Rec date: January 6, 2016, Acc date: February 24, 2016, Pub date: February 29, 2016
Copyright: © 2016 Arya A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Endothelial dysfunction is regarded as an important factor in the pathogenesis of diabetes. Endothelial dysfunction has been posited to play an important role in the pathogenesis of diabetic nephropathy (DN).The vascular complications of diabetes impose a huge burden on the management of this disease. The higher incidence of cardiovascular complications and the unfavorable prognosis among diabetic individuals who develop such complications have been correlated to the hyperglycemia-induced oxidative stress and associated endothelial dysfunction. Both hyperglycemia, as well as the metabolic consequences of glucose dysregulation, are thought to lead to endothelial cell dysfunction. In this regard, endothelial nitric oxide synthase (eNOS) plays a central role in dysfunction. This review will focus on the mechanisms and therapeutics that specifically target endothelial dysfunction in the context of a diabetic setting.
Endothelial nitric oxide synthase; Vascular endothelial dysfunction; Nitric oxide
An epidemic of diabetic nephropathy
Diabetic nephropathy (DN) is a leading cause of mortality and morbidity in patients with diabetes. It is the single major cause of renal failure in many countries. This complication reflects a complex pathophysiology, whereby various genetic and environmental factors determine susceptibility and progression to end-stage renal disease. End-stage renal disease (ESRD) due to diabetes has been estimated to be 30-47% of all incident cases worldwide . Disparities in the incidence of ESRD from diabetes among ethnic groups have existed for many years, but the magnitude has been increasing. The World Health Organization (WHO) has estimated that there are currently 346 million people affected by diabetes worldwide and anticipates that diabetes-related deaths would double by 2030 . These figures highlight the importance of continued research and the need for novel methods to both prevent and treat this pandemic .
There is compelling evidence that endothelial dysfunction serves as a key event in the development and progression of diabetic vascular complications, including nephropathy [4-6]. Endothelial cells maintain vascular function and homeostasis by generating paracrine factors that regulate vascular tone, preventing coagulation and platelet aggregation, inhibiting adhesion of leukocytes, and limiting proliferation of vascular smooth muscle cells as well as by constituting a selective barrier to the diffusion of macromolecules into the interstitial space. Further, it was shown that Nitric Oxide (NO) produced by endothelial cells through the endothelial Nitric Oxide Synthase (eNOS) plays a major role for many of these endothelial functions  and that decreased NO production and bioavailability largely contribute to endothelial dysfunction in diabetes .
Endothelium is a type of epithelium that lines the interior surface of blood vessels and lymphatic vessels. Vascular endothelium is an innermost layer of blood vessels responsible for regulation of vascular tone and free flow of blood in vessels .
The multiple functions of vascular endothelium are summarized in Figure 1 and include regulation of vessel integrity, vascular growth and remodeling, tissue growth and metabolism, immune responses, cell adhesion, angiogenesis, hemostasis and vascular permeability. The endothelium plays a pivotal role in the regulation of vascular tone, controlling tissue blood flow and inflammatory responses and maintaining blood fluidity [8,9].
Figure 1: Functions of vascular endothelium.
Vascular endothelium is the inner layer of the blood vessels, which serves as an important role to regulate the vascular functions and blood flow in vessels .
Vascular endothelium maintains the vascular tone and exerts anticoagulant, antithrombotic, antiplatelet and fibrinolytic properties . Endothelium is a multifunctional organ and it control the release of endothelium derived relaxing factors (EDRF), endothelium derived contracting factors (EDCF), endothelium derived hyperpolarizing factors (EDHF) [11,12] inflammatory mediators such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), nuclear factor kappa-B (NF-kB) and various growth factors like vascular endothelial growth factors (VEGF) and transforming growth factor-β (TGF-β) [4,11].
Nitric oxide is a small gaseous and lipophilic molecule that participates of several biological processes. It is synthesized from Endothelial NOS (eNOS), also known as nitric oxide synthase 3 (NOS3) or constitutive NOS (cNOS), is an enzyme that in humans is encoded by the NOS3 gene located in the 7q35-7q36 region of chromosome 7 . Nitric oxide (NO) is an important protective molecule in the vasculature, and endothelial NO synthase (eNOS) is responsible for most of the vascular NO produced.
eNOS has a protective function in the cardiovascular and renal system, which is attributed to NO production.
Regulation of the vascular tone: Once the nitric oxide diffuses from the endothelial cells, it activates the enzyme soluble guanylate cyclase (sGC), which catalyses the conversion of guanosine triphosphate into cyclic guanosine monophosphate (cGMP) which in turn activates protein kinase G (PKG) and promotes the phosphorylation of cellular targets and promotes the vascular relaxation.
Antiproliferative property: NO exerts antiproliferative effects by cGMP-dependent inhibiting Ca2+ influx or by directly inhibiting the activity of arginase and ornithine decarboxylase, decreasing the generation of polyamides required for DNA synthesis [14,15].
Antithrombotic effects: Nitric oxide exerts antithromic effects by diffusing across the platelet membrane and resulting in inhibition of platelet aggregation.
Leukocyte adhesion: Nitric oxide (NO) is a biologically active compound produced by vascular endothelium and is rapidly inactivated by superoxide. There is circumstantial evidence in the literature that NO may interfere with the ability of polymorphonuclear leukocytes to adhere to microvascular endothelium . It is well established that NO prevents the adhesion of platelets to endothelial monolayers. Additionally, NO inhibits neutrophil aggregation in vitro, an effect that is potentiated by superoxide dismutase. Moreover, NO affects leukocyte adhesion to the vascular endothelium by inhibiting the nuclear factor kappa B (NF-κB), which induces vascular endothelial expression of chemokines and adhesion molecules [17,18].
Attributable to up-regulation of heme-oxygenase-I and ferritin expression, which reduce superoxide anion concentrations in blood vessels .
Vascular endothelial dysfunction
In patients with diabetes, endothelial dysfunction appears to be a consistent finding; indeed, there is general agreement that hyperglycemia and diabetes lead to an impairment of NO production and activity. Endothelial dysfunction is a systemic pathological condition which can be broadly defined as an imbalance between vasodilating and vasoconstricting substances produced by the endothelium or overall functions of the endothelium . Normal functions of endothelial cells include production of nitric oxide (NO), regulation of platelet adhesion, coagulation, immune function, control of volume, and electrolyte content of the intravascular and extravascular spaces [21,22]. Endothelial dysfunction is primarily due to reduction in NO bioavailability, and a marker for vascular health. Endothelial dysfunction can result from and/or contribute to several disease processes, as occurs in diabetes mellitus, hypercholesterolemia and hypertension, and also due to environmental factors, such as smoking tobacco products and exposure to air pollution [23-25].
Additionally, endothelial dysfunction is characterized by one or more of the following features: reduced endotheliummediated vasorelaxation, hemodynamic deregulation, impaired fibrinolytic ability, enhanced turnover, overproduction of growth factors, increased expression of adhesion molecules and inflammatory genes, excessive generation of ROS, increased oxidative stress, and enhanced permeability of the cell layer [26,27].
Vascular endothelial dysfunction cause reduced activation of endothelial nitric oxide synthase, decreased generation and bioavailability of nitric oxide (NO). VED leads to increase in production of reactive oxygen species (ROS) and proinflammatory mediators are implicated in pathogenesis of various disorders like hypertension, atherosclerosis and diabetic nephropathy [28-30].
Diabetic nephropathy and vascular endothelial dysfunction
Hyperglycemia causes vascular endothelial dysfunction via micro and macrovascular complications . Diabetic nephropathy is defined as partial loss of nephrons followed by nephrotic syndrome and glomerulosclerosis. Nephropathy is characterized by persistent elevated albuminuria, declined glomerular filtration rate, and elevated arterial blood pressure and oedema [32,33]. Although several other factors may mediate in the development and progression of diabetic nephropathy.
Insulin resistance usually precedes the development of Type II diabetes and is often accompanied by a cluster of cardiovascular risk factors, notably obesity, hypertension, high triacylglycerol (triglyceride) levels, low HDL (high-density lipoprotein)-cholesterol levels, abnormal LDL composition, hyperinsulinaemia, insulin resistance and inflammation, all of these impair endothelial function. Hyperglycemia is the major causal factor in the development of endothelial dysfunction in diabetes mellitus .
Diabetes mellitus (DM) is a complex metabolic syndrome characterized by absolute insulin deficiency or development of insulin resistance that leads to altered glucose, fat and protein metabolism . Retinopathy, neuropathy, cardiomyopathy, and nephropathy are long term complications due to diabetes mellitus and endothelial nitric oxide synthase imbalance. Both genetic and environmental factors are involved in the development of endothelial dysfunction during DN .
High concentration of glucose has been noted to scavenge nitric oxide and induce vascular endothelial dysfunction finally leads to diabetic nephropathy  which is followed by formation of advance glycation end-products (AGEs), reactive oxygen species (ROS) and increase in the oxidative stress [37,38].
Nevertheless, there is no doubt that chronic hyperglycemia and the subsequent metabolic derangements play a major role in diabetic EC injury and can lead to the over production of advanced glycation end products (AGE), activated protein kinase C (PKC) signaling cascades and accumulated reactive oxygen species (ROS) . Hemodynamic alterations along with renin-angiotensin system (RAS) regulation seem to be another pivotal contributor to dysfunction of renal endothelium in both glomerular afferent and efferent arteries .
Diabetic nephropathy which is characterized by glomerular hypertrophy, accumulation of extracellular matrix protein, increased basement membrane thickness, mesangial expansion, podocyte loss, and vascular endothelial dysfunction progressively leading to glomerulosclerosis, tubulointerstitial fibrosis, and proteinuria .
It has been noted in various studies that hyperglycemia induce endothelium cell death by activating bax-caspase proteases pathway .
Further, diabetic nephropathy is associated with activation of various intracellular signaling mechanisms and transcription factors, i.e. protein kinase C (PKC), mitogen activated protein kinase (MAPKs), nuclear factor kappa B (NFκB) . Furthermore, overexpression of various growth factors, i.e. transforming growth factor (TGF-β), vascular endothelial growth factor (VEGF), and cytokines, i.e. tumor necrosis factor a (TNF-α), interleukin 1 (IL1), and insulin-like growth factor-1 (IGF-1), stimulates proliferation of mesangial cells contributing to glomerulosclerosis and tubulointerstitial fibrosis .
Various endogenous modulators such as Ang-II, ET-I, caveolin, resistin and Rho-kinase are upregulated in VED [45,46] whereas adiponectin and apelin are downregulated in VED . Ang-II stimulates the release of VEGF and develops proteinuria in diabetic nephropathy .
The experimental evidences suggests that hyperlipidemia may mediate renal injury by increasing the expression of sterol regulatory element-binding protein (SREBP), which is responsible for increasing the synthesis of triglycerides and cholesterol in the kidney, that are associated with increased expression of TGF-β, VEGF, extracellular matrix proteins, type IV collagen and fibronectin resulting in glomerular hypertrophy. Further, SREBP stimulates podocyte injury, glomerulosclerosis and tubulointerstitial fibrosis to produce nephropathy [48,49].
In addition, diabetes upregulates the generation of AGEs, which contributes to endothelial cell death and vascular endothelial dysfunction . Moreover, the expression and activity of eNOS is down regulated through glucose mediated production of reactive oxygen species (ROS) in diabetes . The ROS thus generated during diabetes play a major role in the pathogenesis of diabetic nephropathy 
Oxidative stress and endothelial cell dysfunction
Oxidative stress describes the condition wherein an excessive production of ROS overwhelms endogenous antioxidant defense mechanisms. Oxidative stress is caused by three factors: (1) an increase in oxidant generation, (2) a decrease in antioxidant protection, (3) a failure to repair oxidative damage [53-55]. There are numerous risk factors that can cause endothelial cell damage under diabetes such as hyperglycemia, insulin resistance, dyslipidemia, increased oxidative stress, inflammation, and hypertension [56,57].
The vascular endothelium, which regulates the passage of macromolecules and circulating cells from blood to tissues, is a major target of oxidative stress, playing a critical role in the pathophysiology of several vascular diseases and disorders . Specifically, oxidative stress increases vascular endothelial permeability and promotes leukocyte adhesion, which is coupled with alterations in endothelial signal transduction and redox-regulated transcription factors .
Endothelial dysfunction is associated with decreased NO availability, either through loss of NO production or through loss of NO biological activity . NO production is diminished in cells which are subject to oxidative stress. Hyperglycemiainduced eNOS impairment leads to increased oxidative stress and scavenging of NO, which represents initiation event(s) for development of endothelial dysfunction .
A decline in NO bioavailability may be caused by decreased expression of the endothelial cell NO synthase (eNOS), a lack of substrate or cofactors for eNOS, alterations of cellular signaling such that eNOS is not appropriately activated and finally accelerate NO degradation by ROS [62-65].
The imbalance between NO and reactive oxygen species (ROS) generation is a central pathophysiologic denominator in diabetic endothelial dysfunction. High glucose increases ROS production in ECs  and reduces endogenous antioxidant systems  resulting in oxidative stress.
It is known that under diabetic conditions there are increased oxidative stress levels . Increased ROS prompts the EPCs to produce pathologic cytokines such as monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), NF-κB, interleukin-8 (IL-8), elevated levels of iNOS, and decreased eNOS. Diabetes which is a metabolic disorder characterized by impaired endogenous insulin secretion and activity, reduced NO production and increased production of free radicals, or impaired antioxidant defenses. The predominant factor in diabetes-mediated complications is endothelial dysfunction. The mechanisms that lead to endothelial dysfunction in diabetes are complex [69,70].
Both endothelial cells and vascular smooth muscle cells are capable of producing reactive oxygen species from a variety of enzymatic sources. In disease states such as diabetes, vascular production of reactive oxygen metabolites can increase substantially . Increased production of the superoxide anion can lead to decreased tissue bioavailability of nitric oxide (NO) via a facile radical/radical reaction that occurs more rapidly than the reaction of with superoxide dismutase (SOD) . This phenomenon alters endothelial regulation in a variety of disease conditions. Importantly, this endothelial dysfunction is due to vascular production of superoxide. There are several enzymes that involve generating ROS such as NADPH oxidase, aldehyde oxidase, xanthine oxidase, and glucose oxidase . Decreased endothelium-dependent vasodilation in diabetic subjects is associated with the impaired action of NO secondary to its inactivation resulting from increased oxidative stress, rather than decreased NO production from vascular endothelium and that abnormal NO metabolism is related to advanced diabetic microvascular complications .
Potential Therapeutic Strategies
The risk of cardiovascular death in diabetic patients with microalbuminuria is some 7-40 times that of an age matched general population. Lifestyle changes are required to prevent the onset of diabetes. They include cessation of smoking and require regular exercise, weight loss and controlled sodium intake.
Glycemic control and Renin Angiotensin Aldosterone system (RAAS) inhibition have long been mainstays of therapy in patients with diabetic nephropathy .
The natriuretic peptides (NPs), which are secreted from cardiomyocytes in response to cardiac wall stress, play an important role in the regulation of blood pressure, intravascular volume, and cardiac remodeling. The NPs consists of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C type natriuretic peptide, where ANP and BNP are secreted by the heart in response to increased volume and pressure load .
Natriuretic peptides are mainly involved in water and sodium balance and cardiovascular homeostasis, several neurohormones such as endothelin-1 (ET-1), Catecholamines stimulate the secretion of natriuretic peptides [75,76]. The effects of ANP are mediated by the transmembrane guanylylcyclase receptor type A, which promotes intracellular cGMP formation .
Administration of ANP drugs (5 μg/kg/i.p.) and (10 μg/kg/ i.p.) inhibit the renin-angiotensin II-aldosterone system .
Diabetic nephropathy is a potentially fatal endpoint of uncontrolled diabetes. Reduced level of nitric oxide is most negative factor involved in diabetic nephropathy. In hyperglycemia due to up regulation of endothelial dysfunction mediators such as Ang-II, Endothelin-I, Caveolin are actively involved in diabetic nephropathy .
Regarding the endothelial dysfunction, Single therapy may not adequately improve endothelial function, so it is necessary to target multiple factors for therapeutic intervention of endothelial dysfunction. Targeting more than one risk factor of endothelial damage only can improve endothelial functions.
Treatments that improve endothelial function systemically, like ACE inhibitors, statins, metformin, antioxidants, folate, PKC-inhibitors, and supplements like L-arginine, BH4, folic acid, and polyphenols also appear to provide protection from diabetes mediated vascular events .10747