Abstract 368: Dynamic Receptor-Ligand Activation of Nox1 Regulates Blood Flow via a Novel TSP1-CD47 Signaling Axis

Abstract

Reactive oxygen species (ROS) play an important role in ischemia-reperfusion injury but the mechanisms controlling this phenomenon remain elusive. Our previous studies demonstrated that matrix protein thrombospondin-1 (TSP1) at concentrations found in human disease (2.2 nM) is a rapid and potent stimulator of superoxide (O2•–) production in vascular smooth muscle cells (VSMC). However, the mechanism by which TSP1 mediates this response and its role in reperfusion-induced impairment of tissue perfusion are unknown. Here, we show that blockade of integrin-associated CD47 signaling (pmol O2•–/mg protein/min: 6.7±0.6, 10.2±0.7*, 6.0±0.5, and 7.1±0.7 for isotype control + vehicle, isotype control + TSP1, CD47 blocking antibody + vehicle and CD47 blocking antibody + TSP1; *p<0.05 vs isotype control + vehicle) and gene silencing of NADPH oxidase 1 (Nox1) abrogated TSP1-stimulated O2•– in VSMC. By luciferase reporter array, we identified myocyte enhancer factor 2 (MEF2) as a downstream effector of TSP1 and show that knockdown of MEF2A suppresses TSP1-stimulated O2•– (pmol O2•–/mg protein/min: 4.2±0.3, 6.3±0.4*, 4.1±0.3, and 4.6±0.3 for scrambled siRNA + vehicle, scrambled siRNA + TSP1, MEF2A siRNA + vehicle and MEF2A siRNA + TSP1;*p<0.05 vs scrambled siRNA + vehicle). Further, TSP1 rapidly phosphorylated the Nox organizer subunit p47phox in VSMC. Electron paramagnetic resonance demonstrated that TSP1 stimulates O2•– in wild type (WT), but not in Nox1y/-, endothelium-free aortic rings (CM• radical formation (x10 3): 20.9±11.1, 86.8±20.5*, 20.2±28.6 and 29.6±22.2 for WT + vehicle, WT + TSP1, Nox1y/- + vehicle, and Nox1y/- + TSP1; *p<0.05 vs WT + vehicle). Finally, intravenous TSP1 acutely decreased recovery of hindlimb blood flow following ischemia-reperfusion as measured by real time laser Doppler in control, but not in Nox1 gene silenced, rats. Our findings suggest a highly-regulated role of matricellular TSP1 as a stimulator of a novel CD47-MEF2A-Nox1 signaling axis and ROS and support a role for the matrix protein as a blood flow attenuator in cardiovascular disease.