Role of heme and PGP matrikines in lung inflammation

项目概要： 脓毒症相关的急性肺损伤（ALI），其特征是肺通透性增加和炎症， 危重患者的重要死因。几种炎症介质和相关机制 已经确定了肺渗漏增加，这虽然导致了治疗的改进，但已经达到稳定状态， 提示尚未确定的机制和/或不同介质之间的相互作用。在本提案中，我们 建立在我们的初步数据表明，脯氨酸-甘氨酸-脯氨酸（PGP）肽来源于基质， 蛋白质胶原蛋白，在人类和小鼠败血症模型中增加，并且这些基质因子促进肺 通过CXCR 2激活泄漏。在本申请中，我们解决以下三个问题i）什么机制 规范PGP的形成？我们推测，游离血红素，一种新发现的脓毒症发病机制的介质， 刺激含有活性脯氨酰内肽酶（PE）的外泌体的释放，所述活性脯氨酰内肽酶（PE）是一种蛋白酶， 胶原在细胞外室中分解为PGP的终末步骤。初步数据显示 血红素结合素降低体内循环PGP水平; ii）内源性PGP介导脓毒症吗 诱导肺通透性？初步数据显示施用Arg-Thr-Arg（RTR），一种 选择性结合和抑制PGP，在体外和体内防止肺渗漏; iii）PGP是否与肺渗漏的严重程度相关？ 疾病与急性肺损伤的关系初步数据显示，在人类败血症中循环PGP肽增加 并证明这能够引发促渗透性信号传导。我们提出3个目标来检验我们的假设 PGP matrikines介导脓毒症-ALI，SA 1：确定游离血红素的作用和机制， 介导PGP肽形成。SA 2：确定PGP介导脓毒症-ALI的作用和机制 和SA 3：确定游离血红素、PE-外来体和PGP肽的血浆水平是否与两者相关。 脓毒症患者的临床结局和肺损伤的发展。我们建议使用以下组合 原代人上皮细胞、肺微血管内皮细胞和盲肠结扎穿孔模型 使用C57 Bl/6雄性和雌性小鼠来测试我们的假设，并将其与生化和 分子方法来测量蛋白酶活性、游离血红素水平、外来体和PGP肽水平。 使用血红素、PGP和产生PGP的蛋白酶的选择性抑制剂将获得机制见解， 以及为所提出的研究产生的新的内皮靶向CXCR 2-/-小鼠模型。成功 这些目标的完成将显著提高我们对脓毒症-ALI的潜在机制的理解， 特别强调含有蛋白酶的外泌体和基质因子作为新的介质和治疗靶点 在这种令人衰弱的疾病中。

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.