The Impact of Gclm on Efferocytosis and Atherosclerotic Progression

Abstract

Atherosclerosis is the leading cause of coronary artery disease (CAD) and stroke which are the primary contributors to cardiovascular disease related deaths in the world. This is an inflammatory disease characterized by the accumulation of lipid and fibrous elements in large arteries resulting in the development of a plaque. Progression of the disease leads to the accumulation of apoptotic cells and formation of a necrotic core making the plaque unstable and prone to rupture. Rupture can lead to artery occlusion resulting in stroke or myocardial infarction (MI). One of the main contributors to a large necrotic core is macrophages that are unable to clear the accumulated apoptotic cells and as a result undergo secondary necrosis. This clearance process, referred to as efferocytosis, is impaired in advanced atherosclerotic lesions. It has been shown that the process that leads to a thin fibrous cap (characteristic of an unstable plaque) and a large necrotic core are accelerated by a decrease in glutathione (GSH) availability due to a deletion on the modifier subunit of Glutamate Cysteine Ligase (GCLM). The objective of this study is to identify the role and mechanism of GSH during the formation of the necrotic core in atherosclerosis. We hypothesize that availability of GSH is important in macrophages for adequate efferocytosis of apoptotic cells during atherosclerosis progression.MethodsTriglycerides, total cholesterol, and HDL levels were measured on PCSK9 virus driven atherosclerosis in wild type and heterozygous GCLM mice. Sections of the aortic root from these mice were stained with Movat Pentachrome to measure plaque area and percent necrosis. An efferocytic index was measured using PHK26 labelled apoptotic cells (AC). AC were added to cultured bone marrow derived macrophages (BMDM) allowing for their uptake. Percent positive macrophages containing a fluorescently labelled AC were quantified. BMDM from WT, Het, and KO GCLM mice were collected for total protein, surface receptor expression, and mRNA analysis.ResultsEfferocytosis was affected in KO mice as compared to WT and Het BMDM. RNA analysis has showed a decrease in efferocytosis receptors such as MerTK, confirmed by cell surface expression.ConclusionThese results suggest that a loss of GCLM and GSH during efferocytosis impairs adequate efferocytosis.