In VSMCs, Beta-1 Integrin but not Syndecan-4 Gene Expression is Dependent on Matrix Stiffness

Abstract

Mechanotransduction is the process by which cells sense and convert mechanical stimuli into biochemical signals, and has been implicated in the development of certain pathologies. During atherosclerosis, the vessel wall experiences progressive stiffening, particularly at the site of developing plaques, and vascular smooth muscle cells are intimately involved in the development and maintenance of these plaques. In vitro, VSMCs have also been shown to be mechanosensing: cell spreading area, migration speed, and expression of characteristic cytoskeletal components is mediated by substrate stiffness. It is likely, then, that changes in arterial mechanics during atherosclerosis contribute to the pathological behavior of VSMCs, but our understanding of this process remains inadequate.The beta-1 integrin subunit has been implicated in mechanosensing, but its uniqueness as a mechanotransducer has not been established. Syndecan-4 acts in synergy with the alpha5beta1 integrin, though its role in mechanotransduction is unknown.We hypothesized that beta-1, but not syndecan-4, gene expression is regulated by substrate stiffness. We used polyacrylamide gels functionalized with fibronectin which mimic the mechanical properties of healthy (∼30kPa) and diseased (∼80kPa) arterial walls. Primary rat VSMCs were grown to 60% confluence on 18kPa, 35kPa, and 80kPa gels with uniform FN surface content, and collected for quantitative PCR analysis. Gene expression of beta-1 increased 1.5x with each increment in stiffness, while syndecan-4 levels were not affected. These results suggest that beta-1 integrin gene expression is sensitive to substrate mechanics, while syndecan-4 expression is not.Because cell-cell contacts are also capable of mechanotransduction, we also looked at the effects of cell density on beta-1 integrin expression. Preliminary results suggest that increasing cell density eliminates the substrate-mediated increase in beta-1 expression, which suggests that VSMCs integrate mechanical stimuli from various sources to achieve physical homeostasis.