NOVEL PARACRINE MECHANISMS FOR CELL-BASED THERAPY OF INJURED LUNGS

标题：肺损伤细胞治疗的新旁分泌机制 总结。我们最近描述了一种潜在的范式转换信号通路，用于损伤的修复 肺泡上皮由间充质干细胞(基质)细胞(MSCs)诱导。在受伤的远端空隙中，PAI-1增加 血药浓度会抑制局部纤溶活性。我们证明了MSC释放的角质形成细胞生长因子(KGF) 上调纤溶酶，裂解上皮钠通道(ENaC)。根据我们的初步数据，我们 假设间充质干细胞中KGF激活的纤溶蛋白构成一种新的正常化信号级联 受损肺的肺泡液动态平衡和再上皮化。我们建议， KGF/uPA/纤溶酶/ENaC通路在KGF介导的水肿液消退中起关键作用。这一假设是 一系列最新的观察结果支持：1)MSC培养基能恢复阿米洛利可抑制的液体 2)KGF刺激骨髓间充质干细胞中主要的纤溶酶uPA的表达，以及 修复过程中的细胞外纤溶活性；3)上皮细胞的去极化通过以下方式促进伤口愈合 促进迁移，这种去极化主要由ENaC活性决定；4)敲除小鼠uPA 抑制跨上皮液重吸收；5)uPA和纤溶酶均通过裂解激活ENaC功能 ENaC的底物样胞外基序；最后，纤溶酶促进人肺泡液的清除 UPA气管内给药可改善酸吸入损伤小鼠的水肿液消退。目标1 将测试这一新的KGF/纤溶酶/ENaC通路在基于MSC的修复中的有益作用。这一目标 将利用细胞因子/低氧挑战原代人肺泡上皮2型细胞的体外3D模型 (AT2)，酸损伤小鼠，以及感染活细菌的体外灌流的人肺；Aim 2将确定 KGF/uPA/纤溶酶/ENaC途径促进血管内皮细胞重吸收的分子机制 肺泡水肿液。这一目标将利用临床相关的活体小鼠肺，酸损伤，3D原发 AT2细胞暴露于细胞加低氧条件下，以及表达系统。我们将检验假设1) 间充质干细胞来源的KGF为受损的肺上皮重建纤溶利基，2)提高纤溶蛋白的裂解 ENAC在物理分子间调节下的蛋白水解性，以及3)细胞外裂解位点在 ENAC蛋白与纤溶酶最受欢迎的催化底物的序列相同。这一目标还将检验是否 KGF/uPA/纤溶酶/ENaC通路调控AT2细胞的增殖、迁移和分化。这个 这些研究的可靠和公正的结果将确定MSC的新机制和因果关系 和KGF介导的肺损伤模型中正常肺泡液清除的恢复，并确定如何 纤溶蛋白水解酶激活ENaC功能，以提高水肿液的分辨率。我们的研究既是基础的，也是 翻译，因为结果将加强我们对基于MSC的旁分泌因素的理解 临床治疗，并应为基于MSC的细胞治疗的系统机制提供新的见解 治疗急性呼吸窘迫综合症。

TITLE: NOVEL PARACRINE MECHANISMS FOR CELL-BASED THERAPY OF INJURED LUNGS SUMMARY. We have recently described a potentially paradigm-shifting signal pathway for repair of injured alveolar epithelium by mesenchymal stem (stromal) cells (MSCs). In injured distal airspaces, increased PAI-1 levels depress local fibrinolytic activity. We demonstrated that MSC-released keratinocyte growth factor (KGF) up-regulates fibrinolysins, which cleave epithelial sodium channels (ENaC). Based on our Preliminary Data, we hypothesize that KGF-activated fibrinolysins in MSCs constitute a novel signal cascade for normalizing alveolar fluid homeostasis and re-epithelialization in injured lungs. We propose that the KGF/uPA/plasmin/ENaC pathway is critical in KGF-mediated edema fluid resolution. This hypothesis is supported by a body of very recent observations: 1) MSC culture medium restores amiloride-inhibitable fluid re- absorption in injured lungs; 2) KGF stimulates expression of uPA, a predominate fibrinolysin in MSCs, and extracellular fibrinolytic activity during repair; 3) depolarization of epithelial cells improves wound healing by facilitating migration, and this depolarization is mainly determined by ENaC activity; 4) knockout of mouse uPA inhibits transepithelial fluid re-absorption; 5) both uPA and plasmin activate ENaC function by cleaving substrate-like extracellular motifs of ENaC; and finally, 6) plasmin augments alveolar fluid clearance in human lungs, and intratracheal delivery of uPA improves edema fluid resolution in acid aspiration injured mice. Aim 1 will test the beneficial role of this novel KGF/fibrinolysins/ENaC pathway in MSC-based repair. This aim will utilize an in vitro 3D model of cytokine/hypoxia-challenged primary human alveolar epithelial type 2 cells (AT2), acid injured mice, and ex vivo perfused human lungs infected with live bacteria; Aim 2 will determine the molecular mechanisms by which the KGF/uPA/plasmin/ENaC pathway enhances reabsorption of alveolar edema fluid. This aim will utilize a clinically relevant in vivo mouse lungs injured with acid, 3D primary AT2 cultures exposed to cytomix plus hypoxia, and expression systems. We will test the hypotheses 1) that MSC-derived KGF rebuilds the fibrinolytic niche for injured lung epithelium, 2) that elevated fibrinolysins cleave ENaC proteolytically following physical intermolecular regulation, and 3) that extracellular cleavage sites in ENaC proteins are identical to favorite catalytic substrates of fibrinolysins in sequence. This aim will also test if the KGF/uPA/plasmin/ENaC pathway regulates proliferation, migration, and differentiation of AT2 cells. The robust and unbiased results of these studies will identify novel mechanisms and causal relationships for MSC and KGF-mediated restoration of normal alveolar fluid clearance in lung injury models, and determine how fibrinolytic proteases activate ENaC function to improve edema fluid resolution. Our studies are both basic and translational, as the results will enhance our understanding of the paracrine factors involved in MSC-based clinical therapy, and should provide novel insights into the systematic mechanisms of MSC-based cell therapy for acute respiratory distress syndrome.