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.


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 gp91(phox) and p47(phox) 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.
Jin
Si
S
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, 410 N 12th, Richmond, VA 23298, USA.
Zhang
Yang
Y
Yi
Fan
F
Li
Pin-Lan
PL
D000704:Analysis of Variance; D000818:Animals; D001920:Bradykinin; D000001:Calcimycin; D002417:Cattle; D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism; D016000:Cluster Analysis; D003327:Coronary Disease / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D003331:Coronary Vessels / Q000378:metabolism* / Q000473:pathology*; D004108:Dilatation, Pathologic / Q000139:chemically induced / Q000378:metabolism; D043169:Endostatins; D042783:Endothelial Cells / Q000378:metabolism*; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D018613:Microscopy, Confocal; D019255:NADPH Oxidases / Q000378:metabolism; D010084:Oxidation-Reduction; D011336:Probability; D012313:RNA / Q000032:analysis; D015398:Signal Transduction
D002518:Ceramides; D043169:Endostatins; D000001:Calcimycin; D012313:RNA; D019255:NADPH Oxidases; D001920:Bradykinin