Role of Endothelial Purinergic Receptor-TRPV4 Channel Signaling in Lung Ischemia-Reperfusion Injury

Background: Lung ischemia-reperfusion injury (IRI) is a major cause of primary graft dysfunction after transplant. IRI is characterized by robust inflammation, increased vascular permeability, and lung edema. Endothelial cell (EC) transient receptor potential vanilloid 4 (TRPV4 EC ) channels are crucial regulators of endothelial barrier function. We recently reported that endothelial TRPV4 -/- (TRPV4 EC -/- ) mice are protected against lung edema and dysfunction after IR. However, activity of TRPV4 EC channels and TRPV4 EC -dependent mechanisms have not been studied in lung IRI. In this regard, extracellular adenosine triphosphate (ATP) was shown to activate pulmonary TRPV4 EC channels through purinergic P2Y2 receptors (P2Y2R). Moreover, studies in a mouse model showed that pannexin1 (Panx1), an ATP efflux pathway in ECs, plays a critical role in the pathogenesis of lung IRI. We hypothesized that activation of an endothelial Panx1-P2Y2R-TRPV4 signaling axis mediates lung IRI endothelial barrier disruption. Methods: A left lung hilar ligation model of lung IRI was utilized in tamoxifen-inducible TRPV4 EC -/- , P2Y2R EC -/- , Panx1 EC -/- , and tamoxifen-treated control (TRPV4 fl/fl , P2Y2R fl/fl , and Panx1 fl/fl ) mice. Mice undergoing IR were subjected to left lung ischemia for 1 h followed by 2 h of reperfusion. A separate group of mice underwent sham surgery with no hilar ligation. Partial pressure of arterial oxygen (PaO 2 ) and lung compliance were measured to assess lung function, and edema (lung wet-to-dry weight ratio), neutrophil infiltration, and cytokine concentrations in bronchoalveolar lavage fluid were assessed. TRPV4 EC activity was assessed as TRPV4 EC sparklets (unitary calcium influx signals through TRPV4 channels) in en face 4th-generation pulmonary arteries (PAs, ~50mm) loaded with fluo-4AM (10 mM). PAs as well as primary pulmonary microvascular endothelial cells (PMVECs) were exposed to hypoxia-reoxygenation (HR) to simulate IRI, and TRPV4 EC sparklet activity recorded. Results: Compared with control mice, PaO 2 and compliance after IR was significantly improved in P2Y2R EC -/- mice. Pulmonary edema, neutrophil infiltration, lipid peroxidation, and proinflammatory cytokines (TNFα, CXCL1, IL-6, CCL3/MIP-1α) were significantly reduced after IR in P2Y2R EC -/- mice. IR significantly induced TRPV4 EC activity in PAs, which was absent in PAs from P2Y2R EC -/- , TRPV4 EC -/- , and Panx1 EC -/- mice. IR-induced TRPV4 EC activity was also inhibited by apyrase (10 U/mL), which degrades extracellular ATP, confirming a role for extracellular ATP in IR-induced TRPV4 EC activity. Exposure to HR also increased TRPV4 EC sparklet activity in PAs and PMVECs, which was inhibited by P2Y2R inhibitor AR-C (10 mM). Exposure to HR also increased ionic currents through TRPV4 EC channels in PMVECs. Conclusion: An endothelial Panx1-P2Y2R-TRPV4 signaling axis plays a key role in mediating lung edema, inflammation, and dysfunction after IR. P2Y2R may be a potential therapeutic target for lung IRI. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.