Human mesenchymal stem cell microvesicles for the treatment of acute lung injury

描述（由申请人提供）：急性肺损伤（ALI）仍然是一种毁灭性的综合征，在美国每年影响超过20万例患者，死亡率接近40%。目前，没有降低死亡率的药物治疗。因此，需要进一步研究转化疗法。间充质干细胞（MSC）的干细胞治疗是一种有吸引力的新方法。MSC具有分泌多种旁分泌因子的能力，这些因子可以调节肺内皮和上皮的通透性，减少炎症，增强组织修复，并抑制细菌生长。在clinicaltrials.gov注册的150多项使用MSC作为治疗的临床试验中，超过2000名患者接受了细胞，没有任何重大并发症。尽管具有良好的安全性，但是，MSC在体外多次传代后具有自发恶性转化的能力以及促进体内肿瘤生长的能力。最近，一些研究人员发现，由人MSC释放的微泡（MV）具有与干细胞一样的生物活性，部分通过在损伤组织床中转移和表达MV mRNA。在这个应用中，我建议研究生物学和测试潜在的治疗用途的人骨髓来源的MSC MV作为替代细胞治疗模型的ALI。总体假设是人MSC MV具有生物活性，并且其治疗活性主要通过将mRNA从MV转移到受损的肺上皮和肺内皮来介导。在目的1中，主要目的是研究MSC MV的生物学，并使用RNA和蛋白质合成和转运的抑制剂确定MSC MV的哪些组分具有功能活性。我假设MSC MV需要使用细胞膜受体（如CD44）将关键旁分泌可溶性因子的mRNA从MV转移到受损的肺上皮或内皮，以实现其治疗效果。在目标2中，我将测试功能 人MSC MV对人肺泡上皮II型细胞中的净液体转运的活性和对由炎症损伤损伤的人肺微血管内皮细胞中的肺内皮对蛋白质的渗透性的活性，这是ALI的主要病理特征。我假设MSC MV将通过恢复主要上皮钠通道的顶膜表达来防止受损II型细胞中净液体转运的减少，并且将通过防止肌动蛋白应力纤维的形成来减少受损肺内皮细胞中蛋白质通透性的增加。在目标3中，我将确定人MSC MV在大肠杆菌内毒素诱导的ALI损伤的小鼠中是否具有生物活性。我推测MSC MV将通过恢复肺内皮和上皮蛋白通透性、肺液体平衡和减少肺泡炎症来减少小鼠内毒素诱导的ALI。这些研究将提供新的见解，了解如何释放的MV和潜在的机制，解释为什么MSC MV可能是有效的组织修复。此外，这些结果可能提供临床前数据，可以促进MSC MV作为ALI治疗的发展。 公共卫生相关性：由人类间充质干细胞释放的微泡在急性肺损伤的各种临床前模型中具有生物活性，急性肺损伤是危重患者急性呼吸衰竭的重要原因。有益的机制是通过mRNA从微泡转移到受损的肺上皮和内皮细胞，导致几种蛋白质的表达，这些蛋白质可以减少肺损伤并增强修复。

DESCRIPTION (provided by applicant): Acute lung injury (ALI) remains a devastating syndrome affecting more than 200,000 patients annually in the U.S. with a mortality rate approaching 40%. Currently, there are no pharmacologic therapies that reduce mortality. Consequently, further research into translational therapies is needed. Stem cell-based therapy with mesenchymal stem cells (MSC) is one attractive new approach. MSC have the capacity to secrete multiple paracrine factors that can regulate lung endothelial and epithelial permeability, decrease inflammation, enhance tissue repair, and inhibit bacterial growth. In over 150 clinical trials registered with clinicaltrials.gov using MSC as therapy, over 2000 patients have received the cells without any major complications. Despite a favorable safety profile, however, MSC have the capacity for spontaneous malignant transformation following multiple passages in vitro as well as the ability to promote tumor growth in vivo. Recently, some investigators have found that microvesicles (MV) released by human MSC are as biologically active as the stem cells in part through the transfer and expression of MV mRNA in the injured tissue bed. In this application, I propose to study the biology and test the potential therapeutic use of human bone-marrow derived MSC MV as an alternative to cellular therapy in models of ALI. The overall hypothesis is that human MSC MV are biologically active, and that their therapeutic activity is primarily mediated through transfer of mRNA from the MV to injured lung epithelium and lung endothelium. In Aim 1, the primary objective is to study the biology of MSC MV and determine which components of the MSC MV are functionally active, using inhibitors of RNA and protein synthesis and transport. I hypothesize that MSC MV require the transfer of mRNA for key paracrine soluble factors from the MV to the injured lung epithelium or endothelium using a cell membrane receptor, such as CD44, for their therapeutic effect. In Aim 2, I will test the functional activity of human MSC MV on net fluid transport in human alveolar epithelial type II cells and on lung endothelial permeability to protein in human lung microvascular endothelial cells injured by an inflammatory insult, the main pathological features of ALI. I hypothesize that MSC MV will prevent the decrease in net fluid transport in injured type II cells by restoring the apical membrane expression of the major epithelial sodium channel, ¿ENaC, and will reduce the increase in protein permeability in injured lung endothelial cells by preventing the formation of actin stress fibers. In Aim 3, I will determine if human MSC MV are biologically active in mice injured with E.coli endotoxin-induced ALI. I hypothesize that MSC MV will reduce endotoxin-induced ALI in mice by restoring lung endothelial and epithelial protein permeability, lung fluid balance and by reducing alveolar inflammation. These studies will provide novel insights into how MVs are released and the underlying mechanisms that explain why MSC MVs may be effective in tissue repair. Furthermore, the results may provide preclinical data that could facilitate development of MSC MV as a therapy for ALI. PUBLIC HEALTH RELEVANCE: Microvesicles released by human mesenchymal stem cells are biologically active in various pre-clinical models of acute lung injury, an important cause of acute respiratory failure in critically ill patients. The mechanism of benefit is through the transer of mRNA from the microvesicles to the injured lung epithelial and endothelial cells, leading to the expression of several proteins that can reduce lung injury and enhance repair.