Molecular determinants of smooth muscle phenotype in pulmonary hypertension

肺动脉高压平滑肌表型的分子决定因素

• 批准号: 8051637
• 负责人: William T Gerthoffer
• 金额: $ 18.56万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2013-03-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8051637
• 关键词: Anabolism; Apoptosis; Arteries; Biological Assay; Blood Vessels; Bone Morphogenetic Proteins; Cell Proliferation; Cell Survival; Cell physiology; Cells; Chronic; Chronic Disease; Clinical; Contractile Proteins; Development; Disease; Down-Regulation; Endothelial Cells; Epigenetic Process; Family; Fibroblasts; Functional disorder; Future; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Goals; Human; Hypoxia; In Situ Hybridization; Lead; Lentivirus Vector; Lesion; Lung; Measures; Mechanical Stress; Mediating; MicroRNAs; Modeling; Molecular; Monocrotaline; Mutation; Myofibroblast; Organ; Pathogenesis; Pathology; Pattern; Phenotype; Physiological; Proliferating; Protein Family; Proteins; Pulmonary Hypertension; Pulmonary artery structure; RNA; Rattus; Recording of previous events; Regulator Genes; Role; Sampling; Serum Response Factor; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Stem cells; Structure; Structure of parenchyma of lung; Testing; Therapeutic Agents; Time; Transcript; Transcriptional Regulation; Transforming Growth Factor beta; Up-Regulation; Vascular Endothelial Growth Factor Receptor; Vascular Smooth Muscle; Vascular remodeling; Western Blotting; bone morphogenetic protein 4; bone morphogenetic protein receptors; cell motility; human TGFB1 protein; human subject; human tissue; locked nucleic acid; novel; paraform; public health relevance; pulmonary arterial hypertension; pulmonary function

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.

描述（由申请人提供）：该探索性建议的目的是确定microRNA（miRNA）在动脉肌肉化机制和肺动脉高压（PAH）中的作用。主要的假设是，在PAH中发生的血管重塑期间，miRNA介导的基因沉默的miRNA表达和模式在内皮，肌纤维细胞，平滑肌和祖细胞中动态变化。提出改变基因沉默可以显着促进动脉肌肉化和阻塞性血管病变的形成。为了测试这种假设的miRNA在肺血管中的表达，将在两种大鼠模型的PAH模型中测定，暴露于慢性缺氧加上SU -5146（VEGF受体拮抗剂），并用单蛋白治疗的大鼠。三个特定目的是：1。定义PAH发育过程中肺动脉中miRNA表达的时间依赖性变化。 miRNA表达将在正常大鼠和暴露于缺氧加人的大鼠和大鼠中，在0、7、21和35天的大（> 1mm）和小（200-400 5M）肺动脉中测定。在用单蛋白治疗的大鼠中，将在0和21d分析miRNA表达。在这些时间差异表达的miRNA将通过定量实时PCR验证。 PCR验证的miRNA的细胞定位将通过与PAH人类的固定肺样本中的原位杂交评估。 2。比较由TGFBETA和骨形态发生蛋白（BMP）调节的miRNA与在PAH中改变的miRNA。由TGF BetA1，TGFBETA3和BMP-4调节的miRNA将在培养的大鼠肺动脉组织和人类肺动脉平滑肌细胞中测定。将miRNA表达的时间和浓度依赖性模式与大鼠模型和人肺组织中PAH发育过程中动态变化的miRNA进行比较。然后，将测试选定的miRNA对肺动脉平滑肌的功能作用。 3。为了测试miRNA的足够，可以改变平滑肌表型的原代miRNA转录本和反义miRNA，将在培养的大鼠和人类肺动脉平滑肌细胞中表达。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。结果将确定所选miRNA在血管平滑肌中的生理意义，并将鉴定候选miRNA，这些miRNA可以作为治疗剂开发，以逆转严重PAH的血管重塑。 公共卫生相关性：在称为microRNA的小核糖核酸研究中令人兴奋的新发展改变了我们对器官如何发展以及如何治疗慢性疾病的理解。我们提出了microRNA在确定肺脉管系统中细胞的结构和功能方面的重要新作用。发现这些分子如何控制肺血管的功能可能导致肺动脉高压的新治疗方法。