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BMP2 Gene Regulation in Calcific Aortic Valve Disease

BMP2 基因在钙化主动脉瓣疾病中的调控

基本信息

批准号：
8852685
负责人：
DOUGLAS P MORTLOCK
金额：
$ 38.41万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-23 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8852685
关键词：
3&apos Untranslated Regions Adult Animal Model Area BMP2 gene Bacterial Artificial Chromosomes Binding Biological Markers Biology Calcified Cells Cellular biology Cholesterol Chromosome Mapping Cis Tests Collection Consensus Conserved Sequence DNA Sequence Defect Development Diagnosis Diagnostic Diet Disease Disease Progression Disease model Early Diagnosis Electrophoretic Mobility Shift Assay Elements Enhancers FGF2 gene Fishes Future Gene Expression Regulation Genes Genetic Genetic Polymorphism Genetic Transcription Goals Individual Knockout Mice Knowledge Life Mammals Maps Medical MicroRNAs Modeling Molecular Morphogenesis Mus Mutate Operative Surgical Procedures Osteoblasts Osteogenesis Pattern Predisposition Proteins RNA RNA Sequences Reagent Regulation Regulatory Element Reporter Reporter Genes Research Research Infrastructure Shapes Signal Pathway Signal Transduction Site Staging Surveys Testing Therapeutic Therapeutic Intervention Transcript Transfection abstracting aortic valve aortic valve disorder bicuspid aortic valve bone bone morphogenetic protein 2 calcification chromatin immunoprecipitation feeding gene repression genetic element improved interest interstitial cell malformation mouse model new therapeutic target notch protein novel strategies osteoblast differentiation osteogenic prevent recombinase response therapeutic target transcription factor transcriptome sequencing

项目摘要

DESCRIPTION (provided by applicant): Congenital valve defects promote Calcific Aortic Valve Disease (CAVD). Bone morphogenetic protein 2 (BMP2) is a key signal in valve development. In diseased adult valves, the pro-calcific BMP2 and downstream responses stimulate calcification. Thus, aberrant BMP2 patterns and levels influence CAVD indirectly by disrupting valve shape and directly by inducing ectopic bone formation. Our overall hypothesis is that controlling BMP2 levels would be an effective medical therapy for CAVD. Our strategy is to elucidate how BMP2 levels are controlled in the aortic valve during normal valve development and under conditions that promote CAVD. Aim 1. To test DNA sequences that control BMP2 synthesis at the transcriptional level in the valves of mice that model CAVD and during valve development. This aim will use an existing collection of reagents and mice that were created to survey bone regulatory sequences. One region includes a conserved bone enhancer that binds the master bone differentiation transcription factor RUNX2 and other potential regulators. We will test ECR1 and map other cis-elements that control Bmp2 expression in mice that develop CAVD when fed a high cholesterol diet and during the early stages of normal aortic valve development. Aim 2. To test RNA sequences that control BMP2 synthesis at the post-transcriptional level in developing valves and in the valves of mice that model CAVD. Both positive and negative mechanisms influence BMP2 synthesis in the aortic valve. A sequence conserved between mammals and fishes strongly represses BMP2 reporter gene repression in the developing and adult aortic valve. We will test the hypothesis that this element and its flanking RNA prevent BMP2 synthesis and ectopic bone in aortic valves in the Notch-compromised CAVD model. We will also test this regulatory mechanism during normal aortic valve development. Aim 3. To identify trans-regulatory molecules that control Bmp2 transcription through ECR1. The trans-regulatory factors that bind ECR1 may promote BMP2 synthesis in calcifying aortic valves. Molecular studies will be used to define factors interacting with the ECR1 sequence. The association of RUNX2 and Hes1 will be examined in MC3T3-E1 pre-osteoblasts that express BMP2 in response to FGF2. These findings will be confirmed in primary aortic valve interstitial cells and diseased valves from the CAVD mouse model. Aim 4. To identify trans-regulatory molecules that control BMP2 synthesis at the post- transcriptional level. We will test the influence of microRNAs that target the BMP2 transcript MC3T3-E1 cells and primary valve interstitial cells. This study will provide fundamental knowledge regarding the spatial and temporal control of Bmp2 in a new model of CAVD and in the developing aortic valve. These studies will direct future tests of anti-BMP2 therapies in CAVD. This proposal explores these Specific Areas of Research Interest: Genetics of CAVD and bicuspid aortic valve, Infrastructure (improved animal models), Cell biology (signaling pathways, mechanisms, and regulation of calcification), and Diagnosis. (End of Abstract)

描述(由申请人提供)：先天性瓣膜缺陷可导致钙性主动脉瓣病(CAVD)。骨形态发生蛋白2(BMP2)是瓣膜发育过程中的关键信号。在患病的成人瓣膜中，促钙化的BMP2和下游反应刺激钙化。因此，BMP2的异常模式和水平通过破坏瓣膜的形状间接影响CAVD，并通过诱导异位骨形成直接影响CAVD。我们的总体假设是，控制BMP2水平将是治疗CAVD的有效药物。我们的策略是阐明在正常瓣膜发育期间和在促进CAVD的条件下，BMP2水平是如何在主动脉瓣中被控制的。目的1.在CAVD模型小鼠的瓣膜和瓣膜发育过程中，在转录水平上检测控制BMP2合成的DNA序列。这一目标将使用现有的试剂和小鼠集合，这些试剂和小鼠是为了调查骨骼调节序列而创建的。一个区域包括一个保守的骨增强子，它结合了主要的骨分化转录因子RUNX2和其他潜在的调节因子。我们将测试ECR1并定位其他控制BMP2表达的顺式元件，这些顺式元件在高胆固醇饮食和正常主动脉瓣发育的早期阶段发生CAVD的小鼠中。目的2.在发育中的瓣膜和CAVD模型小鼠的瓣膜中，在转录后水平上测试控制BMP2合成的RNA序列。正性和负性机制都会影响主动脉瓣BMP2的合成。哺乳动物和鱼类之间保守的序列强烈抑制BMP2报告基因在发育中的和成年的主动脉瓣中的抑制。我们将在Notch受损的CAVD模型中验证该元件及其侧翼RNA阻止BMP2合成和主动脉瓣异位骨的假设。我们还将在正常的主动脉瓣发育过程中测试这种调节机制。目的3.鉴定通过ECR1调控BMP2转录的反式调节分子。结合ECR1的反式调节因子可能促进钙化的主动脉瓣中BMP2的合成。分子研究将被用来定义与ECR1序列相互作用的因素。RUNX2和Hes1的相关性将在表达BMP2的MC3T3-E1前成骨细胞中进行检测。这些发现将在CAVD小鼠模型的主主动脉瓣间质细胞和病变瓣膜中得到证实。目的4.鉴定在转录后水平控制BMP2合成的反式调节分子。我们将测试靶向BMP2转录产物MC3T3-E1细胞和原代瓣膜间质细胞的microRNAs的影响。这项研究将为BMP2在新的CAVD模型和发育中的主动脉瓣中的空间和时间控制提供基础知识。这些研究将指导未来CAVD中抗BMP2疗法的测试。这项建议探索了这些特定的研究领域：CAVD和二尖瓣主动脉瓣的遗传学、基础结构(改进的动物模型)、细胞生物学(信号通路、机制和钙化调节)和诊断。 (摘要结束)