PROJECT SUMMARY/ABSTRACT Sepsis is a catastrophic systemic inflammatory host response to infection, which can lead to vascular leak, edema, organ failure, and death. Despite intense study, few therapeutic strategies other than nonspecific supportive care have been developed and death rates remain as high as 60-70% in cases of septic shock. More than 750,000 Americans contract sepsis each year and more of these patients die than those that succumb to breast cancer, prostate cancer, and AIDS combined. It is known that agonists found in septic patients, such as inflammatory cytokines, VEGF, thrombin, microparticles, bacterial toxins, and bacteria themselves induce the vascular instability and edema that help trigger septic pathophysiology. Our preliminary data suggest that the direct and immediate effects of these endothelial-disrupting agents may be mediated by diverse receptors that signal via a common convergence point, the intracellular GTPase ARF6. ARF6 appears to control trafficking of cell-cell junction proteins and is distinct from the canonical inflammatory pathways that regulate transcription (e.g., those activating NF-κB). In animal models of inflammatory disease, inhibiting the activation of ARF6 either through conditional genetic ablation or chemical inhibition stabilizes the vasculature, decreases inflammation, and increases survival rates. Therefore, we hypothesize that activation of ARF6 during sepsis induces pathologic vascular leak, which contributes to multi-organ failure and death and that pharmacologic inhibition or genetic ablation of ARF6 or its activating ARF-GEFs will stabilize human and mouse endothelium exposed to septic insults and increase survival rates in animal models of sepsis. We realize that the septic response in mice may not completely mimic the human response. Therefore, we will assess the similarities and differences between the species in regards to ARF-GEF—ARF6 pathway and its control of vascular integrity. In Aim 1, we will determine whether ARF6 represents a convergence point for regulating vascular permeability induced by agonists generated in the septic milieu. We will use defined agonists that are present in the plasma of sepsis patients to determine whether these agonists signal through ARF6 or other ARF family members to induce paracellular permeability of both human and mouse endothelium. We will identify the ARF-GEFs and adaptor proteins involved in these signaling processes. In Aim 2, we will individually ablate Arf6 and Arno (a known ARF6-GEF) in mice and use chemical inhibition of ARF6 in several animal models of sepsis to determine whether removal or inhibition of ARF6 activity stabilizes the vasculature and increases survival rates. To more closely mimic clinical situations, we will also use ARF6 inhibition as an adjuvant to antibiotics to assess whether combination therapy can reduce vascular leak and mortality rates in these animal models. In Aim 3, we examine the efficacy of ARF6 inhibition i...