Microfluidic approaches to study mechanotransduction in cell division and migration

在发育形态发生、正常生理功能和病理条件下，施加在细胞上的物理力驱动表型的变化。机械转导这个术语被用来描述细胞将机械输入转化为生物反应的过程。血管系统中的细胞持续暴露于外部物理力，包括血流施加的剪切应力和血压引起的切向应力。在血管系统中的机械转导的一些方面已经被广泛研究。例如，血流剪切应力对内皮细胞的影响已经研究了几十年。血管平滑肌细胞（VSMC）通常驻留在被细胞外基质包围的3D环境中的中膜内，在那里它们经历切向压力应力，这是另一种已经被很好地研究的机械刺激。然而，在动脉损伤和动脉粥样硬化期间发生的内皮剥脱后，VSMC经历表型转换并从中膜迁移到内膜层。因此，细胞的微环境发生了巨大的变化。当VSMC迁移通过内弹性膜中的紧密窗孔时，VSMC经历压缩和变形，并且一旦在内膜表面上，它们就暴露于来自流动血液的剪切应力。这些力对VSMC的影响还不清楚，因为这些力是通过细胞骨架转导的，它们可能对迁移和细胞分裂有重要影响。这个NSERC发现补助金的重点是研究VSMCs暴露于流体剪切和压缩应力的机械转导机制，使用微流体装置模拟体内微环境。使用详细的共聚焦显微镜分析，我们已经表明，有畸变的细胞迁移和分裂的血管平滑肌细胞包括增厚的新生内膜层后，内皮剥脱血管损伤在体内。新到达内膜的VSMC与血流方向平行排列，表明它们对流动血液施加的剪切应力敏感。此外，在迁移的新生内膜血管平滑肌细胞，极化的细胞骨架被改变。在体外研究的大多数迁移细胞中，微管组织中心（MTOC）位于细胞核的前面，以促进微管阵列的组装和将货物运送到细胞的前缘。然而，在体内迁移的新生内膜VSMC中，MTOC位于细胞核后面。VSMC在迁移过程中也经历了机械力的深刻变化，因为它们很容易通过中膜中松散的胶原纤维网络，但在它们从中膜到内膜的过程中必须挤压通过内弹性膜中的紧密开窗。VSMC的平均直径为10 µm，而窗孔的范围为1-10 µm，因此细胞及其细胞核必须变形才能通过。我发现了一个肌动蛋白网覆盖在新生内膜VSMCs的细胞核的顶面，并提出这个肌动蛋白网作为一个mechanosensor细胞和核变形迁移过程中。细胞骨架承受施加在细胞上的物理力的冲击，并控制细胞内细胞器的位置。我推测，在新生内膜VSMC细胞骨架组织的改变是由于两个不同的物理力量，细胞暴露，因为他们迁移到内膜层：1。血流对内膜表面VSMC施加的剪切力。2.当VSMC和细胞核挤压通过内弹性膜中分离中膜和内膜层的小孔时，使VSMC和细胞核变形的压缩力。

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.