Diabetic retinopathy and atherosclerosis: is there a link?

Diabetic retinopathy (DR) is the leading cause of blindness amongst the working-age population, and diabetes accelerated cardiovascular disease (CVD) the commonest cause of death in diabetic patients. Although, there is evidence suggesting a close association between DR and CVD, particularly in patients with Type 2 diabetes, the pathophysiology underlying the link is unclear. Here we review common risk factors and pathogenic mechanisms linking DR and CVD, and aim to highlight the need for a more holistic view of the management of diabetes and its complications. The understanding of the link between the two complications could eventually lead to refined management strategies and improved patient outcomes in the expanding diabetes epidemic.

Pathophysiology of diabetic retinopathy.

Diabetes is now regarded as an epidemic, with the population of patients expected to rise to 380 million by 2025. Tragically, this will lead to approximately 4 million people around the world losing their sight from diabetic retinopathy, the leading cause of blindness in patients aged 20 to 74 years. The risk of development and progression of diabetic retinopathy is closely associated with the type and duration of diabetes, blood glucose, blood pressure, and possibly lipids. Although landmark cross-sectional studies have confirmed the strong relationship between chronic hyperglycaemia and the development and progression of diabetic retinopathy, the underlying mechanism of how hyperglycaemia causes retinal microvascular damage remains unclear. Continued research worldwide has focussed on understanding the pathogenic mechanisms with the ultimate goal to prevent DR. The aim of this paper is to introduce the multiple interconnecting biochemical pathways that have been proposed and tested as key contributors in the development of DR, namely, increased polyol pathway, activation of protein kinase C (PKC), increased expression of growth factors such as vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1), haemodynamic changes, accelerated formation of advanced glycation endproducts (AGEs), oxidative stress, activation of the renin-angiotensin-aldosterone system (RAAS), and subclinical inflammation and capillary occlusion. New pharmacological therapies based on some of these underlying pathogenic mechanisms are also discussed.

Retinopathy in diabetes.

With the incidence, and prevalence of diabetes mellitus increasing worldwide, diabetic retinopathy is expected to reach epidemic proportions. The aim of this chapter is to introduce diabetic retinopathy, a leading cause of blindness in people of the working age. The clinical course of retinopathy, anatomical changes, its pathogenesis and current treatment are described, followed by an overview of the emerging drug therapies for the potential treatment of this sight-threatening complication of diabetes.

Cellular crosstalk between TNF-α, NADPH oxidase, PKCß2, and C2GNT in human leukocytes.

Increasing evidence suggests that chronic, sub-clinical inflammation plays an important role in the pathogenesis of diabetic retinopathy. We have established the potential role of the inflammatory enzyme, core 2 ß-1, 6-N-acetylglucosaminyltransferase (C2GNT) in diabetic retinopathy. The present study was designed to explore the NADPH oxidase signaling pathway in the tumor necrosis factor-alpha (TNF-α)-induced activity of C2GNT in leukocytes. Human leukocytes (U937 cells) and an Epstein-Barr-transformed lymphoblastoid cell line deficient in p47phox (F10007 cells) were used for the study. Cells were exposed to TNF-α for 24h in the presence and absence of 1) NADPH oxidase inhibitors (apocynin and scrambled and unscrambled gp91ds-tat), 2) LY379196 (specific protein kinase C ß1/2 (PKCß1/2) inhibitor), and 3) the antioxidant tiron. Subsequent C2GNT and NADPH activity was measured and the adhesion of U937 and F10007 cells to endothelial cells was assessed. TNF-α-induced C2GNT activity (1813±326 pmol/h/mg protein) (mean±SEM) in human leukocytes was significantly reversed with apocynin (153±82 pmol/h/mg protein), unscrambled gp91ds-tat (244±122 pmol/h/mg protein) and tiron (756±87 pmol/h/mg protein). We further supported this C2GNT-NADPH oxidase link using p47phox-deficient leukocytes. The deficiency in p47phox prevented TNF-α-induced NADPH oxidase and C2GNT activity and adherence to endothelial cells. The response to TNF-α was restored by transfection with an expression plasmid containing a p47phox cDNA inserted in the sense direction. Our results demonstrate for the first time a novel signaling crosstalk between TNF-α, NADPH oxidase, PKCß1/2 and C2GNT in leukocytes.

Introduction to diabetes mellitus.

The chronic metabolic disorder diabetes mellitus is a fast-growing global problem with huge social, health, and economic consequences. It is estimated that in 2010 there were globally 285 million people (approximately 6.4% of the adult population) suffering from this disease. This number is estimated to increase to 430 million in the absence of better control or cure. An ageing population and obesity are two main reasons for the increase. Furthermore it has been shown that almost 50% of the putative diabetics are not diagnosed until 10 years after onset of the disease, hence the real prevalence of global diabetes must be astronomically high. This chapter introduces the types of diabetes and diabetic complications such as impairment of immune system, periodontal disease, retinopathy, nephropathy, somatic and autonomic neuropathy, cardiovascular diseases and diabetic foot. Also included are the current management and treatments, and emerging therapies.

NADPH Oxidase versus Mitochondria-Derived ROS in Glucose-Induced Apoptosis of Pericytes in Early Diabetic Retinopathy.

Objectives. Using apocynin (inhibitor of NADPH oxidase), and Mitoquinol 10 nitrate (MitoQ; mitochondrial-targeted antioxidant), we addressed the importance of mitochondria versus NADPH oxidase-derived ROS in glucose-induced apoptosis of pericytes. Methods. NADPH oxidase was localised using Western blot analysis and cytochrome C reduction assay. Apoptosis was detected by measuring caspase-3 activity. Intracellular glucose concentration, ROS formation and Nepsilon-(carboxymethyl) lysine (CML) content were measured using Amplex Red assay kit, dihydroethidium (DHE), and competitive immunoabsorbant enzyme-linked assay (ELISA), respectively. Results. NADPH oxidase was localised in the cytoplasm of pericytes suggesting ROS production within intracellular compartments. High glucose (25 mM) significantly increased apoptosis, intracellular glucose concentration, and CML content. Apoptosis was associated with increased gp91phox expression, activity of NADPH oxidase, and intracellular ROS production. Apocynin and not MitoQ significantly blunted the generation of ROS, formation of intracellular CML and apoptosis. Conclusions. NADPH oxidase and not mitochondria-derived ROS is responsible for the accelerated apoptosis of pericytes in diabetic retinopathy.

Is inflammation a common retinal-renal-nerve pathogenic link in diabetes?

The global diabetes burden is predicted to rise to 380 million by 2025 and would present itself as a major health challenge. However, both Type 1 and Type 2 diabetes increase the risk of developing micro-vascular complications and macro-vascular complications which in turn will have a devastating impact on quality of life of the patients and challenge health services Worldwide. The micro-vascular complications that affect small blood vessels are the leading cause of blindness (diabetic retinopathy) in the people of the working-age, end-stage renal disease (diabetic nephropathy) the most common cause of kidney failure today, and foot amputation (diabetic neuropathy) in patients with Type 1 and Type 2 diabetes. It is accepted that hyperglycemia is a major causative factor for the development of these complications, there is also growing evidence for the role of inflammation. Here we discuss low-grade inflammation as a common retinal-renal-nerve pathogenic link in patients with Type 1 and Type 2 diabetes. This review summarizes evidence showing a link between circulating and locally produced inflammatory biomarkers, such as cell adhesion molecules (vascular adhesion cell molecule-1, VCAM-1; intracellular adhesion molecule-1, ICAM-1), pro-inflammatory cytokines (interleukin-6, IL-6; tumour necrosis factor-alpha, TNF-α; C-reactive protein, CRP) with the development and progression of diabetic micro-vascular complications.

Leukocytes in diabetic retinopathy.

Diabetic retinopathy is one of the most common diabetic complications, and is a major cause of new blindness in the working-age population of developed countries. Progression of vascular abnormalities, including the selective loss of pericytes, formation of acellular capillaries, thickening of the basement membrane, and increased vascular permeability characterizes early nonproliferative diabetic retinopathy (NPDR). Capillary occlusion, as shown on fluorescein angiograms, is also one of the earliest clinically recognizable lesion of NPDR. In response to capillary non-perfusion, there is dilation of neighbouring capillaries, leading to early blood-retinal barrier breakdown, capillary non-perfusion, and endothelial cell injury and death. The resulting ischemia leads to increased production of growth factors, and the development of proliferative diabetic retinopathy (PDR), which is characterized by growth of new vessels and potential severe and irreversible visual loss. The exact pathogenic mechanism by which capillary non-perfusion occurs is still unclear but growing evidence now suggests that increased leukocyte-endothelial cell adhesion and entrapment (retinal leukostasis) in retinal capillaries is an early event associated with areas of vascular non-perfusion and the development of diabetic retinopathy. The leukocytes in diabetic patients are less deformable more activated, and demonstrate increased adhesion to the vascular endothelium. This review summarizes the current literature on the role of leukocytes in the pathogenesis of capillary occlusion, and discusses the potential of leukostasis as a new promising target in the treatment of diabetic retinopathy.

21st Century treatment of diabetic retinopathy.

Despite marked improvements in the treatment of diabetes and its retinal complications, diabetic retinopathy remains a leading cause of blindness and vision impairment in working-age adults. Control of blood glucose and blood pressure will remain important means to prevent the onset and progression of diabetic retinopathy. Current and improved surgical treatments, such as laser therapy and vitrectomy, have also proved highly effective in preventing major visual loss in advanced stages of retinopathy. In this review, emerging drug-based therapies (corticosteroids, somatostatin analogues, anti-VEGF agents, a specific PKCß1/2 inhibitor [ruboxistaurin]), more effective inhibitors of aldose reductase, inhibitors of the renin-angiotensin system and anti-inflammatory agents that could help to preserve sight in the growing population of diabetic patients into the 21st Century are discussed.

Glycated proteins stimulate reactive oxygen species production in cardiac myocytes: involvement of Nox2 (gp91phox)-containing NADPH oxidase.

BACKGROUND: Nonenzymatic glycation that results in the production of early-glycation Amadori-modified proteins and advanced-glycation end products may be important in the pathogenesis of diabetic complications. However, the effects of early-glycated proteins, such as glycated serum albumin (Gly-BSA), are poorly defined. In this study, we investigated the effects of Gly-BSA on reactive oxygen species (ROS) production by cardiomyocytes. METHODS AND RESULTS: Cultured neonatal rat cardiomyocytes were incubated with Gly-BSA or vehicle (bovine serum albumin [BSA]) for up to 48 hours. Gly-BSA dose-dependently increased in situ ROS production (whole-cell dichlorodihydrofluorescein fluorescence), with an optimum effect at 400 microg/mL after 24-hour incubation (152+/-10% versus BSA 100%; P<0.01). Treatment with the NADPH oxidase inhibitor apocynin, a Nox2 (gp91phox) antisense oligonucleotide (Nox2 AS), or the peptide gp91ds-tat significantly reduced Gly-BSA-induced ROS production at 24 hours (68.5+/-2.2%, 61.4+/-8.3%, and 53.2+/-5.4% reduction, respectively). NADPH-dependent activity in cell homogenates was also significantly increased by Gly-BSA at 24 hours (161+/-8% versus BSA) and was inhibited by diphenyleneiodonium, apocynin, NOX2AS, and the protein kinase C inhibitor bisindolylmaleimide I but not by a nitric oxide synthase inhibitor or mitochondrial inhibitors. Furthermore, bisindolylmaleimide I prevented Gly-BSA-stimulated Rac1 translocation, an essential step for NADPH oxidase activation. Gly-BSA-induced increases in ROS were associated with apocynin-inhibitable nuclear translocation of nuclear factor-kappaB and an increase in atrial natriuretic factor mRNA expression. CONCLUSIONS: Gly-BSA stimulates cardiomyocyte ROS production through a protein kinase C-dependent activation of a Nox2-containing NADPH oxidase, which results in nuclear factor-kappaB activation and upregulation of atrial natriuretic factor mRNA. These findings suggest that early-glycated Amadori products may play a role in the development of diabetic heart disease.

Tumor necrosis factor-alpha in diabetic plasma increases the activity of core 2 GlcNAc-T and adherence of human leukocytes to retinal endothelial cells: significance of core 2 GlcNAc-T in diabetic retinopathy.

A large body of evidence now implicates increased leukocyte-endothelial cell adhesion as a key early event in the development of diabetic retinopathy. We recently reported that raised activity of the glycosylating enzyme core 2 beta 1,6-N-acetylglucosaminyltransferase (GlcNAc-T) through protein kinase C (PKC)beta2-dependent phosphorylation plays a fundamental role in increased leukocyte-endothelial cell adhesion and capillary occlusion in retinopathy. In the present study, we demonstrate that following exposure to plasma from diabetic patients, the human promonocytic cell line U937 exhibits a significant elevation in core 2 GlcNAc-T activity and increased adherence to cultured retinal capillary endothelial cells. These effects of diabetic plasma on enzyme activity and cell adhesion, mediated by PKCbeta2-dependent phosphorylation of the core 2 GlcNAc-T protein, were found to be triggered by increased plasma levels of tumor necrosis factor (TNF)-alpha. Levels of enzyme activity in plasma-treated U937 cells were closely dependent on the severity of diabetic retinopathy, with the highest values observed upon treatment with plasma of patients affected by proliferative retinopathy. Furthermore, we noted much higher correlation, as compared with control subjects, between increased values of core 2 GlcNAc-T activity and cell adhesion properties. Based on the prominent role of TNF-alpha in the development of diabetic retinopathy, these observations further validate the significance of core 2 GlcNAc-T in the pathogenesis of capillary occlusion, thereby enhancing the therapeutic potential of specific enzyme inhibitors.

Protein kinase C beta2-dependent phosphorylation of core 2 GlcNAc-T promotes leukocyte-endothelial cell adhesion: a mechanism underlying capillary occlusion in diabetic retinopathy.

Increased leukocyte-endothelial cell adhesion is a key early event in the development of retinopathy and atherogenesis in diabetic patients. We recently reported that raised activity of glycosylating enzyme [beta]1,6 acetylglucosaminyltransferase (core 2 GlcNAc-T) is responsible for increased leukocyte-endothelial cell adhesion and capillary occlusion in retinopathy. Here, we demonstrate that elevated glucose increases the activity of core 2 GlcNAc-T and adhesion of human leukocytes to retinal capillary endothelial cells, in a dose-dependent manner, through diabetes-activated serine/threonine protein kinase C beta2 (PKCbeta2)-dependent phosphorylation. This regulatory mechanism, involving phosphorylation of core 2 GlcNAc-T, is also present in polymorphonuclear leukocytes isolated from type 1 and type 2 diabetic patients. Inhibition of PKCbeta2 activation with the specific inhibitor, LY379196, attenuated serine phosphorylation of core 2 GlcNAc-T and prevented increased leukocyte-endothelial cell adhesion. Raised activity of core 2 GlcNAc-T was associated with a threefold increase in O-linked glycosylation of P-selectin glycoprotein ligand-1 on the surface of leukocytes of diabetic patients compared with age-matched control subjects. PKCbeta2-dependent phosphorylation of core 2 GlcNAc-T may thus represent a novel regulatory mechanism for activation of this key enzyme in mediating increased leukocyte-endothelial cell adhesion and capillary occlusion in diabetic retinopathy.

Impairment of vascular endothelial nitric oxide synthase activity by advanced glycation end products.

Endothelial damage is believed to play a key role in the development of both micro- and macrovascular disease in diabetes, and advanced glycation end products (AGEs) may contribute importantly to this. To determine whether glucose-derived AGEs can cause endothelial dysfunction, we examined the effects of albumin AGE-modified by glucose (AGE-Glu) both in vivo, after injection into rabbit femoral artery, and in vitro on rabbit aortic rings and cultured human umbilical vein endothelial cells (HUVEC). Exposure of blood vessels to AGE-Glu, in vivo and in vitro, inhibited endothelium-dependent vasorelaxation, whereas unmodified albumin did not. In isolated rabbit aorta, this effect was reversible after AGE-Glu washout, and the response to the endothelium-independent vasodilator sodium nitroprusside was unaffected by AGE-Glu. In HUVEC, AGE-Glu inhibited endothelial nitric oxide synthase activity, and this was associated with a decrease in serine phosphorylation of this enzyme. Longer term (72 h) incubation decreased HUVEC viability. Use of specific antibodies demonstrated that these effects were mediated by N(epsilon)-(carboxymethyl)lysine (CML), an important AGE found in vivo, and by the AGE-R1 receptor. Furthermore, these effects all occurred at CML concentrations similar to those found in the plasma of diabetic patients. These results suggest an important role of AGE in the pathogenesis of diabetic vasculopathy.