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

描述(申请人提供)：急性肺损伤(ALI)仍然是一种毁灭性的综合征，每年在美国影响20多万名患者，死亡率接近40%。目前，还没有降低死亡率的药物疗法。因此，需要对翻译疗法进行进一步的研究。以间充质干细胞(MSC)为基础的干细胞治疗是一种很有吸引力的新方法。MSC具有分泌多种旁分泌因子的能力，可调节肺内皮细胞和上皮细胞的通透性，减轻炎症反应，促进组织修复，抑制细菌生长。在使用MSC作为治疗方法的150多项临床试验中，超过2000名患者接受了这种细胞，没有任何重大并发症。然而，尽管MSC具有良好的安全性，但它在体外多代传代后仍具有自发恶性转化的能力，并具有促进体内肿瘤生长的能力。最近，一些研究人员发现，人骨髓间充质干细胞释放的微囊泡(microvesicles，MV)具有与干细胞一样的生物学活性，部分原因是通过在损伤的组织床上转移和表达微囊泡(microvesicles，MV)。在这一应用中，我建议研究人骨髓来源的MSC MV的生物学，并测试其作为ALI模型细胞治疗的替代方案的潜在治疗用途。总的假设是，人MSC MV具有生物学活性，其治疗活性主要是通过将mRNA从MV转移到受损的肺上皮和肺内皮细胞来实现的。在目标1中，主要目的是研究MSC MV的生物学，并使用RNA和蛋白质合成和运输的抑制剂来确定MSC MV的哪些成分具有功能活性。我推测，MSC MV需要使用细胞膜受体(如CD44)将关键旁分泌溶解因子的mRNA从MV转移到受损的肺上皮或内皮细胞，以发挥其治疗作用。在目标2中，我将测试功能 人MSC MV对人肺泡II型上皮细胞液体净转运和炎性损伤的人肺微血管内皮细胞蛋白通透性的影响，这是ALI的主要病理特征。我推测MSC MV通过恢复主要上皮钠通道ENaC的顶膜表达来阻止损伤的II型细胞液体净转运的减少，并通过防止肌动蛋白应激纤维的形成来减少损伤的肺内皮细胞蛋白通透性的增加。在目标3中，我将确定在大肠杆菌内毒素诱导的ALI损伤的小鼠中，人MSC MV是否具有生物活性。我推测MSC MV可能通过恢复肺内皮细胞和上皮蛋白的通透性、肺液平衡和减轻肺泡炎来减轻内毒素诱导的小鼠ALI。这些研究将为MVS的释放提供新的见解，并解释为什么MSC MVS在组织修复中可能有效的潜在机制。此外，这些结果可能为MSC MV作为ALI治疗方法的发展提供临床前数据。

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.