Microfluidic approaches to study mechanotransduction in cell division and migration

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Physical forces exerted on cells drive changes in phenotype during developmental morphogenesis, normal physiologic function, and under pathologic conditions. The term mechanotransduction has been coined to describe the process by which cells transduce mechanical inputs into biological responses. Cells in the vascular system are continuously exposed to external physical forces including shear stress imposed by blood flow, and tangential stress due to blood pressure. Some aspects of mechanotransduction in the vascular system have been extensively studied. For example the effects of blood flow shear stress on endothelial cells have been studied for decades. Vascular smooth muscle cells (VSMCs) normally reside within the media in a 3D environment surrounded by extracellular matrix, where they experience tangential pressure stress, another mechanical stimulus which has been very well studied. However, after endothelial denudation, which occurs during arterial injury and atherosclerosis, VSMCs undergo a phenotypic switch and migrate from the media to the intimal layer. As a result the microenvironment of this cell changes dramatically. VSMCs undergo compression and deformation as they migrate through tight fenestrae in the internal elastic lamina, and once on the intimal surface, they are exposed to shear stress from flowing blood. The effects of these forces on VSMCs are not well understood, and because such forces are transduced via the cell cytoskeleton, they are likely to have important effects on migration and cell division. The focus of this NSERC discovery grant is to investigate mechanisms of mechanotransduction in VSMCs exposed to fluid shear and compressive stresses using microfluidic devices to mimic the in vivo microenvironment. Using detailed confocal microscopic analysis we have shown that there are aberrations in cell migration and division in VSMCs comprising the thickened neointimal layer following endothelial-denuding vascular injury in vivo. The VSMCs newly arriving in the intima align in parallel with the direction of blood flow, suggesting that they are sensitive to shear stresses exerted by flowing blood. Also, during migration of the neointimal VSMCs, polarization of the cytoskeleton is altered. In most migrating cells studied in vitro, the microtubule organizing center (MTOC) is oriented in front of the nucleus to facilitate assembly of the microtubule array and delivery of cargoes to the leading edge of the cell. However, in neointimal VSMCs migrating in vivo, the MTOC is positioned behind the nucleus. VSMCs also experience profound changes in mechanical forces during migration, as they pass easily through the loose collagen fibril network in the media, but must squeeze through tight fenestrations in the internal elastic lamina during their passage from media to intima. The average VSMC is 10 µm in diameter, while the fenestrae range from 1-10 µm, so cells and their nuclei must deform to pass through. I have discovered an actin net covering the apical surface of the nucleus in neointimal VSMCs, and propose that this actin net acts as a mechanosensor for cell and nuclear deformation during migration. The cytoskeleton bears the brunt of physical force imposed upon the cell, and governs the position of intracellular organelles. I hypothesize that alterations in the cytoskeletal organization in neointimal VSMCs are due to two distinct physical forces to which the cells are exposed as they migrate into the intimal layer: 1. Shear force exerted by blood flow on VSMCs at the intimal surface. 2. Compressive force which deforms the VSMC and nucleus as it squeezes through small fenestrae in the internal elastic lamina separating the medial and the intimal layers.