ACTIN FENCE THERAPY FOR ACUTE LUNG INJURY

Background: Acute lung injury (ALI), which leads to the acute respiratory distress syndrome (ARDS), causes substantial morbidity and mortality. There is no pharmaco-specific cure for ALI. Our goal is to develop mechanistic understanding of ALI in the context of a protective biologic that we developed to provide rapid cure of ALI.Rationale: Our project is based on the notion that ALI is a disease characterized by severe inflammation that causes loss of alveolar F-actin and breakdown of the fluid barrier formed by the alveolar epithelium, resulting in pulmonary edema. Alveolar proinflammatory receptors (PIRs) are ligated by cytokines released during inflammation, sustaining lung injury. The disease is of rapid onset, requiring intensive care support within hours. Our goals are to understand the role of F-actin in the inflammatory process and to develop intracellular protein therapy to enhance alveolar actin as rapid therapeutic intervention.Hypothesis: We will determine the role of alveolar F-actin in alveolar display of the PIRs, TNFR1, and IL-1betaR, which are cytokine receptors. We propose that F-actin forms a fence that potentially impedes alveolar display of the PIRs. ALI occurs following a sequence of events in which proinflammatory cytokines such as TNFalpha and IL-1beta released by activated alveolar macrophages, activate PIRs, increasing alveolar cytosolic Ca2+ (cytCa2+), inducing Ca2+-dependent activation of the phosphatase, calcineurin. Calcineurin dephosphorylates, hence activates the F-actin severing protein, cofilin to cause actin depolymerization. This lowers the cortical F-actin fence, increasing alveolar display of PIRs. The resulting proinflammatory signaling cascade weakens the alveolar fluid barrier, causing ALI. In mouse models of ALI, intracellular delivery of our novel biologic consisting of the actin polymerizing agent Rac1 enhanced F-actin and abrogated alveolar PIR display. We propose that enhancement of the alveolar F-actin fence will be therapeutic in ALI.Specific Aims and Objectives: We will determine the physiological regulation of the alveolar F-actin fence (Aim 1), role of the actin fence in determining expression of alveolar PIRs (Aim 2), and the extent to which enhancement of alveolar F-actin fence is protective in ALI (Aim 3). We will evaluate underlying molecular mechanisms, and we will apply novel methods for alveolar delivery of activated Rac1 to reinforce the F-actin fence as a strategy for lung repair.Study Design: We will achieve these aims by live confocal microscopy of isolated perfused mouse lungs and by studies in isolated alveolar epithelial cells. Our experiments will include: (1) Dynamic quantifications of F-actin expression and the cytosolic Ca2+ in intact alveoli of live lungs; (2) assays of calcineurin-cofilin activation by optical imaging and western blot; (3) development of methods for delivery of the actin polymerizing agent Rac1 to the alveolar epithelium; and (4) assays of ALI induced by lung infection and ischemia-reperfusion.Short-Term and Long-Term Impact: In the short term, this proposal will provide a systematic determination of the mechanisms regulating alveolar F-actin and the extent to which this regulation determines proinflammatory receptor expression in alveoli. In the long term, new therapeutic strategies for ALI will be realized. Overall, our findings will be highly novel and of high impact in the understanding and therapy of ALI.Relevance of the Project to at Least One Fiscal Year 2015 (FY15) Peer Reviewed Medical Research Program (PRMRP) Topic Area: The proposed project relates directly to the FY15 PRMRP topic area 'Acute Lung Injury.'