O69	Diabetes-induced overproduction of reactive oxygen species impairs post-ischemic neovascularization.
1T.Ebrahimian, 1Chr.Heymes, 1D.You, 1O.Blanc-Brude, 1L.Waeckel, 1J.Vilar, 1B.Lévy, 2A.Shah, 1J-S.Silvestre
1INSERM U689, Paris, FR; 2King's College, London, GB.

Introduction The mechanisms by which diabetes limits neovascularization remain largely undefined. Diabetes is associated with overproduction of reactive oxygen species (ROS) including those derived from NADPH oxidase. We hypothesized that diabetes-induced oxidative stress may affect post-ischemic neovascularization. Methods and Results After femoral artery ligation, wildtype or gp91phox-deficient control or diabetic mice (streptozotocin, 60 mg/kg/day) were treated with or without the anti-oxidant N-acetyl-L-cysteine (NAC, 200mg/kg/day), or by in vivo electrotransfer of a plasmid encoding dominant–negative Rac1 (dnRac1, 50 mg) for 21 days (n=7 per group). In diabetic mice, ROS accumulation was upregulated in ischemic tissues compared to control animals, as assessed by oxidative fluorescent dye dihydroethidium or anti-nitrotyrosine antibody staining. gp91phox and Rac1 protein levels were increased by 1.4- and 1.3-fold, respectively in diabetic ischemic hindlimbs compared to ischemic controls (p<0.05). Angiography scores, capillary numbers and foot perfusion were decreased in diabetic animals, by 1.4-, 2.4-, and 1.6-fold, respectively compared to control animals. Blockade of ROS overproduction by NAC administration, by gp91phox deficiency, or by administration of dnRac1 restored post-ischemic neovascularization in diabetic animals to non diabetic levels. This effect was associated with upregulated VEGF protein levels and restored collagenolysis in ischemic diabetic mice. Finally, diabetes reduced the ability of adherent bone marrow derived mononuclear cells (BMC) to differentiate into endothelial progenitor cells (EPC). Treatment with NAC (3mM), apocynin (1mM) or a p38MAPK inhibitor (LY333351, 10µM) upregulated the number of EPC colonies derived from diabetic BMC by 1.5, 1.6-and 1.5-fold, respectively (p<0.05). In the ischemic hindlimb model, injection of diabetic BMC isolated from NAC-treated or gp91phox deficient diabetic mice increased neovascularization by about 1.5-fold over untreated diabetic BMC (p<0.05). Conclusion Inhibition of NADPH oxidase-derived ROS overproduction improved the angiogenic and vasculogenic processes and restored post-ischemic neovascularization in type I diabetic mice.

Copyright © 2005 S. Karger AG, Basel. Any further use of this abstract requires written permission from the publisher.