UTILIZING SOLUBLE VIMENTIN AND ITS COMPONENTS TO ATTENUATE INFLAMMATION

项目摘要

Project Abstract I am an Assistant Professor of Pediatrics at the Baylor College of Medicine (BCM) with a long- standing interest in the complex interaction between neutrophils, platelets, and endothelium in the microcirculation. As a Pediatric Critical Care Medicine physician, my main career goal is to become a successful independent investigator with a focus on pathology inflammation and how it contributes to the morbidity and mortality in critically-ill patients, such as in acute lung injury and sepsis (severe infection). As a post-doctoral clinical fellow, I investigated the proinflammatory effect of platelets on neutrophil transmigration that lay the groundwork for this proposal. The primary objective of this K08 proposal is to obtain additional expertise in protein biochemistry and animal studies to be able to discover and test novel therapies for these devastating conditions. This proposal will allow me to have protected time to attend classes in molecular biology and biochemistry as well as hone my technical skills in the laboratory. Additionally, it will allow me to enroll in the Clinical Scientist Training Program at BCM, which will improve my knowledge in experimental design, scientific writing, research ethics, and biostatistics. My primary research mentor, Dr. Miguel Cruz, is a renowned researcher with successful funding on studying microvascular thrombosis in systemic inflammation. He has been successful in creating recombinant proteins, including the soluble vimentin used in this proposal. In addition to Dr. Cruz, my research advisory committee consists of Drs. Timothy Palkzill (Pharmacology), David Corry (Murine models of lung inflammation), C Wayne Smith (leukocyte trafficking), and Perumal Thiagarajn (platelet biology), who are all experts in their respective fields related to this proposal. We have observed that soluble vimentin decreases inflammation by blocking leukocyte adhesion to both platelets and endothelial cells through blocking the interaction between P-selectin and its counter-part, P-selectin glycoprotein ligand-1 (PSGL-1). Our preliminary studies in mice suggest that soluble vimentin decreases injury to the lung in one model of sepsis. Whether soluble vimentin results in decreasing lung injury and mortality due to sepsis is unknown. My proposal will test the central hypothesis that soluble vimentin attenuates inflammation by blocking P-selectin-PSGL-1 interactions to decrease leukocyte adhesion and transmigration across endothelium. I will test this hypothesis through the following 3 specific aims: (1) Identify the active motif and binding kinetics of vimentin-P- selectin interactions, (2) Evaluate the effect of soluble vimentin on platelet-enhanced neutrophil transmigration across inflamed endothelium in vitro, and (3) Evaluate the efficacy of soluble vimentin, and its components, in reducing secondary acute lung injury in mice in two models of experimental sepsis, endotoxemia and cecal-ligation and perforation. In Aim 1, I will use different biophysical techniques, such as surface plasmon resonance and circular dichroism, to evaluate the structure and binding kinetics of soluble vimentin to P-selectin. In Aim 2, I will use in vitro dynamic and static adhesion assays to measure the effect of soluble vimentin on neutrophil transendothelial migration, a novel observation on which I had previously published. Finally, in Aim 3, I will study two different models of experimental sepsis because they have different mechanisms of inflammation. I will also study the safety of soluble vimentin administration. I anticipate that soluble vimentin will attenuate inflammation by blocking leukocyte-platelet-endothelial interactions and that it will decrease lung injury in sepsis and improve mortality, which will lead to novel therapies to prevent and treat critically ill patients.