Abstract 3692: A Novel Mechanism of Endothelial Activation by Cholesterol: Promoting the Translocation of Adhesion Molecules Out of Membrane Caveolae
Hypercholesterolemia and inflammation are the two most predominant risk factors in atherosclerosis. Membrane microdomain caveolae and its structural protein caveolin-1 are abundant in vascular endothelial cells (ECs) and have been suggested to contribute to cholesterol trafficking and atherogenesis. In this study, we assessed the hypothesis that cholesterol and inflammation synergistically promote monocyte adhesion to ECs, in which membrane caveolae plays a pivotal role. In human umbilical vein ECs, the treatment of cholesterol enhanced bacterial endotoxin LPS-induced monocytes adhesion as demonstrated by co-culture with fluorescence-labeled THP-1 monocyte-like cells. The protein levels of the cell adhesion molecules (CAMs), including ICAM-1 and VCAM-1, were not altered by the treatment of cholesterol for 2 hours. Using sucrose density gradient centrifugation to isolate caveolae-enriched membranes, we found that CAMs partially localized in caveolae and LPS increased their expression and localization proportionally. Interestingly, cholesterol exposure decreased the amount of CAMs in caveolae, whereas cholesterol deprivation revised the process. Results from co-immunoprecipitation and confocal microscopy revealed that ICAM-1 was physically associated with caveolin-1, and that cholesterol exposure decreased such association and promoted ICAM-1 moving out of caveolae. Further study showed that cholesterol dose-dependently induced the disassociation of ICAM-1 with caveolin-1 in a GST-caveolin-1 pull down experiment. In vivo, we fed ApoE−/− mice with high fat diet to induce hypercholesterolemia and then treated with LPS for 8 hrs. Comparing with control, a decrease in the co-localization of ICAM-1 and caveolin-1 was observed in aortic endothelium from ApoE−/−mice by en face immunostaining. These results suggest that caveolin-1 negatively regulates the monocyte adhesion by directly binding with CAMs in caveolae, and that cholesterol modulates the function of CAMs through disturbing their caveolae localization.