Molecular determinants of smooth muscle phenotype in pulmonary hypertension

描述（由申请人提供）：本探索性提案的目标是确定microrna （miRNA）在肺动脉高压（PAH）动脉肌肉化和过度收缩机制中的作用。主要的假设是，在PAH发生的血管重塑过程中，内皮细胞、肌成纤维细胞、平滑肌细胞和祖细胞中miRNA的表达和miRNA介导的基因沉默模式发生了动态变化。改变的基因沉默被认为对动脉肌肉化和阻塞性血管病变的形成有重要作用。为了验证这一假设，我们将在两种PAH大鼠模型中检测肺血管中mirna的表达——暴露于慢性缺氧加VEGF受体拮抗剂SU-5146的大鼠和服用单芥碱的大鼠。三个具体目标是：1。确定PAH发展过程中肺动脉miRNA表达的时间依赖性变化。在正常大鼠和缺氧加SU-5146大鼠的大（bbb1mm）和小（200- 4005m）肺动脉中，于0、7、21和35天检测miRNA的表达。在给药后第0和21天检测miRNA的表达。这些时间差异表达的mirna将通过实时荧光定量PCR进行验证。pcr验证的mirna的细胞定位将通过PAH患者固定肺样本的原位杂交来评估。2. 比较tgfβ和骨形态发生蛋白（BMP）调控的miRNAs与PAH改变的miRNAs。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检测TGF β 1、TGF β 3和BMP-4调控的mirna。miRNA表达的时间和浓度依赖性模式将与大鼠模型和人肺组织中PAH发展过程中动态变化的miRNA进行比较。然后将测试选定的mirna对肺动脉平滑肌的功能影响。3. 为了测试miRNA是否足以改变平滑肌表型，将在培养的大鼠和人肺动脉平滑肌细胞中表达初级miRNA转录本和反义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和增殖表型的读数。该结果将确定血管平滑肌中选定的mirna的生理意义，并将确定候选mirna，这些mirna可以开发为逆转严重PAH血管重塑的治疗药物。

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.