Abstract
Abstract: :
Purpose: PARP is a nuclear DNA nick–sensor enzyme involved in protein poly(ADP–ribosyl)ation. Activation of PARP ((poly(ADP–ribose) polymerase–1) has been reported to both protect against, and cause, cell death under various conditions. We initiated a long–term study to investigate the role of PARP in hyperglycemia–induced cell death in vitro, and in the development of diabetic retinopathy in vivo. Methods: Lewis rats were randomly assigned to be made diabetic (streptozotocin) or remain as a nondiabetic controls. Experimentally diabetic rats were treated with or without the PARP inhibitor, PJ–34 (20mg/Kg BW in food). At 2 months of diabetes, PARP activity was assessed using an antibody against poly(ADP–ribosyl)ated protein, the product of PARP. At 9 months of diabetes, death of the retinal vascular cells was determined using the TUNEL assay, and obliterated, nonperfused capillaries ("acellular capillaries") were quantitated from retinal vessel preparations (prepared by the trypsin digest method). In vitro, bovine retinal endothelial cells and pericytes were incubated in 5 and 25mM glucose for 5 days with or without PJ–34, and cell death was assessed using a trypan blue exclusion assay. Results: Diabetes was found to increase activity of PARP in retina, and PJ–34 inhibited this increase. PARP activation was detectable also in a subset of nuclei from retinal capillary endothelial cells and pericytes. In vitro, 25 mM glucose significantly increased death of retinal capillary endothelial cells, and PJ–34 significantly inhibited this hyperglycemia–induced cell death. In vivo, diabetes of 9 months duration significantly increased the number of both TUNEL–positive capillary cells and acellular capillaries (a marker of degenerate capillaries), and PJ–34 significantly inhibited these lesions without altering glycemic control. PJ–34 also inhibited a diabetes–induced up–regulation of ICAM (a protein regulated by NF–kappa B that mediates adhesion of leukocytes to vascular walls) and leukostasis within the retinal vasculature. Conclusions: PARP plays a role in the development of diabetic retinopathy, and offers a novel target for the pharmacologic inhibition of the retinopathy.
Keywords: diabetic retinopathy • apoptosis/cell death • pharmacology