Abstract W MP77: The Role of Ischemia/Reperfusion Generated Nitrative Stress on VEGF Angiogenic Signaling in Diabetic rats.

Abstract

Diabetes worsens the outcome and impairs recovery after stroke. We have previously shown that diabetes caused a nitration-dependent dysfunctional cerebral neovascularization via increased vascular endothelial growth factor (VEGF) signalling. Our recent study showed that ischemia/reperfusion (I/R) impaired vascular repair in the post-stroke period in diabetes. Yet, the molecular mechanism is unclear. Our present hypothesis is that I/R-mediated excess peroxynitrite formation impairs VEGF survival and angiogenic signal in a nitration-dependent manner. Methods: Acute ischemic stroke was induced via 90 min middle cerebral artery occlusion in Wistar (Wis) and diabetic Goto-Kakizaki rats (GK). 14 days later brain sections were examined for peroxynitrite generation (nitrotyrosine), survival markers (Akt and P38 activation), and apoptosis markers (caspase-3 and cleaved PARP). Brain micro-vascular endothelial cells (BMVEC) were isolated from Wis and GK rats and subjected to 6 hr hypoxia (0.2% oxygen) followed by 18 h normoxia. Nitrotyrosine, P85 nitration, Akt and P38 activity and caspase-3 cleavage were determined. VEGF signal was assessed via cell proliferation, tube formation and cell migration assay. Results: There was increased basal tyrosine nitration in diabetes. I/R increased nitrotyrosine in control (*2-fold) and diabetic (*3-fold) rats (*p<0.05). This increase was associated with decreased survival signal (20%, *60%) and increased apoptic signal (1.5-fold and *6-fold in Wis and GK, respectively, *p<0.05). In parallel, BMVEC isolated from GK rats showed increased nitrative stress compared to controls (*p<0.05). A second hit by hypoxia/reoxygenation dramatically increased the nitration of P85 subunit of PI3kinase* and activation of p38 MAPK* while decreased Akt activation* (*p<0.05). These effects were associated with impairment of VEGF-induced migration* and restored by treatments with peroxynitrite scavenger FeTPPS* (5 uM) or nitration inhibitor Epicatechin* (200uM) (*p<0.05). Conclusion: Our results demonstrate that I/R diverts prosurvial/angiogenic effects of VEGF to a proapoptotic pathway and suggest peroxynitrite as a novel therapeutic target for improvement of reparative angiogenesis after stroke in diabetes.