Basic Mechanisms of Human Calcific Aortic Valve Disease

描述(由申请人提供)： 进行性钙化性主动脉瓣病(CAVD)的特征是瓣膜尖端钙沉积和明显的骨形成。出生时患有二尖瓣主动脉瓣(BAV)的人，一种常见的先天性心脏缺陷，在壮年时可能会发展为严重的CAVD。BAV和CAVD受遗传因素的调控，包括Notch1基因的突变。我们分析了人类遗传数据集，以确定与BAV和/或CAVD相关的基因变异。我们对Tuft BAV Genetics研究的遗传分析确定了WNT/b-catenin和TGFb信号通路中的基因变异，已知这两种基因变异有助于主动脉瓣发育和瓣膜钙化。我们未发表的Framingham心脏研究的全基因组关联分析进一步确定了与CAVD相关的细胞周期蛋白依赖激酶(CDK)、软组织钙化和细胞-细胞通讯途径中的基因变异。CDK7基因SNPs与主动脉瓣钙化的关系具有刺激性，因为我们先前已经证明，视网膜母细胞瘤蛋白(PRB)和CDK途径通过Runx2转录因子和调节细胞对Notch1的反应来调节骨形成。综上所述，我们提出了一个重要的假设，即pRb-CDK途径通过转录作用于软组织钙化以及由Runx2和Notch1调控的细胞-细胞通讯基因来调控CAVD。我们将在我们的第一个特定目标中通过比较pRB缺陷和对照瓣膜间质细胞(VIC)生长以及骨和钙化基因标记物的表达对刺激矿化的因素的反应来检验这一假说。接下来，我们将比较pRb缺乏和对照VIC细胞外基质的僵硬特性和钙化。最后，我们将确定pRb是否调节软组织钙化基因启动子在VICS中的Runx2占位。在我们的第二个目标中，我们将研究具有显着骨骼和矿化表型的CDK6功能丧失或功能获得的小鼠。进一步，我们将确定CDK6在调节VIC对Notch的成骨反应中的作用，我们将考察pRB-cdk小鼠主动脉瓣钙化模型。在我们的第三个目标中，我们将分析人钙化的主动脉瓣组织中pRB-CDK组分的表达和活性。最后，为了广泛研究pRB-cdk途径的表达，以及调节软组织钙化和细胞间通讯的基因，我们将对CAVD转录组(RNA-seq)进行测序。我们组建了一支在CAVD遗传学、成骨转录程序的调控、pRB-CDK途径的钙化以及心脏细胞外基质方面经验丰富的专家团队。我们预计，我们的研究将为CAVD的生物学提供独特的见解，这项工作将对理解钙化性主动脉瓣疾病有意义，并有助于指导潜在的治疗方案或提高对主动脉瓣潜在不良药物影响的认识。 (摘要结束)

DESCRIPTION (provided by applicant): Progressive calcific aortic valve disease (CAVD) is characterized by calcium deposition in the valve cusps as well as frank bone formation. People born with a bicuspid aortic valve (BAV), a common congenital heart defect, can develop significant CAVD during the prime of life. BAV and CAVD are regulated by genetic factors including mutations in the Notch1 gene. We analyzed human genetic datasets to identify gene variants associated with BAV and/or CAVD. Our genetic analysis of the Tufts BAV Genetics Study identified gene variants in the WNT/b-catenin and TGFb signaling pathways, which are known to contribute to aortic valve development as well as valve calcification. Our unpublished genome-wide association analysis of the Framingham Heart Study has further identified gene variants in the cyclin dependent kinase (CDK), the soft tissue calcification and the cell-cell communication pathways associated with CAVD. The association of CDK7 gene SNPs with aortic valve calcification is provocative because we have previously demonstrated that the retinoblastoma protein (pRb) and CDK pathway regulate bone formation through the runx2 transcription factor and by regulating cellular responses to Notch1. Taken together we put forth an over-arching hypothesis that the pRb-CDK pathway regulates CAVD through transcriptional effects on soft tissue calcification and cell-cell communication genes regulated by runx2 and Notch1. We will test this hypothesis in our first specific aim by comparing pRb-deficient and control valve interstitial cell (VIC) growh and expression of bone and calcification gene markers in response to factors that stimulate mineralization. Next, we will compare pRb-deficient and control VIC extracellular matrix stiffness characteristics and calcification. Finally we will determine whether pRb regulates runx2 occupancy of soft tissue calcification gene promoters in VICs. In our second aim we will study mice engineered to have a loss- or a gain-of-function in CDK6 that have significant bone and mineralization phenotypes. Further, we will define the role of cdk6 in regulation of VIC osteogenic responses to Notch, and we will survey pRb-cdk mouse models for aortic valve calcification. In our third aim we will analyze expression and activity of pRb-cdk components in human calcified aortic valve tissue. Finally, to broadly survey expression of the pRb-cdk pathway, as well as genes that regulate soft-tissue calcification and cell-cell communication, we will sequence the CAVD transcriptome (RNA-seq). We have assembled a team of experts with experience in CAVD genetics, the regulation of the osteogenic transcriptional program and calcification by the pRb-CDK pathway, and the heart extracellular matrix. We anticipate that our studies will provide unique insights to the biology of CAVD and that this work will translate meaningfully to the understanding of calcific aortic valve disease as well as help guide potential therapeutic options or raise awareness of potential adverse drug effects on the aortic valve. (End of Abstract)