The role of ACAT2 in the initiation and treatment of atherosclerosis.

摘要

Hepatic acyl coenzyme A:cholesterol acyltransferase 2 (ACAT2) synthesizes cholesteryl ester (CE) from free cholesterol and fatty acyl-CoA. ACAT2 is responsible for the packaging of cholesteryl oleate (CO) into VLDL secreted from the liver and studies have shown a relationship between both ACAT2 activity and CO content of apoB-containing lipoproteins with atherosclerosis. The purpose of the studies was to extend our understanding of ACAT2 and primarily to elucidate its role in the initiation and treatment of atherosclerosis. To elucidate it's role in the initiation of atherosclerosis we proposed that as a result of elevated ACAT2 activity, more CO would be packaged into the low-density lipoprotein (LDL) core and resulting in a particle with a greater likelihood to be retained in the artery wall. To determine it's role in the treatment of atherosclerosis, we utilized an antisense oligonucleotide (ASO) to knockdown ACAT2 expression in the liver to determine how depleting atherogenic particles of CO would effect atherosclerotic lesion regression.;To study the effects of ACAT2 dependent enrichment of LDL particles in CO, we utilized B-100 only, LDLr-/- mice that had a functional or dysfunctional ACAT2 gene. We fed the mice diets enriched in either cis-monounsaturated fatty acids (Mono) or n-3 polyunsaturated fatty acids (Fish) to yield four distinct LDL particles of varying composition. We then undertook the task of developing a novel real-time binding assay using the properties of surface plasmon resonance (SPR) from a Biacore machine to measure the affinity of the particles to interact with immobilized Biglycan (BGN), a proteoglycan known to interact and retain LDL particles in the artery wall. We found that particles enriched in CO, produced by the mice fed the Mono diet had the highest binding affinity to BGN. When the LDL particle was depleted in CO by genetic deletion of ACAT2 or administration of the Fish diet, there was a substantial reduction in binding to BGN. Furthermore, when taken out to 16 weeks diet, the experimental mice producing LDL particles with the highest binding affinity developed significantly more atherosclerosis. We concluded that enrichment of the LDL particle with CO, the product of hepatic ACAT2, results prolonged retention in artery wall and resulting in more atherosclerosis.;To determine the role of ACAT2 in regression of atherosclerosis, we again utilized the B-100 only, LDLr-/- mouse model. Mice were placed on the Mono diet for a period of 24 weeks to induce the formation of a complicated lesion. After 24 weeks mice either continued on the Mono diet with no treatment, or received biweekly injections of a nonspecific ASO or an ACAT2 ASO to knockdown ACAT2 expression in the liver. A fourth group of mice were switched to the Fish diet and the ASO and dietary treatment periods lasted for 16 weeks. Important for this study was the development of a novel imaging protocol that utilized a 7T MRI to non-invasively track both plaque progression and regression in the aortic arch of the mice. We found that 24 weeks on the Mono diet successfully induced complicated plaques in the arch that were detectable by MRI as early as 12 weeks into the progression phase. Treatment for 16 weeks with ACAT2 ASO was unsuccessful at regressing the size of the plaque compared to baseline; however, the final four weeks of treatment showed a significant reduction in plaque size back to baseline values just prior to treatment. There were no differences in macrophage or lipid content of the lesions amongst groups as evaluated by histology; however, CE measurements in the aorta revealed no additional CE deposition in mice treated with ACAT2 ASO or fish oil compared to aortas after induction. We concluded that hepatic knockdown of ACAT2 successfully depleted the LDL particles of CO and arrested plaque development.