Objectives Endothelial cell injury is a key event in the pathogenesis of atherosclerosis. There is now increasing evidence that excess lipid accumualtion can induce apoptosis which is thought to be triggered through the endoplasmic reticulum (ER) stress pathway in macrophages. ATP binding cassette transporter G1 (ABCG1) is a regulator of cholesterol efflux to HDL and highly expressed in endothelial cells. In the present study, we were focused on whether ABCG1 deficiency was involved in endothelial apoptosis, and whether it was associated with intracellular lipid content and ER stress.
Methods Human umbilical artery endothelial cells (HUAEC ) were transfected with ABCG1 siRNA or scramble siRNA and ABCG1 expression plasmid or empty vector. Twenty four hours after transfection, ABCG1, GRP78 and CHOP protein expression were measured by Western blot. Cholesterol efflux to HDL was determined by means of scintillation counting. Total intracellular cholesterol was determined by gas-liquid chromatography. Endothelial apoptosis was detected by flow cytometry analysis.
Results We observed that silencing of endothelial ABCG1 reduced cholesterol efflux to HDL by 40% and increased intracellular lipid content by 20%. Moreover, reduction of ABCG1 promoted endothelial apoptosis and increased ER stress-related molecules GRP78 and CHOP. In contrast, transfection of ABCG1 overxpression plasmid reversed endothelial apoptosis (70% decrease) and intracellular lipid accumulation as well as decreased expression of GRP78 and CHOP in ABCG1-deficient EC. In addition, endothelial apoptosis and ER stress-related molecules were induced when EC were loaded with soluble cholesterol using cholesterol-loaded cyclodextrin, otherwise endothelial apoptotic response and expression of GRP78 and CHOP were suppressed when depletion of cellular cholesterol by cyclodextrin in ABCG1-deficient endothelial cells. Furthermore, PBA, which has been shown to alleviate ER stress, and U18666A, which has been shown to inhibit cholesterol traffic to ER, both protected against apoptosis showed in ABCG1-deficient EC in vitro.
Conclusions The present results suggest that downregulation of ABCG1 induces endothelial apoptosis, which seems associated with intracellular cholesterol accumulation regulated by ABCG1 deficiency and subsequent ER stress activition.