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IG Obrosova, AG Minchenko, A Kennedy, OI Abatan, RN Frank, MJ Stevens; Poly(ADP-ribose) Synthetase Inhibitors Counteract Diabetes and Hypoxia-induced Retinal VEGF Formation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1311.
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Purpose: Increased aldose reductase activity and resulting oxidative stress have been implicated in diabetes-induced retinal VEGF production, but the mechanism(s) remains unknown. ROS activate multiple signal transduction pathways including poly(ADP-ribosyl)ation, the DNA single strand breakage-stimulated, poly(ADP-ribose)synthetase(PARS)-dependent transfer of ADP-ribose moieties from nicotinamide adenine nucleotide(NAD) to nuclear proteins. PARS activation alters metabolism and gene expression. We evaluated the role for PARS in diabetes- and hypoxia-induced retinal VEGF formation. Methods: In vivo experiments were performed in control (C) and streptozotocin-diabetic (D) rats treated with/without one of two structurally unrelated PARS inhibitors, 3-aminobenzamide(ABA,30 mg/kg/day) or 1,5-isoquinolinediol(ISO, 3 mg/kg/day) administered i.p. for 2 wks after 2 wks of diabetes. Retinal VEGF protein was assayed by ELISA (R&D Systems), VEGF mRNA by ribonuclease protection assay, NAD and sorbitol pathway intermediates spectrofluorometrically by enzymatic methods, and lipid peroxidation products [malondialdehyde (MDA)+4-hydroxyalkenals (4-HA)] colorimetrically with the kits from Oxis Int. In vitro studies were performed in human retinal pigment epithelial cells (RPEs) exposed to normoxia or hypoxia for 48 h. VEGF protein was assayed in the cell culture media. Results: Retinal NAD concentration was decreased by 24% in D vs C (p<0.05), and this reduction was prevented by both ABA and ISO thus indicating that 1)PARS activation is present, and 2) both PARS inhibitors penetrate in the diabetic retina. VEGF protein, but not mRNA expression, was increased by 90% in D vs C (53.48.6 vs 28.35.1 pg/mg prot, p<0.01), and this increase was corrected by both ABA (29.61.4, p<0.01 vs D) and ISO (34.84.1, p<0.05 vs D).Glucose, MDA+4-HA and sorbitol pathway intermediate concentrations were elevated in D vs C, and none of these variables were affected by PARS inhibitors. Hypoxia caused about 3-fold increase in VEGF protein production by RPEs, and this increase was completely arrested by ABA (40 mM) or ISO (4 µM). Conclusion: Poly(ADP-ribosyl)ation is involved in hypoxia- and diabetes-induced retinal VEGF production downstream consequent to sorbitol pathway hyperactivity and oxidative stress.The study provides the rationale for further evaluation of PARS inhibitors in the states with abnormal vascular growth e.a.retinopathy of prematurity and diabetic retinopathy.
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