Mechanotransduction analysis in a microengineered lung-on-a-chip

 描述：本申请的总体目标是证明使用微工程“芯片上的肺”微流控设备来探索人类肺部机械力化学信号的分子机制的可行性，并利用这一知识开发新的和改进的肺水肿发展抑制剂。由微环境通过细胞外基质粘连从微环境到细胞的力传递所触发的最快(&lt；5毫秒)机械信号事件之一涉及到应激激活的膜离子通道TRPV4的整合素依赖激活，这似乎在许多疾病的发展过程中至关重要，包括肺水肿。作用于整合素的力介导这种“早期-即时”的机械信号反应，从而激活TRPV4并导致肺部疾病的分子机制还不是很清楚。为了在体外研究这一过程，我们将使用最近开发的人类单芯片肺微流控装置，该装置包含人工肺泡-毛细血管界面，由活的人肺泡和毛细血管细胞组成，在体外经历生理呼吸运动并再生功能性血管通透性屏障。重要的是，我们之前使用了这种微工程肺芯片来证明TRPV4活性的特定化学抑制剂可以防止由白细胞介素2和机械变形(呼吸运动)引起的肺血管渗漏。此外，我们的初步结果表明，跨膜蛋白CD98可以与1-整合素和TRPV4结合，并且它是机械激活TRPV4所必需的，而不是化学激活TRPV4所必需的。因此，在这个项目中，我们建议使用我们的微工程化的人类单芯片肺装置来描述施加在整合素上的力激活TRPV4的分子机制，并开发针对这一分子机制的肺水肿的新疗法。其具体目的包括：1)明确CD98介导人微血管内皮细胞内整合素依赖的TRPV4的机械激活的分子机制；2)开发通过TRPV4的机械信号的多肽调节剂，以防止单芯片肺水肿模型中的血管渗漏；3)通过在体外小鼠肺水肿模型中证明其预防血管渗漏的能力来验证多肽抑制剂的有效性。

 DESCRIPTION: The overall goal of this application is to demonstrate the feasibility of using a microengineered `Lung-on-a-Chip" microfluidic device to probe the molecular mechanism of mechano-chemical signaling in the human lung, and to use this knowledge to develop new and improved inhibitors of pulmonary edema development. One of the most rapid (< 5 msec) mechanical signaling events triggered by force transmission from the microenvironment to the cell via their extracellular matrix adhesions involves integrin-dependent activation of the stress-activated membrane ion channel TRPV4, which appears to be critical for the development of many disease processes, including pulmonary edema. The molecular mechanism by which forces applied to integrin mediate this `early- immediate' mechanical signaling response that activates TRPV4 and lead to pulmonary disease is not well understood. To study this process in vitro, we will use a recently developed human Lung-on-a-Chip microfluidic device that contains an artificial alveolar-capillary interface lined by living human lung alveolar and capillary cells hat experiences physiological breathing motions and regenerates a functional vascular permeability barrier in vitro. Importantly, we previously used this microengineered lung chip to show that a specific chemical inhibitor of TRPV4 activity can prevent pulmonary vascular leakage induced by both interleukin-2 and mechanical deformation (breathing motions). In addition, our preliminary results have revealed that the transmembrane protein CD98 binds to both ß1-integrin and TRPV4, and that it is required for mechanical, but not chemical, activation of TRPV4. Thus, in this project, we propose to use our microengineered human Lung- on-a-Chip device to delineate the molecular mechanism by which forces applied to integrins activate TRPV4, and to develop new therapeutics for pulmonary edema that targets this molecular mechanism. The specific aims include: 1) to define the molecular mechanism by which CD98 mediates ß1-integrin-dependent mechanical activation of TRPV4 in human microvascular endothelial cells, 2) to develop peptide modulators of mechanical signaling through TRPV4 that prevent vascular leakage in the lung-on-a-chip pulmonary edema model, and 3) to validate the peptide inhibitors by demonstrating their ability to prevent vascular leakage in an ex vivo mouse pulmonary edema model.