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.

Chan, Yuet Wa. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 217-232). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Cheung, Wing Shing David. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 201-217). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Ng, Chun Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 134-150). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vii / Table of Contents --- p.ix / Abbreviations --- p.xii / List of Figures --- p.xv / List of Tables --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- "Introduction to Cardiovascular Disease, Hypertension and Atherosclerosis" --- p.1 / Chapter 1.1.1 --- Cardiovascular Disease --- p.1 / Chapter 1.1.2 --- Hypertension --- p.2 / Chapter 1.1.2.1 --- Background --- p.2 / Chapter 1.1.2.2 --- Causes of Hypertension --- p.3 / Chapter 1.1.2.3 --- Current Western Management and Medication --- p.6 / Chapter 1.1.3 --- Atherosclerosis --- p.9 / Chapter 1.1.3.1 --- Background --- p.9 / Chapter 1.1.3.2 --- Pathogenesis of Atherosclerosis --- p.10 / Chapter 1.1.3.3 --- Current Western Treatment and Medication --- p.12 / Chapter 1.2 --- Selection and Introduction of Current Chinese Medicine Formula --- p.16 / Chapter 1.2.1 --- Cardiac Syndrome in Traditional Chinese Medicine --- p.16 / Chapter 1.2.2 --- Traditional Chinese Medicine as an Complementary or Alternative Medicine --- p.17 / Chapter 1.2.3 --- Selection of TCM Formula from Pharmacopoeia --- p.18 / Chapter 1.2.3.1 --- Compound Formula --- p.18 / Chapter 1.2.4 --- Introduction to Constitutional Herbal Medicine --- p.19 / Chapter 1.2.4.1 --- Danshen (Radix Salviae miltiorrhizae) --- p.19 / Chapter 1.2.4.2 --- Gegen (Radix Puerariae lobatae) --- p.20 / Chapter 1.2.4.3 --- Yanhusuo (Rhizoma Corydalis) --- p.21 / Chapter 1.2.4.4 --- Composition of the Final Formula Used in the Present Study --- p.21 / Chapter 1.2.5 --- Previous work on Danshen-Gegen Formula and its limitations --- p.22 / Chapter 1.3 --- Objectives of the Present Study --- p.25 / Chapter 1.3.1 --- Research Plan --- p.26 / Chapter Chapter 2 --- Experimental Design and General Methodology --- p.27 / Chapter 2.1 --- Source and Authentication of Raw Herbs --- p.27 / Chapter 2.2 --- Materials --- p.29 / Chapter 2.3 --- Ethical Approval --- p.31 / Chapter 2.4. --- General Methods --- p.32 / Chapter 2.4.1 --- Blood Pressure Measurement --- p.32 / Chapter 2.4.2 --- Blood Profile Measurement --- p.33 / Chapter 2.4.3 --- Vascular Reactivity Studies --- p.36 / Chapter 2.5 --- Statistical Analysis --- p.38 / Chapter Chapter 3 --- Anti-hypertensive Studies of Danshen-Gegen Formula on Rat --- p.39 / Chapter 3.1 --- Introduction --- p.39 / Chapter 3.1.1 --- In vivo Anti-Hypertensive Studies --- p.39 / Chapter 3.1.1.1 --- Spontaneously Hypertensive Rat (SHR) --- p.40 / Chapter 3.1.1.2 --- Tail-cuff Blood Pressure Measurement --- p.41 / Chapter 3.1.2 --- Detailed Underlying Mechanistic Studies --- p.42 / Chapter 3.1.2.1 --- Nitric Oxide-mediated Vasodilation --- p.42 / Chapter 3.1.2.2 --- Prostacyclin-mediated Vasodilation --- p.43 / Chapter 3.1.2.3 --- Hyperpolarization-mediated Vasodilation --- p.43 / Chapter 3.1.2.4 --- Endothelium-dependent/-independent Vasodilation --- p.46 / Chapter 3.1.3 --- Long Term Underlying Mechanistic Studies --- p.48 / Chapter 3.2 --- Methods --- p.49 / Chapter 3.2.1 --- In vivo Anti-Hypertensive Studies --- p.49 / Chapter 3.2.2 --- Detailed Underlying Mechanistic Studies --- p.51 / Chapter 3.2.3 --- Long Term Underlying Mechanistic Studies --- p.53 / Chapter 3.2.4 --- Statistical analysis --- p.56 / Chapter 3.3 --- Results --- p.58 / Chapter 3.3.1 --- In vivo Anti-Hypertensive Studies --- p.58 / Chapter 3.3.1.1 --- Preventive Effect in Hypertension --- p.58 / Chapter 3.3.1.2 --- Therapeutic Effect in Hypertension --- p.62 / Chapter 3.3.2 --- Detailed Underlying Mechanistic Studies --- p.66 / Chapter 3.3.2.1 --- DG extract-induced Vasodilation --- p.66 / Chapter 3.3.2.2 --- Endothelium-independent Vasodilation --- p.67 / Chapter 3.3.2.3 --- Nitric Oxide-mediated Vasodilation --- p.68 / Chapter 3.3.2.4 --- Prostacyclin-mediated Vasodilation --- p.69 / Chapter 3.3.2.5 --- Hyperpolarization-mediated Vasodilation --- p.70 / Chapter 3.3.3 --- Long Term Underlying Mechanistic Studies --- p.74 / Chapter 3.4 --- Discussion --- p.79 / Chapter Chapter 4 --- Anti-atherosclerosis Studies of Danshen-Gegen Formula in Rabbits --- p.89 / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.1.1 --- Intima-Media Thickening --- p.89 / Chapter 4.1.2 --- Effect of High Cholesterol Diet in Rabbit --- p.90 / Chapter 4.1.3 --- Thiobarbituric Acid Reactive Substances --- p.91 / Chapter 4.2 --- Methods --- p.93 / Chapter 4.2.1 --- Pilot Study for Establishment of Experimental Protocol --- p.93 / Chapter 4.2.2 --- Effect of DG extract on Intima-media Thickening --- p.97 / Chapter 4.2.3 --- Statistical analysis --- p.99 / Chapter 4.3 --- Result --- p.100 / Chapter 4.3.1 --- Study of the Anti-atherosclerosis Effect of DG extract - First Run --- p.100 / Chapter 4.3.2 --- Study of the Anti-atherosclerosis Effect of DG extract - Second Run --- p.108 / Chapter 4.4 --- Discussion --- p.117 / Chapter Chapter 5 --- General Discussion and Conclusion --- p.122 / Chapter 5.1 --- Significance of the Study --- p.122 / Chapter 5.2 --- Limitations and Future work --- p.127 / Chapter 5.3 --- Clinical Implication of the Use of the DG Preparations for Patients with CVD --- p.132 / References --- p.134

Indiana University-Purdue University Indianapolis (IUPUI) / Cardiovascular disease is the number one cause of death for Americans. New treatments are needed for serious conditions like atherosclerosis, as it can lead to stroke and heart attack. Many types of cells contribute to the progression of cardiovascular disease, including smooth muscle cells that comprise the middle layers of arteries. Inappropriate growth and migration of smooth muscle cells into the lumen of arteries has been implicated in vascular diseases. Death associated protein kinase (DAPK) is a protein that has been found to regulate the survival and migration of cancer cells, but has not been well characterized in vascular cells. The objective of this work was to determine the signaling pathways that DAPK regulates in smooth muscle cells. These studies have focused on smooth muscle cells isolated from human coronary arteries (HCASM cells). We have determined that HCASM cells depleted of DAPK exhibit more rapid migration, showing that DAPK negatively regulates migration of vascular cells. Results from a focused RT-PCR array identified matrix metalloproteinase 9 (MMP9) as a gene that is increased in cells depleted of DAPK. MMP9 is an important enzyme that degrades collagen, a component of the extracellular matrix through which smooth muscle cells migrate during atherosclerosis. We found that DAPK regulates phosphorylation of the NF-kappa B transcription factor p65 at serine 536, a modification previously found to correlate with increased nuclear levels and activity of p65. In DAPK-depleted HCASM cells, there was more phosphorylation of p65, which causes increased MMP9 promoter activity. Additional experiments were conducted using transgenic mice in which the DAPK gene has been deleted. By studying these mice, we have determined that under some circumstances DAPK augments maximal MMP9 levels in mouse carotid arteries which have been injured by ligation surgery via other signaling pathways. MMP9 has been previously implicated as a protein that promotes vascular diseases such as atherosclerosis. Our research in identifying DAPK as a regulator of MMP9 expression identifies a new target for treatment of vascular diseases like atherosclerosis.