Role of heme and PGP matrikines in lung inflammation

点击翻译按钮获取中文摘要

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.