Critical role of lipid raft redox signaling platforms in endostatin-induced coronary endothelial dysfunction

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, gp91(phox) and p47(phox) 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 vasodilation in isolated small coronary arteries, which could be partially reversed by LR disruptors. Conclusions - The early injury effect of EST on the vascular endothelium is associated with the formation of redox signaling platforms via lipid raft clustering.