MicroRNAs regulate proliferative signaling pathways in pulmonary hypertension

描述（由申请人提供）： 肺动脉高压（PH）是一种导致严重发病率和死亡率的进行性疾病，与退伍军人患者常见的多种疾病有关。虽然新的PH疗法改善了PH发病率，但这些疗法的成本和剩余PH相关死亡率仍然高得不可接受。这些观察结果表明，新的见解PH发病机制和确定新的治疗靶点，在这种疾病是迫切需要的。不断发展的证据表明，激活配体激活的核激素转录因子，过氧化物酶体增殖物激活受体γ（PPAR），提供了一个新的潜在的治疗目标，在PH管理。在实验动物和人类研究中，PPAR表达或功能的丧失与PH相关。相比之下，在几个实验动物模型中，刺激PPAR减弱PH。PPAR在PH中发挥作用的机制仍有待确定，并构成本提案的重点。PH的特征是肺血管壁细胞增殖增强。导师实验室发表的观察结果表明，PPAR激活减弱了缺氧诱导的抗增殖介质，10号染色体上缺失的磷酸酶和张力蛋白同源物（PTEN）的改变，其表达减少可能参与PH发病机制。为了进一步研究PPAR调节这种抗增殖介质的机制，本提案将重点关注microRNA（miRNA）在PH中的作用。miRNA是小的非编码（20-22个核苷酸）RNA，有助于转录控制的微调。基于初步数据和计算机模拟分析，PI假设， 低氧增加了miRNA-21诱导的PH和肺血管壁细胞的增殖，并且PPAR活化减弱了低氧诱导的miRNA-21表达的改变以降低PH。为了探讨这一假设，下面的具体目标将检查miRNAs在缺氧中的作用-体外诱导肺血管壁细胞增殖和体内PH发病机制，并通过调节miRNA表达。目的1研究miRNA-21在体外缺氧暴露的肺血管平滑肌细胞增殖信号中的作用以及在体内PH中的作用。暴露于刺激人肺动脉平滑肌细胞（HPASMC）增殖的缺氧条件后，将采用qRT-PCR测定miRNA-21水平。miRNA-21在HPASMC增殖中的作用将通过miRNA敲低或过表达方法结合缺氧HPASMC增殖的功能测定来证实。将确定推定的miRNA- 21靶标PTEN水平的相应改变。这些体外研究结果将在缺氧诱导PH的体内小鼠模型中得到证实。总之，这些研究将确定缺氧诱导的miRNA-21表达改变对体外和体内肺血管壁细胞增殖的调节作用。目标2将治疗靶向miRNA-21与PPAR配体，以减弱缺氧诱导的细胞增殖和PH。这一目标将采用目标1中使用的体外和体内模型，除了实验将包括一系列剂量和持续时间的药理学PPAR配体的治疗，这些配体先前由导师的实验室建立。选定的研究将证实在细胞靶向的PPAR过表达或敲除模型中，PPAR调节的miRNA表达和功能。这些创新研究的成功实施不仅将为miRNA调控在PH发病机制和治疗中的作用提供重要的新见解，而且将为申请人提供肺血管生物学方面的广泛培训，这将促进他成为VA系统中成功的医生科学家的长期职业目标。

DESCRIPTION (provided by applicant): Pulmonary hypertension (PH), a progressive disorder causing significant morbidity and mortality, is associated with a variety of diseases commonly afflicting veteran patients. While new PH therapies have improved PH morbidity, the costs of these therapies and the residual PH-associated mortality remain unacceptably high. These observations indicate that new insights into PH pathogenesis and the identification of new therapeutic targets in this disorder are urgently needed. Evolving evidence indicates that activation of the ligand-activated nuclear hormone transcription factor, peroxisome proliferator-activated receptor gamma (PPAR), provides a new potential therapeutic target in PH management. Loss of PPAR expression or function is associated with PH in experimental animal and human studies. In contrast, stimulating PPAR attenuated PH in several experimental animal models. The mechanisms by which PPAR exerts its effects in PH remain to be defined and constitute the focus of this proposal. PH is characterized by enhanced proliferation of pulmonary vascular wall cells. Published observations from the mentor's lab demonstrated that PPAR activation attenuates hypoxia-induced alterations in the antiproliferative mediator, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), whose reduced expression may participate in PH pathogenesis. To further examine the mechanisms by which PPAR regulates this antiproliferative mediator, this proposal will focus on the role of microRNAs (miRNA) in PH. miRNAs are small, non-coding (20-22 nucleotide) RNAs that contribute to fine tuning of transcriptional control. Based on preliminary data and in silico analysis, the PI hypothesizes that hypoxic increases in miRNA-21 induce PH and the proliferation of pulmonary vascular wall cells and that PPAR activation attenuates hypoxia-induced alterations in miRNA-21 expression to reduce PH. To explore this hypothesis, the following specific aims will examine the role of miRNAs in hypoxia-induced pulmonary vascular wall cell proliferation in vitro and in PH pathogenesis in vivo and determine the ability of PPAR ligands to attenuate PH by modulating miRNA expression. Aim 1 will examine the role of miRNA-21 in proliferative signaling in hypoxia-exposed pulmonary vascular smooth muscle cells in vitro and in PH in vivo. Following exposure to hypoxic conditions that stimulate human pulmonary artery smooth muscle cell (HPASMC) proliferation, qRT-PCR will be employed to determine miRNA-21 levels. The role of miRNA-21 in HPASMC proliferation will be confirmed with miRNA knockdown or overexpression approaches coupled with functional assays of hypoxic HPASMC proliferation. Corresponding alterations in levels of the putative miRNA- 21 target, PTEN, will be determined. These in vitro findings will be confirmed in an in vivo mouse model of hypoxia-induced PH. Collectively, these studies will determine the role of hypoxia-induced alterations in miRNA-21 expression on the regulation of pulmonary vascular wall cell proliferation in vitro and in vivo. Aim 2 will therapeutically target miRNA-21 with PPAR ligands to attenuate hypoxia-induced cell proliferation and PH. This aim will employ the in vitro and in vivo models used in Aim 1, except experiments will include treatments with a range of doses and durations of pharmacological PPAR ligands previously established by the mentor's lab. Selected studies will confirm PPAR -regulated miRNA expression and function in models with cell- targeted PPAR overexpression or knockout. The successful execution of these innovative studies will not only provide critical new insights into the role of miRNA regulation in PH pathogenesis and therapy but will provide the applicant broad-based training in pulmonary vascular biology that will facilitate his long-term career goals of becoming a successful physician-scientist in the VA system.