Although it is clear that there are significant alterations of blood vessels in diabetes, data thus far has been conflicting whether neuropathy promotes the changes in the microvasculature or whether it is the changes in the microvessels of the nerves that lead to neuropathy. Whatever the case, evidence is growing that re-establishing more normal patterns of blood flow to the nerves results in improved neurological function.
Protein kinase C (PKC) and diacylglycerol (DAG) are intracellular signaling molecules that regulate vasculature by endothelial permeability and vasodilation. The PKC isozymes are a family of 12 related serine/threonine kinases (25) whose normal function is the activation of essential proteins and lipids in cells essential for cell survival. PKC-P is expressed in the vasculature (26,27) and belived to be involved in cell proliferation, differentiation, and apoptosis. PKC is activated by oxidative and osmolar stress, both of which are a consequence of the dysmetabolism of diabetes. Increased polyol pathway activity and pro-oxidants bind to the catalytic domain of PKC and it is disin-hibited. PKC-p overactivation is induced by hyperglycemia or fatty acids through receptor-mediated activation by phospholipase C. It is hypothesized that AGEs and oxidants produced by nonenzymatic glycation and the polyol pathway, respectively, increase the production of DAG (28). Increased DAG and calcium promotes the overactivation of PKC-P (29). Activation of PKC-P activates MAP kinase and, subsequently, phosphorylation of transcription factors that are involved in angiogenesis, increased stress-related genes, c-Jun kinases and heat shock proteins, all of which can damage cells and vascular endothelial growth factor (VEGF) (30), which is known to play a critical role in nerve development (31). Diabetic animal models have shown high levels of PKC-P in a number of tissues (28), including nerves and endothelium (32). Activation of PKC-P causes vasoconstriction and tissue ischemia, whereas high levels may impair neurochemical regulation. PKC-P hyperactivity leads to increased vascular permeability, nitric oxide dysregulation (33), increased leukocyte adhesion (34), and altered blood flow (35). Furthermore, PKC-P hyperactivity in the neural microvessels causes vasoconstriction, which might lead to decreased blood flow, resulting in nerve dysfunction and hypoxia (35). Nerve hypoxia, oxidative nitrosative stress, and an increase in NFkB causes endothelial damage, leading to depletion of nerve growth factors, VEGF, and TGF-a autoimmunity and may further accelerate the loss of nerve conduction (31). Both animal models and human clinical trials investigating complications of diabetes have shown that blockade of PKC-P slows the progression of complications (33,36-47).
Multiple studies using a specific PKC-P inhibitor, ruboxistaurin mesylate (LY333531), have shown improvements in diabetic neuropathy. One study in obese rats observed that ruboxistaurin increased resting nitric oxide concentration, and reduced nitric oxide by 15%, indicating that this action is a PKC-P dependent phenomenon (33). Ruboxistaurin has been shown to improve nitric oxide-dependent vascular and autonomic nerve dysfunction in diabetic mice (46). In addition to improving nitric oxide levels, ruboxis-taurin improves nerve function and blood flow. Ruboxistaurin corrected the diabetic reduction in sciatic endoneurial blood flow, sciatic motor, and saphenous sensory nerve conduction velocity in diabetic rats (40,43). In another study, the investigators measured sciatic nerve, superior cervical ganglion blood flow, and nerve conduction velocity in STZ treated rats. After 8 weeks, the authors observed that diabetes reduced sciatic nerve and superior cervical ganglion blood flow by 50% and produced deficits in saphenous nerve sensory conduction velocity (48). After 2 weeks of treatment with ruboxistaurin, the sciatic nerve, and ganglion blood flow were improved. Additionally, nerve dysfunction is commonly attributed to alterations of the nerve transporters. Other studies demonstrated that a specific inhibitor of the PKC-P, (ruboxistaurin), prevents PMA-dependent activation of Na+, K+-ATPase in rats (44,45). In addition to improvements with blood flow nerve function and ion transport, ruboxistaurin corrected thermal hyperalgesia (48,49).
These observations have been supported in preliminary clinical studies. In healthy humans, ruboxistaurin blocked the reduction in endothelium-dependent vasodilation induced by acute hyperglycemia (47), suggesting that the hyperglycemic effects on vasodilation are mediated through PKC-p. More recently, a 1-year double-blind, parallel clinical trial with 205 patients with type 1 or 2 diabetes and DPN was performed to assess the impact of ruboxistaurin on vibration perception in patients with DPN compared with placebo. In patients with DPN, ruboxistaurin treatment improved symptoms and vibratory sensation with a significant correlation between the two compared with placebo group (50). Another recent report indicates that ruboxistaurin is particularly effective in neuropathy patients with intact sural nerve amplitudes (51). A phase II study using NTSS-6 to assess the intensity and frequency of sensory neuropathy symptoms further suggested that ruboxistaurin slows the progression of hyperglycemia-induced microvascular damage (52). Together, these studies support the belief underlying a role for PKC-p in the etiology of diabetes-induced neuropathy.
The most potent stimulus for angiogenesis is VEGF. If the pathogenesis of diabetic neuropathy goes through loss of vasa nervorum, it is likely that appropriate application of VEGF would reverse the dysfunction. Normally, VEGF activity is induced by tissue hypoxia (53,54). In diabetes it is just such hypoxia that results in increased VEGF activity in the retina, with subsequent pathological angiogenesis (55). Conflicting reports indicated that VEGF in diabetes goes up (56) and goes down (57). One possibility to explain this is whether the animals were treated with insulin, which can reduce VEGF expression (56). Only recently it has been demonstrated that there is a reduction in VEGF activity in STZ-diabetic mice that results in failure of neovascularization in hypoxic tissue in the lower limb (57). Furthermore, in the same study it was shown that intramuscular injection of an adenoviral vector encoding for VEGF could induce normal neovascularization in the hindlimb. There have been no human studies of VEGF, and caution is the best approach given the demonstrated pathological effects of VEGF in the retina in diabetes.
Microvascular insufficiency, endoneurial blood flow, and hemodynamic factors lead to nerve damage in patients with DPN (58-62). Although, the sequence of events is not well understood, investigators propose that microvascular vasoconstriction, edema, and ischemia play a role in DPN development. Endoneurial edema increases endoneurial pressure (63), thereby causing capillary closure and subsequent nerve ischemia and damage (64,65). A diminished regulation of the endoneurial blood flow and ischemia may result from decreased nerve density and innervation of vessels (66), measured with laser Doppler (67). Nerve ischemia stimulates VEGF production exacerbating DPN through overactivation of PKC-P (56,68-72). As a result, ischemia and low blood flow reduces both endothelial dependent and nitric-oxide dependent vasorelaxation nangle
(46,73,74). Vascular defects also result in changes in endoneurial vessels. Epineurial changes include arteriolar attenuation, venous distension, arteriovenous shunting that leads to new vessel formation (75). Neural regulation of blood flow is complicated by arteriovenous anastomoses and shunting, which deviate the blood flow from the skin creating an ischemic microenvironment (76). There is thickening and deposition of substances in the vessel wall associated with endothelial cell growth, pericyte loss (in eyes), and occlusion (77) Changes in blood flow correlate with changes in oxygen saturation (60,78) and reduced sural nerve endoneurial oxygen tension (79). These changes are followed by increased expression or action of vasoconstrictors, such as endothelin and angiotensin and decreased activity of vasodilators, such as prostacyclin, substance P, CGRP, endothelial derived hyperpolarizing factor, and bradykinin (80,81). Based on this theory, investigators have given oxygen and vasodilatory agents to patients, however, these therapies have not improved DPN (82,83). Additionally, methods of assessing skin blood flow have demonstrated that diabetes disturbs microvasculature, tissue PO2, and vascular permeability. In particular, in patients with DPN there is disruption in vasomotion, the rhythmic contraction exhibited by arterioles, and small arteries (84,85). In Type 2 diabetes, skin blood flow is abnormal and the loss of neurogenic vasodilative mechanism in hairy skin may precede lower limb microangiopathic processes and C-fiber dysfunction (85,86). Changes in endoneurial blood flow often are reflected by changes in nerve conduction (8,87-89). In addition, impaired blood flow can predict ulceration (2,90-93). Therefore, both vascular or endoneural alterations may cause damage over time in the peripheral nerves of patients with diabetes (Fig. 3).
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