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

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 path-

At cellular level, hyperglycemic damage occurs within a few days and induces compensatory and repair mechanisms that may have consequences in the cell population. Vascular endothelium covers a huge surface in the body and possesses a huge capacity to compensate for any damage that occurs over longer periods, thus changes in vascular function may occur with a

In experimental models, glucose levels are often above 20–30 mmol/L and vascular dysfunction develops over weeks or within a few months [37]. The development of hyperglycemiainduced endothelial cell damage is neither instantaneous *in vitro*, it usually takes a few days of exposure to high glucose levels to induce a significant increase in the mitochondrial membrane potential and oxidant production [35, 38]. Hyperglycemia-induced ROS production induces RNA and DNA damage that may be responsible for the reduced proliferation rate observed in endothelial cells [39]. Reduced proliferation and senescence occur after more than

On the other hand, diabetic vascular complications occur after years of hyperglycemic exposure and poor glycemic control accelerates the development of the disease [40, 41]. Although complications usually first appear some years after clinical diagnosis, retinopathy and nephropathy were often present (in 10–37% of patients) at the time of clinical diagnosis or within the first year after diagnosis [42]. Glucose levels that induce endothelial damage are moderately elevated in most patients due to improved diabetes care and diabetes self-

Endothelial cell senescence and reduced proliferation are the dominant features in diabetes, still pathological proliferation of blood vessels occurs in diabetic retinopathy [45]. This controversy is explained by the fact that progressive retinal angiogenesis is preceded by a series of events that is characterized by reduced cell proliferation and stimulates neovascularization in the retina [45]. Proliferative diabetic retinopathy is not the primary pathogenic response to hyperglycemia but a compensatory response to retinal hypoxia. Diabetic retinopathy starts with the loss of two cell types of the retinal capillaries: the endothelial cells and the vessel supporting pericytes and the earliest pathologic signs are acellular, nonperfused capillary segments in the retina [45]. Pericyte loss may precede the endothelial damage in the retina and it is caused by angiotensin II overexpression induced by oxidative stress in diabetes. However, the increased number of migrating pericytes and loss of pericytes from the straight

10 doublings of endothelial cells exposed to 25 mmol/L glucose *in vitro* [25].

management education and support (DSME/S) [43, 44].

ways has not been clarified [36].

delay.

**2.3. Time course of hyperglycemic injury**

182 Endothelial Dysfunction - Old Concepts and New Challenges

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 term and result in accelerated progression of diabetic complications.

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 concentration ranges.

While earlier studies confirmed that the risk of cardiovascular complications corresponds to the average increase in glucose level (measured as glycated hemoglobin, HbA1c), more recent studies also found independent associations with the postprandial peaks [49]. These results highly suggest the action of secondary mediators that are rather induced by the fluctuations in blood glucose (glycemic variability) than by an absolute increase. Experimental models confirmed that glycemic swings caused at least as severe tissue damage as constant hyperglycemia, and persistence of high-glucose memory was postulated in cells and animals that were exposed to normoglycemic conditions following a hyperglycemic exposure [50– 52]. Endothelial cells when returned to normal glucose concentration after exposure to high glucose showed increased ROS production and activation of poly(ADP-ribose) polymerase (PARP) even a week following the normalization of the glucose level and in this respect they showed similar characteristics to cells maintained at high glucose [52]. The persistence of oxidative stress in endothelial cells *in vitro* confirms that 'glucose memory' is an inherent feature of these cells. It also means that once hyperglycemia activates the various ROS-producing pathways they continue to produce oxidants for multiple days or weeks in endothelial cells even if the glucose level is fully normalized. Oxidative stress is the key feature of the changes induced by hyperglycemia, and 'metabolic memory' is another term used that refers to the characteristic metabolic changes [51]. The length of high glucose memory is unknown in humans but it is suspected to last longer than *in vitro* because (1) inflammatory pathways are also involved and (2) the response is not limited by the life cycle of single cells but it is possibly carried over to multiple cell generations.

tissue damaging actions. Oxidative stress pathways act as executors of tissue damage but the linkage between hyperglycemia and the sustained activation of oxidative pathways still

Hyperglycemia-Induced Endothelial Dysfunction http://dx.doi.org/10.5772/intechopen.71433 185

Alterations in the metabolome in diabetes are suspected to maintain the metabolic changes for extended periods even if there is little change in the expression profile of proteins [57]. Excess glucose load induces changes in a series of metabolite levels and the normalization of these levels may not occur as rapidly as glucose lowering. Apart from glucose, the concentrations of glucose-1-phosphate, lactate, glucosamine, mannose, mannosamine, hydroxybutyrate and glyoxalate also elevate in the plasma in diabetes [58]. All of the above intermediates and the increased fatty acids increase the tricarboxylic acid (TCA) cycle flux in the cells. Perturbation of the TCA cycle flux is also supported by other metabolomics studies in diabetes [59]. Associations between diabetes risk and the plasma levels of branched chain (BCAA, isoleucine, leucine and valine) and aromatic (phenylalanine, tyrosine) amino acids have been found suggesting that the changes not only involve the carbohydrate and lipid metabolism but also the catabolism of proteins and amino acids [60]. Catabolism of BCAAs provides intermediates for the TCA cycle and potentially drives the TCA flux. Apart from the systemic changes that affect the milieu of the cells, specific changes of amino acid levels have been observed in endothelial cells: hyperglycemia increases the concentration of alanine, proline, glycine, serine and glutamine within the cells and induces elevation of the aminoadipate, cystathionine and hypotaurine levels [61]. Whether these changes are only markers of hyperglycemia or

they play a pathogenic role in oxidative stress induction is still undetermined.

(that can be converted to ATP) and further five ATP molecules from NADH<sup>+</sup>

Changes in glucose metabolism are presumed to be directly responsible for provoking oxidant production in endothelial cells. Endothelial cells predominantly use glucose as energy source and rely on glycolysis to generate ATP molecules [14]. Glycolytic flux exceeds the rate of oxidative phosphorylation (OXPHOS) by two orders of magnitude in endothelial cells *in vitro* and similar ratio is suspected *in vivo* [62, 63]. The contribution of fatty acid oxidation to energy production is thought to be negligible in capillary endothelial cells, though endothelial cells take up fatty acids and transport them to the neighboring cells, thus play important role in transendothelial fatty acid delivery [64]. Since the function of capillary endothelial cells is to deliver oxygen and fuel sources to other cells in tissues, they do not consume much oxygen or store energy but preserve them to other perivascular cells. Thus, excess glucose is not converted to glycogen for storage in endothelial cells, but is pushed toward glycolysis [65]. Glutamine is a further energy source in endothelial cells via glutaminolysis that directly produces one GTP molecule

OXPHOS. Glutamine is the most abundant amino acid in the plasma, and glutaminolysis is a valuable energy source if glycolytic output is low since it feeds alpha-ketoglutarate to the TCA cycle and similarly produces lactate (or pyruvate). However, all the energy producing steps in

and FADH2

via

**4. Mechanisms of ROS production in hyperglycemia**

**4.1. Glucose-induced oxidative stress pathways**

remains rather elusive.

Blockade of the early changes has been confirmed to prevent or slow down the progression of complications but the reversal at a later phase may not be achieved by glycemic control [53]. Benefits of intensive glucose control can be detected after 3 years of treatment if no retinopathy or mild disease is present at the start of the treatment strategy in type 1 diabetes [54]. The importance of blocking the glucose-induced damage early on in type 2 diabetes has been confirmed by the results of the United Kingdom Prospective Diabetes Study (UKPDS) [41]. On the other hand, there is little benefit of strict glucose control if established cardiovascular disease is already present at the start of the treatment regimen [53]. Similarly, in diabetic rats, a 6-month long period of good glycemic control following 2 months of poor glycemic control results in significantly reduced progression, whereas no benefit is observed on retinopathy if good glycemic control was started after 6 months of poor glycemic control: both nitrosative stress and tissue damage were similarly advanced as with 12 months of poor glycemic control [55, 56]. These suggest that the processes started by hyperglycemia may be partially reversed if normoglycemia follows a shorter period of high glucose exposure. It is still unclear whether the detrimental effects of transient hyperglycemia is buffered within the individual cells or it is the entire population of endothelial cells that compensate for the changes and the reason why progressive damage occurs following an extended hyperglycemic period is the loss of the renewal capacity of the cells.

#### **3.2. Downstream molecules responsible for the damage**

The mechanism of high glucose memory is still obscure and little is known about the pathways involved. Hyperglycemia modifies the metabolism of the cells and is suspected to induce various downstream pathways or molecules that are responsible for maintaining the tissue damaging actions. Oxidative stress pathways act as executors of tissue damage but the linkage between hyperglycemia and the sustained activation of oxidative pathways still remains rather elusive.

Alterations in the metabolome in diabetes are suspected to maintain the metabolic changes for extended periods even if there is little change in the expression profile of proteins [57]. Excess glucose load induces changes in a series of metabolite levels and the normalization of these levels may not occur as rapidly as glucose lowering. Apart from glucose, the concentrations of glucose-1-phosphate, lactate, glucosamine, mannose, mannosamine, hydroxybutyrate and glyoxalate also elevate in the plasma in diabetes [58]. All of the above intermediates and the increased fatty acids increase the tricarboxylic acid (TCA) cycle flux in the cells. Perturbation of the TCA cycle flux is also supported by other metabolomics studies in diabetes [59]. Associations between diabetes risk and the plasma levels of branched chain (BCAA, isoleucine, leucine and valine) and aromatic (phenylalanine, tyrosine) amino acids have been found suggesting that the changes not only involve the carbohydrate and lipid metabolism but also the catabolism of proteins and amino acids [60]. Catabolism of BCAAs provides intermediates for the TCA cycle and potentially drives the TCA flux. Apart from the systemic changes that affect the milieu of the cells, specific changes of amino acid levels have been observed in endothelial cells: hyperglycemia increases the concentration of alanine, proline, glycine, serine and glutamine within the cells and induces elevation of the aminoadipate, cystathionine and hypotaurine levels [61]. Whether these changes are only markers of hyperglycemia or they play a pathogenic role in oxidative stress induction is still undetermined.
