Thus the need and rationale for improved diabetes control is clear but the effectiveness and practicality of islet transplants, whether from human, porcine or other sources, has yet to be firmly established. As with any transplanted tissue, organ or cells, whether from human or animal, the host immune system must be considered. Immunosuppression has been studied and used extensively in islet transplant patients. An increasingly successful alternative is immune-isolation, that is to isolate the transplanted cells in capsules or devices that exclude immune cells and antibodies, but allow the free diffusion of glucose, insulin, nutrients and dissolved oxygen and carbon dioxide. The latter approach eliminates the risks associated with immunosuppression.

Some of the **key issues** in implementing **porcine islet xenotransplantation** in the treatment of patients with Type 1 diabetes are:

**1.1.1.1** Have 'proof of concept' pre-clinical experiments demonstrated sufficiently effective improvement in the control of Type 1 diabetes in experimental animals?

**1.1.1.2** Have the risks to the patient's safety been sufficiently evaluated, including: (a) the risk of transmission of infectious diseases; and (b) risks to the individual from the transplant procedure itself, including allergic reactions and other immunological responses that might compromise the success of the procedure.

**1.1.1.3** How rejection of the cells by the recipient's immune system can be effectively prevented.

**1.1.1.4** Whether porcine islet xenotransplants can restore, at least partially, the normal regulation of blood glucose (as reflected in decreased insulin requirements and decrease in HbA1c), and the number of islets needed to achieve this.

**1.1.1.5** The duration of effectiveness of the transplanted islets (i.e. whether they remain effective over a sufficiently prolonged period to justify the inconvenience and cost of the procedure), and the extent to which the patient's well-being is enhanced and long-term diabetes complications are prevented.

**1.1.1.6** Ethical considerations, including cultural, ethical and spiritual dimensions, informed consent issues, and measures to ensure animal welfare.

**1.1.1.7** If clinical trials are judged successful by rigorous independent review, will resources to expand the availability of the treatment be provided by commercial and/or government investment?

#### **2. Pre-clinical studies of porcine islet xenotransplantation in non-human primates and rodents**

The main body of information from our laboratory on the efficacy and safety of current preparations of porcine islets is derived from studies with islets sourced from Auckland

Cell Replacement Therapy: The Rationale for Encapsulated Porcine Islet Transplantation 395

Fig. 1. Clusters of encapsulated porcine islets attached to the mesentery of a cynomolgus

cynomolgus monkeys provided comprehensive immunosuppression was given.

**2.2 Studies in small animal models of diabetes** 

successful, albeit with stringent conditions attached.

In contrast, Hering et al (2006) demonstrated that unencapsulated naked porcine islets, injected into the portal vein of the liver, could be used to reverse diabetes for 100 days in

The biocompatibility of encapsulated porcine islets and a dose-dependent effect on glycaemic control have been demonstrated in STZ-diabetic rats. Those that developed diabetes were separated into four groups that received intraperitoneal transplants of encapsulated neonatal porcine islets at increasing dosage. The doses, measured in IEQs, were 3000 IEQ (n = 7), 6000 IEQ (n = 6), 12,000 IEQ (n = 6) or 18,000 IEQ (n = 6) of the standard preparation or empty alginate microcapsules (n = 12), insulin requirements were significantly reduced in rats given the higher doses. Prior to transplantation, all rats had comparable blood glucose control with daily isophane insulin injections. At week 12 after transplantation, the reduction in the weekly average daily insulin dose was significantly greater in rats given doses of 18,000 IEQs (p < 0.01) or 12,000 IEQs (p < 0.05) compared with control animals that received empty alginate microcapsules. Insulin independence was attained in 3 of 6 rats given 18,000 IEQs, 3 of 6 given 12,000 IEQs, 2 of 6 given 6000 IEQs. 1 of the 12 diabetic rats given empty alginate microcapsules did recover, a case of spontaneous islet regeneration which is a known confounding factor in small animal

Similar encouraging results were seen when streptozotocin-induced diabetic mice and

Thus, immunosuppression may not be necessary when treating diabetes if the islets are efficiently sealed inside capsules or devices with semipermeable membranes. The capsule or device outer membranes exclude immune cells and immunoglobulins but must allow efficient diffusion of glucose, insulin, all nutrients and gases (oxygen, CO2) necessary for

This information about the efficacy of encapsulated islets and their tolerance by animal recipients, including some earlier human clinical studies, were assembled in great detail for the submission of applications to the National Regulatory Clinical Trial Agencies of several countries including New Zealand, Russia and Argentina, where the applications were

genetically predisposed diabetic NOD mice were treated with encapsulated islets.

monkey.

studies (Figure 2).

cell nutrition.

Islands Pigs. The Auckland Islands (AI) strain is unique due to its isolation for more than 150 years on a sub-antarctic island and is free of pathogens commonly found in other pig herds. These animals are being bred in custom-constructed pathogen-free facilities for transplant purposes and are discussed in detail later in this chapter.

#### **2.1 Studies in non-human primates**

The first exploration of primate diabetes treatments with encapsulated porcine islets was undertaken by Sun Y et al (1996) in Toronto, Canada. This pioneering group prepared islets and enclosed them in alginate-polylysine capsules in the early 1980s (Sun AM et al, 1984; Sun Y et al, 1993). They were able to find a number of diabetic cynomolgus monkeys that were ideal for testing these encapsulated porcine islets. These animals responded very well to their encapsulated porcine islets transplant, with significant improvement of their diabetes for 120-800 days. This remarkable achievement was based on decades of research by AM Sun, Y Sun and their colleagues that began in the early 1970s.

In a later non-human primate study the clinical efficacy and safety of an encapsulated neonatal porcine islet preparation was investigated in cynomolgus monkeys with streptozotocin (STZ) induced diabetes. The islet toxin streptozotocin is injected into the monkeys and causes insulin deficiency. Sixteen monkeys with the disease established were separated into two groups of 8: one group was given microcapsules containing living porcine islets and the other was given the same microcapsules but without islets in them. They received the microcapsule transplants in two doses three months apart by injection into the peritoneal cavity (Elliott et al, 2005). No immunosuppressive drugs were administered.

In the group that received capsules containing islets, evidence of clinical activity was noted at 12 and 24 weeks after the first transplant; the reduction of the mean weekly insulin requirement relative to the control was 36% (p=.02) and 43% (p=.01), respectively at these time points. Blood glucose in the two groups were maintained to similar levels indicating that the reduced requirement for insulin injections in the treated group was significant. Both the islet-treated and control groups tolerated the transplant procedures well. No hypoglycaemic episodes or other adverse events were observed in the islet-treated group. There were no differences between the two groups of monkeys in body weight or hematology and liver enzyme parameters. Two deaths occurred, one in the islet-treated group from lobar pneumonia with disseminated lung abscesses at 13 weeks after the first transplant (despite systemic antibiotic therapy), and the other in the control (empty microcapsules) group as a result of a stroke that followed the development of hypoglycaemia due to failure to consume food after regular insulin therapy.

At terminal autopsy of these monkeys (Figure 1), no gross inflammatory reactions to either encapsulated islets or the empty microcapsules were noted in the animals' peritoneal cavities. The organs (liver, spleen, stomach, intestines, kidney, heart, lungs and brain) appeared normal .

In another streptozotocin diabetic monkey study, where encapsulated adult porcine islets were used, and thus more similar to the adult human islet isolations, diabetes was successfully treated without immunosuppression (Dufrane et al, 2010). However, although efficacy was demonstrated with encapsulated islets, they lost their function after 2 weeks. Successful glucose control was achieved for 6 months using 'monolayer cellular devices' containing up to 30,000 Islet Equivalents (IEQ)/Kg of monkey weight, implanted in the abdominal subcutaneous tissue.

In contrast, Hering et al (2006) demonstrated that unencapsulated naked porcine islets, injected into the portal vein of the liver, could be used to reverse diabetes for 100 days in cynomolgus monkeys provided comprehensive immunosuppression was given.

#### **2.2 Studies in small animal models of diabetes**

394 Type 1 Diabetes Complications

Islands Pigs. The Auckland Islands (AI) strain is unique due to its isolation for more than 150 years on a sub-antarctic island and is free of pathogens commonly found in other pig herds. These animals are being bred in custom-constructed pathogen-free facilities for

The first exploration of primate diabetes treatments with encapsulated porcine islets was undertaken by Sun Y et al (1996) in Toronto, Canada. This pioneering group prepared islets and enclosed them in alginate-polylysine capsules in the early 1980s (Sun AM et al, 1984; Sun Y et al, 1993). They were able to find a number of diabetic cynomolgus monkeys that were ideal for testing these encapsulated porcine islets. These animals responded very well to their encapsulated porcine islets transplant, with significant improvement of their diabetes for 120-800 days. This remarkable achievement was based on decades of research

In a later non-human primate study the clinical efficacy and safety of an encapsulated neonatal porcine islet preparation was investigated in cynomolgus monkeys with streptozotocin (STZ) induced diabetes. The islet toxin streptozotocin is injected into the monkeys and causes insulin deficiency. Sixteen monkeys with the disease established were separated into two groups of 8: one group was given microcapsules containing living porcine islets and the other was given the same microcapsules but without islets in them. They received the microcapsule transplants in two doses three months apart by injection into the peritoneal cavity (Elliott et al, 2005). No

In the group that received capsules containing islets, evidence of clinical activity was noted at 12 and 24 weeks after the first transplant; the reduction of the mean weekly insulin requirement relative to the control was 36% (p=.02) and 43% (p=.01), respectively at these time points. Blood glucose in the two groups were maintained to similar levels indicating that the reduced requirement for insulin injections in the treated group was significant. Both the islet-treated and control groups tolerated the transplant procedures well. No hypoglycaemic episodes or other adverse events were observed in the islet-treated group. There were no differences between the two groups of monkeys in body weight or hematology and liver enzyme parameters. Two deaths occurred, one in the islet-treated group from lobar pneumonia with disseminated lung abscesses at 13 weeks after the first transplant (despite systemic antibiotic therapy), and the other in the control (empty microcapsules) group as a result of a stroke that followed the development of

At terminal autopsy of these monkeys (Figure 1), no gross inflammatory reactions to either encapsulated islets or the empty microcapsules were noted in the animals' peritoneal cavities. The organs (liver, spleen, stomach, intestines, kidney, heart, lungs and brain)

In another streptozotocin diabetic monkey study, where encapsulated adult porcine islets were used, and thus more similar to the adult human islet isolations, diabetes was successfully treated without immunosuppression (Dufrane et al, 2010). However, although efficacy was demonstrated with encapsulated islets, they lost their function after 2 weeks. Successful glucose control was achieved for 6 months using 'monolayer cellular devices' containing up to 30,000 Islet Equivalents (IEQ)/Kg of monkey weight, implanted in the

hypoglycaemia due to failure to consume food after regular insulin therapy.

transplant purposes and are discussed in detail later in this chapter.

by AM Sun, Y Sun and their colleagues that began in the early 1970s.

**2.1 Studies in non-human primates** 

immunosuppressive drugs were administered.

appeared normal .

abdominal subcutaneous tissue.

The biocompatibility of encapsulated porcine islets and a dose-dependent effect on glycaemic control have been demonstrated in STZ-diabetic rats. Those that developed diabetes were separated into four groups that received intraperitoneal transplants of encapsulated neonatal porcine islets at increasing dosage. The doses, measured in IEQs, were 3000 IEQ (n = 7), 6000 IEQ (n = 6), 12,000 IEQ (n = 6) or 18,000 IEQ (n = 6) of the standard preparation or empty alginate microcapsules (n = 12), insulin requirements were significantly reduced in rats given the higher doses. Prior to transplantation, all rats had comparable blood glucose control with daily isophane insulin injections. At week 12 after transplantation, the reduction in the weekly average daily insulin dose was significantly greater in rats given doses of 18,000 IEQs (p < 0.01) or 12,000 IEQs (p < 0.05) compared with control animals that received empty alginate microcapsules. Insulin independence was attained in 3 of 6 rats given 18,000 IEQs, 3 of 6 given 12,000 IEQs, 2 of 6 given 6000 IEQs. 1 of the 12 diabetic rats given empty alginate microcapsules did recover, a case of spontaneous islet regeneration which is a known confounding factor in small animal studies (Figure 2).

Similar encouraging results were seen when streptozotocin-induced diabetic mice and genetically predisposed diabetic NOD mice were treated with encapsulated islets.

Thus, immunosuppression may not be necessary when treating diabetes if the islets are efficiently sealed inside capsules or devices with semipermeable membranes. The capsule or device outer membranes exclude immune cells and immunoglobulins but must allow efficient diffusion of glucose, insulin, all nutrients and gases (oxygen, CO2) necessary for cell nutrition.

This information about the efficacy of encapsulated islets and their tolerance by animal recipients, including some earlier human clinical studies, were assembled in great detail for the submission of applications to the National Regulatory Clinical Trial Agencies of several countries including New Zealand, Russia and Argentina, where the applications were successful, albeit with stringent conditions attached.

Cell Replacement Therapy: The Rationale for Encapsulated Porcine Islet Transplantation 397

Martin et al. 1998a) and that immune-incompetent SCID (severe combined immunodeficiency) mice may develop either microchimerism or infection in vivo (van der Laan et al. 2000). The in vitro findings have, however, been shown to be strain-specific (Patience 2001; Clemenceau et al. 2001; Oldmixon et al. 2002) and cells from animals studied by LCT have not shown retrovirus infectivity. Moreover, no evidence of PERV transmission has been detected over 200 patients who have been exposed to pig cells or tissues and tested for evidence of PERV infection using sensitive detection methods (Wynyard et al. 2011; Garkavenko et al. 2008b; Denner 2003; Dinsmore et al. 2000; Heneine et al. 1998; Heneine et al. 2001; Irgang et al. 2003; Martin et al. 1998b; Paradis et al. 1999; Patience et al. 1998; Tacke et al. 2001). In 2 New Zealand diabetic patients who received encapsulated porcine islet xenotransplants, no evidence of PERV proviral DNA or RNA was detectable in white blood cells and plasma up to 6 years after the transplant, and neither patient was found to have suffered any ill health as a result of the procedure (Elliott et al. 2000; Garkavenko et al. 2004a). Similarly, in 4 other patients who received unencapsulated islets in similar studies in NZ, no infection has been found in a follow-up time of up to 9 years (Garkavenko et al. 2004a). More recently one patient shown to have some functional transplanted porcine islets

In several other studies, no evidence of PERV transmission was found among recipients of porcine clotting factor VIII (Hyate:C) in a study of 88 haemophiliacs, despite the fact that all manufactured batches of porcine factor VIII concentrate used by patients were subsequently tested and shown to contain PERV RNA (Heneine et al. 2001). Similarly, no evidence of PERV DNA was found in 2 renal dialysis patients whose circulation had been linked

A further refinement of the source herd has been obtained by the use of pigs derived from a colony abandoned on the Auckland Islands about 150 years ago. These animals are free of all measured viruses except the retrovirus (PERV), and are being bred for xenotransplant purposes in a purpose-built facility. Although PERV cannot be removed from the porcine genome, there is strong evidence that it is not functional in the Auckland Island Pig strain and is not expressed under any of the recommended testing conditions. This strain is now termed a "Null Pig Strain" suitable as a source of tissues for xenotransplantation (Wynyard

Nevertheless, the selection of donor animals that do not transmit infectious PERV continues to be important and has to be linked with the selection of donor animals bred in isolation and screened to exclude infection with other exogenous microbes. The use of donor neonatal piglets rather than adult pigs also has the advantage of limiting the exposure time of donor

The selection of piglets without genetic modification as a source of tissue offers another advantage. In attempting to prevent immune rejection of porcine tissue, genetically-modified

after 9.5 years was shown to be free of PERV.

extracorporeally to pig kidneys (Patience et al. 1998).

newborn animals to acquired infections as they age.

**3.2 Porcine endogenous retrovirus** 

**3.3 Auckland Islands pigs** 

et al, 2011).

**3.4 Neonatal pigs** 

**3.5 No genetic modification** 

Fig. 2. **Percentage reductions in weekly averages of daily insulin doses 12 weeks after transplantation.** Each point represents individual rats. Reduction of the insulin dose is calculated as the percentage of the baseline average daily insulin dose in the week prior to transplantation. In the three higher doses eight animals were insulin-independent at 12 weeks. The bars represent the average % reduction in insulin dose for the respective groups.

#### **3. Xenotransplantation: Minimizing risks to patient's safety**

The introduction of porcine islet transplantation has been delayed by concerns relating to the possible transmission of pig diseases to humans via the transplanted cells and the risk of introducing micro-organisms during cell processing and encapsulation. There is also a potential risk of transmitting porcine disease to close contacts of the human recipient and to the wider community. The risks of infection from the donor animal can be minimized by controlling the breed, source of and health status of the donor animals, with ongoing screening and quarantining. The risk of introducing micro-organisms during cell processing can be minimized by ensuring strict aseptic technique when isolating and encapsulating the islets.

#### **3.1 Risk of infections ('xenoses') resulting from transfer of pig micro-organisms to human recipients**

The risk of bacterial, fungal and parasitic infections can be minimized by the use of Designated Pathogen-Free pig herds and by monitoring and treating sows for such infections before pancreatic islets are extracted from their progeny (Garkavenko et al. 2008a). However, this approach will not eliminate porcine endogenous retrovirus sequences (PERVs), which are present in the germline of all pigs (including common New Zealand pig breeds) but cause no known infection in the species. The possible transmission of these retroviruses to humans, and their as yet unknown consequences in recipients, have given rise to some concerns over the safety of xenotransplantation, particularly in view of reports that PERVs from certain pig strains can infect human cells in vitro (Patience et al. 1997; Martin et al. 1998a) and that immune-incompetent SCID (severe combined immunodeficiency) mice may develop either microchimerism or infection in vivo (van der Laan et al. 2000). The in vitro findings have, however, been shown to be strain-specific (Patience 2001; Clemenceau et al. 2001; Oldmixon et al. 2002) and cells from animals studied by LCT have not shown retrovirus infectivity. Moreover, no evidence of PERV transmission has been detected over 200 patients who have been exposed to pig cells or tissues and tested for evidence of PERV infection using sensitive detection methods (Wynyard et al. 2011; Garkavenko et al. 2008b; Denner 2003; Dinsmore et al. 2000; Heneine et al. 1998; Heneine et al. 2001; Irgang et al. 2003; Martin et al. 1998b; Paradis et al. 1999; Patience et al. 1998; Tacke et al. 2001). In 2 New Zealand diabetic patients who received encapsulated porcine islet xenotransplants, no evidence of PERV proviral DNA or RNA was detectable in white blood cells and plasma up to 6 years after the transplant, and neither patient was found to have suffered any ill health as a result of the procedure (Elliott et al. 2000; Garkavenko et al. 2004a). Similarly, in 4 other patients who received unencapsulated islets in similar studies in NZ, no infection has been found in a follow-up time of up to 9 years (Garkavenko et al. 2004a). More recently one patient shown to have some functional transplanted porcine islets after 9.5 years was shown to be free of PERV.

#### **3.2 Porcine endogenous retrovirus**

In several other studies, no evidence of PERV transmission was found among recipients of porcine clotting factor VIII (Hyate:C) in a study of 88 haemophiliacs, despite the fact that all manufactured batches of porcine factor VIII concentrate used by patients were subsequently tested and shown to contain PERV RNA (Heneine et al. 2001). Similarly, no evidence of PERV DNA was found in 2 renal dialysis patients whose circulation had been linked extracorporeally to pig kidneys (Patience et al. 1998).

#### **3.3 Auckland Islands pigs**

396 Type 1 Diabetes Complications

**STZ Rat Study 2005**

Control Gp 3000IEQ Gp 6000IEQGp 12000IEQGp 18000IEQ

Fig. 2. **Percentage reductions in weekly averages of daily insulin doses 12 weeks after transplantation.** Each point represents individual rats. Reduction of the insulin dose is calculated as the percentage of the baseline average daily insulin dose in the week prior to transplantation. In the three higher doses eight animals were insulin-independent at 12 weeks. The bars represent the average % reduction in insulin dose for the respective groups.

The introduction of porcine islet transplantation has been delayed by concerns relating to the possible transmission of pig diseases to humans via the transplanted cells and the risk of introducing micro-organisms during cell processing and encapsulation. There is also a potential risk of transmitting porcine disease to close contacts of the human recipient and to the wider community. The risks of infection from the donor animal can be minimized by controlling the breed, source of and health status of the donor animals, with ongoing screening and quarantining. The risk of introducing micro-organisms during cell processing can be minimized by ensuring strict aseptic technique when isolating and encapsulating the

**3.1 Risk of infections ('xenoses') resulting from transfer of pig micro-organisms to** 

The risk of bacterial, fungal and parasitic infections can be minimized by the use of Designated Pathogen-Free pig herds and by monitoring and treating sows for such infections before pancreatic islets are extracted from their progeny (Garkavenko et al. 2008a). However, this approach will not eliminate porcine endogenous retrovirus sequences (PERVs), which are present in the germline of all pigs (including common New Zealand pig breeds) but cause no known infection in the species. The possible transmission of these retroviruses to humans, and their as yet unknown consequences in recipients, have given rise to some concerns over the safety of xenotransplantation, particularly in view of reports that PERVs from certain pig strains can infect human cells in vitro (Patience et al. 1997;

**3. Xenotransplantation: Minimizing risks to patient's safety** 

**0**

**25**

**50**

**%Drop Insulin**

islets.

**human recipients** 

**75**

**100**

A further refinement of the source herd has been obtained by the use of pigs derived from a colony abandoned on the Auckland Islands about 150 years ago. These animals are free of all measured viruses except the retrovirus (PERV), and are being bred for xenotransplant purposes in a purpose-built facility. Although PERV cannot be removed from the porcine genome, there is strong evidence that it is not functional in the Auckland Island Pig strain and is not expressed under any of the recommended testing conditions. This strain is now termed a "Null Pig Strain" suitable as a source of tissues for xenotransplantation (Wynyard et al, 2011).

#### **3.4 Neonatal pigs**

Nevertheless, the selection of donor animals that do not transmit infectious PERV continues to be important and has to be linked with the selection of donor animals bred in isolation and screened to exclude infection with other exogenous microbes. The use of donor neonatal piglets rather than adult pigs also has the advantage of limiting the exposure time of donor newborn animals to acquired infections as they age.

#### **3.5 No genetic modification**

The selection of piglets without genetic modification as a source of tissue offers another advantage. In attempting to prevent immune rejection of porcine tissue, genetically-modified

Cell Replacement Therapy: The Rationale for Encapsulated Porcine Islet Transplantation 399

face not only Immediate Blood Mediated Immune Rejection (IBMIR) but also immune attack from a variety of cells which may result in loss of function and cell death. Two approaches

The administration of immunosuppressive drugs before a cell transplant and for the rest of the life of the patient. These procedures to manage the immune competence of the patient are essential in greater or lesser forms for all transplants of naked cells except perhaps those that are sourced from the patient (autotransplantation) or from a perfectly matched donor. Even then, any patient-derived cells (e.g. endogenous adult stem cells) that may be induced to mature into functional islet cells may be attacked by auto-immune processes engendered during the auto-immune destruction of the patient's original pancreatic islets, since the

A suppressed immune system, with lower surveillance of foreign antigens, can provide an opportunity for infection. Immunosuppressive agents may also prevent the essential immune surveillance that detects and destroys most cellular chance mutations that lead to cancer cell growth. This has been a serious concern about stem cell transplants that may contain a small number of viable undifferentiated cells that can exhibit uncontrolled

Immunoprotection of the transplanted cells via the use of a semipermeable membrane that acts as a protective barrier (Figures 3a and 3b). The latter technique appears to be a viable approach, the principle being that the permeability of the outer capsule membrane allows smaller molecules such as glucose, dissolved oxygen and carbon dioxide and all nutrients to penetrate into the capsule and reach the islets. The membrane is constructed to allow insulin and most small proteins to be released out into the bloodstream, but it does not allow the passage of large immune cells or antibodies that would cause rejection of the islets. Indeed, the islets can survive well inside such systems. There are several approaches to providing

 Encapsulation of the islets within a bead of alginate gel, which is then coated with poly-L-ornithine, poly-L-lysine or some other material to provide perm-selectivity and

Tubular diffusion chambers which consist of long (up to 20mm) tubular membranes of

 Perfusion devices (also known as vascularised artificial pancreases) which consist of an outer housing 90 mm in diameter and 20 mm in width that is connected surgically to the patient's vascular system so that it is fed by an artery and drained by a vein. Islets in both perfusion devices and diffusion chambers are usually immobilised with alginate or agar to prevent settling and to provide uniform distribution of nutrients and dissolved

 Co-transplantation with 'nursery' cells such as testicular Sertoli cells which have been claimed to protect against immune-mediated rejection via the production of the immunomodulator TGF-beta1 (transforming growth factor-beta 1) (Suarez-Pinzon et al.

newly matured islet cells carry essentially the same antigens (Zhao et al, 2011).

are currently used to overcome this:

replication and form tumours (Bauer SR, 2010).

**5.2 Immunoisolation** 

this immunoprotection which are:

5-6mm inner diameter.

2000; Valdes-Gonzalez, 2005).

strength (Calafiore 2006; Weir & Bonner-Weir 1997).

oxygen and carbob-dioxide (Maki et al. 1995).

**5.1 Immunosuppressive drugs** 

pigs with the gene for the xeno-antigen alpha-gal eliminated have been developed. However, with the use of alpha-gal gene knock-out pig donor cells, PERV-exiting cells are not enveloped (coated) with the alpha-gal antigen and hence are not recognised as 'foreign' by the recipient's blood complement system (Fujita et al. 2003). It is known that normal human serum contains natural anti-alpha-gal antibodies that inactivate retrovirus (Rother et al. 1995).

#### **3.6 Intact immune system**

The maintenance of an intact immune system is an important safety factor. Immunosuppressive drugs are commonly used to prevent immune rejection of transplanted organs and cells. However, the use of alginate-encapsulated islets is intended to allow the survival of transplanted islets and their continued secretion of insulin in the recipient, without the need for life-long immunosuppressive drugs.

#### **3.7 Surveillance**

Long-term surveillance of recipients of porcine islet xenotransplants and testing of the transplant material for the presence of PERVs, using highly specific and highly sensitive assays developed for this purpose will always be an integral part of strategies proposed for future clinical trials. This safeguard, which is a necessary precaution recommended for clinical trials of xenotransplantation by authorities in various countries including the USA (US Department of Health & Human Services 2002, 2003), Australia (National Health & Medical Research Council 2002), UK (United Kingdom Xenotransplantation Interim Regulatory Authority 1998, 1999), and Canada (Therapeutic Products Programme, Health Protection Branch, Health Canada 1999), is designed to allow early detection of infectious disease transmission, and includes standard hospital infection control measures to limit the spread of such an infection should one be detected. These carefully structured and independently monitored precautionary measures are now mandatory for all patients enrolled in clinical trials of porcine islet (or other cells) xenotransplantation and are put in place before the trials begin.

#### **4. Minimizing risks of contaminating with infective agents during product preparation**

After the pancreas is removed from the donor animal it must be treated with a series of procedures to isolate the insulin-producing islets, substantially free of the proteolytic exocrine tissue which makes up the majority of the pancreas. It is then kept in cell culture medium and tested for microbiological contamination and for its ability to produce insulin in response to stimulation by exposure to a glucose challenge. This must be done with every batch of islets produced for clinical use. The manufacturing facility, records and staff training need to be regularly inspected by expert teams from government health agencies. Samples of islets from every batch and biopsies of pancreas, heart, lung, spleen, kidney and brain tissues, also blood samples, from every donor animal must also be stored frozen at -80 Centigrade as historical resources that can be retrospectively analyzed, in case of later complications in patients.

#### **5. Preventing rejection of islets by the recipient's immune system**

The vulnerability of transplanted islets to the recipient's immune system has been a major scientific challenge and a barrier to successful islet transplantation. The transplanted cells face not only Immediate Blood Mediated Immune Rejection (IBMIR) but also immune attack from a variety of cells which may result in loss of function and cell death. Two approaches are currently used to overcome this:

#### **5.1 Immunosuppressive drugs**

398 Type 1 Diabetes Complications

pigs with the gene for the xeno-antigen alpha-gal eliminated have been developed. However, with the use of alpha-gal gene knock-out pig donor cells, PERV-exiting cells are not enveloped (coated) with the alpha-gal antigen and hence are not recognised as 'foreign' by the recipient's blood complement system (Fujita et al. 2003). It is known that normal human serum contains

The maintenance of an intact immune system is an important safety factor. Immunosuppressive drugs are commonly used to prevent immune rejection of transplanted organs and cells. However, the use of alginate-encapsulated islets is intended to allow the survival of transplanted islets and their continued secretion of insulin in the recipient,

Long-term surveillance of recipients of porcine islet xenotransplants and testing of the transplant material for the presence of PERVs, using highly specific and highly sensitive assays developed for this purpose will always be an integral part of strategies proposed for future clinical trials. This safeguard, which is a necessary precaution recommended for clinical trials of xenotransplantation by authorities in various countries including the USA (US Department of Health & Human Services 2002, 2003), Australia (National Health & Medical Research Council 2002), UK (United Kingdom Xenotransplantation Interim Regulatory Authority 1998, 1999), and Canada (Therapeutic Products Programme, Health Protection Branch, Health Canada 1999), is designed to allow early detection of infectious disease transmission, and includes standard hospital infection control measures to limit the spread of such an infection should one be detected. These carefully structured and independently monitored precautionary measures are now mandatory for all patients enrolled in clinical trials of porcine islet (or other cells) xeno-

**4. Minimizing risks of contaminating with infective agents during product** 

**5. Preventing rejection of islets by the recipient's immune system** 

After the pancreas is removed from the donor animal it must be treated with a series of procedures to isolate the insulin-producing islets, substantially free of the proteolytic exocrine tissue which makes up the majority of the pancreas. It is then kept in cell culture medium and tested for microbiological contamination and for its ability to produce insulin in response to stimulation by exposure to a glucose challenge. This must be done with every batch of islets produced for clinical use. The manufacturing facility, records and staff training need to be regularly inspected by expert teams from government health agencies. Samples of islets from every batch and biopsies of pancreas, heart, lung, spleen, kidney and brain tissues, also blood samples, from every donor animal must also be stored frozen at -80 Centigrade as historical resources that can be retrospectively analyzed, in case of later

The vulnerability of transplanted islets to the recipient's immune system has been a major scientific challenge and a barrier to successful islet transplantation. The transplanted cells

natural anti-alpha-gal antibodies that inactivate retrovirus (Rother et al. 1995).

without the need for life-long immunosuppressive drugs.

transplantation and are put in place before the trials begin.

**3.6 Intact immune system** 

**3.7 Surveillance** 

**preparation** 

complications in patients.

The administration of immunosuppressive drugs before a cell transplant and for the rest of the life of the patient. These procedures to manage the immune competence of the patient are essential in greater or lesser forms for all transplants of naked cells except perhaps those that are sourced from the patient (autotransplantation) or from a perfectly matched donor. Even then, any patient-derived cells (e.g. endogenous adult stem cells) that may be induced to mature into functional islet cells may be attacked by auto-immune processes engendered during the auto-immune destruction of the patient's original pancreatic islets, since the newly matured islet cells carry essentially the same antigens (Zhao et al, 2011).

A suppressed immune system, with lower surveillance of foreign antigens, can provide an opportunity for infection. Immunosuppressive agents may also prevent the essential immune surveillance that detects and destroys most cellular chance mutations that lead to cancer cell growth. This has been a serious concern about stem cell transplants that may contain a small number of viable undifferentiated cells that can exhibit uncontrolled replication and form tumours (Bauer SR, 2010).

#### **5.2 Immunoisolation**

Immunoprotection of the transplanted cells via the use of a semipermeable membrane that acts as a protective barrier (Figures 3a and 3b). The latter technique appears to be a viable approach, the principle being that the permeability of the outer capsule membrane allows smaller molecules such as glucose, dissolved oxygen and carbon dioxide and all nutrients to penetrate into the capsule and reach the islets. The membrane is constructed to allow insulin and most small proteins to be released out into the bloodstream, but it does not allow the passage of large immune cells or antibodies that would cause rejection of the islets. Indeed, the islets can survive well inside such systems. There are several approaches to providing this immunoprotection which are:


Cell Replacement Therapy: The Rationale for Encapsulated Porcine Islet Transplantation 401

These capsules, as shown in **Figure 3**, have been the method of choice for many laboratories including our own (Opara & Kendall, 2002; Hernandez et al, 2010; Elliott, 2011). Their biocompatibility is critical to their in vivo efficacy and durability. Alginate is derived from seaweed and has to be highly purified to remove contaminating heavy metals, proteins and endotoxins. Different sources of alginate vary in their chemical composition with different ratios of their main constituents of mannuronic acid and guluronic acids and hence their physical gel properties. The consistency of the microcapsules with respect to their size, wall thickness, compressibility and cell occupancy has to be ensured from batch to batch. The central core containing the islets is liquefied alginate and the outer surface is a membrane formed by ionic interaction between positively charged poly-L-ornithine (or poly-L-lysine) and negatively charged alginate. Properly made, the membrane is remarkably robust

Since up to a million capsules may be required for treatment, the production must be reproducible, using durable equipment to Good Manufacturing Practice (GMP) standards. The main components are carefully engineered flow-through needles which generate reproducible sized droplets impelled by air 'knife' flows or electrostatic mechanisms

Embodied in the wider concepts of informed consent are considerations that young people and the old or infirm are not exploited. Peoples with poor understanding of the language and scientific concepts involved are not to be misled. Religious and cultural feelings are not to be disregarded in potential recipients who find it difficult to express their reluctance to

The ethical issues of xenotransplantation include concerns over the cultural, ethical and spiritual dimensions of xenotransplantation; the ethical acceptability of using animals to provide tissue for human transplantation; how the welfare of donor animals can be adequately protected and their suffering reduced; and how the welfare and interests of patients in early clinical xenotransplantation trials can be protected. These issues have been addressed by the Nuffield Council on Bioethics in the United Kingdom (1996) and, in New Zealand, the Bioethics Council, Toi te Taiao (2005). The latter body concluded that prohibition of xenotransplantation could not be justified, given the compelling human need argument, and that it should be allowed to develop in New Zealand, with that development being demonstrably shaped by the resolution and management of safety issues by a competent authority; the relationship between the majority European culture and indigenous Maori people and the cultural, ethical and spiritual factors that matter to most

In recent years, a number of recommendations to protect the ethical integrity of future human research have been made by various regulatory authorities. Key issues in the conduct of clinical trials of xenotransplantation include the requirement to provide patients with an explanation of the likely success, its attendant risks, and the subsequent quality-oflife that can be expected when obtaining their informed consent, and informing patients that their consent to the procedure includes consent to ongoing post-transplantation

participate in the presence of those whom they perceive as medical authorities.

**6. Alginate-poly-l-ornithine capsules** 

(Skinner et al, 2009).

(Hernandez et al, 2010).

New Zealanders.

microbiological monitoring.

**7. Ethical considerations** 

Of these approaches, the strategy selected by Living Cell Technologies for intensive investigation is encapsulation of the islets in 'minimal volume' alginate microcapsules developed at the Department of Internal Medicine and Endocrine & Metabolic Services, University of Perugia in Italy (Calafiore 1997; Calafiore et al. 1999). Alginate-encapsulated porcine islets have been extensively investigated in experimental animals both with and without diabetes, and in a small number of human diabetic subjects.

Fig. 3a. **The concept of encapsulation:** enclosure of the islet in an alginate/poly-L-ornithine or poly-L-lysine membrane that is permeable to glucose, nutrients and insulin, but not to lymphocytes and antibodies, provides protection against immune destruction of the cells.

Fig. 3b. **Islets isolated from neonatal porcine pancreas in alginate-polyornithine capsules.** Green fluorescent stain (acridine orange) demonstrating viable cells and red tetra-methylrhodamine-ethyl ester (TMRE) stain for mitochodria of viable cells.

#### **6. Alginate-poly-l-ornithine capsules**

400 Type 1 Diabetes Complications

Of these approaches, the strategy selected by Living Cell Technologies for intensive investigation is encapsulation of the islets in 'minimal volume' alginate microcapsules developed at the Department of Internal Medicine and Endocrine & Metabolic Services, University of Perugia in Italy (Calafiore 1997; Calafiore et al. 1999). Alginate-encapsulated porcine islets have been extensively investigated in experimental animals both with and

> Poly-L-ornithine or Poly-L-lysine

Alginate

**Pancreatic islet** Insulin Glucose

Fig. 3a. **The concept of encapsulation:** enclosure of the islet in an alginate/poly-L-ornithine or poly-L-lysine membrane that is permeable to glucose, nutrients and insulin, but not to lymphocytes and antibodies, provides protection against immune destruction of the cells.

Insulin, Glucose, Gases and Nutrients

 Fig. 3b. **Islets isolated from neonatal porcine pancreas in alginate-polyornithine capsules.** Green fluorescent stain (acridine orange) demonstrating viable cells and red tetra-methyl-

rhodamine-ethyl ester (TMRE) stain for mitochodria of viable cells.

without diabetes, and in a small number of human diabetic subjects.

Lymphocytes and Antibodies These capsules, as shown in **Figure 3**, have been the method of choice for many laboratories including our own (Opara & Kendall, 2002; Hernandez et al, 2010; Elliott, 2011). Their biocompatibility is critical to their in vivo efficacy and durability. Alginate is derived from seaweed and has to be highly purified to remove contaminating heavy metals, proteins and endotoxins. Different sources of alginate vary in their chemical composition with different ratios of their main constituents of mannuronic acid and guluronic acids and hence their physical gel properties. The consistency of the microcapsules with respect to their size, wall thickness, compressibility and cell occupancy has to be ensured from batch to batch. The central core containing the islets is liquefied alginate and the outer surface is a membrane formed by ionic interaction between positively charged poly-L-ornithine (or poly-L-lysine) and negatively charged alginate. Properly made, the membrane is remarkably robust (Skinner et al, 2009).

Since up to a million capsules may be required for treatment, the production must be reproducible, using durable equipment to Good Manufacturing Practice (GMP) standards. The main components are carefully engineered flow-through needles which generate reproducible sized droplets impelled by air 'knife' flows or electrostatic mechanisms (Hernandez et al, 2010).

#### **7. Ethical considerations**

Embodied in the wider concepts of informed consent are considerations that young people and the old or infirm are not exploited. Peoples with poor understanding of the language and scientific concepts involved are not to be misled. Religious and cultural feelings are not to be disregarded in potential recipients who find it difficult to express their reluctance to participate in the presence of those whom they perceive as medical authorities.

The ethical issues of xenotransplantation include concerns over the cultural, ethical and spiritual dimensions of xenotransplantation; the ethical acceptability of using animals to provide tissue for human transplantation; how the welfare of donor animals can be adequately protected and their suffering reduced; and how the welfare and interests of patients in early clinical xenotransplantation trials can be protected. These issues have been addressed by the Nuffield Council on Bioethics in the United Kingdom (1996) and, in New Zealand, the Bioethics Council, Toi te Taiao (2005). The latter body concluded that prohibition of xenotransplantation could not be justified, given the compelling human need argument, and that it should be allowed to develop in New Zealand, with that development being demonstrably shaped by the resolution and management of safety issues by a competent authority; the relationship between the majority European culture and indigenous Maori people and the cultural, ethical and spiritual factors that matter to most New Zealanders.

In recent years, a number of recommendations to protect the ethical integrity of future human research have been made by various regulatory authorities. Key issues in the conduct of clinical trials of xenotransplantation include the requirement to provide patients with an explanation of the likely success, its attendant risks, and the subsequent quality-oflife that can be expected when obtaining their informed consent, and informing patients that their consent to the procedure includes consent to ongoing post-transplantation microbiological monitoring.

Cell Replacement Therapy: The Rationale for Encapsulated Porcine Islet Transplantation 403

data shows a reduction in daily insulin dose and reduction in HbA1c compared with preimplant values following the first implant (Tables 1 and 2) in the majority of patients. Two patients became insulin independent for a period, the maximum being 32 weeks. At the repeat implant, 6 months after the first implant, intact microcapsules were retrieved and subsequently found to contain viable cells. Porcine insulin was also detected in the

**Pre 1st Tx** 

**1 5,000 113 32 34 2 5,000 22 100 32 3 5,000 60 -7 5 4 5,000 30 13 13 5 5,000 68 15 16 6 10,000 41 ? 0 7 10,000 37 73 100 8 10,000 83 0 0** 

**Mean 57 32 25** 

Table 1. **Reduction in daily insulin dose after first implant (Tx) in 8-patient pilot study.** Patient 2 was insulin-independent at 3 months but subsequently needed insulin support.

Table 2. Reduction in Haemoglobin A1c (HbA1c) after first implant (Tx) in 8-patient pilot

**Insulin Dose % Dose Reduction** 

**Post-1st Tx 3-month 6-month** 

circulation following glucagon stimulation.

**(IEQ/kg)** 

Patient 7 became insulin-independent at 6 months.

study.

**Patient ID Dose 1st Tx** 

Other international spiritual organisations have given their opinions about xenotransplantation. The Vatican in Rome, Italy have considered xenotransplantation in some depth and suggest it is worthy of serious consideration (Vatican, 2011). Jewish organisations have given qualified support to xenotransplantation as a life enhancing procedure while Muslim opinion is unclear but has generally been negative because of the status of pigs in the religious context.

#### **8. Clinical experiences with xenotransplantation in treating type 1 diabetes**

Many of the considerations in the preceding part of this chapter have been opinion and experiment seeking to determine the safety and efficacy of xenotransplantation for Type 1 diabetes in animal models. These animal models are perhaps more or less imperfect as true reflections of the human condition of Type 1 diabetes. It is therefore of critical relevance to review all data and experiences relating to the small number of human exposures to xenotransplanted porcine islets and other cells.

Six patients were treated in 1995-6 with either encapsulated or unencapsulated neonatal porcine islets. One of these, a 41-yr-old Caucasian male with type 1 diabetes for 18 years was given an intraperitoneal transplant of alginate-encapsulated porcine islets at the dose of 15,000 islet equivalents (IEQs)/kg bodyweight (total dose 1,305,000 IEQs) via laparoscopy. By 12 weeks following the transplant, his insulin dose was significantly reduced by 30% (p = 0.0001). The insulin dose returned to the pre-transplant level at week 49. Improvement in glycaemic control continued as reflected by total glycated haemoglobin of 7.8% at 14 months from a pre-transplant level of 9.3%. Urinary porcine C-peptide, derived from the porcine pro-insulin precursor, peaked at 4 months (9.5 ng/ml) and remained detectable for 11 months (0.6 ng/ml). The patient was followed as part of a long-term microbiologic monitoring program which subsequently showed no evidence of porcine viral or retroviral infection.

The patient opted for elective laparoscopy 9.5 yr after transplantation. Abundant nodules were seen throughout the peritoneum. Biopsies of the nodules showed they contained capsules still protecting living cell clusters. Immunohistology noted sparse insulin and moderate glucagon staining cells. The retrieved capsules produced a small amount of insulin when placed in high glucose concentrations in vitro. An oral glucose tolerance test induced a small rise in serum of immuno-reactive insulin, identified as porcine by reversed phase high pressure liquid chromatography (Elliott et al, 2007).

With this demonstration it was clear that this form of xenotransplantation treatment has the potential for sustained benefit in human type 1 diabetics.

Since then two further human studies have started using porcine islets in microcapsules and without immunosuppressive drugs. In 2007, a pilot study with 8 patients was approved by the Scientific and Ethics Committees of the Sklifosovsky Institute, Moscow where islets were obtained from biocertified designated pathogen free pigs and encapsulated under GMP conditions in New Zealand. Adult patients were aged 23-63 with type 1 diabetes as defined by the American Diabetes Association criteria. They were insulin dependent for 5 -15 years. Before the implants, patients had to have stimulated plasma c-peptide levels < 0.2 ng/ml to confirm insulin deficiency. Their diabetes had to be inadequately controlled with HbA1c of > 7% pre-implant. Patients were administered 5,000 or 10,000 islet equivalents per kilogram body weight (IEQ/kg). There were no significant adverse events and no evidence of zoonosis. Patients were also given repeat implants with no untoward effects. Preliminary

Other international spiritual organisations have given their opinions about xenotransplantation. The Vatican in Rome, Italy have considered xenotransplantation in some depth and suggest it is worthy of serious consideration (Vatican, 2011). Jewish organisations have given qualified support to xenotransplantation as a life enhancing procedure while Muslim opinion is unclear but has generally been negative because of the

**8. Clinical experiences with xenotransplantation in treating type 1 diabetes**  Many of the considerations in the preceding part of this chapter have been opinion and experiment seeking to determine the safety and efficacy of xenotransplantation for Type 1 diabetes in animal models. These animal models are perhaps more or less imperfect as true reflections of the human condition of Type 1 diabetes. It is therefore of critical relevance to review all data and experiences relating to the small number of human exposures to

Six patients were treated in 1995-6 with either encapsulated or unencapsulated neonatal porcine islets. One of these, a 41-yr-old Caucasian male with type 1 diabetes for 18 years was given an intraperitoneal transplant of alginate-encapsulated porcine islets at the dose of 15,000 islet equivalents (IEQs)/kg bodyweight (total dose 1,305,000 IEQs) via laparoscopy. By 12 weeks following the transplant, his insulin dose was significantly reduced by 30% (p = 0.0001). The insulin dose returned to the pre-transplant level at week 49. Improvement in glycaemic control continued as reflected by total glycated haemoglobin of 7.8% at 14 months from a pre-transplant level of 9.3%. Urinary porcine C-peptide, derived from the porcine pro-insulin precursor, peaked at 4 months (9.5 ng/ml) and remained detectable for 11 months (0.6 ng/ml). The patient was followed as part of a long-term microbiologic monitoring program which subsequently showed no evidence of porcine viral or retroviral

The patient opted for elective laparoscopy 9.5 yr after transplantation. Abundant nodules were seen throughout the peritoneum. Biopsies of the nodules showed they contained capsules still protecting living cell clusters. Immunohistology noted sparse insulin and moderate glucagon staining cells. The retrieved capsules produced a small amount of insulin when placed in high glucose concentrations in vitro. An oral glucose tolerance test induced a small rise in serum of immuno-reactive insulin, identified as porcine by reversed

With this demonstration it was clear that this form of xenotransplantation treatment has the

Since then two further human studies have started using porcine islets in microcapsules and without immunosuppressive drugs. In 2007, a pilot study with 8 patients was approved by the Scientific and Ethics Committees of the Sklifosovsky Institute, Moscow where islets were obtained from biocertified designated pathogen free pigs and encapsulated under GMP conditions in New Zealand. Adult patients were aged 23-63 with type 1 diabetes as defined by the American Diabetes Association criteria. They were insulin dependent for 5 -15 years. Before the implants, patients had to have stimulated plasma c-peptide levels < 0.2 ng/ml to confirm insulin deficiency. Their diabetes had to be inadequately controlled with HbA1c of > 7% pre-implant. Patients were administered 5,000 or 10,000 islet equivalents per kilogram body weight (IEQ/kg). There were no significant adverse events and no evidence of zoonosis. Patients were also given repeat implants with no untoward effects. Preliminary

phase high pressure liquid chromatography (Elliott et al, 2007).

potential for sustained benefit in human type 1 diabetics.

status of pigs in the religious context.

xenotransplanted porcine islets and other cells.

infection.

data shows a reduction in daily insulin dose and reduction in HbA1c compared with preimplant values following the first implant (Tables 1 and 2) in the majority of patients. Two patients became insulin independent for a period, the maximum being 32 weeks. At the repeat implant, 6 months after the first implant, intact microcapsules were retrieved and subsequently found to contain viable cells. Porcine insulin was also detected in the circulation following glucagon stimulation.


Table 1. **Reduction in daily insulin dose after first implant (Tx) in 8-patient pilot study.** Patient 2 was insulin-independent at 3 months but subsequently needed insulin support. Patient 7 became insulin-independent at 6 months.


Table 2. Reduction in Haemoglobin A1c (HbA1c) after first implant (Tx) in 8-patient pilot study.

Cell Replacement Therapy: The Rationale for Encapsulated Porcine Islet Transplantation 405

that this form of treatment for Type 1 diabetes, without immunosuppression, is worthwhile. There is the concern that the treatment may be too expensive to be a practical treatment for the large numbers of patients who are expected to benefit from porcine islet implants. However, a health economic analysis of quality adjusted life years suggests that this approach may be cost effective taking into account the current cost of treating the disease and complications of cardiovascular disease, blindness, limb amputation, end-stage kidney

The work embodied in this chapter could not have been possible without the integrated energy and persistence of all staff at Living Cell Technologies. Leaders in support are Isobel Cooper, Sandy Ferguson, Peter Hosking and Colleen Pilcher. Previous contributors were

http://www.diabetes.org/diabetes-basics/diabetes-statistics/ Accessed June 2011.

USA. http://www.diabetes.org/diabetes-basics/diabetes-statistics/ Accessed June

http://www.fda.gov/BiologicsBloodVaccines/ScienceResearch/BiologicsResearch

P. (2006) Standard technical procedures for microencapsulation of human islets for graft into nonimmunosuppressed patients with type 1 diabetes mellitus. Transplant

minimal volume microcapsules in diabetic high mammalians. Ann N Y Acad Sci

vitro transmission of full-length porcine endogenous retroviruses from specific

screening for transmission in several clinical trials and in experimental models

human cells with porcine endogenous retrovirus (PERV) after exposure to porcine

American Diabetes Association (2011) Diabetes statistics. Available from:

American Diabetes Association (2011) Food and fitness. Available from:

http://www.diabetes.org/food-and-fitness/ Accessed June 2011.

American Diabetes Association (2011) Economic consequences of diabetes mellitus in the

Bauer SR (2010) Assuring safety and efficacy of Stem-Cell Based products.

Beckwith J, Nyman JA, Flanagan B, Schrover R, Schuurman H-J.(2010) A health-economic analysis of porcine islet xenotransplantation. Xenotransplantation 17: 233-242. Calafiore R. Perspectives in pancreatic and islet cell transplantation for the therapy of

Calafiore R, Basta G, Luca G, Lemmi A, Racanicchi L, Mancuso F, Montanucci MP, Brunetti

Calafiore R, Basta G, Luca G, et al.(1999) Transplantation of pancreatic islets contained in

Clemenceau B, Jegou D, Martignat L, Saï P. (2001) Long-term follow-up failed to detect in

Denner J. (2003) Porcine endogenous retroviruses (PERVs) and xenotransplantation:

Dinsmore JH, Manhart C, Raineri R, Jacoby DB, Moore A.(2000) No evidence of infection of

pathogen-free pig islets to human cells. Diabetologia 44:2044-55.

using non-human primates. Ann Transplant 8(3):39-48.

fetal neuronal cells. Transplantation 70:1382-9.

disease and neuropathy (Beckwith et al, 2010).

**10. Acknowledgements** 

**11. References** 

2011.

Trevor Speight and Michelle Tatnell.

Areas/ucm127182.htm

Proc. 38(4):1156-7

875:219-32.

IDDM. Diabetes Care 1997;20:889-96.

In 2009, a Phase I/IIa study approved by the New Zealand government following international peer review and Ethics Committee Approval was commenced. It will include 14 adult patients. Unlike the preceding clinical studies, these patients were required to have unstable type 1 diabetes accompanied with hypoglycemic episodes. The patients are to be administered a single intra-abdominal implant of 5,000, 10,000, 15,000 or 20,000 IEQ/kg. To date no significant adverse events have been attributable to the treatment. At this early stage of the open label study, episodes of hypoglycemic unawareness (Figure 4) and hypoglycemic convulsions have been eliminated in the first patient. This was associated with significant reduction in the severity of hypoglycemic scores. A full one year follow-up of all patients is expected to be completed at the end of 2011. A third trial will be conducted in Argentina in 2011/2. The clinical studies are thus still at the stage of dose finding to determine the optimum dose and dosing regimen.

Weeks (time of implant at week 0)

Fig. 4. Example of Elimination of Episodes of Unaware Hypoglycemia (Patient #1, Phase I/IIa Auckland, New Zealand)

#### **9. Conclusion**

Xenotransplantation is not yet standard treatment. There is current research into the feasibility of using donor animal organs such a kidney and liver. However, unlike cells, organs are difficult to screen against potential infectious agents. If clinical trials with cell transplants for diabetes are successful, porcine islet transplants will lead the way for the use of other cells for the treatment of nervous system disorders and enzyme deficiencies (Skinner et al, 2009).

Porcine islet xenotransplantation has the potential to be beneficial for those with absolute insulin deficiency. There is now a consensus that the procedure is relatively safe from xenotic infections. The results of several non-human primate and rodent studies indicate significant efficacy may be achieved. The early results of human clinical trials also suggest that this form of treatment for Type 1 diabetes, without immunosuppression, is worthwhile. There is the concern that the treatment may be too expensive to be a practical treatment for the large numbers of patients who are expected to benefit from porcine islet implants. However, a health economic analysis of quality adjusted life years suggests that this approach may be cost effective taking into account the current cost of treating the disease and complications of cardiovascular disease, blindness, limb amputation, end-stage kidney disease and neuropathy (Beckwith et al, 2010).

#### **10. Acknowledgements**

The work embodied in this chapter could not have been possible without the integrated energy and persistence of all staff at Living Cell Technologies. Leaders in support are Isobel Cooper, Sandy Ferguson, Peter Hosking and Colleen Pilcher. Previous contributors were Trevor Speight and Michelle Tatnell.

#### **11. References**

404 Type 1 Diabetes Complications

In 2009, a Phase I/IIa study approved by the New Zealand government following international peer review and Ethics Committee Approval was commenced. It will include 14 adult patients. Unlike the preceding clinical studies, these patients were required to have unstable type 1 diabetes accompanied with hypoglycemic episodes. The patients are to be administered a single intra-abdominal implant of 5,000, 10,000, 15,000 or 20,000 IEQ/kg. To date no significant adverse events have been attributable to the treatment. At this early stage of the open label study, episodes of hypoglycemic unawareness (Figure 4) and hypoglycemic convulsions have been eliminated in the first patient. This was associated with significant reduction in the severity of hypoglycemic scores. A full one year follow-up of all patients is expected to be completed at the end of 2011. A third trial will be conducted in Argentina in 2011/2. The clinical studies are thus still at the stage of dose finding to

Weeks (time of implant at week 0) Fig. 4. Example of Elimination of Episodes of Unaware Hypoglycemia (Patient #1, Phase

Xenotransplantation is not yet standard treatment. There is current research into the feasibility of using donor animal organs such a kidney and liver. However, unlike cells, organs are difficult to screen against potential infectious agents. If clinical trials with cell transplants for diabetes are successful, porcine islet transplants will lead the way for the use of other cells for the treatment of nervous system disorders and enzyme deficiencies

Porcine islet xenotransplantation has the potential to be beneficial for those with absolute insulin deficiency. There is now a consensus that the procedure is relatively safe from xenotic infections. The results of several non-human primate and rodent studies indicate significant efficacy may be achieved. The early results of human clinical trials also suggest

determine the optimum dose and dosing regimen.

I/IIa Auckland, New Zealand)

**9. Conclusion** 

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**Part 5** 

**Diabetes and Oral Health** 


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## **Part 5**

**Diabetes and Oral Health** 

408 Type 1 Diabetes Complications

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**19** 

*Hungary* 

S. Mikó and M. G. Albrecht

**Dental Conditions and Periodontal Disease in** 

The importance of type 1 (diabetes mellitus) DM lies in the function it plays in dentition and oral health for children and adolescents. The consequences of DM concerning oral health are in connection with the systemic changes1 caused by the disease, though the results are in certain cases conflicting. Oral manifestations related to DM may have a strong inclination to periodontal disease23, as well as an increased incidence of dental caries, mucosal lesions and dry mouth (xerostomia). However, as mentioned before, the results are not entirely in accord456. Children and adolescents with type 1 DM are more susceptible to infections in the dental connective tissues than those without type 1 DM. The dentist's liablility is to evaluate the dangers and maintain adequate oral hygiene to prevent the occurrence of disfunctional

Periodontitis is a chronic multifactorial plaque induced Gram-negative anaerobic infection of the periodontium that results in the destruction of periodontal tissues and loss of alveolar bone.7 Periodontitis is to be treated by the mechanical removal of supra- and subgingival bacterial plaque with scalers, curettes or ultrasonic devices (scaling and root planing [SRP]), and by instructing the parient about oral hygiene. The development of new dental plaque deposits and re-infection of the subgingival tissues can only be prevented in case of a nearideal oral hygiene. For the improvement of clinical periodontal status the regular use of systemic or local antibiotics as an adjunctive therapy to SRP is still concerned problematic.8910 Deep residual periodontal lesions can usually be reduced or eliminated by

There is an extensive debate on the influence of DM on the risk of periodontal disease. According to most of the experts, patients suffering from diabetes mellitus are highly susceptible to develop periodontal disease.111213141516 In 1993, periodontal disease was termed as the sixth complication of diabetes17, while in the report of the Expert Comittee on the Diagnosis and Classification of Diabetes Mellitus issued in 1997, periodontal disease was mentioned as a disease of very high incidence in case of patients with diabetes.18 Many papers on the subject have outlined that the inclination, extent, severity and development of

**1. Introduction** 

oral effects of type 1 DM.

**2. Periodontal disease** 

**2.1 Diabetes and periodontal disease** 

surgery.

**Adolescents with Type 1 Diabetes Mellitus** 

*Semmelweis University, Department of Conservative Dentistry,* 

### **Dental Conditions and Periodontal Disease in Adolescents with Type 1 Diabetes Mellitus**

S. Mikó and M. G. Albrecht

*Semmelweis University, Department of Conservative Dentistry, Hungary* 

#### **1. Introduction**

The importance of type 1 (diabetes mellitus) DM lies in the function it plays in dentition and oral health for children and adolescents. The consequences of DM concerning oral health are in connection with the systemic changes1 caused by the disease, though the results are in certain cases conflicting. Oral manifestations related to DM may have a strong inclination to periodontal disease23, as well as an increased incidence of dental caries, mucosal lesions and dry mouth (xerostomia). However, as mentioned before, the results are not entirely in accord456. Children and adolescents with type 1 DM are more susceptible to infections in the dental connective tissues than those without type 1 DM. The dentist's liablility is to evaluate the dangers and maintain adequate oral hygiene to prevent the occurrence of disfunctional oral effects of type 1 DM.

#### **2. Periodontal disease**

Periodontitis is a chronic multifactorial plaque induced Gram-negative anaerobic infection of the periodontium that results in the destruction of periodontal tissues and loss of alveolar bone.7 Periodontitis is to be treated by the mechanical removal of supra- and subgingival bacterial plaque with scalers, curettes or ultrasonic devices (scaling and root planing [SRP]), and by instructing the parient about oral hygiene. The development of new dental plaque deposits and re-infection of the subgingival tissues can only be prevented in case of a nearideal oral hygiene. For the improvement of clinical periodontal status the regular use of systemic or local antibiotics as an adjunctive therapy to SRP is still concerned problematic.8910 Deep residual periodontal lesions can usually be reduced or eliminated by surgery.

#### **2.1 Diabetes and periodontal disease**

There is an extensive debate on the influence of DM on the risk of periodontal disease. According to most of the experts, patients suffering from diabetes mellitus are highly susceptible to develop periodontal disease.111213141516 In 1993, periodontal disease was termed as the sixth complication of diabetes17, while in the report of the Expert Comittee on the Diagnosis and Classification of Diabetes Mellitus issued in 1997, periodontal disease was mentioned as a disease of very high incidence in case of patients with diabetes.18 Many papers on the subject have outlined that the inclination, extent, severity and development of

Dental Conditions and Periodontal Disease in Adolescents with Type 1 Diabetes Mellitus 413

Recently many studies have proposed that periodontal disease is a substantial aggravating factor in restoring the health of patients with diabetes.56 It is mainly due to the fact that it maintains a chronic systemic inflammatory process. However, the causality between periodontitis and altered glycaemic control can only be justified epidemiologically with prospective interventional studies.57 The control of periodontal disease could be an essential part of the overall treatment of diabetic patients: the periodontal therapy may improve

Concerning the adolescents, the oral hygiene seems to be better in case of the diabetic patients than in case of the other group of young patients who have shown highly variable work shifts leading to poor oral hygiene. The difference is reflected by a lesser bacterial plaque among the diabetics. This is in conflict with the results of other authors who observed similar oral hygiene status and plaque indexes in the two groups, or even comparatively increased plaque accumulation among the diabetics.58 The reason for this may be the following: although the diabetics were controlled, they showed a high gingival response to the irrigation caused by bacterial plaque retention.59 Regularly diabetics have a greater incidence regarding periodontal disease than healthy individuals6061, though other studies have not detected such relation between periodontal disease and diabetes.626364 They didn't confirm any association between diabetes and periodontal disease in adolescents. They believe that there is no difference between on the one part type 1 DM adolescents and children, and on the other healthy individuals regarding clinical periodontal status. However Fructosamine value, used to diagnose and monitor diabetes, was observed to be in correlation with the gingival index score in case of type 1 DM adolescents and children, but not in case of healthy control subjects.65 Bay et al.34 found no difference in the degree resolution of gingivitis following scaling and root planning between young diabetics and healthy control group. Barnett et al.35 studied periodontitis prevalence in young diabetics

Despite the comparatively better control of bacterial plaque among the diabetics, a change in their periodontal response was detected. Therefore it seems that local immune response may possibly change among these patients, and the lesions caused by microbial agents in case of periodontal disease, which can be associated with a lesser tissue repair capacity, might be the reason for the increased deterioration of periodontal structures shown in the diabetic

It is possible, that the diversities observed by the different authors result from local health care habits. In an area with a tradition of good oral hygene, the oral hygiene habits of diabetes are not better than those of healthy people. Therefore in case of similar oral hygiene habits, diabetical patients show lower periodontal values than healthy persons because of the above mentioned immune response. In areas where the dental treatment habits of the population are poorer, the greater attention of well-controlled DM patients to

Since there are conflicting reports in the literature, the aim of this study was analyse the periodontal disease in adolescents with type 1 diabetes mellitus.66 Characterization of risk factors, local such as oral hygiene-, general (duration of DM, age, degree of metabolic control of DM) and contributory factors-, such as toothbrushing, have been identified that determining the risk factors place people enhanced risk in development and progression of

glycaemic control, and could lead to a significant lowering of HbA1c values.40

but none of the individuals showed radiographic sign of periodontitis.

oral hygiene results in higher values than those of healthy individuals.

**2.2 Prevalence of gingivitis, periodontitis in adolescents with type 1 DM** 

group.4

periodontal diseases.

periodontal disease are significantly increased in case of patients with diabetes.1920 Impaired immune response, different bacterial microflora and collagen metabolism are involved in the pathogenesis of diabetic periodontal disease21. This is especially true if the diabetes is poorly controlled22 and there is a lasting periodontal disease in case of type 1 DM individuals23.

Gingival bleeding is a sign of inflammation. Vascular changes in diabetes mellitus may result in increased gingival bleeding. If however the diabetes is adequately treated, there are fewer vascular changes.24 Gingival bleeding is in positive correlation with the accumulation of plaque and calculus.25 Diabetics showed more plaque and higher calculus index scores than the control groups.26 Calcium concentration was high in both the parotid and submandibular saliva of patients with type 1 DM.27 This result may explain the regularly experienced increase in calculus formation in the case of these patients.2829 Individuals with a decreased glucose tolerance showed a higher rate of pocket formation, presence of calculus, increased tooth mobility and tooth loss.30 Variable models have been developed to give reason to this correlation such as periodontal chronic inflammation which results in increased circulating cytokines and inflammatory mediators, autoimmune response to the chronic periodontal infection that gives way to endothelial dysfunction, or the presence of certain factors that leads to increased inclination both to periodontal disease and diabetes vascular diseases at the same time.31

Statistically significant relation has been detected between the HbA1C levels of the patients and the biomass, haemolysin activity and proteinase activity. The biofilm formation is likely to have an effect on the pathogenicity of oral candidosis.32

There is, however, a minority of experts who are sceptical about the high risk of type 1 DM.33343536

Today Glickman's theory is the most widely accepted one. According to Glickman, the father of modern periodontology, diabetes are not the direct cause, but a predisposing condition for periodontal disease.37 There is a bidirectional relationship between diabetes and periodontal diseases38. Diabetic adolescents have more prevalence and more severe gingival inflammation than healthy control persons of the same age39, despite similar plaque scores51. Metabolic imbalances in the tissues may reduce the resistance of diabetics to infection,40 and thus affect the initiation, developement and progression of periodontal disease.41 Adolescents with type 1 DM develop an earlier and higher gingival inflammatory response to a similar bacterial challenge than non diabetics.3442 In accordance with this fact, extended studies have shown that severe periodontal disease in diabetic patients is primarily related to poor metabolic control and other diabetes complications occurring during the follow-up process.4344 Still, long-duration diabetics resulted in more severe periodontitis than short-duration diabetics.45 It has been proposed that fine periodontal treatment of diabetic patients may lead to the lowering of diabetic complications.464748 Chronic Gram-negative infections and chronic endotoxaemia, which arise in case of periodontal disease, may also increase insulin resistance and reduce metabolic control in case of diabetic patients. This entails the hypothesis that elimination or control of periodontal infection may improve the metabolic control of diabetes.4950 It has been proposed that a microbiological imbalance in the gut may increase the gram-negative bacterial load, which would also increase the systematic inflammatory burden through the lipopolysaccharides leakage into the circulation. Increased inflammation leads to insulin resistance.515253 According to a study, insulin requirements were lowered following periodontal treatment and reduction of inflammation in diabetic patients.54 It has been demonstrated that the interaction of periodontal bacterial byproducts with mononuclear phagocytic cells and fibroblasts induces the chronic release of cytokines (IL-1β, IL-6, TNF-α), PGE2 and CRP.55

periodontal disease are significantly increased in case of patients with diabetes.1920 Impaired immune response, different bacterial microflora and collagen metabolism are involved in the pathogenesis of diabetic periodontal disease21. This is especially true if the diabetes is poorly controlled22 and there is a lasting periodontal disease in case of type 1 DM individuals23. Gingival bleeding is a sign of inflammation. Vascular changes in diabetes mellitus may result in increased gingival bleeding. If however the diabetes is adequately treated, there are fewer vascular changes.24 Gingival bleeding is in positive correlation with the accumulation of plaque and calculus.25 Diabetics showed more plaque and higher calculus index scores than the control groups.26 Calcium concentration was high in both the parotid and submandibular saliva of patients with type 1 DM.27 This result may explain the regularly experienced increase in calculus formation in the case of these patients.2829 Individuals with a decreased glucose tolerance showed a higher rate of pocket formation, presence of calculus, increased tooth mobility and tooth loss.30 Variable models have been developed to give reason to this correlation such as periodontal chronic inflammation which results in increased circulating cytokines and inflammatory mediators, autoimmune response to the chronic periodontal infection that gives way to endothelial dysfunction, or the presence of certain factors that leads to increased inclination both to periodontal disease and diabetes

Statistically significant relation has been detected between the HbA1C levels of the patients and the biomass, haemolysin activity and proteinase activity. The biofilm formation is likely

There is, however, a minority of experts who are sceptical about the high risk of type 1

Today Glickman's theory is the most widely accepted one. According to Glickman, the father of modern periodontology, diabetes are not the direct cause, but a predisposing condition for periodontal disease.37 There is a bidirectional relationship between diabetes and periodontal diseases38. Diabetic adolescents have more prevalence and more severe gingival inflammation than healthy control persons of the same age39, despite similar plaque scores51. Metabolic imbalances in the tissues may reduce the resistance of diabetics to infection,40 and thus affect the initiation, developement and progression of periodontal disease.41 Adolescents with type 1 DM develop an earlier and higher gingival inflammatory response to a similar bacterial challenge than non diabetics.3442 In accordance with this fact, extended studies have shown that severe periodontal disease in diabetic patients is primarily related to poor metabolic control and other diabetes complications occurring during the follow-up process.4344 Still, long-duration diabetics resulted in more severe periodontitis than short-duration diabetics.45 It has been proposed that fine periodontal treatment of diabetic patients may lead to the lowering of diabetic complications.464748 Chronic Gram-negative infections and chronic endotoxaemia, which arise in case of periodontal disease, may also increase insulin resistance and reduce metabolic control in case of diabetic patients. This entails the hypothesis that elimination or control of periodontal infection may improve the metabolic control of diabetes.4950 It has been proposed that a microbiological imbalance in the gut may increase the gram-negative bacterial load, which would also increase the systematic inflammatory burden through the lipopolysaccharides leakage into the circulation. Increased inflammation leads to insulin resistance.515253 According to a study, insulin requirements were lowered following periodontal treatment and reduction of inflammation in diabetic patients.54 It has been demonstrated that the interaction of periodontal bacterial byproducts with mononuclear phagocytic cells and fibroblasts induces the chronic

vascular diseases at the same time.31

DM.33343536

to have an effect on the pathogenicity of oral candidosis.32

release of cytokines (IL-1β, IL-6, TNF-α), PGE2 and CRP.55

Recently many studies have proposed that periodontal disease is a substantial aggravating factor in restoring the health of patients with diabetes.56 It is mainly due to the fact that it maintains a chronic systemic inflammatory process. However, the causality between periodontitis and altered glycaemic control can only be justified epidemiologically with prospective interventional studies.57 The control of periodontal disease could be an essential part of the overall treatment of diabetic patients: the periodontal therapy may improve glycaemic control, and could lead to a significant lowering of HbA1c values.40

Concerning the adolescents, the oral hygiene seems to be better in case of the diabetic patients than in case of the other group of young patients who have shown highly variable work shifts leading to poor oral hygiene. The difference is reflected by a lesser bacterial plaque among the diabetics. This is in conflict with the results of other authors who observed similar oral hygiene status and plaque indexes in the two groups, or even comparatively increased plaque accumulation among the diabetics.58 The reason for this may be the following: although the diabetics were controlled, they showed a high gingival response to the irrigation caused by bacterial plaque retention.59 Regularly diabetics have a greater incidence regarding periodontal disease than healthy individuals6061, though other studies have not detected such relation between periodontal disease and diabetes.626364 They didn't confirm any association between diabetes and periodontal disease in adolescents. They believe that there is no difference between on the one part type 1 DM adolescents and children, and on the other healthy individuals regarding clinical periodontal status. However Fructosamine value, used to diagnose and monitor diabetes, was observed to be in correlation with the gingival index score in case of type 1 DM adolescents and children, but not in case of healthy control subjects.65 Bay et al.34 found no difference in the degree resolution of gingivitis following scaling and root planning between young diabetics and healthy control group. Barnett et al.35 studied periodontitis prevalence in young diabetics but none of the individuals showed radiographic sign of periodontitis.

Despite the comparatively better control of bacterial plaque among the diabetics, a change in their periodontal response was detected. Therefore it seems that local immune response may possibly change among these patients, and the lesions caused by microbial agents in case of periodontal disease, which can be associated with a lesser tissue repair capacity, might be the reason for the increased deterioration of periodontal structures shown in the diabetic group.4

It is possible, that the diversities observed by the different authors result from local health care habits. In an area with a tradition of good oral hygene, the oral hygiene habits of diabetes are not better than those of healthy people. Therefore in case of similar oral hygiene habits, diabetical patients show lower periodontal values than healthy persons because of the above mentioned immune response. In areas where the dental treatment habits of the population are poorer, the greater attention of well-controlled DM patients to oral hygiene results in higher values than those of healthy individuals.

#### **2.2 Prevalence of gingivitis, periodontitis in adolescents with type 1 DM**

Since there are conflicting reports in the literature, the aim of this study was analyse the periodontal disease in adolescents with type 1 diabetes mellitus.66 Characterization of risk factors, local such as oral hygiene-, general (duration of DM, age, degree of metabolic control of DM) and contributory factors-, such as toothbrushing, have been identified that determining the risk factors place people enhanced risk in development and progression of periodontal diseases.

Dental Conditions and Periodontal Disease in Adolescents with Type 1 Diabetes Mellitus 415

**Prevalence of Gingivitis and Periodontitis**

61.03

16.5

girl boy Total girl boy Total girl boy Total girl boy Total girl boy Total girl boy Total Diabetes Control Diabetes Control Diabetes Control Healthy Periodontium Gingivitis Periodontitis

**Oral Hygiene and PI**

1.73

0 0-3 3--6 OHI-S

28.1

18.4

35.7

43.4

39

0.7

2.66

3.1 1.1

1.8

0

0.5

1

1.5

**PI**

2

2.5

3

**Percentage %**

4.3 2.6

Fig. 1. Prevalence of gingivitis and periodontis

0.81

Fig. 2. The mean value of PI according to oral hygiene (OHI-S)

98.6

68.5

80.5

62.5

52.2

A dental clinical cross-sectional examination was carried out on 259 adolescents of ages 14-19 with type 1 DM in comparison with a non-DM group as control group. Children who had been under the age of eight at the onset of DM were excluded, as where those with any additional disease or taking other chronic medication. The control group comprised of metabollically healthy individuals. The DM patients were classified according to the categories recommended by the World Health Organisation (1999). The Greene-Vermillion OHI-S index was used to determine the level of oral hygiene. Periodontal changes were rated by the Russell's periodontal index (PI), which estimates the degree of periodontal disease occurring in the mouth by measuring both bone loss around the teeth and gingival inflammation. This method is regularly used in the epidemiological investigation of the disease.67 Alveolar bone loss was tested according to Schei et al (1959), and Hirschman et al (1994) with panoramic radiograph and vertical or posterior bitewings radiographs.

The DM subjects were characterised according to the following criteria: mean postprandial blood glucose level during the period of 6 months prior to the dental examination; glycosylated haemoglobin (HbA1C) level; duration of the DM; the age at the onset of DM. The ADA (2005) criteria were used concerning the DM control. Diabetes mellitus was wellcontrolled (in 210 cases), if six months before the dental examination the mean postprandial blood glucose level of the patients was normal or near normal (below 7.5 mmol/l), there was no glycosuria HbA1C≤ 6.5%, and severe hypoglicaemia did not occur. Control was taken to be poor when the postprandial blood sugar was ≥ 7.5 mmol/l and/or haemoglobin was > 6.5%, or in case of patients with asymptomatic hypoglicaema.

The incidence of gingivitis and periodontitis was much higher in case of diabetic adolescents than in case of metabolically healthy persons (p< 0. 0001) and especially in case of girls ( p< 0. 001). Healthy periodontium was only found in case of 2.6% of the DM adolescents and the rest of DM patients had either gingivitis (61.03%) or periodontitis (39%), while a high rate of metabolically healthy persons (80.5%) has shown a healthy periodontium. Gingivitis and periodontitis was experienced in case of 18.4% and 1.1% of the patients. (Fig.1.)

With the fall of oral hygiene (OHI-S) index (p< 0. 0001) the intensity of gingivitis and periodontitis (PI) has become more severe. (Fig.2.)

An important positive correlation has been shown between the control level of the disease and the intensity of gingivitis and periodontitis. In case of well-controlled 'Type 1' diabetic adolescents the PI mean value was lower than in case of patients with poor glycemic control ( p< 0. 001).

In the diabetic group the periodontal disease was detected to be severe in case of those who had a lasting DM (>5 years) (p<0.001).

The majority of the control persons showed no alveolar bone loss (83% had intact alveolar bone) and those who had alveolar bone loss, had primarily that of horizontal type. In case of diabetic adolescents prevalence of intact alveolar bone was lower (61.8%) than in case of the control group. Therefore higher degree of alveolar bone resorption (both horizontal and horizontal+vertical type) was experienced in case of diabetic patients than in case of the control group (p<0.001).

The mean values of alveolar bone resorption were higher in case of diabetic boys than in case of diabetic girls (p<0.0001).

**Prevalence of Gingivitis and Periodontitis**

Fig. 1. Prevalence of gingivitis and periodontis

414 Type 1 Diabetes Complications

A dental clinical cross-sectional examination was carried out on 259 adolescents of ages 14-19 with type 1 DM in comparison with a non-DM group as control group. Children who had been under the age of eight at the onset of DM were excluded, as where those with any additional disease or taking other chronic medication. The control group comprised of metabollically healthy individuals. The DM patients were classified according to the categories recommended by the World Health Organisation (1999). The Greene-Vermillion OHI-S index was used to determine the level of oral hygiene. Periodontal changes were rated by the Russell's periodontal index (PI), which estimates the degree of periodontal disease occurring in the mouth by measuring both bone loss around the teeth and gingival inflammation. This method is regularly used in the epidemiological investigation of the disease.67 Alveolar bone loss was tested according to Schei et al (1959), and Hirschman et al (1994) with panoramic radiograph and vertical or

The DM subjects were characterised according to the following criteria: mean postprandial blood glucose level during the period of 6 months prior to the dental examination; glycosylated haemoglobin (HbA1C) level; duration of the DM; the age at the onset of DM. The ADA (2005) criteria were used concerning the DM control. Diabetes mellitus was wellcontrolled (in 210 cases), if six months before the dental examination the mean postprandial blood glucose level of the patients was normal or near normal (below 7.5 mmol/l), there was no glycosuria HbA1C≤ 6.5%, and severe hypoglicaemia did not occur. Control was taken to be poor when the postprandial blood sugar was ≥ 7.5 mmol/l and/or haemoglobin

The incidence of gingivitis and periodontitis was much higher in case of diabetic adolescents than in case of metabolically healthy persons (p< 0. 0001) and especially in case of girls ( p< 0. 001). Healthy periodontium was only found in case of 2.6% of the DM adolescents and the rest of DM patients had either gingivitis (61.03%) or periodontitis (39%), while a high rate of metabolically healthy persons (80.5%) has shown a healthy periodontium. Gingivitis and periodontitis was experienced in case of 18.4% and 1.1% of

With the fall of oral hygiene (OHI-S) index (p< 0. 0001) the intensity of gingivitis and

An important positive correlation has been shown between the control level of the disease and the intensity of gingivitis and periodontitis. In case of well-controlled 'Type 1' diabetic adolescents the PI mean value was lower than in case of patients with poor glycemic control

In the diabetic group the periodontal disease was detected to be severe in case of those who

The majority of the control persons showed no alveolar bone loss (83% had intact alveolar bone) and those who had alveolar bone loss, had primarily that of horizontal type. In case of diabetic adolescents prevalence of intact alveolar bone was lower (61.8%) than in case of the control group. Therefore higher degree of alveolar bone resorption (both horizontal and horizontal+vertical type) was experienced in case of diabetic patients than in case of the

The mean values of alveolar bone resorption were higher in case of diabetic boys than in

was > 6.5%, or in case of patients with asymptomatic hypoglicaema.

periodontitis (PI) has become more severe. (Fig.2.)

had a lasting DM (>5 years) (p<0.001).

posterior bitewings radiographs.

the patients. (Fig.1.)

control group (p<0.001).

case of diabetic girls (p<0.0001).

( p< 0. 001).

Fig. 2. The mean value of PI according to oral hygiene (OHI-S)

Dental Conditions and Periodontal Disease in Adolescents with Type 1 Diabetes Mellitus 417

Two groups of bacteria may cause caries: *Streptococcus mutans*69 and *Lactobacillus*2570. Bacteria gather around the teeth and gum in a sticky, creamy-coloured mass called plaque, which serves as a biofilm. Bacteria in an individual's mouth transform glucose, fructose, and most regularly sucrose into acids such as lactic acid through a glycolytic process called fermentation.71 In direct relation with the tooth, these acids may lead to demineralization, i.e. the dissolution of its mineral content. However the process is dynamic. If the acid is neutralized by saliva or mouthwash, remineralization may arise. Fluoride toothpaste or dental varnish can help remineralization.72 If the demineralization process is continuous, enough mineral content could be lost so that the soft organic material left behind disintegrates, forming a cavity or hole. Caries is one of the most common diseases of the world at present.

Epidemiological studies do not seem to be in accord on the characteristics of diabetes mellitus (DM) concerning the incidence of dental caries in case of both children and adults737475767778. Although adolescents may be more caries-prevalent than individuals of other ages, only few studies deal with this age group. In case of type 1 diabetics an increased prevalence was detected79, located particularly in the root or dental neck regions.70 However studies differ whether control of DM or sucrose-free diet of DM patients is more likely to promote or inhibit the development of dental caries. Opinions do not match concerning the dental condition for patients with DM or the outcome of early DM manifestation regarding the dental condition. Cross-sectional studies have reported a low prevalence of caries in case of children and adolescents with type 1 DM, and this has mainly been explained by the sucrose-restricted diet, which is a part of the lifelong treatment.8081 According to Wegner,82 the frequency of caries in DM childen is at least not lower than in non-DM children. Wegner83 also observed that, directly after the diagnosed onset of their disease, some young DM patients displayed a higher activity of caries than healthy subjects of the same age, but the frequency of caries gradually diminished in association with dietary restriction and treatment with insulin. Other researchers proposed a relationship between the development of caries and the level of metabolic control.298485 These researches showed a higher prevalence of caries in case of poorly controlled DM than in case of well-controlled disease. The reason is that higher glucose content in oral fluids adds to bacterial proliferation, enhancing the formation of dental plaque. It has been shown for all concerning age groups that children with DM have less caries than non-diabetic children.26 However, these experiments also demonstrated that the result was due to an abundance of sites where teeth were lost without replacement. The complications were not symptoms of type 1 DM, they might be caused by poor medical and dental care. Some studies proposed that salivary secretion rates should be substantially lowered in case of children with type 1 DM when compared to healthy children.86 Reduced salivary secretion increases the probability of caries, although adequate metabolic control prevents the most dangerous salivary alterations such as high glucose content and low pH, while a fine diabetic diet, rich in fiber and poor in simple carbohydrates, can decelerate the development of plaque and the proliferation of acidogenic bacterial microflora.2987 Individuals at ages of adolescence generally clean their teeth less frequently than persons at ages after adolescence.88 Behavioral factors such as dental selfefficacy are in relation with DM self-efficacy and adherence.71 Adequate oral health habits may lead to lower incidence of caries. On the other hand, Moore et al. observed similar regularities concerning the use of dentifrice and dental floss between diabetics and non-diabetics89. However other studies demonsrtated that diabetic patients used dental floss more frequently than non-diabetics.90 It seems that there are contradicting studies on the dental condition in case of patients with DM as well as on the effect of early DM manifestation on the dental condition.

**3.1 Diabetes and caries** 

The intensity of gingivitis and periodontitis, as well as the increased alveolare bone loss was more prevalent and more severe in diabetic adolescents than in healthy individuals. Several authors suggested a correlation between diabetes mellitus and periodontal diseases. According to them periodontal disease is one of the most prevalent and rapidly progressive complications3738 of DM in case of adults.

Sappala et al. (1993) has shown that adults with Type 1 diabetes mellitus have higher degree of attachment loss and bone loss than control subjects under similar dental plaque conditions. The authors received the same results for diabetic adolescents. The oral hygiene (OHI-S), especially the debris index was worse in case of diabetic individuals than in case of the control persons and correlation was found between on the one part the intensity of gingivitis and periodontitis and on the other the oral hygiene. However De Pommereu et al.57 found more serious gingivitis in diabetics than in control persons, although plaque scores were the same in both groups. Interestingly, according to the present study diabetic adolescents, especially boys have more severe alveolare bone destruction than non-diabetics.

The present study demonstrates that severity of periodontal disease increases with the duration of diabetes mellitus, in agreement with the results in case of adults measured by Pavez23 and Greene Vermillion (1964), Rosenthal et al. (1988), and Lopey et al. (2002).

This study shows that the poorly controlled type 1 DM with elevated blood glucose and glycosylated hemoglobin (HgA 1c) levels have more incidental and serious periodontal diseases with alveolare bone loss in case of adolescents with lasting diabetes mellitus. Adequate metabolic control of diabetes mellitus lowers inclination to infection. This is also crucial for the prevention of periodontal disease in case of adolescents with diabetes mellitus. In case of adult diabetic patients with poorly controlled diabetes mellitus, increased metabolic control may improve periodontal condition (Miller et al. 1992, Yki-Jarvinen 1989, Harris 1995, RST/AAP 1999).

Periodontitis is a complex multifactorial disease. It can especially cause a problem for adolescents with DM. Inadequate oral hygiene is responsible for high oral debris in case of diabetic adolescents, being in correlation with the status of periodontium.

Several other charcteristics such as long duration, early onset (under 14 years), and degree of metabolic control, have been determined as factors increasing the risk of disease. In case of diabetic girls the monthly hormonal level alterations of the gingival may play a role in the development and progression of gingivitis, periodontitis and alveolare bone resorption.

Frequent dental treatments may help maintain good oral health. Treatment is especially crucial at the onset of the disease. Dental care at the early stages of the disease, reducing susceptibility to infection, is also significant for the prevention of periodontal disease in case of adolescents with type 1 diabetes mellitus. Children should also be checked regularly for bleeding gums or inflammation for to prevent the alveolare bone loss, which leads to irreversible changes of periodontium.

The education of youngsters on proper home oral care is the basic method for periodontal greatment and prevention. In most cases the gingivitis can be resolved by plaque control.

#### **3. Caries**

Dental caries is a chronical bacterial disorder where bacterial processes damage hard tooth structure (enamel, dentin, and cementum).68 The tissues break down progressively, resulting in dental caries (cavities, holes in the teeth). The basic factors are the causal microorganism, the host (tooth), the substrate (diet), and the immune capacity of the patient. Two groups of bacteria may cause caries: *Streptococcus mutans*69 and *Lactobacillus*2570. Bacteria gather around the teeth and gum in a sticky, creamy-coloured mass called plaque, which serves as a biofilm. Bacteria in an individual's mouth transform glucose, fructose, and most regularly sucrose into acids such as lactic acid through a glycolytic process called fermentation.71 In direct relation with the tooth, these acids may lead to demineralization, i.e. the dissolution of its mineral content. However the process is dynamic. If the acid is neutralized by saliva or mouthwash, remineralization may arise. Fluoride toothpaste or dental varnish can help remineralization.72 If the demineralization process is continuous, enough mineral content could be lost so that the soft organic material left behind disintegrates, forming a cavity or hole. Caries is one of the most common diseases of the world at present.

#### **3.1 Diabetes and caries**

416 Type 1 Diabetes Complications

The intensity of gingivitis and periodontitis, as well as the increased alveolare bone loss was more prevalent and more severe in diabetic adolescents than in healthy individuals. Several authors suggested a correlation between diabetes mellitus and periodontal diseases. According to them periodontal disease is one of the most prevalent and rapidly progressive

Sappala et al. (1993) has shown that adults with Type 1 diabetes mellitus have higher degree of attachment loss and bone loss than control subjects under similar dental plaque conditions. The authors received the same results for diabetic adolescents. The oral hygiene (OHI-S), especially the debris index was worse in case of diabetic individuals than in case of the control persons and correlation was found between on the one part the intensity of gingivitis and periodontitis and on the other the oral hygiene. However De Pommereu et al.57 found more serious gingivitis in diabetics than in control persons, although plaque scores were the same in both groups. Interestingly, according to the present study diabetic adolescents, especially boys

The present study demonstrates that severity of periodontal disease increases with the duration of diabetes mellitus, in agreement with the results in case of adults measured by

This study shows that the poorly controlled type 1 DM with elevated blood glucose and glycosylated hemoglobin (HgA 1c) levels have more incidental and serious periodontal diseases with alveolare bone loss in case of adolescents with lasting diabetes mellitus. Adequate metabolic control of diabetes mellitus lowers inclination to infection. This is also crucial for the prevention of periodontal disease in case of adolescents with diabetes mellitus. In case of adult diabetic patients with poorly controlled diabetes mellitus, increased metabolic control may improve periodontal condition (Miller et al. 1992,

Periodontitis is a complex multifactorial disease. It can especially cause a problem for adolescents with DM. Inadequate oral hygiene is responsible for high oral debris in case of

Several other charcteristics such as long duration, early onset (under 14 years), and degree of metabolic control, have been determined as factors increasing the risk of disease. In case of diabetic girls the monthly hormonal level alterations of the gingival may play a role in the development and progression of gingivitis, periodontitis and alveolare bone resorption. Frequent dental treatments may help maintain good oral health. Treatment is especially crucial at the onset of the disease. Dental care at the early stages of the disease, reducing susceptibility to infection, is also significant for the prevention of periodontal disease in case of adolescents with type 1 diabetes mellitus. Children should also be checked regularly for bleeding gums or inflammation for to prevent the alveolare bone loss, which leads to

The education of youngsters on proper home oral care is the basic method for periodontal greatment and prevention. In most cases the gingivitis can be resolved by plaque control.

Dental caries is a chronical bacterial disorder where bacterial processes damage hard tooth structure (enamel, dentin, and cementum).68 The tissues break down progressively, resulting in dental caries (cavities, holes in the teeth). The basic factors are the causal microorganism, the host (tooth), the substrate (diet), and the immune capacity of the patient.

diabetic adolescents, being in correlation with the status of periodontium.

Pavez23 and Greene Vermillion (1964), Rosenthal et al. (1988), and Lopey et al. (2002).

complications3738 of DM in case of adults.

have more severe alveolare bone destruction than non-diabetics.

Yki-Jarvinen 1989, Harris 1995, RST/AAP 1999).

irreversible changes of periodontium.

**3. Caries** 

Epidemiological studies do not seem to be in accord on the characteristics of diabetes mellitus (DM) concerning the incidence of dental caries in case of both children and adults737475767778. Although adolescents may be more caries-prevalent than individuals of other ages, only few studies deal with this age group. In case of type 1 diabetics an increased prevalence was detected79, located particularly in the root or dental neck regions.70 However studies differ whether control of DM or sucrose-free diet of DM patients is more likely to promote or inhibit the development of dental caries. Opinions do not match concerning the dental condition for patients with DM or the outcome of early DM manifestation regarding the dental condition. Cross-sectional studies have reported a low prevalence of caries in case of children and adolescents with type 1 DM, and this has mainly been explained by the sucrose-restricted diet, which is a part of the lifelong treatment.8081 According to Wegner,82 the frequency of caries in DM childen is at least not lower than in non-DM children. Wegner83 also observed that, directly after the diagnosed onset of their disease, some young DM patients displayed a higher activity of caries than healthy subjects of the same age, but the frequency of caries gradually diminished in association with dietary restriction and treatment with insulin. Other researchers proposed a relationship between the development of caries and the level of metabolic control.298485 These researches showed a higher prevalence of caries in case of poorly controlled DM than in case of well-controlled disease. The reason is that higher glucose content in oral fluids adds to bacterial proliferation, enhancing the formation of dental plaque. It has been shown for all concerning age groups that children with DM have less caries than non-diabetic children.26 However, these experiments also demonstrated that the result was due to an abundance of sites where teeth were lost without replacement. The complications were not symptoms of type 1 DM, they might be caused by poor medical and dental care. Some studies proposed that salivary secretion rates should be substantially lowered in case of children with type 1 DM when compared to healthy children.86 Reduced salivary secretion increases the probability of caries, although adequate metabolic control prevents the most dangerous salivary alterations such as high glucose content and low pH, while a fine diabetic diet, rich in fiber and poor in simple carbohydrates, can decelerate the development of plaque and the proliferation of acidogenic bacterial microflora.2987 Individuals at ages of adolescence generally clean their teeth less frequently than persons at ages after adolescence.88 Behavioral factors such as dental selfefficacy are in relation with DM self-efficacy and adherence.71 Adequate oral health habits may lead to lower incidence of caries. On the other hand, Moore et al. observed similar regularities concerning the use of dentifrice and dental floss between diabetics and non-diabetics89. However other studies demonsrtated that diabetic patients used dental floss more frequently than non-diabetics.90 It seems that there are contradicting studies on the dental condition in case of patients with DM as well as on the effect of early DM manifestation on the dental condition.

Dental Conditions and Periodontal Disease in Adolescents with Type 1 Diabetes Mellitus 419

Fig. 3. The mean number of the DMF-T and D+F according to the age of the patient at the

Fig. 4. The mean number of the DMF-T and D+F according to the age of the patient at the

onset of type 1 DM in patients with good oral hygiene (OHI-S=0)

onset of type 1 DM

#### **3.2 Dental caries in adolescent with type 1 DM**

Since there are conflicting reports regarding the dental condition in patients with DM or the effect of early DM manifestation on dental condition, the aim was to analyse the dental status DMF-T and the prevalence of dental caries in adolescents with type 1 DM and compare the findings with those from metabolically healthy individuals, in an effort to determine the risk factors play a role in the development of dental caries in DM adolescents.91

A dental clinical cross-sectional examination was carried out on 259 adolescents aged 14-19 years with type 1 DM in comparison with a non-DM group as control. Children who had been under the age of eight at he onset of DM were excluded, as where those with any additional disease or taking other chronic medication. The control group comprised metabollically healthy individuals. The DM patients were classified according to the categories recommended by the World Health Organisation (WHO). Caries was assessed by the DMF-T index. Within this index, F was used for the filled teeth, D denoted the number of untreated carious teeth without regard to whether the lesion was enamel or of root. Extractions for orthodontics or periodontal reasons were excluded. The Greene-Vermillion OHI-S index was used to determine the level of oral hygiene. The DM subjects were characterised according to the following criteria: mean postprandial blood glucose level and during the period of 6 months prior to the dental examination; glycosylated haemoglobin (HbA1C) level; the duration of the DM; and the age at the onset of DM: Concerns by DM control, the ADA (2005) criteria were offered. Diabetes mellitus was well-controlled (210 cases) if six months before the dental examination the mean postprandial blood glucose level of the patients was normal or near normal (below 7.5 mmol/l) there was no glycosuria HbA1C≤ 6.5% and severe hypoglicaemia is did not occur. Control was taken as poor when the postprandial blood sugar was ≥ 7.5 mmol/l and/ or haemoglobin was > 6.5%, or in cases of patients with asymptomatic hypoglicaema.

The DM adolescents had a slightly higher mean DMF-T score than the control subjects. The difference was found to be statistically significant (p<0.001).

When the components of DMFT were considered, there were more filled (F) (p<0.001) and fewer decayed teeth (D) (p<0.0001) among the DM adolescents than in the healthy controls. When the number of missing teeth was considered, there were no significant differences between the DM patients and the healthy controls.

The DM boys had a slightly higher mean DMFT score than the DM girls (p<0.001). More decayed (D) (p<0.001) and filled (F) teeth were found among the DM boys than among the DM girls (p<0.001). As regard the number of extracted (M) teeth, there were no significant differences between the boys and the girls.

The age of the patient at the onset of DM was correlated to the caries condition (D). This suggested that the early onset of DM (before the age of 14) was related to significantly fewer decayed and filled teeth compared with the patients in whom the DM had developed after age 14 years (p< 0.01). (Fig.3.)

However the adolescents with good oral hygiene (OHI-S=0), there was a significant differences (P<0.0001). (Fig.4.) A positive correlation was found between of the level of control of the DM and the dental condition. In the well-controlled DM adolescents the mean number of decayed (D) teeth was lower (p< 0.0001), but the numbert of filled (F) teeth was higher than in patients with poorer glycemic control(P<0.001). (Fig.5.)

Since there are conflicting reports regarding the dental condition in patients with DM or the effect of early DM manifestation on dental condition, the aim was to analyse the dental status DMF-T and the prevalence of dental caries in adolescents with type 1 DM and compare the findings with those from metabolically healthy individuals, in an effort to determine the risk factors play a role in the development of dental caries in DM

A dental clinical cross-sectional examination was carried out on 259 adolescents aged 14-19 years with type 1 DM in comparison with a non-DM group as control. Children who had been under the age of eight at he onset of DM were excluded, as where those with any additional disease or taking other chronic medication. The control group comprised metabollically healthy individuals. The DM patients were classified according to the categories recommended by the World Health Organisation (WHO). Caries was assessed by the DMF-T index. Within this index, F was used for the filled teeth, D denoted the number of untreated carious teeth without regard to whether the lesion was enamel or of root. Extractions for orthodontics or periodontal reasons were excluded. The Greene-Vermillion OHI-S index was used to determine the level of oral hygiene. The DM subjects were characterised according to the following criteria: mean postprandial blood glucose level and during the period of 6 months prior to the dental examination; glycosylated haemoglobin (HbA1C) level; the duration of the DM; and the age at the onset of DM: Concerns by DM control, the ADA (2005) criteria were offered. Diabetes mellitus was well-controlled (210 cases) if six months before the dental examination the mean postprandial blood glucose level of the patients was normal or near normal (below 7.5 mmol/l) there was no glycosuria HbA1C≤ 6.5% and severe hypoglicaemia is did not occur. Control was taken as poor when the postprandial blood sugar was ≥ 7.5 mmol/l and/ or haemoglobin was > 6.5%, or in

The DM adolescents had a slightly higher mean DMF-T score than the control subjects. The

When the components of DMFT were considered, there were more filled (F) (p<0.001) and fewer decayed teeth (D) (p<0.0001) among the DM adolescents than in the healthy controls. When the number of missing teeth was considered, there were no significant differences

The DM boys had a slightly higher mean DMFT score than the DM girls (p<0.001). More decayed (D) (p<0.001) and filled (F) teeth were found among the DM boys than among the DM girls (p<0.001). As regard the number of extracted (M) teeth, there were no significant

The age of the patient at the onset of DM was correlated to the caries condition (D). This suggested that the early onset of DM (before the age of 14) was related to significantly fewer decayed and filled teeth compared with the patients in whom the DM had developed after

However the adolescents with good oral hygiene (OHI-S=0), there was a significant differences (P<0.0001). (Fig.4.) A positive correlation was found between of the level of control of the DM and the dental condition. In the well-controlled DM adolescents the mean number of decayed (D) teeth was lower (p< 0.0001), but the numbert of filled (F) teeth was

higher than in patients with poorer glycemic control(P<0.001). (Fig.5.)

**3.2 Dental caries in adolescent with type 1 DM** 

cases of patients with asymptomatic hypoglicaema.

between the DM patients and the healthy controls.

differences between the boys and the girls.

age 14 years (p< 0.01). (Fig.3.)

difference was found to be statistically significant (p<0.001).

adolescents.91

Fig. 4. The mean number of the DMF-T and D+F according to the age of the patient at the onset of type 1 DM in patients with good oral hygiene (OHI-S=0)

Dental Conditions and Periodontal Disease in Adolescents with Type 1 Diabetes Mellitus 421

Adler et al.96 proposed that metabolic disorders cause the acceleration and delay experienced in dentition. A biphasic effect of the diabetic state on dental development was detected: on the one hand acceleration in the early diabetes, and on the other retardation in lasting diabetes.92 The result conflicted with common knowledge: acceleration was observed in dentition until the age of 10 and delay after the age of 10 (especially for the eruption of canine and the premolars).26 This may be in correlation with diseases containing metabolical

Other studies showed that children with diabetes in the late mixed dentition period (ages 10-14) had a higher inclination for advanced tooth eruption than those without diabetes. Alterations in tooth eruption were not detected in the early mixed dentition group (ages 6- 10). Individuals with higher height showed higher propensity for expedited tooth eruption. The expetited eruption among patients with higher height was more characteristic in the

Diabetes is a chronic metabolic disease which affects the entire organism, disturbing especially the oral health. Health habits are substantial for preventing dental and periodontal diseases and maintaining oral health in a population of patients with type 1

It is a vital task for dentists to foster good oral health habits, executing periodic dental examinations and ensuring sufficient oral hygiene. These conditions significantly affect the diabetic patients' oral health. Dentists must minimize the risk factors of periodontal disease, caries and oral soft tissue pathologies.88 Dentists should continually instruct and motivate

Dental practice showed that health promoting methods reduce the patient's smoking habits.98 It is possible to use pharmacological and behavioral strategies in a private practitioner's office to help patients quit smoking.99 However inadequate undergraduate dental education and practitioner continuing education threaten to be an obstacle for the daily use of these practices.100 There are positive signs conserning the improvement of educational opportunities, nevertheless the incorporation of these technics in the clinical

There are two kinds of professional oral hygiene promotion: active and sustaining procedure. The initial or causal promotion is to eliminate the plaque and the plaque retentional factors and to remove the supra- and subgingival calculus, as well as the root planing and the curettage and to eliminate the carious lesions. The goal of these procedures is to terminate the inflamation of the gingiva and to stop further development of the gingival recession, thus eliminating the bacterial elements responsible for inflammation. The most important condition of efficient private oral hygiene is the smooth cleanable tooth surface. For it is useless to instruct the patient even on the most refined tooth brushing technique or on the use of dental floss if the teeth are covered with calculus or there are overfilled interdental spaces. Dental practice should include dental check within one or two months, oral hygiene treatment, repeated fixing of gingival and periodontal indexes, as well as their comparison with the initial values, and in certain cases causal surgical periodontal interventions. Nontheless, further instructions and

**5. Diabetes and dental, periodontal prevention in adolescents** 

instabilities, such as diabetes.

DM.

older group than in the younger one.97

the patient concerning oral hygiene.

practice must still be highlighted.101

motivations are also essential.

Fig. 5. The control level of DMF-T and its components (D, M, F)

This study of adolescents with type 1 DM demonstrated that none of the subjects has intact dentitions. Poor glicaemic control and the early onset of DM may increase the risk of dental caries but appropriate oral hygiene together with satisfactory metabolic control may prevent the development of dental caries in adolescents with type 1 DM.

#### **4. Tooth eruption**

Eruption is the phisiological process of tooth development and growth during which the teeth enter the mouth and become visible. Normally they cut themselves throught the oral mucosa without any inflammation.

Mixed dentition starts when the first permanent molar appears in the mouth, usually at five or six years, and ends when the last primamry tooth is lost, usually at ten, eleven, or twelve years.92 Afterwards the permanent dentition begins and lasts as long as the person lives or until all of the teeth are lost (edentulism).

#### **4.1 Diabetes mellitus and accelerated eruption**

It has been demonstrated that diseases containing metabolic instabilities, such as diabetics, weaken the resistance to inflammation93. As a result, the gingival inflammation accompanies eruption in the diabetic patients at an increased rate as compared to the non-diabetic individuals.

Information on dentition concerning the age group from 5 to 9 is limited to about 60 type 1 DM children. Ziskin94 et al. detected a small and insubstantial affect of diabetes on dental development. Studies concerning accelerated dental development in case of diabetic children who are less than 11.5 years old are not in accord. Older diabetic children manifested delayed dental development*.*<sup>95</sup> The edentulous interval was longer in case of diabetic children than in case of the control population.

This study of adolescents with type 1 DM demonstrated that none of the subjects has intact dentitions. Poor glicaemic control and the early onset of DM may increase the risk of dental caries but appropriate oral hygiene together with satisfactory metabolic control may prevent

Eruption is the phisiological process of tooth development and growth during which the teeth enter the mouth and become visible. Normally they cut themselves throught the oral

Mixed dentition starts when the first permanent molar appears in the mouth, usually at five or six years, and ends when the last primamry tooth is lost, usually at ten, eleven, or twelve years.92 Afterwards the permanent dentition begins and lasts as long as the person lives or

It has been demonstrated that diseases containing metabolic instabilities, such as diabetics, weaken the resistance to inflammation93. As a result, the gingival inflammation accompanies eruption in the diabetic patients at an increased rate as compared to the non-diabetic

Information on dentition concerning the age group from 5 to 9 is limited to about 60 type 1 DM children. Ziskin94 et al. detected a small and insubstantial affect of diabetes on dental development. Studies concerning accelerated dental development in case of diabetic children who are less than 11.5 years old are not in accord. Older diabetic children manifested delayed dental development*.*<sup>95</sup> The edentulous interval was longer in case of

Fig. 5. The control level of DMF-T and its components (D, M, F)

the development of dental caries in adolescents with type 1 DM.

**4. Tooth eruption** 

individuals.

mucosa without any inflammation.

until all of the teeth are lost (edentulism).

**4.1 Diabetes mellitus and accelerated eruption** 

diabetic children than in case of the control population.

Adler et al.96 proposed that metabolic disorders cause the acceleration and delay experienced in dentition. A biphasic effect of the diabetic state on dental development was detected: on the one hand acceleration in the early diabetes, and on the other retardation in lasting diabetes.92 The result conflicted with common knowledge: acceleration was observed in dentition until the age of 10 and delay after the age of 10 (especially for the eruption of canine and the premolars).26 This may be in correlation with diseases containing metabolical instabilities, such as diabetes.

Other studies showed that children with diabetes in the late mixed dentition period (ages 10-14) had a higher inclination for advanced tooth eruption than those without diabetes. Alterations in tooth eruption were not detected in the early mixed dentition group (ages 6- 10). Individuals with higher height showed higher propensity for expedited tooth eruption. The expetited eruption among patients with higher height was more characteristic in the older group than in the younger one.97

#### **5. Diabetes and dental, periodontal prevention in adolescents**

Diabetes is a chronic metabolic disease which affects the entire organism, disturbing especially the oral health. Health habits are substantial for preventing dental and periodontal diseases and maintaining oral health in a population of patients with type 1 DM.

It is a vital task for dentists to foster good oral health habits, executing periodic dental examinations and ensuring sufficient oral hygiene. These conditions significantly affect the diabetic patients' oral health. Dentists must minimize the risk factors of periodontal disease, caries and oral soft tissue pathologies.88 Dentists should continually instruct and motivate the patient concerning oral hygiene.

Dental practice showed that health promoting methods reduce the patient's smoking habits.98 It is possible to use pharmacological and behavioral strategies in a private practitioner's office to help patients quit smoking.99 However inadequate undergraduate dental education and practitioner continuing education threaten to be an obstacle for the daily use of these practices.100 There are positive signs conserning the improvement of educational opportunities, nevertheless the incorporation of these technics in the clinical practice must still be highlighted.101

There are two kinds of professional oral hygiene promotion: active and sustaining procedure. The initial or causal promotion is to eliminate the plaque and the plaque retentional factors and to remove the supra- and subgingival calculus, as well as the root planing and the curettage and to eliminate the carious lesions. The goal of these procedures is to terminate the inflamation of the gingiva and to stop further development of the gingival recession, thus eliminating the bacterial elements responsible for inflammation. The most important condition of efficient private oral hygiene is the smooth cleanable tooth surface. For it is useless to instruct the patient even on the most refined tooth brushing technique or on the use of dental floss if the teeth are covered with calculus or there are overfilled interdental spaces. Dental practice should include dental check within one or two months, oral hygiene treatment, repeated fixing of gingival and periodontal indexes, as well as their comparison with the initial values, and in certain cases causal surgical periodontal interventions. Nontheless, further instructions and motivations are also essential.

Dental Conditions and Periodontal Disease in Adolescents with Type 1 Diabetes Mellitus 423

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**20** 

*Brazil* 

**Impact of Hyperglycemia on Xerostomia and** 

Carlos Cesar Deantoni, Rosângela Réa and Luciana Reis Azevedo-Alanis

Xerostomia is defined as a subjective sensation of having a dry mouth (Fox et al., 1987) and is reported by the patient (Guggenheimer & Moore, 2003; Moore et al., 2001). The subjective feeling of dry mouth (xerostomia) is one of the oral manifestations of diabetes (Sreebny et al., 2006; von Bültzingslöwen et al., 2007). Xerostomia results from a reduction in saliva secretion, although it may occur in spite of the presence of a normal salivary flow rate (Guggenheimer & Moore, 2003; Scully, 2003). Altered saliva composition rather than the quantity of saliva may

Type 1 diabetes mellitus (DM1) is a metabolic dysfunction characterized by hyperglycemia resulting from definitive deficiency in insulin secretion caused by autoimmune illness and genetic factors (ADA, 2004). The American Diabetes Association (ADA) reports that 75% of DM1 cases are diagnosed in persons under the age of 18 years (ADA, 2006). Glycemic control is fundamental to the management of diabetes and is associated with sustained decreased rates of microvascular (retinopathy and nephropathy) as well as neuropathic complications (ADA, 2008). Glycemic control has a modifying effect on the relation between

Patients with DM1, particularly those who have poor glycemic control, may have decreased salivary flow rate (Guggenheimer & Moore, 2003). Many clinical problems develop in the presence of xerostomia, such as: difficulty in swallowing and speech, high susceptibility to oral infections (mainly candidiasis and dental caries), gingivitis and mucositis (Anttila et al., 1998). Furthermore, xerostomia was shown to have a negative impact on the quality of life

The relationship among DM1, salivary composition and xerostomia has been widely investigated (Swanljung et al., 1992; Moore et al., 2001; López et al., 2003; Siudikiene et al., 2006; Siudikiene et al., 2008; Orbak et al., 2008). It has been found that most DM1 patients have salivary dysfunction as well as differences in biochemical salivary composition compared with healthy subjects (Swanljung et al., 1992; Moore et al., 2001; López et al., 2003; Siudikiene et al., 2006; Siudikiene et al., 2008; Orbak et al., 2008). Moreover, there is a lack of studies showing the relationship among hyperglycemia, xerostomia and salivary factors, especially in

play a role in the induction of xerostomia (Anttila et al., 1998; Fox, 1996).

dental caries and salivary factors in young patients (Syjälä et al., 2003).

of adolescents with DM1 (Busato et al., 2009).

**1. Introduction** 

**Adolescents with Type 1 Diabetes Mellitus** 

**Salivary Composition and Flow Rate of** 

Ivana Maria Saes Busato, Maria Ângela Naval Machado, João Armando Brancher, Antônio Adilson Soares de Lima,

*Pontifical Catholic University of Paraná* 


### **Impact of Hyperglycemia on Xerostomia and Salivary Composition and Flow Rate of Adolescents with Type 1 Diabetes Mellitus**

Ivana Maria Saes Busato, Maria Ângela Naval Machado, João Armando Brancher, Antônio Adilson Soares de Lima, Carlos Cesar Deantoni, Rosângela Réa and Luciana Reis Azevedo-Alanis *Pontifical Catholic University of Paraná Brazil* 

#### **1. Introduction**

426 Type 1 Diabetes Complications

[84] Twetman S, Nederfors T, Ståhl B, Aronson S. Two-year longitudinal observations of

[85] Twetman S, Johansson I, Birkhed D, Nederfors T. Caries Incidence in young Type 1

[86] Ben-Aryeh H, Serouya R, Kanter Y, Szargel R, Laufer D. Oral health and salivary composition in diabetic patients. *J Diabetes Complications.*1993;7:57-62.

[88] Kneckt M C, Syrjälä A M H S, Knuuttila M L E. Attributions to dental and diabetes

[89] Moore PA, Orchard T, Guggenheimer J, Weyant RJ. Diabetes and oral health promotion: a survey of disease prevention behaviors. *JADA.* 2000;131:1333-1341. [90] Alves C, Márcia B, Andion J, Menezes R. Oral health knowledge and habits in children

[91] Miko S, Ambrus S J, Sahafian S, Dinya E, Tamas G, Albrecht M G. Dental caries and adolescents with type 1 diabetes. *British Dental Journal* 2010;208:(6) p. E12. [92] Ash, Major M, Stanley J. Nelson. Wheeler's Dental Anatomy, Physiology and Occlusion.

[93] American Academy of Pediatric Dentistry Clinical Affairs Comittee, Developing

[94] Ziskin DE, Siegel EH, Loughlin WC, Diabetes in relation to certain oral and systemic

[95] Bohatka L, Wegner H, Adler P. Parameters of the mixed dentition in diabetic children. *J* 

[96] Adler P, Wegner H, Bohatka L. Influence of age and duration of diabetes an diabetes on dental development in diabetic children. *J Dent Res.* 1973;52:535-537. [97] Lal S, Cheng B, Kaplan S, Softness B, Greenberg E, Goland RS, Lalla E, Lamster IB.

[98] Hastreiter RJ, Bakdash B, Roesch MH, Walseth J. Use of tobacco prevention and

[99] Christen AG, Klein JA,Christen JA, McDonald JL Jr, Guba CJ. How-to-do-it quit-

[100] Gerbert B, Coates T, Zahnd E, Richard RJ, Cummings SR. Dentists as smoking

[101] Curriculum guidelines for predoctoral preventive dentistry. *J Dent Educ.*

occlusion in pediatric dentistry. Pediatr Dent. 2005;27:143-155.

Dentition Subcomittee; American Academy of Pediatric Dentistry Council on Clinical Affairs. Guideline on management of the developing dentition and

problems. Part I: clinical study of dental caries, tooth eruption, gingival changes, growth phenomena and related observations in juveniles. *J Dent Res.* 1944;23:317-331.

Accelerated Tooth Eruption in Children With Diabetes Mellitus. *Pediatrics.*

cessation strategies and techniques int he dental office. *JADA.* 1994;125(11):1475-84.

smoking strategies for the dental office team: an eight-step program. *JADA.*

[87] Got I, Fontaine A. Teeth and diabetes. *Diabete Metab.*1993;19:467-471.

with type 1 diabetes mellitus. *Braz Dent.* 2009;20:70-73.

health outcomes. *J Clin Periodontol*; 27: 205–211.

mellitus. *Pediatr Dent* 1992;14: 184–188.

factors. *Caries Res* 2002; 36: 31–35.

8th edition. 2003.P.41.

*Dent Res.* 1973;52:131-135.

2008;121:1139-1143

1990;(suppl)S20-S7*.*

1991;55(11):746-50.

cessation counselors. *JADA.* 1989;118(1):29-32.

salivary status and dental caries in children with insulindependent diabetes

diabetes mellitus patients in relation to metabolic control and cariesassociated risk

Xerostomia is defined as a subjective sensation of having a dry mouth (Fox et al., 1987) and is reported by the patient (Guggenheimer & Moore, 2003; Moore et al., 2001). The subjective feeling of dry mouth (xerostomia) is one of the oral manifestations of diabetes (Sreebny et al., 2006; von Bültzingslöwen et al., 2007). Xerostomia results from a reduction in saliva secretion, although it may occur in spite of the presence of a normal salivary flow rate (Guggenheimer & Moore, 2003; Scully, 2003). Altered saliva composition rather than the quantity of saliva may play a role in the induction of xerostomia (Anttila et al., 1998; Fox, 1996).

Type 1 diabetes mellitus (DM1) is a metabolic dysfunction characterized by hyperglycemia resulting from definitive deficiency in insulin secretion caused by autoimmune illness and genetic factors (ADA, 2004). The American Diabetes Association (ADA) reports that 75% of DM1 cases are diagnosed in persons under the age of 18 years (ADA, 2006). Glycemic control is fundamental to the management of diabetes and is associated with sustained decreased rates of microvascular (retinopathy and nephropathy) as well as neuropathic complications (ADA, 2008). Glycemic control has a modifying effect on the relation between dental caries and salivary factors in young patients (Syjälä et al., 2003).

Patients with DM1, particularly those who have poor glycemic control, may have decreased salivary flow rate (Guggenheimer & Moore, 2003). Many clinical problems develop in the presence of xerostomia, such as: difficulty in swallowing and speech, high susceptibility to oral infections (mainly candidiasis and dental caries), gingivitis and mucositis (Anttila et al., 1998). Furthermore, xerostomia was shown to have a negative impact on the quality of life of adolescents with DM1 (Busato et al., 2009).

The relationship among DM1, salivary composition and xerostomia has been widely investigated (Swanljung et al., 1992; Moore et al., 2001; López et al., 2003; Siudikiene et al., 2006; Siudikiene et al., 2008; Orbak et al., 2008). It has been found that most DM1 patients have salivary dysfunction as well as differences in biochemical salivary composition compared with healthy subjects (Swanljung et al., 1992; Moore et al., 2001; López et al., 2003; Siudikiene et al., 2006; Siudikiene et al., 2008; Orbak et al., 2008). Moreover, there is a lack of studies showing the relationship among hyperglycemia, xerostomia and salivary factors, especially in

Impact of Hyperglycemia on Xerostomia and

(Banderas-Tarabay et al., 1997).

**1.1.5 Statistical analysis** 

**2. Results and discussion** 

mg/dL (SD = 108.09).

concentrations.

(Table 1).

concentrations was performed three times.

considering statistically significant values (p ≤ 0.05 and CI 95%).

Salivary Composition and Flow Rate of Adolescents with Type 1 Diabetes Mellitus 429

produced during the first minute of stimulation was discarded. During the following five minutes, the patient expelled saliva into a sterilized universal collecting recipient that had been previously weighed using Marte® analytical scales, model AL 500 (São Paulo-SP/Brazil). The saliva was collected between 8 a.m. and 10 a.m. Stimulated saliva flow rate (SSFR) was evaluated by means of the gravimetric method and expressed in mL/min

The remaining saliva samples were centrifuged (3,000 g for 10 min). Total protein and calcium salivary concentrations were determined using a colorimetric method (LABTEST® kits/Vista Alegre-MG/Brazil). Amylase salivary concentrations were determined by a kinetic colorimetric method (LABTEST® kits/Vista Alegre-MG/Brazil). Urea salivary concentrations were determined by an enzymic colorimetric method (LABTEST® kits/Vista Alegre-MG/Brazil). Salivary glucose was analysed by an enzymic colorimetric method (BIOCLIN® kits/Belo Horizonte-MG/Brazil). The determination of the salivary

The data were analysed using SPSS version 15.0 for Windows. Normality analysis was performed using the Kolmogorov-Smirnov Test, and the Levene test was used to analyse variance homogeneity. The other tests used were Mann-Whitney test and Fisher's exact test

A total of 102 subjects were included in this study: 51 patients with DM1 (DM1 group) and 51 subjects without DM1 (control group). Twenty-seven subjects were female (52.9%) and 24 were male (47.1%) in each group (DM1 group and control group). Average age was 17 years (14-19, SD = 1.4) in both groups. In DM1 group, average CG was 200.5

In the present study, hyperglycemia (CG > 130 mg/dL) was observed in 33 (65%) adolescents with DM1 (DM1-B group) while 18 (35%) showed good glycemic control (DM1-A group). DM1, regardless of glycemic control, was a risk factor for higher xerostomia prevalence and increased glucose salivary concentrations. Hyperglycemia was a risk factor for SSFR reduction and increased urea and calcium salivary

The presence of xerostomia 1 (dry mouth) was indicated by 27/51 (53%) subjects in DM1 group, and 8/51 (16%) subjects in control group (P < 0.001) (Table 1). A total of 12 subjects (24%) stated the need to drink liquids during meals in DM1 group in contrast to 2 (4%) subjects in control group (*P* = 0.004). There were no significant differences between DM1 group and control group for the following questions: difficulty in swallowing food and amount of saliva perceived (P > 0.05). Only DM1 group subjects presented xerostomia 2 (n=10, 20%) and xerostomia 3 (n=5, 10%). There were significant differences between DM1 group and control group regarding xerostomia 2 (*P* = 0.001) and xerostomia 3 (*P* = 0.028)

Among well-controlled adolescents (DM1-A group), 11/18 (61%) subjects reported xerostomia 1, in contrast to 16/33 (48%) hyperglycemic adolescents (DM1-B group). There were significant differences between DM1-A and control group, and DM1-B and controls for

adolescents with DM1. Thus, the aim of this study was to evaluate the association among hyperglycemia, xerostomia, salivary flow and composition of adolescents with DM1.

#### **1.1 Materials and methods**

This study was approved by the Research Ethics Committee of the Pontifical Catholic University of Paraná and by the Management of the Paraná Federal University Teaching Hospital. Patients and their parents or guardians were informed about the objective and the other aspects of the study and signed a Term of Independent Informed Consent.

#### **1.1.1 Study groups and study design**

A case-control epidemiologic study was performed on adolescents, allocated between two groups: control group, comprised of 51 non-diabetic subjects who were recruited from public high schools, and DM1 group, comprised of 51 adolescents with DM1, who receive follow-up at the Diabetes Outpatients Department of the Paraná Federal University Teaching Hospital. DM1 group and control group were paired regarding gender and age (14 – 19 years). DM1 diagnosis using the ADA (ADA, 2004) classification was established as a criterion for inclusion in DM1 group. The criterion for inclusion in control group was that of non-diabetic adolescents who had not used any medication for at least one month. The exclusion criteria used for both groups were: presence of systemic conditions that could influence salivary gland physiology, psychotropic drugs users, smokers, illicit drugs users or alcohol users (Busato et al., 2009).

#### **1.1.2 Glycemic control**

The results of postprandial capillary glucose (CG) tests performed at the time of saliva collection were recorded. Patients with good glycemic control were considered to be those with CG values of ≤ 130 mg/dL (DM1-A group), whereas hyperglycemic patients were considered to be those with CG values of > 130 mg/dL (DM1-B group) (ADA, 2006; 2008).

#### **1.1.3 Xerostomia**

Xerostomia was defined as a dry mouth sensation, reported by the subject. The subjects were asked if they had had a dry mouth sensation in the last six months (question A). If the answer was positive to xerostomia, they were also asked if it had occurred constantly during the last six months. Xerostomia was considered to exist if it had occurred daily during the six-month period. This evaluation was completed by the following questions: How would you describe the amount of saliva in your mouth? (question B). Do you have difficulty in swallowing food? (question C). Do you need to have something to drink in order to be able to swallow your food? (question D) (Carda et al., 2006).

Xerostomia was weighted according to three scales of perception: xerostomia 1 (dry mouth), when the answer to question A was "yes"; xerostomia 2, when there was a positive answer for question A and one other question (B, C or D); xerostomia 3, when there was a positive answer to question A and to two or more questions relating to xerostomia (B, C or D).

#### **1.1.4 Saliva collection and treatment**

Salivary flow was evaluated by means of stimulated saliva collection. The method used was that of mechanical masticatory stimulation, using a piece of sterile rubber tourniquet of a standardized size (1.5 cm), masticated continuously by the patient for six minutes. Saliva produced during the first minute of stimulation was discarded. During the following five minutes, the patient expelled saliva into a sterilized universal collecting recipient that had been previously weighed using Marte® analytical scales, model AL 500 (São Paulo-SP/Brazil). The saliva was collected between 8 a.m. and 10 a.m. Stimulated saliva flow rate (SSFR) was evaluated by means of the gravimetric method and expressed in mL/min (Banderas-Tarabay et al., 1997).

The remaining saliva samples were centrifuged (3,000 g for 10 min). Total protein and calcium salivary concentrations were determined using a colorimetric method (LABTEST® kits/Vista Alegre-MG/Brazil). Amylase salivary concentrations were determined by a kinetic colorimetric method (LABTEST® kits/Vista Alegre-MG/Brazil). Urea salivary concentrations were determined by an enzymic colorimetric method (LABTEST® kits/Vista Alegre-MG/Brazil). Salivary glucose was analysed by an enzymic colorimetric method (BIOCLIN® kits/Belo Horizonte-MG/Brazil). The determination of the salivary concentrations was performed three times.

#### **1.1.5 Statistical analysis**

428 Type 1 Diabetes Complications

adolescents with DM1. Thus, the aim of this study was to evaluate the association among

This study was approved by the Research Ethics Committee of the Pontifical Catholic University of Paraná and by the Management of the Paraná Federal University Teaching Hospital. Patients and their parents or guardians were informed about the objective and the

A case-control epidemiologic study was performed on adolescents, allocated between two groups: control group, comprised of 51 non-diabetic subjects who were recruited from public high schools, and DM1 group, comprised of 51 adolescents with DM1, who receive follow-up at the Diabetes Outpatients Department of the Paraná Federal University Teaching Hospital. DM1 group and control group were paired regarding gender and age (14 – 19 years). DM1 diagnosis using the ADA (ADA, 2004) classification was established as a criterion for inclusion in DM1 group. The criterion for inclusion in control group was that of non-diabetic adolescents who had not used any medication for at least one month. The exclusion criteria used for both groups were: presence of systemic conditions that could influence salivary gland physiology, psychotropic drugs users, smokers, illicit drugs users

The results of postprandial capillary glucose (CG) tests performed at the time of saliva collection were recorded. Patients with good glycemic control were considered to be those with CG values of ≤ 130 mg/dL (DM1-A group), whereas hyperglycemic patients were considered to be those with CG values of > 130 mg/dL (DM1-B group) (ADA, 2006; 2008).

Xerostomia was defined as a dry mouth sensation, reported by the subject. The subjects were asked if they had had a dry mouth sensation in the last six months (question A). If the answer was positive to xerostomia, they were also asked if it had occurred constantly during the last six months. Xerostomia was considered to exist if it had occurred daily during the six-month period. This evaluation was completed by the following questions: How would you describe the amount of saliva in your mouth? (question B). Do you have difficulty in swallowing food? (question C). Do you need to have something to drink in order to be able

Xerostomia was weighted according to three scales of perception: xerostomia 1 (dry mouth), when the answer to question A was "yes"; xerostomia 2, when there was a positive answer for question A and one other question (B, C or D); xerostomia 3, when there was a positive answer to question A and to two or more questions relating to xerostomia (B, C or D).

Salivary flow was evaluated by means of stimulated saliva collection. The method used was that of mechanical masticatory stimulation, using a piece of sterile rubber tourniquet of a standardized size (1.5 cm), masticated continuously by the patient for six minutes. Saliva

hyperglycemia, xerostomia, salivary flow and composition of adolescents with DM1.

other aspects of the study and signed a Term of Independent Informed Consent.

**1.1 Materials and methods** 

**1.1.1 Study groups and study design** 

or alcohol users (Busato et al., 2009).

to swallow your food? (question D) (Carda et al., 2006).

**1.1.4 Saliva collection and treatment** 

**1.1.2 Glycemic control** 

**1.1.3 Xerostomia** 

The data were analysed using SPSS version 15.0 for Windows. Normality analysis was performed using the Kolmogorov-Smirnov Test, and the Levene test was used to analyse variance homogeneity. The other tests used were Mann-Whitney test and Fisher's exact test considering statistically significant values (p ≤ 0.05 and CI 95%).

#### **2. Results and discussion**

A total of 102 subjects were included in this study: 51 patients with DM1 (DM1 group) and 51 subjects without DM1 (control group). Twenty-seven subjects were female (52.9%) and 24 were male (47.1%) in each group (DM1 group and control group). Average age was 17 years (14-19, SD = 1.4) in both groups. In DM1 group, average CG was 200.5 mg/dL (SD = 108.09).

In the present study, hyperglycemia (CG > 130 mg/dL) was observed in 33 (65%) adolescents with DM1 (DM1-B group) while 18 (35%) showed good glycemic control (DM1-A group). DM1, regardless of glycemic control, was a risk factor for higher xerostomia prevalence and increased glucose salivary concentrations. Hyperglycemia was a risk factor for SSFR reduction and increased urea and calcium salivary concentrations.

The presence of xerostomia 1 (dry mouth) was indicated by 27/51 (53%) subjects in DM1 group, and 8/51 (16%) subjects in control group (P < 0.001) (Table 1). A total of 12 subjects (24%) stated the need to drink liquids during meals in DM1 group in contrast to 2 (4%) subjects in control group (*P* = 0.004). There were no significant differences between DM1 group and control group for the following questions: difficulty in swallowing food and amount of saliva perceived (P > 0.05). Only DM1 group subjects presented xerostomia 2 (n=10, 20%) and xerostomia 3 (n=5, 10%). There were significant differences between DM1 group and control group regarding xerostomia 2 (*P* = 0.001) and xerostomia 3 (*P* = 0.028) (Table 1).

Among well-controlled adolescents (DM1-A group), 11/18 (61%) subjects reported xerostomia 1, in contrast to 16/33 (48%) hyperglycemic adolescents (DM1-B group). There were significant differences between DM1-A and control group, and DM1-B and controls for

Impact of Hyperglycemia on Xerostomia and

DM1-group n = 51

> 27 (53) 24 (47)

Variables N (%) or mean (SD)

Xerostomia 1

Total Protein (mg/dL)

Calcium (mmol/L)

Glucose (mmol/L)

X control group

SSFR (stimulated salivary flow rate)

Yes No

Salivary Composition and Flow Rate of Adolescents with Type 1 Diabetes Mellitus 431

DM1-B n=33

16 (48) 17 (52)

218 (386) 139 (67) 262 (460) 239 (144) 0.009a Ŧ

0.752 (0.496) 0.562 (0.366) 0.803 (0.515) 0.401(0.338) 0.001a Ŧ

0.174 (0.183) 0.158 (0.154) 0.170 (0.189) 0.098 (0.115) 0.021a Ŧ

Control group n = 51

8 (16) 43 (84) *P* value

<0.001a c † 0.001b †

> NSb 0.002c Ŧ

0.007b Ŧ NSc

0.042c Ŧ

NSb <0.001c Ŧ

0.024b Ŧ 0.038c Ŧ

DM1-A n=18

11 (61) 7 (39)

SSFR (mL/min) 0.932 (0.537) 1.140 (0.688) 0.812 (0.361) 1.224 (0.577) 0.003a Ŧ

Amylase (U/dL) 758 (33) 767 (35) 757 (31) 778 (9) NSa b c

Urea (mmol/L) 5.340 (2.157) 4.662 (1.565) 5.769 (2.140) 4.957 (2.040) NSa b

DM1-group: adolescents with DM1; DM1-A: adolescents with DM1 (CG ≤ 130mg/dL); DM1-B:

a p value of DM1-group X control group; b p value of DM1-A X control group; and c p value of DM1-B

In the present study, xerostomia 1 prevalence was demonstrated in 27 (53%) adolescents with DM1 (DM1 group): 11 (61%) with good glycemic control and 16 (48%) with hyperglycemia, in contrast to 8 (16%) non-diabetes ones (control group) (Tables 1 and 2). Xerostomia was significantly associated with DM1 (Table 1) regardless of hyperglycemia (Table 2). Xerostomia 2 and xerostomia 3 only occurred in DM1 group, demonstrating that xerostomia is one of the oral manifestations of diabetes (Sreebny et a.l, 2006; von Bültzingslöwen et al., 2007). Xerostomia prevalence in elderly diabetic patients varies from

adolescents with DM1 (CG > 130mg/dL); and Control group: adolescents without DM1

Table 2. Salivary characteristics and xerostomia of the studied population

Ŧ Mann-Whitney U test, † Fisher's exact test, NS non-significant (P > 0.05)

xerostomia 1 (*P* < 0.05) (Table 2). Table 2 shows the mean and the standard deviations of the salivary concentrations of total protein, amylase, urea, calcium and glucose in DM1, DM1-A, DM1-B and control groups. There were significant differences between DM1 and control groups for salivary concentrations of total protein (*P* = 0.009), calcium (*P* = 0.001), and glucose (*P* = 0.021). There were significant differences regarding total proteins (*P* = 0.007) and glucose (*P* = 0.024) salivary concentrations when DM1-A group was compared with control group. DM1-B group (adolescents with hyperglycemia) showed higher urea (*P =*  0.042), calcium (*P* < 0.001), and glucose (*P =* 0.038) salivary concentrations compared with controls.


DM1-group: adolescents with DM1, Control group: adolescents without DM1

† Fisher's exact test P ≤ 0.05, NS non-significant (P > 0.05)

Table 1. Characteristics of xerostomia of the studied population


DM1-group: adolescents with DM1; DM1-A: adolescents with DM1 (CG ≤ 130mg/dL); DM1-B: adolescents with DM1 (CG > 130mg/dL); and Control group: adolescents without DM1 SSFR (stimulated salivary flow rate)

Ŧ Mann-Whitney U test, † Fisher's exact test, NS non-significant (P > 0.05)

Impact of Hyperglycemia on Xerostomia and

430 Type 1 Diabetes Complications

xerostomia 1 (*P* < 0.05) (Table 2). Table 2 shows the mean and the standard deviations of the salivary concentrations of total protein, amylase, urea, calcium and glucose in DM1, DM1-A, DM1-B and control groups. There were significant differences between DM1 and control groups for salivary concentrations of total protein (*P* = 0.009), calcium (*P* = 0.001), and glucose (*P* = 0.021). There were significant differences regarding total proteins (*P* = 0.007) and glucose (*P* = 0.024) salivary concentrations when DM1-A group was compared with control group. DM1-B group (adolescents with hyperglycemia) showed higher urea (*P =*  0.042), calcium (*P* < 0.001), and glucose (*P =* 0.038) salivary concentrations compared with

n = 51

No 24 (47) 43 (84)

No 39 (76) 49 (96)

Normal 46 (90) 49 (96)

No 49 (94) 50 (98)

No 41 (80) 51 (100)

No 46 (90) 51 (100)

Yes 12 (24) 2 (4) 0.004 †

Low 5 (10) 2 (4) NS

Yes 3 (6) 1 (2) NS

Yes 10 (20) 0 (0) 0.001 †

Yes 5 (10) 0 (0) 0.028 †

Xerostomia 1 (dry mouth) Yes 27 (53) 8 (16) <0.001 †

Control group

n = 51 *P* value

Variables n (%) DM1-group

DM1-group: adolescents with DM1, Control group: adolescents without DM1

Table 1. Characteristics of xerostomia of the studied population

† Fisher's exact test P ≤ 0.05, NS non-significant (P > 0.05)

controls.

Need to drink

Amount of saliva

Difficulty in swallowing

perceived

Xerostomia 2

Xerostomia 3

 a p value of DM1-group X control group; b p value of DM1-A X control group; and c p value of DM1-B X control group

Table 2. Salivary characteristics and xerostomia of the studied population

In the present study, xerostomia 1 prevalence was demonstrated in 27 (53%) adolescents with DM1 (DM1 group): 11 (61%) with good glycemic control and 16 (48%) with hyperglycemia, in contrast to 8 (16%) non-diabetes ones (control group) (Tables 1 and 2). Xerostomia was significantly associated with DM1 (Table 1) regardless of hyperglycemia (Table 2). Xerostomia 2 and xerostomia 3 only occurred in DM1 group, demonstrating that xerostomia is one of the oral manifestations of diabetes (Sreebny et a.l, 2006; von Bültzingslöwen et al., 2007). Xerostomia prevalence in elderly diabetic patients varies from

Impact of Hyperglycemia on Xerostomia and

al., 1998; Iughetti et al., 1999; Mata et al., 2004).

adolescents with DM1 (Busato et al., 2009).

studies difficult.

477932/2007-0.

**5. References** 

**4. Acknowledgment** 

showed higher urea salivary concentrations compared with controls.

adolescents with hyperglycemia, renal dysfunction and diet.

al., 2004).

**3. Conclusion** 

Salivary Composition and Flow Rate of Adolescents with Type 1 Diabetes Mellitus 433

of calcium (Table 2), and calcium salivary concentration in DM1 group was significantly higher compared with that of control group, in consonance with a previous study (Mata et

In this study, the glucose salivary concentration was significantly higher in DM1, DM1-A and DM1-B groups when each one was compared with control group. Some studies (Belazi et al., 1998; López et al., 2003; Moreira et al., 2009) have shown this difference, whereas others studies (Swanljung et al., 1992; Carda et al., 2006) have not found difference in glucose salivary concentrations between subjects with and without DM1. The increased concentrations of glucose in the saliva of adolescents with DM1 may be important for controlling and monitoring the disease. It may possibly be related to blood glucose (Belazi et

There were no significant differences for urea salivary concentrations between adolescents with DM1 (DM1 group) and without DM1 (control group), which is in accordance with a previous study (Meurman et al., 1998) and contradicts others (López et al., 2003; Carda et al., 2006). Moreover, in the latters (López et al., 2003; Carda et al., 2006), subjects with DM1

In the present study, hyperglycemia was associated with an increased urea salivary concentration, with significant difference between DM1-B and control groups (Table 2). Hyperproteic diet and dysfunction of urea excretion due to renal failure may increase urea values in plasma and urine (Searcy et al., 1964). Future studies are needed to further investigate the relationship among the increased values of urea salivary concentration in

The significant difference in salivary composition and SSFR between adolescents with and without DM1 and the significantly higher xerostomia prevalence noted in adolescents who have DM1 may suggest an increased risk of dental caries and oral disease in DM1 patients. Furthermore, xerostomia has been shown to have a negative impact on the quality of life of

Moreover, there are limited accounts in the literature regarding the prevalence of hyperglycemia and its association with salivary composition, flow rate and xerostomia in adolescents with DM1, which makes direct comparisons between our study and other

Authors would like to thank the Director and the employees of the Hospital de Clínicas, Universidade Federal do Paraná. This study was supported by MCT/CNPq grant n°

American Diabetes Association-ADA. (2004). Diagnosis and classification of diabetes mellitus. *Diabetes Care,* Vol. 27, No. Suppl 1, (jan), pp. (S5-10), ISSN 1935-5548 American Diabetes Association-ADA. (2006). Standards medical care in diabetes-2006. *Diabetes Care*, Vol. 29, No. Suppl 1, (jan), pp. (S4-42), ISSN 1935-5548

24.1% in patients with DM1 (Moore *et al* 2001) up to 76.4% in patients with type 2 DM (Carda et al., 2006). Moreover, there are limited accounts in the literature regarding the prevalence of xerostomia in adolescents with DM1, which makes direct comparisons between our study and other studies in adults difficult.

The need to drink liquids during meals was reported by 12 (24%) adolescents with DM1 and by 2 (4%) adolescents without diabetes (*P =* 0.004, Table 1). Nevertheless, in spite of this relationship between DM1 and "need to drink", it should be emphasized that family and individual habits may be related to this relationship. The habit of drinking juices, soft drinks or even water during meals is very common and frequently does not indicate a real necessity to drink in order to be able to swallow food. The clinical importance of the need to drink during meals among adolescents with DM1 needs to be further investigated in other studies.

In this study, average SSFR was 0.932 mL/min in DM1 group and 1.224 mL/min in control group (*P* = 0.003). In DM1-A group, average SSFR was 1.140 mL/min, presenting no significant difference compared with controls (*P* > 0.05) (Table 2). In the hyperglycemic subjects group (DM1-B), average SSFR was 0.812 mL/min. There was significant difference for SSFR between DM1-B and control groups (*P* = 0.002) (Table 2). Average SSFR values vary from 0.79 mL/min in children and adolescents with DM1 (Belazi et al., 1998) reaching 1.17 mL/min in adolescents with DM1 (Siudikiene et al., 2006). The latter value (Siudikiene et al., 2006) is similar to the average SSFR in well-controlled adolescents in the present study (DM1-A group, 1.140 mL/min, Table 2). The average SSFR value was significantly different between DM1 group (0.932 mL/min) and control group (1.224 mL/min), which is in consonance with previous studies with adolescents with DM1 (Siudikiene et al., 2006, 2008). In the present study, hyperglycemia was associated to a reduction in salivary flow (Table 2). This result agrees with a previous study, where it was suggested that it might be that the overall dehydration associated with hyperglycemia decreased the volume of saliva excreted (Karjalainen et al., 1996). Low salivary flow can influence increased caries experience in DM patients (Siudikiene et al., 2006, Márton et al., 2008). Furthermore, the subjective feeling of dry mouth (xerostomia) may result from a reduction in saliva secretion and was shown to have a negative impact on the quality of life of adolescents with DM1 (Busato et al., 2009).

Saliva contains immunological and non-immunological proteins with antibacterial properties (Humphrey & Williamson, 2001). In this study, good glycemic control (DM1- A group) was associated to a decrease in total proteins salivary concentration compared with controls. Conversely, there was no significant difference for salivary concentration of total proteins in the presence of hyperglycemia (DM1-B group) compared with nondiabetic subjects (control group). Moreover, amylase salivary concentration in adolescents with DM1 did not show significant differences compared with controls. Previous studies (Twetman et al., 2002; Mata et al., 2004; Carda et al., 2006; Moreira et al., 2009) have shown significant differences in total proteins salivary concentrations between subjects with and without DM1. Others studies are needed to further investigate the association of hypoglycemia with total proteins salivary concentrations in adolescents with DM1.

Salivary calcium concentration has a fundamental role in maintaining tooth integrity though the modulation of remineralization and demineralization (Humphrey & Williamson, 2001). In the present study, hyperglycemia was associated to an increase in salivary concentration of calcium (Table 2), and calcium salivary concentration in DM1 group was significantly higher compared with that of control group, in consonance with a previous study (Mata et al., 2004).

### **3. Conclusion**

432 Type 1 Diabetes Complications

24.1% in patients with DM1 (Moore *et al* 2001) up to 76.4% in patients with type 2 DM (Carda et al., 2006). Moreover, there are limited accounts in the literature regarding the prevalence of xerostomia in adolescents with DM1, which makes direct comparisons

The need to drink liquids during meals was reported by 12 (24%) adolescents with DM1 and by 2 (4%) adolescents without diabetes (*P =* 0.004, Table 1). Nevertheless, in spite of this relationship between DM1 and "need to drink", it should be emphasized that family and individual habits may be related to this relationship. The habit of drinking juices, soft drinks or even water during meals is very common and frequently does not indicate a real necessity to drink in order to be able to swallow food. The clinical importance of the need to drink during meals among adolescents with DM1 needs to be further investigated in other

In this study, average SSFR was 0.932 mL/min in DM1 group and 1.224 mL/min in control group (*P* = 0.003). In DM1-A group, average SSFR was 1.140 mL/min, presenting no significant difference compared with controls (*P* > 0.05) (Table 2). In the hyperglycemic subjects group (DM1-B), average SSFR was 0.812 mL/min. There was significant difference for SSFR between DM1-B and control groups (*P* = 0.002) (Table 2). Average SSFR values vary from 0.79 mL/min in children and adolescents with DM1 (Belazi et al., 1998) reaching 1.17 mL/min in adolescents with DM1 (Siudikiene et al., 2006). The latter value (Siudikiene et al., 2006) is similar to the average SSFR in well-controlled adolescents in the present study (DM1-A group, 1.140 mL/min, Table 2). The average SSFR value was significantly different between DM1 group (0.932 mL/min) and control group (1.224 mL/min), which is in consonance with previous studies with adolescents with DM1 (Siudikiene et al., 2006, 2008). In the present study, hyperglycemia was associated to a reduction in salivary flow (Table 2). This result agrees with a previous study, where it was suggested that it might be that the overall dehydration associated with hyperglycemia decreased the volume of saliva excreted (Karjalainen et al., 1996). Low salivary flow can influence increased caries experience in DM patients (Siudikiene et al., 2006, Márton et al., 2008). Furthermore, the subjective feeling of dry mouth (xerostomia) may result from a reduction in saliva secretion and was shown to have a negative impact on the quality of

Saliva contains immunological and non-immunological proteins with antibacterial properties (Humphrey & Williamson, 2001). In this study, good glycemic control (DM1- A group) was associated to a decrease in total proteins salivary concentration compared with controls. Conversely, there was no significant difference for salivary concentration of total proteins in the presence of hyperglycemia (DM1-B group) compared with nondiabetic subjects (control group). Moreover, amylase salivary concentration in adolescents with DM1 did not show significant differences compared with controls. Previous studies (Twetman et al., 2002; Mata et al., 2004; Carda et al., 2006; Moreira et al., 2009) have shown significant differences in total proteins salivary concentrations between subjects with and without DM1. Others studies are needed to further investigate the association of hypoglycemia with total proteins salivary concentrations in

Salivary calcium concentration has a fundamental role in maintaining tooth integrity though the modulation of remineralization and demineralization (Humphrey & Williamson, 2001). In the present study, hyperglycemia was associated to an increase in salivary concentration

between our study and other studies in adults difficult.

life of adolescents with DM1 (Busato et al., 2009).

adolescents with DM1.

studies.

In this study, the glucose salivary concentration was significantly higher in DM1, DM1-A and DM1-B groups when each one was compared with control group. Some studies (Belazi et al., 1998; López et al., 2003; Moreira et al., 2009) have shown this difference, whereas others studies (Swanljung et al., 1992; Carda et al., 2006) have not found difference in glucose salivary concentrations between subjects with and without DM1. The increased concentrations of glucose in the saliva of adolescents with DM1 may be important for controlling and monitoring the disease. It may possibly be related to blood glucose (Belazi et al., 1998; Iughetti et al., 1999; Mata et al., 2004).

There were no significant differences for urea salivary concentrations between adolescents with DM1 (DM1 group) and without DM1 (control group), which is in accordance with a previous study (Meurman et al., 1998) and contradicts others (López et al., 2003; Carda et al., 2006). Moreover, in the latters (López et al., 2003; Carda et al., 2006), subjects with DM1 showed higher urea salivary concentrations compared with controls.

In the present study, hyperglycemia was associated with an increased urea salivary concentration, with significant difference between DM1-B and control groups (Table 2). Hyperproteic diet and dysfunction of urea excretion due to renal failure may increase urea values in plasma and urine (Searcy et al., 1964). Future studies are needed to further investigate the relationship among the increased values of urea salivary concentration in adolescents with hyperglycemia, renal dysfunction and diet.

The significant difference in salivary composition and SSFR between adolescents with and without DM1 and the significantly higher xerostomia prevalence noted in adolescents who have DM1 may suggest an increased risk of dental caries and oral disease in DM1 patients. Furthermore, xerostomia has been shown to have a negative impact on the quality of life of adolescents with DM1 (Busato et al., 2009).

Moreover, there are limited accounts in the literature regarding the prevalence of hyperglycemia and its association with salivary composition, flow rate and xerostomia in adolescents with DM1, which makes direct comparisons between our study and other studies difficult.

#### **4. Acknowledgment**

Authors would like to thank the Director and the employees of the Hospital de Clínicas, Universidade Federal do Paraná. This study was supported by MCT/CNPq grant n° 477932/2007-0.

#### **5. References**

American Diabetes Association-ADA. (2004). Diagnosis and classification of diabetes mellitus. *Diabetes Care,* Vol. 27, No. Suppl 1, (jan), pp. (S5-10), ISSN 1935-5548

American Diabetes Association-ADA. (2006). Standards medical care in diabetes-2006. *Diabetes Care*, Vol. 29, No. Suppl 1, (jan), pp. (S4-42), ISSN 1935-5548

Impact of Hyperglycemia on Xerostomia and

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Moreira AR., Passos LA., Sampaio FC., Soares MSM. & Oliveira RJ. (2009). Flow rate, pH

Orbak R., Simsek S., Orbak Z., Kavrut F. & Colak M. (2008). The influence of type-1 diabetes

Scully C. (2003). Drugs effects on salivary glands: dry mouth. *Oral Dis,* Vol. 9, No.4, (jul), pp.

Searcy RL., Korotzer JL., Douglas GL. & Bergquist LM. (1964). Quantitation of serum urea as

Siudikiene J., Machiulskiene V., Nyvad B., Tenovuo J. & Nedzelskiene I. (2006). Dental

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Syjälä A-M H., Niskanen MC., Ylöstalo P. & Knuuttila MLE. (2003). Metabolic control

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**21** 

*1Japan* 

*3Egypt* 

*2Saudia Arabia* 

**Craniofacial Complex** 

*King AbdulAziz University, 3Alexandria University* 

Mona Abbassy1,2,3, Ippei Watari1 and Takashi Ono1

**The Effect of Type 1 Diabetes Mellitus on the** 

Diabetes mellitus (DM) is one of the systemic diseases affecting a considerable number of patients (Bensch *et al.*, 2003). Numerous experimental and clinical studies on the complications of diabetes mellitus have demonstrated extensive alterations in bone and mineral metabolism, linear growth, and body composition (Giglio and Lama, 2001). DM is a metabolic disorder characterized by disturbed glucose metabolism, manifesting primarily as

Bone metabolism is impaired in Type 1 Diabetes Mellitus (T1DM), either through the direct effect of insulin deficiency or hyperglycaemia or via the more long-term effects of vascular disease. Previous data suggested that osteoblast function can be corrected by restoration of glycaemic control, indicating that hyperglycaemia produces direct impairment of bone

Growth is a highly complex mechanism resulting not only in a major change in size, but also in minor changes in shape. The changes in shape of the craniofacial skeleton are of particular interest because pathological growth of certain anatomical regions may lead to functional and esthetic problems. In the craniofacial skeleton, it has been well known that growth involves a mosaic of growth sites that grow at different rates and mature at different times (Vandeberg *et al.*, 2004a; VandeBerg *et al.*, 2004b), its growth and by analogy, the response to growth disruption is much more complex than that of the appendicular skeleton (Vandeberg *et al.*, 2004a). Different parts of the craniofacial complex might be expected to respond differently to the same hormonal or biomechanical stimulus (VandeBerg *et al.*, 2004b). T1DM has been shown to affect the general growth of patients due to insulin

deficiency and consequently leads to delayed skeletal maturation (Schwartz, 2003).

Bone forms in two ways, resulting in two types of mature bone – intramembranous and cartilage. Cartilage bone forms in a replacement process within the cartilage models of the

**2. Types of bone formation in craniofacial complex** 

**1. Introduction** 

chronic hyperglycemia.

metabolism (Shyng *et al.*, 2001).

*1Orthodontic Science, Department of Orofacial Development and Function, Division of Oral Health Sciences, Tokyo Medical and Dental University, 2Preventive Dental Sciences Department, Orthodontic Department Devision,* 

von Bültzingslöwen I., Sollecito TP., Fox PC., Daniels T., Jonsson R., Lockhart PB., Wray D., Brennan MT., Carrozzo M., Gandera B., Fujibayashi T., Navazesh M., Rhodus NL. & Schiødt M. (2007). Salivary dysfunction associated with systemic diseases: systematic review and clinical management recommendations. *Oral Surg Oral Med Oral Pathol Oral Radiol Endod,* Vol. 103, No.suppl 1, (mar), pp. (S57.e1-15), ISSN 1528-395X

### **The Effect of Type 1 Diabetes Mellitus on the Craniofacial Complex**

Mona Abbassy1,2,3, Ippei Watari1 and Takashi Ono1

*1Orthodontic Science, Department of Orofacial Development and Function, Division of Oral Health Sciences, Tokyo Medical and Dental University, 2Preventive Dental Sciences Department, Orthodontic Department Devision, King AbdulAziz University, 3Alexandria University 1Japan 2Saudia Arabia 3Egypt* 

#### **1. Introduction**

436 Type 1 Diabetes Complications

von Bültzingslöwen I., Sollecito TP., Fox PC., Daniels T., Jonsson R., Lockhart PB., Wray D.,

1528-395X

Brennan MT., Carrozzo M., Gandera B., Fujibayashi T., Navazesh M., Rhodus NL. & Schiødt M. (2007). Salivary dysfunction associated with systemic diseases: systematic review and clinical management recommendations. *Oral Surg Oral Med Oral Pathol Oral Radiol Endod,* Vol. 103, No.suppl 1, (mar), pp. (S57.e1-15), ISSN

> Diabetes mellitus (DM) is one of the systemic diseases affecting a considerable number of patients (Bensch *et al.*, 2003). Numerous experimental and clinical studies on the complications of diabetes mellitus have demonstrated extensive alterations in bone and mineral metabolism, linear growth, and body composition (Giglio and Lama, 2001). DM is a metabolic disorder characterized by disturbed glucose metabolism, manifesting primarily as chronic hyperglycemia.

> Bone metabolism is impaired in Type 1 Diabetes Mellitus (T1DM), either through the direct effect of insulin deficiency or hyperglycaemia or via the more long-term effects of vascular disease. Previous data suggested that osteoblast function can be corrected by restoration of glycaemic control, indicating that hyperglycaemia produces direct impairment of bone metabolism (Shyng *et al.*, 2001).

> Growth is a highly complex mechanism resulting not only in a major change in size, but also in minor changes in shape. The changes in shape of the craniofacial skeleton are of particular interest because pathological growth of certain anatomical regions may lead to functional and esthetic problems. In the craniofacial skeleton, it has been well known that growth involves a mosaic of growth sites that grow at different rates and mature at different times (Vandeberg *et al.*, 2004a; VandeBerg *et al.*, 2004b), its growth and by analogy, the response to growth disruption is much more complex than that of the appendicular skeleton (Vandeberg *et al.*, 2004a). Different parts of the craniofacial complex might be expected to respond differently to the same hormonal or biomechanical stimulus (VandeBerg *et al.*, 2004b). T1DM has been shown to affect the general growth of patients due to insulin deficiency and consequently leads to delayed skeletal maturation (Schwartz, 2003).

#### **2. Types of bone formation in craniofacial complex**

Bone forms in two ways, resulting in two types of mature bone – intramembranous and cartilage. Cartilage bone forms in a replacement process within the cartilage models of the

The Effect of Type 1 Diabetes Mellitus on the Craniofacial Complex 439

Numerous experimental and clinical studies on the complications of DM have demonstrated extensive alterations in bone and mineral metabolism, linear growth, and body composition. Investigators in the fields of bone biology including orthodontics have long been interested in the general causes that affect the normal growth of the craniofacial region. T1DM has been shown to affect the general growth of patients with earlier onset of the disease,

In general, growth of the craniofacial complex is controlled by genetic and environmental factors (Giglio and Lama, 2001; Yonemitsu *et al.*, 2007). Regulatory mechanisms responsible for normal morphogenesis of the face and head involve hormones, nutrients, mechanical forces, and various local growth factors. The poor growth and alterations in bone metabolism have been associated with T1DM in both humans and experimental animals (Giglio and Lama, 2001). It is of prime importance investigating the changes in craniofacial bone structure and dynamic bone formation in DM condition to explore the impact of the

Investigating the effects of juvenile diabetes on general craniofacial growth and skeletal

Analyzing the pattern of association between craniofacial morphology and skeletal

Determination of the changes in bone morphology in diabetic rat mandible using micro-

Determination of the mineral apposition rate and the bone formation rate in diabetic rat

The animal studies using diabetic model presents various advantages when compared to studies carried out on human diabetic cases. Human studies can be limited by small sample sizes, cross-sectional designs, uncontrolled variables, and often retrospective nature, animal

Experimental diabetic models include the streptozotocin-induced diabetic rat and the spontaneously diabetic BioBreeding rat. The occurrence of different abnormalities indicating altered bone formation after inducing DM with streptozotocin (STZ) is well documented (Giglio and Lama, 2001; Hough *et al.*, 1981; Tein *et al.*, 1998). Streptozotocin-induced diabetes mellitus (STZ-DM), caused by the destruction of pancreatic β-cells and is similar to T1DM in human, is characterized by mild to moderate hyperglycemia, glucosuria, polyphagia, hypoinsulinemia, hyperlipidemia, and weight loss. STZ-DM also exhibits many of the complications observed in human DM including enhanced susceptibility to infection and cardiovascular disease, retinopathy, alterations in angiogenesis, delayed wound healing, diminished growth factor expression, and reduced bone formation (Lu *et al.*, 2003).

In the usual clinical situation, although T1DM patient is treated with insulin, patient may still suffer from an overall poor diabetic metabolic state with an uncontrollable blood glucose level and a high and sometimes changing insulin requirement (Follak *et al.*, 2004).

models have been used to yield more rigorous analyses (Singleton *et al.*, 2006).

**5.2 Importance of testing the uncontrolled diabetic condition** 

especially onset before or around the circumpubertal growth spurts (Chew, 1991).

diabetic condition on various mandibular growth elements and bone quality. **The following parts of this chapter are going to focus on these points:** 

mandible using histomorphometric analysis.

**5. Animal and experimental diabetic model** 

maturation.

maturation.

**5.1 Diabetic model** 

C.T.

embryo and infant. Intramembranous bone forms through the activation of the osteoblast cell or specialized bone forming cells in one of the layers of fetal connective tissue. The bones of the cranial vault, the face, and the clavicle are intramembranous in origin. All other bones of the body form from cartilage. Intramembranous bone include the mandible, the maxilla, the premaxilla, the frontal bone, the palatine bone, the squamous part of temporal bone, the zygomatic bone, the medial plate of the pterygoid process, the vomer, the tympanic part of the temporal bone, the nasal bone, the lacrimal bone, and the parietal bone. The original pattern of intramembranous bone changes with progressive maturative growth when these bones begin to adapt to environmental influences. This accounts for deformities due to malfunction, disease and other environmental factors (Salzmann, 1979).

### **3. Causes of growth problems**

Growth disturbances can be associated with specific anatomic or functional defects. They may be of endocrine or non endocrine origin and may result from genetic, nutritional or environmental factors. Disturbances in somatic growth show themselves in retardation or acceleration of the skeletal system, including the facial and cranial bones. Causes for growth problems usually fall into the following categories (Kumar, 2009):

**Familial short stature**

Familial short stature is a tendency to follow the family's inherited short stature (shortness).


#### **4. Outline of studying the effect of diabetes mellitus on craniofacial growth**

Approximately 60% of adult bone mass including craniofacial bone is gained during the peak of the growth period which coincides with the onset of T1DM condition affecting the bone formation process (Eastell and Lambert, 2002). It is worth mentioning here that although T1DM condition exact etiological factors are totally unknown however; understanding the course of T1DM condition and its impact on craniofacial development may lead to improving the oral health for a large sector of the population worldwide.

Numerous experimental and clinical studies on the complications of DM have demonstrated extensive alterations in bone and mineral metabolism, linear growth, and body composition. Investigators in the fields of bone biology including orthodontics have long been interested in the general causes that affect the normal growth of the craniofacial region. T1DM has been shown to affect the general growth of patients with earlier onset of the disease, especially onset before or around the circumpubertal growth spurts (Chew, 1991).

In general, growth of the craniofacial complex is controlled by genetic and environmental factors (Giglio and Lama, 2001; Yonemitsu *et al.*, 2007). Regulatory mechanisms responsible for normal morphogenesis of the face and head involve hormones, nutrients, mechanical forces, and various local growth factors. The poor growth and alterations in bone metabolism have been associated with T1DM in both humans and experimental animals (Giglio and Lama, 2001). It is of prime importance investigating the changes in craniofacial bone structure and dynamic bone formation in DM condition to explore the impact of the diabetic condition on various mandibular growth elements and bone quality.

#### **The following parts of this chapter are going to focus on these points:**


#### **5. Animal and experimental diabetic model**

The animal studies using diabetic model presents various advantages when compared to studies carried out on human diabetic cases. Human studies can be limited by small sample sizes, cross-sectional designs, uncontrolled variables, and often retrospective nature, animal models have been used to yield more rigorous analyses (Singleton *et al.*, 2006).

#### **5.1 Diabetic model**

438 Type 1 Diabetes Complications

embryo and infant. Intramembranous bone forms through the activation of the osteoblast cell or specialized bone forming cells in one of the layers of fetal connective tissue. The bones of the cranial vault, the face, and the clavicle are intramembranous in origin. All other bones of the body form from cartilage. Intramembranous bone include the mandible, the maxilla, the premaxilla, the frontal bone, the palatine bone, the squamous part of temporal bone, the zygomatic bone, the medial plate of the pterygoid process, the vomer, the tympanic part of the temporal bone, the nasal bone, the lacrimal bone, and the parietal bone. The original pattern of intramembranous bone changes with progressive maturative growth when these bones begin to adapt to environmental influences. This accounts for deformities

Growth disturbances can be associated with specific anatomic or functional defects. They may be of endocrine or non endocrine origin and may result from genetic, nutritional or environmental factors. Disturbances in somatic growth show themselves in retardation or acceleration of the skeletal system, including the facial and cranial bones. Causes for growth

Familial short stature is a tendency to follow the family's inherited short stature

A child who tends to be shorter than average and who enters puberty later than average, but is growing at a normal rate. Most of these children tend to eventually grow

Constant malnutrition, digestive tract diseases, kidney disease, heart disease, lung

Adequate production of the thyroid hormone is necessary for normal bone growth. Cushing's syndrome can be caused by a myriad of abnormalities that are the result of hypersecretion of corticosteroids by the adrenal gland. Growth hormone deficiency involves a problem with the pituitary gland (small gland at the base of the brain) that

A condition called intrauterine growth restriction (IUGR), slow growth within the uterus occurs during a pregnancy. The baby is born smaller in weight and length than

**4. Outline of studying the effect of diabetes mellitus on craniofacial growth**  Approximately 60% of adult bone mass including craniofacial bone is gained during the peak of the growth period which coincides with the onset of T1DM condition affecting the bone formation process (Eastell and Lambert, 2002). It is worth mentioning here that although T1DM condition exact etiological factors are totally unknown however; understanding the course of T1DM condition and its impact on craniofacial development

may lead to improving the oral health for a large sector of the population worldwide.

disease, hepatic disease, diabetes, and severe stress can cause growth problems.

**Congenital (present at birth) problems in the tissues where growth occurs**

**Constitutional growth delay with delayed adolescence or delayed maturation**

due to malfunction, disease and other environmental factors (Salzmann, 1979).

problems usually fall into the following categories (Kumar, 2009):

to approximately the same height as their parents.

secretes several hormones, including growth hormone.

normal, in proportion to his/her short stature.

**Illnesses that affect the whole body (Also called systemic diseases)**

**3. Causes of growth problems** 

**Endocrine (hormone) diseases**

**Familial short stature**

(shortness).

Experimental diabetic models include the streptozotocin-induced diabetic rat and the spontaneously diabetic BioBreeding rat. The occurrence of different abnormalities indicating altered bone formation after inducing DM with streptozotocin (STZ) is well documented (Giglio and Lama, 2001; Hough *et al.*, 1981; Tein *et al.*, 1998). Streptozotocin-induced diabetes mellitus (STZ-DM), caused by the destruction of pancreatic β-cells and is similar to T1DM in human, is characterized by mild to moderate hyperglycemia, glucosuria, polyphagia, hypoinsulinemia, hyperlipidemia, and weight loss. STZ-DM also exhibits many of the complications observed in human DM including enhanced susceptibility to infection and cardiovascular disease, retinopathy, alterations in angiogenesis, delayed wound healing, diminished growth factor expression, and reduced bone formation (Lu *et al.*, 2003).

#### **5.2 Importance of testing the uncontrolled diabetic condition**

In the usual clinical situation, although T1DM patient is treated with insulin, patient may still suffer from an overall poor diabetic metabolic state with an uncontrollable blood glucose level and a high and sometimes changing insulin requirement (Follak *et al.*, 2004).

The Effect of Type 1 Diabetes Mellitus on the Craniofacial Complex 441

\* \*

\*

Control DM

Fig. 2. Line graph represents the blood glucose levels for the control and DM group. The blood glucose level in DM group increased significantly 48h post-STZ injection and during the entire experimental period. Values are mean±SD. Significant differences between the

Day 0 Day 2 Day 7 Day 14 Day 21 Day 28 **Experimental duration after STZ injection**

**6. Analytic studies conducted to test the effect of diabetic condition on** 

Cephalometric measurements are still one of the most widely spread diagnostic aids crucial for the diagnosis of various abnormalities in the craniofacial complex (Chidiac *et al.*, 2002). The protocol for examining the cephalometric measurements in DM rats involves the

 Prior to each radiographic session, the rats are anaesthetized with diethyl ether and intraperitoneal injection of 8% chloral hydrate using 0.5ml/100g of body weight. Each animal is then placed in this specially-designed apparatus (Fig. 3) to maintain standardized head posture and contact with the film (SGP-3, Mitsutoy, Tokyo, Japan) where the head of each rat is fixed firmly with a pair of ear rods oriented vertically to

 The settings of lateral and dorsoventral cephalometric radiographs are 50/55kVp, 15/10mA, and 20/60-sec impulses respectively (Singleton *et al.*, 2006; Vandeberg *et al.*,

A 10 mm steel calibration rod is incorporated into the clear acrylic table on which the

All the radiographs are developed and scanned at high resolution by the same operator

The cephalometric landmarks (Table 1; Fig. 4) were derived from previous studies on rodents (Engstrom *et al.*, 1988; Kiliaridis and Thilander, 1985; Singleton *et al.*, 2006; Vandeberg *et al.*, 2004a; VandeBerg *et al.*, 2004b). Selected linear measurements were then

the sagittal plane and the incisors are fixed into a plastic ring.

two groups are marked with asterisks (*p*< 0.05).

0

100

200

300

**Blood glucose level in mg/dl**

400

500

600

\*

\*

**craniofacial growth** 

(b)

following steps:

**6.1 Cephalometric analysis** 

2004a; VandeBerg *et al.*, 2004b).

(Singleton *et al.*, 2006).

animals are positioned for the radiographs.

#### **5.3 Inducing diabetic condition**

All the experimental protocols followed had been approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University, and the experiments were carried out under the control of the University's Guidelines for Animal Experimentation. In our investigation we explored the various effects of DM using the streptozotocin DM model. Twelve 3-week old male Wistar rats were used for this study. They were randomly divided into two groups, the control group and the diabetes group (DM group), each group consists of 6 rats. The rats in the control group were injected intraperitoneal with a single dose of 0.1M sodium citrate buffer (pH 4.5), while the rats in the DM group were injected intra-peritoneal with a single dose of citrate buffer containing 60mg/kg body weight of streptozotocin (Sigma Chemical Co., St. Louis, MO, USA).(Alkan *et al.*, 2002; McCracken *et al.*, 2006; Shyng *et al.*, 2001; Tein *et al.*, 1998) All animals were fed on standard Rodent diet (Rodent Diet CE-2; Japan Clea Inc., Shizuoka, Japan) with free access to water. Body weights, the presence of glucose in urine and blood glucose levels were recorded on day 0, 2, 7, 14, 21 and 28 after STZ injection.

Diabetes condition was determined by the presence of glucose in urine and blood. The urine of the rats was tested using reagent strips (Uriace Ga; TERUMO). (Abdus Salam *et al.*, 2004; Matin *et al.*, 2002) Blood samples of the rats were obtained via vein puncture of a tail vein, and blood glucose levels were determined using a glucometer (Ascensia Brio. Bayer Medical). Positive urine test and a blood glucose level greater than 200 mg/dl was considered DM.(Giglio and Lama, 2001).

Fig. 1. shows the weights of the rats (mean±SD) in both groups. DM group showed a significant decrease in weight. After STZ injection by 48 hours the urine test showed that the entire DM group had a high glucose level and this was confirmed by the high blood glucose measurements as shown in Fig. 2. A Student's t-test was used to compare the mean of weights and blood glucose levels in both groups.

Fig. 1. Comparison between the changes of the rat's weight in the control and DM group. The weights of the DM group are significantly decreased as compared to the weights of the control group (\*:*p<0.05*).

All the experimental protocols followed had been approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University, and the experiments were carried out under the control of the University's Guidelines for Animal Experimentation. In our investigation we explored the various effects of DM using the streptozotocin DM model. Twelve 3-week old male Wistar rats were used for this study. They were randomly divided into two groups, the control group and the diabetes group (DM group), each group consists of 6 rats. The rats in the control group were injected intraperitoneal with a single dose of 0.1M sodium citrate buffer (pH 4.5), while the rats in the DM group were injected intra-peritoneal with a single dose of citrate buffer containing 60mg/kg body weight of streptozotocin (Sigma Chemical Co., St. Louis, MO, USA).(Alkan *et al.*, 2002; McCracken *et al.*, 2006; Shyng *et al.*, 2001; Tein *et al.*, 1998) All animals were fed on standard Rodent diet (Rodent Diet CE-2; Japan Clea Inc., Shizuoka, Japan) with free access to water. Body weights, the presence of glucose in urine and blood glucose levels were recorded on

Diabetes condition was determined by the presence of glucose in urine and blood. The urine of the rats was tested using reagent strips (Uriace Ga; TERUMO). (Abdus Salam *et al.*, 2004; Matin *et al.*, 2002) Blood samples of the rats were obtained via vein puncture of a tail vein, and blood glucose levels were determined using a glucometer (Ascensia Brio. Bayer Medical). Positive urine test and a blood glucose level greater than 200 mg/dl was

Fig. 1. shows the weights of the rats (mean±SD) in both groups. DM group showed a significant decrease in weight. After STZ injection by 48 hours the urine test showed that the entire DM group had a high glucose level and this was confirmed by the high blood glucose measurements as shown in Fig. 2. A Student's t-test was used to compare the mean

Fig. 1. Comparison between the changes of the rat's weight in the control and DM group. The weights of the DM group are significantly decreased as compared to the weights of the

0 day 2 day 7 day 14 day 21 day 28 day **Experimental duration after STZ injection**

\* \* \*

Control DM

**5.3 Inducing diabetic condition** 

day 0, 2, 7, 14, 21 and 28 after STZ injection.

considered DM.(Giglio and Lama, 2001).

0

100 200 300

**W eig h t (g )**

(a)

control group (\*:*p<0.05*).

of weights and blood glucose levels in both groups.

Fig. 2. Line graph represents the blood glucose levels for the control and DM group. The blood glucose level in DM group increased significantly 48h post-STZ injection and during the entire experimental period. Values are mean±SD. Significant differences between the two groups are marked with asterisks (*p*< 0.05).

#### **6. Analytic studies conducted to test the effect of diabetic condition on craniofacial growth**

#### **6.1 Cephalometric analysis**

Cephalometric measurements are still one of the most widely spread diagnostic aids crucial for the diagnosis of various abnormalities in the craniofacial complex (Chidiac *et al.*, 2002). The protocol for examining the cephalometric measurements in DM rats involves the following steps:


The cephalometric landmarks (Table 1; Fig. 4) were derived from previous studies on rodents (Engstrom *et al.*, 1988; Kiliaridis and Thilander, 1985; Singleton *et al.*, 2006; Vandeberg *et al.*, 2004a; VandeBerg *et al.*, 2004b). Selected linear measurements were then

The Effect of Type 1 Diabetes Mellitus on the Craniofacial Complex 443

*On the sagittal radiograph*

*On the transverse radiograph*

palatal width

N: The most anterior point on the nasal bone

Po: The most posterior and superior point on the skull

Go: The most posterior point on the mandibular ramus

E: The intersection of the frontal bone and floor of anterior cranial fossa

Mn: The most concave portion of the concavity on the inferior border of the mandibular corpus Gn: The most inferior point on the ramus that lies on a perpendicular bisector of the line Go-Mn

So: The intersection of the most anterior tympanic bulla and the superior border of the sphenoid bone

Il: The most anterior and superior point on the alveolar bone of the mandibular incisor

CB1: The most anterior point on the occipital bone at the spheno-occipital synchondrosis CB1′: The most posterior point on the sphenoid bone at the spheno-occipital synchondrosis CB2: The most anterior point on the sphenoid bone at the spheno-basispheno synchondrosis CB2′: The most posterior point on the basisphenoid bone at the spheno-basispheno-synchondrosis

Ml: The junction of the alveolar bone and the mesial surface of the first mandibular molar Mu1: The junction of the alveolar bone and the mesial surface of the first maxillary molar Mu2: The junction of the alveolar bone and the distal surface of the third maxillary molar Iu: The most anterior-inferior point on the maxilla posterior to the maxillary incisors

Z1 & Z2: The points on the lateral portion of the zygomatic arch that produce the widest width

P1 & P2: The most anterior and medial points within the temporal fossae that produce the most narrow

Go1 & Go2: The points on the angle of the mandible that produce the widest width

C1 & C2: The points on the cranium that produce the widest cranial width

Table 1. Definition of radiographic points

Ba: The most posterior and inferior point on the occipital condyle Co: The most posterior and superior point on the mandibular condyle

obtained (Table 2). To ensure reliability and replicability of each measurement, each distance was digitized twice and the two values were averaged.

Fig. 3. Apparatus for roentgenographic cephalometry

Fig. 4. Location of cephalometric points on radiographs: (A) Sagittal and (B) transverse.

#### *On the sagittal radiograph*

442 Type 1 Diabetes Complications

obtained (Table 2). To ensure reliability and replicability of each measurement, each

P1

I1

E

M1

Mu2 Mu1

CB2'

N

Iu

Z1

Gn Mn

CB2

C1

Go1

Go

Co So CB1 CB1'

**A**

(a)

Ba

(b)

Z2

Fig. 4. Location of cephalometric points on radiographs: (A) Sagittal and (B) transverse.

C2

Go2

P2

distance was digitized twice and the two values were averaged.

Fig. 3. Apparatus for roentgenographic cephalometry

Po

N: The most anterior point on the nasal bone


Ba: The most posterior and inferior point on the occipital condyle

Co: The most posterior and superior point on the mandibular condyle

Go: The most posterior point on the mandibular ramus

Mn: The most concave portion of the concavity on the inferior border of the mandibular corpus

Gn: The most inferior point on the ramus that lies on a perpendicular bisector of the line Go-Mn

Il: The most anterior and superior point on the alveolar bone of the mandibular incisor

So: The intersection of the most anterior tympanic bulla and the superior border of the sphenoid bone

CB1: The most anterior point on the occipital bone at the spheno-occipital synchondrosis

CB1′: The most posterior point on the sphenoid bone at the spheno-occipital synchondrosis

CB2: The most anterior point on the sphenoid bone at the spheno-basispheno synchondrosis

CB2′: The most posterior point on the basisphenoid bone at the spheno-basispheno-synchondrosis

Ml: The junction of the alveolar bone and the mesial surface of the first mandibular molar

Mu1: The junction of the alveolar bone and the mesial surface of the first maxillary molar

Mu2: The junction of the alveolar bone and the distal surface of the third maxillary molar

Iu: The most anterior-inferior point on the maxilla posterior to the maxillary incisors

#### *On the transverse radiograph*

Z1 & Z2: The points on the lateral portion of the zygomatic arch that produce the widest width

Go1 & Go2: The points on the angle of the mandible that produce the widest width


The Effect of Type 1 Diabetes Mellitus on the Craniofacial Complex 445

Po-N Po-E Ba-E So-E Ba-CB1 Ba-So

\*

\* \*

Control STZ

**Neurocranial measurements**

Fig. 6. Changes in the neurocranial measurements of the control and DM group. All the significant measurements are shown in this figure. Values are mean±S.D. Significant

All measurements of the viscerocranium, including the nasal length (E-N), palatal length (Mu2-Iu), midface length (CB2-Iu), and viscerocranial height (E-Mu1) showed a statistically

**Po-N** total skull length **Po-E** cranial vault length **Ba-E** total cranial base length **So-E** anterior cranial base length **Ba-CB1** occipital bone length

**Ba-So** posterior cranial base length

Table 3. Significant changes in the total skull and neurocranium

\*

differences between the two groups are marked with asterisks (*p*<0.05).

\*

\*

Fig. 5. Neurocranium

**6.1.3 Changes in the viscerocranium** 

significant decrease in DM group (Table 4, Figs. 7,8)

**Linear measurements in mm**

#### *Neurocranium*

Po-N: total skull length Po-E: cranial vault length Ba-E: total cranial base length So-E: anterior cranial base length Ba-CB1: occipital bone length CB1′-CB2: sphenoid bone length Ba-So: posterior cranial base length Po-Ba: posterior neurocranium height *Viscerocranium* E-N: nasal length Mu2-Iu: palate length CB2-Iu: midface length E-Mu1: viscerocranial height *Mandible* Go-Mn: posterior corpus length Ml-Il: anterior corpus length Co-Il: total mandibular length Co-Gn: ramus height *Transverse X-ray* Go1-Go2: Bigonial width C1-C2: Maximum cranial width P1-P2: Palatal width Z1-Z2: Bizygomatic width

Table 2. Measurements of craniofacial skeleton

In our studies, evaluation of the craniofacial growth of diabetic rats at the age of 7 weeks was done using lateral and dorsoventral cephalometric radiographs. All of the data in each experiment were confirmed the normal distribution, so a Student's t-test was used to compare the mean of each data recorded in the control group and in the DM group. All statistical analyses were performed at a 5% significance level using statistic software (v. 10; SPSS, Chicago, IL, USA).

#### **6.1.1 Changes in the total skull**

The size of total skull, denoted by Po-N, was significantly smaller in the DM group than in the control group.

#### **6.1.2 Changes in the neurocranium**

Cranial vault length (Po-E), total cranial base length (Ba-E), anterior cranial base length (So-E), Occipital bone length (Ba-CB1), and posterior cranial base length (Ba-So) showed statistically significant decrease in DM group (Table 3, Figs. 5, 6), while other dimensions exhibited no significant differences.


Table 3. Significant changes in the total skull and neurocranium

Fig. 5. Neurocranium

444 Type 1 Diabetes Complications

In our studies, evaluation of the craniofacial growth of diabetic rats at the age of 7 weeks was done using lateral and dorsoventral cephalometric radiographs. All of the data in each experiment were confirmed the normal distribution, so a Student's t-test was used to compare the mean of each data recorded in the control group and in the DM group. All statistical analyses were performed at a 5% significance level using statistic software (v. 10;

The size of total skull, denoted by Po-N, was significantly smaller in the DM group than in

Cranial vault length (Po-E), total cranial base length (Ba-E), anterior cranial base length (So-E), Occipital bone length (Ba-CB1), and posterior cranial base length (Ba-So) showed statistically significant decrease in DM group (Table 3, Figs. 5, 6), while other dimensions

*Neurocranium*

*Viscerocranium* E-N: nasal length Mu2-Iu: palate length CB2-Iu: midface length E-Mu1: viscerocranial height

Go-Mn: posterior corpus length Ml-Il: anterior corpus length Co-Il: total mandibular length

Co-Gn: ramus height *Transverse X-ray*

P1-P2: Palatal width Z1-Z2: Bizygomatic width

Table 2. Measurements of craniofacial skeleton

SPSS, Chicago, IL, USA).

the control group.

**6.1.1 Changes in the total skull** 

**6.1.2 Changes in the neurocranium** 

exhibited no significant differences.

Go1-Go2: Bigonial width C1-C2: Maximum cranial width

*Mandible*

Po-N: total skull length Po-E: cranial vault length Ba-E: total cranial base length So-E: anterior cranial base length Ba-CB1: occipital bone length CB1′-CB2: sphenoid bone length Ba-So: posterior cranial base length Po-Ba: posterior neurocranium height

Fig. 6. Changes in the neurocranial measurements of the control and DM group. All the significant measurements are shown in this figure. Values are mean±S.D. Significant differences between the two groups are marked with asterisks (*p*<0.05).

#### **6.1.3 Changes in the viscerocranium**

All measurements of the viscerocranium, including the nasal length (E-N), palatal length (Mu2-Iu), midface length (CB2-Iu), and viscerocranial height (E-Mu1) showed a statistically significant decrease in DM group (Table 4, Figs. 7,8)

The Effect of Type 1 Diabetes Mellitus on the Craniofacial Complex 447

Go-Mn Co-I1 Co-Gn

\* \* \*

Control DM

**Mandible measurements**

Fig. 10. Changes in the mandible measurements of the control and DM group. Values are mean ± S.D. Significant differences between the two groups are marked with asterisks

In transverse X-ray only the maximum cranial width (C1-C2) and the bizygomatic width

All other linear measurements showed no significant differences between both groups

(Z1-Z2) were statistically decreased in DM group (Table 6, Figs. 11, 12).

**C1-C2** Maximum cranial width **Z1-Z2** Bizygomatic width

Table 6. Significant changes in the transverse X-ray

**Go-Mn** Posterior corpus length **Co-Il** Total mandibular length

**Co-Gn** Ramus height

Table 5. Significant changes in the mandible

Fig. 9. Mandible

(*p<*0.05).

**6.1.5 Changes in the transverse X-ray** 

**Linear measurements (mm )**


Table 4. Significant changes in the viscerocranium

Fig. 7. Viscerocranium

Fig. 8. Changes in the viscerocranial measurements of the control and DM group. All the viscerocranial measurements are significant. Values are mean±S.D. Significant differences between the two groups are marked with asterisks (*p<*0.05).

#### **6.1.4 Changes in the mandible**

In the DM group, the posterior corpus length (Go-Mn), total mandibular length (Co-Il) and the ramus height (Co-Gn) were significantly shorter than in the control group (Table 5, Figs. 9, 10), whereas no remarkable differences were found in the remaining dimensions.


Table 5. Significant changes in the mandible

Fig. 9. Mandible

446 Type 1 Diabetes Complications

E-N Mu2-Iu CB2-Iu E-Mu1 **Viscerocranial measurements**

Fig. 8. Changes in the viscerocranial measurements of the control and DM group. All the viscerocranial measurements are significant. Values are mean±S.D. Significant differences

In the DM group, the posterior corpus length (Go-Mn), total mandibular length (Co-Il) and the ramus height (Co-Gn) were significantly shorter than in the control group (Table 5, Figs.

9, 10), whereas no remarkable differences were found in the remaining dimensions.

\* \* \*

between the two groups are marked with asterisks (*p<*0.05).

Control DM

\*

**E-N** Nasal length **Mu2-Iu** Palate length **Cb2-Iu** midface length

Table 4. Significant changes in the viscerocranium

Fig. 7. Viscerocranium

**6.1.4 Changes in the mandible** 

**Linear measurements ( mm )**

**E-Mu1** Posterior viscerocranial height

Fig. 10. Changes in the mandible measurements of the control and DM group. Values are mean ± S.D. Significant differences between the two groups are marked with asterisks (*p<*0.05).

#### **6.1.5 Changes in the transverse X-ray**

 In transverse X-ray only the maximum cranial width (C1-C2) and the bizygomatic width (Z1-Z2) were statistically decreased in DM group (Table 6, Figs. 11, 12).

All other linear measurements showed no significant differences between both groups


Table 6. Significant changes in the transverse X-ray

The Effect of Type 1 Diabetes Mellitus on the Craniofacial Complex 449

*DM*

**Mean SD Mean SD**

*C*

**Linear measurements**

*Neurocranium* Po-N Po-E Ba-E So-E Ba-CB1 CB1′-CB2 Ba-So

M1-I1

*Mandible* Go-Mn

Co-I1

Co-Gn *Transverse X-ray* Go1-Go2 C1-C2

> P1-P2 Z1-Z2

(DM) and controls (C)

\**p*<0.05 as compared to control values

**6.2 Histomorphometric analysis** 

combined with calcium (Stuart and Smith, 1992).

Po-Ba *Viscerocranium*

> E-N Mu2-Iu CB2-Iu E-Mu1

41.67 0.59\* 55.61 9.48 26.86 0.6\* 37.12 6.35 27.74 0.64\* 38.87 7.19 17.91 0.31\* 24.41 3.72 8.23 1.08\* 12.3 2.34 4.78 0.74 5.93 1.18 8.44 0.7\* 13.61 4.23 10.03 0.83 13.14 2.81

13.07 1.56\* 19.57 3.48 19.34 0.95\* 25.01 4.5 20.16 1.29\* 29 5.07 8.93 0.69\* 16.43 5.36

10.27 1.04\* 16 2.39 5.4 0.88 6.97 1.54 22.95 0.34\* 31.13 5.51 12.22 1.2\* 17.9 4.04

18.42 0.42 19.77 1.32 15.43 0.67\* 17.03 1.16 8.5 0.41 9.19 0.52 21.9 0.33\* 24 1.4

Table 7. Comparision of skeletal cephalometric measurements for Type 1 Diabetes mellitus

Fluorochromes are calcium binding substances that are preferentially taken up at the site of active mineralization of bone known as the calcification front, thus labeling sites of new bone formation. They are detected using fluorescent microscopy on undecalcified sections. Labeling bones with fluorochrome markers provides a means to study the dynamics of bone formation. The rate and extent of bone deposition and resorption can be determined using double and triple fluorochrome labeling sequences. The sequential use of fluorochromes of clearly contrasting colors permits a more detailed record of events relating to calcification. Flurochromes commonly used in mammals include tetracycline, calcein green, xylenol orange, alirazin red, and hematoporphyrin. Calcein is a fluoresces bright green when

**6.2.1 Fluorescent dyes used for double labeling in histomorphometric analysis**

Fig. 11. Transverse X-ray measurements

Fig. 12. Changes in the transverse X-ray measurements of the control and DM group. Two measurements in the transverse X-ray were significant. Values are mean±S.D. Significant differences between the two groups are marked with asterisks (*p*<0.05).

Z1

C1

Go1

Z2

C1-C2 Z1-Z2 **Transverse X-ray measurements**

Fig. 12. Changes in the transverse X-ray measurements of the control and DM group. Two measurements in the transverse X-ray were significant. Values are mean±S.D. Significant

C2

\*

differences between the two groups are marked with asterisks (*p*<0.05).

Go2

Fig. 11. Transverse X-ray measurements

**Linear measurements ( m m )**

P1

P2

\*

Control DM


\**p*<0.05 as compared to control values

Table 7. Comparision of skeletal cephalometric measurements for Type 1 Diabetes mellitus (DM) and controls (C)

#### **6.2 Histomorphometric analysis**

#### **6.2.1 Fluorescent dyes used for double labeling in histomorphometric analysis**

Fluorochromes are calcium binding substances that are preferentially taken up at the site of active mineralization of bone known as the calcification front, thus labeling sites of new bone formation. They are detected using fluorescent microscopy on undecalcified sections. Labeling bones with fluorochrome markers provides a means to study the dynamics of bone formation. The rate and extent of bone deposition and resorption can be determined using double and triple fluorochrome labeling sequences. The sequential use of fluorochromes of clearly contrasting colors permits a more detailed record of events relating to calcification. Flurochromes commonly used in mammals include tetracycline, calcein green, xylenol orange, alirazin red, and hematoporphyrin. Calcein is a fluoresces bright green when combined with calcium (Stuart and Smith, 1992).

The Effect of Type 1 Diabetes Mellitus on the Craniofacial Complex 451

bone surfaces; that is, CLS ═ dL + 0.5sL (Keshawarz and Recker, 1986). The mineral apposition rate (MAR, in μ/day) is determined by dividing the mean of the width of the double labels by the interlabel time (7 days). The bone formation rate (BFR) is calculated by multiplying MAR by CLS (Sheng *et al.*, 1999). Based on the reference line along the long axis of the buccal root, the area superior to the root apex was considered alveolar bone, while the area inferior to the root apex was considered the jaw bone. The lingual side of the bone is excluded,

The periosteal surfaces of the mandible are divided into four regions for analysis (Fig. 13):

The obtained results in our study showed that in the alveolar bone (region 2), there was a significant decrease in the MAR (Fig. 15A) BFR (Fig. 15B) recorded in the DM group compared to the control group. However, in the alveolar crest (region 1), the MAR and the BFR in the control and the DM groups were not significantly different. (*p*<0.05). In the buccal surface (region 3) and inferior borders (region 4) of the jaw bone the MAR (Fig. 15A) and BFR (Fig. 15B) were significantly suppressed compared with those in the control group (*p*<0.05). Most of the periosteal surfaces in the mandibular regions of the control group showed significantly higher values recorded for the mineral apposition rate and the bone formation rate when compared to the DM group. These results agree with previous studies that recorded diminished lamellar bone formation in DM rats' femur and may suggest an association between the DM condition and the decreased number and function of osteoblasts (Follak *et al.*, 2004; Shyng *et al.*, 2001). The alveolar crest region was the only region that did not show a significant difference in the mineral apposition rate and the bone formation rate parameters among the two groups; this may be attributed to the unique nature of this region exhibiting a highly intensive bone remodeling process especially during the teeth eruption that decreases toward the base of the socket (Gerlach *et al.*, 2002), however further studies are needed to elaborate the detailed pattern

Fig. 14. Frontal sections of the mandibular second molar area. (A) Control; (B) DM. Fluorescent labeling on the periosteal surface indicates new bone formation.

DM

because the existence of the incisor root may influence bone formation.

Region 3: buccal surface of the jaw bone Region 4: inferior border of the jaw bone

of bone growth at the alveolar crest region.

Control

**6.2.4 Histomorphometric indices** 

Region 1: alveolar crest (upper 1/2 of the tooth root, near the tooth crown) Region 2: alveolar bone (lower 1/2 of the tooth root, near the root apex)

#### **6.2.2 Calcein administrations and sections preparation**

The steps needed for detecting the double labeling involves the following:


Region 4: Inferior Border

Fig. 13. Schematic drawing of observation regions for dynamic bone histomorphometry. The periosteal surfaces were delimited into 4 areas as alveolar crest (region 1), alveolar bone (region 2), buccal surface of the jaw bone (region 3), and inferior border of the jaw bone (region 4).

#### **6.2.3 Method of analysis**

The bone around the lower second molar is centrally located within the mandibular arch, and because of the parallel alignment of the buccal and lingual roots this made a precise reference when frontal sections are produced (Shimomoto *et al.*, 2005) . To conduct the histomorphometric analysis it is essential to use a digitizing morphometry system to measure bone formation indices. The system consists of a confocal laser scanning microscope (LSM510, Carl Zeiss Co. Ltd., Jena, Germany), and a morphometry program (LSM Image Browser, Carl Zeiss Co. Ltd., Jena, Germany). Bone formation indices of the periosteal surfaces of the alveolar/jaw bone include mineral apposition rate (μm/day) and bone formation rate (μm3/μm2/day), according to the standard nomenclature described by (Parfitt *et al.*, 1987). The calcein-labeled surface (CLS, in mm) is calculated as the sum of the length of double labels (dL) plus one half of the length of single labels (sL) along the entire endosteal or periosteal bone surfaces; that is, CLS ═ dL + 0.5sL (Keshawarz and Recker, 1986). The mineral apposition rate (MAR, in μ/day) is determined by dividing the mean of the width of the double labels by the interlabel time (7 days). The bone formation rate (BFR) is calculated by multiplying MAR by CLS (Sheng *et al.*, 1999). Based on the reference line along the long axis of the buccal root, the area superior to the root apex was considered alveolar bone, while the area inferior to the root apex was considered the jaw bone. The lingual side of the bone is excluded, because the existence of the incisor root may influence bone formation.

The periosteal surfaces of the mandible are divided into four regions for analysis (Fig. 13):

Region 1: alveolar crest (upper 1/2 of the tooth root, near the tooth crown)

Region 2: alveolar bone (lower 1/2 of the tooth root, near the root apex)

Region 3: buccal surface of the jaw bone

Region 4: inferior border of the jaw bone

#### **6.2.4 Histomorphometric indices**

450 Type 1 Diabetes Complications

 Rats are subcutaneously injected with 50 mg/kg body weight calcein fluorescent marker on day 21 and day 28 after STZ injection. The time difference between the 2 injections is one week to be able to compare the amount of bone formed during this

Sacrifice of all animals by transcardiac perfusion under deep anesthesia using 4%

All specimens are embedded in polystyrene resin (Rigolac, Nisshin EM Co. Ltd., Tokyo,

 Undemineralized ground frontal sections are processed to show the crown and both apices of buccal and lingual roots of the lower second molar (Shimomoto *et al.*, 2007).

Fig. 13. Schematic drawing of observation regions for dynamic bone histomorphometry. The periosteal surfaces were delimited into 4 areas as alveolar crest (region 1), alveolar bone (region 2), buccal surface of the jaw bone (region 3), and inferior border of the jaw bone

The bone around the lower second molar is centrally located within the mandibular arch, and because of the parallel alignment of the buccal and lingual roots this made a precise reference

histomorphometric analysis it is essential to use a digitizing morphometry system to measure bone formation indices. The system consists of a confocal laser scanning microscope (LSM510, Carl Zeiss Co. Ltd., Jena, Germany), and a morphometry program (LSM Image Browser, Carl Zeiss Co. Ltd., Jena, Germany). Bone formation indices of the periosteal surfaces of the alveolar/jaw bone include mineral apposition rate (μm/day) and bone formation rate (μm3/μm2/day), according to the standard nomenclature described by (Parfitt *et al.*, 1987). The calcein-labeled surface (CLS, in mm) is calculated as the sum of the length of double labels (dL) plus one half of the length of single labels (sL) along the entire endosteal or periosteal

Reference Line

Region 4: Inferior Border

. To conduct the

**6.2.2 Calcein administrations and sections preparation** 

paraformaldehyde in 0.1 M phosphate buffer (pH 7.4).

Region 1: Alveolar Crest

Region 2: Alveolar Bone

Region 3: Buccal surface

when frontal sections are produced (Shimomoto *et al.*, 2005)

period (Fig. 14).

Japan).

(region 4).

**6.2.3 Method of analysis** 

The steps needed for detecting the double labeling involves the following:

Mandibles are dissected and fixed in the same solution for 24 hours.

The obtained results in our study showed that in the alveolar bone (region 2), there was a significant decrease in the MAR (Fig. 15A) BFR (Fig. 15B) recorded in the DM group compared to the control group. However, in the alveolar crest (region 1), the MAR and the BFR in the control and the DM groups were not significantly different. (*p*<0.05). In the buccal surface (region 3) and inferior borders (region 4) of the jaw bone the MAR (Fig. 15A) and BFR (Fig. 15B) were significantly suppressed compared with those in the control group (*p*<0.05). Most of the periosteal surfaces in the mandibular regions of the control group showed significantly higher values recorded for the mineral apposition rate and the bone formation rate when compared to the DM group. These results agree with previous studies that recorded diminished lamellar bone formation in DM rats' femur and may suggest an association between the DM condition and the decreased number and function of osteoblasts (Follak *et al.*, 2004; Shyng *et al.*, 2001). The alveolar crest region was the only region that did not show a significant difference in the mineral apposition rate and the bone formation rate parameters among the two groups; this may be attributed to the unique nature of this region exhibiting a highly intensive bone remodeling process especially during the teeth eruption that decreases toward the base of the socket (Gerlach *et al.*, 2002), however further studies are needed to elaborate the detailed pattern of bone growth at the alveolar crest region.

Fig. 14. Frontal sections of the mandibular second molar area. (A) Control; (B) DM. Fluorescent labeling on the periosteal surface indicates new bone formation.

The Effect of Type 1 Diabetes Mellitus on the Craniofacial Complex 453

 For observation and analysis of reconstructed 3D images, 3D trabecular structure analysis software (TRI/3D-BON; RATOC System Engineering, Tokyo, Japan) is used (Takada *et al.*, 2006). Reconstructed 3D images were prepared from slice images using the volume rendering method, to analyze the microstructure of the bone (Fig. 16). The following parameters are measured: tissue volume (TV), bone volume (BV), bone surface (BS), bone surface / bone volume (BS/BV), bone-volume fraction (BV/TV). Four properties of the trabeculae are evaluated: trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), and Trabecular space (Tb.S) (Nakano *et* 

The quantification of micro-CT trabecular bone changes (mean±SD) is shown for the DM and the control groups in (Table 4). All trabecular parameters in both alveolar bone and buccal surface of jaw bone showed significant changes. Compared with the control group, bone volume fraction (BV/TV) was significantly decreased only in the alveolar bone; however, trabecular thickness (Tb.Th) and trabecular numbers (Tb.N) were significantly decreased both in alveolar and buccal surface of jaw bone, in the DM group. Correspondingly, significantly higher trabecular separation (Tb.Sp) and trabecular space (Tb.S) were revealed both in alveolar and buccal surface of jaw bone for the DM group when compared with that of the control group. Also, the bone surface / bone volume (BS/BV) was significantly increased only in alveolar bone (*p*<0.05). These findings indicate deterioration of the bone quality in the DM group. These results agree with other research work suggesting that the glycaemic levels play an important role in modulating the

The DM condition resulted in alteration of the trabecular distance and thickness as compared to the control group indicating profound impact on the histological integrity of the bone. The reduction in trabecular bone volume accompanied by the expansion of the bone marrow space is in agreement with another investigation (Duarte *et al.*, 2005). In this context, these results may describe a state of osteopenia in experimental diabetic rats, which

trabecular architecture especially in mandibular bone (Thrailkill *et al.*, 2005).

might be the result of an imbalance between bone formation and resorption

*al.*, 2004; Takada *et al.*, 2006).

Fig. 16. The left mandible was imaged by micro-CT

**6.3.2 Microtomography of the DM mandible** 

Fig. 15. (A) The changes in mineral apposition rate (MAR) of the mandible between the control group and the DM group. Alveolar crest (region 1, upper 1/2 of the tooth root, near the tooth crown). Alveolar bone (region 2, lower 1/2 of the tooth root, near the root apex). Buccal surface of the jaw bone (region 3). Inferior border of the jaw bone (region 4). The data are expressed as means ± SD. n = 5 for each group. \*:*p*<0.05. (B) The changes in the bone formation rate (BFR/BS) of the mandible between the control group and the DM group. Alveolar crest (region 1, upper 1/2 of the tooth root, near the tooth crown). Alveolar bone (region 2, lower 1/2 of the tooth root, near the root apex). Buccal surface of the jaw bone (region 3). Inferior border of the jaw bone (region 4). The data are expressed as means ± S.D. n = 5 for each group. \*:*p*<0.05.

#### **6.3 Microtomography of the mandible (micro-CT)**

Micro-computed tomography (micro-CT) has rapidly become a standard technique for the visualization and quantification of the 3D structure of trabecular bone. Bone architecture and mineralization are generally considered to be important components of bone quality, and determine bone strength in conjunction with bone mineral density.

#### **6.3.1 Protocol adopted to examine the mandible using micro-CT**

In our study all specimens were imaged by micro-CT (inspeXio SMX-90CT; Shimadzu Science East Corporation, Tokyo, Japan)


Control DM

A B Fig. 15. (A) The changes in mineral apposition rate (MAR) of the mandible between the control group and the DM group. Alveolar crest (region 1, upper 1/2 of the tooth root, near the tooth crown). Alveolar bone (region 2, lower 1/2 of the tooth root, near the root apex). Buccal surface of the jaw bone (region 3). Inferior border of the jaw bone (region 4). The data are expressed as means ± SD. n = 5 for each group. \*:*p*<0.05. (B) The changes in the bone formation rate (BFR/BS) of the mandible between the control group and the DM group. Alveolar crest (region 1, upper 1/2 of the tooth root, near the tooth crown). Alveolar bone (region 2, lower 1/2 of the tooth root, near the root apex). Buccal surface of the jaw bone (region 3). Inferior border of the jaw bone (region 4). The data are expressed as means ± S.D.

Micro-computed tomography (micro-CT) has rapidly become a standard technique for the visualization and quantification of the 3D structure of trabecular bone. Bone architecture and mineralization are generally considered to be important components of bone quality,

In our study all specimens were imaged by micro-CT (inspeXio SMX-90CT; Shimadzu

After removing only the soft tissue, the mandibular plane is set orthogonal to the

Three dimensional images of each hemi mandible are acquired with a resolution voxel

 Raw data are obtained by rotating the sample stage 360 degrees. Then, slice images are prepared using multi-tomographic image reconstruction software (MultiBP;

The resulting gray-scale images are segmented using a low-pass filter to remove noise

 The volume of interest is drawn on a slice-based method starting from the first slice containing the crown of the first molar and moving dorsally 100 slices (Laib and Ruegsegger, 1999; Zhang *et al.*, 2008), in the area of the alveolar crest (Between the buccal and lingual roots of the second molar at the cervical region); and the buccal surface of the jaw bone (Shimomoto *et al.*, 2007). Trabecular bone was carefully contoured on the first and the last slice, while the intermediate slices were first interpolated by morphing.

Bone mineral apposition rate (µm/day)

Alveolar crest Alveolar bone Buccal surface Inferior border

\*

**6.3 Microtomography of the mandible (micro-CT)**

Science East Corporation, Tokyo, Japan)

sample stage.

size of 15 μm / pixel.

Imagescript, Tokyo, Japan).

and determine bone strength in conjunction with bone mineral density.

**6.3.1 Protocol adopted to examine the mandible using micro-CT** 

and a fixed threshold to extract the mineralized bone phase.

\*

\*

n = 5 for each group. \*:*p*<0.05.


Fig. 16. The left mandible was imaged by micro-CT

#### **6.3.2 Microtomography of the DM mandible**

The quantification of micro-CT trabecular bone changes (mean±SD) is shown for the DM and the control groups in (Table 4). All trabecular parameters in both alveolar bone and buccal surface of jaw bone showed significant changes. Compared with the control group, bone volume fraction (BV/TV) was significantly decreased only in the alveolar bone; however, trabecular thickness (Tb.Th) and trabecular numbers (Tb.N) were significantly decreased both in alveolar and buccal surface of jaw bone, in the DM group. Correspondingly, significantly higher trabecular separation (Tb.Sp) and trabecular space (Tb.S) were revealed both in alveolar and buccal surface of jaw bone for the DM group when compared with that of the control group. Also, the bone surface / bone volume (BS/BV) was significantly increased only in alveolar bone (*p*<0.05). These findings indicate deterioration of the bone quality in the DM group. These results agree with other research work suggesting that the glycaemic levels play an important role in modulating the trabecular architecture especially in mandibular bone (Thrailkill *et al.*, 2005).

The DM condition resulted in alteration of the trabecular distance and thickness as compared to the control group indicating profound impact on the histological integrity of the bone. The reduction in trabecular bone volume accompanied by the expansion of the bone marrow space is in agreement with another investigation (Duarte *et al.*, 2005). In this context, these results may describe a state of osteopenia in experimental diabetic rats, which might be the result of an imbalance between bone formation and resorption

The Effect of Type 1 Diabetes Mellitus on the Craniofacial Complex 455

Histological sections are incubated for 30–60 min at 37ºC in a mixture of 0.8% naphthol AS-BI phosphate (Sigma, St Louis, MO, USA), 0.7% fast red violet salt (Sigma, St Louis, MO, USA) and 50mM sodium tartate diluted in 0.2M sodium acetate buffer (pH 5.4) (Tsuchiya and Kurihara, 1995). Sections were examined under a light microscope. For the histomorphometric assessment of resorption, the number of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar were counted in each 540 μm x 120 μm area in five consecutive sections, at the middle third of the root selected at least 25μm apart

Bone-resorption activity was assessed by counting the number of tartrate-resistant acid phosphatase-positive multinucleate cells (osteoclasts) on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar (Fig. 18A-D). Statistical analysis

Fig. 18. Osteoclast counts in a horizontal section of the mandibular second molar region stained with Tartarate-resistant acid phosphatase (TRAP). (A) Low magnification photograph of the three roots of the second molar stained with TRAP stain. The black rectangle (540 X 120 μm) indicates the area on the distal surface of the alveolar bone adjacent to the middle third of the mesio-buccal root of the second molar in which the osteoclast cells were counted. Bu, buccal; Li, lingual; Me, mesial; Di, distal. (B) The mesio-buccal root of the control rat (original magnification 100X). (C) The mesio-buccal root of the DM rat (original magnification 100X). (D) A schematic drawing showing the observation area on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar in which the osteoclast cells were counted. (E) The number of TRAP-positive cells on the distal surface of the mesio-buccal root of the mandibular second

TRAP positive cell number / area

100 μm 100 μm

DM Control

\*

D E

Control 

A B C

from each specimen (*n* = 5) of each group (Misawa *et al.*, 2007; Mishima *et al.*, 2002).

**6.4.2 TRAP staining** 

**6.4.3 Histological analysis** 

**Bu**

First molar

**Li**

**D**

**Me**

(M1) Second molar (M2)

Lingual

molar. Values are mean ± SD. \*:(*p*<0.05), bars = 100 µm.

Mesial Distal

Buccal

Third molar (M3)

**Di**

DM


\**p*<0.05 as compared to control values

Table 8. Microarchitectural properties in the mandible as measured by micro-CT

#### **6.4 Histological analysis**

#### **6.4.1 Procedure for preparing mandibles for histological analysis**


Fig. 17. Experimental procedure for histological section preparation

#### **6.4.2 TRAP staining**

454 Type 1 Diabetes Complications

Trabecular Space (µm) 87.6±4.3\* 71.2±11.5 67.7±7.9\* 53.1±2.0

23.2±2.1\* 34.3±4.5 21.5±2.9\* 26.1±2.1

Serial horizontal sections were prepared using a microtome (5 µm thick parallel to the occlusal plane)

Embedded in paraffin

DM Control DM Control

Alveolar Bone Buccal Surface Of The Jaw Bone

Trabecular Separation (µm) 37.5±1.0\* 25.5±2.4 38.1±9.1\* 25.9±2.4

Mandibles for all groups were decalcified in 10% EDTA solution pH 7.4 for 5 weeks at

Specimens are then dehydrated in an ascending ethanol series and embedded in

Serial horizontal sections (5 μm thick parallel to the occlusal plane) are prepared using a

15.2±1.7\* 18.9±0.7

87.8±5.6 70.0±15.5

55.2±16.4

22.1±11.6\*

**6.4.1 Procedure for preparing mandibles for histological analysis** 

microtome (Leica RM 2155, Nussloch, Germany) (Figs. 12, 13)

Fig. 17. Experimental procedure for histological section preparation

First m olar ( M1) Second m olar (M 2) Thir d m olar (M3)

**D**

in EDTA

Buc cal Mesial Distal L in g u a l

Dehydration in ascending ethanol series

Bone Volume/Tissue Volume (%) 46.2±10.2 37.3±4.2

Table 8. Microarchitectural properties in the mandible as measured by micro-CT

Trabecular Number (1/mm) 10.8±1.2\* 14.4±2.6

Bone Surface/Bone Volume (1/mm) 36.8±9.5\* 63.93±15.3

Trabecular Thickness (µm)

\**p*<0.05 as compared to control values

4ºC (Yokoyama *et al.*, 2009).

paraffin (Figs. 9, 10, 11).

Fi rs t m o la r ( M1 ) Se co nd m o la r ( M2) T h ir d mo l ar (M 3) Bu cc a l M e s ia l Di s ta l L in g u a l **D**

TRAP Stain

Left side of mandible Decalcification

**6.4 Histological analysis** 

Histological sections are incubated for 30–60 min at 37ºC in a mixture of 0.8% naphthol AS-BI phosphate (Sigma, St Louis, MO, USA), 0.7% fast red violet salt (Sigma, St Louis, MO, USA) and 50mM sodium tartate diluted in 0.2M sodium acetate buffer (pH 5.4) (Tsuchiya and Kurihara, 1995). Sections were examined under a light microscope. For the histomorphometric assessment of resorption, the number of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar were counted in each 540 μm x 120 μm area in five consecutive sections, at the middle third of the root selected at least 25μm apart from each specimen (*n* = 5) of each group (Misawa *et al.*, 2007; Mishima *et al.*, 2002).

#### **6.4.3 Histological analysis**

Bone-resorption activity was assessed by counting the number of tartrate-resistant acid phosphatase-positive multinucleate cells (osteoclasts) on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar (Fig. 18A-D). Statistical analysis

Fig. 18. Osteoclast counts in a horizontal section of the mandibular second molar region stained with Tartarate-resistant acid phosphatase (TRAP). (A) Low magnification photograph of the three roots of the second molar stained with TRAP stain. The black rectangle (540 X 120 μm) indicates the area on the distal surface of the alveolar bone adjacent to the middle third of the mesio-buccal root of the second molar in which the osteoclast cells were counted. Bu, buccal; Li, lingual; Me, mesial; Di, distal. (B) The mesio-buccal root of the control rat (original magnification 100X). (C) The mesio-buccal root of the DM rat (original magnification 100X). (D) A schematic drawing showing the observation area on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar in which the osteoclast cells were counted. (E) The number of TRAP-positive cells on the distal surface of the mesio-buccal root of the mandibular second molar. Values are mean ± SD. \*:(*p*<0.05), bars = 100 µm.

The Effect of Type 1 Diabetes Mellitus on the Craniofacial Complex 457

2005) and this mechanism of action may explain the retardation of craniofacial growth in

Diabetes has a deleterious effect on osseous turnover due to decreased osteoblast and osteoclast activities and numbers and, a lower percentage of osteoid surface and osteocalcin synthesis, as well as increased time for mineralization of osteoid (Duarte *et al.*, 2005). It was reported that the influence of diabetes on discrete stages of matrix-induced endochondral bone formation could have profound effects on the biomechanical behavior of bone. Also, chondrogenesis and calcification of bone were reduced by 50% in diabetic animals (Reddy *et al.*, 2001). This was evident in the current study results that showed a significant decrease in

In addition to this, insulin may exert synergistic effects with other anabolic agents in bone, such as parathyroid hormone (PTH) (Thomas *et al.*, 1998; Thrailkill *et al.*, 2005). An animal model of T1DM has frequently demonstrated alteration in bone turnover, retarded growth, increased concentration of PTH, and reduced concentration of 1,25-dihydroxivitamin D (Duarte *et al.*, 2005; Tsuchida *et al.*, 2000). The effects of PTH on the bones are complex; it stimulates resorption or bone formation depending on the concentration used, the duration of the exhibition, and the administration method (Duarte *et al.*, 2005; Toromanoff *et al.*, 1997; Tsuchida *et al.*, 2000). Also, 1,25-dihydroxivitamin D, like PTH, belongs to the most important group of bone regulatory hormones. It regulates osteoclastic differentiation from hematopoietic mononuclear cells, and osteoblastic functions and activity (Collins *et al.*, 1998;

Moreover, Insulin may indirectly regulate the enhancement of growth hormone serum concentration by direct regulation of the hepatic growth hormone receptor, this results in abnormalities in the insulin growth factor-1 in T1DM (Chiarelli *et al.*, 2004) which consequently may have lead to the retarded growth in uncontrolled DM in the current

In the present study most of the periosteal surfaces in the mandibular regions of the control group showed significantly higher values recorded for the mineral apposition rate and the bone formation rate when compared to the DM group. These results agree with previous studies that recorded diminished lamellar bone formation in DM rats' femur and may suggest an association between the DM condition and the decreased number and function of osteoblasts (Follak *et al.*, 2004; Shyng *et al.*, 2001). The alveolar crest region was the only region that did not show a significant difference in the mineral apposition rate and the bone formation rate parameters among the two groups; this may be attributed to the unique nature of this region exhibiting a highly intensive bone remodeling process especially during the teeth eruption that decreases toward the base of

Micro CT analysis showed a significant decrease of bone volume fraction, trabecular thickness, and trabecular number. Also, it showed a significant increase of the trabecular separation and the trabecular space in the DM group when compared with the control group. This finding indicates deterioration of the bone quality in the DM group. These results agree with other research work suggesting that the glycaemic levels play an important role in modulating the trabecular architecture especially in mandibular bone (Thrailkill *et al.*, 2005). In this context, these results may describe a state of osteopenia in experimental diabetic rats, which might be the result of an imbalance between bone

the craniofacial linear measurements of the DM group.

STZ-DM.

Duarte *et al.*, 2005).

the socket (Gerlach *et al.*, 2002).

formation and resorption.

study.

demonstrated a significantly higher number of osteoclast cells in the control group when compared with the DM group (*p<*0*.*05) (Fig. 18). Results revealed that the number of osteoclasts was significantly lower in the DM rats than in the controls, in line with previous studies on DM rats' mandible (Mishima *et al.*, 2002) and long bones (Glajchen *et al.*, 1988; Shires *et al.*, 1981). These results confirm that the decreased rate of bone turnover may be associated with the DM condition.

#### **7. Suggested mechanisms for the effect of diabetic condition on craniofacial complex**

Growth of the craniofacial complex is controlled by genetic and environmental factors (Giglio and Lama, 2001; Yonemitsu *et al.*, 2007). Regulatory mechanisms responsible for normal morphogenesis of the face and head involve hormones, nutrients, mechanical forces, and various local growth factors. The poor growth and alterations in bone metabolism have been associated with T1DM in both humans and experimental animals (Giglio and Lama, 2001). Because human studies can be limited by small sample sizes, cross-sectional designs, uncontrolled variables, and often retrospective nature, animal models have been used to yield more rigorous analyses (Singleton *et al.*, 2006). In our studies we observed the rat growth from the age of 3 weeks old till 7 weeks old. This time period is corresponding to early growth stage in human according to previous craniofacial growth studies (Losken *et al.*, 1994; Siegel and Mooney, 1990). Consequently, in the current study STZ-DM model was used to investigate the effect of T1DM on craniofacial growth.

The studied STZ-DM rats showed significantly reduced growth in most of the craniofacial skeletal units but no significant differences were observed between controls and DM group as regards the remaining craniofacial skeletal units (Sphenoid bone length, posterior neurocranium height, anterior corpus length, bigonial width and palatal width). Craniofacial growth as a whole was also significantly lower in DM group compared to controls in all three dimensions. Previous study investigated the DM effect exclusively on the growth of the mandible and suggested that the diabetic condition had a differential effect on the osseous components and / or its associated non-skeletal tissues. They found that the disharmonious growth of the mandible was due to DM condition and might not be associated with diabetic condition complications such as renal failure, anemia, body weight change or alteration in the food intake qualities (Giglio and Lama, 2001). Thus we hypothesize that the deficiency in the craniofacial growth in our experiment might be attributed to the diabetic condition in the DM group as it was reported that specific alterations in bone metabolism are associated with DM. Moreover, several pathogenic possibilities have been proposed, such as insulinopenia, bone microangiopathy, impaired regulation of mineral metabolism, alterations in local factors that regulate bone remodeling, and even an intrinsic disorder associated with T1DM (Duarte *et al.*, 2005; Ward *et al.*, 2001). The aforementioned insulin hormone deficiency that is associated with T1DM cases may have direct effect on bone metabolism. It was mentioned in literature that normal insulin hormone level exerts direct anabolic effects on bone cells (Duarte *et al.*, 2005). Multiple osteoblast-like cell lines express insulin receptors on the cell surface and have a high capacity for insulin binding (Pun *et al.*, 1989). Moreover, osteoclasts exhibit reduced bone resorption in response to insulin stimulation (Thomas *et al.*, 1998). These findings support the idea that the actions of insulin in bone could be mediated directly via stimulation of osteoblasts in combination with inhibition of osteoclasts, (Thomas *et al.*, 1998; Thrailkill *et al.*,

demonstrated a significantly higher number of osteoclast cells in the control group when compared with the DM group (*p<*0*.*05) (Fig. 18). Results revealed that the number of osteoclasts was significantly lower in the DM rats than in the controls, in line with previous studies on DM rats' mandible (Mishima *et al.*, 2002) and long bones (Glajchen *et al.*, 1988; Shires *et al.*, 1981). These results confirm that the decreased rate of bone turnover may be

**7. Suggested mechanisms for the effect of diabetic condition on craniofacial** 

Growth of the craniofacial complex is controlled by genetic and environmental factors (Giglio and Lama, 2001; Yonemitsu *et al.*, 2007). Regulatory mechanisms responsible for normal morphogenesis of the face and head involve hormones, nutrients, mechanical forces, and various local growth factors. The poor growth and alterations in bone metabolism have been associated with T1DM in both humans and experimental animals (Giglio and Lama, 2001). Because human studies can be limited by small sample sizes, cross-sectional designs, uncontrolled variables, and often retrospective nature, animal models have been used to yield more rigorous analyses (Singleton *et al.*, 2006). In our studies we observed the rat growth from the age of 3 weeks old till 7 weeks old. This time period is corresponding to early growth stage in human according to previous craniofacial growth studies (Losken *et al.*, 1994; Siegel and Mooney, 1990). Consequently, in the current study STZ-DM model was

The studied STZ-DM rats showed significantly reduced growth in most of the craniofacial skeletal units but no significant differences were observed between controls and DM group as regards the remaining craniofacial skeletal units (Sphenoid bone length, posterior neurocranium height, anterior corpus length, bigonial width and palatal width). Craniofacial growth as a whole was also significantly lower in DM group compared to controls in all three dimensions. Previous study investigated the DM effect exclusively on the growth of the mandible and suggested that the diabetic condition had a differential effect on the osseous components and / or its associated non-skeletal tissues. They found that the disharmonious growth of the mandible was due to DM condition and might not be associated with diabetic condition complications such as renal failure, anemia, body weight change or alteration in the food intake qualities (Giglio and Lama, 2001). Thus we hypothesize that the deficiency in the craniofacial growth in our experiment might be attributed to the diabetic condition in the DM group as it was reported that specific alterations in bone metabolism are associated with DM. Moreover, several pathogenic possibilities have been proposed, such as insulinopenia, bone microangiopathy, impaired regulation of mineral metabolism, alterations in local factors that regulate bone remodeling, and even an intrinsic disorder associated with T1DM (Duarte *et al.*, 2005; Ward *et al.*, 2001). The aforementioned insulin hormone deficiency that is associated with T1DM cases may have direct effect on bone metabolism. It was mentioned in literature that normal insulin hormone level exerts direct anabolic effects on bone cells (Duarte *et al.*, 2005). Multiple osteoblast-like cell lines express insulin receptors on the cell surface and have a high capacity for insulin binding (Pun *et al.*, 1989). Moreover, osteoclasts exhibit reduced bone resorption in response to insulin stimulation (Thomas *et al.*, 1998). These findings support the idea that the actions of insulin in bone could be mediated directly via stimulation of osteoblasts in combination with inhibition of osteoclasts, (Thomas *et al.*, 1998; Thrailkill *et al.*,

used to investigate the effect of T1DM on craniofacial growth.

associated with the DM condition.

**complex** 

2005) and this mechanism of action may explain the retardation of craniofacial growth in STZ-DM.

Diabetes has a deleterious effect on osseous turnover due to decreased osteoblast and osteoclast activities and numbers and, a lower percentage of osteoid surface and osteocalcin synthesis, as well as increased time for mineralization of osteoid (Duarte *et al.*, 2005). It was reported that the influence of diabetes on discrete stages of matrix-induced endochondral bone formation could have profound effects on the biomechanical behavior of bone. Also, chondrogenesis and calcification of bone were reduced by 50% in diabetic animals (Reddy *et al.*, 2001). This was evident in the current study results that showed a significant decrease in the craniofacial linear measurements of the DM group.

In addition to this, insulin may exert synergistic effects with other anabolic agents in bone, such as parathyroid hormone (PTH) (Thomas *et al.*, 1998; Thrailkill *et al.*, 2005). An animal model of T1DM has frequently demonstrated alteration in bone turnover, retarded growth, increased concentration of PTH, and reduced concentration of 1,25-dihydroxivitamin D (Duarte *et al.*, 2005; Tsuchida *et al.*, 2000). The effects of PTH on the bones are complex; it stimulates resorption or bone formation depending on the concentration used, the duration of the exhibition, and the administration method (Duarte *et al.*, 2005; Toromanoff *et al.*, 1997; Tsuchida *et al.*, 2000). Also, 1,25-dihydroxivitamin D, like PTH, belongs to the most important group of bone regulatory hormones. It regulates osteoclastic differentiation from hematopoietic mononuclear cells, and osteoblastic functions and activity (Collins *et al.*, 1998; Duarte *et al.*, 2005).

Moreover, Insulin may indirectly regulate the enhancement of growth hormone serum concentration by direct regulation of the hepatic growth hormone receptor, this results in abnormalities in the insulin growth factor-1 in T1DM (Chiarelli *et al.*, 2004) which consequently may have lead to the retarded growth in uncontrolled DM in the current study.

In the present study most of the periosteal surfaces in the mandibular regions of the control group showed significantly higher values recorded for the mineral apposition rate and the bone formation rate when compared to the DM group. These results agree with previous studies that recorded diminished lamellar bone formation in DM rats' femur and may suggest an association between the DM condition and the decreased number and function of osteoblasts (Follak *et al.*, 2004; Shyng *et al.*, 2001). The alveolar crest region was the only region that did not show a significant difference in the mineral apposition rate and the bone formation rate parameters among the two groups; this may be attributed to the unique nature of this region exhibiting a highly intensive bone remodeling process especially during the teeth eruption that decreases toward the base of the socket (Gerlach *et al.*, 2002).

Micro CT analysis showed a significant decrease of bone volume fraction, trabecular thickness, and trabecular number. Also, it showed a significant increase of the trabecular separation and the trabecular space in the DM group when compared with the control group. This finding indicates deterioration of the bone quality in the DM group. These results agree with other research work suggesting that the glycaemic levels play an important role in modulating the trabecular architecture especially in mandibular bone (Thrailkill *et al.*, 2005). In this context, these results may describe a state of osteopenia in experimental diabetic rats, which might be the result of an imbalance between bone formation and resorption.

The Effect of Type 1 Diabetes Mellitus on the Craniofacial Complex 459

complex procedure when compared to the treatment of normal patients, moreover it is suggested that it is of prime importance to keep close attention to the general systemic condition of these patients and administer the proper hormonal therapy for these patients when needed to avoid any detrimental effects on bone resulting from any hormonal

1. T1DM reduces craniofacial growth, resulting in retardation of skeletal development. 2. DM in the rat affects the bone architecture, as shown by micro-CT, and impairs the rate of the mandibular bone formation, as examined by the dynamic histomorphometric

3. All of these results were verified on the cellular level by a histological study that showed the diminished number of osteoclasts on the alveolar wall, suggesting that the

4. These findings should be considered when conducting any treatment in the craniofacial region in T1DM since the better understanding of how diabetes affects bone will

5. Good control of diabetes mellitus should be considered before orthodontic treatment by

Abbassy MA, Watari I, Soma K (2008). Effect of experimental diabetes on craniofacial growth in rats. *Archives of Oral Biology,* Vol.53, No.9, (September 2008), pp. 819-825 Abbassy MA, Watari I, Soma K (2010). The effect of diabetes mellitus on rat mandibular

Abdus Salam M, Matsumoto N, Matin K, Tsuha Y, Nakao R, Hanada N, Senpuku H (2004).

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bone formation and microarchitecture *European Journal of Oral Sciences,* Vol.118,

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in patients with diabetes mellitus. *American Journal of Orthodontics and Dentofacial* 

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density. *Osteoporosis International,* Vol.8, No.2, (March 1998), pp. 110-114, ISSN

Within the limitations of this *in vitro* study, it was concluded that:

early stage of DM resulted in low bone turnover.

a long time inorder to obtain the best outcome.

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improve our ability to protect bone health in diabetic patients.

imbalance.

analysis.

**9. References** 

pp.898-904

0937-941X

A histometric evaluation of bone resorption was performed by counting the number of osteoclast cells on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar. These evaluations revealed that the number of osteoclasts was significantly lower in the DM rats than in the controls, in line with previous studies on DM rats' mandible (Mishima *et al.*, 2002) and long bones (Glajchen *et al.*, 1988; Shires *et al.*, 1981). These studies confirm that the decreased rate of bone turnover may be associated with the DM condition.

This deteriorating effect on mandibular bone structure and dynamic bone formation might be be attributed to several pathogenic possibilities, such as insulinopenia, bone microangiopathy, impaired regulation of mineral metabolism, alterations in local factors that regulate bone remodeling, and even an intrinsic disorder associated with DM (Abbassy *et al.*, 2008; Duarte *et al.*, 2005). However, the detrimental effects observed may not be associated with the significant loss of rats` weights observed in the diabetic group starting from day 14 because previous research (Abbassy *et al.*, 2008; Giglio and Lama, 2001; Shires *et al.*, 1981; Thrailkill *et al.*, 2005) showed that the mandibular growth was not affected in normal rats supplied with restricted diet and having same pattern of weight loss resembling weight loss pattern observed in DM rats.

#### **8. Conclusions**

It is obvious that the T1DM condition significantly affects craniofacial growth, bone formation mechanism and the quality of the bone formed which may alter many aspects of planning and treatment of orthodontic patients affected by this globally increasing hormonal disturbance.

There should be a new strategy for treating orthodontic patients suffering from metabolic disorders specially those disorders having direct and indirect effects on bone growth as the diabetic condition. The orthodontic craniofacial linear measurements were significantly decreased in the T1DM cases when compared to normal cases. These facts should be considered when orthodontic problems are diagnosed and treated in T1DM, because this may alter the orthodontic treatment period and the final treatment outcome. In this chapter it was suggested that a better understanding of how diabetes affects bone will improve our ability to protect bone health in diabetic patients. Also, it was recommended to conduct further studies in order to elucidate the exact mechanism by which diabetes produces alterations in most of the craniofacial units.

Thus, T1DM condition had detrimental effects on the different bone regions of the mandible, that shed the light on the complexity of the craniofacial structure which is affected by many hormonal disorders suggesting further examination for the overlying soft tissues which are directly affected by any underlying boney changes. Diabetes condition had detrimental effect on bone architecture, as shown by micro-CT, and impaired the rate of the mandibular bone formation, as examined by the dynamic histomorphometric analysis. All of these results were verified on the cellular level by a histological study that showed the diminished number of osteoclasts on the alveolar wall, suggesting that the early stage of T1DM resulted in low bone turnover. These results may suggest that more care is needed during the consideration of the force needed for treatment of orthodontic patients affected by T1DM, also, the retention period and rate of success in these patients may be affected.

These comprehensive studies done on bone and craniofacial growth suggest that planning the treatment in craniofacial region for patients affected with hormonal disorders is a more complex procedure when compared to the treatment of normal patients, moreover it is suggested that it is of prime importance to keep close attention to the general systemic condition of these patients and administer the proper hormonal therapy for these patients when needed to avoid any detrimental effects on bone resulting from any hormonal imbalance.

Within the limitations of this *in vitro* study, it was concluded that:


#### **9. References**

458 Type 1 Diabetes Complications

A histometric evaluation of bone resorption was performed by counting the number of osteoclast cells on the distal surface of the alveolar bone adjacent to the mesio-buccal root of the second molar. These evaluations revealed that the number of osteoclasts was significantly lower in the DM rats than in the controls, in line with previous studies on DM rats' mandible (Mishima *et al.*, 2002) and long bones (Glajchen *et al.*, 1988; Shires *et al.*, 1981). These studies confirm that the decreased rate of bone turnover may be associated with the

This deteriorating effect on mandibular bone structure and dynamic bone formation might be be attributed to several pathogenic possibilities, such as insulinopenia, bone microangiopathy, impaired regulation of mineral metabolism, alterations in local factors that regulate bone remodeling, and even an intrinsic disorder associated with DM (Abbassy *et al.*, 2008; Duarte *et al.*, 2005). However, the detrimental effects observed may not be associated with the significant loss of rats` weights observed in the diabetic group starting from day 14 because previous research (Abbassy *et al.*, 2008; Giglio and Lama, 2001; Shires *et al.*, 1981; Thrailkill *et al.*, 2005) showed that the mandibular growth was not affected in normal rats supplied with restricted diet and having same pattern of weight loss resembling

It is obvious that the T1DM condition significantly affects craniofacial growth, bone formation mechanism and the quality of the bone formed which may alter many aspects of planning and treatment of orthodontic patients affected by this globally increasing

There should be a new strategy for treating orthodontic patients suffering from metabolic disorders specially those disorders having direct and indirect effects on bone growth as the diabetic condition. The orthodontic craniofacial linear measurements were significantly decreased in the T1DM cases when compared to normal cases. These facts should be considered when orthodontic problems are diagnosed and treated in T1DM, because this may alter the orthodontic treatment period and the final treatment outcome. In this chapter it was suggested that a better understanding of how diabetes affects bone will improve our ability to protect bone health in diabetic patients. Also, it was recommended to conduct further studies in order to elucidate the exact mechanism by which diabetes produces

Thus, T1DM condition had detrimental effects on the different bone regions of the mandible, that shed the light on the complexity of the craniofacial structure which is affected by many hormonal disorders suggesting further examination for the overlying soft tissues which are directly affected by any underlying boney changes. Diabetes condition had detrimental effect on bone architecture, as shown by micro-CT, and impaired the rate of the mandibular bone formation, as examined by the dynamic histomorphometric analysis. All of these results were verified on the cellular level by a histological study that showed the diminished number of osteoclasts on the alveolar wall, suggesting that the early stage of T1DM resulted in low bone turnover. These results may suggest that more care is needed during the consideration of the force needed for treatment of orthodontic patients affected by T1DM,

These comprehensive studies done on bone and craniofacial growth suggest that planning the treatment in craniofacial region for patients affected with hormonal disorders is a more

also, the retention period and rate of success in these patients may be affected.

DM condition.

**8. Conclusions** 

hormonal disturbance.

weight loss pattern observed in DM rats.

alterations in most of the craniofacial units.


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**22** 

M.G.K. Albrecht

*Hungary* 

**The Role of Genetic Predisposition in Diagnosis** 

The prevalence of diabetes mellitus (DM) and its probable influence on periodontal disease suggests that DM patients will very likely probably become an increasing proportion of the patient population seen by both general dentists and periodontists. Many investigators have studied the oral manifestations involving periodontitis as a complicating factor in the periodontal therapy of DM patients, whose disease may be more prevalent and more severe and progress rapidly.1 2 3 Periodontal disease makes chewing difficult or painful, thereby leading to an improper diet. On the other hand, uncontrolled periodontal disease may upset

Until the past 15 years, the management was based on the periodontology model of care, and the aim of these methods was to diagnose the problem and resolve it via treatment. Consequently, repairs were made, but the periodontitis generally recurred or progressed unabated. Disease prevention was not practised. It is clear that the risk of periodontal

Today many practitioners are providing better and more complete service to their patients because they are beginning to incorporate the principles of the information-intense medical model.9 Changes in our political and social structure have affected how health care is being managed. With increasing evidence of the influence of periodontal disease on systemic health, dentist and hygienists are taking a more intensive look at the risk factors associated with its onset and progression. Bacteriology, immunology, genetics, and systemic cofactors are often used to determine what is wrong with the patients. Genetic knowledge is an important part of the medical model because it allows for a complete and comprehensive picture of all of the factors contributing to the patient's past, current, and

Gingivitis is an inflammatory pathologic alteration affecting the gingival epithelium and connective tissue (Fig.1). Clinical symptoms comprise reddness and swelling of the gingiva,

**2. Classification and characterization of periodontal diseases** 

bleeding on probing, and a periodontal pocket deph ≥1mm.

**1. Introduction** 

metabolic control of DM.2

future status.

**2.1 Gingivitis** 

disease varies greatly from one patient to another.4-8

**and Therapy of Periodontal Diseases in** 

*Department of Conservative Dentistry Semmelweis University, Budapest,* 

**Type 1 Diabetes Mellitus** 


### **The Role of Genetic Predisposition in Diagnosis and Therapy of Periodontal Diseases in Type 1 Diabetes Mellitus**

M.G.K. Albrecht *Department of Conservative Dentistry Semmelweis University, Budapest, Hungary* 

#### **1. Introduction**

462 Type 1 Diabetes Complications

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(1998). Bone mineral density and composition in rat pregnancy: effects of streptozotocin-induced diabetes mellitus and insulin replacement. *Experimental* 

Insulin receptor expression in primary and cultured osteoclast-like cells. *Bone,* 

Carver AA, Fowlkes JL, Aronson J, Lumpkin CK, Jr. (2005). Bone formation is impaired in a model of type 1 diabetes. *Diabetes,* Vol.54, No.10, (October 2005), pp. 2875-2881 Toromanoff A, Ammann P, Mosekilde L, Thomsen JS, Riond JL (1997). Parathyroid hormone

increases bone formation and improves mineral balance in vitamin D-deficient female rats. *Endocrinology,* Vol.138, No.6, (June 1997), pp. 2449-2457, ISSN 0013-7227

Histomorphometric evaluation of the recovering effect of human parathyroid hormone (1-34) on bone structure and turnover in streptozotocin-induced diabetic

demonstration of tartarate -resistant acid phosphatase (TRAP) activity in osteoclastlike multinucleate cells. *Journal of Bone and Mineral Metabolism,* Vol.13, pp. 71-76 Vandeberg JR, Buschang PH, Hinton RJ (2004a). Craniofacial growth in growth hormone-

deficient rats. *The Anatomical Record. Part A, Discoveries in Molecular, Cellular and* 

craniofacial skeleton. *Archives of Oral Biology,* Vol.49, No.6, (June 2004), pp. 477-484, ISSN 0003-9969Ward DT, Yau SK, Mee AP, Mawer EB, Miller CA, Garland HO, Riccardi D (2001). Functional, molecular, and biochemical characterization of streptozotocin-induced diabetes. *Journal of the American Society of Nephrology,* Vol.12,

metabolism in the rat temporomandibular joint after molar extraction using bone scintigraphy. *Europen Journal of Oral Sciences,* Vol.117, No. 4, (August 2009), pp. 374-

mandibular growth. *Archives of Oral Biology,* Vol.52, No.5, (May 2007), pp. 487-493,

changes of mineral composition in femurs of diabetic rats by insulin. *Biological Trace* 

*Evolutionary Biology,* Vol.278, No.2, (June 2004), pp. 561-570, ISSN 1552-4884 VandeBerg JR, Buschang PH, Hinton RJ (2004b). Absolute and relative growth of the rat

Yokoyama M, Atsumi T, Tsuchiya M, Koyama S, Sasaki K (2009). Dynamic changes in bone

Yonemitsu I, Muramoto T, Soma K (2007). The influence of masseter activity on rat

Zhang X, Fei Y, Zhang M, Wei D, Li M, Ding W, Yang J (2008). Reversal of osteoporotic

*Element Research, Vol.*121, No.3, (March 2008), pp. 233-242

The prevalence of diabetes mellitus (DM) and its probable influence on periodontal disease suggests that DM patients will very likely probably become an increasing proportion of the patient population seen by both general dentists and periodontists. Many investigators have studied the oral manifestations involving periodontitis as a complicating factor in the periodontal therapy of DM patients, whose disease may be more prevalent and more severe and progress rapidly.1 2 3 Periodontal disease makes chewing difficult or painful, thereby leading to an improper diet. On the other hand, uncontrolled periodontal disease may upset metabolic control of DM.2

Until the past 15 years, the management was based on the periodontology model of care, and the aim of these methods was to diagnose the problem and resolve it via treatment. Consequently, repairs were made, but the periodontitis generally recurred or progressed unabated. Disease prevention was not practised. It is clear that the risk of periodontal disease varies greatly from one patient to another.4-8

Today many practitioners are providing better and more complete service to their patients because they are beginning to incorporate the principles of the information-intense medical model.9 Changes in our political and social structure have affected how health care is being managed. With increasing evidence of the influence of periodontal disease on systemic health, dentist and hygienists are taking a more intensive look at the risk factors associated with its onset and progression. Bacteriology, immunology, genetics, and systemic cofactors are often used to determine what is wrong with the patients. Genetic knowledge is an important part of the medical model because it allows for a complete and comprehensive picture of all of the factors contributing to the patient's past, current, and future status.

#### **2. Classification and characterization of periodontal diseases**

#### **2.1 Gingivitis**

Gingivitis is an inflammatory pathologic alteration affecting the gingival epithelium and connective tissue (Fig.1). Clinical symptoms comprise reddness and swelling of the gingiva, bleeding on probing, and a periodontal pocket deph ≥1mm.

The Role of Genetic Predisposition in

**2.3 Refractory marginal periodontitis** 

tissue the affected teeth are often lost (Fig.3).

**2.4 Localised Juvenile Periodontitis (LJP)** 

considered to be the marker pathogen for LJP.

**2.5 Rapidly Progressive Periodontitis (RP)** 

**3. The oral cavity ecosystem** 

adapted to their enviroments.

Diagnosis and Therapy of Periodontal Diseases in Type 1 Diabetes Mellitus 465

Refractory or therapy-resistant AP shows a progressive loss of attachment even with diligent mechanical therapy and positive compliance of the patients. This type of periodontitis is characterized by a progressive loss of supporting tissue even if treated thoroughly. In most cases the lesions compromise more than one tooth and are infected by high concentration s of periodontal pathogens. After a massive degradation of supporting

Fig. 3. Periodontitis (degradation of supporting tissue the affected teeth are often lost)

This type usually affects healthy adolescents at the age of 10 to 20 years. It is distinguished by a severe, but localized loss of bone tissue combined with the development of deep pockets at the first molars and/or the incisors. *Actinobacillus actinomycetemcomitans* is

RPP is mostly seen in patient's at the age of 20 to 35 years. The patients medical history frequently shows a prior LJP. This form of periodontitis is generalized and progresses intermittently with severe loss of bone tissue, gingival bleeding and acute inflammation.The subgingival oral microflora is characterized by a high concentration of periodontal pathogens.

The human oral cavity accomodates about 50 billion bacteria belonging to about 400 different species.10 The various habitats are occupied by microbial populations specifically

The deep periodontal lesion is a unique eco-system within the oral cavity providing particular living conditions. It is the only place within the oral cavity not being flushed by saliva. Instead is filled with crevicular fluid. Moreover, the acigen concentration decreases progressively with pocket depth creating optimal growth conditions for anaerobic bacteria.1

Fig. 1. Gingivitis

Gigivitis is characterized by a subgingival microflora that is slightly shifted in favor of gramm-negativ, anaerobic bacteria without any periodontopathogenic microorganisms. ANUG is the acronym of the acute necrotic ulcerative gingivitis.

#### **2.2 Chronic Adult Periodontitis (AP)**

The slowly progressing AP is clinically characterized by persistent loss of attachment, a positive bleeding on probing (BOP), periodontal pockets depths of 1-3 mm, and slight loss of alveolar bone tissue (Fig.2). This is the most common type of periodontitis and generally occurs at the age of 30 years or later. It may be either generalized or limited to molars and/or incisors. The subgingival microbial panel is slightly shifted in favor of gramnegative, anaerobic bacteria with an increased amount of periopathogenic microorganizms.

Fig. 2. Periodontitis (loss of alveolar bone tissue)

#### **2.3 Refractory marginal periodontitis**

464 Type 1 Diabetes Complications

Gigivitis is characterized by a subgingival microflora that is slightly shifted in favor of gramm-negativ, anaerobic bacteria without any periodontopathogenic microorganisms.

The slowly progressing AP is clinically characterized by persistent loss of attachment, a positive bleeding on probing (BOP), periodontal pockets depths of 1-3 mm, and slight loss of alveolar bone tissue (Fig.2). This is the most common type of periodontitis and generally occurs at the age of 30 years or later. It may be either generalized or limited to molars and/or incisors. The subgingival microbial panel is slightly shifted in favor of gramnegative, anaerobic bacteria with an increased amount of periopathogenic microorganizms.

ANUG is the acronym of the acute necrotic ulcerative gingivitis.

**2.2 Chronic Adult Periodontitis (AP)** 

Fig. 2. Periodontitis (loss of alveolar bone tissue)

Fig. 1. Gingivitis

Refractory or therapy-resistant AP shows a progressive loss of attachment even with diligent mechanical therapy and positive compliance of the patients. This type of periodontitis is characterized by a progressive loss of supporting tissue even if treated thoroughly. In most cases the lesions compromise more than one tooth and are infected by high concentration s of periodontal pathogens. After a massive degradation of supporting tissue the affected teeth are often lost (Fig.3).

Fig. 3. Periodontitis (degradation of supporting tissue the affected teeth are often lost)

#### **2.4 Localised Juvenile Periodontitis (LJP)**

This type usually affects healthy adolescents at the age of 10 to 20 years. It is distinguished by a severe, but localized loss of bone tissue combined with the development of deep pockets at the first molars and/or the incisors. *Actinobacillus actinomycetemcomitans* is considered to be the marker pathogen for LJP.

#### **2.5 Rapidly Progressive Periodontitis (RP)**

RPP is mostly seen in patient's at the age of 20 to 35 years. The patients medical history frequently shows a prior LJP. This form of periodontitis is generalized and progresses intermittently with severe loss of bone tissue, gingival bleeding and acute inflammation.The subgingival oral microflora is characterized by a high concentration of periodontal pathogens.

#### **3. The oral cavity ecosystem**

The human oral cavity accomodates about 50 billion bacteria belonging to about 400 different species.10 The various habitats are occupied by microbial populations specifically adapted to their enviroments.

The deep periodontal lesion is a unique eco-system within the oral cavity providing particular living conditions. It is the only place within the oral cavity not being flushed by saliva. Instead is filled with crevicular fluid. Moreover, the acigen concentration decreases progressively with pocket depth creating optimal growth conditions for anaerobic bacteria.1

The Role of Genetic Predisposition in

with different results.27,28

have been tested for this genotype.

periodontal therapy.35

predictable by currently known mechanisms.

increased severity of several chronic inflammatory diseases.

Diagnosis and Therapy of Periodontal Diseases in Type 1 Diabetes Mellitus 467

others. In adults, these bacteria routinely colonize the teeth when tooth cleaning is not performed on a regular basis. Although bacteria are essential for the initiation of periodontitis, there is currently no mechanism for determing the clinical trajectory of the disease for individual patients, i.e., differentiating those patients who will have a mild to moderate form of disease and respond well to simple professional care from those who are likely to develop a more severe periodontitis that demands extensive therapy and results in tooth mobility. Individual differences in disease progression are dramatic and are often not

Identification of a risk factors may explain why individual patients do not respond uniformly to standard treatment. For example, a patient who is a heavy smoker may not heal as soon or as well as expected after treatment. Patients react differently to bacterial stimulation. This is a result not only of the type and amount of bacteria but also of the underlying genetic characteristics of the patient's immune system.The cytokines tumor necrosis factor alpha(TNFά) and interleukin 1 (IL-1) are key mediators of the inflammatory process and modulate the extracellular matrix components and bone which comprise the periodontal tissues.The genes encode pro-inflammatory proteins (IL 1A and IL-1B producing IL-1 ά and IL-1β). Several genetic polimorphisms have been described in the genes of the IL-1 cluster and, in case control studies, associations have been reported with

The genetic risk of developing periodontitis has been investigated by studying families and populations as well as twin.21-26 The studies conducted on twins have reported a significant genetic component explaining the variation in clinical attachment loss, probing depth and gingivitis. An association between the severe chronic form of the disease and a composite genotype in the interleukin IL-1ά and IL-1β genes has been reported.21 However, this association was found only in non-smokers. Other authors have subsequently investigated the IL-1ά - IL-1β genotypes with a chronic form of periodontitis

The genetics influence resistance on periodontal disease has been determined from a wide variety of sources.29-32 Genotype positive patients had significantly more clinical expression of inflammation, as determined by bleeding on probing. In healthy patients 46.7 % of genotype positive patients had bleeding as compared with only 8.6% of genotype-negative patients.32,33 Their results from genetic susceptibility testing have the real potential to improve patient management. Many of the genetic markers for common disease involve polymorphisms in gene sequences involved in cytokine biological activity. Researchers know that in healthy subject IL-1 plays a very important role in inflammation and the expression of periodontitis. Patients with this genotype progress more rapidly toward severe periodontitis and have statistically significant increased inflammation.32 It has been established that this genotype occurs in approximately 30% of most of the populations that

Cells from people with a positive genotype produced up to four times more IL-1 in response to the same bacterial challenge.34 Because IL-1 in high concentrations is involved with destruction of tissues, this increased IL-1 response may explain the more rapid progression of periodontal disease in genotype-positive patients when faced with a bacterial challenge in their plaque. For patients with this genetic susceptibility to periodontitis, tooth loss was be minimized by good plaque control and definitive

These bacteria lack the enzymes necessary to detoxify oxigen radicals resulting in a severely reduced growth or even death in the presence of oxigen. Dispite the great variety of the microbial flora only a fraction of the bacterial species is etiologically connected to the development of periodontitis. The flora of a healthy sulcus usually consists of aerobic grampositive cocci and rods. These bacteria known as "beneficial flora" show no pathogenic potential but, by their presence, are able to prevent the colonization is progressing the microbial spectrum is shifted in favor of anaerobic gram-negative rods.

The marker pathogens of periodontitis belong to the group of obligatory anaerobic, black pigmented Bacteroides species as *Porphyromonas gingivalis* and *Prevotella intermedia* as well as *Bacteroides forsythus, Treponema denticola, and Actinobacillus actinomycetemcomitans*.11,12 A strict correlation of the alveolar pocket depth and presence of periodontopathogens could be proven by several clinical studies.13,14,15

Several metabolites produced by the periodontitis-associated pathogens either destroy the sorrounding periodontal tissue or inactivate the humoral host defense system. The most important virulence factors produced by the three marker pathogens *P. gingivalis*, *P. intermedia* and *A. actinomycetemcomitans*.14

#### **4. Aetiology of periodontitis**

The development of plaque is considered to be the primary cause of periodontitis. Especially at the gingival margin plaque hardens to tartar resulting in a mechanical irritation. Exotoxins produced by the plaque bacteria diffuse into adjacent tissue and give rise to reddening and swelling -the typical clinical characteritics- of gingivitis. After professional removal of plaque and dental calculus a healthy periodontium is quickly restored indicating that gingivitis is a reversible condition. In case no professional removal of the dental calculus is performed the infection will progress and a periodontitis becomes established showing the typical clinical characteristics like bleeding on probing (BOP), increasing pocket deph and loss of alveolare bone tissue.11

At the age play a roule development of periodontitis16, and the age of 40 far more teeth are lost by periodontitis than by caries.

Periodontitis associated bacteria are found in low concentration even in the healthy sulcus. Therefore, additional factors must exist that determine the onset and the progress of disease. In case of an impaired immune system e.g. by stress, medication, hormonal imbalances, diabetes or smoking the pathogens utilize the selective advantages for prompt proliferation leading to the establishment of an manifest infection of the periodontium.17-20

#### **5. Genetic component to periodontitis**

For approximately the past 15 years, dental researchers have been focusing on dental plaque. Clinicians have made treatment decisions as though the plaque-disease interrelationship was quantitative: the more plaque, the more bacteria, the more inflammation, the more disease. However, clinical experience demonstrates that not all people respond the same way to similar accumulations of plaque. There are patients with a lot of plaque who have moderate and advanced disease. Some types of plaque simply are more virulent than others. However, clinical experience demonstrates that not all people respond the same way to plaque accumulations. Because so much variability respond to plaque, and respond to treatment. Some type of plaque symply are more virulent than

These bacteria lack the enzymes necessary to detoxify oxigen radicals resulting in a severely reduced growth or even death in the presence of oxigen. Dispite the great variety of the microbial flora only a fraction of the bacterial species is etiologically connected to the development of periodontitis. The flora of a healthy sulcus usually consists of aerobic grampositive cocci and rods. These bacteria known as "beneficial flora" show no pathogenic potential but, by their presence, are able to prevent the colonization is progressing the

The marker pathogens of periodontitis belong to the group of obligatory anaerobic, black pigmented Bacteroides species as *Porphyromonas gingivalis* and *Prevotella intermedia* as well as *Bacteroides forsythus, Treponema denticola, and Actinobacillus actinomycetemcomitans*.11,12 A strict correlation of the alveolar pocket depth and presence of periodontopathogens could be

Several metabolites produced by the periodontitis-associated pathogens either destroy the sorrounding periodontal tissue or inactivate the humoral host defense system. The most important virulence factors produced by the three marker pathogens *P. gingivalis*, *P.* 

The development of plaque is considered to be the primary cause of periodontitis. Especially at the gingival margin plaque hardens to tartar resulting in a mechanical irritation. Exotoxins produced by the plaque bacteria diffuse into adjacent tissue and give rise to reddening and swelling -the typical clinical characteritics- of gingivitis. After professional removal of plaque and dental calculus a healthy periodontium is quickly restored indicating that gingivitis is a reversible condition. In case no professional removal of the dental calculus is performed the infection will progress and a periodontitis becomes established showing the typical clinical characteristics like bleeding on probing (BOP), increasing pocket

At the age play a roule development of periodontitis16, and the age of 40 far more teeth are

Periodontitis associated bacteria are found in low concentration even in the healthy sulcus. Therefore, additional factors must exist that determine the onset and the progress of disease. In case of an impaired immune system e.g. by stress, medication, hormonal imbalances, diabetes or smoking the pathogens utilize the selective advantages for prompt proliferation

For approximately the past 15 years, dental researchers have been focusing on dental plaque. Clinicians have made treatment decisions as though the plaque-disease interrelationship was quantitative: the more plaque, the more bacteria, the more inflammation, the more disease. However, clinical experience demonstrates that not all people respond the same way to similar accumulations of plaque. There are patients with a lot of plaque who have moderate and advanced disease. Some types of plaque simply are more virulent than others. However, clinical experience demonstrates that not all people respond the same way to plaque accumulations. Because so much variability respond to plaque, and respond to treatment. Some type of plaque symply are more virulent than

leading to the establishment of an manifest infection of the periodontium.17-20

microbial spectrum is shifted in favor of anaerobic gram-negative rods.

proven by several clinical studies.13,14,15

*intermedia* and *A. actinomycetemcomitans*.14

deph and loss of alveolare bone tissue.11

**5. Genetic component to periodontitis** 

lost by periodontitis than by caries.

**4. Aetiology of periodontitis** 

others. In adults, these bacteria routinely colonize the teeth when tooth cleaning is not performed on a regular basis. Although bacteria are essential for the initiation of periodontitis, there is currently no mechanism for determing the clinical trajectory of the disease for individual patients, i.e., differentiating those patients who will have a mild to moderate form of disease and respond well to simple professional care from those who are likely to develop a more severe periodontitis that demands extensive therapy and results in tooth mobility. Individual differences in disease progression are dramatic and are often not predictable by currently known mechanisms.

Identification of a risk factors may explain why individual patients do not respond uniformly to standard treatment. For example, a patient who is a heavy smoker may not heal as soon or as well as expected after treatment. Patients react differently to bacterial stimulation. This is a result not only of the type and amount of bacteria but also of the underlying genetic characteristics of the patient's immune system.The cytokines tumor necrosis factor alpha(TNFά) and interleukin 1 (IL-1) are key mediators of the inflammatory process and modulate the extracellular matrix components and bone which comprise the periodontal tissues.The genes encode pro-inflammatory proteins (IL 1A and IL-1B producing IL-1 ά and IL-1β). Several genetic polimorphisms have been described in the genes of the IL-1 cluster and, in case control studies, associations have been reported with increased severity of several chronic inflammatory diseases.

The genetic risk of developing periodontitis has been investigated by studying families and populations as well as twin.21-26 The studies conducted on twins have reported a significant genetic component explaining the variation in clinical attachment loss, probing depth and gingivitis. An association between the severe chronic form of the disease and a composite genotype in the interleukin IL-1ά and IL-1β genes has been reported.21 However, this association was found only in non-smokers. Other authors have subsequently investigated the IL-1ά - IL-1β genotypes with a chronic form of periodontitis with different results.27,28

The genetics influence resistance on periodontal disease has been determined from a wide variety of sources.29-32 Genotype positive patients had significantly more clinical expression of inflammation, as determined by bleeding on probing. In healthy patients 46.7 % of genotype positive patients had bleeding as compared with only 8.6% of genotype-negative patients.32,33 Their results from genetic susceptibility testing have the real potential to improve patient management. Many of the genetic markers for common disease involve polymorphisms in gene sequences involved in cytokine biological activity. Researchers know that in healthy subject IL-1 plays a very important role in inflammation and the expression of periodontitis. Patients with this genotype progress more rapidly toward severe periodontitis and have statistically significant increased inflammation.32 It has been established that this genotype occurs in approximately 30% of most of the populations that have been tested for this genotype.

Cells from people with a positive genotype produced up to four times more IL-1 in response to the same bacterial challenge.34 Because IL-1 in high concentrations is involved with destruction of tissues, this increased IL-1 response may explain the more rapid progression of periodontal disease in genotype-positive patients when faced with a bacterial challenge in their plaque. For patients with this genetic susceptibility to periodontitis, tooth loss was be minimized by good plaque control and definitive periodontal therapy.35

The Role of Genetic Predisposition in

influence on the test results.

 **test** 

**7.1 MicroDent<sup>R</sup>**

authors.50

duration (Fig 5).

Diagnosis and Therapy of Periodontal Diseases in Type 1 Diabetes Mellitus 469

Like GenotypR PRT the microDentR test is a molecular biological diagnostic device. Since it is based on the analysis of nucleic acids, there is no need for viable bacteria to perform the test and no special precautions are requireed during transport. The microDentR test a highly sensitive and highly specific molecular bological PCR-DNA-probe method. Due to the high specificity of the PCR, any potential contamination of the probe by concomitant flora has no

A defined cut-off ensures that every positive test result is of clinical relevance and that bacterial concentrations present in a healthy sulcus lead to a negative result. Sampling is performed from the gingival pocket with strile paper points. These marker species can be detected with the microDentR test: *Actinobacillus actinomycetemcomitans, Porphyromonas* 

Periodontopathogenic bacteria activate inflammatory mechanisms within the local periodontal tissue throught the production of toxins and other metabolites. The degree of this response depens on the general health and immunologic state of the patients. Besides that, exogenic risk factors such as haevy smoking, stress and medication can negatively influence the progression of periodontal disease. Patients who in addition are PRT-positive suffer from an overproduction of IL-1 leading to a significantly increased immunologic response to the presence of periodontopathogenic bacteria. This individuals therefore are at an even higher risk for developing severe disease and losing teeth. Knowledge of the IL—1 genotype, the bacterial load, and possible additional risk factors allow for the prediction of

A number of studies have demonstrated a relationship between DM and periodontal diseases, which are among the most prevalent complications of DM.38-57 Individuals with DM tend to have a higher prevalence of periodontal diseases and more severe and rapidly progressing forms than those who do not have DM.41,48 DM is a known risk factor for periodontitis in adults. Seppälä et al.49 demonstrated that patients with type 1 DM exhibit a higher degree of attachment loss and bone loss than control subjects under similar dental plaque conditions. This finding was confirmed in a follow-up site-by-site study by the same

The changes in the periodontal conditions are mostly expressed in the first year of the disease, and the damage to the periodontium which develops at this time is not greatly

It is an interesting result that younger DM subjects display more periodontal destruction than do non-DM subjects at a later age. In the all-age groups, the periodontal status varied according to the age of the patient at the onset of DM. This suggests that the early onset of DM (before 14) is a much greater risk factor for periodontal diseases than mere disease

Earlier investigators51,52 noted that the duration of DM was greater in groups with severe periodontal disease. Our results53 indicated that DM is associated with an increased risk of the development of periodontal disease in the event of an increased duration of the DM, and the level of oral hygiene is considered to be a contributory factor rather than the primary etiologic factor in the initiation of gingivitis and periodontitis in those with DM. In agreement with Gusberti et al.57 the findings of our study53 demonstrate that poorly controlled type 1 DM patients with elevated blood glucose and HbA1c levels have a greater

*gingivalis, Prevotella intermedia, Bacteroides forsythus, Treponema denticola.* 

the patient's future periodontal status including the risk of further tooth loss.

**8. Risk factor influence on periodontitis in type 1 DM** 

influeced in the further course of the disease (Fig 4).

#### **6. Genetic test for susceptibility to periodontal disease**

Two polymorphisms within the IL-1 gene cluster show a close association with periodontitis. One polymorphism is located at position -899 of the Interleukin 1ά gene, the other at position +3953 of the Interleukin 1β gene.32,36 Within both polymorphisms allele 1 harbors a cytidin c, whereas allele 2 carries a thymidin (T) at the respective position. Allele 2 of the +3953 polymorphism of the IL-1β gene leads to an alteration of the corresponding protein resulting in an overproduction of IL-1β.37 This overproduction of IL-1 seems to override the feedback mechanisms which normally limit inflammation resulting in the development of massive gingival pockets and degradation of periodontal tissue. These data allow a risk assessment, defining a patients as PRT-positive or PRT negative, the presence of periodontitis risk alleles at positions IL-1ά-889 and IL-1β +3953.

#### **6.1 Genotyp<sup>R</sup> PRT test**

With the GenotypR PRT test (Hain Lifescience) the base composition and allelic combination of the two IL1 loci can be analysed. The test is a molecular biological assay based on the identification of gene loci associated with an elevated risk in developing periodontitis by means of highly specific DNA probes. It is based on the analysis of nucleic acids, there is no need for viable bacteria to perform the test and no special precautions are required during transport. A detailed sequence analysis has to be performed by additional examinations. The GenotypR PRT test is not a diagnostic test for periodontal disease. It is rather a test determing the patient's genetic susceptibility to developing severe, generalized periodontitis in the future and helps to plan a comprehensive therapy. Processing and interpretation of the test is performed in clinical laboratories from a buccal swab containing cells of the mucous membrane of the patient's cheek.

#### **7. Indications for microbiological testing of the subgingival flora**

It is generally accepted that periodontitis is initiated by the establishment of a specific subgingival bacterial flora. Some of the marker pathogens belong to the group of obligatory anaerobic, black pigmented *Bacteroides species* such as *Porphyromonas gingivalis* and *Prevotella intermedia.* In addition, the bacterial species *Actinobacillus actinomycetemcomitans*  (*Haemophilus a.*), *Bacteroidea forsythus,* and *Treponema denticola* play a pivotal role in the initiation of periodontal disease (Table 1).


Table 1. Specific Bacteria Associated with Periodontal Disease (Annals of Periodontology 1:928,1996)

#### **7.1 MicroDent<sup>R</sup> test**

468 Type 1 Diabetes Complications

Two polymorphisms within the IL-1 gene cluster show a close association with periodontitis. One polymorphism is located at position -899 of the Interleukin 1ά gene, the other at position +3953 of the Interleukin 1β gene.32,36 Within both polymorphisms allele 1 harbors a cytidin c, whereas allele 2 carries a thymidin (T) at the respective position. Allele 2 of the +3953 polymorphism of the IL-1β gene leads to an alteration of the corresponding protein resulting in an overproduction of IL-1β.37 This overproduction of IL-1 seems to override the feedback mechanisms which normally limit inflammation resulting in the development of massive gingival pockets and degradation of periodontal tissue. These data allow a risk assessment, defining a patients as PRT-positive or PRT negative, the presence of

With the GenotypR PRT test (Hain Lifescience) the base composition and allelic combination of the two IL1 loci can be analysed. The test is a molecular biological assay based on the identification of gene loci associated with an elevated risk in developing periodontitis by means of highly specific DNA probes. It is based on the analysis of nucleic acids, there is no need for viable bacteria to perform the test and no special precautions are required during transport. A detailed sequence analysis has to be performed by additional examinations. The GenotypR PRT test is not a diagnostic test for periodontal disease. It is rather a test determing the patient's genetic susceptibility to developing severe, generalized periodontitis in the future and helps to plan a comprehensive therapy. Processing and interpretation of the test is performed in clinical laboratories from a buccal swab containing cells of the

It is generally accepted that periodontitis is initiated by the establishment of a specific subgingival bacterial flora. Some of the marker pathogens belong to the group of obligatory anaerobic, black pigmented *Bacteroides species* such as *Porphyromonas gingivalis* and *Prevotella intermedia.* In addition, the bacterial species *Actinobacillus actinomycetemcomitans*  (*Haemophilus a.*), *Bacteroidea forsythus,* and *Treponema denticola* play a pivotal role in the

> Prevotella intermedia Peptostreptococcus micros

Treponema denticola

Table 1. Specific Bacteria Associated with Periodontal Disease (Annals of Periodontology

Streptococcus intermedius-complex

**7. Indications for microbiological testing of the subgingival flora** 

**Strong evidence for etiology Moderate evidence for etiology** 

actinomycetemcomitans Campylobacter rectus Porphyromonas gingivalis Eubacterium nodatum Bacteroides forsytus Fusobacterium nucleatum

**6. Genetic test for susceptibility to periodontal disease** 

periodontitis risk alleles at positions IL-1ά-889 and IL-1β +3953.

**6.1 Genotyp<sup>R</sup>**

 **PRT test** 

mucous membrane of the patient's cheek.

initiation of periodontal disease (Table 1).

Actinobacillus

1:928,1996)

Like GenotypR PRT the microDentR test is a molecular biological diagnostic device. Since it is based on the analysis of nucleic acids, there is no need for viable bacteria to perform the test and no special precautions are requireed during transport. The microDentR test a highly sensitive and highly specific molecular bological PCR-DNA-probe method. Due to the high specificity of the PCR, any potential contamination of the probe by concomitant flora has no influence on the test results.

A defined cut-off ensures that every positive test result is of clinical relevance and that bacterial concentrations present in a healthy sulcus lead to a negative result. Sampling is performed from the gingival pocket with strile paper points. These marker species can be detected with the microDentR test: *Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Bacteroides forsythus, Treponema denticola.* 

Periodontopathogenic bacteria activate inflammatory mechanisms within the local periodontal tissue throught the production of toxins and other metabolites. The degree of this response depens on the general health and immunologic state of the patients. Besides that, exogenic risk factors such as haevy smoking, stress and medication can negatively influence the progression of periodontal disease. Patients who in addition are PRT-positive suffer from an overproduction of IL-1 leading to a significantly increased immunologic response to the presence of periodontopathogenic bacteria. This individuals therefore are at an even higher risk for developing severe disease and losing teeth. Knowledge of the IL—1 genotype, the bacterial load, and possible additional risk factors allow for the prediction of the patient's future periodontal status including the risk of further tooth loss.

#### **8. Risk factor influence on periodontitis in type 1 DM**

A number of studies have demonstrated a relationship between DM and periodontal diseases, which are among the most prevalent complications of DM.38-57 Individuals with DM tend to have a higher prevalence of periodontal diseases and more severe and rapidly progressing forms than those who do not have DM.41,48 DM is a known risk factor for periodontitis in adults. Seppälä et al.49 demonstrated that patients with type 1 DM exhibit a higher degree of attachment loss and bone loss than control subjects under similar dental plaque conditions. This finding was confirmed in a follow-up site-by-site study by the same authors.50

The changes in the periodontal conditions are mostly expressed in the first year of the disease, and the damage to the periodontium which develops at this time is not greatly influeced in the further course of the disease (Fig 4).

It is an interesting result that younger DM subjects display more periodontal destruction than do non-DM subjects at a later age. In the all-age groups, the periodontal status varied according to the age of the patient at the onset of DM. This suggests that the early onset of DM (before 14) is a much greater risk factor for periodontal diseases than mere disease duration (Fig 5).

Earlier investigators51,52 noted that the duration of DM was greater in groups with severe periodontal disease. Our results53 indicated that DM is associated with an increased risk of the development of periodontal disease in the event of an increased duration of the DM, and the level of oral hygiene is considered to be a contributory factor rather than the primary etiologic factor in the initiation of gingivitis and periodontitis in those with DM. In agreement with Gusberti et al.57 the findings of our study53 demonstrate that poorly controlled type 1 DM patients with elevated blood glucose and HbA1c levels have a greater

The Role of Genetic Predisposition in

0

OHI-S = oral hygiene OHI-S= 0 / PI: p<0.0001

PI = The intensity of gingivitis and periodontitis

with severe ophthalmic complications in type 1 DM.

Duration time: < 1 year > 5 year

(OHI-S) and the duration of DM

0,5

1

1,5

2

2,5

3

3,5

Diagnosis and Therapy of Periodontal Diseases in Type 1 Diabetes Mellitus 471

Type 1 DM to increase the prevalence and severity of periodontitis independent of the effects of oral hygiene, and duration time of DM.42 However the severity of periodontal disease increased with the duration of DM only among those with an adequate level of oral hygiene (OHI-S = 0) The association between periodontal disease and the duration of diabetes mellitus is consistent with trends seen in other complications of DM whereas the longer duration of diabetes mellitus is in direct proportion of the prevalence and severity of periodontal disease. The development of systemic complications of diabetes such as retinopathy, nephropathy, is also is relationship with the duration of diabetes mellitus

OHI-S PI

Fig. 6. The intensity of gingivitis and periodontitis(PI) according to the level of oral hygiene

On the other hand, the presence of severe periodontal infection may also increase the risk for microvascular and macrovascular complications. DM patients with severe periodontal disease demonstrate a significantly higher prevalence of proteinuria and a greater number of cardiovascular complications.31,54,55 Karjalainen et al.56, Genco,31 Lopez et al.52, Albrecht59 examined the association between the severity of periodontal disease and organ complications (retinopathy) and found that advanced periodontal disease was associated

A more pronounced incidence of poor glycemic control in subjects with a shorter duration of DM would be consistent with the hypothesis that hyperglycemia increases linearly with time, but at different rates in different people. This hypothesis suggests that patients with

agreement with Rylander58, Galea et al.59, Rosenthal et al.60 and Lopez.52

prevalence to more severe periodontal diseases. The severity of periodontal disease was observed to decrease as the control of the DM improved, in agreement with Tervonen and Knuuttila55, Rylander et al.58

Duration of DM: < 1year / > 1year: \*P<0.001

Duration of DM: <1 year, >1 year

Fig. 4. Periodontal conditions (PI) in patients with short and long histories of DM.


Fig. 5. Age of the patient at the onset of DM

prevalence to more severe periodontal diseases. The severity of periodontal disease was observed to decrease as the control of the DM improved, in agreement with Tervonen and

15-19 20-24 25-29 30-34 35-44 45-54 55+

15-19 20-24 25-29 30-34 35-44 45-54 55+

Fig. 4. Periodontal conditions (PI) in patients with short and long histories of DM.

**Age/years**

**Age/years**

Knuuttila55, Rylander et al.58

**PI**

\*

Duration of DM: < 1year / > 1year: \*P<0.001 Duration of DM: <1 year, >1 year

**PI**

< 14 year / > 14 year: P<0.0001

< 14 year, ▧ 15-25 year, > 25 year

Fig. 5. Age of the patient at the onset of DM

\*

Type 1 DM to increase the prevalence and severity of periodontitis independent of the effects of oral hygiene, and duration time of DM.42 However the severity of periodontal disease increased with the duration of DM only among those with an adequate level of oral hygiene (OHI-S = 0) The association between periodontal disease and the duration of diabetes mellitus is consistent with trends seen in other complications of DM whereas the longer duration of diabetes mellitus is in direct proportion of the prevalence and severity of periodontal disease. The development of systemic complications of diabetes such as retinopathy, nephropathy, is also is relationship with the duration of diabetes mellitus agreement with Rylander58, Galea et al.59, Rosenthal et al.60 and Lopez.52

PI = The intensity of gingivitis and periodontitis OHI-S = oral hygiene OHI-S= 0 / PI: p<0.0001 Duration time: < 1 year > 5 year

Fig. 6. The intensity of gingivitis and periodontitis(PI) according to the level of oral hygiene (OHI-S) and the duration of DM

On the other hand, the presence of severe periodontal infection may also increase the risk for microvascular and macrovascular complications. DM patients with severe periodontal disease demonstrate a significantly higher prevalence of proteinuria and a greater number of cardiovascular complications.31,54,55 Karjalainen et al.56, Genco,31 Lopez et al.52, Albrecht59 examined the association between the severity of periodontal disease and organ complications (retinopathy) and found that advanced periodontal disease was associated with severe ophthalmic complications in type 1 DM.

A more pronounced incidence of poor glycemic control in subjects with a shorter duration of DM would be consistent with the hypothesis that hyperglycemia increases linearly with time, but at different rates in different people. This hypothesis suggests that patients with

The Role of Genetic Predisposition in

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

PAG positive/PAG negative: p<0.001

negative subjects in type 1 DM adolescents shows the Fig. 8.

Diagnosis and Therapy of Periodontal Diseases in Type 1 Diabetes Mellitus 473

To control the effect of age on disease severity, data were analyzed separately for type 1 DM adolescents aged 14-19 years. In this age range, the composite genotype was present in 22,7 % of DM adolescents and 8,57% of healthy individuals were estimated to carry the IL-1 risk genotype. Distribution of the PAG (Periodontitis Associated Genotype)21 positive and

Type 1DM

Fig. 8. Frequency of PAG positive and negative adolescents with type 1 DM

Gingivitis was more severe in those adolescents with positive GenoTypeRPRT test (Fig.9).

GI

PAG pos PAG neg PAG pos PAG neg Typ1 DM Type1 DM Control Control

Fig. 9. The intensity of gingivitis (GI) of PAG positiv and in negative adolescents with type 1 DM.

PAG pos Pag NEG

■ PAG positive □ PAG negative

rapidly increasing hyperglycemia would have more severe periodontal disease at the onset of DM, resulting in damage to the periodontium.

A positive correlation between the level of control of the disease and the intensity of gingivitis and periodontitis. In the well-controlled type 1 DM patients the intensity of gingivitis and periodontitis was lower than in those with poor glycemic control agreement with Gusberti et al.57, Albrecht et al.41 Good metabolic control of DM reduces the susceptibility to infection and is therefore also important for the prevention of periodontal disease in people with type 1 DM. In patients with poorly controlled DM, an improvement of the metabolic control may improve the periodontal condition.61-64 Conversely, periodontal disease can interfere with the control of DM and can increase the insulin requirements in previously stable patients.65-67,56,68,2,69

Smoking is associated with an increased intensity of periodontitis. Very light or occasional smokers did not show statistically significant differences compared to non-smokers respect to the prevalence and intensity of gingivitis or periodontitis. No periodontally healthy subjects who has been, or who use to be heavy smokers. Tobacco contains cytotoxic substances such as nicotine which may also have a negative effect on the cellular turnover and repair of the periodontium.68,70,53

#### **9. Genetic predisposition and periodontal disease in type 1 DM**

Recently several study demonstrates that specific genetic markers, that have been associated with increased IL-1 production, are a strong indicator of susceptibility to severe periodontitis in healthy adults. 22,29,31 The study presented here was to explore a possible association between IL-1A and IL-1-B genotypes in patient with type 1 DM and controls with periodontitis. The frequency -in type 1 DM and controls- of the composite genotype that comprises allele 2 of the IL-1A plus IL-1B is shown in Fig.7. All subject were non-smokers.

Fig. 7. Frequency of the GenotypeR PRT positive and negative type 1 DM adult patients and control with periodontitis.

rapidly increasing hyperglycemia would have more severe periodontal disease at the onset

A positive correlation between the level of control of the disease and the intensity of gingivitis and periodontitis. In the well-controlled type 1 DM patients the intensity of gingivitis and periodontitis was lower than in those with poor glycemic control agreement with Gusberti et al.57, Albrecht et al.41 Good metabolic control of DM reduces the susceptibility to infection and is therefore also important for the prevention of periodontal disease in people with type 1 DM. In patients with poorly controlled DM, an improvement of the metabolic control may improve the periodontal condition.61-64 Conversely, periodontal disease can interfere with the control of DM and can increase the insulin requirements in

Smoking is associated with an increased intensity of periodontitis. Very light or occasional smokers did not show statistically significant differences compared to non-smokers respect to the prevalence and intensity of gingivitis or periodontitis. No periodontally healthy subjects who has been, or who use to be heavy smokers. Tobacco contains cytotoxic substances such as nicotine which may also have a negative effect on the cellular turnover

Recently several study demonstrates that specific genetic markers, that have been associated with increased IL-1 production, are a strong indicator of susceptibility to severe periodontitis in healthy adults. 22,29,31 The study presented here was to explore a possible association between IL-1A and IL-1-B genotypes in patient with type 1 DM and controls with periodontitis. The frequency -in type 1 DM and controls- of the composite genotype that comprises allele 2 of the IL-1A plus IL-1B is shown in Fig.7. All subject were non-smokers.

positive negative positive negative

Fig. 7. Frequency of the GenotypeR PRT positive and negative type 1 DM adult patients and

Type 1DM control

**9. Genetic predisposition and periodontal disease in type 1 DM** 

of DM, resulting in damage to the periodontium.

previously stable patients.65-67,56,68,2,69

and repair of the periodontium.68,70,53

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

control with periodontitis.

**%**

To control the effect of age on disease severity, data were analyzed separately for type 1 DM adolescents aged 14-19 years. In this age range, the composite genotype was present in 22,7 % of DM adolescents and 8,57% of healthy individuals were estimated to carry the IL-1 risk genotype. Distribution of the PAG (Periodontitis Associated Genotype)21 positive and negative subjects in type 1 DM adolescents shows the Fig. 8.

Fig. 8. Frequency of PAG positive and negative adolescents with type 1 DM

Gingivitis was more severe in those adolescents with positive GenoTypeRPRT test (Fig.9).

PAG positive/PAG negative: p<0.001

Fig. 9. The intensity of gingivitis (GI) of PAG positiv and in negative adolescents with type 1 DM.

The Role of Genetic Predisposition in

and losing teeth.

Td: *Treponema denticola* Bf: *Bacteroides forsythus*

Actinobacillus

Pg: *Porphyromonas gingivalis*

Aa: *Actinobacillus actinomycetemcomitans*

actinomycetemcomitans(Aa)

population with type 1DM though they are not risk factors.

Diagnosis and Therapy of Periodontal Diseases in Type 1 Diabetes Mellitus 475

cause disease, is not sufficient. According our findings there were a significant difference between the severity of the gingivitis, parodontitis and the bacteria identified from the gingival pocket of patients with type 1 DM (Fig.11, Table 2). In case of gingivitis alone, the most prevalent bacterium was *Bacteroides forsythus* (11.11%), whereas in parodontitis it was *Treponema denticola* (75.92%). In type 1 DM the presence of *Treponema denticola* no additional risk of developing agressive periodontitis, despite the fact its presence is necessary for the disease to develop. The *Bacteriodes forsythus, Prevotella intermedia, Porphyromonas gingivalis, Actinobacillus actinomicetemcomitans* may be risk indicators for periodontal disease in

Patients who in addition are PRT positive suffer from an overproduction of IL-1 leading to a significantly increased immunologic response to the presence of periodontopathogenic bacteria. These individuals therefore are at an even higher risk for developing severe disease

Fig. 11. The distribution of the subgingival microflora in adult patient with type 1 DM.

Porphyromonas gingivalis (Pg)

5% 15% 14% 31% 35%

Table 2. Distribution of the Subgingival Microflora in Type 1 DM with Periodontal Disease

Subgingival bacterial flora in type 1 DM

Prevotella inermedia(Pi) Bacteroides forsythus (Bf)

Treponema denticola (Td)

Td Bf Aa Pg Pi

In type 1 DM there was significant more extracted teeth in GenotypeR PRT positive subjects than in negative group or non-diabetic people (p< 0.001) (Fig.10).

#### P< 0.001

Fig. 10. The mean value of extracted teeth (M) in DM patients and metabolically healthy individuals with positive Genotype.

Periodontitis involves multiple clinical patterns including various severities of periodontitis, uncommon early onset forms that affect children and young adults with type 1 DM, and patients who do not respond predictably to conventional therapy refractory periodontitis.

Guzman et al.71 have shown a possible interactions between genetic an enviromental factors that there is interplay between genetic an environmental factors that results in periodontal disease.

The finding that a specific genotype in the IL-1 gene cluster correlates with severe periodontitis suggest a genetic mechanism by which some individuals, if challenged by bacterial accumulations, may have a more vigorous immuno-inflammatory response leading to more severe periodontitis in type 1 DM. The lack of reliable markers for type 1 DM patient susceptibility to severe periodontitis has prevented the early identification of those at most risk and has prevented delivery of therapy appropriate for the degree of the risk.

#### **10. The modification of the subgingival microflora in patients with type 1 DM**

Periodontopathogenic bacteria activate inflammatory mechanisms within the local periodontal tissue throught the production of toxins and other metabolites. The degree of this response depends on the general health and immunologic state of the patients. Besides that, exogenic risk factors such as heavy smoking, stress, and medication can negatively influence the progression of periodontal disease. Of all of the various microorganism that colonize the mouth in healthy subjects, there are three, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Bacteroides forsythus have been implicated as etiologic agents in periodontitis. The presence of periodontal pathogen, though necessary to

In type 1 DM there was significant more extracted teeth in GenotypeR PRT positive subjects

type1 DM pos type1DM neg control pos control neg

type 1 DM positive type 1 DM negative control positive control negative

pos neg pos neg

type1 DM type1DM control control

Fig. 10. The mean value of extracted teeth (M) in DM patients and metabolically healthy

Periodontitis involves multiple clinical patterns including various severities of periodontitis, uncommon early onset forms that affect children and young adults with type 1 DM, and patients who do not respond predictably to conventional therapy refractory periodontitis. Guzman et al.71 have shown a possible interactions between genetic an enviromental factors that there is interplay between genetic an environmental factors that results in periodontal

The finding that a specific genotype in the IL-1 gene cluster correlates with severe periodontitis suggest a genetic mechanism by which some individuals, if challenged by bacterial accumulations, may have a more vigorous immuno-inflammatory response leading to more severe periodontitis in type 1 DM. The lack of reliable markers for type 1 DM patient susceptibility to severe periodontitis has prevented the early identification of those at most risk

**10. The modification of the subgingival microflora in patients with type 1 DM**  Periodontopathogenic bacteria activate inflammatory mechanisms within the local periodontal tissue throught the production of toxins and other metabolites. The degree of this response depends on the general health and immunologic state of the patients. Besides that, exogenic risk factors such as heavy smoking, stress, and medication can negatively influence the progression of periodontal disease. Of all of the various microorganism that colonize the mouth in healthy subjects, there are three, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Bacteroides forsythus have been implicated as etiologic agents in periodontitis. The presence of periodontal pathogen, though necessary to

and has prevented delivery of therapy appropriate for the degree of the risk.

than in negative group or non-diabetic people (p< 0.001) (Fig.10).

individuals with positive Genotype.

**M**

P< 0.001

disease.

cause disease, is not sufficient. According our findings there were a significant difference between the severity of the gingivitis, parodontitis and the bacteria identified from the gingival pocket of patients with type 1 DM (Fig.11, Table 2). In case of gingivitis alone, the most prevalent bacterium was *Bacteroides forsythus* (11.11%), whereas in parodontitis it was *Treponema denticola* (75.92%). In type 1 DM the presence of *Treponema denticola* no additional risk of developing agressive periodontitis, despite the fact its presence is necessary for the disease to develop. The *Bacteriodes forsythus, Prevotella intermedia, Porphyromonas gingivalis, Actinobacillus actinomicetemcomitans* may be risk indicators for periodontal disease in population with type 1DM though they are not risk factors.

Patients who in addition are PRT positive suffer from an overproduction of IL-1 leading to a significantly increased immunologic response to the presence of periodontopathogenic bacteria. These individuals therefore are at an even higher risk for developing severe disease and losing teeth.

Td: *Treponema denticola* Bf: *Bacteroides forsythus* Aa: *Actinobacillus actinomycetemcomitans* Pg: *Porphyromonas gingivalis*

Fig. 11. The distribution of the subgingival microflora in adult patient with type 1 DM.

#### Subgingival bacterial flora in type 1 DM


Table 2. Distribution of the Subgingival Microflora in Type 1 DM with Periodontal Disease

The Role of Genetic Predisposition in

are informed about their condition.

minimized.

pockets.

**12. Conclusion** 

not be as good as outcomes for those who do not smoke.

Diagnosis and Therapy of Periodontal Diseases in Type 1 Diabetes Mellitus 477

Treatment decisions will be affected if a patient has risk factors, for example, haevy smokers. Haevy smokers are counseled that the treatment outcome from regenerative therapy will

In all cases where patient motivation and compliance are major obstacles to efficient prophylactic measures the situation can be dramatically improved when high risk patients

Knowledge of the IL-1 genotype, the bacterial load, and possible additional risk factors allow for the prediction of the patient's future periodontal status including the risk of further tooth loss. For the first time, these data enable the dentist to plan an individual therapy matching the patient's needs. Knowledge of the IL-1 genotype also allows a more efficient therapy from an economic point of view because over- and under-treatment can be

Periodontitis is a complex multifactorial disease. Similarly, Type 1 DM is a complex metabolic syndrome. Periodontal disease can be especially problematic for individuals with type 1 DM, in whom the disease may have an early onset or may progress more rapidly. Many characteristics and local factors such as dental calculus, the smoking habits and general factors (the duration of DM, the age, the degree of metabolic control, and the complications of DM) have been identified as factors that put people et an enhanced risk. The initial dental therapy for patients with type 1 DM, as for all patients, must be directed toward the control of acute oral infections at the onset of DM. It is important to advise the phisician of the periodontal status, since the presence of infections including advanced periodontal disease may increase the insulin resistance and cotribute to a worsening of the DM state. Regular dental care may help maintain good oral health and it is especially important at the onset of the disease. Patients should also be checked regularly for bleeding gums or inflammation. Educating the patient in proper home oral care is a standard routine of periodontal treatment and prevention. Plaque control and scaling procedures frequently resolve gingivitis. However, where more tissue destruction has occurred, it may still be difficult or impossible for the patient to remove plaque deposits from the periodontal

Periodontitis is a disease leading to destruction of connective tissue and bone that support the teeth. Due to the multifunctional etiology of the disease its clinical appearence and progression greatly vary resulting in difficulties in planing an effective therapy. Smoking should be avoided because it is associated with an increased intensity of periodontitis. Patients who are genotype positive do not lose all of their teeth to periodontitis because, for the most part, it is a treatable and preventable disease. If patients are identified as having the genetic susceptibility factor and begin a treatment or prevention plan, there are high expectations for favorable outcomes and there is no known risk from being tested. Determining the patient's genetic susceptibility in the future helps to plan a comprehensiv therapy and regular dental control to improve patients care prevent to developing severe, generalized periodontitis and toothlos. This IL-1 genotype does not cause periodontal disease directly, this marker is not a causative factor, instead it is a severity risk factor for susceptibility or predisposition to periodontitis. Optimal prophylaxis and efficient therapy of the polymorphisms defining the periodontitis risk genotype (PRT-positive) patients a careful microbiological testing of the subgingival bacterial flora for allows for the timely

#### **11. Diagnosis and therapy of periodontal disease in type 1 DM**

A considerable fraction of periodontal diseases can be stabilized for years using classical mechanical treatments as root planing or deep scaling. However, this treatment often is not sufficient for elimination of the tissue invading periodontal pathogens. Subsequently, progressive loss of attachment and bone tissue might occur in spite of diligent treatment. In these cases a specific concomitant therapy with antibiotics promises to be more efficient -of course only after careful microbiological testing-.

The choice of medication and mode of application depends on the composition of the subgingival flora and the clinical manifestation of the periodontitis. Where tissue-invasive, periopathogenic bacteria such as *Treponema denticola* are present, mechanical methods like root-planing or deep-scaling are often ineffective in eliminating the pathogen. Despite careful treatment, the result is progressive attachment loss and bone resorption. In such cases, a one-of antimicrobial concomittal therapy – only undertaken after microbiological diagnostics, of course – is much more effective while causing less side effects.

In the main, both local and systemic antibiotic applications are available. In the case of a generalized periodontal disease, an adjuvant systemic therapy is indicated. If the infection focus is limited to individual sites, a local treatment is a sensible alternative.

Antibiotic therapies should in any case only be implemented after microbiological diagnostics (e.g.microDentR test) have been completed, in order to avoid both excessive and under treatment.

In most cases a negative bacterium test result can be equated with periodontal stability. However, the presence of periodontal marker organisms indicates an increased risk for progressive destruction of the periodontium. It is obvious that a therapy with antibiotics should be initiated only after thorough microbiological test.

A sustained success of the therapy depends on an optimal compliance of the patients and regular recall sessions. Regular control examinations of the subgingival flora are rather helpful in early diagnosis of potential rezidives.

Genetics factors should also be determined because they play a key role in providing information to make better treatment decisions. A genotype result is always important to consider when making treatment decisions, even if the patient is genotype-negative. A negative result does not mean that a patient will be periodontal-disease-free. Genotype negative individuals must still be cautious about other risk factors, such as stress, smoking, bad oral hygiene-, and diabetes control.

The genetic predictive test for periodontitis complements the dentist's full scope of services by providing additional wanted and useful information. Indications for the genetic predictive test may be of value to the following patients groups:


Treatment decisions will be affected if a patient has risk factors, for example, haevy smokers. Haevy smokers are counseled that the treatment outcome from regenerative therapy will not be as good as outcomes for those who do not smoke.

In all cases where patient motivation and compliance are major obstacles to efficient prophylactic measures the situation can be dramatically improved when high risk patients are informed about their condition.

Knowledge of the IL-1 genotype, the bacterial load, and possible additional risk factors allow for the prediction of the patient's future periodontal status including the risk of further tooth loss. For the first time, these data enable the dentist to plan an individual therapy matching the patient's needs. Knowledge of the IL-1 genotype also allows a more efficient therapy from an economic point of view because over- and under-treatment can be minimized.

#### **12. Conclusion**

476 Type 1 Diabetes Complications

A considerable fraction of periodontal diseases can be stabilized for years using classical mechanical treatments as root planing or deep scaling. However, this treatment often is not sufficient for elimination of the tissue invading periodontal pathogens. Subsequently, progressive loss of attachment and bone tissue might occur in spite of diligent treatment. In these cases a specific concomitant therapy with antibiotics promises to be more efficient -of

The choice of medication and mode of application depends on the composition of the subgingival flora and the clinical manifestation of the periodontitis. Where tissue-invasive, periopathogenic bacteria such as *Treponema denticola* are present, mechanical methods like root-planing or deep-scaling are often ineffective in eliminating the pathogen. Despite careful treatment, the result is progressive attachment loss and bone resorption. In such cases, a one-of antimicrobial concomittal therapy – only undertaken after microbiological

In the main, both local and systemic antibiotic applications are available. In the case of a generalized periodontal disease, an adjuvant systemic therapy is indicated. If the infection

Antibiotic therapies should in any case only be implemented after microbiological diagnostics (e.g.microDentR test) have been completed, in order to avoid both excessive and

In most cases a negative bacterium test result can be equated with periodontal stability. However, the presence of periodontal marker organisms indicates an increased risk for progressive destruction of the periodontium. It is obvious that a therapy with antibiotics

A sustained success of the therapy depends on an optimal compliance of the patients and regular recall sessions. Regular control examinations of the subgingival flora are rather

Genetics factors should also be determined because they play a key role in providing information to make better treatment decisions. A genotype result is always important to consider when making treatment decisions, even if the patient is genotype-negative. A negative result does not mean that a patient will be periodontal-disease-free. Genotype negative individuals must still be cautious about other risk factors, such as stress, smoking,

The genetic predictive test for periodontitis complements the dentist's full scope of services by providing additional wanted and useful information. Indications for the genetic

 DM patients exhibiting refractory, therapy-resistent periodontitis. A positive test result might explain previous treatment failures and is an indicator for planing an alternative

DM patients exhibiting progressive periodontitis. A positive test result might indicate

 Type 1 DM adolescens -under 14 years- exhibiting early clinical sign of periodontitis. Before starting treatment, the test helps to plan an individual therapy maching the patient's t.i. a therapy stopping progress of the disease without risking over-treatment.

the necessity for a more agressive therapy and shortes recall intervals.

**11. Diagnosis and therapy of periodontal disease in type 1 DM** 

diagnostics, of course – is much more effective while causing less side effects.

focus is limited to individual sites, a local treatment is a sensible alternative.

should be initiated only after thorough microbiological test.

predictive test may be of value to the following patients groups:

helpful in early diagnosis of potential rezidives.

bad oral hygiene-, and diabetes control.

Haewy smokers patients with type 1 DM.

course only after careful microbiological testing-.

under treatment.

therapy.

Periodontitis is a complex multifactorial disease. Similarly, Type 1 DM is a complex metabolic syndrome. Periodontal disease can be especially problematic for individuals with type 1 DM, in whom the disease may have an early onset or may progress more rapidly. Many characteristics and local factors such as dental calculus, the smoking habits and general factors (the duration of DM, the age, the degree of metabolic control, and the complications of DM) have been identified as factors that put people et an enhanced risk.

The initial dental therapy for patients with type 1 DM, as for all patients, must be directed toward the control of acute oral infections at the onset of DM. It is important to advise the phisician of the periodontal status, since the presence of infections including advanced periodontal disease may increase the insulin resistance and cotribute to a worsening of the DM state. Regular dental care may help maintain good oral health and it is especially important at the onset of the disease. Patients should also be checked regularly for bleeding gums or inflammation. Educating the patient in proper home oral care is a standard routine of periodontal treatment and prevention. Plaque control and scaling procedures frequently resolve gingivitis. However, where more tissue destruction has occurred, it may still be difficult or impossible for the patient to remove plaque deposits from the periodontal pockets.

Periodontitis is a disease leading to destruction of connective tissue and bone that support the teeth. Due to the multifunctional etiology of the disease its clinical appearence and progression greatly vary resulting in difficulties in planing an effective therapy. Smoking should be avoided because it is associated with an increased intensity of periodontitis.

Patients who are genotype positive do not lose all of their teeth to periodontitis because, for the most part, it is a treatable and preventable disease. If patients are identified as having the genetic susceptibility factor and begin a treatment or prevention plan, there are high expectations for favorable outcomes and there is no known risk from being tested. Determining the patient's genetic susceptibility in the future helps to plan a comprehensiv therapy and regular dental control to improve patients care prevent to developing severe, generalized periodontitis and toothlos. This IL-1 genotype does not cause periodontal disease directly, this marker is not a causative factor, instead it is a severity risk factor for susceptibility or predisposition to periodontitis. Optimal prophylaxis and efficient therapy of the polymorphisms defining the periodontitis risk genotype (PRT-positive) patients a careful microbiological testing of the subgingival bacterial flora for allows for the timely

The Role of Genetic Predisposition in

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### *Edited by David Wagner*

This book is a compilation of reviews about the complication of Type 1 Diabetes. T1D is a classic autoimmune disease. Genetic factors are clearly determinant but cannot explain the rapid, even overwhelming expanse of this disease. Understanding etiology and pathogenesis of this disease is essential. The complications associated with T1D cover a range of clinical obstacles. A number of experts in the field have covered a range of topics for consideration that are applicable to researcher and clinician alike. This book provides apt descriptions of cutting edge technologies and applications in the ever going search for treatments and cure for diabetes.

Type 1 Diabetes Complications

Type 1 Diabetes

Complications

*Edited by David Wagner*

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