Abstract 4914: Extracellular SOD-derived H2O2 Promotes VEGF Signaling via Oxidative Inactivation of Protein Tyrosine Phosphatases Localized in Caveolae/Lipid Rafts
Reactive oxygen species (ROS), in particular, H2O2, is essential for full activation of VEGF receptor2 (VEGER2) signaling involved in endothelial cell (EC) proliferation and migration. Extracellular superoxide dismutase (ecSOD) is a major extracellular antioxidant enzyme in the vasculature that converts superoxide to H2O2. It is secreted and anchored to the endothelial surface through binding to the heparan sulfate proteoglycan (HSPG), which binds to VEGFR2 to facilitate its signaling. Mice lacking ecSOD show impaired postnatal angiogenesis. However, underling mechanism remains unknown. Here we show that adenovirus-mediated ecSOD overexpression in cultured medium in ECs enhances VEGF-induced tyrosine phosphorylation of VEGFR2 (VEGFR2-pY) (2.9-fold). This ecSOD-induced effect is prevented by short-term pretreatment with catalase which scavenges extracellular H2O2. Of note, either exogenous H2O2 or mutant ecSOD lacking heparin binding domain (ecSOD-deltaHBD) has no effect on VEGF-induced VEGFR2-pY, suggesting that H2O2 needs to be produced close to VEGFR2 for its activation. Mechanistically, VEGFR2 as well as protein tyrosine phosphatases (PTPs) PTP1B and DEP1, which negatively regulate VEGFR2, but can be inactivated by ROS, are colocalized in caveolae/lipid rafts. VEGF-induced VEGFR2-pY in these microdomains is enhanced by ecSOD (2.3-fold), which is associated with oxidation of PTP1B and DEP1, and decrease in PTPs activity (48.9%) in caveolae/lipid rafts. PTPs inhibitor orthovanadate; or siRNAs for PTP1B and DEP1; or disruption of caveolae/lipid rafts by methyl-b-cyclodextrin enhances VEGF-induced VEGFR2-pY, which prevents ecSOD-induced effect. Functionally, ecSOD significantly promotes VEGF-stimulated EC migration (1.7-fold) and proliferation (1.4-fold). In vivo, adenovirus ecSOD injection into adductor muscle improves blood flow recovery and increases capillary formation after hindlimb ischemia, while mutant ecSOD-deltaHBD has no effects. In summary, H2O2 produced proximity to VEGFR2 by ecSOD inactivates PTPs in caveolae/lipid rafts via oxidation, thereby promoting VEGF-induced VEGFR2-pY and angiogenesis. Thus, ecSOD is important therapeutic target for angiogenesis-dependent cardiovascular diseases.
This research has received full or partial funding support from the American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).