MicroRNAs regulate proliferative signaling pathways in pulmonary hypertension

描述(由申请人提供)： 肺动脉高压(PH)是一种进行性疾病，导致显著的发病率和死亡率，与多种疾病有关，通常困扰退伍军人患者。虽然新的PH疗法改善了PH发病率，但这些疗法的成本和与PH相关的残余死亡率仍然高得令人无法接受。这些观察表明，迫切需要对PH的发病机制有新的认识，并确定这种疾病的新治疗靶点。越来越多的证据表明，配体激活的核激素转录因子过氧化体增殖物激活受体γ(PPAR)的激活为PH治疗提供了一个新的潜在治疗靶点。在实验动物和人类研究中，PPAR表达或功能的丧失与PH有关。相反，在几个实验动物模型中，刺激PPAR可降低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的特点是肺血管壁细胞增殖增强。来自导师实验室的已发表的观察表明，PPAR的激活可以减轻低氧诱导的抗增殖介质、10号染色体缺失的磷酸酶和张力蛋白同源物(PTEN)的变化，它们的表达减少可能参与了PH的发病。为了进一步研究PPAR调节这种抗增殖介质的机制，本提案将重点研究microRNAs(MiRNA)在PH中的作用。MiRNAs是一种小的、非编码(20-22个核苷酸)的RNA，有助于转录控制的微调。根据初步数据和计算机分析，PI假设 低氧增加miRNA-21诱导PH和肺血管壁细胞的增殖，PPAR激活减弱低氧诱导的miRNA-21表达变化以降低PH。为了探索这一假说，以下特定目的将检测miRNAs在体外低氧诱导的肺血管壁细胞增殖中的作用以及在体内PH发病机制中的作用，并确定PPAR配体通过调节miRNA表达来减轻PH的能力。目的1研究miRNA-21在体外和体内低氧暴露的肺血管平滑肌细胞增殖信号中的作用。在低氧条件下刺激人肺动脉平滑肌细胞(HPASMC)增殖后，将使用qRT-PCR来检测miRNA-21的水平。MiRNA-21在HPASMC增殖中的作用将通过miRNA下调或过表达的方法结合缺氧性HPASMC增殖的功能分析来证实。将确定可能的miRNA-21靶标PTEN水平的相应变化。这些体外发现将在体内低氧诱导的PH小鼠模型中得到证实。总之，这些研究将确定在体外和体内低氧诱导的miRNA-21表达变化在调节肺血管壁细胞增殖中的作用。目的2将以PPAR配体为靶点治疗miRNA-21，以减轻低氧诱导的细胞增殖和PH。这个目标将使用目标1中使用的体外和体内模型，除了实验将包括由导师的实验室先前建立的一系列药理PPAR配体的治疗剂量和持续时间。选定的研究将证实PPAR调节的miRNA在细胞靶向PPAR过度表达或敲除的模型中的表达和功能。这些创新性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关键的新见解，还将为申请者提供广泛的肺血管生物学培训，这将有助于他成为VA系统中成功的内科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.