published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

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Atherosclerotic cardiovascular disease is a leading cause of death in the United States and worldwide. While much progress has been made in investigating dyslipidemia and inflammation regarding atherosclerotic disease, much is still unknown about the role of endogenous vascular cells in atherosclerosis. More importantly, as targeting dyslipidemia and inflammation has yielded successful therapies, can therapeutically targeting vascular dysfunction enhance existing therapies for treating cardiovascular disease? In this thesis, I sought to investigate the role of the matrix metalloproteinase, ADAMTS7, a gene implicated in atherosclerosis by genome-wide association studies (GWAS). Subsequent to the human genetic studies associating ADAMTS7 with atherosclerotic cardiovascular disease, in vivo investigations demonstrated that ADAMTS7 is proatherogenic and induced in response to vascular injury. However, the mechanisms governing ADAMTS7's function and the causal cell type responsible for producing ADAMTS7 remain unclear. To determine where ADAMTS7 expression occurs in atherosclerosis, we interrogated the largest single-cell RNA sequencing dataset of human carotid atherosclerosis. We found ADAMTS7 expression in endothelial cells, smooth muscle cells (SMCs), fibroblasts, and mast cells. We subsequently created both endothelial and SMC-specific Adamts7 conditional knockout and transgenic mice. The conditional knockout of Adamts7 in either cell type is insufficient to reduce atherosclerosis, but transgenic induction in either cell type increases peripheral atherosclerosis. In SMC transgenic mice, this increase coincides with decreased plaque stability and an expansion of lipid-laden SMC foam cells. RNA sequencing in SMCs revealed an upregulation of lipid uptake genes typically assigned to macrophages. Subsequent experiments demonstrated that Adamts7 increases SMC oxLDL uptake through Cd36. Furthermore, Cd36 expression is increased due to an Adamts7-mediated increase in Spi1, a known myeloid cell fate transcription factor. In summary, Adamts7 is expressed by multiple vascular cell types during atherosclerosis, and in SMCs, Adamts7 promotes oxLDL uptake, thereby increasing SMC foam cell and atherosclerosis. While investigating ADAMTS7, we sought to identify a cell surface persistent marker of SMCs to aid investigations into ADAMTS7. SMCs play a central role in the development of atherosclerosis due in part to their capability to phenotypically transition into either a protective or harmful state. However, the ability to identify and trace SMCs and their progeny in vivo is limited due to the lack of well-defined SMC cell surface markers. Therefore, investigations into SMC fate must utilize lineage-tracing mouse models, which are time-consuming and challenging to generate and not feasible in humans. We, thus, employed CITE-seq to phenotypically characterize the expression of 119 cell surface proteins in mouse atherosclerosis. We found that CD200 is a highly expressed and specific marker of SMCs, which persists even with phenotypic modulation. We validated our findings using a combination of flow cytometry, qPCR, and immunohistochemistry, all confirming that CD200 can identify and mark SMCs and their derived cells in early to advanced mouse atherosclerotic lesions. Additionally, we describe a similar expression pattern of CD200 in human coronary and carotid atherosclerosis. Thus, CD200 is a lineage marker for SMCs and SMC-derived cells in mouse and human atherosclerosis. In conclusion, this body of work investigated the role of vascular cells in atherosclerosis. We have identified a new marker of SMCs, adding an additional tool that can be broadly employed to investigate the vasculature. In addition, we have mechanistically unraveled how one vascular GWAS hit, ADAMTS7, can perpetuate atherosclerosis. Our findings demonstrate that ADAMTS7 can promote foam cell expansion in atherosclerosis. While more work is needed to understand the role of these SMC foam cells in atherosclerosis, our investigations thus far have demonstrated that ADAMTS7 can greatly expand these cells. As such, our work supports the development of a drug to inhibit ADAMTS7 for treating atherosclerotic cardiovascular disease.

[Truncated abstract] The low density lipoprotein receptor-related protein (LRP), a member of the low-density lipoprotein (LDL) receptor gene family is involved in numerous biological processes including lipoprotein metabolism. This thesis concerns investigations into some aspects of LRP metabolism/regulation and possible roles in coronary artery disease (CAD). Specific aims were: to investigate the association between polymorphisms in the LRP gene and in its associated protein, the lipoprotein receptor-associated protein (RAP), with the risk of CAD; to extensively examine the influence of the LRP exon 22 C200T polymorphism on lipid metabolism; to develop and characterise assays for the mRNA expression of LRP and 2 other genes relevant to lipid metabolism, the LDL receptor (LDLR), and HMG CoA reductase (HMGCR); and finally, to apply the latter techniques to studies on the influence of genetic variation in LRP, and dietary and drug interventions, on LRP, LDLR and HMGCR mRNA expression in nucleated blood cells from healthy human subjects. Six hundred CAD subjects and 700 similarly aged controls were genotyped for 8 LRP gene polymorphisms as well as for the RAP V311M polymorphism. ... In the final phase of my studies, I examined the influence of 4 weeks therapy with a cholesterol lowering drug, an HMGCR inhibitor, atorvastatin (20mg daily), on the mRNA expression of LDLR, LRP and HMGCR in human nucleated blood cells. Twelve normal Caucasian male subjects aged 49 ? 5 (SD) years were studied. Plasma total cholesterol and LDL-C decreased by averages of 29 % and 41 % after the 4 week period. This was accompanied by an elevation in LDLR mRNA expression by approximately 30 35 %. In contrast, there was no significant effect on LRP and HMGCR mRNA expression. In conclusion, the original findings in this thesis included: demonstration of a strong influence of the LRP exon 22 C200T polymorphism on coronary artery disease and LDLR expression, but without a clear effect on fasting or postprandial lipid levels; data on the biological variation in LDLR and LRP gene expression in nucleated blood cells from normal subjects; the influence of an oral fat load on the expression viii of these genes, finding that LDLR was significantly depressed; and finally, the observation that statin therapy upregulated LDLR in nucleated blood cells.