MicroRNA Shuttling during Acute Respiratory Distress Syndrome

项目名称 急性呼吸窘迫综合征期间的microRNA穿梭 项目摘要 本申请涉及以下NHLBI特别感兴趣(TOSI)主题：HL-142-exosome as 心血管、肺和血液疾病中的旁分泌信号介质(R01)。这项研究的主要目标 建议以microRNA穿梭为靶点来预防或治疗围手术期急性呼吸窘迫综合征 (ARDS)。ARDS是一种危及生命的疾病，是一种常见的术后并发症，发生于 超过7%的手术患者处于危险之中。它的特点是急性呼吸衰竭的设置在非 心源性肺水肿，对外科病人的发病率和死亡率有很大影响。 ARDS的特征包括炎性细胞-特别是中性粒细胞(PMN)在 伴随着上皮损伤和失控的肺部炎症。中性粒细胞是第一批免疫细胞。 它进入受损的肺部，并与肺泡上皮细胞密切接触。这里, 我们考虑了以microRNAs(MiRNAs)形式的遗传信息可以被 从中性粒细胞到肺泡上皮细胞。MiRNAs是一种小RNA，可以抑制mRNA靶标的表达。研究 已经显示出miRNAs在调节外科和重症护理患者的炎症结果方面的功能，以及 细胞间串扰中含有miRNAs的微泡。 为了检测miRNA从PMN穿梭到肺泡上皮细胞，我们最初使用的是体外方法， 将活化的人PMN与原代人肺泡上皮细胞(HPAEpiC)共同孵育6h， 被一层可渗透的膜隔开。一种专注于PMN依赖的miRNA的靶向miRNA阵列被揭示 共培养后，miR-223的选择性增加(超过100倍)。同样，我们观察到 利用小鼠PMN和小鼠肺泡上皮细胞共培养系统中的肺泡上皮miR223水平。 然而，从miR-223产生的PMN完全消除了miR-223对肺泡上皮细胞的促进作用 采用缺陷型小鼠。随后对人PMN的研究表明，miR-1的释放依赖于激活 223进入其上清液，并在PMN来源的miR-223的转移中涉及细胞外小泡。 肺泡上皮细胞。为了解决依赖miR-223的miRNA在ARDS中的功能作用，我们 将小鼠暴露于呼吸机诱导的肺损伤(VILI)。事实上，我们观察到在 VILI染毒小鼠肺泡上皮细胞的分离。相比之下，这一反应完全被废除了 在PMN耗尽Ly6G抗体后，表明miR-223从PMN向肺泡穿梭 活体ARDS时的上皮细胞。功能研究表明，miR-223基因靶向的小鼠经历了 VILI期间更严重的表型，表明依赖miR-223的miRNA穿梭具有保护作用 在急性呼吸窘迫综合征期间。因此，我们假设依赖miR-223的miRNA穿梭代表了一种抗炎 可作为ARDS预防或治疗靶点的途径。我们提出三个目标来解决我们的 假设。在第一个目标中，我们将研究PMN依赖的miR-223在肺泡上皮细胞中的转移。 阿兹。在第二个目标中，我们将确定miR-223在减弱肺上皮细胞中的功能作用。 炎症通过研究假定的miR-223靶基因多聚(ADP-核糖)聚合酶-1(PARP-1)。在AIM 3、我们将以miR-223为靶点治疗ARDS。这些研究旨在确定新的治疗方法 急性呼吸窘迫综合征患者的治疗方法。特别是在选择性围手术期环境中，靶向 MiR223代表了一种预防ARDS风险患者术后ARDS的潜在预防性治疗方法， 或接受“高风险”手术的患者。

Project Title MicroRNA Shuttling during Acute Respiratory Distress Syndrome Project Summary This application addresses the following NHLBI Topic of Special Interest (TOSI): HL-142 - Exosomes as Paracrine Signal Mediators in Cardiovascular, Lung, and Blood Disease (R01).The main goal of this proposal is to target microRNA shuttling to prevent or treat perioperative acute respiratory distress syndrome (ARDS). ARDS is a life threatening disease that represents a frequent postoperative complication, occurring in over 7% of surgical patients at risk. It is characterized by acute respiratory failure in the setting of non- cardiogenic pulmonary edema, and contributes significantly to morbidity and mortality of surgical patients. Characteristic features of ARDS include accumulation of inflammatory cells – particularly neutrophils (PMN), in conjunction with epithelial injury and uncontrolled lung inflammation. PMNs are among the first immune cells that traffic into the injured lungs and come into close spatial contact specifically with alveolar epithelia. Here, we considered the possibility that genetic information in the form of microRNAs (miRNAs) could be transferred from PMNs to alveolar epithelia. MiRNAs are small RNAs that inhibit the expression of mRNA targets. Studies have shown functions of miRNAs in regulating inflammatory outcomes in surgical and critical care patients, and implicate micro-vesicle contained miRNAs in intercellular crosstalk. To examine miRNA shuttling from PMN into alveolar-epithelia, we initially used an in vitro approach where activated human PMN were co-incubated with primary human alveolar epithelial cells (HPAEpiC) for 6h, separated by a permeable membrane. A targeted miRNA array focusing on PMN-dependent miRNAs revealed a selective increase of miR-223 (over 100-fold) following co-culture. Similarly, we observed robust increases in alveolar-epithelial miR223 levels in a co-culture system utilizing murine PMN and murine alveolar epithelia. However, alveolar epithelial increases of miR-223 were completely abolished when PMN from miR-223 deficient mice were used. Subsequent studies of human PMN showed activation-dependent release of miR- 223 into their supernatant, and implicate extracellular vesicles in the transfer of PMN-derived miR-223 into alveolar epithelia. To address the functional role of miR-223-dependent miRNA-shuttling during ARDS, we exposed mice to ventilator-induced lung injury (VILI). Indeed, we observed increased miR-223 levels in isolated alveolar epithelial cells of mice exposed to VILI. In contrast, this response was completely abolished following Ly6G antibody depletion of PMN, indicating that miR-223 is shuttled from PMN towards alveolar epithelia during ARDS in vivo. Functional studies revealed that gene-targeted mice for miR-223 experienced a more severe phenotype during VILI, suggesting a protective role of miR-223-dependent miRNA shuttling during ARDS. Thus, we hypothesize that miR-223-dependent miRNA shuttling represents an anti-inflammatory pathway that can be targeted for ARDS prevention or treatment. We propose 3 Aims to address our hypothesis. In the first aim, we will study PMN-dependent miR-223 transfer into alveolar epithelial cells during ARDS. In the second aim, we will identify the functional role of miR-223 in attenuating pulmonary epithelial inflammation by studying the putative miR-223 target gene poly (ADP-ribose) polymerase-1 (PARP-1). In Aim 3, we will target miR-223 for the treatment of ARDS. These studies are designed to identify novel treatment approaches for patients suffering from ARDS. Particularly in the elective perioperative setting, targeting miR223 represents a potential prophylactic treatment to prevent postoperative ARDS in patients at ARDS risk, or in patients undergoing “high risk” surgery.