Abstract 1381: Novel Role of Extracellular SOD as a Positive Regulator for H2O2-dependent VEGF Signaling Linked to Angiogenesis in Endothelial Cells
The extracellular superoxide dismutase (ecSOD), a secretory enzyme that converts superoxide to H2O2, is highly expressed in the vessel wall, and is anchored to the endothelial surface through binding to the heparan sulfate proteoglycan (HSPG). We previously demonstrated that ecSOD plays an essential role in post-ischemic neovascularization in vivo; however, its underlying mechanism is poorly understood. VEGF activates NADPH oxidase to produce H2O2 which promotes autophosphorylation VEGF receptor2 (VEGFR2) that binds to HSPG, thereby stimulating endothelial cell (EC) migration and proliferation. We thus examined the role of ecSOD in VEGF signaling linked to angiogenesis in ECs. Here we show that ecSOD expression is increased (2.3-fold) in a mouse hindlimb ischemia model of angiogenesis. Conditioned media from ECs overexpressing ecSOD using adenovirus (overexpression of ecSOD) significantly enhances VEGF-induced VEGFR2 autophosphorylation (2.1-fold), which is prevented by pretreatment with catalase (500 U/ml, 1 hr) that scavenges extracellular H2O2. VEGF induces phosphorylation of VEGFR2 at Tyr951, Tyr1054, Tyr1175 and Tyr1214, all of which are augmented by ecSOD-overexpression (2.7, 1.9, 1.5 and 1.6-fold, respectively). Consistent with this, overexpession of ecSOD significantly increases downstream VEGFR2 signaling linked to EC proliferation such as phosphorylation of PLCgamma (2.0-fold) and ERK1/2 (1.7-fold) as well as migratory signaling including paxillin, p130cas and p38 MAPK (1.4-, 1.9-, 1.7-fold, respectively). Functionally, modified Boyden chamber assay reveals that ecSOD overexpression significantly promotes VEGF-stimulated EC migration (1.7-fold, n=4), which is prevented by catalase pretreatment. In ecSOD-deficient mice, defective ischemia-induced neovascularization is associated with reduction of H2O2 production in ischemic tissue (95%), as measured by Amplex Red which detects extracellular H2O2. In summary, extracellular H2O2 generated by ecSOD facilitates VEGFR2 signaling at the site of receptor activation, thereby promoting angiogenesis in ECs. These findings provide insight into novel mechanism by which ecSOD promotes postnatal neovascularization in ischemic cardiovascular diseases.
This research has received full or partial funding support from the American Heart Association, AHA National Center.