Access Type

Open Access Dissertation

Date of Award

January 2017

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biochemistry and Molecular Biology

First Advisor

Li Li

Second Advisor

David Evans

Abstract

Background: The vascular smooth muscle cells(VSMC) possess the ability to differentiate into a synthetic phenotype in response to stress. This phenotypic modulation may be accompanied by inflammatory or osteogenic response in chronic stress. The synthetic state is characterized by low levels of contractile markers unlike the differentiated state.

Hypothesis: Endoplasmic reticulum (ER) stress causes phenotypic modulation in VSMCs leading to apoptosis. Many transcription factors induced by ER stress contribute to the downregulation of Sm22α. Perturbation in cytoskeletal dynamics exacerbates the ER stress response.

Methods: Ex-vivo culture was used to establish importance of Sm22 in ER stress. In vitro analysis was carried out in PAC1 cells to elucidate the signaling induces by ER stress in smooth muscle cells and its contribution to the downregulation of Sm22α.

Results: ER stress caused a transient upregulation of smooth muscle markers during 1-6 hrs. of treatment with the ER stress inducer thapsigargin. The downregulation of contractile markers at 24hour of thapsigargin treatment was accompanied by upregulation of phenotypic regulators- Klf4, Mmp9 and Pdgf-bb, inflammatory markers Vacm-1 and Ccl-2 and osteogenic markers-Runx2, Msx2, Ocn and Pit-1. Overexpression of SM22 reduced the expression of ER stress markers (Atf4 and Chop) and inflammation marker (Ccl-2). Myocardin overexpression could reduce the downregulation of SM22α. Promoter analysis of Sm22α showed that all the cis acting elements were involved in its downregulation under ER stress. As our results indicated a protected role of SM22α under ER stress conditions, a drug screen was carried out to identify potential regulators of Sm22α.The screen identified isoflavonoids as positive regulators for Sm22α expression. Biochanin A, a candidate from the drug screen was subsequently validated in the fibroblast cell line

Conclusion: SM22α plays a protective role in the ER stress condition. Many of the ER stress induced factors associate with different cis acting elements in the Sm22α promoter to repress it. Sm22α could be a potential drug target to reduce the pathogenic effects of ER stress in cardiovascular diseases. Preliminary drug screen suggests that the phytoestrogens – flavonoids and isoflavonoids could be potential candidates with therapeutic benefits.

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