Molecular Cell Biology Of Endothelial Dysfunction In Diabetes

General remarks

Endothelial dysfunction in diabetes originates from three main sources [1,3,6876]. Firstly, hyperglycaemia and its immediate biochemical sequelae directly alter endothelial function. Glucose transport into endothelial and vascular smooth muscle cells is insulin-independent and is autoregulated in smooth muscle cells, but not in endothelial cells, in which an increase in blood glucose concentration will thus increase the intracellular accumulation of glucose and its metabolites.

High Glucose Insulin Pathway

Figure 2. Postulated pathways linking cardiovascular risk factors, insulin resistance and microvascular function

Thus, endothelial cells exposed to high glucose in vitro increase the production of extracellular matrix components, such as collagen and fibronectin, and of procoagulant proteins, such as vWF and tissue factor, and show decreased proliferation, migration and fibrinolytic potential, and increased apoptosis. Secondly, high glucose influences endothelial cell functioning indirectly by the synthesis of growth factors (e.g., transforming growth factor-^ (TGF-P) and vascular endothelial growth factor (VEGF), cytokines (e.g., TNF-a) and vasoactive agents in other cells. Thirdly, the components of the metabolic syndrome can affect endothelial function.

Hyperglycaemia and its immediate biochemical sequelae

An increase in intracellular glucose will lead to an increase in the flux of glucose to sorbitol via the polyol pathway, an increase in glucosamine-6-phosphate via the hexosamine pathway, and the activation of protein kinase C

(PKC) via de novo synthesis of diacylglycerol (DAG). In addition, glucose and glucose-derived dicarbonyl compounds react non-enzymatically with the basic amino acids, lysine and arginine, in proteins to form advanced glycation endproducts (AGEs) both extra- and intracellularly. Figure 3 shows how, intracellularly, these four biochemical mechanisms may all be the consequence of hyperglycaemia-induced overproduction of reactive oxygen species in mitochondria [68,74].

HYPERGLYCAEMIA

OXIDATIVE STRESS

HYPERGLYCAEMIA

OXIDATIVE STRESS

SORBITAL

DAG-PKC

HEXOSAMINE

AGE

PATHWAY

PATHWAY

PATHWAY

PATHWAY

ENDOTHELIAL DYSFUNCTION

ENDOTHELIAL DYSFUNCTION

VASCULAR COMPLICATIONS

Figure 3. Four biochemical pathways through which hyperglycaemia can cause endothelial dysfunction and vascular complications

The sorbitol pathway. In most cells, excess glucose can be metabolised to sorbitol and fructose by aldose reductase and sorbitol dehydrogenase, which is accompanied by increased oxidation of NADPH to NADP+ and increased reduction of NAD+ to NADH. This pathway is thought to impair endothelial function because the increase in the cytosolic NADH/NAD+ ratio results in a redox imbalance that resembles that which occurs in tissue hypoxia and therefore is termed hyperglycaemic pseudohypoxia. It increases the formation of methylglyoxal and AGEs and enhances oxidative stress [1]. The full impact of the sorbitol pathway in vascular dysfunction is, however, not completely understood and the role of inhibition of aldose reductase in the prevention and treatment of diabetic complications remains unclear [1].

The DAG-PKC pathway. The cellular pathogenic consequences of hyperglycaemia-induced synthesis of DAG and activation of PKC are multiple and include dysregulation of vascular permeability directly or indirectly (the latter through the induction of VEGF in smooth muscle cells); dysregulation of blood flow by decreasing endothelial nitric oxide synthase activity and (or) increasing endothelin-1 synthesis; basement membrane thickening through TGF-^-mediated increased synthesis of type IV collagen and fibronectin; impaired fibrinolysis through increased expression of PAI-1; and increased oxidative stress by the regulation of several NADPH oxidases.

In vascular cells, the PKC^II isoform appears preferentially activated. In diabetic animals, an oral PKCP inhibitor prevented diabetes-induced abnormalities in mRNA expression of TGF-^1, type IV collagen and fibronectin, ameliorated increases in glomerular filtration rate and accelerated glomerular mesangial expansion, and partly corrected urinary albumin excretion. Studies to evaluate the importance of the DAG-PKC pathway in humans are underway [1].

The hexosamine pathway. The vascular effects of the hexosamine pathway, in which fructose-6-phosphate is converted to glucosamine-6-phosphate by the enzyme glutamine:fructose-6-phosphate amidotransferase, are just beginning to be understood but may be profound. In aortic endothelial cells, hyperglycaemia was shown to increase levels of hexosamine-6-phosphate and subsequently N-acetylglucosamine (GlcNAc). This, by the addition of GlcNAc to serine and threonine residues, increased O-linked glycosylation of the transcription factor SP-1, which decreased SP-1 phosphorylation and increased SP-1 activity, which in turn can increase transcription of PAI-1 and TGF-^1 [74]. Other proteins, such as PKC and endothelial cell nitric oxide synthase can be modified in a similar way. For example, such a modification of the Akt site of endothelial cell nitric oxide synthase has been shown to decrease enzyme activity [75].

Non-enzymatic glycation. Non-enzymatic glycation of proteins is the condensation reaction of the carbonyl group of sugar aldehydes with the N-

terminus of free amino acids of proteins and initially leads to a Schiff base, which then undergoes rearrangement to early glycation Amadori-adducts such as fructosamine. Amadori-adducts are relatively stable and only a small fraction undergoes rearrangements to irreversible AGEs. AGEs are a mixture of different moieties. When oxidation is involved in their formation, so-called glycoxidation products such as pentosidine and NE-(carboxymethyl)lysine result. Initially, AGEs were thought to form only on long-lived extracellular molecules, because of the slow rate of reaction of glucose with proteins. However, intracellular and short-lived molecules have now also been shown to be targets for AGE formation through reactions with other sugars such as glucose-6-phosphate and glyceraldehyde-3-phosphate, which form AGEs at a much faster rate than glucose. In addition, the highly reactive dicarbonyl compounds methylglyoxal, glyoxal and 3-deoxyglucosone, which are formed from the degradation of glycolytic intermediates, are believed to contribute importantly to the formation of AGEs in vivo. This so-called carbonyl stress has been implicated in the accelerated vascular damage in both diabetes and uraemia. In endothelial cells, methylglyoxal is probably the main AGE-forming compound and can be degraded to D-lactate by the glyoxylase system [69,70].

The introduction of AGEs in the extracellular matrix can interfere with endothelial function in several ways. AGE-modified type I and IV collagen inhibit normal matrix formation and cross-linking, and decrease arterial elasticity; AGE-modified matrix stimulates interactions with mononuclear cells and macromolecules such as LDL; and AGEs may act as oxidants. In addition, AGE-modified plasma proteins can bind to the receptor for AGE (RAGE) [76], which has been shown to mediate signal transduction via a receptor-mediated induction of reactive oxygen species and activation of the transcription factors NF-kB and p21ras. In animal models, blockade of RAGE inhibited the development of macrovascular disease and diabetic nephropathy [76].

Many aspects of diabetic complications are thus potentially related to the effect of Amadori-adducts [77] and AGEs. Clinical trials with aminoguanidine, an AGE formation inhibitor that had shown promise in animal experiments, have unfortunately been halted because of unforeseen side effects, but trials with other AGE formation inhibitors and with AGE cross-link breakers are underway or are being planned.

Oxidative stress as a final common pathway of hyperglycaemia-induced vascular dysfunction. Hyperglycaemic pseudohypoxia, glucose autooxidation and AGE

formation increase oxidative stress. In addition, hyperglycaemia impairs endothelial free radical scavenging by reducing the activity of the pentose phosphate pathway and thus decreasing the availability of NADPH to the glutathione redox cycle [70].

Reactive oxygen species can affect many signalling pathways, such as G-proteins, protein kinases, ion channels and transcription factors, and may modify endothelial function by a variety of mechanisms. These include direct effects on the endothelium such as peroxidation of membrane lipids, activation of NF-kB, and interference with the availability of nitric oxide; and indirect effects such as increasing the oxidation of LDL and the activation of platelets and monocytes. On the other hand, endothelial cells can respond to high glucose levels by increasing the expression of antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase.

It is not known whether oxidative stress causes endothelial dysfunction in human diabetes. It has been difficult to assess the presence of increased oxidative stress in vivo, mainly because of questionable specificity and reproducibility of the methods used. In short-term experiments, high doses of vitamin C can improve some aspects of endothelial dysfunction in diabetes [1].

On the other hand, randomised clinical trials of anti-oxidants have failed to show a decrease in macrovascular disease. The reasons for this discrepancy are unclear.

Growth factors and cytokines: TGF-fi, VEGF and TNF-a TGF-fi. TGF-^ plays a major role in diabetic nephropathy [78]. It mediates glomerular capillary basement thickening and mesangial matrix expansion. Its role in the thickening of capillary basement membranes elsewhere (e.g., in the retina) is less well established. TGF-^1 is increased through hyperglycaemia-induced PKC activation; through Amadori-albumin and AGEs; through stretch and angiotensin-II; and through cytokine activation of endothelial cells. TGF-P1 stimulates the production of matrix components such as type I and IV collagen, fibronectin, laminin, and proteoglycans in cultured glomerular mesangial cells and epithelial cells; is involved in the regulation of glomerular endothelial, epithelial and mesangial proliferation; and also has potent anti-inflammatory effects on vascular cells, down-regulating cytokine-induced expression of E-selectin, VCAM-1 and monocyte chemotactic protein-1 [78].

VEGF. VEGF is a potent and apparently endothelium-specific mitogenic cytokine, whose expression can be induced by hypoxia through hypoxia-inducible factor-1, but also by insulin-like growth factor-1 and TGF-^1. VEGF can induce the proliferation and migration of vascular endothelial cells leading to angiogenesis, neovascularisation and increased vascular permeability. Several studies have shown increased vitreous VEGF levels in patients with proliferative diabetic retinopathy, and antagonists of VEGF and its receptors have been shown to reduce retinopathy in animal models. The role of increased or decreased VEGF in other diabetic complications is the subject of much ongoing research [79].

TNF-a and inflammation. TNF-a is an inflammatory cytokine produced by neutrophils, macrophages and, importantly, adipocytes. TNF-a can induce other powerful cytokines such as interleukin-6, which in turn regulates the expression of CRP. These mediators alone or in combination can impair endothelial function and contribute to atherothrombosis. In addition, TNF-a can induce insulin resistance, which may at least in part explain why insulin resistance, endothelial dysfunction and atherothrombosis are so closely related (see above). Finally, recent studies have shown that TNF-a and inflammation in general can contribute to the pathogenesis of diabetic nephropathy.

Vascular cells are both a target for cytokines and a source. The spectrum of endothelial cell responses elicited by cytokines is varied. Briefly, inflammatory cytokines increase vascular permeability; alter vasoregulatory responses; increase leukocyte adhesion to endothelium; and facilitate thrombus formation by inducing procoagulant activity, by inhibiting anticoagulant pathways and by impairing fibrinolysis via stimulation of PAI-1. Activation of the transcription factor NF-kB is crucial in cytokine regulation of gene expression in endothelial cells [12]. NF-kB is activated not only by TNF-a and interleukin-1, but also by hyperglycaemia, AGEs, angiotensin II, oxidised lipids and insulin. Taken together, these data suggest that NF-kB pathway is an important contributor to the pathogenesis of vascular disease in diabetes mellitus.

The metabolic syndrome: insulin resistance, insulin, hypertension, dyslipidaemia, and obesity

Insulin resistance. How metabolic and endothelial insulin resistance occur and why they are closely related is not fully understood (see above). Endothelial insulin resistance, whether primary or secondary (Figure 2), can be regarded as a form of endothelial dysfunction and conceivably contributes to both atherothrombosis and microangiopathy. Both TNF-a and free fatty acids can cause metabolic and endothelial insulin resistance. TNF-a may induce endothelial insulin resistance through its ability to impair intracellular signalling by inhibition of insulin-stimulated autophosphorylation and phosphorylation of insulin receptor substrate-1. How non-esterified fatty acids impair insulin's endothelial actions is not clear.

Insulin. Both type 2 and type 1 diabetes are usually accompanied by chronic hyperinsulinaemia. Whether insulin has atherogenic effects is controversial, mainly because it is not clear whether effects such as increased vascular permeability to macromolecules and increased vascular smooth muscle cell proliferation occur at physiological concentrations. In addition, insulin can increase nitric oxide synthesis and may have anti-inflammatory and anti-atherogenic effects [80]. Taken together, these data raise the possibility that insulin may have adverse effects when insulin signalling in vascular cells is abnormal.

Hypertension. Hypertension is a major determinant of microangiopathy and atherothrombosis in diabetes. Hypertension causes endothelial activation and impaired nitric oxide availability (see above); whether the latter then contributes to increased blood pressure is not clear. Experimental data indicate that decreased nitric oxide availability in the kidney may contribute to vasoconstriction and decreased glomerular filtration; impaired tubuloglomerular feedback; decreased medullary blood flow and impaired pressure natriuresis; and progressive proteinuria. Salt sensitivity of blood pressure may denote an inability to increase nitric oxide availability in response to increased blood pressure [81].

Dyslipidaemia. The effects of LDL cholesterol (see above) may be enhanced in type 2 diabetes, which is associated with increased small dense LDL particles. In addition, type 2 diabetes, especially when glycaemic control is poor, is characterised by increased postprandial triglyceride-rich lipoproteins (chylomicrons and VLDL particles), which can enhance oxidative stress and impair endothelial function both directly and indirectly (by increasing the production of small dense LDL particles and by reducing HDL [82,83]). These changes contribute to atherothrombosis and may also play a role in nephropathy, as such dyslipidaemia can damage glomerular podocytes and mesangial cells [84]. Dyslipidaemia has been associated with increases in urinary albumin excretion in both type 1 and type 2 diabetes [85,86].

Obesity. Obesity, especially visceral obesity, is associated with increased risk of atherothrombosis and microangiopathy. These effects may be mediated through the associations of obesity with hypertension, dyslipidaemia and insulin resistance, and also through mediators directly secreted by adipocytes, such as TNF-a, leptin and PAI-1. For example, obesity-associated proteinuria may be related to hyperfiltration, increased renal venous pressure, glomerular hypertrophy and increased matrix production through increased synthesis of vasoactive and fibrogenic mediators, such as angiotensin-II, insulin, leptin and TGF-p1 [87].

Asymmetric dimethylarginine (ADMA). There is some evidence that levels of ADMA, an endogenous inhibitor of nitric oxide, are associated with the metabolic syndrome [88], which may in part be caused by hyperglycaemia-induced impairment of ADMA breakdown [89]. This is an area of active investigation.

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