Role of heme and PGP matrikines in lung inflammation

项目总结： 脓毒症相关性急性肺损伤(ALI)，以肺通透性增加和炎症为特征 危重病人的重要死因。几种炎性介质及其作用机制 肺渗漏的增加已被发现，这在导致治疗方法改进的同时，已趋于平稳 暗示尚不明确的机制，和/或不同调解人之间的相互作用。在这项提案中，我们 根据我们的初步数据显示，从基质中衍生的脯氨酸-甘氨酸-脯氨酸(PGP)肽 蛋白胶原蛋白，在人类和小鼠脓毒症模型中增加，这些基质因子促进肺 通过激活CXCR2进行泄漏。在本应用程序中，我们解决了以下三个问题：i)什么机制 调控PGP的形成？我们假设，游离态的血红素，一种新发现的脓毒症发病机制的介体， 刺激含有活性的Pro内肽酶(PE)的外切体的释放，PE是一种催化 胶原蛋白在细胞外腔分解为Pgp的最后一步。初步数据显示拾荒者 血凝素降低体内循环Pgp水平；ii)内源性Pgp是否介导败血症 诱导肺通透性？初步数据显示，给予Arg-Thr-Arg(RTR)，一种能够 选择性地结合和抑制Pgp，在体外和体内防止肺渗漏；iii)Pgp是否与 人类败血症中的疾病和ALI？初步数据显示脓毒症患者循环中的PGP多肽增加 并证明了这能够引发有利于渗透的信号。我们提出三个目标来检验我们的假设 PGP基质因子介导脓毒症-ALI、SA1：确定游离血红素的作用和机制 调节PGP多肽的形成。SA2：确定PGP在脓毒症-ALI中的作用和机制 和SA3：确定血浆中游离血红素、PE外切体和PGP多肽水平是否两者相关 脓毒症患者的临床转归和肺损伤的发展。我们建议结合使用 原代人肺上皮细胞、肺微血管内皮细胞与盲肠结扎穿刺术模型 用C57BL/6雄性和雌性小鼠来验证我们的假设，并将其与生化和 测量蛋白酶活性、游离血红素水平、外切体和PGP多肽水平的分子方法。 使用选择性的血红素、Pgp和产生Pgp的蛋白酶的抑制剂，将获得机理上的见解， 以及为拟议的研究而建立的一种新的内皮靶向CXCR2/-小鼠模型。成功 这些目标的完成将显著提高我们对脓毒症潜在机制的理解-ALI 特别强调将含有外切体和基质因子的蛋白酶作为新的介体和治疗靶点 在这种令人衰弱的混乱中。

PROJECT SUMMARY: Sepsis-related acute lung injury (ALI), characterized by increased lung permeability and inflammation remains a significant cause of death in critically ill individuals. Several inflammatory mediators and mechanisms underlying increased lung leak have been identified, which while leading to improved therapeutics, have plateaued suggesting as yet undefined mechanisms, and / or interactions between distinct mediators. In this proposal, we build upon our preliminary data showing that proline-glycine-proline (PGP) peptides derived from the matrix protein collagen, are increased in human and murine models of sepsis, and that these matrikines promote lung leak via CXCR2 activation. In this application, we address the following three questions i) What mechanisms regulate PGP formation? We hypothesize that free heme, a newly identified mediator of sepsis pathogenesis, stimulates the release of exosomes containing active prolyl endopeptidase (PE), a protease that catalyzes the terminal step of collagen breakdown to PGP in the extracellular compartment. Preliminary data show scavenging of heme by hemopexin decreases circulating PGP levels in vivo; ii) Does endogenous PGP mediate sepsis induced lung permeability? Preliminary data show that administration of Arg-Thr-Arg (RTR), a peptide that selectively binds and inhibits PGP, prevents lung leak in vitro and vivo; iii) Does PGP associate with severity of disease and ALI in human sepsis? Preliminary data show increases in circulating PGP peptides in human sepsis and demonstrate that this is able to elicit pro-permeability signaling. We propose 3 aims to test our hypothesis that PGP matrikines mediate sepsis-ALI, SA1: Determine the role and mechanisms by which free heme mediates PGP peptide formation. SA2: Determine the role and mechanisms by which PGP mediates sepsis-ALI and SA3: Determine whether plasma levels of free heme, PE-exosomes, and PGP peptides correlate both clinical outcomes and development of lung injury in patients with sepsis. We propose to use a combination of primary human epithelial cells, pulmonary microvascular endothelial cells and the cecal-ligation puncture model of sepsis using C57Bl/6 male and female mice to test our hypothesis, and couple this with biochemical and molecular approaches to measure protease activity, free heme levels, exosomes and PGP peptide levels. Mechanistic insights will be gained using selective inhibitors of heme, PGP and proteases that generate PGP, as well as a novel endothelial targeted CXCR2-/- mouse model generated for the proposed studies. Successful completion of these aims will significantly improve our understanding of mechanisms underlying sepsis-ALI with a specific emphasis on protease containing exosomes and matrikines as new mediators and therapeutic targets in this debilitating disorder.