Human Mesenchymal Stem Cell Microvesicles for the Treatment of Acute Lung Injury

项目总结/摘要 急性呼吸窘迫综合征仍然是一种影响20多万患者的毁灭性综合征 在美国每年发生一次，死亡率接近40%。目前，没有药物治疗 可以降低死亡率。因此，需要创新疗法。在各种临床前模型中， 在急性肺损伤时，间充质干细胞（MSC）已显示分泌多种旁分泌因子， 可以降低肺内皮和上皮的通透性，减少炎症，增强组织修复， 抑制细菌生长，最终降低死亡率。尽管在早期临床试验中具有良好的安全性， 然而，MSC在体外多次传代后具有自发恶性转化的能力， 培养以及在小动物模型中促进肿瘤生长的能力。在过去的一段时间里，我们 发现人类间充质干细胞释放的微囊泡（MV）与母体干细胞一样具有生物活性， 急性肺损伤的炎症和感染原因。曾经被认为是细胞碎片，MV，无核 大小为50 nm至200 nm的环形膜结合囊泡，其由许多细胞类型组成性释放， 现在被认为是细胞间通讯的重要机制。我们的总体假设是 目前的建议是MSC MV具有生物学活性，并且其在严重的细菌感染中的治疗活性 肺炎是通过从肺组织转移mRNA、microRNA、蛋白质和/或细胞器介导的。 对受伤的肺泡进行MV。在目标1中，我们将通过研究 人MSC MVs对脂质代谢和ATP结合盒转运蛋白活性的影响， 耐药相关蛋白（MRP）1，在小鼠严重肺炎损伤。我们假设MSC MV 将抑制单核细胞/巨噬细胞中的MRP 1活性，并减少 白三烯（LT）A4向LTC/D/E4的转化，白三烯参与肺蛋白的通透性，并增加转化率 转化为LTB 4，白三烯参与细菌的杀伤。在目标2中，我们将测试人MSC MV对净细胞的影响， 严重细菌性肺炎损伤的新型离体灌注人肺和 由炎症损伤损伤的人肺泡上皮II型细胞的原代培养物。我们假设 MSC MV将通过恢复肺动脉内皮细胞的顶膜表达来防止肺水肿的形成。 主要上皮钠通道αENaC。在目标3中，我们将确定人MSC MV是否在治疗中具有治疗性。 绵羊烟雾吸入性脓毒症和铜绿假单胞菌肺炎模型。我们假设 MSC MV将通过恢复肺泡液体清除率、减少肺蛋白质， 增加细菌杀灭，降低肺血管通透性， 通过释放一氧化氮和恢复内皮上的糖萼层来降低压力。的总目标 目前的提交是为了进一步发展在急性呼吸道疾病中使用MSC MV的生物学原理， 焦虑综合征，等待可能的FDA IND申请。

PROJECT SUMMARY/ABSTRACT Acute respiratory distress syndrome 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 pharmacological treatments available that reduce mortality. Consequently, innovative therapies are needed. In various preclinical models of acute lung injury, mesenchymal stem cells (MSC) have been shown to secrete multiple paracrine factors that can reduce lung endothelial and epithelial permeability, decrease inflammation, enhance tissue repair, and inhibit bacterial growth, ultimately decreasing mortality. Despite a favorable safety profile in early clinical trials, however, MSC have the capacity for spontaneous malignant transformation following multiple passages in culture as well as the ability to promote tumor growth in small animal models. In the prior grant period, we found that microvesicles (MV) released by human MSCs were as biologically active as the parent stem cells in both inflammatory and infectious causes of acute lung injury. Once considered cellular debris, MVs, anuclear circular membrane bound vesicles 50 nm to 200 nm in size that are constitutively released by many cell types, are now recognized as important mechanisms for cell-cell communication. Our overall hypothesis for the current proposal is that MSC MVs are biologically active, and its therapeutic activity in severe bacterial pneumonia is mediated through transfer of mRNAs, microRNAs, proteins and/or organelles from the MVs to the injured alveolus. In Aim 1, we will further uncover the therapeutic mechanisms by studying the effect of human MSC MVs on lipid metabolism and the activity of ATP-binding cassette transporter, multidrug resistance associated protein (MRP)1, in mice injured with severe pneumonia. We hypothesize that MSC MVs will inhibit MRP1 activity in monocytes/macrophages and decrease the enzymatic conversion and secretion of leukotriene (LT)A4 to LTC/D/E4, leukotrienes involved in lung protein permeability, and increase the conversion into LTB4, leukotriene involved in bacterial killing. In Aim 2, we will test the effect of human MSC MV on net fluid transport in both a novel ex vivo perfused human lung injured with severe bacterial pneumonia and in primary cultures of human alveolar epithelial type II cells injured by an inflammatory insult. We hypothesize that MSC MVs will prevent pulmonary edema formation by restoring the apical membrane expression of the major epithelial sodium channel, αENaC. In Aim 3, we will determine if human MSC MVs are therapeutic in a sheep model of sepsis following smoke inhalation and Pseudomonas aeruginosa pneumonia. We hypothesize that MSC MVs will improve oxygenation (PaO2/FiO2) by restoring alveolar fluid clearance, reducing lung protein permeability and alveolar inflammation, increasing bacterial killing, and decreasing pulmonary vascular pressure by releasing nitric oxide and restoring the glycocalyx layer on the endothelium. The overall goal of the current submission is to further develop the biological rationale for using MSC MVs in acute respiratory distress syndrome in anticipation for a possible FDA IND submission.