Mechanisms Underlying Endothelial Dysfunction in Diabetes

Endothelial dysfunction in diabetes stems from three main sources: chronic hyperglycemia, oxidative stress, and the increased production of cytokines and growth factors.

Hyperglycemia and Its Immediate Biochemical Sequelae

High concentrations of glucose have been shown to be associated with endothelial dysfunction both in vivo (75) and in vitro (76). Underlying mechanisms contributing to this process include decreased activity and/or expression of eNOS, decreased activity and/or expression of the NO downstream target soluble guanylyl cyclase (sGC), and increased degradation of NO due to enhanced superoxide production (77).

It has been reported that in a hyperglycemic environment, expression of eNOS was upregulated, with resultant increases in NO production. Furthermore, they also found an overall decrease in the bioavailable NO, in concert with dramatic increases in superoxide production (78). It was postulated that, in a diabetic milieu, uncoupled eNOS becomes a significant source of superoxide. Other in vivo studies corroborated the earlier findings indicating that superoxide in diabetic vessels may either overwhelm NO production by the upregulated eNOS, or that the eNOS itself may be uncoupled, thereby contributing directly to the superoxide production (79).

Two conditions leading to uncoupling of eNOS have been described. These include BH4 (eNOS co-factor) deficiency and intracellular L-arginine (eNOS substrate) depletion (77). In conditions of BH4 deficiency, eNOS remains in an uncoupled state and preferentially produces superoxide rather than NO. NO in turn is thought to be a superoxide scavenger. Superoxide product peroxynitrite has been shown to rapidly oxidize the active eNOS cofactor BH4 to inactive dihydrobiopterin (BH2) (80). In addition, uncoupled eNOS and L-arginine depletion is characteristically found in conditions where high oxidative stress is encountered, as observed in patients with diabetes (77), hypercholesterolemia (81), and in chronic smokers (82). Thus, hyperglycemia-induced uncoupling of eNOS leads to increased formation of ROS resulting in increased oxidative stress, which has been shown to be a strong stimulus for PKC activation (see Fig. 3).

1s Triglycerides

1s Triglycerides eg. superoxide anion t HDL levels eg. superoxide anion eNOS uncoupling

FFA: free fatty acids HDL: high-density lipoproteins NOS: nitric oxide synthase PKC: protein kinase C ROS: reactive oxygen species

Fig. 3. Hyperglycemia and its biochemical sequelae. ROS reactive oxygen species, FFA free fatty acids, HDL high density lipoproteins, PKC protein kinase C, eNOS endothelial nitric oxide synthase (Source: Found on Web search; no source listed).

Hyperglycemia and Sex-specific Differences in EC Dysfunction

As discussed previously, hyperglycemia has also been shown to decrease estradiol-mediated NO production in women, perhaps contributing to the increased CVD risk in women than in men with diabetes. NO-dependent vascular tone and endothelial-dependent vasodilation are enhanced in nondiabetic premenopausal women than in men. The interaction between hyperglycemia and estradiol-mediated NO production has been well documented. Hyperglycemia decreases estradiol-mediated NO production from cultured EC (83). Men with DM2 do not appear to have reduced endothelium-dependent vasodilation beyond that observed with obesity alone in contrast to women with DM2. Thus, hyperglycemia appears to negate the protective effects of estradiol in part by decreasing vascular and perhaps platelet NO production.

The DAG/PKC Pathway

PKC activation in diabetes has been well established. It occurs primarily via increased levels of DAG, a common scenario in a hyperglycemic state (84). A specific PKC isoform, the PKC-b, is implicated in hyperglycemia-induced endothelial dysfunction. The role of PKC in mediating endothe-lial dysfunction was evidenced by the observation that incubation of aortic rings with high glucose led to endothelial dysfunction, which improved by a simultaneous incubation with a PKC inhibitor (76). Furthermore, a large body of evidence exists suggesting that PKC inhibition has beneficial effects on eNOS and has inhibitory effects on superoxide production, leading to a marked increase in NO bioavailability (85).

The activity of the most important superoxide-producing enzyme in vascular tissue, NADPH oxidase, has been shown to be increased by fatty acid stimulation as well as by glucose, in a PKC-dependent manner (86). This stimulation further increases the vascular superoxide burden. NADPH oxidase, as well as mitochondrial-formed superoxide, may combine with NO to form the highly reactive intermediate peroxynitrite, which as mentioned earlier, inactivates the eNOS cofactor BH4, leading to eNOS uncoupling and perpetuating the vicious cycle of superoxide formation. The resultant increase in oxidative stress also leads to accumulation of asymmetrical dimethylarginine (ADMA), which is a competitive inhibitor of eNOS. Elevated levels of ADMA and intracellular BH4 depletion act to maintain an environment of enhanced oxidative stress within the vascular tissue (77).

Hyperglycemia-Induced Oxidative Stress and Vascular Dysfunction

Hyperglycemia-induced oxidative stress also involves the sorbitol and hexosamine pathways. These have been implicated in leading to a redox imbalance, simulating an environment similar to tissue hypoxia and augmented oxidative stress. The hexosamine pathway in particular has been associated with increased transcription of PAI-1 and TGF-b (87), which are commonly associated with endothelial dysfunction.

The diabetic state, which is typified by an increased tendency for oxidative stress, leaves the EC very susceptible to damage. High levels of oxidized lipoproteins, fatty acids, and hyperglycemia have all been shown to induce oxidation of phospholipids and proteins, leading to impairment of EC function. ROS can affect many signaling pathways, including G proteins, protein kinases, ion channels, and transcription factors, which may modify endothelial function via various mechanisms. The end result is a decrease in the production of NO, VSMC hyperreactivity to vasoconstrictive stimuli, and an increase in proinflammatory and adhesion molecules, culminating in an extremely prothrombotic state. Oxidative stress is thought to be the final common pathway of hyperglycemia-induced endothelial dysfunction.

Nonenzymatic Glycation

As mention earlier, chronic hyperglycemia has been shown to promote nonenzymatic glycation of proteins (88) and macromolecules, producing AGE. Nonenzymatic glycation of proteins is a condensation reaction of the carbonyl group of sugar aldehydes with the N-terminus of free amino acids of proteins (62). Changes in the properties of protein and DNA have been documented to occur as a result of nonenzymatic glycation.

Formation of AGE may lead to endothelial activation. These products act to neutralize NO and increase the susceptibility of LDL to oxidation. The binding of the AGE to their receptors also activates the receptors for the cytokines interleukin-1 (IL-1), tumor necrosis factor-a (TNF-a), and growth factors, leading to the migration and proliferation of smooth muscle cells (89). Furthermore, the introduction of AGE into the extracellular matrix can interfere with EC function. AGE-modified type I and IV collagen inhibit normal matrix formation and cross-linking and decrease arterial elasticity (62). Hence, AGE may potentiate EC dysfunction by attracting proinflammatory factors including adhesion molecules as a result of immune-mediated damage, thereby disrupting the overall homeos-tasis of the vasculature.

The Role of Growth Factors and Cytokines

Hyperglycemia and the associated state of oxidative stress results in excess production of cytokines and growth factors. Overexpression of growth factors has been implicated in diabetes-related proliferation of EC and VSM, resulting in increased angiogenesis (90, 91 ). Among those studied include TGF-b, vascular endothelial growth factor (VEGF), and TNF-a. C-reactive protein (CRP), another marker of inflammation, has also been found to be significantly elevated in both DM1 and DM2 (92).

TGF-b is increased via hyperglycemia-induced PKC stimulation, AGEs, Ang II, and other cytokines. TGF-b in particular has been proposed as the major candidate to mediate diabetic nephro-pathy (93). VEGF is thought to be involved in differentiation, proliferation, and vascular permeability of the endothelium. This is likely mediated via Ang II as well. Elevated VEGF has been associated with proliferative diabetic retinopathy (94). TNF-a is produced by neutrophils, macrophages, and adipocytes.

It is an important inflammatory cytokine, which also regulates the expression of CRP. These mediators have been implicated in inducing insulin resistance, thereby causing endothelial dysfunction and contributing to atherothrombosis.

Thus, endothelial dysfunction, caused by the deleterious effects of hyperglycemia, is the primary lesion leading to vascular complications associated with diabetes. The mechanisms are several, as outlined earlier, which culminate into a prothrombotic environment with an increased risk of atherosclerosis and CVD. This dysfunction is more pronounced in women with DM than in men, as suggested by several studies. Therapeutic interventions should, therefore, target various stages of the proposed mechanisms in an attempt to restore the overall homeostasis of the vasculature. Additionally screening, diagnosis, and treatment of CVD should be at least as aggressive in women with DM as it is in men.

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