Abstract 453: Investigating The Role Of Glutathione During Macrophage Apoptotic Cell Clearance

Abstract

Atherosclerosis (ASVD) is characterized by the buildup of lipids and fibrous elements in large arteries creating a plaque. As it progresses, apoptotic cells (AC) accumulate forming a necrotic core making the plaque unstable and prone to rupture. Rupture can lead to artery occlusion and moreover to stroke or myocardial infarction (MI). A main contributor to a large necrotic core is the inability of macrophages to clear accumulated AC 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 and a large necrotic core are accelerated by a decrease in glutathione (GSH) availability by a deletion of the modifier subunit of Glutamate Cysteine Ligase (GCLM) in mice. Additionally, it has also been shown that a single nucleotide polymorphism (SNP) in the promoter region of human GCLM (-588 C/T) has an increased risk for MI. Our objective is to identify the role and mechanism of the GCLM SNP during the formation of the necrotic core in atherosclerosis. We hypothesize that proper function of GCLM and GSH is important in macrophages for adequate function and clearance of AC in ASVD. Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from blood donors, genotyped to test for the presence of SNP, and used for RNA sequencing. Efferocytic index was measured using labelled AC in GCLM WT, Het, and KO murine bone marrow derived macrophages (BMDM) and PBMCs. Percent positive macrophages containing fluorescently labelled AC were quantified for each sample. Cells were collected for total protein, surface receptor expression, and mRNA analysis. Results: Efferocytosis was affected in macrophages with defective GCLM as compared to control. RNA analysis has showed a decrease in efferocytosis receptors such as MerTK, confirmed by cell surface expression. Conclusion: These results suggest that loss of GCLM and GSH in macrophages impairs adequate efferocytosis.