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，每年在美国影响超过20万患者，死亡率接近40%。目前，还没有降低死亡率的药物疗法。因此，需要进一步研究转化疗法。以间充质干细胞（MSC）为基础的干细胞治疗是一种有吸引力的新方法。MSC能够分泌多种旁分泌因子，调节肺内皮细胞和上皮细胞的通透性，减少炎症，增强组织修复，抑制细菌生长。在clinicaltrials.gov注册的150多个使用MSC作为治疗的临床试验中，超过2000名患者接受了干细胞治疗，没有出现任何重大并发症。然而，尽管具有良好的安全性，MSC在体外多次传代后具有自发恶性转化的能力，以及在体内促进肿瘤生长的能力。近年来，一些研究者发现，人间充质干细胞释放的微囊泡（MV）具有与干细胞一样的生物活性，部分原因是微囊泡mRNA在损伤组织床上的转移和表达。在这个应用中，我建议研究生物学并测试人类骨髓来源的MSC MV作为ALI模型中细胞治疗的替代疗法的潜在治疗用途。总体假设是，人间充质干细胞MV具有生物活性，其治疗活性主要是通过mRNA从MV转移到损伤的肺上皮和肺内皮介导的。在Aim 1中，主要目标是研究MSC MV的生物学特性，并使用RNA和蛋白质合成和运输抑制剂确定MSC MV的哪些成分具有功能活性。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mRNA从MV转移到受损的肺上皮或内皮细胞，以达到治疗效果。在Aim 2中，我将测试该功能

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.