Critical Role of Lipid Raft Redox Signaling Platforms in Endostatin-Induced Coronary Endothelial Dysfunction

Abstract

Objective— Endostatin (EST) was found to initiate a redox signaling cascade associated with activation of NADPH oxidase in endothelial cells (ECs). The present study tested whether EST stimulates clustering of ceramide-enriched lipid rafts (LRs), which assembles and activates NADPH oxidase to form redox signaling platforms. Methods and Results— Using confocal microscopy, we first demonstrated a colocalization of LR clusters with NADPH oxidase subunits, gp91phox and p47phox in the ECs membrane on EST stimulation. Immunoblot analysis of floated detergent-resistant membrane fractions found that in LR fractions NADPH oxidase subunits gp91phox and p47phox are enriched and that the activity of this enzyme increased dramatically, as measured by electron spin resonance (ESR) spectrometry. This EST-increased LR platform formation was shown to be attenuated by inhibition or RNA interference of acid sphingomyelinase (A-SMase). Functionally, EST pretreatment significantly impaired bradykinin or A23187-induced vasodil... Besides its proapototic effects, EST is also able to induce endothelial dysfunction. This early-stage action of EST is associated with LR clustering and consequent assembling and activation of NADPH oxidase.