Abstract 3515: VEGF And Peroxynitrite Interplay Regulates Cerebral Angiogenic Mediators In Diabetes
Background: Diabetes targets micro and macrovascular endothelium differently leading to vascular complications. Although there are extensive studies on retinal and coronary neovascularization, little is known about cerebral neovascularization. Growing evidence suggests that reactive oxygen and nitrogen species such as peroxynitrite can interact in growth factor signaling and mediate VEGF’s angiogenic properties. This study tests the hypothesis that diabetes stimulates cerebral angiogenesis via activation of VEGF and matrix metalloproteases (MMP) in a peroxynitrite-dependent manner.
Methods: Cerebral microvascular endothelial cells (CMEC) were isolated using immunomagnetic beads from control and diabetic Goto-Kakizaki (GK) rats. Cell proliferation, migration and tube formation assays were used as the indices of angiogenic potential. Protein expression and activity were determined using immunoblotting, slot blots and zymography techniques.
Results: Angiogenic potential was pronounced in the diabetic group and significantly inhibited by peroxynitrite decomposition catalyst FeTPPS (2.5µM), MMP inhibitor minocycline (50µg/ml) and src kinase inhibitor PP2(1µM) compared to control. Diabetic endothelial conditioned media pronounced angiogenic potential in control CMEC while VEGF neutralizing antibody abrogated this response. Diabetes increased the expression of soluble VEGF A isoforms and activity of cognate receptors. Basal levels of phospho-c-src and peroxynitrite were also increased in the diabetic group. Secreted active MMP-2 and pro-MMP-2 was lower in diabetes while the cellular active MMP-2 and MT1-MMP was higher compared to control. VEGF (30ng/ML) stimulation activated its cognate receptor biophysically, dysregulated c-src activity and increased tyrosine nitration in diabetes. Peroxynitrite inhibition reduced VEGFR2, phosphor-c-src and MT1-MMP even in the presence of VEGF stimulation.
Conclusion: Peroxynitrite interacts with VEGF signaling in diabetes and dysregulates c-src and MMPs. Understanding cerebral neovascularization mechanisms will not only open new paths towards developing therapeutic angiogenesis but also prevention of hemorrhagic complications of stroke. N=3-8 * p≤ 0.05, ** p≤ 0.005, *** p≤ 0.001.
- © 2012 by American Heart Association, Inc.