The Cellular Response to Elafin in PAH

项目1建立在令人兴奋的临床前研究的基础上，这些研究显示了每日皮下注射 弹性蛋白酶抑制剂，人重组Elafin，逆转大鼠严重肺动脉高压模型。 Elafin还诱导了肺动脉（PA）中新生内膜病变的显著消退， 肺动脉高压（PAH）患者的肺，并意外改善BMPR2缺陷 PAH患者的内皮细胞（EC）中通过募集BMPR2受体至小窝的信号传导。 此外，Elafin预期抑制的中性粒细胞弹性蛋白酶循环水平升高， 有利的毒性和药代动力学特征，使我们能够解决Elafin的生物学， 我们假设，将Elafin在PAH中的疗效归因于其颠覆不良反应的特性。 中性粒细胞（PMN）的功能和逆转血管细胞功能障碍。在目标1中，我们将研究 Elafin在静态条件下控制异常PAH PMN功能和PAH PMN-PAEC相互作用， 流动以及层流和扰动流。为了研究所涉及的机制，我们将使用候选人 方法，以及RNA Seq和转座酶可接近染色质的测定（ATAC Seq），适用于 在静态、层流和扰动流条件下的少量PA EC。在目标2中，我们将确定 是否有希望的初步研究表明，天然PAEC中的弹性蛋白应答表型， 在相同患者的iPSC-EC中复制的结果对于更大的队列是真实的。然后我们将生物学 用Elafin对基因表达标记进行改进。我们将从阶段中的患者产生iPSC-EC。 项目3中的1和2项临床试验，以及iPSC-EC对Elafin反应的相关生物学测量 与基因表达和临床结果相关，因为这涉及Elafin应答者与非应答者。 在目标3中，我们将对细胞中的Elafin功能进行更广泛的研究，因为我们描述了PMN中的Elafin相互作用组， PAEC。这将通过标记Elafin的免疫沉淀（IP）和质谱法来完成， 识别相互作用的蛋白质。一旦通过co-IP验证，Elafin对功能的影响 将评估相互作用蛋白。在目标4中，我们将以令人信服的初步数据为基础， 新型质谱-流式细胞术混合技术（CyTOF）在鉴定泛PAH中的影响 提示CD4 + CD25hi细胞活化的信号，并将决定这种和其他 在急性和慢性PAH后PAH患者中， 治疗我们将把CyTOF与血管周围龛内细胞的生物学和激活状态联系起来， 使用多重离子束成像（MIBI）。拟议的研究提供了前所未有的机会， 在PMN-EC相互作用，流动依赖性EC基因和表观遗传的更广泛领域的先进知识 调节和PBMC活化与血管周围生态位变化的相关性。我们也将是独一无二的 定位于识别和预测对Elafin的阳性反应，以及非反应者的替代策略。

Project 1 builds upon exciting preclinical studies showing efficacy of daily subcutaneous administration of an elastase inhibitor, human recombinant Elafin, in reversing a severe model of pulmonary hypertension in rats. Elafin also induces significant regression of neointimal lesions in pulmonary arteries (PA) in explants from lungs of patients with pulmonary arterial hypertension (PAH), and unexpectedly improved deficient BMPR2 signaling in endothelial cells (EC) from patients with PAH by recruiting BMPR2 receptors to caveolae. Moreover, the elevated circulating levels of neutrophil elastase that Elafin would be expected to inhibit and the favorable toxicity and pharmacokinetic profile of Elafin, have led us to address the biology of Elafin, in a manner, we hypothesize, that will attribute Elafin efficacy in PAH to its properties of subverting the adverse functions of neutrophils (PMN) and of reversing vascular cell dysfunction. In Aim 1, we will investigate the role of Elafin in controlling abnormal PAH PMN function and PAH PMN-PAEC interactions under static conditions, of flow as well as laminar and disturbed flow. To investigate the mechanisms involved we will use a candidate approach, as well as RNA Seq and assays of transposase accessible chromatin (ATAC Seq), applicable to small numbers of PA EC under conditions of static, laminar and disturbed flow. In Aim 2, we will determine whether promising preliminary studies that show an Elafin-responsive phenotype in native PAEC that is reproduced in iPSC-EC from the same patients is true for a larger cohort. We will then relate the biologic improvement with Elafin to a gene expression signature. We will generate iPSC-EC from patients in the Phase 1 and 2 Clinical Trials in Project 3, and correlate biologic measurements of the iPSC-EC response to Elafin with gene expression and with clinical outcome as this relates to Elafin responders vs. non-responders. In Aim 3 we will cast a wider net for Elafin function in cells as we delineate the Elafin interactome in PMN and PAEC. This will be accomplished by immunoprecipitation (IP) with labeled Elafin and mass spectrometry to identify the interacting proteins. Once validated by co-IP, the impact of Elafin has on the function of the interacting protein will be assessed. In Aim 4, we will build upon compelling preliminary data showing the impact of a novel mass spectrometry-flow cytometry hybrid technology (CyTOF) in identifying a pan-PAH signature that suggests activation of CD4+CD25hi cells, and will determine the extent to this and other abnormalities can be normalized ex vivo by Elafin both acutely and in patients with PAH following chronic treatment. We will relate CyTOF to the biology and the state of activation of cells within the perivascular niche, using multiplex ion beam imaging (MIBI). The studies proposed allow an unprecedented opportunity to advance knowledge in the broader fields of PMN-EC interaction, flow dependent EC gene and epigenetic regulation, and correlation of PBMC activation with changes in the perivascular niche. We will also be uniquely positioned to identify and predict a positive response to Elafin, and alternative strategies for the non-responder.