Molecular determinants of smooth muscle phenotype in pulmonary hypertension

描述(由申请人提供)：这项探索性建议的目标是确定microRNAs(MiRNA)在肺动脉高压(PAH)的动脉肌化和高收缩机制中的作用。主要假设是在PAH血管重塑过程中，血管内皮细胞、肌成纤维细胞、平滑肌细胞和前体细胞中miRNA的表达和miRNA介导的基因沉默模式发生动态变化。基因沉默的改变被认为对动脉肌化和阻塞性血管病变的形成有重要贡献。为了验证这一假设，我们将检测两种PAH模型大鼠肺血管中miRNAs的表达--慢性低氧加血管内皮生长因子受体拮抗剂SU-5146的大鼠，以及野百合碱治疗的大鼠。本研究的三个具体目标是：1.明确肺动脉高压形成过程中肺组织miRNA表达的时间依赖性变化。在正常大鼠和低氧加SU-5146暴露大鼠的大(&gt；1 mm)和小(200-400 5m)肺动脉中，分别于0、7、21和35天检测miRNA的表达。在用野百合碱治疗的大鼠，将在0和21d检测miRNA的表达。在这些时间点差异表达的miRNAs将通过实时定量聚合酶链式反应进行验证。经PCR验证的miRNAs的细胞定位将通过原位杂交在来自患有PAH的人的固定肺样本中进行评估。2.比较转化生长因子β和骨形态发生蛋白(BMP)调节的miRNAs和PAH中改变的miRNAs。在体外培养的大鼠肺血管组织和人肺血管平滑肌细胞中，检测受转化生长因子β1、转化生长因子β3和骨形态发生蛋白-4调控的miRNAs。在大鼠模型和人类肺组织中，miRNA表达的时间和浓度依赖模式将与在PAH发展过程中动态变化的miRNAs进行比较。然后，将测试选定的miRNAs对肺动脉平滑肌的功能影响。3.为了验证miRNAs是否足以改变血管的表型，我们将在培养的大鼠和人的肺动脉平滑肌细胞中表达原代miRNA转录本和反义miRNAs。平滑肌收缩蛋白、细胞收缩、细胞增殖和细胞迁移将作为收缩和增殖表型的读数而被测量。这些结果将确定血管平滑肌中选定的miRNAs的生理学意义，并将确定候选的miRNAs，这些miRNAs可能被开发为逆转重度PAH血管重构的治疗药物。 与公共卫生相关：称为microRNAs的小核糖核酸研究取得了令人兴奋的新进展，改变了我们对器官如何发育以及慢性疾病可能如何治疗的理解。我们提出了MicroRNAs在确定肺血管中细胞的结构和功能方面的一个重要的新作用。发现这些分子是如何控制肺血管功能的，可能会带来治疗肺动脉高压的新方法。

DESCRIPTION (provided by applicant): The goal of this exploratory proposal is to define the role of microRNAs (miRNA) in mechanisms of arterial muscularization and hypercontractility in pulmonary arterial hypertension (PAH). The major hypothesis is that miRNA expression and patterns of miRNA-mediated gene silencing change dynamically in endothelial, myofibroblast, smooth muscle and progenitor cells during vessel remodeling that occurs in PAH. Altered gene silencing is proposed to contribute significantly to arterial muscularization and to formation of obstructive vascular lesions. To test this hypothesis expression of miRNAs in pulmonary blood vessels will be assayed in two rat models of PAH - rats exposed to chronic hypoxia plus SU-5146, a VEGF receptor antagonist, and rats treated with monocrotaline. The three Specific Aims are: 1. Define the time-dependent changes in miRNA expression in pulmonary artery during development of PAH. miRNA expression will be assayed at 0, 7, 21 and 35 days in large (>1mm) and small (200-400 5m) pulmonary arteries in normal rats and rats exposed to hypoxia plus SU-5146. miRNA expression will be assayed at 0 and 21d in rats treated with monocrotaline. miRNAs differentially expressed at these times will be verified by quantitative real-time PCR. Cellular localization of PCR-verified miRNAs will be assessed by in situ hybridization in fixed lung samples from humans with PAH. 2. Compare miRNAs regulated by TGFbeta and bone morphogenetic proteins (BMP) to miRNAs altered in PAH. miRNAs regulated by TGF beta1, TGFbeta3 and BMP-4 will be assayed in cultured rat pulmonary artery tissues and human pulmonary artery smooth muscle cells in culture. Time- and concentration- dependent patterns of miRNA expression will be compared to miRNAs that change dynamically during development of PAH in the rat models and in human lung tissues. Selected miRNAs will then be tested for functional effects on pulmonary artery smooth muscle. 3. To test the sufficiency of miRNAs to alter smooth muscle phenotype primary miRNA transcripts and antisense miRNAs will be expressed in cultured rat and human pulmonary artery smooth muscle cells. Smooth muscle contractile proteins, cell contraction, cell proliferation and cell migration will be measured as readouts of the contractile vs proliferating phenotypes. The results will establish the physiological significance of selected miRNAs in vascular smooth muscle, and will identify candidate miRNAs that could be developed as therapeutic agents to reverse vessel remodeling in severe PAH. PUBLIC HEALTH RELEVANCE: Exciting new developments in studies of small ribonucleic acids called microRNAs have changed our understanding of how organs develop and how chronic diseases might be treated. We are proposing an important novel role for microRNAs in determining the structure and function of cells in the pulmonary vasculature. Discovering how these molecules control the function of pulmonary blood vessels could lead to new treatments of pulmonary hypertension.