The Contribution of Superoxide Influx via LRRC8 Anion Channels to Infectious Vascular Dysfunction

About This Grant

PROJECT SUMMARY Sepsis, the systemic inflammatory response of a host to a pathogen, is a leading cause of death in modern intensive care units. It induces significant vascular dysfunction that manifests as altered vascular tone, leading to hypotension and capillary leak, leading to edema and acute respiratory distress syndrome. Therapies that target vascular pathophysiology in sepsis are lacking, and clinical care remains primarily supportive. We have found that leucine-rich repeat containing 8 A (LRRC8A), the required isoform for all volume-regulated anion channels (VRACs), significantly modulates both the endothelial cell (EC) and vascular smooth muscle cell (VSMC) response to multiple acute inflammatory challenges. Our data demonstrate that LRRC8A, in partnership with another specific isoform, LRRC8C, functions as both regulators of superoxide (O2-•) production by NADPH oxidases (NOXes), and a portal for O2-• influx. Movement of O2-• into the cytoplasm sets off a series of intracellular redox signals that enhance vascular contraction, impair relaxation, reduce endothelial barrier function, and cause significant alterations in metabolism. Mice with VSMC-specific knockout of LRRC8A have preserved vascular reactivity and LRRC8A knockdown EC cells have reduced permeability in response to inflammatory challenges. Importantly, our data also suggests that FDA-approved LRRC8 inhibitors administered after infectious challenge can reduce EC inflammation, indicating LRRC8 blockade may serve as a post-infectious rescue. In this proposal, we will test the central hypothesis that LRRC8A/C channels regulate vascular inflammation during infectious challenge. O2-• influx alters Rho GTPase-mediated regulation of the cytoskeleton, leading to impaired metabolism and altered cellular contractility. Further, we hypothesize that blocking LRRC8 channels with known FDA-approved pharmaceuticals after the onset of sepsis can improve vascular function and outcomes. Aim #1. Influx of O2-• controls VSMC and EC cytoskeletal function via LRRC8-dependent signaling. We will define mechanisms by which NOX/LRRC8/O2-• drives inflammation-induced changes in VSMC vascular contractility and EC barrier function via a multi-protein complex that controls small GTPases. We will further assess how O2-• bioavailability affects the composition and function of this complex, alters inflammatory signaling, and regulates the cytoskeleton. Aim #2. O2-• influx alters metabolism to promote inflammation-induced vascular dysfunction. The impact of O2-• on inflammation-induced changes in metabolism (Seahorse, 13C-labelling) will be correlated with vasomotor and barrier function (wire myography, transendothelial resistance). Aim #3. Impaired vascular function in sepsis is abrogated by reduced LRRC8-dependent signaling. Vasculopathy, as examined by wire myography and indices of capillary permeability, will be assessed in murine models of sepsis to determine if genetic or pharmacological impairment of LRRC8 channels improves outcomes. These integrated aims will increase our understanding of fundamental mechanisms that cause infection-mediated vascular dysfunction and assess the potential of targeting LRRC8 channels for the treatment of patients with sepsis.