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

 产品说明：本申请的总体目标是证明使用微工程化的“Lung-on-a-Chip”微流体装置来探测人肺中机械化学信号传导的分子机制的可行性，并使用该知识来开发新的和改进的肺水肿发展抑制剂。由从微环境经由细胞外基质粘附到细胞的力传递触发的最快速（< 5毫秒）的机械信号传导事件之一涉及应激活化的膜离子通道TRPV 4的整合素依赖性活化，其似乎对于许多疾病过程（包括肺水肿）的发展至关重要。施加于整联蛋白的力介导这种激活TRPV 4并导致肺部疾病的“早期-立即”机械信号传导反应的分子机制还不清楚。为了在体外研究这一过程，我们将使用最近开发的人类Lung-on-a-Chip微流控装置，该装置包含由活的人类肺泡和毛细血管细胞排列的人工肺泡-毛细血管界面，所述细胞经历生理呼吸运动并在体外再生功能性血管渗透屏障。重要的是，我们以前使用这种微工程肺芯片来证明TRPV 4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CD 98与β 1-整联蛋白和TRPV 4结合，并且它是TRPV 4的机械活化而非化学活化所必需的。因此，在该项目中，我们建议使用我们的微工程化人肺芯片装置来描绘施加于整合素的力激活TRPV 4的分子机制，并开发针对该分子机制的肺水肿的新疗法。具体目标包括：1）确定CD 98介导人微血管内皮细胞中TRPV 4的β 1-整联蛋白依赖性机械活化的分子机制，2）开发通过TRPV 4的机械信号传导的肽调节剂，其防止肺芯片肺水肿模型中的血管渗漏，和3）通过证明肽抑制剂在离体小鼠肺水肿模型中预防血管渗漏的能力来验证肽抑制剂。

 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.