Complement Protein C1q in Atherosclerosis

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PROJECT SUMMARY Complement Protein C1q in Atherosclerosis Atherosclerosis is a chronic inflammatory disorder which in early stages is characterized by the migration of modified lipoproteins and macrophages into the arterial intima and the formation and apoptosis of macrophage foam cells. In late stages of disease, inadequate/defective apoptotic foam cell removal by macrophages leads to their secondary necrosis and plaque formation. Damage to this plaque by pro-inflammatory cytokines, proteases and oxygen radicals can cause rupture and thrombus formation, and acute clinical complications such as myocardial infarction and ischemic stroke. Thus, inflammation, macrophages and vascular integrity are key in progression of this disease. Activation of complement has been shown to contribute to inflammation and exacerbate pathology. However, studies in mice deficient in the first protein in the classical complement pathway, C1q, suggest that this protein actually has a protective role in the early atherosclerotic lesion. Innate immune protein C1q is not only able to trigger the inflammatory complement cascade, but is also a pattern recognition receptor that opsonizes targets and directly interacts with phagocytes and other cells. Interaction activates responses including phagocytosis of targets such as apoptotic cells or damaged molecules, and modulation of cytokine and gene expression. Therefore, our central hypothesis is that complement-independent actions of C1q program protective, anti-atherosclerotic cellular responses in atherosclerosis. Our recent studies have demonstrated C1q modulation of macrophage inflammatory polarization in vitro and in vivo models of atherosclerosis and identified several molecular mechanisms involved. These studies provided preliminary data that C1q modulates vascular endothelial responses in atherosclerosis. In addition, data suggest that C1q increases levels of bioactive oxysterols 24- and 25- hydroxycholesterol by macrophages in response to hyperlipidemic conditions in vitro and in vivo. The goal of this project is to broaden our understanding of C1q molecular interactions beyond macrophages to the entire lesional environment. Specific aims are: 1: Investigate C1q modulation of vascular responses in atherosclerosis. We will test the hypothesis that C1q reduces monocyte chemotaxis and improves vascular integrity in atherosclerosis. This will be tested using primary human monocytes and vascular endothelial cells to perform chemotaxis, transendothelial migration and permeability assays. In addition, a comprehensive exploration of chemokines and adhesion molecules produced by macrophages and vascular endothelial cells in response to modified LDL in the presence or absence of C1q will be performed using human cells and in plasma harvested from C1q sufficient and deficient hyperlipidemic mice. Specific Aim 2: Investigate C1q modulation of lipid metabolism in atherosclerosis. These studies will test the hypothesis that C1q modulation of oxysterols is involved in macrophage foam cell survival and polarization. Oxysterol levels will be measured in vitro, in primary human macrophages, and in vivo in plasma from a mouse model of hyperlipidemia, using mass spectrometry. Survival and polarization assays will be performed in specific pathway-deficient macrophages to identify their relative importance in these biological responses. Overall, these studies aim to explore the dual role that C1q plays in atherosclerosis, and should assist in identifying novel molecular pathways for therapeutic targeting in this disease.