Date of Award

5-2009

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Bioengineering

Advisor

Nagatomi, Jiro

Committee Member

Simionescu , Dan

Committee Member

Ramamurthi , Anand

Abstract

Previous research has demonstrated that bladder smooth muscle cells (BSMC) respond to various forms of mechanical stimuli, including stretch and hydrostatic pressure, by increases of cell proliferation, activation of intracellular signaling pathways, and alteration of contractile and synthetic marker protein expression. These cellular/molecular level changes are all indicative of a BSMC phenotypic shift that can negatively impact the bladder function at the tissue and organ level. The objective of the present study is to test a hypothesis that bladder SMCs shift their phenotype from contractile to synthetic in response to elevated hydrostatic pressure. Rat bladder SMC cultures were exposed to 7.5 cm H2O of hydrostatic pressure in custom-made columns up to 48 hours. Following exposure to pressure, the SMCs were fixed, stained, and imaged using fluorescence microscopy. Image analyses revealed that, compared to the control, SMCs exposed to hydrostatic pressure for 4 hours exhibited a more spread morphology, which was quantitatively confirmed when examining the aspect ratio of the cell population. Moreover, cell density of BSMCs exposed to hydrostatic pressure exhibited an increase after 24 and 48 hours when compared to their respective controls. Additionally, total proteins collected from these cells were analyzed using the Western blotting technique to quantify extracellular signal-regulated kinase (ERK_) activation as well as phenotype marker proteins, alpha-smooth muscle actin (α-SMA) and SM-22 in SMCs. Compared to control (0 minutes), the expression of activated ERK _ was up to two-fold when these cells were exposed to hydrostatic pressure (7.5 cm H2O) for up to 180 minutes. In contrast, α-SMA and SM-22 expression was similar in the control and cells exposed to hydrostatic pressure for 48 hours. While the proliferative and morphological responses suggest a possible ERK _ mediated phenotypic shift from a contractile phenotype toward a synthetic phenotype under mechanical stimulus, the expression of contractile proteins did not corroborate the other aspects of SMC phenotypic modulation in the time points examined in this study (48 hours). These results suggest that contractile protein expression in response to mechanical stimulus could possibly be mediated by ERK _ at earlier or later time points not detected in this study, or by a different signaling pathway altogether. Future study recommendations include, but are not limited to, exploring the expression of contractile proteins at various time points and their relationship with ERK _ activation.

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