title: Extracellular SOD-derived H2O2 promotes VEGF signaling in caveolae/lipid rafts and post-ischemic angiogenesis in mice.
abstract: Reactive oxygen species (ROS), in particular, H(2)O(2), is essential for full activation of VEGF receptor2 (VEGFR2) signaling involved in endothelial cell (EC) proliferation and migration. Extracellular superoxide dismutase (ecSOD) is a major secreted extracellular enzyme that catalyzes the dismutation of superoxide to H(2)O(2), and anchors to EC surface through heparin-binding domain (HBD). Mice lacking ecSOD show impaired postnatal angiogenesis. However, it is unknown whether ecSOD-derived H(2)O(2) regulates VEGF signaling. Here we show that gene transfer of ecSOD, but not ecSOD lacking HBD (ecSOD-DeltaHBD), increases H(2)O(2) levels in adductor muscle of mice, and promotes angiogenesis after hindlimb ischemia. Mice lacking ecSOD show reduction of H(2)O(2) in non-ischemic and ischemic limbs. In vitro, overexpression of ecSOD, but not ecSOD-DeltaHBD, in cultured medium in ECs enhances VEGF-induced tyrosine phosphorylation of VEGFR2 (VEGFR2-pY), which is prevented by short-term pretreatment with catalase that scavenges extracellular H(2)O(2). Either exogenous H(2)O(2) (<500 microM), which is diffusible, or nitric oxide donor has no effect on VEGF-induced VEGFR2-pY. These suggest that ecSOD binding to ECs via HBD is required for localized generation of extracellular H(2)O(2) to regulate VEGFR2-pY. Mechanistically, VEGF-induced VEGFR2-pY in caveolae/lipid rafts, but non-lipid rafts, is enhanced by ecSOD, which localizes at lipid rafts via HBD. One of the targets of ROS is protein tyrosine phosphatases (PTPs). ecSOD induces oxidation and inactivation of both PTP1B and DEP1, which negatively regulates VEGFR2-pY, in caveolae/lipid rafts, but not non-lipid rafts. Disruption of caveolae/lipid rafts, or PTPs inhibitor orthovanadate, or siRNAs for PTP1B and DEP1 enhances VEGF-induced VEGFR2-pY, which prevents ecSOD-induced effect. Functionally, ecSOD promotes VEGF-stimulated EC migration and proliferation. In summary, extracellular H(2)O(2) generated by ecSOD localized at caveolae/lipid rafts via HBD promotes VEGFR2 signaling via oxidative inactivation of PTPs in these microdomains. Thus, ecSOD is a potential therapeutic target for angiogenesis-dependent cardiovascular diseases.
authors.affiliation: Department of Pharmacology, University of Illinois at Chicago, Center for Lung and Vascular Biology, Illinois, United States of America.
authors.forename: Jin
authors.initials: J
authors.lastname: Oshikawa
authors.forename: Norifumi
authors.initials: N
authors.lastname: Urao
authors.forename: Ha Won
authors.initials: HW
authors.lastname: Kim
authors.forename: Nihal
authors.initials: N
authors.lastname: Kaplan
authors.forename: Masooma
authors.initials: M
authors.lastname: Razvi
authors.forename: Ronald
authors.initials: R
authors.lastname: McKinney
authors.forename: Leslie B
authors.initials: LB
authors.lastname: Poole
authors.forename: Tohru
authors.initials: T
authors.lastname: Fukai
authors.forename: Masuko
authors.initials: M
authors.lastname: Ushio-Fukai
mesh_terms: D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D002465:Cell Movement; D049109:Cell Proliferation; D042783:Endothelial Cells / Q000166:cytology; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007511:Ischemia / Q000473:pathology*; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D018919:Neovascularization, Physiologic; D010084:Oxidation-Reduction; D010766:Phosphorylation; D015398:Signal Transduction; D013482:Superoxide Dismutase / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D042442:Vascular Endothelial Growth Factors / Q000378:metabolism*
chemical_list: D042442:Vascular Endothelial Growth Factors; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D040301:Vascular Endothelial Growth Factor Receptor-2