Study On Therapy And Mechanisms Of The Role Of Reactive Oxygen Species In Diabetic Retinopathy

Purpose: To elucidate the mechanisms of the role of reactive oxygen species (ROS) in diabetic retinopathy (DR), investigate the effects of pigment epithelium-derived factor (PEDF) on the apoptosis of hyperglycemia-incubated cells, and explore statins simvastatin and angiotensin-converting enzyme inhibitors (ACEI) perindopril on retinopathy in diabetic rats.Method: (1) The primary culture approach of bovine retinal capillary endothelial cells(BRECs) were incubated with either normal glucose concentration (5 mM), high glucose concentration (30 Mm) in the presence or absence of 100 nM PEDF, 10μM diphenyleneiodonium (DPI), 10 mM N-acetylcysteine (NAC), or 10μM AG490. The rate of the apoptosis of BRECs was measured by TUNEL, and ROS and NADPH oxidase in the BRECs were analysed with fluoroanalyzer. RT-PCR and western blot were respectively used to assay vascular endothelial growth factor(VEGF) mRNA and protein,Janus kinase 2 / signal transducer and activator of transcription 3 (JAK2/STAT3) and phospho-JAK2/phospho-STAT3(p-JAK2/p-STAT3) protein expression in the cells. (2) Eight-week-old male Sprague-Dawley rats were randomly assigned into groups receiving either 60 mg/kg streptozotocin (STZ) intraperitoneally or citrate buffer alone. After 6 months, retinas in rats were collected. p-JAK2/p-STAT3 in the retinas were measured by immunohistochemistry and western blot, and VEGF mRNA and protein by RT-PCR and western blot. Furthermore, a correlation analysis between VEGF and p-STAT3 protein expression was performed. (3) Control and STZ-induced diabetic rats were treated with or without the simvastatin (4mg/kg/day) for 6 months starting from induction of diabetes for 1 week. To elucidate the mechanism, BRECs were incubated with either normal glucose or high glucose combined with or without simvastatin (5μM). Retinal trypsin digestion was performed to identify the number of pericytes and acellular capillaries, and immunoprecipitation was used to analyse poly(ADP-ribose) polymerase (PARP) activation from retinas and BRECs. Angiopoietin-2 (Ang-2) and VEGF mRNA and protein from retinas and BRECs, and uncoupling protein-2 (UCP-2) mRNA and protein from BRECs were measured respectively by RT-PCR and western blot. In addition, flow cytometry was used to evaluate mitochondrial membrane potential. As described above, ROS and NADPH oxidase were analysed with fluoroanalyzer. (4) Vitreous samples were collected from 21 eyes of 21 patients with proliferative diabetic retinopathy (PDR) and 16 eyes of 16 patients with an idiopathic macular hole (IMH), who underwent pars plana vitrectomy for the treatment of retinal disorders. ELISA was used to assay vitreous VEGF and PEDF levels. Control and STZ-induced diabetic rats were treated with or without the perindopril (2mg/kg/day) for 6 months starting from induction of diabetes for 1 week. To elucidate the mechanism, BRECs were incubated with either normal glucose or high glucose combined with or without perindopril (10μM). Retinal capillary basement membrane thickness (BMT) in rats was measured by transmission electron microscopy, and PKC activities in membranous and cytosolic fractions in the cells were determined using ELISA. VEGF and PEDF mRNA and protein expression from the retinas and the BRECs, and UCP-2 and PPARγmRNA and protein from the BRECs were respectively measured by RT-PCR and western blot. In addition, the number of pericytes and acellular capillaries in the retinas,ROS,NADPH oxidase and mitochondrial membrane potential in the BRECs were assayed according to description above.Resμlts: (1) In vitro hyperglycemia induced the upregulation of VEGF mRNA and protein expression was regulated by p-JAK2/p-STAT3 in BRECs (p<0.01). The treatment with PEDF inhibited high-glucose-derived NADPH oxidase activation and subsequently decreased ROS generation. This resulted in the inhibition of p-JAK2/p-STAT3 expression and the concomitant downregulation of VEGF levels, and was followed by the arrest of BREC apoptosis (p<0.05). (2) Retinal p-JAK2/p-STAT3 levels increased significantly in diabetic rats compared with those of nondiabetic rats, and were accompanied by the upregulation of VEGF mRNA and protein expression(p < 0.01). Furthermore, retinal VEGF protein levels had a correlation with p-STAT3 protein levels(p=0.01). Retinal p-JAK2 and p-STAT3 protein immunostaining was predominantly present in the ganglion cell and inner nuclear layers. (3) Ang-2 and VEGF mRNA and protein expression were up-regulated in diabetic rats, accompanied by an increase in PARP activity. Here, their expression and activity closely paralleled that of the loss of pericytes and formation of acellular vessels. A marked increase was observed in poly(ADP-ribosyl)ation of the proteins obtained from the retinal extract of 6-month diabetic rats as compared to the nondiabetic controls and immunostaining to detect PARP activity sites revealed that PARP activity slightly increased in the nuclei of the ganglion cell layer, inner nuclear layer, and outer nuclear layer of diabetic rats. Simvastatin ameliorates retinopathy in diabetic rats, in association with down-regulation of Ang-2 and VEGF expression involving PARP activity inhibition via UCP-2 expression contributing to a decrease in mitochondrial membrane hyperpolarization, in addition to its inhibiting NADPH oxidase activation(p<0.05). (4) Up-regulation of VEGF and down-regulation of PEDF in the vitreous from the eyes with PDR were found as compared with that of the eyes with IMH(p<0.01). In contrast, in diabetic rats, although the increase in the retinal VEGF to PEDF ratio, VEGF and PEDF expression significantly up-regulated simultaneously(p< 0.01). Perindopril decreased the ratio and promoted a concomitant amelioration of the retinal histopathological lesions in diabetic rats(p<0.05). In vitro, the results indicated that perindopril decreased the VEGF to PEDF ratio(down-regulation of VEGF and up-regulation of PEDF)through inhibiting ROS-mediated PKC activation via PPAR-gamma activation up-regulating uncoupling protein-2 expression in BRECs incubated by high glucose.Conclusions(1)ROS plays a critical role in the angiogenesis of retinopathy in diabetic rats through the activation of JAK2/STAT3/VEGF, PARP/Ang-2 and VEGF, and PKC/VEGF and PEDF pathways. The results suggest that the ROS signaling pathways might represent important effective targets of therapeutic intervention for DR.(2)PEDF prevents the apoptosis of capillary endothelial cells induced by high glucose through the inhibition of ROS/JAK2/STAT3 axis by reducing NADPH oxidase activation. JAK2/STAT3 Pathway might represent a target for PEDF treatment. (3)Statins and ACEI ameliorate the retinopathy histopathologicol lesions in rats involving the suppression of ROS/PARP or ROS/PKC axis regulated by the NADPH oxidase and the mitochondrial pathways. Taken together, the findings in our current study highlight the potential importance of treatment with statins and ACEI in preventing loss of vision in diabetic patients, which provide scientific theoretical basis for the prevention and treatment of DR.