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，并定义组织和组织模式。机械转导这个术语指的是细胞将机械输入转化为生物反应的过程。血管系统为研究机械转导提供了一个理想的模型，因为细胞不断地暴露在外部物理力中，包括血流施加的剪应力和由于血压产生的切向应力。血管平滑肌细胞(VSMCs)通常存在于细胞外基质包围的3D环境中。然而，在内皮剥离和暴露于生长因子如血小板衍生生长因子B(PDGF-B)后，VSMCs经历了从收缩到合成的表型转换，并从中膜增殖并迁移到内膜层。因此，细胞的微环境发生了巨大的变化：VSMCs在穿过内弹力板的紧密隔板时受到压缩和变形，一旦到达内膜表面，它们就暴露在流动的血液中的剪应力下。调控VSMC迁移和增殖的生化信号已被研究多年。然而，这些物理力量对损伤后VSMCs的影响尚不清楚，我们试图了解细胞如何将机械转导信号与细胞骨架的变化结合起来，以影响迁移和增殖的行为决定。这项资助的重点是利用微流体设备模拟体内微环境来研究VSMC在流体剪切和压应力作用下的机械转导机制。 理论基础和假设：细胞骨架在VSMCs迁移的机械转导中起重要作用：它承受施加在细胞上的物理力，是表型转换的目标，并控制细胞内细胞器的位置，完成增殖和迁移所需的极性。我推测，VSMCs细胞骨架组织的改变是由于细胞迁移到内膜层时所受到的两种不同的物理作用力：1.血流对内膜表面细胞施加的剪切力。2.当细胞和细胞核挤过分隔内层和内膜层的内弹力板中的小窗孔时，使细胞和细胞核变形的压力。

Physical forces exerted on cells drive changes in phenotype and define organization and tissue patterning during developmental morphogenesis, normal physiologic function, and under pathologic conditions. The term mechanotransduction refers to the process by which cells transduce mechanical inputs into biological responses. The vascular system provides an ideal model to study mechanotransduction, because cells are continuously exposed to external physical forces including shear stress imposed by blood flow, and tangential stress due to blood pressure. Vascular smooth muscle cells (VSMCs) normally reside within the media in a 3D environment surrounded by extracellular matrix. However after endothelial denudation and with exposure to growth factors like platelet derived growth factor B (PDGF-B) VSMCs undergo a phenotypic switch from contractile to synthetic, and they proliferate and migrate from the media to the intimal layer. As a result the microenvironment of the 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 biochemical signals regulating VSMC migration and proliferation have been studied for many years. However the effects of these physical forces on VSMCs after injury are not well understood, and we seek to understand how cells integrate mechanotransduction signals with changes in the cytoskeleton to influence behavioural decisions to migrate and proliferate. The focus of this 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. Rationale and Hypotheses: The cytoskeleton plays an important role in mechanotransduction in migrating VSMCs: it bears the brunt of physical forces imposed upon the cell, is the target of the phenotypic switch, and governs the position of intracellular organelles to accomplish polarity required for proliferation and migration. I hypothesize that alterations in the cytoskeletal organization in 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 cells at the intimal surface. 2. Compressive force which deforms the cell and nucleus as it squeezes through small fenestrae in the internal elastic lamina separating the medial and the intimal layers.