Targeting Latexin Signaling for Endothelial Barrier Dysfunction in Inflammatory Lung Injury

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Project Summary The Acute Respiratory Distress Syndrome (ARDS) is a common cause of respiratory failure in the critically ill, accounting for ~75000 deaths/year and 3.6 million hospital days. The existing paradigm is that the ARDS results from widespread dysfunction of the pulmonary endothelium, leading to microvascular thrombosis, interstitial edema, alveolar flooding and respiratory failure, although the mechanisms leading to endothelial dysfunction remain unknown. This proposal arises from novel observations linking the upregulation of Latexin to endothelial dysfunction in the lung. We found that healthy lung endothelium expresses low levels of Latexin whereas levels markedly increase in response to turbulent flow or bacterial LPS. Further, we found that endothelial barrier function was enhanced by reducing Latexin expression in LPS-exposed lung endothelial cells and that global deficiency of Latexin in mice reduced LPS-induced pulmonary edema, lung inflammation and mortality. Mechanistically, we found that Latexin mediated its effects through complex mechanisms, including binding to the enzyme Src kinase and facilitating its membrane translocation and phosphorylation of VE-cadherin at adherens junctions (AJ). Additionally, mass spectroscopy revealed that Latexin physically interacts with several other proteins, including clathrin and AP2, suggesting a role in endocytic trafficking, and syntaxin-3 and SNAP3, suggesting a role in exocytosis. Consistent with this latter mechanism, we found that Latexin deficiency reduced von Willenbrand factor secretion from lung endothelium. Taken together, these observations lead us to propose the following central hypothesis regarding the role of Latexin in lung endothelial biology and the pathogenesis of endothelial dysfunction in ARDS. We hypothesize that Latexin plays a pathogenic role in driving agonist- induced endothelial dysfunction in the lung and that inhibiting its interactions with other key proteins will reduce the severity of pulmonary edema, lung inflammation and microvascular thrombotic complications in experimentally-induced ARDS. To test this hypothesis, we propose 3 independent but mechanistically linked Specific Aims. In Aim 1, we will establish that endothelial-specific deletion of Latexin enhances endothelial barrier protection and reduces microvascular thrombosis and mortality to LPS in mice. In Aim 2, we will delineate the molecular mechanisms by which Latexin regulates VE-cadherin membrane bioavailability at endothelial AJs by performing various in vitro and in vivo loss- and gain-of-function studies for genes linked to Src-mediated VE-cadherin phosphorylation and clathrin-mediated endocytosis in ECs. In Aim 3, we will delineate the molecular mechanisms by which Latexin mediates vWF secretion from endothelial cells, focusing on its role in SNARE complex formation. In toto, this proposal will establish the mechanisms by which Latexin regulates key pathological behaviors in lung endothelium and provide direction for developing novel therapeutic approaches for inflammatory vascular diseases of the lung, such as the ARDS.