**2. Characteristics of the damage**

#### **2.1. Glucose and oxidative stress in diabetic vascular damage**

Endothelial dysfunction is a pathological state of the endothelium and can be defined as an aberration of the normal endothelial function of vascular relaxation, blood clotting and immune function. In general, it means impaired endothelium-dependent vasodilation as a result of imbalance between vasodilating and vasoconstricting substances produced by (or acting on) the endothelium. Endothelial dysfunction can be a significant predictor of coronary artery disease and atherosclerosis and it increases the risk of stroke and heart attack [8]. In basic science and in clinical research, endothelial function is commonly assessed by the use of the acetylcholine-mediated vasodilatation test or by flow-mediated vasodilation, and this methodology is considered the 'gold standard' at this moment [8, 9]. Endothelial dysfunction is primarily responsible for the impaired vasorelaxation in diabetes but it is closely followed by the development of vascular smooth muscle cell dysfunction [10, 11]. Impaired relaxation may be caused by diminished production or increased destruction of vasodilating factors or impaired response to them in diabetes. Oxidative stress is considered as one of the major underlying mechanisms, which leads to endothelial dysfunction in hyperglycemia, since the therapeutic supplementation of antioxidants or antioxidant enzymes can restore the endothelium-dependent vasodilation in experimental models of diabetes [10].

Glucose-induced damage is apparently controversial: glucose is a major source of energy and a small increase in blood glucose, which has no obvious ill effect on the short term, can cause serious long-term complications in diabetes. Glucose uptake is noninsulin dependent in endothelial cells and it occurs via GLUT1 (glucose transporter 1), thus high blood glucose level results in similarly high intracellular glucose concentration in endothelial cells [12, 13]. Endothelial cells have few mitochondria and primarily use glycolysis to produce ATP molecules, which suggests low oxygen consumption and relatively low level of oxidant production [14]. Furthermore, higher glucose concentration would allow even higher rate of anaerobic metabolism to produce the necessary amount of ATP and limit aerobic metabolism, oxygen consumption and reactive oxygen species (ROS) production in the cells. Still, hyperglycemia is associated with the activation of various ROS-producing pathways and increased oxidant production in endothelial cells [15, 16]. Oxidants play a significant role in the destruction of nitric oxide and other signaling molecules and result in impaired vasoreactivity [10, 17, 18]. Inflammatory pathways may be implicated in the early stages of the injury and they are typically involved in the later stages of the disease and contribute to oxidant production and inflammatory cytokine secretion, which can also change the vascular function [19]. Oxidative stress also induces DNA damage that triggers endothelial cell senescence that might have an impact on vascular function in the later stages of the injury [20]. There are approximately 2–10 trillion (2–10 × 1012) endothelial cells in the human body and they form the endothelial surface of 500 m2 of blood vessels and require constant renewal [21–23]. Mostly, the resident stem cells (located in the vessel wall) take part in the repair processes but also circulating progenitor cells that arise from the bone marrow are involved in the process [22]. In diabetes, endothelial cell turnover is impaired and it might be a consequence of accelerated aging or reduced renewal of cells [24, 25]. While ROS-mediated injury dominates in the earlier stages of hyperglycemia-induced damage, cell senescence and impairment of endothelial cell turnover may play the leading part in the later stages.

#### **2.2. Target cells of hyperglycemia**

25% above 80 years of age [1, 2]. The (direct and indirect) medical costs for patients with diabetes are double the amount compared to expenses for nondiabetic individuals and three times higher in case of cardiovascular diseases such as myocardial infarction or stroke [3]. Currently, diabetes-related healthcare expenditure accounts for 10% of the total healthcare costs and it is estimated to increase by 70% over the next 25 years leading to a serious societal and economic burden [4]. Diabetes complications are responsible for the majority of the associated costs and excess costs gradually increase with the duration of the disease leading to substantially higher expenses after 8–10 years [3, 5]. Hyperglycemia-induced endothelial dysfunction is the major contributor to the development of vascular disease in diabetes mellitus [6]. While insulin resistance may be present in patients with no increase in plasma glucose level and it may contribute to endothelial dysfunction, the major pathway that is responsible

Endothelial dysfunction is a pathological state of the endothelium and can be defined as an aberration of the normal endothelial function of vascular relaxation, blood clotting and immune function. In general, it means impaired endothelium-dependent vasodilation as a result of imbalance between vasodilating and vasoconstricting substances produced by (or acting on) the endothelium. Endothelial dysfunction can be a significant predictor of coronary artery disease and atherosclerosis and it increases the risk of stroke and heart attack [8]. In basic science and in clinical research, endothelial function is commonly assessed by the use of the acetylcholine-mediated vasodilatation test or by flow-mediated vasodilation, and this methodology is considered the 'gold standard' at this moment [8, 9]. Endothelial dysfunction is primarily responsible for the impaired vasorelaxation in diabetes but it is closely followed by the development of vascular smooth muscle cell dysfunction [10, 11]. Impaired relaxation may be caused by diminished production or increased destruction of vasodilating factors or impaired response to them in diabetes. Oxidative stress is considered as one of the major underlying mechanisms, which leads to endothelial dysfunction in hyperglycemia, since the therapeutic supplementation of antioxidants or antioxidant enzymes can restore the endothe-

Glucose-induced damage is apparently controversial: glucose is a major source of energy and a small increase in blood glucose, which has no obvious ill effect on the short term, can cause serious long-term complications in diabetes. Glucose uptake is noninsulin dependent in endothelial cells and it occurs via GLUT1 (glucose transporter 1), thus high blood glucose level results in similarly high intracellular glucose concentration in endothelial cells [12, 13]. Endothelial cells have few mitochondria and primarily use glycolysis to produce ATP molecules, which suggests low oxygen consumption and relatively low level of oxidant production [14]. Furthermore, higher glucose concentration would allow even higher rate of anaerobic metabolism to produce the necessary amount of ATP and limit aerobic metabolism,

for endothelial damage is glucose-induced oxidative stress in diabetes [6, 7].

**2.1. Glucose and oxidative stress in diabetic vascular damage**

lium-dependent vasodilation in experimental models of diabetes [10].

**2. Characteristics of the damage**

180 Endothelial Dysfunction - Old Concepts and New Challenges

Hyperglycemia induces damage in a select cell population in the body, including mainly the mesangial cells in the kidney, neurons and Schwann cells in peripheral nerves and a subset of endothelial cells: only the microvascular and the arterial endothelial cells show impairment [26]. Interestingly, this dichotomy in the vulnerability is often preserved in *in vitro* experiments: microvascular endothelial cells are more susceptible to glucose-induced injury, whereas venous endothelial cells show reduced oxidant production and damage. This suggests that differences in the pressure, blood flow or vessel function in various parts of the circulation may not be accounted for the susceptibility. It is rather an inherent difference between the cells that explain the vulnerability of the microvasculature [27]. There are differences in the protein and RNA expression patterns, including the miRNA expression profiles, and the different responses of microvascular and macrovascular endothelial cells to various metabolic stimuli may be attributed to this difference [28].

Differences in glucose uptake may be partially responsible for the susceptibility: most cells tightly regulate the glucose transport rate and prevent the unrestricted uptake, but endothelial and mesangial cells are unable to decrease the transport rate [29, 30]. Glucose overload induces a gradual increase in the mitochondrial membrane potential and the elevated protonic potential increases the superoxide generation by the respiratory chain [31]. The mitochondrial membrane potential is regulated by uncoupling proteins in the cells: these channels release excess protons from the intermembrane space to the matrix and protect against mitochondrial hyperpolarization. Endothelial cells express uncoupling protein 2 (UCP2) and its transport capacity is controlled by oxidative stress: high levels of oxidants open the channel, whereas the absence of oxidants closes the channel [32, 33]. In venous endothelial cells, hyperglycemia upregulates the expression of UCP2 and it effectively protects against mitochondrial hyperpolarization and ROS production [34, 35]. This process does not work in microvascular endothelial cells: there is no change in UCP2 expression in response to elevated glucose concentration resulting in mitochondrial hyperpolarization with a simultaneous rise in mitochondrial superoxide generation [35]. In many cases, endothelial cells were found to produce excess levels of mitochondrial oxidants in response to hyperglycemia only in the presence of pro-inflammatory cytokines, suggesting further mechanisms to be involved in the hyperglycemia-induced cell-damaging processes but the potential implication of inflammatory pathways has not been clarified [36].

parts of capillaries may also occur as a result of hypoxia, and thus might be a consequence of prior endothelial damage. On the other hand, the loss of pericytes results in reduced proliferation of stalk endothelial cells leading to fewer phalanx cells and promotes hypoxia in the retina. Hypoxia is the main stimulus of uncontrolled proliferation in diabetic vessels and both angiotensin II and vascular endothelial growth factor (VEGF) are involved in the neovascularization. In the pathological angiogenesis, not only the retinal endothelial cells take part but also the bone marrow-derived progenitor cells that may explain how enhanced proliferation

Hyperglycemia-Induced Endothelial Dysfunction http://dx.doi.org/10.5772/intechopen.71433 183

Glucose-induced endothelial damage is not only caused by constantly high glucose concentration but also by transiently elevated glucose levels. In experimental models, damage induced by intermittent high glucose is comparable or more severe than the injury induced by constantly high glucose concentration. Glucose levels studied in most experimental models are often much higher than the values that cause irreversible damage in humans on the long

Diagnostic criteria for diabetes are based on the relationship between plasma glucose values and the risk of diabetes-specific microvascular complications: blood glucose concentration that causes diabetic vascular damage has been empirically determined and diagnostic criteria were established. The World Health Organization (WHO) introduced new diagnostic criteria in 1980, which were globally accepted, but had to lower the cut-off values for diabetes in 1999 since growing body of evidence supported the development of complications at lower blood glucose levels [46, 47]. The updated threshold values has raised considerable dispute and are often criticized for not preventing complications but further lowering has not been achieved because of the risk of hypoglycemia. The definition of hyperglycemia is challenging, since blood glucose values show a physiological increase after a meal and this calls for separate normal values for fasting, postprandial and random blood glucose levels. Still, it is evident that "high" glucose levels that induce damage in endothelial cells in the long term are very close to the normal blood glucose values, less than a twofold increase in the blood glucose level triggers injury in the cells. In the past, osmotic damage was presumed to play a pathogenic role in glucose-induced cellular injury but the minor changes in osmolality rule out this possibility. In healthy human subjects, the rise in blood glucose levels after a meal typically reaches or goes beyond these values, making the definition of hyperglycemia rather confusing [48]. From the pathogenic viewpoint of hyperglycemia, absolute cut-off values cannot be established to separate normoglycemic and hyperglycemic

While earlier studies confirmed that the risk of cardiovascular complications corresponds to the average increase in glucose level (measured as glycated hemoglobin, HbA1c), more

capacity replaces the cell loss at the later stage.

**3.1. Hyperglycemia and 'glucose memory'**

concentration ranges.

**3. Triggers of endothelial dysfunction and damage**

term and result in accelerated progression of diabetic complications.
