Measuring the effect of extracellular mechanics on smooth muscle contractility

描述(申请人提供)：动脉存在于一个动态的环境中，需要血管平滑肌细胞(VSMCs)的持续适应，以维持机械内稳态。在健康的动脉中，血管张力的维持主要是通过VSMCs的收缩和松弛来实现的，而收缩能力的丧失是VSMC功能障碍的标志，这可能在动脉粥样硬化斑块形成和再狭窄的发病机制中发挥重要作用。斑块形成或支架植入可显著扰乱机械环境，无论是由于动脉粥样硬化中的脂质池导致的组织软化，还是由于斑块钙化区域或植入的支架而导致的硬化。最近的研究结果发现，细胞周围环境的力学对其功能行为起着重要作用，这表明由于斑块或支架而导致的非稳态组织力学也可能影响收缩功能。我们假设周围组织的机械性能直接影响血管平滑肌细胞的收缩功能。在目前的技术中，很难评估组织硬度对VSMC功能的影响，因为现有的力学独立可控的体外系统都局限于狭窄的基质硬度范围，在这个范围内，它们可以用来测量细胞或组织的应力产生。因此，为了验证这一假设，我们需要开发一种新的方法来测量组织应力，这种方法可以用于广泛的底物力学。我们之前开发了一种名为血管肌肉薄膜(VMTF)的方法，在这种方法中，我们在细橡胶梁上将细胞微图案化成高度有序的组织，并通过测量光束的曲率来确定细胞应力。我们将对这种方法进行调整，以包括可变模数的底物，使我们能够测量模拟广泛机械环境的VSMC的功能收缩性能，例如软脂池和硬支架。然后，我们将把这种收缩能力与VSMC表型标志物的表达联系起来，以确定表型转换在VSMC底物调节的机械适应中的作用。表型转换与许多VSMC功能障碍有关。这项工作的完成将为动脉病理中VSMCs机械环境的演变提供更清晰的功能图景。

DESCRIPTION (provided by applicant): Arteries exist in a dynamic environment, necessitating constant adaptation by vascular smooth muscle cells (VSMCs), in order to maintain mechanical homeostasis. In a healthy artery, maintenance of vessel tension is primarily achieved by contraction and relaxation of VSMCs, and loss of contractility is indicative of VSMC dysfunction, which may play an important role in pathogenesis of atherosclerotic plaque formation and restenosis. The mechanical environment can be significantly perturbed by plaque development or stent implantation, either by softening of the tissue, due to lipid pools in atherosclerosis, or stiffening due to calcified regions of the plaque, or an implanted stent. Recent results have found that the mechanics of a cell's surroundings play a significant role in its functional behavior, suggesting that non-homeostatic tissue mechanics, due to plaques or stents, could also affect contractile function. We hypothesize that the mechanics of the surrounding tissue directly affects vascular smooth muscle cells' contractile functionality. With current techniques, it is difficult to assess the effects of tissue stiffness on VSMC function because existing in vitro systems in which mechanics are independently controllable are all limited to narrow ranges of substrate stiffness over which they can be used to measure cell or tissue stress generation. So, to test this hypothesis we will need to develop a new method for measuring tissue stress that can be employed with a wide range of substrate mechanics. We have previously developed a method called vascular muscular thin films (vMTFs), where we micropatterned cells into highly organized tissues on a thin rubber beam, and the cell stress was determined by measuring the curvature of the beam. We will adapt this method to include a variable modulus substrate, allowing us to measuring functional contractility of VSMCs mimicking a wide range of mechanical environments, such as soft lipid pools and stiff stents. We will then correlate this contractility with VSMC phenotype marker expression to determine how phenotype switching, which has been implicated in a number of VSMC dysfunctions, plays a role in VSMC substrate-modulated mechano-adaptation. Completion of this work will provide a sharper picture of the functional implications of the evolving mechanical environment of VSMCs in arterial pathologies.