Cell-cell interactions and extracellular matrix presentation mediate the effects of substrate stiffness on vascular smooth muscle cell behavior
Abstract: In the blood vessel wall, the development of atherosclerotic lesions involves phenotypic changes among resident vascular smooth muscle cells (VSMCs) that reflect an attempt to repair already-damaged tissue. Often, these changes contribute to a cascade of inflammation at the site of injury and are correlated with stiffening of the blood vessel wall. It is thus likely that altered extracellular matrix (ECM) mechanics play a role in VSMC response to injury. However, the biochemical and mechanical factors that control VSMC behavior remain poorly understood. The central goal of this dissertation is to further the understanding of VSMC response to extracellular mechanics (mechanotransduction). To this end, we created polyacrylamide (PAAM) substrates with tunable mechanics and ligand presentation and investigated the effect of substrate stiffness on VSMC mRNA and protein expression. We also studied whether cell-cell interactions can mediate these effects by correlating stiffness-induced changes in expression with varying cell density. At low cell density, we observed a strong correlation between increasing substrate stiffness and the expression of β1 integrin, syndecan-4, and several other proteins critical to cell-ECM adhesion and cytoskeletal integrity. Culturing VSMCs at high cell density eliminated this trend for focal adhesion but not cytoskeletal proteins. Fluorescent microscopy studies of cell and focal adhesion morphology showed that: 1) VSMC spreading decreases with increasing cell-cell interactions in a stiffness-dependent manner, and 2) individual cell area is strongly correlated with focal adhesion number. Studies of VSMCs spreading on PAAM functionalized with fibronectin, laminin, or type I collagen revealed that the effects of stiffness and cell-cell interactions on cell area are ECM-dependent, demonstrating the relevance of ECM biochemistry in VSMC mechanotransduction. Our findings highlight the influential role of cell-cell contacts in VSMC response to mechanics, demonstrating that communication through cadherin-based contacts can mute specific effects of substrate stiffening through a mechanism that involves the regulation of cell morphology. Given the likely role of tissue mechanics in atherosclerosis, our findings inform how the microenvironment of the vascular lesion can influence VSMC behavior and point to cell-cell contacts as critical mediators of VSMC response to injury
Thesis, Dissertation, English, 2011