Date of Award

12-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Advisor

Vyavahare, Narendra

Committee Member

LaBerge , Martine

Committee Member

Ramamurthi , Anand

Committee Member

Webb , Ken

Abstract

Cardiovascular diseases are the leading causes of mortality in the United States and will cost around $500 billion this year alone. Elevated proteolytic activity, increased proliferation and migration of vascular smooth muscle cells are hallmarks of atherosclerosis, stenosis and aortic aneurysms. These diseases often manifest the transdifferentiation of vascular smooth muscle cells into osteoblast-like cells followed by deposition of hydroxyapatite–like mineral in the arterial walls.
Currently, there are no standard treatments available for vascular calcification or aneurysms. Atherosclerosis treatment options are limited to statins while balloon angioplasty and stenting – surgical procedures for stenosis, often end in restenosis. Therefore, we investigated pentagalloylglucose (PGG), a polyphenolic compound, as a therapeutic agent that can inhibit excess proteolytic activity, mitigate proliferation and disrupt the transformation of vascular smooth muscle cells into osteoblast-like cells. Previous experiments conducted in our lab have shown that PGG has elastoprotective properties in a rat aneurismal model. Studies conducted by other researchers have shown that PGG also has anti-cancer and anti-inflammatory properties.
Our results show that PGG effectively decreased the level of cathepsins K, L and S, and the activity of MMP-2 in tumor necrosis factor activated rat aortic smooth muscle cells (RASMCs) in vitro. Transcription levels of cathepsins K and S were dramatically decreased by PGG. Scratch test assay showed that PGG treatment resulted in visibly reduced migration and proliferation. PGG treatment also reduced the expression of osteogenic markers in activated RASMCs. Gene expressions of CBFA–1 and MSX–2 were downregulated. Alkaline phosphatase activity was significantly reduced at days 1, 3 and 6. Addition of PGG 3 days past activation of RASMCs also resulted in decreased alkaline phosphatase activity, signifying that PGG could potentially reverse osteogenic differentiation of RASMCs.
We also conducted studies to verify if PGG could possibly increase elastin production in primary RASMCs by potentially inhibiting proteolytic activity. We found that levels of both tropoelastin and insoluble elastin were significantly increased in cells treated with PGG.
In order to deliver PGG locally to a diseased vascular site, we investigated the possibility of using nanoparticles. Poly(lactic–co–glycolic acid) nanoparticles encapsulated with PGG were prepared and their in vitro release profiles were studied. Sonication times during emulsion steps were varied and resulting encapsulation efficiencies were studied.
We conclude that PGG could potentially be a valuable therapeutic agent in vascular pathologies. Excess proteolytic activity, migration and proliferation of RASMCs were effectively controlled by PGG. PGG also inhibited the osteogenic signaling in smooth muscle cells through potentially affecting cell cycle progression by down regulating the gene expression of c–Fos. PGG could be used alone or with other existing treatments to control or reverse vascular diseases discussed above. Further optimization needs to be performed in order to determine the dose and mode of PGG delivery in vivo.

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