Activation of the unfolded protein response by cholesterol enrichment in human endothelium

Abstract

Elevated blood cholesterol is a major risk factor for atherosclerosis, but how cholesterol affects the endothelium has not been well studied. The object of the present study was to determine whether hypercholesterolemia alters endothelial cell (EC) membrane cholesterol content in vivo, and whether excess membrane cholesterol might activate the unfolded protein response (UPR), also know as the ER stress response. In rabbits fed cholesterol for up to 12 days, EC membrane cholesterol content increased 2.3-fold, paralleling the increase in blood cholesterol, demonstrating that hypercholesterolemia leads to EC accumulation of excess cholesterol in-vivo. We mimicked this response in human umbilical vein EC by enriching cells in culture with cholesterol using a mixture of β-cyclodextrins and cholesterol-rich liposomes. This resulted in comparable enrichment with cholesterol and resulted in a 1.5-fold increase in the ER chaperon protein BiP, a hallmark of activation of the UPR, along with an increase in BiP mRNA determined using real-time PCR. We then measured the levels of the three known downstream UPR signaling proteins ATF4, ATF6 and XBP-1. While ATF4 and AFT6 protein and mRNA levels were decreased, spliced XBP-1 (active form of XBP-1) protein levels were elevated 13.8-fold. Thus, mobilization of the ER chaperone BiP and subsequent modulation of downstream UPR signaling proteins demonstrates activation of the UPR in EC by accumulation of cholesterol in EC membranes. These findings implicate a direct role for cholesterol in inducing ER stress in endothelium during the development of hypercholesterolemia and may contribute to EC participation in atherogenesis.