Extracellular Matrix Presentation Modulates Vascular Smooth Muscle Cell Mechanotransduction

The development of atherosclerotic lesions involves phenotypic changes among resident vascular smooth muscle cells (VSMCs) that often contribute to inflammation at the site of injury and are correlated with stiffening of the vessel wall. Studies have shown that there are major alterations in extracellular matrix (ECM) composition and mechanical properties during atherosclerosis that likely contribute to VSMC pathology, yet precisely how such changes lead to regulation of VSMC behavior remains poorly understood. In this study, we used substrates with tunable mechanics to investigate the combined influence of ECM stiffness and composition on VSMC adhesion, spreading, proliferation, and traction force generation. To model the stiffening ECM, we synthesized 25kPa and 135kPa polyacrylamide substrates functionalized with equal mass quantities of fibronectin, laminin, type I collagen, or a combination of fibronectin and laminin. On fibronectin and collagen substrates, we observed that increasing stiffness stimulates VSMC adhesion, spreading, and proliferation, whereas on laminin substrates, the effect is reversed, with 135kPa substrates supporting 35% less attachment (p<0.05), 25% smaller cell area (p<0.05), and 10% less proliferation than 25kPa substrates. We also examined attachment on gels containing varying ratios of fibronectin and laminin, and found that cell number on 135kPa versus 25kPa substrates was a direct function of the proportion of each ligand, i.e., gels with more fibronectin supported higher attachment at 135kPa, while gels with more laminin supported higher attachment at 25kPa. We then quantified single cell traction forces on 10kPa substrates containing fibronectin or laminin and found that total force per area on laminin was 55% less than on fibronectin (p<0.05). Collectively, our results demonstrate that VSMC response to substrate stiffness is critically dependent on ligand biochemistry, and have broad implications for the study of VSMC physiology and mechanotransduction in other cell types.