MicroRNA Shuttling during Acute Respiratory Distress Syndrome

项目名称 MicroRNA在急性呼吸窘迫综合征中的穿梭 项目摘要 本申请涉及以下NHLBI特别关注主题（TOSI）：HL-142 -外来体， 心血管、肺和血液疾病中的旁分泌信号介质（R 01）。 一项针对microRNA穿梭的建议是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， 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非- 心源性肺水肿，并显著增加手术患者的发病率和死亡率。 ARDS的特征性特征包括炎性细胞-特别是中性粒细胞（PMN）-的积聚， 并伴有上皮损伤和不受控制的肺部炎症。中性粒细胞是最早的免疫细胞之一 它们进入受伤的肺部，并与肺泡上皮细胞进行密切的空间接触。在这里， 我们考虑了以microRNAs（miRNAs）形式存在的遗传信息可以被转移的可能性， 从中性粒细胞到肺泡上皮细胞。miRNA是抑制mRNA靶点表达的小RNA。研究 已经显示了miRNAs在调节外科和重症监护患者的炎症结果中的功能， 暗示微泡含有细胞间串扰中的miRNA。 为了检测miRNA从PMN穿梭进入肺泡上皮细胞，我们最初使用体外方法， 将活化的人PMN与原代人肺泡上皮细胞（HPAEpiC）共孵育6小时， 被渗透膜隔开。一个针对PMN依赖性miRNA的靶向miRNA阵列显示， 共培养后miR-223的选择性增加（超过100倍）。同样，我们观察到， 在利用鼠PMN和鼠肺泡上皮的共培养系统中的肺泡上皮miR 223水平。 然而，当PMN从miR-223中去除时，肺泡上皮细胞miR-223的增加被完全消除。 使用缺陷型小鼠。随后对人中性粒细胞的研究表明，miR-121的激活依赖性释放， 223转移到它们的上清液中，并暗示细胞外囊泡将PMN衍生的miR-223转移到它们的上清液中。 肺泡上皮为了阐明miR-223依赖的miRNA穿梭在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miR-223水平增加， 暴露于VILI的小鼠的分离的肺泡上皮细胞。相比之下，这一回应被彻底废除 在Ly 6 G抗体耗尽PMN后，表明miR-223从PMN穿梭到肺泡 在体内的ARDS期间的上皮。功能性研究显示，miR-223基因靶向小鼠经历了一系列的功能性变化。 VILI期间更严重的表型，表明miR-223依赖性miRNA穿梭的保护作用 在ARDS期间因此，我们假设miR-223依赖性miRNA穿梭代表了抗炎作用 这是一种可以用于ARDS预防或治疗的靶向途径。我们提出三个目标，以解决我们的 假说.在第一个目标中，我们将研究PMN依赖的miR-223在肺损伤过程中向肺泡上皮细胞的转移。 ARDS。在第二个目标中，我们将确定miR-223在减弱肺上皮细胞凋亡中的功能作用。 通过研究推定的miR-223靶基因聚（ADP-核糖）聚合酶-1（PARP-1）来研究炎症。在Aim中 3、我们将靶向miR-223用于治疗ARDS。这些研究旨在确定新的治疗方法 为患有ARDS的患者提供帮助。特别是在择期围手术期， miR 223代表了一种潜在的预防性治疗，以预防有ARDS风险的患者的术后ARDS， 或在经历“高风险”手术的患者中。

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.