Duplex miR-223 and Exosomes in Sepsis

描述(由申请人提供)：心血管功能障碍是重症患者败血症导致死亡的主要原因。尽管进行了数十年的密集研究，但基本机制仍然难以捉摸。特别是，针对单一炎症介质或途径的治疗干预未能改善脓毒症患者的心功能和生存结局。因此，继续寻找针对多种因素的有效治疗试剂是 非常需要。最近发现的microRNAs可能为有效的脓毒症治疗提供新的靶点。我们的最新工作发现，例如：1)miR-223(-5p和-3p)在严重脓毒症后小鼠心脏显著减少；2)双链miR-223的全局缺失加剧了脓毒症所致的心功能障碍和死亡率；以及3)miR-223-5p抑制了巨噬细胞和心肌细胞中IL-6、STAT3、MIP-1和�的表达。鉴于这些分子中的每一个都可能导致脓毒症引起的心功能障碍和死亡率，测试双重miR-223的全球升高对脓毒症引起的损伤的影响将具有重要意义。我们的试点数据还表明：1)与健康小鼠相比，从脓毒症小鼠血液中分离出的双链miR-223被较少的双链miR-223包裹(称为败血症外切体)，2)脓毒症外切体会损害心肌细胞的收缩能力，并刺激巨噬细胞释放促炎细胞因子(即肿瘤坏死因子-�IL-6)。因此，重要的是要测试双链miR-223是否是一个关键的限制因素，它的减少有助于败血症外切体对心功能和死亡率的不利影响。最后，提出了一个重要的翻译问题：工程外切体是否有任何积极的治疗作用，以包含额外的miR-223对脓毒症？在这项提议中，我们假设，双链miR-223的全球升高通过在多个水平上靶向不同的介质来限制脓毒症引起的心功能障碍和死亡率，而双链miR-223的缺失导致败血症外切体触发的心功能障碍。这些想法将通过追求三个特定的目标来验证：1)利用转基因小鼠模型，我们将测试双链miR-223的全球上调是否可以减轻脓毒症引起的心肌抑制和死亡率；2)利用从miR-223基因敲除小鼠和CLP手术小鼠的血液中分离的二聚体miR-223缺失的外切体和败血症外切体来测试败血症外切体介导的心功能障碍和死亡率是否归因于双链miR-223的减少；3)我们将以骨髓间充质干细胞(MSCs)作为miR-223-exosome的来源，以确定双链miR-223工程exosome对脓毒症所致心功能障碍和死亡率的治疗作用。总之，拟议的研究有望揭示一个以前未被认识到的双重miR-223在脓毒症所致心力衰竭中的作用。此外，他们有望提供新的见解，导致原始的基于外切体的治疗策略的发展，以减少败血症死亡。

DESCRIPTION (provided by applicant): Cardiovascular dysfunction is a major contributor to sepsis-induced death in critically ill patients. Despite decades of intensive study, the basic mechanisms remain elusive. In particular, therapeutic interventions aimed at a single mediator or pathways of inflammation have failed to improve cardiac function and survival outcome in sepsis. Thus, continuing search for effective therapeutic reagents which target multiple factors is greatly needed. Recent identified microRNAs may provide new targets for effective sepsis therapy. Our latest work has discovered, for example, that: 1) miR-223 (-5p & -3p) are significantly reduced in mouse hearts after severe sepsis; 2) global loss of duplex miR-223 aggravated sepsis-induced cardiac dysfunction and mortality; and 3) miR-223-5p inhibited the expression of TNFR1 and Sema3A (a ligand of TLRs), while miR-223-3p repressed the expression of IL-6, STAT3, MIP-1�MIP-2 in macrophages and myocytes. Given that each of these molecules can potentially contribute to sepsis-induced cardiac dysfunction and mortality, it will be significant to test the effects of global elevation of duplex miR-223 on sepsis-caused injury. Our pilot data also indicate that: 1) lesser amounts of duplex miR-223 are encased within exosomes isolated from the blood of septic mice (referred to as septic exosomes), compared to healthy exosomes and 2) septic exosomes impaired cardiomyocyte contractility and stimulated macrophages to release pro-inflammatory cytokines (i.e. TNF-�IL-6). Thus, it will be important to test whether duplex miR-223 is a critical limiting factor whose reduction contributes to septic exosome-induced detrimental effects on cardiac function and mortality. Finally, an important translational question is raised: are there any positive therapeutic effects of engineering exosomes to contain extra miR-223 on sepsis? In this proposal, we hypothesize that global elevation of duplex miR-223 limits sepsis-induced cardiac dysfunction and mortality through targeting diverse mediators at multiple levels, and loss of duplex miR-223 contributes to septic exosome-triggered cardiac dysfunction. These ideas will be tested by pursuing three specific aims: 1) Using a transgenic mouse model, we will test whether global elevation of duplex miR-223 attenuates sepsis-induced myocardial depression and mortality; 2) Utilizing duplex miR-223-null exosomes and septic exosomes isolated from the blood of miR-223-knockout mice and CLP-operated mice to test whether septic exosome-mediated cardiac dysfunction and mortality is ascribed to the reduction of duplex miR-223; and 3) We will use bone marrow-derived mesenchymal stem cells (MSCs) as a source of miR-223-exosomes to determine the therapeutic effects of duplex-miR-223-engineered exosomes on sepsis-induced cardiac dysfunction and mortality. Together, the proposed studies are expected to unveil a previously unrecognized role of duplex miR-223 in sepsis-induced heart failure. Additionally, they are expected to provide novel insights that lead to the development of original exosome-based therapeutic strategies for reducing septic death.