Diabetic Microvascular Disease: An Endocrine Society Scientific Statement

Eugene J. Barrett; Zhenqi Liu; Mogher Khamaisi; George L. King; Ronald Klein; Barbara E. K. Klein; Timothy M. Hughes; Suzanne Craft; Barry I. Freedman; Donald W. Bowden; Aaron I. Vinik; Carolina M. Casellini

Disclosures

J Clin Endocrinol Metab. 2017;102(12):4343-4410. 

In This Article

Biochemical Pathways of Microvascular Injury

Introduction

Vascular complications are the major cause of morbidity and mortality in diabetic patients. These result from interactions between systemic metabolic abnormalities, such as hyperglycemia, dyslipidemia, genetic and epigenetic modulators, and local tissue responses to toxic metabolites. Macrovascular complications involve atherosclerotic/thrombotic obstructions, such as those that occur in coronary, cerebral, and peripheral artery diseases. Classic microvascular pathologies include retinopathy, nephropathy, and neuropathy, but brain, myocardium, skin, and other tissues are also affected. In this work, we focus on cellular/molecular mechanisms causing diabetic microvascular pathologies.

Hyperglycemia is the major systemic risk factor for diabetic microvascular complications. The Diabetes Control and Complications Trial (DCCT) in T1DM and the United Kingdom Prospective Diabetes Study (UKPDS) in T2DM clearly demonstrated that intensive blood glucose control delays the onset and retards the progression of diabetic microvascular complications.[1,2]

Hyperglycemia alone, however, is not sufficient to trigger generalized diabetic microvascular pathologies (e.g., only 20% to 40% of diabetic patients will ultimately develop chronic renal failure), suggesting that as yet unidentified genetic or other endogenous protective factors play important roles.[3,4] The Joslin Diabetes Center 50-Year Medalist Study of patients surviving >50 years with T1DM has shown that 30% to 35% are without significant microvascular complications, regardless of their hemoglobin A1c (HbA1c) levels and other classical risk factors thought to predict diabetic vascular complications.[3] These patients may possess endogenous tissue factors that diminish the adverse microvascular effects of hyperglycemia.

Research has suggested that multiple biochemical pathways link the adverse effects of hyperglycemia with vascular complications. Cellular mechanisms include the following: nonenzymatic glycation and the formation of advanced glycation end products (AGEs); enhanced reactive oxygen production and actions; endoplasmic reticulum (ER) stress; and the activation of the polyol pathway, the diacylglycerol (DAG)–protein kinaseC (PKC) pathway,[5] Src homology-2 domain-containing phosphatase-1 (SHP-1), and the reninangiotensin system (RAS) and kallikrein-bradykinin (BK) systems. It is likely that hyperglycemia-induced intracellular and extracellular changes alter signal transduction pathways, thus affecting gene expression and protein function and causing cellular dysfunction and damage.

Molecular Mechanisms of Injury

Research has described multiple abnormalities in cell signaling, gene expression, and the regulation of cell biology and physiology in diabetes, and it is likely that many of these abnormalities operate concurrently to cause various diabetic microvascular complications. These mechanisms may be active preferentially in some (although probably not all) vascular tissues or organs, but generally they are associated with the development of complications in several organs (Figure 1). We will discuss seven mechanistic pathways that appear to be involved in diabetic microvascular injury, as well as several potentially protective factors. The emphasis in this study is on cellular mechanisms; we will cite mechanism-based efforts at clinical interventions but not analyze them in detail.

Figure 1.

Schema of hyperglycemia's induced pathways to microvascular complications. MAP, mitogen-activated protein. Figure 2. Interplay of hyperglycemia's toxic mechanisms and tissues' endogenous protective properties. IGF, insulinlike growth factor.

The activation of PKC in vascular tissues. PKC is a family of serine/threonine-related protein kinases that includes multiple isoforms and affects many cellular functions and signal transduction pathways.[6] Phosphatidyl serine, calcium, DAG, and phorbol esters (such as phorbol-12-myristate-13-acetate) activate the conventional PKC isoforms PKCα, β1, β2, and γ. Phosphatidyl serine and DAG (but not calcium) also activate the novel PKC isoforms PKCδ, ε, ø, and η. Neither calcium nor DAG activates the atypical PKC isoforms PKCζ and ι/λ. Hyperglycemia, per se, modulates PKC activation. In addition, oxidants (e.g., H2O2 and superoxide) can also activate PKC in a manner unrelated to lipid second messengers.[7,8] Many abnormal vascular and cellular processes, including endothelial dysfunction, vascular permeability, angiogenesis, cell growth and apoptosis, vessel dilation, basement membrane (BM) thickening, extracellular matrix (ECM) expansion, and altered enzymatic activity of mitogen-activated protein kinase (MAPK), cytosolic phospholipase A2, Na+–K+–adenosine trisphosphatase (ATPase), and several transcription factors, are attributed to the activation of several PKC isoforms. Diabetes increases PKC activity in skeletal muscle and the renal glomeruli, retina, myocardium, and liver. Among the isoforms of PKC, the α, β, and δ isoforms are most consistently implicated in diabetic vascular complications.

The activation of DAG– PKC pathway and diabetes. DAG levels are elevated chronically in the hyperglycemic diabetic environment due to increased levels of glycolytic intermediate dihydroxyacetone phosphate. This intermediate is reduced to glycerol-3-phosphate, which subsequently increases de novo synthesis of DAG.[9] In diabetes, studies reported that total DAG levels were elevated in the retina[10] and renal glomeruli.[11] However, there is no consistent change in DAG levels in the central nervous system (CNS) or peripheral nerves.[12] Cell culture studies have shown that as glucose levels rise from 5.5 to 22 mmol/L, DAG levels increase in a time-dependent manner in aortic endothelial cells,[13] retinal pericytes,[14] smooth muscle cells,[9] kidney proximal tubular cells,[15] and renal mesangial cells.[16] Increased DAG synthesis can also occur from dihydroxyacetone phosphate that accumulates when poly adenosine 5′-diphosphate (ADP) ribosylation inhibits glyceraldehyde-3-phosphate dehydrogenase in the presence of high glucose concentrations.[17] Elevated cytosolic glucose levels promote the accumulation of glyceraldehyde-3-phosphate, which can increase DAG and activate PKC.[18] In an experimental model of diabetes, large doses of thiamine and thiamine monophosphate derivative (benfotiamine) appear to decrease the formation of DAG and mitigate PKC activation.[19]

PKC activation and the development of diabetic nephropathy. Experiments in diabetic rodents support a role for PKC in the pathogenesis of diabetic nephropathy (DN). PKCα, β, and δ isoforms are activated in renal glomeruli isolated from streptozotocin (STZ)-induced diabetic rats[20] and mice, and 50% of the increase in PKC activity in renal glomeruli is prevented in PKCβ knockout mice.[21] PKCα activation can upregulate vascular endothelial growth factor (VEGF) expression through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.[22] PKCα knockout mice are protected against BM proteoglycan losses induced by VEGF.[23] In wild-type mice, diabetes increases NADPH oxidase activity and induces the expression of endothelin 1 (ET-1), VEGF, transforming growth factor β (TGF-β), connective tissue growth factor, and collagen types IV and VI. These changes are partly prevented in PKCβ knockout mice.[21] Mesangial expansion and albuminuria in mice with STZ-induced diabetes are reduced in both PKCβ[21] and PKCδ[24] knockout vs wild-type mice.

General PKC isoform inhibitors can interact with other kinases and can have significant toxic side effects. The PKCβ inhibitor ruboxistaurin (RBX) is a bisindolylmaleimide class agent that selectively inhibits PKCβ1 and PKCβ2.[25] Rottlerin (mallotoxin) has higher affinity for PKCδ but also inhibits other isoforms of PKC[26] and other non-PKC kinases, such as MAPKs, protein kinase A, and glycogen synthase kinase-3.[27] Orally administered RBX reversed glomerular hyperfiltration and reduced urinary albumin excretion in diabetic rodents without a change in DAG content.[28] In addition, glomerular TGF-β1 expression, mesangial expansion, glomerulosclerosis, tubule-interstitial fibrosis, and renal function all improved.[28] In Ren-2 diabetic rats, RBX attenuated macrophage recruitment, tubulointerstitial injury associated with TGF-β activation, and increases in PKC-induced osteopontin expression in tubular epithelial cells of the renal cortex.[29]

Remarkably, PKCε may have effects on DN that are opposite to the effects of PKCα, β, and δ. One study showed that knockout of PKCε upregulated renal TGF-β1 and its downstream signaling and increased the expression of fibronectin and collagen type IV, which caused glomerular and tubulointerstitial fibrosis and the development of albuminuria.[30] These changes were further aggravated by diabetes.[30] Therefore, PKCε may act as a protective factor by reducing kidney damage.

Supporting the relevance of these findings to human DN, one study associated polymorphisms of the PKCβ gene with accelerated kidney disease (KD) in Japanese T2DM subjects,[31] and another study associated polymorphisms in the PKCβ-1 gene with end-stage KD (ESKD) in Chinese patients with T2DM.[32] However, efforts to treat DN by inhibiting PKC activation with RBX have generally been disappointing, as illustrated by a secondary analysis of three DN trials,[33] which showed no differences in kidney outcomes with RBX treatment.

PKC activation and the development of diabetic retinopathy. The early stages of diabetic retinopathy (DR) are characterized by the loss of pericytes in capillaries of the retina, followed by weakness in the capillary wall, microaneurysm formation and fluid leakage, and increased adhesion of leukocytes and monocytes to the endothelium.[34] Hyperglycemia activates PKCα, β, δ, and ε[18] in retinal tissues and alters ET-1 and VEGF activity and nitric oxide (NO) levels in endothelial cells, as well as levels of platelet-derived growth factor (PDGF), reactive oxygen species (ROS), and nuclear factor κB in pericytes.[35] Administering RBX to diabetic rats can reduce retinal PKC activation and normalize retinal blood flow (RBF).[36,37] In vessels isolated from diabetic animals, NO-dependent acetylcholine-induced vessel relaxation is delayed,[38] and the PKC agonist phorbol-12-myristate-13-acetate impaired vascular relaxation in otherwise normal arteries.[39]

The mechanism for reduced RBF mediated by PKCβ involves the upregulation of ET-1 synthesis in the retina of diabetic rats.[40] RBX treatment can block this induction of retinal ET-1.[40] VEGF (through signaling involving PKCβ)[41] helps mediate diabetic macular edema to increase the phosphorylation of occludin (a component of tight junctions), leading to increased vascular permeability[42] and kallikrein activation.[43] Hyperglycemia may also increase endothelial cell permeability via PKCα activation.[44]

Recently, researchers have clarified the actions of PKC on vascular cell proliferation and death. Both PKCβ and PKCδ isoforms are translocated to the membrane fraction in total retinal lysates of diabetic mice, but the consequences of PKCβ, δ, and ε isoform activation are very different. PKCδ induces cell apoptosis,[14] whereas PKCβ enhances cell growth.[45] Accordingly, the elevation of membranous PKCδ levels in diabetes correlated with the appearance of retinal pericyte apoptosis in vitro and acellular capillaries in vivo. In vivo studies reported that the induction of PKCδ in the retinal capillaries of diabetic mice led to PDGF resistance; this was not true with PKCδ knockout mice. Hyperglycemia (through PKCδ action) promotes two distinct important pathways, as follows: (1) increasing ROS production and nuclear factor κ light chain enhancer of activated B cell (NF-κB) activity, and (2) decreasing the survival-signaling pathway of PDGF by upregulating SHP-1 expression. These findings suggest a pivotal role for PKCδ in regulating pericyte apoptosis and the formation of cellular capillaries.[14]

In animal studies, inhibiting PKCβ ameliorated the decline of RBF typically associated with DR and decreased diabetes-induced vascular leakage.[36] Similarly, the stimulus for neovascularization is suppressed in animals with reduced PKCβ levels.[45,46] More recently, Nakamura showed that subcutaneous RBX treatment reduced retinal neovascularization (induced in neonatal mice) by returning the retina to normoxia (21% O2) after exposure to hyperoxia (75% O2). In addition, Nakamura et al.[47] reported that the RBX antiangiogenic effects were due, in part, to suppressed phosphorylation of extracellular signal-regulated protein kinases 1 and 2 and Akt.

In phase II clinical trials (PKC-Diabetic Retinopathy and PKC-Diabetic Macular Edema Studies),[48] RBX failed to alter the primary outcome (loss of visual acuity). However, there was a significant reduction in the secondary endpoint—the progression of diabetic macular edema. A much larger clinical trial (PKC-Diabetic Retinopathy Study 2) that administered a single daily dose (again using the loss of visual acuity, as the primary endpoint)[49] reported that RBX treatment significantly prevented the loss of visual acuity for diabetic patients with moderate vision loss and decreased the onset of diabetic macular edema.[50] These results suggest that PKC activation, especially of the β isoform, could participate in the development of nonproliferative DR. However, RBX did not delay the progression of vascular DR. This suggests that inhibiting the PKCβ isoformalone is not adequate to stop the metabolic changes that drive the progression of proliferative DR.

PKC and the development of diabetic peripheral neuropathy. Neuropathy is one of the most distressing complications of diabetes and involves the entire peripheral nervous system.[51] Healthy nerves receive a rich supply of blood from the vasa nervorum.[52] Hyperglycemia can damage neuronal cells by impairing vasodilation and increasing capillary BM thickening and endothelial hyperplasia, which diminish oxygen tension.[52,53] Additionally, hyperglycemia reduces Na+K+ ATPase activity, which is essential for maintaining normal nerve membrane resting potential, as well as providing neurotrophic support.[54]

The contribution of PKC activation to diabetic peripheral neuropathy (DPN) is still unclear. Hyperglycemia does not increase DAG content in nerve cells, nor is there any consensus as to whether it increases, decreases, or has any effect on PKC activity.[55] One study reported that high glucose concentrations in neurons can decrease phosphatidylinositol, thereby decreasing DAG levels and actually decreasing PKC activity. This diminished activity reduces the phosphorylation of Na+K+ ATPase, leading to a decrease in nerve conduction and regeneration. Immunochemical analysis demonstrated the presence of PKCα, β1, β2, γ, δ, and ε isoforms in nerves.[56] A previous study that directly measured sciatic nerve tissues in STZ diabetic rats also reported a reduction of PKC activity.[57] However, these results contrast with recent studies showing that treating diabetic animals with nonselective PKC isoform inhibitors, as well as selective PKCβ inhibitors, improved neural function.[58] Some animal studies have reported that PKCβ inhibitor treatments improved nerve conduction as well as neuronal blood flow.[59] Indeed, Cameron et al. showed that low-dose RBX treatments improve motor nerve conduction velocity, normalize nerve blood flow, and restore Na+K+ATPase activity in diabetic rats.[60] In humans, 1 year of RBX treatment did not significantly affect vibration detection threshold and Neuropathy Total Symptoms Score-6, but may have benefitted a subgroup of patients with less severe symptomatic DPN.[61,62] More recently, Boyd et al.[63] reported that RBX produced significant improvements in large fiber measures, quality of life (QOL), and Neuropathy Total Symptoms Score-6 in diabetic patents.

In summary, there is substantial evidence that PKCβ mediates some of the micropathologies in the early stages of microvascular complications. However, it is also clear that the effective prevention or treatment of these microvascular complications may involve inhibiting multiple PKC isoforms, including α, β, and δ.

The polyol pathway and the pathogenesis of diabetic microvascular complications. Increased cellular glucose uptake elevates glucose flux through multiple pathways, including the polyol pathway (also known as the sorbitol pathway). Aldose reductase (AR), the first enzyme of this pathway, has a Km between 5 and 10 mM glucose, which allows it to be active only when intracellular glucose is elevated. This pathway consumes NADPH in the AR reaction and reduces nicotinamide adenine dinucleotide (NAD)+ in the sorbitol reductase reaction.[64] A hyperactive polyol pathway may deplete cytosolic NADPH, which is necessary to maintain the primary intracellular antioxidant [glutathione (GSH)] in its reduced state. In mice, deleting AR−/− reduced retinal neovascularization and capillary permeability. Furthermore, the expression of VEGF, p-Erk, p-Akt, and p-IκB was significantly reduced in AR−/− retina.[65] In diabetic mice induced to have retinal ischemia by transient middle cerebral artery occlusion, AR−/− leptin receptor-deficient diabetic (db/db) mice had significantly less retinal swelling than the db/db control mice; this correlated with a reduced expression of the water channel aquaporin 4.[66] Similarly, AR deficiency in the renal glomeruli protects the mice from the diabetes-induced ECM accumulation and collagen IV overproduction. Furthermore, AR deficiency completely or partially prevented diabetes-induced glomerular hypertrophy and activation of renal cortical PKC and TGF-β1. In diabetic AR−/− mice, loss of AR resulted in reduced urinary albumin excretion[67] and protection from the decreased motor and sensory nerve conduction velocities (NCVs) seen in diabetic AR+/+ mice. Sorbitol levels in the sciatic nerves of diabetic AR+/+ mice were increased significantly, whereas sorbitol levels in the diabetic AR−/− mice were significantly lower. In addition, the study reported signs of oxidative stress [such as increased activation of c-Jun NH(2)-terminal kinase, depletion of reduced GSH, increased superoxide formation, and DNA damage] in the sciatic nerves of diabetic AR+/+ mice, but not diabetic AR−/− mice. This indicates that the diabetic AR−/− micewere protected from oxidative stress in the sciatic nerve.[68] Polymorphisms in the promoter gene region of AR are associated with susceptibility to neuropathy, retinopathy, or nephropathy, and these associations have been replicated in patients with either T1DM or T2DM, as well as across several ethnic groups.[69]

Animal studies using AR inhibitors showed promise with regard to an effect on DN or DR, but clinical trials since the 1980s have generally not confirmed such effects in patients with diabetes, except in Japan, where AR inhibitor treatments are approved for DN.

Oxidative stress and the pathogenesis of diabetic microvascular complications. The production of superoxide and other ROS in vascular cells may play an important role in the pathogenesis of vascular diseases in general and particularly in the diabetic state. A major source of superoxide in vascular cells is the NOX family of NADPH oxidases that favors reduced NAD as a substrate.[70] The elevation of oxidants and signaling enzymes, like PKC, can induce NOX 1, 2, 4, and 5 in endothelial and contractile vascular cells.[70] The expression and activity of NOX are increased in the vascular tissue of rodents with T1DM[71] and T2DM.[72] An increase in the reduced NAD/NAD+ ratio may activate NOX. In diabetes this may be caused by an increased flux through the polyol pathway (see previous description) or through the activation of poly (ADP-ribose) polymerase[73] or PKC.[74] In animal models, Baicalein, a NOX inhibitor, reduced vascular hyperpermeability and improved retinal endothelial cell barrier dysfunction.[75] However, the role of NOX isoforms in the pathogenesis of KD in diabetes is unclear. For example, NOX2 deficiency did not protect NOX2 knockout mice against DN, despite a reduction in macrophage infiltration.[76] Administering apocynin, a NOX inhibitor, corrected the vascular conductance deficits and reversed the reduction of sciatic nerve motor conduction velocity and sensory saphenous nerve blood flow induced by diabetes.[77]

Mitochondria are another important source of ROS. The elevated intracellular glucose concentration in diabetes can yield excessive mitochondrial-reducing equivalents, thus increasing the proton gradient. This inhibits the transfer of electrons from reduced coenzyme Q-10 (ubiquinone) to complex III of the electron transport chain.[64] As a result, these electrons are transferred to molecular oxygen, which results in superoxide production.

By promoting DNA strand breaks, oxidative stress can activate poly(ADP-ribose) polymerase, which can activate NF-κB and cause endothelial dysfunction.[73] Oxidative stress can also inhibit the proteasomal degradation of homeo-domain–interacting protein kinase 2, which promotes kidney fibrosis through the activation of p53, TGF-β, and Wnt.[78]

When cultured rat mesangial cells are incubated with high glucose, adding an inhibitor of the tyrosine kinase c-Src (which is activated by oxidative stress) reduces type IV collagen accumulation.[79] Similarly, in STZ-induced diabetic mice, inhibiting c-Src in vivo reduced albuminuria, glomerular collagen accumulation, and podocyte loss.[79] Podocyte injury, a major contributor to the genesis of diabetic glomerulopathy, may (in part) result from excess ROS generation. Khazim et al.[80] reported that the antioxidant plant extract silymarin reduced the high glucose-induced apoptosis of cultured mouse podocytes. In type I diabetic mice, it reduces glomerular podocyte apoptosis and albuminuria. Another study reported that silymarin treatment reduced the urinary excretion of albumin in T2DM patients with macroalbuminuria and suggested silymarin as a treatment of preventing the progression of DN.[81]

ROS overproduction can also cause major retinal metabolic abnormalities associated with the development of DR. NF-E2–related factor 2 (Nrf2) (a redoxsensitive factor) provides cellular defenses against the cytotoxic ROS. In stress conditions, Nrf2 dissociates from its cytosolic inhibitor [Kelch erythroid cell-derived protein with CNC homolog-associated protein (Keap) 1] and moves to the nucleus to regulate the transcription of multiple (>30) antioxidant genes, including the catalytic subunit of glutamyl cysteine ligase, a ratelimiting enzyme for reduced GSH biosynthesis (see section on antioxidant enzymes). Diabetes increased retinal Nrf2 and its binding to Keap1 but decreased the DNA-binding activity of Nrf2l, as well as its binding to the promoter region of glutamyl cysteine ligase. A study reported similar impairments in Nrf2-Keap-glutamyl cysteine ligase in endothelial cells exposed to high glucose and in the retina from donors with DR.[82]

To date, large clinical trials using antioxidants have not shown that the vitamins E, C, or α-lipoic acid (ALA) have a significant effect on definitive clinical endpoints for preventing or treating DR and other vascular complications.[83–85]

Motor and sensory neuron myelination and nerve conduction decline with DPN; however, the mechanisms responsible are poorly understood. Chronic oxidative stress is one potential determinant of demyelination, as lipids and proteins are important structural constituents of myelin and are highly susceptible to oxidation. Using the db/db mouse model of DPN and the superoxide dismutase 1 knockout mouse model of in vivo oxidative stress, Hamilton et al.[86] reported recently that oxidationmediated protein misfolding and the aggregation of key myelin proteins may be linked to demyelination and reduced nerve conduction in peripheral neuropathies.

Some studies have reported high oxidative status and oxidative stress index together with low serum total antioxidant status in serum from DPN patients.[87] In a double-blind placebo-controlled trial of DPN subjects, vitamin E improved electrophysiological parameters of nerve function, including motor NCV and tibial motor nerve distal latency.[88] Furthermore, a meta-analysis of 15 randomized controlled trials (RCTs) reported that the antioxidant ALA significantly improved both NCV and positive neuropathic symptoms.[89] Despite this, it is clear that we will need better antioxidants if they are to significantly delay the progression of diabetic microvascular pathologies.

Protein glycation and diabetic microvascular complications. Sugars, such as pentosidine, carboxymethyllysine, methylglyoxal, and pyraline, can cause AGE formation.[90] AGE formation can occur via a nonenzymatic reaction between glucose and protein through the Amadori product 1-amino-1-deoxyfructose adducts to lysine. However, faster reactions take place between proteins and intracellularly formed dicarbonyls, including 3-deoxyglucosone, glyoxal, and methylglyoxal, which result in the cross-linking of proteins. Due to their long turnover rate, structural extracellular proteins (such as collagen) accumulate more AGE modification. AGEs are probably present in all tissues of diabetic and/or ageing patients. AGE modification of ECM proteins and signaling molecules may alter their function. In addition, AGE-modified extracellular proteinsmay bind to receptors, the most well-characterized being the receptor for AGE (RAGE).[91] Most cells express RAGE—including the following: endothelial cells, mononuclear phagocytes, smooth muscle cells, pericytes, mesangial cells, podocytes, and neurons—and RAGE may play a role in the regulation of these cells in homeostasis and/or their dysfunction in the development of diabetic complications.[92] Binding to RAGE on the endothelial cell surface can stimulate NOX and increase ROS, p21 RAS, and MAPK. The AGE–RAGE interaction may also stimulate signaling via p38 MAPK and Rac/Cdc; however, its exactmechanism is unclear because RAGE is not an enzyme. A key target of RAGE signaling in the endothelium is NF-κB, which is translocated to the nucleus, where it increases the transcription of a number of different proteins, including ET-1, intercellular adhesion molecule-1, E-selectin, and tissue factor.[93] The ability of RAGE signaling to cause diabetic complications has been reported in transgenic mice overexpressing both inducible NO synthase (iNOS) targeted to β cells (providing a model for T1DM) and RAGE in all cells. These double-transgenic mice develop accelerated glomerular lesions,[94] which an AGE inhibitor prevents.[94] Conversely, a soluble RAGE prevents the development of increased vascular permeability and atherosclerosis in experimental diabetes.[95] Furthermore, RAGE fusion protein inhibitor administered to STZ-diabetic rats had beneficial effects on early DR or DN.[96] Clinical trials are ongoing for small molecule antagonists of RAGE.[97] Researchers have used other approaches to inhibit tissue accumulation of AGE in diabetes, including AGE formation inhibitors, such as aminoguanidine, ALT 946, and pyridoxamine, or putative cross-link breakers, such as ALT 711.[98]

Interestingly, not all AGEs or their actions affect vascular cells adversely. Several recent studies have reported inverse correlations of carboxymethyl-lysine and fructose-lysine with vascular complications.[4]

The renin-angiotensin system and the pathogenesis of diabetic microvascular complications. A large number of clinical trials have clearly shown that angiotensin-converting enzyme (ACE) inhibitors, angiotensin type-1 receptor blockers, or the combination may delay the onset of renal disease or progression to renal failure.[99] However, an analysis of renal biopsies from T1DM patients treated with these drugs did not report improved glomerular pathology, indicating that RAS inhibition may only delay the progression of functional impairment in DN.[100] The kidney produces angiotensin I and angiotensin II (Ang II) locally, and part of the renoprotective effect of ACE inhibition (in addition to lowering systemic BP) is a decrease of glomerular capillary pressure. Ang II actions may also lead to kidney damage through the induction of local factors, including ECM protein synthesis via TGF-β and inflammatory cytokines.[101] Ang II receptors mediate angiotensin action, including the activation of RAF kinase/MAPK and multiple inflammatory cytokines, such as tumor necrosis factor-α, interleukin 6, and others.[102] Furthermore, RAS blockade may improve or delay the development of DR and macular edema in diabetic patients[103] and DR in normotensive, normal albuminuric T1DM patients.[104] This suggests their beneficial effects may be more than just the reduction of BP. In animal models of diabetes, the renin inhibitor aliskiren provided similar or greater protection than ACE inhibition alone to decrease nonproliferative DR and proliferative neoangiogenesis in oxygen-induced retinopathy. In transgenic TGR(mRen-2)27 rats, which overexpress mouse renin in extrarenal tissues, aliskiren treatment reduced retinal acellular capillaries and leucostasis and normalized retinal VEGF expression.[105]

ER stress and diabetic microvascular complications. The ER plays an important role in Ca+2 and redox homeostasis, lipid biosynthesis, and protein folding. Increases in protein synthesis, protein misfolding, or perturbations in Ca+2 and redox balance can disturb ER function, causing ER stress. This triggers a coordinated program (the unfolded protein response) that reduces translation and increases protein-folding capacity to restore ER homeostasis. With chronic, unresolved ER stress, the unfolded protein response can initiate signaling that promotes apoptosis. Unfolded protein response genes are upregulated in kidney tissue from patients with diabetes, and ER stress may be a mediator of DN. Mice with STZ-induced diabetes and knockout of C/EBP homologous protein are protected from DN.[106] In the retina of rats with STZ-induced diabetes, ER stress is also involved in increased vascular permeability and the upregulation of inflammatory genes and VEGF.[107] These and other findings have prompted the development of therapeutics to reduce ER stress. These include synthetic chaperones to promote protein folding, as well as inhibitors of CCAAT/enhancer-binding homologous protein and other molecules that interfere with protein folding.[107]

Several studies have also implicated ER dysfunction in the pathogenesis of DPN. In cultured Schwann cells, knockout of antiapoptotic protein ORP150 promoted high glucose-induced Schwann cell apoptosis, whereas knockout of C/EBP homologous protein protected Schwann cells from apoptosis.[108] In rat models of high-fat STZ diabetes, knockout of ORP150 induced DPN in early diabetes and exacerbated DPN after prolonged diabetes, whereas knockout of the proapoptotic protein C/EBP homologous protein ameliorated DPN in rats with prolonged diabetes.

The kallikrein-bradykinin system and the development of diabetic microvascular complications. Plasma kallikrein is a serine protease with wellcharacterized effects in innate inflammation and the intrinsic coagulation cascade.[109] The majority of plasma kallikrein's physiological actions are attributed to the cleavage of factor XII and high-molecular-weight kininogen. The conversion of factor XII to factor XIIa leads to the activation of factor XI and the intrinsic coagulation cascade, which results in fibrin production and thrombus stabilization. Kininogen cleavage releases the nonapeptide BK, which is the ligand for the G protein–coupled BK2 receptor (BK2R). Subsequent BK cleavage by carboxypeptidases generates des-Arg9-BK, which binds and activates the BK1 receptor (BK1R). The activation of BK2R by BK and the activation of BK1R by des-Arg9-BK are associated with nearly all the effects the plasma kallikrein-kinin system (KKS) has on inflammation, vascular function, BP regulation, and nociceptive responses.[110] Plasma KKS is also associated with a variety of coagulation, vascular, and metabolic abnormalities in diabetes. However, most studies have examined the physiological effects of the KKS using BK receptor-targeted approaches.

The kallikrein-kinin system and diabetic retinopathy. Experimental studies have demonstrated that KKS activation can result in biological effects that also occur in DR (e.g., increased vascular permeability and edema); promote changes in vascular diameter and hemodynamics; and affect inflammation, angiogenesis, and neuronal functions.

Retinal vascular permeability and blood flow. Activating the KKS by injecting C1 esterase inhibitor into the vitreous increases retinal vascular permeability (RVP). The coinjection of C1-inhibitor (C1-INH) (a neutralizing antibody against plasma kallikrein) and a small-molecule plasma kallikrein inhibitor (1-benzyl-1Hpyrazole-4-carboxylic acid 4-carbamimidoyl-benzylamide) inhibited this response.[111] Intravitreal plasma kallikrein's effect is greater in diabetic rats compared with nondiabetic rats, suggesting that diabetes enhances the retinal responses to intraocular KKS activation.[111] Systemic administration of ASP-440 decreased RVP both in diabetic rats and in rats subjected to Ang II–induced hypertension.[111,112] Intravitreal BK injection increased RVP in both diabetic and nondiabetic rats, whereas only diabetic rats demonstrated a RVP response to des-Arg9-BK.[112,113] A BK1R antagonist reduced RVP in STZ-induced diabetic rats.[113,114] These data suggest that activating KKS in the circulation, and/or locally in the retina and vitreous, can increase RVP via both BK1R and BK2R mediation, and that diabetes increases actions mediated via BK1R.

KKS can also regulate retinal vessel diameters and hemodynamics. Intravitreal or intravenous BK injections acutely increased retinal vessel diameters and blood flow in adult cats[115] and rats,[116] respectively. Des-Arg9-BK increased vessel diameters in the retinal vessels of diabetic rats, but not in the retinal vessels of nondiabetic controls.[117] These effects of BK1R and BK2R on retinal vessel dilation are dependent on NO and prostaglandin in vascular endothelial cells. BK1R blockade reduces the retinal expression of potential inflammatory mediators (including iNOS and cyclooxygenase-2), NGnitro-L-arginine methyl ester, and indomethacin. In addition, the BK2R antagonist Hoe140 inhibited in vitro BK-induced vasodilation responses.[117,118] BK and Des-Arg9-BK increase intracellular free calcium by coupling Gα q/11 or Gα i/o through the BK2R or BK1R, respectively.[119,120] The increased Ca+2 can stimulate phospholipase A2 to liberate arachidonic acid from membrane phospholipids, which can increase prostacyclin[121] and increase NO synthase (NOS) phosphorylation via Ca+2/calmodulin-dependent activation. However, under inflammatory conditions, BK1R stimulation results in a much higher and prolonged NO production via Gα(i) activation of the MAPK pathway, leading to iNOS activation.[120,122] Endothelial NOS (eNOS) and iNOS activation can independently and additively increase NO production.[120,123] BK also activates the Src kinases and the subsequent vascular endothelial cadherin phosphorylation, leading to the quick and reversible opening of endothelial cell junctions and plasma leakage.[124]

Kallikrein-kinin system inhibitors: novel therapeutic applications to diabetic retinopathy. Targeting the KKS could occur at multiple levels, including the inhibition of the contact system, selective inhibition of plasma kallikrein activity, and blockade of BK receptors. Plasma kallikrein inhibitors include endogenous inhibitors, engineered proteins, and small molecules. C1-INH is a primary physiological inhibitor of plasma kallikrein, factor XIa, factor XIIa, C1r, and C1s proteases. Both plasma-derived and recombinant forms of C1-INH are effective treatments for hereditary angioedema.[125] Intravitreal injection of exogenous C1-INH reduced retinal vascular hyperpermeability induced by diabetes and by intravitreal carbonic anhydrase-1 in rats.[111] Although C1-INH is detected in the vitreous, it is unknown whether intravitreal concentrations of this endogenous serpin protease inhibitor are sufficient to inhibit plasma kallikrein. Exogenously administered C1-INH into the vitreous may provide an opportunity to inhibit the KKS, as well as other proteases in the complement and intrinsic coagulation cascades. Selective plasma kallikrein inhibition could provide increased efficacy in targeting the inflammatory effects of the plasma KKS while preserving the potential beneficial effects of the tissue KKS.

The generation of peptides that can activate BK1Rs and BK2Rs (which are expressed in a variety of ocular cell types and tissues) in large part mediates the effects of the KKS. Because both plasma kallikrein– and tissue kallikrein–mediated pathways activate BK receptors, the antagonism of these receptors blocks the effects of both KKSs. Although both BK1Rs and BK2Rs can induce RVP, BK1R appears to increase plasma extravasation in DR. The selective peptide BK1R antagonist R-954 reduced vascular permeability in a variety of tissues from STZ-induced diabetic rats, including the retina.[114] Treating STZ-induced diabetic rats with R-954 for 5 days at the end of the 4- and 12-week periods of diabetes reduced NO, kallikrein activity, and capillary permeability, whereas retinal Na+, K+-ATPase activity increased.[126] Treating diabetic rats with FOV-2304, a nonpeptide BK1R antagonist administered via eye drops, reduced RVP and normalized the retinal messenger RNA (mRNA) expression of inflammatory mediators.[127] Pouliot et al. have reported that one eye drop of the nonpeptide BK1R antagonist LF22-0542 reversed retinal plasma extravasation and RVP in the diabetic retina. These reports indicated that both local and systemic administrations of BK1R antagonists are effective in ameliorating retinal vascular abnormalities in diabetic rodents, which are similar to findings from studies using plasma kallikrein inhibitors.[128]

Protection factors. Clinical observational studies in patients with longduration diabetes (e.g., the Joslin's Medalist Study) tell us that, in addition to metabolic toxic factors, there may be equally important protective factors that spare the function and survival of cells involved in microvascular disease beyond the effect of glycemic control.[3,4] The finding that over half of diabetic patients with microalbuminuria have regression of this marker over 6 years of follow-up[129] also supports the possibility that endogenous protective factors are common in the general population. Researchers have only recently suggested that some factors with well-established functions were protective (Figure 2).

Figure 2.

Interplay of hyperglycemia's toxic mechanisms and tissues' endogenous protective properties. IGF, insulinlike growth factor.

Insulin: selective insulin resistance on the vessel wall in diabetes. Insulin receptors are present on vascular cells and cells recruited to the vascular wall; these include endothelial cells, vascular smooth muscle cells, pericytes, macrophages, and all the glomerular cells. Insulin signal transduction in these cells occurs primarily by the activation of the insulin receptor substrate 1/2 (IRS1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways, which have been shown to phosphorylate eNOS, induce the expression of VEGF and heme oxygenase-1, and decrease expression of VCAM-1. Insulin also activates the Src/MAPK pathway to induce the expression of ET-1 and the migration (and perhaps proliferation) of vascular contractile cells.[130] In diabetes or insulin resistance, hyperglycemia or free fatty acids (FAs) activate PKCα, β, or δ to phosphorylate IRS2 and p85/PI3K and selectively inhibit the p-Akt pathway in the vessel wall with the loss of insulin's anti-inflammatory and antioxidative effects,[130] whereas insulin activation of the MAPK pathway persists. In the kidney, podocytes are critically important for maintaining the integrity of the glomerular filtration barrier and preventing albuminuria. Insulin receptor signaling has a surprisingly profound effect on podocyte survival. Mice with targeted knockout of the podocyte insulin receptor[131] after 5 weeks of age developed albuminuria, effacement of podocyte foot processes, and increased apoptosis, together with increased deposition of BM components. Some of these glomerular pathologies were similar to those observed in DN. Some animals also developed shrunken kidneys with scar tissue (similar to the macroscopic appearance of kidneys in late-stage DN), accompanied by mild worsening of kidney function. This is notable because kidney function is not affected by STZ-induced diabetes (the most commonly studied rodent model of diabetes), despite albuminuria and histopathological changes. One explanation for the importance of insulin action on podocytes is that insulin increases the expression of VEGF in several cell types, including podocytes.[132] Insulin upregulates VEGF expression mostly via the IRS/Akt pathway, which may act as a survival factor for podocytes, endothelial cells, and mesangial cells. Recently, Hale et al.[132] reported that insulin directly increased VEGF-A mRNA levels and protein production in conditionally immortalized wildtype human and murine podocytes. Furthermore, when podocytes were rendered insulin resistant in vitro (using stable short hairpin RNA knockdown of the insulin receptor) or in vivo (using transgenic podocyte-specific insulin receptor knockout mice), podocyte VEGF-A production was impaired. Insulin could also prevent apoptosis by other mechanisms, including inhibiting proapoptotic molecule caspase-9,[133] inhibiting transcription factor FoxO,[134] or upregulating the antioxidant activity of heme oxygenase-1.[18]

Researchers have also described the selective (IRS1/PI3K/Akt pathway) impairment of insulin action in the glomeruli of diabetic animals and patients, which may contribute to DN development. The IRS/PI3K/Akt pathway mediates many of insulin's protective effects, including the upregulation of eNOS[135,136] and heme oxygenase-1.[18] In contrast, the Ras/MAPK pathway mediates the induction of ET-1.[137] With diabetes or insulin resistance, elevated concentrations of glucose and free FAs can activate PKC and selectively inhibit insulin signaling through the PI3K pathway.[138] Certain threonine/serine residues on IRS2 and on the p85 regulatory subunit of PI3K are substrates for PKC, and phosphorylation of these sites inhibits insulin-stimulated PI3K pathway signaling.[139,140] Hyperinsulinemia in T2DM may promote vascular disease through the induction of ET-1[141] or other factors induced by MAPK signaling.

Antioxidant enzymes. Although extensive evidence from cell- and animalbased studies supports the role of oxidative stress in the development of vascular complications, nearly all clinical trials using antioxidants have failed to show efficacy with clinically significant vascular endpoints.

Nevertheless, tissue-specific endogenous antioxidant enzymes are most likely important to neutralize the increased levels of oxidants seen with hyperglycemia. This idea has stimulated clinical trials using bardoxolonemethyl,[142] a synthetic triterpenoid that activates Nrf2. This nuclear factor upregulates a gene program of molecules with antioxidant activity called phase 2 genes, which includes heme oxygenase 1 and enzymes in the GSH biosynthesis pathway. Keap1, a repressor that binds Nrf2 in the cytoplasm and promotes Nrf2 proteasomal degradation, inhibits Nrf2 translocation to the nucleus. Bardoxolone methyl interacts with cysteine residues on Keap1, preventing Nrf2 repression and allowing phase-2 gene transcription. Results from a trial of bardoxolone methyl in patients with advanced chronic KD (CKD) showed an improvement in glomerular filtration rate (GFR) up to 1 year after start of treatment.[143] However, proteinuria was increased and researchers stopped phase-III trials due to safety issues. Researchers also reported that Nrf2 has a protective role in the retina against neuronal and capillary degeneration in retinal ischemia–reperfusion injury. InNrf2+/+ mice, ischemia–reperfusion injury resulted in leukocyte infiltration of the retina and vitreous and increases in retinal levels of superoxide and proinflammatory mediators. These changes were greatly accentuated in Nrf2−/− mice.[144]

PDGF and VEGF. PDGF expressed by retinal endothelial cells plays a role both in vascular cell survival and proliferative retinopathy.[145] During sprouting angiogenesis, endothelial tip cells produce PDGF, which acts through PDGF receptor-β expressed by pericytes. This signal recruits pericytes to develop blood vessels. Pericytes, in turn, can support endothelial cell survival and inhibit its proliferation. Reports of pericyte loss and endothelial cell proliferation in PDGF knockout mouse embryos demonstrate this process.[146] Mice with heterozygous deletion of the PDGF gene not only have an increased frequency of acellular capillaries (particularly after diabetes induction), but also an increased tendency for retinal neovascularization during ischemic retinopathy.[147] As described previously, we have reported that hyperglycemia can inhibit the survival effects of PDGF by upregulating SHP-1, which causes dephosphorylation of the PDGF receptor in pericytes and possibly also in podocytes.[14]

High glucose concentrations and diabetes can activate SHP-1 (a tyrosine phosphatase) in microvessels, including the retina and renal glomeruli. This leads to the dephosphorylation and deactivation of specific growth factor receptors critical for the survival of pericytes in the retina and podocytes in the kidney.[14] One study reported that SHP-1 regulates AGE-related endothelial cell injury in vitro.[148] In the retina of diabetic rodents, SHP-1 activation can desensitize pericytes to PDGF and cause pericyte apoptosis, an initiating step in the development of DR.[14] In the renal glomeruli, the upregulation of SHP-1 expression can impair VEGF survival signaling and increase podocyte apoptosis and endothelial dysfunction.[24] The upregulation of SHP-1 expression in diabetes depends on the activation of PKCδ and p38MAPKα transcription,[14,24] which is prevented in PKCδ knockout mice. These mice are protected from the apoptosis of retinal pericytes, mesangial expansion, and albuminuria.[14,24] Therefore, inhibiting SHP-1 is a potential novel approach to preserving survival signaling in vascular cells.

TGF-β1. TGF-β1 is a major inducer of profibrotic responses in diabetic kidneys. Diabetes increases the expression of TGF-β in blood vessels in many vascular beds, and one study suggests that it is a causative factor for the development of fibrosis in the kidney and other tissues.[149] An earlier study has shown that treating C57BLKS/J db/db mice with neutralizing monoclonal TGF-β1 antibody decreases plasma TGF-β1, mesangial matrix expansion, and kidney mRNA levels of collagen IV and fibronectin.[150] In addition, this therapy prevented a loss of renal function but had no effect on the elevated albuminuria in db/db mice. More recently, investigators have used an inhibitor of TGF-β receptor kinase activity, GW788388, to treat C57BLKS/J db/db mice.[151] This therapy reduced glomerular collagen staining and kidney mRNA levels of plasminogen activator inhibitor 1 and types I and III collagen, but did not alter albuminuria.[151]

However, numerous studies have reported that TGF-β has potent anti-inflammatory effects on macrophages and is a negative regulator of T cell and B cell activation. Therefore, TGF-β may have protective actions due to an anti-inflammatory effect, and its elevation may be a reaction to the inflammatory stress of diabetes. Thus, it is likely that diabetes-induced overexpression of TGF-β in many tissues could be an endogenous response to the inflammatory actions of hyperglycemia in vascular cells. These paradoxical roles of TGF-β make it a challenging drug target.

VEGF. VEGF expression changes paradoxically with diabetes, it increases in the retina and renal glomeruli, but it decreases in the myocardium, peripheral limbs, and nerves correlating with the extent of angiogenesis. VEGF neutralization is already a treatment of proliferative DR and macular edema, and one study suggests it as a therapy for DN.[152] However, the increased levels of VEGF in both tissues are most likely an appropriate response to hypoxia, which results from loss of capillary function. It has been a longstanding concern that neutralizing VEGF could counteract survival signaling in retinal neurons. Interestingly, injecting low doses of VEGF accelerated the restoration of the physiological capillary bed and prevented preretinal neovascularization in a mouse model of proliferative retinopathy.[153]

In the kidney, podocytes contain the highest level of VEGF expression, and some of the most insightful work describing a role for VEGF as a survival factor comes from studies of renal podocytes. The conditional deletion of VEGF in podocytes resulted in a complete lack of endothelial and mesangial cells in mature glomeruli and death within the first day of life.[154] This finding strongly supports a role for VEGF in the maintenance of glomerular endothelial cells. The heterozygous knockout of VEGF in podocytes of mice resulted in proteinuria and ESKD in young adults[154] and was preceded by the disappearance of endothelial cell fenestrations, increases in necrosis, the effacement of podocyte foot processes, and a dramatic loss of mesangial cells.[154] Inducing STZ diabetes in these mice exacerbated glomerular cell apoptosis, glomerulosclerosis, and proteinuria compared with nondiabetic controls.[155] However, other studies reported that increased podocyte VEGF[156] expression worsens DN, characterized by glomerulosclerosis, microaneurysms, mesangiolysis, glomerular BM thickening, podocyte effacement, and massive proteinuria associated with hyperfiltration.[156]

VEGF also has neuroprotective effects. Primary dorsal root ganglion cultures lacking VEGF-B or fms-like tyrosine kinase 1 (FLT-1) exhibited increased neuronal stress and are more susceptible to paclitaxel-induced cell death, and mice lacking VEGF-B or a functional FLT-1 develop more retrograde degeneration of sensory neurons. Conversely, adding VEGF-B[157] to dorsal root ganglia cultures antagonized neuronal stress, maintained the mitochondrial membrane potential, and stimulated neuronal survival. Mice overexpressing VEGF-B[157] or FLT-1 selectively in neurons were protected against distal neuropathy, whereas exogenous VEGF-B,[157] delivered by either gene transfer or as a recombinant factor, was protective by directly affecting sensory neurons and not the surrounding vasculature.[158] Identifying the prosurvival mechanisms in stressed neuronal cells revealed that protein kinase A functioned concurrently with the VEGF receptor-2 pathway to signal the activation of extracellular signal-regulated protein kinases 1/2 protection against caspase-3/7 activation and subsequent cell death.[159]

Activated protein C. Activated protein C (APC) is an anticoagulant factor that acts as a survival factor for renal glomerular cells.[160] Thrombomodulin, a procoagulant factor that activates protein C, is highly expressed in glomeruli of mice, but downregulated in diabetes.[160] Diabetic mice with a loss-of-function thrombomodulin gene mutation had more albuminuria and more severe glomerular pathology than diabetic wild-type mice, whereas diabetic mice with a gain-of-function mutation of the protein C gene had less.[160] The anticoagulant effects of APC did not account for its protective actions. Rather, APC was shown to counteract the apoptosis of endothelial cells and podocytes through the activation of two of its receptors.[160] Therefore, endothelial-derived APC appears to act as a protective factor with local survival effects for both podocytes and endothelial cells in the glomerulus. The underlying mechanism for APC protection from renal dysfunction is still unknown. However, Li Calzi et al.[161] reported that APC-mediated protease suppressed lipopolysaccharide-induced increases in the vasoactive peptide adrenomedullin, suppressed infiltration of iNOS-positive leukocytes into renal tissue, and activated receptor-1 agonism. The anticoagulant function of APC was responsible for suppressing lipopolysaccharideinduced stimulation of the proinflammatory mediators ACE-1, interleukin-6, and interleukin-18, perhaps accounting for its ability to modulate renal hemodynamics.[162]

Vascular progenitor cells. Bone marrow–derived cells, including endothelial progenitor cells (EPCs) and myeloid progenitors, may contribute to postnatal angiogenesis.[163] EPCs may contribute by incorporating into newly formed blood vessels. However, it is likely the major action of EPCs is to release proangiogenic factors and temporarily associate with neovascular structures. In diabetic patients, both the number and function of EPCs are reduced,[164] impairing the ability of EPCs to repair the vascular endothelium.[165] The angiogenic potential of EPCs was also reduced in diabetic animals.[166]

The differentiation of bone marrow–derived EPCs may also play a role.[166] eNOS is necessary to mobilize EPCs from bone marrow.[167] Uncoupling eNOS that favors superoxide rather than NO production may impair EPC function. Indeed, EPC function improves after eNOS is inhibited ex vivo in EPCs isolated from patients with diabetes.[168] Interestingly, neuropathy in the bone marrow may reduce EPC mobilization. STZ-induced diabetes in rats reduced nerve terminals in bone marrow, and this correlated with increased EPCs in bone marrow and decreased EPCs released into the circulatory system. These abnormalities were associated with an increase in retinal acellular capillaries.[169] Transplanting nondiabetic EPCs into diabetic animals can improve angiogenesis after peripheral ischemic injury.[170]

These studies suggest that it may be possible to promote the repair of ischemic tissue in diabetes by improving the mobilization, differentiation, and function of EPCs or other progenitors. Recently, researchers have suggested that autologous EPC transplantation could be a potential therapy for DN. However, safety concerns regarding possible unwanted proliferation or differentiation of the transplanted stem cells might limit such treatment. An alternative approach is to stimulate endogenous bone marrow–derived EPC recruitment into ischemic lesions by administering stem cell mobilization agents or chemokines.[171] Administering the EPC mobilization agent AMD3100 increased the local expression levels of vasculogenesis-associated factors and the number of newly formed endothelial cells in the sciatic nerve, which restored the sciatic vasa nervorum.[171]

Circulating EPCs are markedly reduced in CKD patients,[172] and EPCs have been shown to improve renal function, attenuate the proinflammatory response associated with renal injury, and improve damaged tubules and renal vascular segments during kidney injury while providing enhanced neoangiogenesis.[173] An intact and healthy EPC niche, residing in the bone marrow but also found locally in renal vascular beds (i.e., in the adventitia layer of vessels), may be able to support normal vascular function, including maintenance and possible replacement of the endothelium.[174]

Emerging studies in animal models suggest that EPCs help revascularize ischemic and injured retinas. Thus, EPCs could be a potential therapy for ischemic retinopathies in humans, which are a leading cause of blindness.[161] In nonproliferative DR, EPCs may be less effective, as they do not recruit other EPCs and repair the acellular capillaries. In proliferative DR, the EPCs take on a proinflammatory phenotype and recruit too many EPCs, leading to pathological neovascularization.

For the last 10 years, many groups have focused on understanding the basic mechanism responsible for the diabetes-associated defect in EPC function. Correcting this defect may allow diabetic patients to use their own EPCs to repair injured retinal and systemic vasculature. Specifically in the retina, correction of this dysfunction may prevent early and intermediate stages of vasodegeneration (thus enhancing vessel repair), reverse ischemia, and prevent the progression to the late stages of DR. However, these findings on the changes of EPCs and their correlation to various complications in diabetes have been inconsistent. Clearly, we need more studies to clarify changes in diabetes and the role EPCs play before patients can use them therapeutically.[161]

Summary

Hyperglycemia initiates its adverse effects by increasing its metabolites in vascular cells; this can cause specific changes in vascular functions, such as those mediated by PKC or ROS activation. However, increases in glucose metabolism can also generate nondiabetic specific toxic products (such as oxidants, AGE, and methylglyoxal), which accelerate the specific toxic actions of hyperglycemia and cause microvascular pathologies. The specific needs of various tissues (such as the retina, glomeruli, and the peripheral neuron), the importance of the various functions that are changed by hyperglycemia, and the protective responses generated by each tissue all modulate specific pathologic manifestations. Thus, treatments that prevent and delay the progression of diabetic microvascular complications must do the following: (1) eliminate hyperglycemia; (2) inhibit the major mechanisms that hyperglycemia activates to induce vascular dysfunction; (3) neutralize accelerants, such as inflammation and oxidative stress; and (4) activate tissuespecific protective factors.

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