Date of Graduation

12-2015

Document Type

Dissertation (PhD)

Program Affiliation

Biomedical Sciences

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Yong-jian Geng,M.D., Ph.D.

Committee Member

Jianping Jin , Ph.D.

Committee Member

Melvin E Klegerman, Ph.D.

Committee Member

Ba-bie Teng, Ph.D.

Committee Member

Chu-Huang "Mendel" Chen, M.D.,Ph.D.

Committee Member

Mikael J,Akesson-Wassler, Ph.D.

Abstract

Background: Calcification occurs often in the atherosclerotic lesions of patients with coronary heart disease and animals with hypercholesterolemia, such as apolipoprotein-E deficient (ApoE-/-) mice. However, the mechanism(s) underlying the development of calcification in atherosclerosis remains unclear. ApoE acts as a lipid transporter, but also has been recognized as a potential regulator of osteogenesis. Little information is available as to whether ApoE has any direct impact on osteogenesis and calcification in vascular smooth muscle cells (VSMC). Several signal transduction pathways play a role in regulation of calcification, including the Wnt/β-catenin system and potentially GTAP, an ubiquitin-conjugating enzyme responsible for protein degradation, which participates in the homeostasis of proteins and metabolites in stem cells. The long-term goal of this study is to delineate the molecular pathway(s) by which ApoE regulates expression of osteogenic genes and calcification in vascular VSMC of atherosclerotic lesions. Hypothesis: ApoE functions as an inhibitor of vascular calcification, through regulation of the function of Wnt/β-catenin and GTAP signaling pathways. 4

Methods and Results: ApoE-/- and wild type control mice were used for histopathology and cell culture studies. Compared to the aortic tissue and VSMCs of wild type C57BL/6J mice, ApoE-/- aortic tissue and VSMCs from ApoE-/- mice showed markedly increased calcification, and enhanced expression of pro-osteogenic genes but lowered expressions of myogenic biomarkers. Incubation of VSMCs with native ApoE protein added to culture or ApoE cDNA delivery by plasmid transfection or lentiviral infection reduced the levels of osteogenic genes (e.g. Runx2, BMP-2 and ALP) as well as calcification in ApoE-/- VSMCs exposed to elevated inorganic phosphate (Pi). The genetically modified VSMCs isolated from ApoE-/- mice displayed inceased activation of the canonical Wnt/β-catenin pathway at the presence of Pi, which could be reversed by incubation with native ApoE protein in culture. The GTAP-deficient aortic rings developed more severe calcification when exposed to Pi. Moreover, the half-life of the osteogenesis-regulator Runx2 shortened in VSMCs with GTAP overexpression.

Conclusion: ApoE serves as a critical inhibitor of vascular calcification through the Wnt/β-catenin pathway. This study provides direct evidence that ApoE regulates calcification by regulating expression of certain pro-osteogenic factors, such as Runx2, BMP2 and ALP. Besides, GTAP is identified as a novel regulator of osteogenesis by mediating Runx2 protein stability.

Keywords

atherosclerosis, vascular calcification, ApoE, Wnt signaling, ube2q1, ubiquitylation

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