Pathogenesis of Microvascular Complications
Risk factors and protective factors in diabetic microvascular complication.
KeywordsAdvanced glycation end products (AGE) Reactive oxygen species (ROS) Protein kinase C (PKC) Vascular endothelial growth factor (VEGF) Activated protein C (APC) Platelet-derived growth factor (PDGF) Transforming growth factor b (TGF) Heme oxygenase-1 (HO-1)
Systemic metabolic factors are clearly the major causal factors for diabetic microvascular diseases. It is the major metabolic dysfunction that is causing microvascular diseases for retinopathy and nephropathy. The results of the Diabetes Control and Complications Trial (DCCT) in type 1 diabetes (T1D) and the United Kingdom Perspective Study (UKPDS) in type 2 diabetes (T2D) have clearly demonstrated that intensive blood glucose control delays the onset and retards the progression of diabetic microvascular complications (DCCT Group 1993). Further, intensive control of hyperglycemia is also helpful to decrease cardiovascular diseases (CVD) in T1D patients (DCCT 2016). In contrast, intensive glycemic control with insulin has not shown a dramatic decrease of CVD in T2D patients (Patel et al. 2008) demonstrating clear differences in the pathogenesis of macro- versus microvascular diseases. The importance of hyperglycemia on microvascular diseases is overwhelming. There is very little evidence that without hyperglycemia such as syndromes of insulin resistance will induce significant risk in the development of DR and DN. The presence of dyslipidemia and insulin resistance probably do not have a major impact on DR and DN, since people with metabolic syndrome and without diagnosis of diabetes have minimal risks for DR and even DN (Keenan et al. 2009; Foster et al. 2008). Thus, it is likely that dyslipidemia and insulin resistance may have additive effects only in the presence of hyperglycemia for DR and DN. Interesting findings from the DCCT and Epidemiology of Diabetes Interventions and Complications (EDIC) have also shown that reversal of hyperglycemia will not rapidly normalize all of the risks of microvascular diseases since after 10 years of intensive glycemic control, the group previously on non-intensive glycemic control continued to experience increased risks for DR and DN, which have led to the concept of metabolic memory (Retinopathy 2000).
Genetic and epigenetic regulation. Extensive studies have shown that only 30% of diabetic patients, T1D and T2D, will experience increased risk of renal failure (Molitch et al. 2004). In addition, the risk for DN also has familial cluster, again supporting a genetic and epigenetic role. The risk for DR appears to be also related to familial clusters; however, genetics for DR has been difficult to establish since more than 90% of diabetic patients will have significant DR with prolonged duration of disease (Aiello et al. 1998). The genetic role of microvascular complications has been studied in detail over the last 20 years; however, definitive role and targets have not been established clearly. This has led to interest in studying the pathogenesis for familial clusters and lack of DN in 60–70% of diabetic populations and in those individuals with T1D of extreme duration, 50–80 years of diabetes, could possibly be due to epigenetic changes. The difficulty of identifying genetic factors for the lack of DN/DR in a large group of diabetic patients has given rise to the idea that the development and establishment of vascular complications of diabetes could be an interplay of increased risk factors induced by hyperglycemia and a decrease in the protective mechanisms that are naturally occurring in the tissues. Further discussions on this idea will be provided below.
Local tissue response. Differential tissue responses are also critical in the development of microvascular complications. A classic example of the importance of local responses is the paradoxical exhibition of increase in neovascularization in proliferative diabetic retinopathy (PDR) versus the decrease in capillary density in response to ischemia as exhibited in the peripheral limbs, myocardium, and wound healing process (Aiello et al. 1994; Chou et al. 2002). At the biochemical and molecular levels, differences of angiogenesis are related to changes in VEGF expression which are clearly increased in PDR and decreased VEGF expression in the peripheral tissues, myocardium, and wounds. It is unknown at this time what are the differential mechanisms that are causing the paradoxical changes in VEGF and other factors that lead to differences in the pathologies.
Thus, the clinical and epidemiological information derived from the microvascular complications exhibited by the diabetic individuals have strongly suggested that protective or neutralizing factors exist to prevent or delay the progression of toxicity due to hyperglycemia. For example, the studies by Krolewski et al. have shown that 2/3 of T1D patients who exhibited microalbuminuria do not progress with loss of significant renal function even after 15 years of duration. In fact, 1/3 of these individuals with diabetes have resolution of their microalbuminuria while maintaining their estimated glomerular filtration rate (GFR) in the same time span, supporting the idea of potentially delaying the progression of DN by neutralizing hyperglycemia’s toxic effects (Perkins et al. 2003). Another example of the presence of endogenous protective factors has been exhibited in the finding that the transcription factor nuclear factor (erythroid-derived 2)–like 2 (Nrf2) can activate over 100 genes for both antitoxin and antioxidant enzymes (Tebay et al. 2015). The activation of Nrf2 has been shown to be important as the body’s defense against environmental toxins and oxidants (Zhang et al. 2015). A great deal of work now is in progress to determine whether activation of Nrf2 could be a therapeutic target for the treatment of DN and DR (de Zeeuw et al. 2013a). The most important and conclusive evidence that endogenous factors exist to neutralize the toxic effect of hyperglycemia and other metabolic factors induced by diabetes to prevent or delay the progression of DR/DN have come from the Joslin Medalist Study (Keenan et al. 2007). The Joslin Medalist Program is a study which contains a cohort of over 1000 individuals with T1D duration for at least 50 years. The Joslin Medalist Study reported that approximately 35% of the Medalists do not experience significant DR or DN (Keenan et al. 2007). Analysis of those Medalists with and without significant DR or DN did not correlate to their history of glycemic control as measured by over 20 years of HbA1c. Detailed analysis of over 100 Medalists and their rate of progression of DR in this subset of close to 150 Medalists exhibited a bimodal distribution. Over 50% of the Medalists experienced PDR after the onset of the disease of more than 17 years. Though more than 30% of these individuals will develop at most one or two microaneurysms, there will be no progression to severe renal or retinal lesions. Furthermore, history of HbA1c for those with and without DR is also not different. These results clearly demonstrated that at the clinical level endogenous protective factors exist to neutralize the toxic effects of hyperglycemia and other dysmetabolites induced by diabetes (Sun et al. 2011).
In the following, we will provide a detailed description on the potential molecular mechanisms which can be induced by hyperglycemia to cause capillary and microvascular pathologies. This will be followed by a discussion on the mechanisms of protective factors which are in play to neutralize the toxic effects of hyperglycemia. At the end, we will summarize the proposal that the development of microvascular disease has to be an interplay between increased risk of glucose toxic metabolites and the loss of protective mechanisms.
Molecular mechanisms of injury. Multiple abnormalities in cell signaling, gene expression, and regulation of cell biology and physiology have been described in diabetes, and it is likely that many of these abnormalities operate concurrently to cause the various diabetic microvascular complications. These mechanisms may be active preferentially in some, though probably not all, vascular tissues or organs, but generally they are relevant for development of complications in several organs (Fig. 1). The description of the molecular mechanisms will be briefly reviewed since many of them have been studied extensively with extremely long lists of publications. In addition, the summary of these molecular mechanisms will focus mainly on microvascular pathologies (Fig. 2).
Role of the Polyol Pathway
Extracellular hyperglycemia can lead to elevation of intracellular free glucose mostly through the transport of GLUT-1, which is a facilitated transporter (Kaiser et al. 1993). Elevated intracellular free glucose will cause increased flux through main glucose metabolic pathways such as glycolysis and glucose-6 (G6) phosphate pathways, but it can also significantly increase glucose metabolism through those pathways that are not normally activated due to their high Km for glucose. This is clearly demonstrated by the increased flux of glucose through the polyol or sorbitol pathway. Aldose reductase (AR), the first enzyme of the polyol pathway, has Km between 5 and 10 mM of glucose, which is activated mostly in the hyperglycemic state (Srivastava et al. 1985). The product of AR is sorbitol which is further metabolized by sorbitol dehydrogenase to fructose which is returned back to the glycolytic metabolism (Jeffery and Jornvall 1983). The hyperactivity of this pathway has been postulated to cause diabetic complications both in micro- and macrovascular tissues (Burg and Kador 1988; Ramasamy and Goldberg 2010). It is believed that the hyperflux of glucose to the polyol pathway could potentially cause complications through two ways. First, it is believed that this pathway can consume nicotinamide adenine dinucleotide phosphate (NADPH) in the AR reaction and increase NADPH and reduce NAD in the processing of the sorbitol via the sorbitol dehydrogenase actions resulting in oxidative stress. In addition, it is also believed that the increased activation of AR could elevate sorbitol levels which could provide a hyperosmolar effect to cause cellular dysfunction. Activation of the polyol pathway may result not only from increased availability of free intracellular glucose but also from inactivation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). This can occur by the addition of adenosine diphosphate (ADP)-ribose moieties to GAPDH by the enzyme poly(ADP-ribose) polymerase (PARP) after its activation by reactive oxygen (Du et al. 2003). Therefore, loss of GAPDH leads to increased levels of glyceraldehyde 3-phosphate (GA3P), which in turn causes increased production of methylglyoxal, an advanced glycated end product (AGE) precursor, or de novo synthesis of diacylglycerol (DAG), a protein kinase C (PKC) activator (Brownlee 2001). However, it should also be noted that the changes in sorbitol levels are in the nanomolar which is unlikely to change cellular or plasma osmolarities significantly. The only site where there is a significant increase in sorbitol levels that could potentially cause pathological changes is the lens, where increase in sorbitol could potentially accelerate cataract formation that is observed in diabetic patients (Lightman 1993). In addition, the rationale for proposing that elevation of sorbitol production can cause complications is the correlation between elevated plasma and tissue sorbitol levels to increased risks of various vascular complications (Gabbay 1973). However, it is also possible that the increased flux through the AR pathway may even be a protective mechanism in order to decrease intracellular free glucose. Therefore, an increased flux through the sorbitol pathway with a combined increase of all of the enzymes in the sorbitol pathway could decrease hyperflux via glycolysis and will be reflected as lower sorbitol levels in those individuals who are protected from complications.
At the mechanistic level, it is suggested that hyperactive polyol pathway may adversely affect cellular homeostasis by depleting cytosolic NADPH, which is necessary to maintain the primary intracellular antioxidant, and glutathione in its reduced state. The deletion of aldose reductase [AR−/−] reduced neovascularization and capillary permeability. Levels of VEGF, p-Erk, p-Akt, and p-IκB were reduced in AR−/− retina (Fu et al. 2012). In mice which were induced to have retinal ischemia by transient middle cerebral artery occlusion, AR−/− db/db mice had significantly lower retinal swelling than the db/db mice (Yeung et al. 2010). Similarly, AR deficiency in the renal glomeruli protects from the diabetes-induced extracellular matrix accumulation and collagen IV overproduction. Furthermore, AR deficiency completely or partially prevented diabetes-induced activation of renal cortical PKC, transforming growth factor (TGF)β1, and glomerular hypertrophy. Loss of AR resulted in a reduction of urinary albumin excretion in the diabetic AR−/− mice (Liu et al. 2011). AR−/− mice were protected from the reduction of motor and sensory nerve conduction velocities observed 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 than those in diabetic AR+/+ mice. Polymorphisms promoter gene region of AR have been associated with susceptibility to neuropathy, retinopathy, or nephropathy. These associations have been replicated in patients with either T1D or T2D as well as across several ethnic groups (Demaine 2003).
Animal studies using AR inhibitors (ARI) showed promise with regard to an effect on diabetic retinopathy or nephropathy, but clinical trials have not confirmed such effects in patients with diabetes. Reports of ARI used in rodents have shown to be effective for preventing capillary abnormalities, especially cataract formation, but not effective in retinopathy in diabetic dogs (Kador et al. 2006; Neuenschwander et al. 1997). One possible explanation for the discrepancy is the high levels of AR expression in rats, especially in the lens.
Clinical trials since the 1980s have generally not confirmed such effects in patients with diabetes except in Japan, where ARI were approved as treatment for diabetic neuropathy. One of these clinical studies, the Aldose Reductase Inhibitor–Diabetes Complications Trial (ARIDCT) was conducted in patients with mild diabetic neuropathy. Among those patients who received ARI, epalrestat treatment showed a reduction in the development of diabetic retinopathy and nephropathy, which may have resulted from the suppressive effect of epalrestat on oxidative and inflammatory stress through inhibition of the polyol pathway. Recently, the efficacy of epalrestat in diabetic retinopathy and nephropathy was examined by re-analysis of the ARIDCT results, with consideration of the influence of patient background factors and severity of DN. The results suggested that epalrestat may have delayed the progression of diabetic retinopathy and nephropathy. Some interesting evidence has been reported that ARI may alter glucose metabolism in the myocardium (Trueblood and Ramasamy 1998).
The Role of the Glycation Modification of Proteins in Diabetic Microvascular Complications
Modification of extracellular and intracellular proteins by sugars can result in the formation of AGE, such as pentosidine, carboxymethyllysine (CML), methylglyoxal, and pyraline (Reddy et al. 2002). Increased concentrations of AGE in plasma and tissues are directly correlated with the level of hyperglycemia (Fleming et al. 2011). AGE formation can occur via a non-enzymatic 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 are prone to accumulate more AGE modification. AGEs have been demonstrated in numerous tissues such as the retina, glomeruli, skin, neurons, and probably all tissues in diabetic states and aging. AGE modification of extracellular matrix proteins and signaling molecules may alter their function. In addition, AGE-modified extracellular proteins may act by binding to receptors, the most well-characterized being receptor for AGE (RAGE) (Chen et al. 2012). RAGE is expressed by most cells including endothelial cells, mononuclear phagocytes, smooth muscle cells, pericytes, mesangial cells, podocytes, and neurons, indicating a potential role in the regulation of their properties in homeostasis and/or their dysfunction in the development of diabetic complications (Schmidt et al. 1994). RAGE is a multi-ligand receptor structurally belonging to the immunoglobulin superfamily. RAGE receptor is composed of extracellular binding domain and a short cellular cytosolic domain (43 amino acids) which binds to diaphanous-1 (DIAPH1) (Hudson et al. 2008). Binding to RAGE on the endothelial cell surface has been reported to stimulate NOX and increase ROS, p21 RAS, and mitogen-activated protein kinase (MAPK). The AGE-RAGE interaction may stimulate signaling via p38 MAPK and Rac/Cdc, although its exact mechanism is unclear since RAGE is not an enzyme. A key target of RAGE signaling is nuclear factor κB (NF-κB), which is translocated to the nucleus where it increases transcription of a number of different proteins, including ET-1, ICAM-1, E-selectin, and tissue factor (Goldin et al. 2006). The ability of RAGE signaling to cause diabetic complications has been reported in transgenic mice overexpressing both inducible nitric oxide synthase (NOS) (iNOS) and RAGE in all cells. These double transgenic mice developed accelerated glomerular lesions (Yamamoto et al. 2001), which could be prevented by an AGE inhibitor. A soluble receptor for AGE prevents development of increased vascular permeability and atherosclerosis (Park et al. 1998). Furthermore, diabetic rats treated with RAGE fusion protein inhibitor displayed beneficial effects on early diabetic retinopathy and signs of neuropathy (Li et al. 2011). RAGE has been implicated to have a role in many diseases where inflammation is important, even in the absence of diabetes, such as cancer, atherosclerosis, insulin resistance, and Alzheimer’s disease (Ramasamy et al. 2009). Thus, it is likely that AGE and RAGE have significant effects in the inflammatory cascade but not unique to diabetes. Thus, the pathophysiological effects of AGE and RAGE are to accelerate the main pathway of inflammation. The enhancement nature of AGE/RAGE for inflammation is further supported by the findings that RAGE knockout (KO) mice do not manifest significant pathologies (Sakaguchi et al. 2003). Clinical trials are ongoing for small molecule antagonists of RAGE (Yan et al. 2010). Other approaches have been used to inhibit tissue accumulation of AGE in diabetes, including inhibitors of AGE formation such as aminoguanidine, ALT 946, and pyridoxamine or putative cross-link breakers such as ALT 711 (Jandeleit-Dahm et al. 2005). Interestingly, not all AGE or their actions affect vascular cells adversely. Several recent studies have reported inverse correlations of CML and fructoselysine with vascular complications (Sun et al. 2011). Further studies have suggested that food with high AGE content such as those cooked on grills could potentially elevate circulating AGEs (Goldberg et al. 2004). A recent clinical report suggested that low AGE diet improved insulin resistance compared to high AGE diets, again suggesting that AGE contributed in the general elevations of inflammatory cytokines systemically (Mark et al. 2014).
Oxidative Stress in the Pathogenesis of Diabetic Microvascular Complications
Production of superoxide and other ROS in vascular cells may play an important role in the pathogenesis of vascular diseases in diabetes. A major source of superoxide in vascular is thought to be from the family of NADPH oxidases (NOX) that favors NADH as a substrate (Lassegue et al. 2012). Elevation of oxidants and signal enzymes such as PKC can induce NOX 1, 2, and 4 with NOX 1, 2, 4, and 5 in endothelial and contractile cells (Lassegue et al. 2012). Expression and activity of NOX are increased in the vascular tissue of rodents with T1D (Hink et al. 2001) and T2D (Kim et al. 2002). NOX may be activated by an increase in the NADH/NAD+ ratio, which in diabetes may be caused by an increased flux through the polyol pathway (see above) or activation of poly(ADP-ribose) polymerase (PARP) (Garcia Soriano et al. 2001). NOX activity may also be increased by hyperglycemia through PKC activation (Inoguchi et al. 2000). In animal models, Baicalein, a NOX inhibitor, reduced vascular hyperpermeability and improved retinal endothelial cell barrier dysfunction (Othman et al. 2013). However, the role of NOX isoforms in the pathogenesis of diabetic kidney disease is unclear and was evaluated by You et al. using the NOX2 knockout mice with insulin-deficient diabetes (You et al. 2013). They reported that lack of NOX2 does not protect against diabetic kidney disease despite a reduction in macrophage infiltration (You et al. 2013). Apocynin, a NOX inhibitor, treatment corrected the reduced sciatic nerve motor conduction velocity, sensory saphenous nerve deficit blood flow, and vascular conductance that are known to be reduced in diabetes (Cotter and Cameron 2003).
Mitochondria are another important source of reactive ROS. The citric acid cycle provides NADH and flavin adenine dinucleotide (FADH2) that can act as electron donors for the electron transport chain, creating a proton gradient over the inner mitochondrial membrane (Brownlee 2001). When intracellular glucose concentration increases, and thereby yields excessive reducing equivalents for this process, the proton gradient increases and inhibits the transfer of electrons from reduced coenzyme Q (ubiquinone) to complex III of the electron transport chain (Brownlee 2001). Instead, electrons are transferred to molecular oxygen, causing production of superoxide.
Nitric oxide (NO) can neutralize ROS, but paradoxically, eNOS can become a source of ROS if an already pro-oxidant redox state favors oxidation of the eNOS cofactor tetrahydrobiopterin (BH4). This leads to uncoupling of electron transport in eNOS and release of superoxide (Laursen et al. 2001). By promoting DNA strand breaks, oxidative stress can activate PARP, which in turn can activate NF-κB and cause endothelial dysfunction (Garcia Soriano et al. 2001). Oxidative stress can also inhibit the proteasomal degradation of homeo-domain-interacting protein kinase 2 (HIPK2), which promotes kidney fibrosis through activation of p53, TGF-β, and WNT (Jin et al. 2012).
In vitro studies suggest oxidative stress may contribute to diabetic nephropathy and retinopathy. Cultured rat mesangial cells (MC) incubated with high glucose in the presence of tyrosine kinase (c-Src) inhibitor, which is sensitive to oxidative stress, showed a reduction in collagen type IV accumulation (Taniguchi et al. 2013). Similar results were obtained in vivo in streptozotocin (STZ)-induced diabetic mice, where treatment with Src inhibitor reduced albuminuria, glomerular collagen accumulation, and podocyte loss (Taniguchi et al. 2013). Podocyte injury, a major contributor to the pathogenesis of diabetic glomerulopathy, may in part be the excessive generation of ROS. Overproduction of superoxide by NOX4 may also have an important role in podocyte injury. Khasim et al. reported that treatment with the plant extract Silymarin, which is known to have antioxidant properties, reduced the high glucose-induced apoptosis in cultures of mouse podocytes. In T2D patients with macroalbuminuria, Silymarin treatment reduces urinary excretion of albumin and has been suggested as a treatment for preventing the progression of diabetic nephropathy (Fallahzadeh et al. 2012).
Increased ROS is reported to cause major retinal metabolic abnormalities associated with the development of diabetic retinopathy. NF-E2-related factor 2 (Nrf2), a redox-sensitive factor, provides cellular defenses against the cytotoxic ROS. In stress conditions, Nrf2 dissociates from its cytosolic inhibitor, Kelch-like-ECH-associated protein 1 (Keap1), and moves to the nucleus to regulate the transcription of several antioxidant genes including the catalytic subunit of glutamyl cysteine ligase (GCLC), a rate-limiting reduced glutathione (GSH) biosynthesis enzyme (see section “Antioxidant Enzymes”). Diabetes increased retinal Nrf2 and its binding with Keap1 but decreased DNA-binding activity of Nrf2 and also its binding at the promoter region of GCLC. Similar impairments in Nrf2-Keap1-GCLC were observed in the endothelial cells exposed to high glucose and in the retina from donors with diabetic retinopathy (Zhong et al. 2013).
So far, large clinical trials using antioxidants, vitamins E or C, for prevention or treatment of diabetic retinopathy and other vascular complications have not shown conclusive evidence for efficacy when definitive endpoints were measured in humans (Kowluru and Zhong 2011). The Heart Outcomes Prevention Evaluation (HOPE) study reported that daily administration of vitamin E for an average of 4.5 years to middle-aged and elderly people with diabetes and cardiovascular disease (CVD) and/or additional coronary risk factor(s) had no effect on nephropathy (Lonn et al. 2002). Likewise, α-lipoic acid treatment failed to show a clinically significant improvement on macular edema (Haritoglou et al. 2011). However, a recent epidemiological study in cohort of T2D patients reported that the risk for diabetic retinopathy declined with increased intake of vitamin C (Tanaka et al. 2013).
Diabetic peripheral neuropathy (DPN) is associated with decrements in motor and sensory neuron myelination and nerve conduction; however, the mechanisms of reduced myelination in diabetes are poorly understood. Chronic elevation of oxidative stress may be one of the potential determinants for demyelination as lipids and proteins are important structural constituents of myelin and highly susceptible to oxidation. Using the leptin receptor-deficient mouse (db/db) model of DPN and the superoxide dismutase 1 knockout [Sod1(−/−)] mouse model of in vivo oxidative stress, Hamilton reported that oxidation-mediated protein misfolding and aggregation of key myelin proteins may be linked to demyelination and reduced nerve conduction in peripheral neuropathies (Hamilton et al. 2013).
Some human studies have reported a high total oxidative status (TOS) and oxidative stress index (OSI) levels together with low levels of serum total antioxidant status (TAS) in serum from diabetic patients with neuropathy (Uzar et al. 2012). In a double-blind placebo-controlled trial in subjects with T2D and DPN, treatment with vitamin E improved electrophysiological parameters of nerve function, including motor nerve conduction velocity (NCV) and tibial motor nerve distal latency (Tutuncu et al. 1998). Furthermore, a meta-analysis reported that treatment with the antioxidant α-lipoic acid significantly improved both nerve conduction velocity and positive neuropathic symptoms (Han et al. 2012).
Activation of Protein Kinase C in Vascular Tissues
PKC is a family of serine/threonine-related protein kinases of multiple isoforms that play key roles in many cellular functions and affects many signal transduction pathways (Newton 2003). The conventional PKC (cPKC) isoforms (PKCα, -β1, -β2, and -γ) are activated by phosphatidylserine, calcium, and DAG or phorbol esters such as phorbol 12-myristate 13-acetate (PMA), whereas novel PKC (nPKC) isoforms (PKCδ, -ε, -ϕ, and -η) are activated by phosphatidylserine and DAG, but not calcium. The atypical PKC (aPKC) isoforms (PKCζ and -ι/λ) are not activated by calcium or DAG. In this review, we highlighted the mechanism by which hyperglycemia modulates PKC activation. PKC can also be activated by oxidants such as H2O2 in a manner unrelated to lipid second messengers (Konishi et al. 1997) and by mitochondrial superoxide induced by elevated glucose levels (Nishikawa et al. 2000). Many abnormal vascular and cellular processes, including endothelial dysfunction, vascular permeability, angiogenesis, cell growth, and apoptosis, changes in vessel dilation, basement membrane thickening, and extracellular matrix (ECM) expansion; enzymatic activity alterations, such as in mitogen-activated protein kinase (MAPK), cytosolic phospholipase A2, Na+/K+–ATPase; and alterations in several transcription factors are attributed to the activation of several PKC isoforms. PKC activity is increased by diabetes in the renal glomeruli, retina, almost all of the vessels and the myocardium, as well as skeletal muscle wounds and liver (Geraldes and King 2010). Among the isoforms of PKC, the α, β, and δ isoforms have been most consistently implicated in diabetic vascular complications.
Evidence for the Activation of Diacylglycerol (DAG)-PKC Pathway in Diabetes
DAG levels are elevated chronically in the hyperglycemic or diabetic environment due to increased levels of glycolytic intermediate dihydroxyacetone phosphate. This intermediate is reduced to glycerol-3-phosphate, which subsequently increased de novo synthesis of DAG (Xia et al. 1994). In diabetes, total DAG levels were found to be elevated in vascular tissues, such as the retina (Shiba et al. 1993), and renal glomeruli. However, there is no consistent change in DAG levels in the central nervous system and peripheral nerves (Ido et al. 1994). Cell culture studies have shown that DAG levels increase in a time-dependent manner as glucose levels elevate from 5.5 to 22 mmol/L in aortic and capillary endothelial cells (Inoguchi et al. 1992), retinal pericytes (Geraldes et al. 2009), smooth muscle cells (Xia et al. 1994), kidney proximal tubular cells (Wu et al. 2000), and renal mesangial cells (Ayo et al. 1991). Alternatively, increased DAG synthesis can occur from the glycolytic intermediate dihydroxyacetone phosphate, which accumulates when the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is inhibited by poly-ADP-ribosylation of high glucose concentrations (Du et al. 2003). Elevated cytosolic glucose levels promote the accumulation of glyceraldehyde 3-phosphate (GA3P), which can increase DAG and activate PKC (Geraldes and King 2010). Large doses of thiamine and thiamine monophosphate derivative, benfotiamine, may decrease the formation of DAG and mitigate PKC activation in an experimental model of diabetes (Babaei-Jadidi et al. 2003).
PKC Activation in the Development of Diabetic Nephropathy
Experiments in diabetic mice and rats support a role for PKC in the pathogenesis of diabetic nephropathy (DN): PKCα, β, and δ isoforms are activated in renal glomeruli isolated from rats (Babazono et al. 1998) and mice with streptozotocin-induced diabetes, and 50% of the increase in PKC activity in renal glomeruli is prevented in PKCβ knockout mice (PKCβKO) (Ohshiro et al. 2006). PKC activity is also increased in mesangial cells and podocytes cultured in high-glucose media (Ayo et al. 1991). Activation of PKCα can upregulate VEGF expression through NADPH oxidase (Thallas-Bonke et al. 2008). PKCα knockout mice are protected against loss of basement membrane proteoglycans induced by VEGF (Menne et al. 2004). In wild-type mice, diabetes increases activity of NADPH oxidase and induces expression of endothelin-1 (ET-1), VEGF, transforming growth factor β (TGF-β), connective tissue growth factor (CTGF), and collagen types IV and VI. These changes are partly prevented in PKCβ knockout mice (Ohshiro et al. 2006). Mesangial expansion and albuminuria in mice with streptozotocin-induced diabetes are reduced in both PKCβ (Ohshiro et al. 2006) and PKCδ (Mima et al. 2012) KO mice compared to wild type.
General PKC isoform inhibitors can interact with other ATP-binding kinases and therefore display significant toxic side effects in vivo. The PKC-β inhibitor, ruboxistaurin (RBX), is a bisindolylmaleimide class agent and selectively inhibits PKCβ1 and PKCβ2 (Jirousek et al. 1996). Rottlerin (mallotoxin) has higher affinity for PKCδ but also inhibits other isoforms of PKC (Parmer et al. 1997) and other non-PKC kinases, such as MAPK, PKA, and glycogen synthase kinase-3 (Soltoff 2007). The oral administration of RBX was reported to reverse glomerular hyperfiltration and reduce urinary albumin excretion in rodent models of diabetes without a change in DAG content (Tuttle and Anderson 2003).
Improvements were also noted in glomerular TGF-β1 expression, mesangial expansion, glomerulosclerosis, tubule-interstitial fibrosis, and renal function.
Remarkably, PKCε may have effects on DN opposite of PKCα, PKCβ, and PKCδ. One study showed that knockout of PKCε upregulated renal TGFβ1 and its downstream signaling and increased expression of fibronectin and collagen type IV, which caused glomerular and tubulointerstitial fibrosis and development of albuminuria (Meier et al. 2007). These changes were further aggravated by diabetes (Meier et al. 2007). Therefore, PKCε may act as a protective factor by reducing kidney damage.
Supporting the relevance of these findings for human disease, polymorphisms of the PKCβ gene accelerated kidney disease in Japanese subjects with T2D without overt proteinuria (Araki et al. 2006), and polymorphisms in the PKCβ1 gene have been associated with end-stage renal disease in Chinese patients with T2D (Ma et al. 2010).
Clinical Studies Using RBX for Treatment of DN
RBX has been described to stabilize the progression of nephropathy in patients with T2D and early diabetic nephropathy. One year of RBX treatment reduced albuminuria and stabilized the estimated glomerular filtration rate (e-GFR). However, renal outcomes that were evaluated in a secondary analysis of three DN trials (Tuttle et al. 2005) showed no differences in kidney outcomes with RBX treatment.
PKC Activation in the Development of Diabetic Retinopathy
Hyperglycemia activates several PKC isoforms in retinal tissues, including PKCα, -β, -δ, and -ε (Geraldes and King 2010). PKC activation causes retinal vascular dysfunction by altering activities of ET-1, VEGF, and nitric oxide (NO) levels in endothelial cells and platelet-derived growth factor (PDGF), reactive oxygen species (ROS), and nuclear factor κB in pericytes (Idris et al. 2001). RBX administration to diabetic rats normalized retinal blood flow (RBF) (Ishii et al. 1996). Furthermore, local intravitreous injection of RBX reduced retinal PKC activation and restored RBF (Bursell et al. 1997). Alterations in NO production and endothelial nitric oxide synthase (eNOS) expression directly influenced vascular hemodynamics, which may affect RBF. In vessels isolated from diabetic animals, acetylcholine-induced vessel relaxation was found to be delayed (Matsumoto et al. 2008), and the PKC agonist, phorbol 12-myristate 13-acetate (PMA), provoked vascular relaxation impairment in normal arteries (Kamata et al. 1995).
The mechanism for reduced RBF mediated by PKCβ involves ET-1, which is upregulated in the retina of diabetic rats (Yokota et al. 2003). This induction of retinal ET-1 can be blocked by treatment with RBX (Yokota et al. 2003). Diabetic macular edema is mediated in part by VEGF through signaling involving PKCβ(Aiello and Cavallerano 1997) to increase phosphorylation of occludin, a component of tight junctions, leading to increased vascular permeability (Murakami et al. 2012) and others like kallikrein activation (Feener et al. 2013). Hyperglycemia may also increase endothelial cell permeability via the activation of PKCα isoform (Hempel et al. 1997).
The impact of hyperglycemia on vascular cell survival has been extensively studied. However, PKC’s actions on vascular cell proliferation and death have been clarified only recently. 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 was found to induce cellular apoptosis (Geraldes et al. 2009) whereas PKCβ enhanced cellular growth (Suzuma et al. 2002). 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 showed that induction of retinal PKCδ in the retinal capillaries of diabetic mice leads to PDGF resistance, which is not observed in PKCδ knockout mice. We showed that hyperglycemia through PKCδ action promotes two distinctly important pathways by (a) increasing ROS production and NF-κB activity and (b) decreasing the important survival signaling pathway of PDGF by upregulating the expression of SHP-1. These findings suggest a pivotal role for PKCδ in regulating pericyte apoptosis and the formation of cellular capillaries (Geraldes et al. 2009).
In animal studies, inhibition of PKCβ ameliorated the decline of retinal blood flow associated with diabetic retinopathy and prevents diabetes-induced vascular leakage (Ishii et al. 1996). Similarly, the stimulus for neovascularization is suppressed in animals with a reduction of PKCβ levels (Suzuma et al. 2002; Danis et al. 1998). More recently, Nakamura showed that retinal neovascularization was reduced by subcutaneous RBX treatment. In addition, the RBX anti-angiogenic effects were found to be exerted partly via suppressing the phosphorylation of ERK1/2 and Akt (Nakamura et al. 2010).
Clinical studies on diabetic retinopathy with ruboxistaurin. Phase II and phase III clinical trials were conducted in late stages of nonproliferative diabetic retinopathy (NPDR), with the loss of visual acuity as the primary endpoint. Phase II clinical trials, PKC-Diabetic Retinopathy Study (PKC-DRS) and PKC-Diabetic Macular Edema Study (PKC-DMES) (Aiello et al. 2011), failed to reach primary outcomes because of multiple factors (underpowered, three treatment arms of differing dosages, high dropout rate of patients). However, there was a significant reduction in the secondary endpoint of the progression of diabetic macular edema. A much larger clinical trial, PKC-DRS2, was undertaken using a single oral dose with the primary endpoint, again, the loss of visual acuity (Aiello et al. 2006). RBX treatment significantly prevented the reduction of visual acuity in diabetic patients with moderate vision loss and decreases the onset of diabetic macular edema (The PKC-DRS Study Group 2005). These clinical results suggest that PKC activation, especially of the β isoform, could participate in the development of NPDR. However, because treatment with RBX preserves visual acuity by decreasing capillary permeability or targeting the neural retina, but does not significantly delay the progression of vascular DR, suggests that inhibition of the PKCβ isoform alone is not adequate to stop the early metabolic changes that are likely driving the progression of proliferative diabetic retinopathy (PDR). Recently, the effect of RBX on vision loss was assessed through a prospectively defined combined analysis of two phase III trials (MBDL and MBCU) and showed a magnitude effect of RBX on vision loss similar to that seen in the DMES and DRS studies. However, event rates were low and statistical significance was not achieved (Sheetz et al. 2013).
Role of PKC in the Development of Diabetic Peripheral Neuropathy (DPN)
Neuropathy is one of the most distressing complications of diabetes and involves the entire peripheral nervous system due to hyperglycemic states (Dyck et al. 1993). Healthy nerves receive a rich supply of blood from surrounding neural microvasculature known as the vasa nervorum (Cameron et al. 2001). This microvascular network is damaged by hyperglycemia (Sytze Van Dam et al. 2013). Hyperglycemia eventually leads to impaired vasodilation and vascular injury, such as capillary basement membrane thickening and endothelial hyperplasia, resulting in diminished oxygen tension and hypoxia leading to damage to neuronal cells (Cameron et al. 2001). Additionally, hyperglycemia reduces Na+/K+–ATPase activation, an enzyme essential in maintaining normal nerve membrane resting potential, as well as providing neurotrophic support (Stevens et al. 1994).
Changes in PKC activation can contribute to diabetic neuropathy by neurovascular mechanisms such as blood flow and conduction velocity. Levels of DAG have not been shown to be increased in nerve cells, and data from studies of PKC activity in nerve cells have been conflicting, which have reported its activity as increased, decreased, or unchanged (Eichberg 2002). Hyperglycemia in neurons has been shown to decrease phosphatidylinositol, thereby decreasing DAG levels and actually decreasing PKC activity. This diminished activity reduces phosphorylation of Na+/K+–ATPase, leading to a decrease in nerve conduction and regeneration. A previous report demonstrated a reduction of PKC activity by direct measurement of sciatic nerve tissues in STZ diabetic rats (Kim et al. 1991). These results contrast with more recent studies showing that treatment with nonselective PKC isoform inhibitor as well as selective PKCβ inhibitor improved neural function in diabetic animals (Yamagishi et al. 2003). Some studies have reported that treatment with the PKCβ inhibitor resulted in improved nerve conduction as well as improved neuronal blood flow (Nakamura et al. 1999). Indeed, Cameron et al. showed that treatment with RBX at low dose improves motor nerve conduction velocity, normalizes nerve blood flow, and restores Na+/K+–ATPase activity in diabetic rats (Cameron and Cotter 2002).
In humans: Vinik et al. conducted an analysis of a 1-year trial that used a standardized clinical neurological examination to assess the effects of RBX on DPN (Vinik et al. 2005). Changes in vibration detection threshold (VDT) and Neuropathy Total Symptoms Score-6 (NTSS-6), total score did not differ among treatment groups at endpoint. However, RBX treatment appeared to be of benefit for the subgroup of patients with less severe symptomatic DPN by relieving sensory symptoms and improving nerve fiber function, as indicated by reductions in VDT and NTSS-6 total score (Vinik et al. 2005). PKCβ inhibition enhanced skin microvascular blood flow at the distal calf, reduced NTSS-6, and improved measures of Norfolk QOL-DN (Casellini et al. 2007). More recently, Boyd et al. reported that RBX produced significant improvement in large fiber measures QOL and NTSS-6 in diabetic patents (Boyd et al. 2011).
PKCδ Activation in Fibroblasts and Wound Healing
Abnormalities of wound healing, as exemplified by chronic foot ulcers, are major complications of diabetes. Poor wound healing in diabetic patients are due to a combination of multiple causes such as neuropathy, delay in recruitment of immune responses, and poor angiogenesis and fibroblasts functions. Recent studies have shown that abnormal functioning fibroblasts from diabetic patients produced less VEGF in response to wounds, hypoxia, and growth factors such as insulin, resulting in less angiogenesis and delay in wound closure. These abnormal functions of the fibroblasts from diabetic patients were associated with PKCδ activation. When PKCδ isoforms were inhibited with small molecules or overexpression of PKCδ dominant-negative using adenoviral vectors, the functions of fibroblasts derived from diabetic patients were normalized with respect to VEGF production, angiogenesis, and wound closure, suggesting that PKCδ isoform activation could be causing fibroblast dysfunction and poor angiogenesis expression in delay in wound closure in diabetic patients (Khamaisi et al. 2016).
In summary, there is substantial evidence that PKCβ and δ inhibition could be mediating some of the micropathologies in early changes of microvascular complications. However, it is also clear that to achieve clinical significance on the prevention or treatment of these microvascular complications, inhibition of multiple PKC isoforms, including α, β, and δ, may be needed.
Renin-Angiotensin System (RAS) in the Pathogenesis of Diabetic Microvascular Complications
A large number of clinical trials have clearly shown that treatment with angiotensin-converting enzyme (ACE) inhibitors, angiotensin type 1 (AT1) receptor blockers, or their combination may delay the onset of renal disease or progression to renal failure (Burnier and Zanchi 2006). However, analysis of renal biopsies from T1D patients treated with these drugs did not exhibit improvement in glomerular pathology, indicating that inhibition of the RAS may only delay the progression of functional impairment in diabetic nephropathy (Mauer et al. 2009). Angiotensin I and II (AngI, AngII) are produced locally in the kidney, and part of the renoprotective effect of ACE inhibition is a decrease of glomerular capillary pressure beyond lowering systemic blood pressure. AngII actions may also lead to kidney damage through induction of local factors, including extracellular matrix protein synthesis via TGF-β and inflammatory cytokines (Kagami et al. 1994). The mechanisms of angiotensin actions are mediated by AngII receptors, leading to the activation of RAF kinase/MAP kinase and multiple inflammatory cytokines such as TNF-α, IL-6, and others (Zou et al. 1998). Furthermore, RAS blockade may improve or delay the development of DR and macular edema in diabetic patients (Wang et al. 2012). Similarly, RAS blockade reduces DR progression in normotensive, normal albuminuric T1D patients (Harindhanavudhi et al. 2011), suggesting their beneficial effects may be more than just the reduction of blood pressure. In animal models of diabetes, renin inhibitor, Aliskiren, provided similar or greater protection than ACE inhibition alone to decrease NPDR and proliferative neoangiogenesis. In transgenic (mRen-2)27 rats, which overexpress mouse renin in extrarenal tissues, Aliskiren treatment reduced retinal acellular capillaries and leukostasis and normalized retinal vascular endothelial growth factor expression (Wilkinson-Berka et al. 2011).
Role of Endoplasmic Reticulum (ER) Stress in Diabetic Microvascular Complications
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 and cause ER stress. In response, a coordinated program referred to as the unfolded protein response (UPR) is initiated to reduce translation and increase protein folding capacity in an attempt to restore ER homeostasis. Under conditions of chronic, unresolved ER stress, the UPR can also initiate signaling events that promote apoptosis. UPR genes are upregulated in kidney tissue from patients with diabetes, and ER stress may be a mediator of diabetic nephropathy. In the retina of diabetic rats, ER stress is involved in upregulation of inflammatory genes and VEGF and increased vascular permeability (Jing et al. 2012). These and other findings have prompted development of therapeutics which can ameliorate ER stress in patients, including synthetic chaperones to promote protein folding and inhibitors of CHOP and other molecules enabling the UPR (Jing et al. 2012) .
A small number of studies have also implicated ER dysfunction in the pathogenesis of diabetic neuropathy. In cultured Schwann cells (SC), knockdown of ORP150 promoted high glucose-induced SC apoptosis, whereas knockdown of CHOP protected SC from apoptosis.
In rat models of high-fat STZ-diabetes, knockdown of anti-apoptotic protein ORP150 induced DPN in early diabetes and exacerbated DPN after prolonged diabetes, whereas knockdown of the pro-apoptotic protein CHOP ameliorated DPN in rats with prolonged diabetes (Wu et al. 2013).
Role of Kallikrein-Bradykinin System in the Development of Diabetic Microvascular Complications
Plasma kallikrein (PK) is a serine protease with well-characterized effects in innate inflammation and the intrinsic coagulation cascade (Sainz et al. 2007). The majority of PK physiological actions have been attributed to cleavage of its two primary substrates and cofactors, namely, FXII and high-molecular-weight kininogen (HK). Conversion of FXII to FXIIa leads to activation of FXI and the intrinsic coagulation cascade resultant in fibrin production and thrombus stabilization. Cleavage of HK releases the nonapeptide bradykinin, which is the ligand for the G protein-coupled B2 receptor (B2R). Subsequent cleavage of bradykinin by carboxypeptidases generates des-Arg9-bradykinin, which binds and activates the B1 receptor (B1R). Activation of B2R and B1R by bradykinin and des-Arg9-bradykinin, respectively, has been implicated in nearly all the effects of the plasma kallikrein-kinin system (plasma KKS) on inflammation, vascular function, blood pressure regulation, and nociceptive responses (Marceau and Regoli 2004). The plasma KKS has been implicated in a variety of coagulation, vascular, and metabolic abnormalities in diabetes. However, most of the physiological effects of the KKS have been examined using bradykinin receptor-targeted approaches.
Role of the Kallikrein-Kinin System in Diabetic Retinopathy
Activation of the KKS exerts a number of biological effects that also occur in DR, including increased vascular permeability and edema, changes in vascular diameter and hemodynamics, and a variety of effects on inflammation, angiogenesis, and neuronal functions.
Retinal vascular permeability: Intraocular activation of the KKS by injection of C1-INH into the vitreous has been shown to increase RVP, and this response was inhibited by the co-injection of C1-INH, a neutralizing antibody against PK, and a small molecule PK inhibitor, 1-benzyl-1 H-pyrazole-4-carboxylic acid 4-carbamimidoyl-benzylamide (ASP-440) (Clermont et al. 2011). Intravitreal injection of PK increased RVP and retinal thickness by a greater extent in diabetic rats, suggesting that diabetes enhances the retinal responses to intraocular KKS activation. Systemic administration of ASP-440 decreased RVP both in diabetic rats and in rats subjected to AngII-induced hypertension (Clermont et al. 2011; Phipps et al. 2009). Intravitreal injection of BK increased RVP in both diabetic and nondiabetic rats, whereas only diabetic rats demonstrated an RVP response to des-Arg 9-bradykinin (DABK) (Phipps et al. 2009; Abdouh et al. 2008). The administration of B1R antagonist reduced RVP in diabetic rats (Abdouh et al. 2008; Lawson et al. 2005). The data in animal models suggest that the activation of the KKS in the circulation and/or locally in the retina and vitreous can increase RVP via both B1R and B2R and that diabetes appears to increase actions mediated via the B1R.
Retinal blood flow and vasodilation: KKS regulate retinal vessel diameters and hemodynamics. Intravitreal injection of BK acutely stimulated increases in retinal vessel diameters and blood flow (Sogawa et al. 2010), whereas intravenous infusion increases retinal vessel diameter (Kojima et al. 2009). DABK increased vessel diameters in the retinal vessels from diabetic rats but not in nondiabetic controls (Abdouh et al. 2003). The effects of B1R and B2R on retinal vessel dilation have been mainly attributed to NO and prostaglandin (PG) generation from vascular endothelial cells. Pouliot et al. have shown that B1R blockade reduces the retinal expression of potential inflammatory mediators, including iNOS and COX-2. In vitro BK-induced vasodilation responses were inhibited by N G-nitro-l-arginine methyl ester, indomethacin, and the B2R antagonist Hoe140, suggesting that the vasodilation induced by BK is mediated by NO and PG (Abdouh et al. 2003; Jeppesen et al. 2002). BK and DABK stimulate increases in the intracellular concentrations of free calcium by coupling G α q/11 or G α i/o through the B2R or B1R, respectively (Busse and Fleming 1996; Kuhr et al. 2010). Ca+2-induced stimulation of phospholipase A2 (PLA2) liberated the arachidonic acid from the membrane phospholipids, which can lead to the synthesis of prostacyclin (PGI2) (Kolte et al. 2011). B2R stimulates NOS phosphorylation via Ca2+-calmodulin-dependent activation, whereas under inflammatory conditions, B1R stimulation results in a much higher and prolonged NO production via G α i activation of the MAP kinase pathway, leading to the activation of iNOS (Kuhr et al. 2010; Brovkovych et al. 2011). The activation of eNOS and iNOS can independently and additively increase NO production (Kuhr et al. 2010; Yayama and Okamoto 2008). BK also activates the Src kinases and the subsequent vascular endothelial cadherin (VEC) phosphorylation, leading to the quick and reversible opening of endothelial cell junctions and plasma leakage (Orsenigo et al. 2012).
KKS inhibitors—A novel therapeutic application to diabetic retinopathy: In both humans and animal models, the genetic deficiency of PPK causes the prolongation of the activated partial thromboplastin time (APTT) without causing an apparent prothrombotic phenotype or bleeding diathesis (Girolami et al. 2010), and this decrease is reversed by the systemic administration of a PK inhibitor. These results suggest that systemic PK activity contributes to APTT shortening in diabetes because increased activities of PK and BK receptors have been linked to vasogenic edema (Plesnila et al. 2001).
Inhibiting contact system activation: Decreasing the contact system activation may provide opportunities to reduce the effects of the KKS in DR. Feener et al. described that carbonic anhydrase (CA)-1 is increased in the vitreous in PDR patients and intravitreal injection of CA-1 into rats increases RVP, and this response is blocked by co-injection with CA inhibitors, a PK-neutralizing antibody, BK receptor antagonists, and a small-molecule PK inhibitor (Feener et al. 2013; Clermont et al. 2011). These findings revealed that increased CA activity in the vitreous leads to KKS activation and suggest that CA-1 inhibitors may reduce DME, in part, via the reduction of the PK activity.
Plasma kallikrein inhibitors: PK inhibitors include endogenous inhibitors, engineered proteins, and small molecules. C1-INH is a primary physiological inhibitor of PK, FXIa, FXIIa, C1r, and C1s proteases. Intravitreal injection of exogenous C1-INH reduced retinal vascular hyperpermeability induced by diabetes and by intravitreal CA-1 in rats (Clermont et al. 2011). Although C1-INH is detected in the vitreous, it is unknown whether intravitreal concentrations of this endogenous serpin protease inhibitor are sufficient to inhibit PK. 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 PK inhibition could provide increased efficacy and targeting of the inflammatory effects of the plasma KKS while preserving the potential beneficial effects of the tissue kallikrein system.
B1 receptor antagonists: The effects of the KKS are mediated in large part via the generation of BK peptides that activate B1 and B2 receptors, which are expressed in a variety of ocular cell types and tissues. Because both PK- and tissue kallikrein-mediated pathways activate BK receptors, the antagonism of these receptors blocks the effects of both kallikrein systems. Although both B1 and B2 receptors can induce RVP, B1R appears to increase plasma extravasations in DR. The selective peptide B1R antagonist, R-954, reduced vascular permeability in a variety of tissues from STZ-induced diabetic rats, including the retina (Lawson et al. 2005). When diabetic rats were treated with R-954, NO, kallikrein activity, and capillary permeability were remarkably reduced and the Na+/K+–ATPase activity in the retina was increased (Catanzaro et al. 2012). Treatment with FOV-2304, a nonpeptide B1R antagonist, reduced RVP and normalized retinal mRNA expression of inflammatory mediators (Pruneau et al. 2010). Pouliot et al. reported that retinal plasma extravasation and RVP were significantly increased in the diabetic retina, and these abnormalities were reversed to control levels when treated with one eye drop of the nonpeptide B1R antagonist LF22-0542. These reports indicated that both local and systemic administrations of B1R antagonists are effective in ameliorating retinal vascular abnormalities in diabetic rodents, which are similar to the findings observed using PK inhibitors (Pouliot et al. 2012).
It has become clear from the clinical observational studies in patients with long duration of diabetes that factors may play a protective role on the function and survival of vascular cells involved in the microvascular complications of diabetes, which may be equally as important as metabolic toxic factors. In the Medalist Study from the Joslin Diabetes Center, we reported that more than 35% of a large group of insulin-requiring diabetic patients with disease duration of 50 years or longer were free from significant retinal and renal dysfunction (Keenan et al. 2007; Sun et al. 2011). The presence of microvascular complications did not correlate with glycemic control, suggesting the presence of endogenous protective factors in this unusual group of patients with diabetes of extreme duration. The possibility that endogenous protective factors are common in the general population of patients with diabetes is supported by the finding that over half of diabetic patients with microalbuminuria have regression of this marker over 6 years of follow-up (Perkins et al. 2003). Some factors with well-established functions have only recently been perceived as protective. In this section, we are summarizing some well-known and potentially protective factors (Fig. 2).
Insulin: Concept of Selective Insulin Resistance on the Vessel Wall in Diabetes
Selective impairment of insulin action via IRS1/pI3K/Akt pathway on glomeruli has been described in diabetic animals and patients and may contribute to the development of diabetic nephropathy. Many of insulin’s protective effects are mediated via the IRS/PI3K/Akt pathway, including upregulation of eNOS (Artwohl et al. 2007; Wang et al. 2009) and HO-1 (Geraldes and King 2010).
Recently, we have reported that insulin can also increase the expression of ETBR in the endothelial cells via the activation of IRS1/Akt pathway. This finding could be important for improving endothelial function since ETB receptors can enhance the activation of eNOS via the calmodulin pathway identifying a new mechanism by which insulin can enhance the activation of eNOS and NO production (Park et al. 2016). Since endothelial dysfunction could be important for both the pathogenesis of DR and DN, it is likely that insulin’s actions on ETB and NO production will have important functions in the retina and glomeruli. For example, activation of ETB receptors in the retinal capillaries is known to increase retinal blood flow which could be important for the delay of progression of DR. In addition, it has been reported that the loss of eNOS function will accelerate and worsen the diabetic nephropathy in several rodent models of DN (Kanetsuna et al. 2007; Nakagawa et al. 2007; Zhao et al. 2006). In contrast, some mechanisms of injury stimulated by insulin are mediated by the Ras/MAPK pathway, such as induction of ET-1 (Oliver et al. 1991). In diabetes or insulin resistance, elevated concentrations of glucose and FFA can activate PKC, causing selective inhibition of insulin signaling through the PI3K pathway (Naruse et al. 2006). Certain threonine/serine residues on IRS2 and on the p85 regulatory subunit of PI3K have recently been identified as substrates for PKC, and phosphorylation of these sites inhibits insulin-stimulated PI3K signaling (Maeno et al. 2012; Park et al. 2013). Hyperinsulinemia in T2D could conceivably promote vascular disease through induction of ET-1 (Motawi et al. 2014) or other factors induced by MAPK signaling. Insulin may also be important in the retina and promote the maturation and survival of photoreceptors in the murine retina. Deletion of IRS2 leads to loss of neural retinal cell layers in IRS2 knockout mice (Yi et al. 2005). Insulin signaling and its activation of eNOS have been reported in the retina of rodents, which appears to diminish in the presence of diabetes.
There is an enormous number of studies supporting the role of oxidative stress in the development of vascular complications (see previous section on “Oxidative Stress”). However, almost all of the clinical trials using antioxidants have not shown efficacy with clinically significant vascular endpoints. Nevertheless, it is likely that tissue specific endogenous antioxidant enzymes are important to neutralize the increased levels of oxidants produced by the enzymatic and nonenzymatic metabolisms of hyperglycemia. This idea has stimulated clinical trials using bardoxolone methyl (BARD), a synthetic triterpenoid that potentially can reduce oxidative stress and inflammation (Ruiz et al. 2013). One of the main mechanisms of action for this drug is to activate Nrf2. This nuclear factor upregulates a gene program of molecules with antioxidant activity called phase 2 genes, including HO-1 and enzymes in the glutathione biosynthesis pathway. Nrf2 translocation to the nucleus is inhibited by kelch-like ECH-associated protein 1 (Keap1), a repressor which binds Nrf2 in the cytoplasm and promotes Nrf2 proteasomal degradation. BARD interacts with cysteine residues on Keap1, making it unable to repress Nrf2, which then activates transcription of phase 2 genes. Results from a trial of BARD in patients with advanced chronic kidney disease showed an improvement in GFR up to 1 year after start of treatment (de Zeeuw et al. 2013b). However, proteinuria was increased and phase III trials were stopped due to safety issues. In the retina, Nrf2 was also reported to have a protective role against neuronal and capillary degeneration in retinal ischemia-reperfusion injury (I/R). I/R resulted in an increase in retinal levels of superoxide and proinflammatory mediators, as well as leukocyte infiltration of the retina and vitreous, in Nrf2 (+/+) mice. These pathologies were greatly accentuated in Nrf2 (−/−) mice (Wei et al. 2011).
SHP-1 Activation and Its Inhibitory Effects of PDGF and VEGF
During neurogenesis, PDGF is reported to play a critical role for maintenance of many specific neuronal cell types together with vascular cells. PDGF modulates neuronal excitability through adjusting various ion channels and affecting synaptic plasticity and function. Furthermore, PDGF stimulates survival signals, mainly via PI3K/Akt pathway but also other ways, rescuing cells from apoptosis (Funa and Sasahara 2014).
Role of SHP-1 in the Development of Diabetic Microvascular Complications
Diabetes and hyperglycemia, via PKCδ/P38MAPK, activates SHP-1, a tyrosine phosphatase in micro vessels including the retina and renal glomeruli (Fig. 4). This leads to the dephosphorylation and deactivation of specific growth factor receptors critical for survival of pericytes in the retina and podocytes in the kidney (Geraldes et al. 2009). In the retina, SHP-1 activation can desensitize pericytes to PDGF and cause pericyte apoptosis, an initiating step in the development of diabetic retinopathy (Geraldes et al. 2009). In the renal glomerular podocytes, impairment of VEGF survival signaling can be induced by upregulation of SHP-1 expression and lead to increased podocyte apoptosis and endothelial dysfunction (Mima et al. 2012). Upregulation of SHP-1 expression in diabetes is dependent upon activation of PKCδ and p38MAPKα (78, 86). The upregulation of p38MAPK and SHP-1 induced by diabetes is prevented in PKCδ knockout mice, which are protected from apoptosis of retinal pericytes, mesangial expansion, and albuminuria (Geraldes et al. 2009; Mima et al. 2012). Although retinal pericyte apoptosis induced by hyperglycemia has been shown to involve activation of NF-κB, the increase of SHP-1 levels in the diabetic retina and glomerulus is independent of NF-κB activation (Geraldes et al., 2009; Mima et al. 2012). Therefore, inhibition of SHP-1 is a potential novel approach to preserve survival signaling in vascular cells.
Expression of TGF-β is increased in blood vessels, monocytes, heart, and many tissues in diabetes and has been viewed as a causative factor for development of fibrosis in the kidney (Ghosh et al. 2013). Administration of a neutralizing monoclonal TGF-β1 antibody to db/db mice decreases plasma TGF-β1, mesangial matrix expansion, and kidney mRNA levels of collagen IV and fibronectin (Ziyadeh et al. 2000). This therapy prevented a loss of renal function but had no effect on the elevated albuminuria. More recently, the TGF-β receptor kinase activity inhibitor, GW788388, reduced glomerular collagen staining and kidney mRNA levels of PAI-1 and collagen (I and III) but did not alter albuminuria (Petersen et al. 2008). However, TGF-β is well known to have potent anti-inflammatory effects on macrophages and is a negative regulator of T and B cells activation (Ruscetti et al. 1993). Therefore, it may have protective actions due to an anti-inflammatory effect, and its elevation is a reaction to the inflammatory stress of diabetes. Thus, it is likely that the overexpression of TGF-β in many tissues by diabetes could be an endogenous response to the inflammatory actions of hyperglycemia in vascular cells. These paradoxical roles of TGF-β are a challenge for using it as a drug target. Development of targeted nanoparticles or other means of tissue-specific drug delivery may allow inhibition of TGF-β signaling in the kidney and not increase inflammatory actions in other tissues. A recent clinical trial using anti-TGF-β was not shown to delay or improve renal function in people with DN (Voelker et al. 2017).
The expressions of VEGF are changed paradoxically by diabetes, with increases in the retina and renal glomeruli but decreases in the myocardium, peripheral limbs, and nerves correlating with the extent of angiogenesis (Aiello et al. 1994; Chou et al. 2002). Neutralization of VEGF is already approved treatment for proliferative diabetic retinopathy and macular edema and has been suggested as a therapy for diabetic nephropathy (Chen and Ziyadeh 2008). However, the increased levels of VEGF in both tissues are likely an appropriate response to hypoxia, which are the results of loss of capillary function induced by hyperglycemia in the retina. It has been a long-standing concern that neutralization of VEGF could counteract survival signaling in retinal neurons. Interestingly, injection of low doses of VEGF accelerated restoration of the physiological capillary bed and prevented preretinal neovascularization (Dorrell et al. 2010).
The highest expression level of VEGF in the kidney is in renal podocytes, and some of the most insightful work describing a role for VEGF as a survival factor in any organ susceptible to diabetes complications has been done in renal podocytes. 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 (Eremina et al. 2003). This finding strongly supports a role for VEGF in the maintenance of glomerular endothelial cells. Heterozygous knockout of VEGF in podocytes resulted in proteinuria and end-stage renal failure (Eremina et al. 2003) and was preceded by disappearance of endothelial cell fenestrations, increase in necrosis, effacement of podocyte foot processes, and a dramatic loss of mesangial cells (Eremina et al. 2003). When diabetes was induced with STZ in these mice, glomerular cell apoptosis, glomerulosclerosis, and proteinuria were exacerbated compared with nondiabetic controls (Sivaskandarajah et al. 2012). However, other studies reported that increased podocyte VEGF164 expression worsens diabetic nephropathy characterized by glomerulosclerosis, microaneurysms, mesangiolysis, glomerular basement membrane thickening, podocyte effacement, and massive proteinuria associated with hyperfiltration (Veron et al. 2011).
VEGF also has been reported to have neuroprotective effects: Primary dorsal root ganglion (DRG) cultures lacking VEGF-B or VEGFR-1 (FLT1) exhibited increased neuronal stress and were more susceptible to paclitaxel-induced cell death. Concurrently, mice lacking VEGF-B or a functional FLT1 developed more retrograde degeneration of sensory neurons of distal neuropathy. On the other hand, the addition of the VEGF-B isoform, VEGF-B (Chen and Ziyadeh 2008), to DRG cultures antagonized neuronal stress, maintained the mitochondrial membrane potential, and stimulated neuronal survival. Mice overexpressing VEGF-B (Chen and Ziyadeh 2008) or FLT1 selectively in neurons were protected against distal neuropathy, whereas exogenous VEGF-B (Chen and Ziyadeh 2008), either delivered by gene transfer or as a recombinant factor, was protective by directly affecting sensory neurons and not the surrounding vasculature (Dhondt et al. 2011). Identifying the prosurvival mechanisms in stressed neuronal cells revealed that protein kinase A functioned concurrently with VEGFR2 pathway to signal the activation of the extracellular signal-regulated protein kinases (ERK1/2) as protection against caspase-3/7 activation and subsequent cell death (Gomes et al. 2007).
Activated Protein C (APC)
Protein C is a well-known anticoagulant factor but more recently was recognized as a survival factor for renal glomerular cells (Isermann et al. 2007). Thrombomodulin, a procoagulant factor which activates protein C, was found to be highly expressed in glomeruli of mice but was downregulated in diabetes (Isermann et al. 2007). 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 albuminuria and glomerular pathology (Isermann et al. 2007). The anticoagulant effects of APC did not account for its protective actions. Rather, APC was shown to counteract apoptosis of endothelial cells and podocytes through activation of two of its receptors (Isermann et al. 2007). Therefore, endothelial-derived APC appears to be 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, but Gupta et al. reported that APC-mediated protease activated receptor-1 agonism suppressed lipopolysaccharides (LPS)-induced increases in the vasoactive peptide adrenomedullin and infiltration of iNOS-positive leukocytes into renal tissue. The anticoagulant function of APC was responsible for suppressing LPS-induced stimulation of the proinflammatory mediators ACE-1, IL-6, and IL-18, perhaps accounting for its ability to modulate renal hemodynamics (Gupta et al. 2009).
Vascular Progenitor Cells (VPC)
Endothelial progenitor cells (EPC) and myeloid progenitors may contribute to postnatal angiogenesis (Bautch 2011). Recent report from the Joslin Medalist Study showed that circulating VPC levels were correlated to CVD in the Medalists and higher than aged-matched T2DM patients and comparable to nondiabetic controls, again suggesting that circulating VPC either are markers of vascular complications or may even contribute to the levels of functions of vascular cells against the adverse effects of diabetes (Hernandez et al. 2014). The mechanism for EPC to improve angiogenesis is currently not well characterized. EPC may contribute by incorporating into newly formed blood vessels. However, it is likely the major action of EPC is to release proangiogenic factors and temporarily associate with neovascular structures. In diabetic patients, both the number and function of EPC are impaired (Loomans et al. 2004) leading to a subsequent reduction in the ability of EPC to repair the vascular endothelium (Jarajapu and Grant 2010), leading to poor collateral circulation in response to ischemia. eNOS is necessary for mobilization of EPC from the bone marrow, as this phenomenon is impaired in eNOS knockout mice (Aicher et al. 2003). Uncoupling of eNOS, with synthesis of superoxide rather than NO by eNOS, could be one mechanism for impaired EPC function. In fact, EPC function is improved after inhibition of eNOS ex vivo in EPC isolated from patients with diabetes (Thum et al. 2007). Interestingly, neuropathy in the bone marrow may cause reduced mobilization of EPC. Thus, diabetic rats had a reduction in nerve terminals in bone marrow and this denervation resulted in an increased number of EPC in the bone marrow, but decreased release of EPC to the circulation. These abnormalities were associated with an increase in retinal acellular capillaries (Busik et al. 2009). Transplantation of nondiabetic EPC has been shown to improve angiogenesis in peripheral ischemia (Yan et al. 2009). These studies suggest that it may be possible to promote repair of ischemic tissue in diabetes by improving mobilization, differentiation, and function of EPC or other progenitors. Recently, autologous EPC transplantation has been suggested as a potential therapy for DN. An alternative approach is the stimulation of endogenous bone-marrow-derived EPC (BM-EPC) recruitment into ischemic lesions by the administration of stem cell mobilization agents or chemokines (Kim et al. 2013). The administration of AMD3100, an EPC mobilization agent, increased local expression levels of vasculogenesis-associated factors and newly formed endothelial cells in the sciatic nerve, resulting in the restoration of the sciatic vasa nervorum (Kim et al. 2013).
Circulating EPC were markedly reduced in chronic kidney disease (CKD) patients (Chen et al. 2013), and EPC delivery has been shown to improve renal function, attenuate the proinflammatory response associated with renal injury, and improve damage to tubules and renal vascular segments during kidney injury while providing enhanced neoangiogenesis (Kale et al. 2003). An intact and healthy EPC niche, residing in the bone marrow but also found locally in renal vascular beds such as in the area of the adventitia layer of vessels, may be able to support normal vascular function including maintenance and possible replacement of the endothelium (Minamino et al. 2002).
Emerging studies suggest the potential of these cells in revascularization of ischemic and injured retinas in animal models of retinal disease. Since ischemic retinopathies are leading causes of blindness, they are a potential disease target for EPC-based therapy (Li Calzi et al. 2010). In NPDR, EPC may have reduced function as they cannot recruit outgrowth of EPC into the retina to repair the acellular capillaries, while in PDR the EPC take on a proinflammatory phenotype and recruit too many EPC 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 the use of a diabetic patient’s own EPC for repair of their injured retinal and systemic vasculature. Specifically in the retina, correction of this dysfunction may prevent early and intermediate stages of vasodegeneration to enhance vessel repair, reverse ischemia, and prevent progression to the late stages of DR. However, these findings on the changes of EPC and their correlation to various complications in diabetes have been inconsistent. Clearly, more studies are needed to clarify their roles and changes in diabetes before they can be used therapeutically.
Elimination of hyperglycemia
Inhibition of major mechanisms, which are activated by hyperglycemia to induce vascular dysfunction
Neutralization of accelerants such as inflammation and oxidative stress
Activation of tissue-specific protective factors
The authors would like to acknowledge funding from the Juvenile Diabetes Research Foundation grant 17-2011-474, National Institutes of Health grants 5 R01 DK053105-12, 5 R24 DK090961-02, 1 DP3 DK094333-01, NIH Diabetes Research Center 2 P30 DK036836-26A1, ADA mentor-based fellowship.
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