cyclic-gmp and Diabetic-Retinopathy

The vasodegenerative phase of diabetic retinopathy is likely caused by endothelial dysfunction and reduced endothelial repair. Migration of endothelial progenitor cells (EPCs) into areas of vascular injury is critical to vascular repair. This key function, often defective in diabetes, is largely mediated by nitric oxide (NO), which is known to be inactivated by superoxide produced by NADPH oxidase. The authors tested the hypothesis that either increasing eNOS expression or inhibiting NADPH oxidase would restore the reparative function in diabetic EPCs.. Peripheral blood was obtained from healthy (n = 27) and diabetic (n = 31) persons, and CD34(+) cells were isolated. Expression and activation of eNOS and NADPH oxidase and intracellular levels of NO, superoxide, and peroxynitrite were evaluated. cGMP production and migration to SDF-1α were also determined. Reparative function was evaluated in a mouse model of retinal ischemia-reperfusion injury.. Diabetic EPCs demonstrate reduced eNOS expression and decreased NO bioavailability and migration in response to SDF-1α. Increasing eNOS expression in diabetic cells by AVE3085 resulted in increased peroxynitrite levels and, therefore, did not enhance NO-mediated functions in vitro and in vivo. Expression of Nox2, NADPH oxidase activity, and superoxide levels were higher in diabetic than in nondiabetic EPCs. Pretreatment with apocynin or gp91ds-tat increased NO bioavailability without increasing eNOS activity in response to SDF-1α. Ex vivo NADPH oxidase inhibition in diabetic cells restored migratory function in vitro and enhanced their homing to ischemic retinal vasculature in vivo.. The NADPH oxidase system is a promising target for correcting vasoreparative dysfunction in diabetic EPCs.

Topics: Acetophenones; Adult; Animals; Antigens, CD34; Benzodioxoles; Chemokine CXCL12; Cyclic GMP; Diabetic Retinopathy; Disease Models, Animal; Endothelium, Vascular; Enzyme Inhibitors; Female; Glycoproteins; Humans; Indans; Male; Mice; Middle Aged; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase Type III; Peroxynitrous Acid; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Superoxides; Young Adult

Asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of nitric oxide synthase, is generated in presence of type 1 protein arginine N-methyltransferase (PRMT-1) and is metabolized by dimethylarginine dimethylaminohydrolases (DDAHs). Reportedly ADMA is associated with endothelial dysfunction. The aim of this study is to investigate whether PRMT-1- and DDAHs-induced ADMA increase in diabetic rat retina and high glucose-treated bovine retinal capillary endothelial cells (BRCECs) is involved in reactive oxygen species (ROS)- and renin-angiotensin system (RAS)-mediated diabetic retinopathy. Rats were divided into four groups: sham-injected group, streptozotocin (STZ)-induced diabetic model group, STZ-induced diabetic model plus 12-week ACEI benazepril treatment group, and STZ-induced diabetic model plus 12-week ARB telmisartan treatment group. BRCECs were exposed to 5mM glucose, 30mM glucose, and 30mM glucose plus benazepril, telmisartan, diphenyliodonium (NADPH oxidase inhibitor, DPI), or N-Acetyl-l-cysteine (antioxidant and free radical scavenger, NAC) until passage four. We found that the concentrations of ADMA were significantly elevated in the plasma of diabetic rat models, and were significantly reduced by benazepril or telmisartan. DDAHs expression was decreased and PRMT-1 expression was increased in diabetic rat retina, which was reversed by benazepril. Telmisartan decreased PRMT-1 expression and increased DDAH II expression, but had no effect on DDAH I expression. In vitro, BRCECs exposed to high glucose had elevated ROS production, decreased cGMP, increased PRMT-1 expression, and decreased DDAH activity and DDAH II expression. Coincubating BRCECs with benazepril, telmisartan, DPI or NAC reversed the effects of high glucose. It can be concluded that PRMT-I and DDAHs-induced upregulation of ADMA levels might be involved in ROS- and RAS-mediated diabetic retinopathy.

Topics: Amidohydrolases; Animals; Arginine; Blotting, Western; Cells, Cultured; Cyclic GMP; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelial Cells; Male; Protein-Arginine N-Methyltransferases; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Renin-Angiotensin System; Up-Regulation

Aminoguanidine inhibits the development of retinopathy in diabetic animals, but the mechanism remains unclear. Inasmuch as aminoguanidine is a relatively selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), we have investigated the effects of hyperglycemia on the retinal nitric oxide (NO) pathway in the presence and absence of aminoguanidine. In vivo studies utilized retinas from experimentally diabetic rats treated or without aminoguanidine for 2 months, and in vitro studies used bovine retinal endothelial cells and a transformed retinal glial cell line (rMC-1) incubated in 5 mm and 25 mm glucose with and without aminoguanidine (100 microg/mL). NO was detected as nitrite and nitrate, and nitrotyrosine and iNOS were detected using immunochemical methods. Retinal homogenates from diabetic animals had greater than normal levels of NO and iNOS (p < 0.05), and nitrotyrosine was greater than normal, especially in one band immunoprecipitated from retinal homogenates. Oral aminoguanidine significantly inhibited all of these increases. Nitrotyrosine was detected immunohistochemically only in the retinal vasculature of non-diabetic and diabetic animals. Retinal endothelial and rMC-1 cells cultured in high glucose increased NO and NT, and aminoguanidine inhibited both increases in rMC-1 cells, but only NT in endothelial cells. Hyperglycemia increases NO production in retinal cells, and aminoguanidine can inhibit this abnormality. Inhibition of diabetic retinopathy by aminoguanidine might be mediated in part by inhibition of sequelae of NO production.

Topics: Animals; Cattle; Cells, Cultured; Cyclic GMP; Diabetes Complications; Diabetes Mellitus; Diabetic Retinopathy; Endothelium, Vascular; Glucose; Guanidines; Hyperglycemia; Immunohistochemistry; Male; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidative Stress; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Retina; Streptozocin; Tyrosine

We examined changes in guanosine triphosphate-dependent signal transduction mechanisms in the retina from the early stages of the streptozotocin-diabetic rat, a model for Type 1 (insulin-dependent) diabetes mellitus. Guanosine triphosphate binding, guanosine triphosphatase activity, and binding of (azido) guanosine triphosphate decreased significantly in the retina as early as 2 weeks after the induction of diabetes. The ability of guanosine triphosphate to inhibit forskolin-stimulatable adenyl cyclase was also abolished. These data suggest functional deterioration of G-proteins, especially Gi, in diabetic retina. Further studies using retinal rod outer segments revealed deterioration in light-sensitive, guanosine triphosphate-dependent functions of transducin in diabetic rats. Pertussis toxin-catalysed ADP ribosylation of the alpha subunit of transducin, a heterotrimeric G-protein of rod outer segments, was also reduced in diabetes. No functional effects were seen in purified subunits of transducin subjected to non-enzymatic glycation in vitro. On the other hand, incubation of non-diabetic rod outer segments with (12-0-tetradeconyl) phorbol-13-acetate, a protein kinase C agonist, in the presence of magnesium and adenosine triphosphate resulted in the reduction of guanosine triphosphate-binding and hydrolysis, thus indicating that protein kinase C may be involved in the regulation of these activities. The significance of these observations in the early visual abnormalities associated with diabetes is discussed.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Animals; Cyclic GMP; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Retinopathy; Glucose; Glycosylation; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Kinetics; Pertussis Toxin; Rats; Rats, Inbred Strains; Retina; Rod Cell Outer Segment; Transducin; Virulence Factors, Bordetella

Cyclic GMP and cyclic AMP are present in lower concentrations in the central (macular) region of the neural retina of the human and monkey than in other areas. This pattern approximates the distribution of rod photoreceptor cells. Surprisingly, an inverse gradient of cyclic GMP concentration is observed in the pigmented epithelium. Levels in the central region are over fourfold higher than in cells in the periphery, offering the first evidence of biochemical differences in this embryologically uniform cell type.

Topics: Adolescent; Adult; Aged; Animals; Child; Cyclic AMP; Cyclic GMP; Diabetic Retinopathy; Female; Humans; Macaca mulatta; Macula Lutea; Male; Microscopy, Electron; Middle Aged; Photoreceptor Cells; Pigment Epithelium of Eye; Retina; Retinitis Pigmentosa

Topics: Animals; Aspirin; Cyclic AMP; Cyclic GMP; Diabetes Mellitus; Diabetic Retinopathy; Disease Models, Animal; Dog Diseases; Dogs; Drug Therapy, Combination; Epoprostenol; Platelet Count