Duplex miR-223 and Exosomes in Sepsis

描述（由申请方提供）：心血管功能障碍是危重患者败血症诱导死亡的主要原因。尽管进行了数十年的深入研究，但基本机制仍然难以捉摸。特别是，针对单一介质或炎症途径的治疗干预未能改善脓毒症患者的心脏功能和生存结局。因此，继续寻找靶向多种因子的有效治疗试剂是必要的。 非常需要。最近鉴定的microRNA可能为有效的脓毒症治疗提供新的靶点。例如，我们的最新工作发现：1）miR-223（-5p & -3p）在严重脓毒症后小鼠心脏中显著降低; 2）双链体miR-223的整体丢失加重脓毒症诱导的心功能障碍和死亡率; 3）miR-223- 5 p抑制TNFR 1和Sema 3A的表达miR-223- 3 p抑制巨噬细胞和心肌细胞IL-6、STAT 3、MIP-1、MIP-2的表达。考虑到这些分子中的每一种都可能导致脓毒症诱导的心功能障碍和死亡率，测试双链体miR-223的总体升高对脓毒症引起的损伤的影响将是重要的。我们的试验数据还表明：1）与健康的外泌体相比，从脓毒症小鼠血液中分离的外泌体（称为脓毒症外泌体）中包裹的双链体miR-223的量较少，2）脓毒症外泌体损害心肌细胞收缩力并刺激巨噬细胞释放促炎细胞因子（即TNF-β IL-6）。因此，重要的是测试双链体miR-223是否是一个关键的限制因素，其减少有助于脓毒性外泌体诱导的对心脏功能和死亡率的有害影响。最后，提出了一个重要的翻译问题：工程化外泌体含有额外的miR-223对脓毒症是否有任何积极的治疗作用？在该提案中，我们假设双链体miR-223的总体升高通过在多个水平靶向不同的介质来限制脓毒症诱导的心功能障碍和死亡率，并且双链体miR-223的丧失有助于脓毒症外泌体触发的心功能障碍。这些想法将通过追求三个具体目标来测试：1）使用转基因小鼠模型，我们将测试双链体miR-223的整体升高是否减弱脓毒症诱导的心肌抑制和死亡率; 2）利用从miR-223敲除小鼠和CLP手术小鼠的血液中分离的双链体miR-223无效外泌体和脓毒性外泌体，以测试脓毒性外泌体是否与miR-223敲除小鼠和CLP手术小鼠的血液中分离的外泌体相关。介导的心功能障碍和死亡率的降低归因于双链体miR-223的减少;和3）我们将使用骨髓来源的间充质干细胞（MSC）作为miR-223-外泌体的来源，以确定经miR-223工程改造的外泌体对脓毒症诱导的心功能障碍和死亡率的治疗效果。总之，拟议的研究有望揭示双链体miR-223在脓毒症诱导的心力衰竭中先前未被认识的作用。此外，他们预计将提供新的见解，导致原始的基于外泌体的治疗策略的发展，以减少脓毒性死亡。

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.