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.
Lung inflammation causes calcium ion influx into pulmonary endothelial cells, leading to gap formation in the vessel wall, increases in vascular permeability, and lung edema. This proposal is designed to test the hypothesis that channel-dependent calcium signals increase the contractile forces within endothelial cells that elicit interendothelial gap formation and increase lung permeability. If the hypothesis is correct, then reducing coordination of interendothelial forces could be a potential therapeutic target for lung edema patients.