Abstract 447: Foam Cell Formation and Triglyceride Production After Incubation of Human Monocytes and Endothelial Cells with MAA-Modified Proteins

Abstract

Introduction: Oxidized proteins and the formation of foam cells are central aspects in the development and progression of atherosclerosis. Malondialdehyde acetaldehyde (MAA) modified low density lipoprotein (LDL) is generated when lipoproteins are exposed to the oxidative product malondialdehyde. These MAA-adducted proteins have been shown to bind scavenger receptors, up-regulate adhesion molecules, induce cytokine expression, and initiate antibody and T-cell responses in both human and animal models of atherosclerotic disease. However, the induction of foam cells using these antigens has been untested. Objective: The purpose of this study was to evaluate the ability of MAA-adducted proteins on LDL to induce foam cell formation and upregulate innate inflammatory mediators of atherosclerosis in human peripheral monocytes, mouse macrophage, and human aortic endothelial cell lines. Methods: Mouse aortic endothelial (2167), mouse macrophage (J774) and human peripheral monocytes were incubated with LDL, oxidized-LDL, MAA-LDL, or MAA-human albumin (25 and 50 ng/ml) for 24 or 48 hours and evaluated. Cells were accessed for uptake using Oil Red O, triglyceride production, and cholesterol esters using filipin. Real-time semi-quantitative RT-PCR and ELISA was performed to evaluate IL-6 and monocyte chemotactic protein-1 (MCP-1) mRNA expression and protein secretion. MAA binding was also assessed using an anti-MAA monoclonal antibody. Results: LDL uptake is increased in aortic endothelial cells, J774 and human monocytes as evidenced by filipin and Oil Red O staining for esterified cholesterol LDL as compared to sham controls. Triglyceride data show a 5.1 fold increase in monocytes and a 6 fold increase in aortic endothelial cells increase following MAA-LDL incubation over control p<0.001. Additionally, IHC for MAA-adducted proteins after incubation with MAA-modified demonstrate surface binding and intracellular uptake of MAA-adducted proteins. Incubation of MAA-albumin and MAA-LDL with aortic endothelial cells results in significant IL-6 and MCP-1 mRNA synthesis and protein expression 5 fold for IL-6 p<0.001 and 3 fold for MCP-1 p<0.01 Conclusions: These data show that MAA-modified proteins bind to and are processed by endothelial cells, macrophage, and human monocytes. MAA-modified proteins result in the production of innate immune mediators of tissue inflammation and inflammation recruitment. The direct effect of MAA in forming “foam cells” as evidenced by increased triglyceride and cholesterol is supportive for a role of MAA-modified proteins in the progression of atherosclerosis. The nature and impact of MAA-modification is hypothesized to be central to cellular apoptosis, autophagy and/or necrosis. Implications: MAA-modified proteins may be directly pathogenic in the development of plaque inflammation and progression of atherosclerosis.