Abstract 13827: p66Shc, a Longevity Adaptor Protein, is Involved in ROS-Dependent Activation of VEGF Receptor 2 and AMP Kinase: Role in VEGF-Induced Energy Metabolism and Angiogenesis
p66Shc is a master regulator of mitochondrial reactive oxygen species (ROS)(mtROS) production involved in aging and cardiovascular diseases.We demonstrated that ROS derived from NADPH oxidase (Nox) are required for VEGF receptor 2 autophosphorylation (VEGFR2-pY) and endothelial cells (ECs) proliferation. AMPK is an oxidant-sensing protein that regulates energy metabolism and VEGF-induced angiogenesis in ECs. However, a role of p66Shc in ROS-dependent VEGFR2-AMPK signaling remains unknown. Here we show that VEGF stimulation increases Ser36-phoshorylation of p66Shc (pS36-p66Shc) within 5 min with a peak at 15min (3.1-fold), which is required for VEGF-induced mtROS production, VEGFR2-pY and EC proliferation. Nox inhibition by apocynin attenuates VEGF-induced mtROS production. Mitochondria-targeted catalase blocks Nox-dependent VEGF-induced VEGFR2-pY (67 %), suggesting that the Nox/pS36-p66Shc/mtROS axis activates VEGFR2. Moreover, depletion of p66Shc with siRNA or overexpression of pS36-p66Shc-defective mutant p66Shc (S36A) significantly inhibits VEGF-induced phosphorylation of AMPK and its downstream acetyl-CoA Carboxylase (ACC), a key enzyme for ATP production, at the later phase (>=15 min), which is associated with inhibition of pS36-p66Shc and mtROS production. Mechanistically, VEGF promotes p66Shc binding to AMPK as well as Cys oxidation of AMPK, as measured by novel Cys-OH trapping reagent. This AMPK oxidation is required for its activation and inhibited by p66Shc siRNA. As its consequence, depletion of p66Shc inhibits VEGF-induced AMPK-ACC downstream energy metabolic pathways including intracellular NAD+/NADH ratio (41%), ATP production (63%), deacetylation PGC-1alpha, a key mediator for angiogenesis and mitochondrial biogenesis. In vivo, p66Shc expression is robustly increased in mice hindlimb ischemia model, and local gene transfer of adenovirus expressing p66Shc(S36A) significantly inhibits ischemia-induced blood flow recovery (35%). In summary, p66Shc functions as a central organizer to link Nox-derived ROS to mtROS, which activate VEGFR2 and AMPK, thereby promoting sustained VEGF signaling involved in energy metabolism and angiogenesis.
- © 2011 by American Heart Association, Inc.