Characterization of anti-proteolytic and anti-proliferative activities of pentagalloylglucose; its potential application as a therapeutic agent in vascular diseases

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

Breaking the In Vitro Alveolar Type II Cell Proliferation Barrier while Retaining Ion Transport Properties

Alveolar type (AT)I and ATII cells are central to maintaining normal alveolar fluid homeostasis. When disrupted, they contribute to the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome. Research on ATII cells has been limited by the inability to propagate primary cells in vitro to study their specific functional properties. Moreover, primary ATII cells in vitro quickly transdifferentiate into nonproliferative “ATI-like” cells under traditional culture conditions. Recent studies have demonstrated that normal and tumor cells grown in culture with a combination of fibroblast (feeder cells) and a pharmacological Rho kinase inhibitor (Y-27632) exhibit indefinite cell proliferation that resembled a “conditionally reprogrammed cell” phenotype. Using this coculture system, we found that primary human ATII cells (1) proliferated at an exponential rate, (2) established epithelial colonies expressing ATII-specific and “ATI-like” mRNA and proteins after serial passage, (3) up-regulated genes important in cell proliferation and migration, and (4) on removal of feeder cells and Rho kinase inhibitor under air–liquid interface conditions, exhibited bioelectric and volume transport characteristics similar to freshly cultured ATII cells. Collectively, our results demonstrate that this novel coculture technique breaks the in vitro ATII cell proliferation barrier while retaining cell-specific functional properties. This work will allow for a significant increase in studies designed to elucidate ATII cell function with the goal of accelerating the development of novel therapies for alveolar diseases