TRPC4-DEPENDENT CYTOSOLIC CALCIUM SIGNALS COORDINATE INTERENDOTHELIAL FORCES THAT ELICIT GAP FORMATION

项目摘要 急性肺损伤是一种急性炎症引起的肺内皮屏障破坏的疾病 并且在重症监护室中造成了显著的发病率和死亡率。内皮屏障完整性 是由血浆激活的内皮细胞胞质钙离子（Ca2+）瞬变决定的 膜库操纵的Ca2+进入（ISOC）通道。通道亚单位之一，典型瞬时受体 潜在的4（TRPC 4）蛋白，关键调节通道激活和Ca2+内流。细胞内Ca2+浓度升高， 足以诱导肺泡外段的内皮间隙并引起肺水肿。但 TRPC 4依赖性Ca2+内流破坏粘附连接和协调的机制 内皮间的力知之甚少，并且是本申请的焦点。 胞浆Ca2+是不稳定的，而是在融合的内皮细胞单层内波动。TRPC4有助于 这些基础的胞质钙瞬变。炎症激动剂通过激活TRPC 4 ISOC诱导Ca2+内流 通道，导致胞质Ca2+的显著升高，这导致内皮细胞收缩和间隙形成。 然而，尚不清楚这些TRPC 4依赖性的胞浆Ca2+瞬时增加是否导致细胞内Ca2+浓度升高。 在协调内皮细胞间力、内皮细胞间间隙形成和肺水肿方面。 炎症性疾病与细胞和组织的机械性质的变化有关。细胞间 力在空间上是异质的，高度合作的，并且排列成力链。在炎症期间， 血管内皮屏障的破坏部分是通过一组 细胞由此产生的增加的收缩力通过细胞间连接传播到相邻细胞， 增加内皮细胞间力协调和相关性，增加细胞内力排列和延伸 迫使链远离差距区域。收缩力的大量积累可能最终超过 粘附连接可以承受并导致内皮间间隙形成的最大力。一个 内皮间间隙也可以在力链被 不对齐，并可能将粘附连接拉开。然而，目前还不清楚TRPC 4依赖性细胞质中 Ca2+瞬变与基线和激动剂激发后的内皮间力相关性有关。这项建议 测试了TRPC 4激活触发独特的细胞溶质Ca2+瞬变的总体假设， 增加内皮细胞间力相关性并促进间隙形成、肺渗透性和肺 水肿

Project Summary Acute lung injury is a disorder of acute inflammation that causes disruption of the pulmonary endothelial barrier and contributes a significant amount of morbidity and mortality in the intensive care unit. Endothelial barrier integrity is critically determined by endothelial cytosolic calcium ion (Ca2+) transients that are activated by the plasma membrane store-operated Ca2+ entry (ISOC) channels. One of the channel subunits, canonical transient receptor potential 4 (TRPC4) protein, critically regulates channel activation and Ca2+ influx. Elevation of cytosolic Ca2+ is sufficient to induce interendothelial gaps in extra-alveolar segments and cause pulmonary edema. However, the mechanism by which TRPC4-dependent Ca2+ influx disrupts adherens junction and coordinates interendothelial forces is poorly understood and is the focus of this application. Cytosolic Ca2+ is not stable but rather fluctuates within a confluent endothelial monolayer. TRPC4 contributes to these basal cytosolic Ca2+ transients. Inflammatory agonists induce Ca2+ influx by activating the TRPC4 ISOC channel leading to a marked elevation of cytosolic Ca2+, which causes endothelial cell retraction and gap formation. However, it is unknown whether these TRPC4-dependent transient increases in cytosolic Ca2+ cause increases in the coordination of interendothelial forces, interendothelial gap formation, and lung edema. Inflammatory diseases are related to changes in mechanical properties of cells and tissues. Intercellular forces are spatially heterogeneous, highly cooperative, and aligned into force chains. During inflammation, disruption of the vascular endothelial barrier is partially initiated through cytoskeletal contraction of a group of cells. The resulting increased contractile forces propagate across neighboring cells via intercellular junctions to increase interendothelial force coordination and correlation, increase intracellular force alignment and extend force chains far outside the gap regions. A large accumulation of contractile forces may eventually exceed the maximum force that an adherens junction can withstand and cause the formation of an interendothelial gap. An interendothelial gap also may form at a tri-junctional (tricellular junction) point where force chains are misaligned and may pull the adherens junction apart. However, it is unknown how TRPC4-dependent cytosolic Ca2+ transients are related to interendothelial force correlation at baseline and after agonist challenge. This proposal tests the overall HYPOTHESIS that TRPC4 activation triggers unique cytosolic Ca2+ transients that are sufficient to increase interendothelial force correlation and promote gap formation, pulmonary permeability, and pulmonary edema.