Molecular determinants of smooth muscle phenotype in pulmonary hypertension

描述（由申请人提供）：本探索性提案的目的是确定microRNA（miRNA）在肺动脉高压（PAH）中动脉肌化和过度收缩机制中的作用。主要假设是，在PAH发生的血管重塑过程中，内皮细胞、肌成纤维细胞、平滑肌和祖细胞中的miRNA表达和miRNA介导的基因沉默模式动态变化。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作用。为了检验这一假设，将在两种PAH大鼠模型中测定肺血管中miRNA的表达-暴露于慢性缺氧加SU-5146（VEGF受体拮抗剂）的大鼠和用野百合碱处理的大鼠。三个具体目标是：1。明确PAH发生过程中肺动脉中miRNA表达的时间依赖性变化。将在正常大鼠和暴露于缺氧加SU-5146的大鼠的大（> 1 mm）和小（200-400 μ m）肺动脉中在0、7、21和35天测定miRNA表达。将在用野百合碱处理的大鼠中在0和21天时测定miRNA表达。将通过定量实时PCR验证在这些时间差异表达的miRNA。将在PAH患者的固定肺样本中通过原位杂交评估PCR验证的miRNA的细胞定位。2.比较TGF β和骨形态发生蛋白（BMP）调节的miRNA与PAH中改变的miRNA。将在培养的大鼠肺动脉组织和培养的人肺动脉平滑肌细胞中测定由TGF β 1、TGF β 3和BMP-4调节的miRNA。将时间和浓度依赖性的miRNA表达模式与大鼠模型和人肺组织中PAH发生期间动态变化的miRNA进行比较。然后测试所选的miRNA对肺动脉平滑肌的功能作用。3.为了测试miRNA改变平滑肌表型的充分性，将在培养的大鼠和人肺动脉平滑肌细胞中表达初级miRNA转录物和反义miRNA。平滑肌收缩蛋白、细胞收缩、细胞增殖和细胞迁移将被测量为收缩与增殖表型的读数。这些结果将确立所选miRNAs在血管平滑肌中的生理意义，并将鉴定可开发为治疗剂的候选miRNAs，以逆转重度PAH的血管重塑。 公共卫生相关性：小核糖核酸（microRNA）研究的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如何治疗的理解。我们提出了一个重要的新作用microRNAs在决定肺血管系统中的细胞的结构和功能。发现这些分子如何控制肺血管的功能可能会导致肺动脉高压的新治疗方法。

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.