Pigment epithelium-derived factor protects cultured retinal pericytes from advanced glycation end product-induced injury through its antioxidative properties

Pigment epithelium-derived factor (PEDF) has recently been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, suggesting that loss of PEDF is involved in the pathogenesis of proliferative diabetic retinopathy. However, a protective role for PEDF in pericyte loss in early diabetic retinopathy remains to be elucidated. In this study, we investigated whether PEDF proteins could protect against advanced glycation end product (AGE)-induced injury in retinal pericytes. Ligand blot analysis revealed that pericytes possessed a membrane protein with binding affinity for PEDF. PEDF proteins were found to significantly inhibit AGE-induced reactive oxygen species (ROS) generation and the subsequent decrease in DNA synthesis and apoptotic cell death in pericytes. Further, PEDF proteins completely restored the down-regulation of bcl-2 gene expression in AGE-exposed pericytes. The results demonstrated that PEDF proteins protected cultured pericytes from AGE-induced cytotoxicity through its anti-oxidative properties. Our present study suggests that substitution of PEDF proteins may be a promising strategy in treatment of patients with early diabetic retinopathy.     

Palmitate-induced apoptosis of microvascular endothelial cells and pericytes

BACKGROUND: Recent observations in the EURODIAB Complications Study demonstrated that markers of insulin resistance are strong risk factors for retinopathy incidence in patients with diabetes. However, the molecular mechanism underlying this remains to be elucidated. In this study, we investigated the influence of palmitate, a major saturated free fatty acid in plasma, on the apoptotic cell death of cultured microvascular endothelial cells (EC) and retinal pericytes. MATERIALS AND METHODS: The intracellular formation of reactive oxygen species (ROS) was detected using the fluorescent probe CM-H(2)DCFDA. DNA synthesis was determined by measuring [(3) H]-thymidine incorporation into cells. DNA fragmentations of EC were quantitatively analyzed in an enzyme-linked immunosorbent assay, and DNA laddering was evaluated on agarose gel electrophoresis. RESULTS: Palmitate increased ROS generation in microvascular EC. Furthermore, palmitate significantly inhibited DNA synthesis and induced apoptotic cell death in EC, which were completely prevented by an antioxidant, N-acetylcysteine. Palmitate up-regulated pericyte mRNA levels of a receptor for advanced glycation end products (AGE), and thereby potentiated the apoptotic effects of AGE on pericytes. CONCLUSIONS: The results suggest that palmitate could induce apoptotic cell death in microvascular EC and pericytes through the overgeneration of intracellular ROS, and thus be involved in the development of diabetic retinopathy.     

Sorbitol dehydrogenase overexpression potentiates glucose toxicity to cultured retinal pericytes

The polyol pathway consists of two enzymes, aldose reductase (AR) and sorbitol dehydrogenase (SDH). There is a growing body of evidence to suggest that acceleration of the polyol pathway is implicated in the pathogenesis of diabetic vascular complications. However, a functional role remains to be elucidated for SDH in the development and progression of diabetic retinopathy. In this study, cultured bovine retinal capillary pericytes were used to investigate the effects of SDH overexpression on glucose toxicity. High glucose modestly increased reactive oxygen species (ROS) generation, decreased DNA synthesis, and up-regulated vascular endothelial growth factor (VEGF) mRNA levels in cultured pericytes. SDH overexpression was found to significantly stimulate ROS generation in high glucose-exposed pericytes and subsequently potentiate the cytopathic effects of glucose. Fidarestat, a newly developed AR inhibitor, and N-acetylcysteine, an antioxidant, completely prevented these deleterious effects of SDH overexpression on pericytes. Furthermore, fidarestat administration was found to significantly prevent vascular hyperpermeability, the characteristic changes of the early phase of diabetic retinopathy, in streptozotocin-induced diabetic rats. Our present results suggest that SDH-mediated conversion of sorbitol to fructose and the resultant ROS generation may play an active role in the pathogenesis of diabetic retinopathy. Blockage of sorbitol formation by fidarestat could be a promising therapeutic strategy for the treatment of early phase of diabetic retinopathy.     

Advanced glycation end products-induced apoptosis and overexpression of vascular endothelial growth factor in bovine retinal pericytes.

The influence of advanced glycation end products (AGEs) on apoptotic cell death and vascular endothelial growth factor (VEGF) gene expression in cultured bovine retinal pericytes was investigated. When pericytes were incubated with three immunochemically distinct AGEs, which were prepared in vitro by incubating bovine serum albumin with glucose, glyceraldehyde, or glycolaldehyde, apoptotic cell death and DNA ladder formation were significantly induced. The cytopathic effects of glyceraldehyde- or glycolaldehyde-derived AGEs were significantly enhanced in AGE receptor-transfected pericytes. Furthermore, all of these AGEs were found to upregulate the secretory forms of VEGF mRNA levels in retinal pericytes. These results suggest that AGEs disturbed retinal microvascular homeostasis by inducing pericyte apoptosis and VEGF overproduction and thus were involved in the pathogenesis of early phase diabetic retinopathy.     

Angiotensin II augments advanced glycation end product-induced pericyte apoptosis through RAGE overexpression

Advanced glycation end product (AGE)-their receptor (RAGE) and angiotensin II (AII) are implicated in diabetic retinopathy. However, a crosstalk between the two is not fully understood. In vivo, AGE injection stimulated RAGE expression in the eye of spontaneously hypertensive rats, which was blocked by an AII-type 1 receptor blocker, telmisartan. In vitro, AII-type 1 receptor-mediated reactive oxygen species generation elicited RAGE gene expression in pericytes through NF-kappaB activation. Further, AII augmented AGE-induced pericyte apoptosis, the earliest hallmark of diabetic retinopathy. Our present study may implicate a crosstalk between AGE-RAGE system and AII in diabetic retinopathy.     

Involvement of caspase-10 in advanced glycation end-product-induced apoptosis of bovine retinal pericytes in culture

Apoptosis appears to be the death mechanism of pericyte loss observed in diabetic retinopathy. We have previously shown that advanced glycation end-products (AGE-MGX) induce apoptosis of retinal pericytes in culture associated with diacylglycerol (DAG)/ceramide production. In the present study, we investigated possible caspase involvement in this process. Bovine retinal pericytes (BRP) were cultured with AGE-MGX and apoptosis examined after annexin V staining. Effects of peptidic inhibitors of caspases were determined on DAG/ceramide production and apoptosis. Pan-caspase inhibitor z-VAD-fmk (50 microM) was able to inhibit both DAG/ceramide production and apoptosis, whereas caspase-3-like inhibitor z-DEVD-fmk (50 microM) or caspase-9 inhibitor z-LEHD-fmk (50 microM) was only active on apoptosis. This differential effect strongly suggests involvement of initiator caspase(s) upstream and effector caspase(s) downstream DAG/ceramide production in AGE-mediated apoptosis. Pericyte treatment with caspase-8 inhibitor z-IETD-fmk (50 microM) did not protect cells against AGE-induced apoptosis and we failed to detect caspase-8 in pericytes by immunoblotting assay. Interestingly, one inhibitor of caspase-10 and related caspases z-AEVD-fmk (50 microM) inhibited both AGE-MGX-induced apoptosis and DAG/ceramide formation in pericytes. Cleavage of caspase-10 precursor into its active subunits was demonstrated by immunoblotting assay in pericytes incubated with AGE-MGX. These results strongly suggest that caspase-10, but not caspase-8, might be involved in the early phase of AGE-induced pericyte apoptosis, in contrast to caspase-9 and -3-like enzymes involved after DAG/ceramide production. This finding may provide new therapeutic perspectives for early treatment in diabetic retinopathy.     

Pigment epithelium-derived factor inhibits advanced glycation end product-induced retinal vascular hyperpermeability by blocking reactive oxygen species-mediated vascular endothelial growth factor expression

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis, suggesting that loss of PEDF contributes to proliferative diabetic retinopathy. However, the role of PEDF against retinal vascular hyperpermeability remains to be elucidated. We investigated here whether and how PEDF could inhibit the advanced glycation end product (AGE) signaling to vascular hyperpermeability. Intravenous administration of AGEs to normal rats not only increased retinal vascular permeability by stimulating vascular endothelial growth factor (VEGF) expression but also decreased retinal PEDF levels. Simultaneous treatments with PEDF inhibited the AGE-elicited VEGF-mediated permeability by down-regulating mRNA levels of p22(phox) and gp91(phox), membrane components of NADPH oxidase, and subsequently decreasing retinal levels of an oxidative stress marker, 8-hydroxydeoxyguanosine. PEDF also inhibited the AGE-induced vascular hyperpermeability evaluated by transendothelial electrical resistance by suppressing VEGF expression. Furthermore, PEDF decreased reactive oxygen species (ROS) generation in AGE-exposed endothelial cells by suppressing NADPH oxidase activity via down-regulation of mRNA levels of p22(PHOX) and gp91(PHOX). This led to blockade of the AGE-elicited Ras activation and NF-kappaB-dependent VEGF gene induction in endothelial cells. These results indicate that the central mechanism for PEDF inhibition of the AGE signaling to vascular permeability is by suppression of NADPH oxidase-mediated ROS generation and subsequent VEGF expression. Substitution of PEDF may offer a promising strategy for halting the development of diabetic retinopathy.     

The effect of antioxidants on glycated albumin-induced cytotoxicity in bovine retinal pericytes

Loss of retinal pericytes is the initial deficit in the early stage of diabetic retinopathy. Glycated albumin (GA) forms under hyperglycemic conditions and exists in the retinal blood vessels of diabetic patients with retinopathy. In this study, using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction test, we investigated whether GA induces cytotoxicity in cultured bovine retinal pericytes, and whether the antioxidants, l-ascorbic acid, Trolox, and probucol, provide any protection from GA-mediated cytotoxicity. GA induced pericyte death in a dose-dependent manner. With increasing time, GA-induced cytotoxicity also increased despite no strict time dependence. Furthermore, this cell death was found to be mediated both by apoptosis, which was confirmed by apoptosis-specific fluorescent staining of nuclei and cell membranes, and mitochondrial damage, as elucidated by electron microscopy. All three antioxidants used in this study partially protected against GA-induced pericyte death, suggesting that oxidative stress plays a role in GA-induced pericyte death. The results indicate that GA induces cell death in cultured bovine retinal pericytes, and that certain antioxidants may reduce this cytotoxicity.     

Diabetes-induced activation of caspase-3 in retina: effect of antioxidant therapy

Apoptosis of retinal endothelial cells and pericytes is postulated to contribute to the development of retinopathy in diabetes. The goal of this study is to investigate diabetes-induced activation of retinal caspase-3, an apoptosis executer enzyme, in retina, and examine the effects of antioxidants on the activation. Caspase-3 activation was determined in the retina of alloxan diabetic rats (2-14 months duration) and in the isolated retinal capillary cells (endothelial cells and pericytes) by measuring cleavage of caspase-3 specific fluorescent substrate, and cleavage of caspase-3 holoenzyme and poly (ADP ribosyl) polymerase. Effect of antioxidants on the activation of caspase-3 was determined by feeding a group of diabetic rats diet supplemented with a comprehensive mixture of antioxidants, including Trolox, alpha-tocopherol, N-acetyl cysteine, ascorbic acid, beta-carotene and selenium for 2-14 months, and also under in vitro conditions by incubating isolated retinal capillary cells with antioxidants with wide range of actions. Caspase-3 was activated in the rat retina at 14 months of diabetes (P < 0.05 vs. normal), but not at 2 months of diabetes, and administration of antioxidants for the entire duration inhibited this activation. In the isolated retinal capillary cells incubated in 25 mM glucose medium, caspase-3 activity was increased by 50% compared to the cells incubated in 5 mM glucose (P < 0.02), and antioxidants or caspase-3 inhibitor inhibited this increase. Our results suggest that increased oxidative stress in diabetes is involved in the activation of retinal caspase-3 and apoptosis of endothelial cells and pericytes. Antioxidants might be inhibiting the development of diabetic retinopathy by inhibiting microvascular apoptosis.     

KIOM-79 Protects AGE-Induced Retinal Pericyte Apoptosis via Inhibition of NF-kappaB Activation In Vitro and In Vivo

KIOM-79 is an herbal mixture of parched Puerariae radix, gingered Magnoliae cortex, Glycyrrhizae radix and Euphorbiae radix. In the present study, we determined the efficacy and possible mechanism of KIOM-79 on the advanced glycation end product (AGE)-modified bovine serum albumin (BSA)-induced apoptosis of cultured bovine retinal pericytes and rat retinal pericytes in Zucker diabetic fatty (ZDF) rats. Seven-week-old male ZDF rats were treated with KIOM-79 (50 mg/kg body weight) once a day orally for 13 weeks. KIOM-79 significantly inhibited pericyte apoptosis which were induced by the AGE-BSA treatment. The KIOM-79 treatment markedly suppressed the activation of nuclear factor-kappaB (NF-κB) through the inhibition of inhibitory κB kinase complex. In addition, the oral administration of KIOM-79 inhibited the changes in retinal vasculature (vascular hyperpermeability, acellular capillary). KIOM-79 strongly inhibited pericyte apoptosis, NF-κB activation and the expression of pro-apoptotic Bax and tumor necrosis factor-α. Our results suggest that KIOM-79 may exert inhibitory effects on AGE-induced pericyte apoptosis by blocking NF-κB activation, thereby ameliorating retinal microvascular dysfunction.     

Changes in oxidative balance in rat pericytes exposed to diabetic conditions

Recent data indicate that the oxidative stress plays an important role in the pathogenesis of diabetes and its complications such as retinopathy, nephropathy and accelerated atherosclerosis. In diabetic retinopathy, it was demonstrated a selective loss of pericytes accompanied by capillary basement membrane thickening, increased permeability and neovascularization. This study was designed to investigate the role of diabetic conditions such as high glucose, AGE-Lysine, and angiotensin II in the modulation of antioxidant enzymes activities, glutathione level and reactive oxygen species (ROS) production in pericytes. The activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total glutathione (GSH) was measured spectrophotometrically. The production of ROS was detected by spectrofluorimetry and fluorescence microscopy after loading the cells with 2'-7' dichlorofluoresceine diacetate; as positive control H2O2 was used. Intracellular calcium was determined using Fura 2 AM assay. The results showed that the cells cultured in high glucose alone, do not exhibit major changes in the antioxidant enzyme activities. The presence of AGE-Lys or Ang II induced the increase of SOD activity. Their combination decreased significantly GPx activity and GSH level. A three times increase in ROS production and a significant impairment of intracellular calcium homeostasis was detected in cells cultured in the presence of the three pro-diabetic agents used. In conclusion, our data indicate that diabetic conditions induce in pericytes: (i) an increase of ROS and SOD activity, (ii) a decrease in GPx activity and GSH level, (iii) a major perturbation of the intracellular calcium homeostasis. The data may explain the structural and functional abnormalities of pericytes characteristic for diabetic retinopathy.     

The effects of aminoguanidine on retinopathy in STZ-induced diabetic rats

BackgroundThe accumulation of advanced glycated end products (AGEs) in retinal blood vessels is one of the major etiological factors contributing to diabetic retinopathy. Aminoguanidine (AG) is one of the most extensively used inhibitors of AGEs formation. The aim of this study was to investigate whether AG could protect the development of diabetic retinopathy through inhibition of AGEs.MethodsRat diabetes was induced by intraperitoneal injection with streptozotocin (STZ). AG was given to rats in drinking water. Retina was extracted 3 and 6 months following STZ and AG administration. Immunochemistry and transmission electron microscope were used to detect the expression of AGEs and retina morphology.ResultsExtensive staining of AGEs was detected in retinal blood vessels of 3- and 6-month diabetic rats, while no significant staining was found in the control non-diabetic retina or AG treated groups. Pericyte loss, endothelial cell proliferation, increased ratio of endothelial cells/pericytes, acellular capillaries and capillary occlusion were observed in the retina of 6-month diabetic rats. The increased electron density of retinal capillary basement membrane, mitochondrial swelling in pericytes and endothelial cells were also found in 6-month diabetic rats. The 3-month diabetic rats and the AG-treated rats did not have similar morphological changes compared to control group. The AGEs staining in AG-treated rats was still weakly positive.ConclusionsAGEs plays pivotal roles in diabetic retinopathy. AGE deposition occurs prior to retinal microvasculature changes. AG could prevent the onset and development of diabetic retinopathy through inhibition of AGEs.     

Diabetes-induced Activation of Caspase-3 in Retina: Effect of Antioxidant Therapy

Apoptosis of retinal endothelial cells and pericytes is postulated to contribute to the development of retinopathy in diabetes. The goal of this study is to investigate diabetes-induced activation of retinal caspase-3, an apoptosis executer enzyme, in retina, and examine the effects of antioxidants on the activation. Caspase-3 activation was determined in the retina of alloxan diabetic rats (2-14 months duration) and in the isolated retinal capillary cells (endothelial cells and pericytes) by measuring cleavage of caspase-3 specific fluorescent substrate, and cleavage of caspase-3 holoenzyme and poly (ADP ribosyl) polymerase. Effect of antioxidants on the activation of caspase-3 was determined by feeding a group of diabetic rats diet supplemented with a comprehensive mixture of antioxidants, including Trolox, &#102 -tocopherol, N -acetyl cysteine, ascorbic acid, &#103 -carotene and selenium for 2-14 months, and also under in vitro conditions by incubating isolated retinal capillary cells with antioxidants with wide range of actions. Caspase-3 was activated in the rat retina at 14 months of diabetes ( P <0.05 vs. normal), but not at 2 months of diabetes, and administration of antioxidants for the entire duration inhibited this activation. In the isolated retinal capillary cells incubated in 25 mM glucose medium, caspase-3 activity was increased by 50% compared to the cells incubated in 5 mM glucose ( P <0.02), and antioxidants or caspase-3 inhibitor inhibited this increase. Our results suggest that increased oxidative stress in diabetes is involved in the activation of retinal caspase-3 and apoptosis of endothelial cells and pericytes. Antioxidants might be inhibiting the development of diabetic retinopathy by inhibiting microvascular apoptosis.     

Immune complex formation in human diabetic retina enhances toxicity of oxidized LDL towards retinal capillary pericytes

Recently it has been shown that levels of circulating oxidized LDL immune complexes (ox-LDL-ICs) predict the development of diabetic retinopathy (DR). This study aimed to investigate whether ox-LDL-ICs are actually present in the diabetic retina, and to define their effects on human retinal pericytes versus ox-LDL. In retinal sections from people with type 2 diabetes, costaining for ox-LDL and IgG was present, proportionate to DR severity, and detectable even in the absence of clinical DR. In contrast, no such staining was observed in retinas from nondiabetic subjects. In vitro, human retinal pericytes were treated with native LDL, ox-LDL, and ox-LDL-IC (0–200 mg protein/l), and measures of viability, receptor expression, apoptosis, endoplasmic reticulum (ER) and oxidative stresses, and cytokine secretion were evaluated. Ox-LDL-IC exhibited greater cytotoxicity than ox-LDL toward retinal pericytes. Acting through the scavenger (CD36) and IgG (CD64) receptors, low concentrations of ox-LDL-IC triggered apoptosis mediated by oxidative and ER stresses, and enhanced inflammatory cytokine secretion. The data suggest that IC formation in the diabetic retina enhances the injurious effects of ox-LDL. These findings offer new insights into pathogenic mechanisms of DR, and may lead to new preventive measures and treatments.     

Advanced glycation end-products induce apoptosis of bovine retinal pericytes in culture: involvement of diacylglycerol/ceramide production and oxidative stress induction

One of the earliest changes observed in retinal microvessels in diabetic retinopathy is the selective loss of intramural pericytes. We tested the hypothesis that AGE might be involved in the disappearance of retinal pericytes by apoptosis and further investigated the signaling pathway leading to cell death. Chronic exposure of pericytes to methylglyoxal-modified bovine serum albumin (AGE-BSA) (3 microM) leads to a 3-fold increase of apoptosis (8.9 +/- 1.1%), associated with an increase in cellular ceramide (185 +/- 12%) and diacylglycerol (194 +/- 9%) levels. Ceramide formation was almost inhibited (95%) by an acidic sphingomyelinase inhibitor, desipramine (0.3 microM). Dual inhibition of ceramide (95%) and diacylglycerol (80%) production was observed with a phosphatidylcholine-phospholipase C inhibitor, D609 (9.4 microM). Taken together, these results suggest activation of phosphatidylcholine-phospholipase C coupled to acidic sphingomyelinase. However, both inhibitors only partially protected pericytes against apoptosis, suggesting another apoptotic pathway independent of diacylglycerol/ceramide production. Treatments with various antioxidants completely inhibited pericyte apoptosis, suggesting oxidative stress induction during this apoptotic process. Inhibition of diacylglycerol/ceramide production by N-acetyl-L-cysteine suggests that oxidative stress acts upstream of the two metabolic pathways. AGE treated with metal chelators were also able to induce pericyte apoptosis, suggesting a specific effect of AGE on intracellular oxidative stress independent of redox-active metal ions bound to AGE. In conclusion, these results identify new biochemical targets involved in pericyte loss, which can provide new therapeutic perspectives in diabetic retinopathy.     

Increased levels of vascular endothelial growth factor and advanced glycation end products in aqueous humor of patients with diabetic retinopathy.

Clinical studies have shown a relationship between diabetic retinopathy and vascular endothelial growth factor (VEGF) levels in ocular fluid. Advanced glycation end products (AGEs) have been implicated in diabetes complications, including diabetic retinopathy. Nepsilon-(carboxymethyl) lysine (CML) is a glycoxidation product that may be a marker of oxidative stress. In this study, we used enzyme-linked immunosorbent assays to determine the levels of VEGF, non-CML AGE and CML in the aqueous humor and serum of 82 Japanese patients with type 2 diabetes and 60 non-diabetic subjects. VEGF, non-CML AGE, and CML concentrations in aqueous humor and serum were then compared with the severity of diabetic retinopathy. Immunohistochemical detection analysis of non-CML AGE and CML was also performed using retinal tissues from patients with progressive diabetic retinopathy. Aqueous levels of VEGF, non-CML AGE and CML increased along with the progression of diabetic retinopathy compared to age-matched controls. After coagulation therapy, the VEGF, non-CML AGE, and CML levels were significantly reduced. Immunostaining showed diffuse co-localization of non-CML AGE and CML around microvessels and in the glial cells of proliferative membranes from patients with progressive diabetic retinopathy. These findings suggest that glycation and glycoxidation reactions (or oxidation, as revealed by CML) may contribute to both the onset and progression of diabetic retinopathy.     

Comparative effects of beraprost, a stable analogue of prostacyclin, with PGE(1), nitroglycerin and nifedipine on canine model of vasoconstrictive pulmonary hypertension

Acute hemodynamic effects of beraprost sodium were tested in a canine vasoconstrictive pulmonary hypertension model induced by the continuous infusion of U-46619, a thromboxane A(2)mimetic. The effects of beraprost were compared with those of prostaglandin E(1), nitroglycerin and nifedipine. Beraprost and nitroglycerin decreased pulmonary arterial pressure. On the other hand, prostaglandin E(1)and nifedipine increased pulmonary arterial pressure. All drugs except nitroglycerin increased cardiac output and decreased pulmonary vascular resistance. Beraprost was selective to pulmonary circulation, while nitroglycerin, prostaglandin E(1), and nifedipine showed poor selectivity for the pulmonary vasculature. These results suggest that the vasodilative effect of beraprost is the most selective for the pulmonary circulation among these four vasodilators.     

Advanced glycation end-products induce apoptosis involving the signaling pathways of oxidative stress in bovine retinal pericytes

One of the histopathologic hallmarks of early diabetic retinopathy is the selective loss of pericytes. Evidences suggest that the pericyte loss in vivo is mediated by apoptosis. However, the underlying cause of pericyte apoptosis is not fully understood. This study investigated the effect of advanced glycation end products (AGEs) on apoptotic cell death in bovine retinal pericytes (BRPs). After incubation of BRPs with 0.47, 1.88, 7.5, 30 microM of AGE-bovine serum albumin (BSA) for 4 days, we assayed the pericytes apoptosis by FACS (fluorescence activated cell sorting), and further measured the signaling pathway involved. The results showed that AGE-BSA could induce significantly the apoptosis of BRPs in a dose-dependent manner compared with controls, associated with an increase in intracellular malondialdehyde level and caspase-3 activity; a decrease in intracellular catalase, SOD activities and Bcl-2/Bax ratio. SOD and selective caspase-3 inhibitor Z-DEVD-fmk can inhibit pericyte apoptosis induced by AGE-BSA. These data suggest that the pericyte loss in diabetic retinopathy involves an apoptotic process, and that elevated AGE observed in diabetes may cause apoptosis in BRPs through an oxidative stress mechanism. The decreased Bcl-2/Bax ratio and activation of caspase-3 are associated with apoptotic process.