UTILIZING SOLUBLE VIMENTIN AND ITS COMPONENTS TO ATTENUATE INFLAMMATION

项目摘要 我是贝勒医学院（Baylor College of Medicine）的儿科助理教授，有着长期的- 中性粒细胞、血小板和内皮细胞之间复杂的相互作用， 微循环作为一名儿科重症监护医生，我的主要职业目标是 成为一名成功的独立研究者，专注于病理学炎症以及如何 它导致了危重患者的发病率和死亡率，例如急性肺损伤 脓毒症（严重感染）。作为一名博士后临床研究员，我调查了 血小板对中性粒细胞迁移的促炎作用为此奠定了基础 提议本K 08提案的主要目标是获得蛋白质方面的额外专业知识 生物化学和动物研究能够发现和测试这些新的疗法， 毁灭性的条件。这个建议将使我有受保护的时间上课， 分子生物学和生物化学以及在实验室磨练我的技术技能。 此外，它将使我能够参加临床科学家培训计划， 将提高我在实验设计，科学写作，研究道德， 生物统计学我的主要研究导师米格尔·克鲁兹博士是一位著名的研究员， 成功资助研究全身性炎症中的微血管血栓形成。他 已经成功地创造了重组蛋白质，包括用于这一研究的可溶性波形蛋白。 提议除了克鲁兹博士，我的研究顾问委员会由蒂莫西博士组成。 Palkzill（药理学），大卫科里（肺部炎症的小鼠模型），C韦恩史密斯 （白细胞运输）和Perumal Thiagarajn（血小板生物学），他们都是他们的专家。 与本提案相关的各个领域。我们观察到可溶性波形蛋白减少 炎症通过阻断白细胞粘附到血小板和内皮细胞， 阻断P-选择素与其对应物P-选择素糖蛋白配体-1之间的相互作用 （PSGL-1）。我们在小鼠中的初步研究表明，可溶性波形蛋白减少了对 肺脓毒症模型。可溶性波形蛋白是否会减少肺损伤以及 脓毒症导致的死亡率尚不清楚。我的建议将测试中心假设，可溶性 波形蛋白通过阻断P-选择素-PSGL-1相互作用来减轻炎症， 白细胞粘附和穿过内皮的迁移。我将通过 （1）确定波形蛋白-P-的活性基序和结合动力学; （2）评价可溶性波形蛋白对血小板增强的中性粒细胞 （3）评估可溶性血管内皮细胞的功效， 波形蛋白及其组分在两种模型中减轻小鼠继发性急性肺损伤的作用 实验性败血症、内毒素血症和盲肠结扎和穿孔。在目标1中，我将使用 不同的生物物理技术，如表面等离子体共振和圆二色性， 评估可溶性波形蛋白与P-选择素的结构和结合动力学。在目标2中，我将使用 体外动态和静态粘附测定以测量可溶性波形蛋白对 中性粒细胞跨内皮迁移，这是我之前发表的一项新观察。 最后，在目标3中，我将研究两种不同的实验性脓毒症模型，因为它们具有 不同的炎症机制。我也会研究可溶性波形蛋白的安全性 局我预计可溶性波形蛋白会通过阻断 白细胞-血小板-内皮细胞相互作用，并将减少脓毒症中的肺损伤， 降低死亡率，这将导致新的治疗方法来预防和治疗重症患者。

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.