Mechanisms of Notch1 in Aortic Valve Calcification

描述(由申请人提供)： 在发达国家，主动脉瓣钙化/狭窄是成人心脏病的第三大原因，也是获得性瓣膜疾病最常见的形式。与钙化性主动脉狭窄关系最密切的危险因素是二尖瓣的存在、年龄增加、男性、吸烟、高血压和脂蛋白升高。对钙化的人主动脉瓣组织的检查表明，这涉及到几种病理途径。然而，涉及瓣膜钙化的分子机制尚未阐明。最近，斯利瓦斯塔瓦实验室报告了两个人类家族，其中NOTCH1突变与主动脉瓣和二尖瓣早期钙化有关。Notch1是高度保守的信号通路的一部分，参与许多细胞事件，包括决定细胞命运和细胞分化。本研究证实了转录抑制因子Hrt2可以调节Runx2的活性，而Runx2是决定成骨细胞命运的中心调节因子。我们的初步数据显示，与野生型年龄匹配的小鼠相比，Notchl杂合子小鼠的主动脉瓣钙化增加。我推测Notchl信号抑制了主动脉瓣间充质中的促成骨通路，Notchl的破坏可能使瓣膜对促进钙化的已知危险因素敏感。具体目标1：确定干扰Notchl信号是否通过激活成骨通路导致小鼠主动脉瓣钙化。在出生前或出生后，主动脉瓣组织瓣膜中的Notchl信号将有条件地中断。将对主动脉瓣进行功能性狭窄、钙化和骨形成相关基因的表达检查。此外，Notchl基因缺陷的小鼠将与高脂血症的小鼠模型杂交，以检查这一危险因素对Notchl介导的主动脉瓣钙化的贡献。具体目的2：阐明Notchl在间充质细胞和心内膜细胞中介导的钙化途径。在各种条件下，培养的间充质细胞和心内膜细胞中Notch1的表达会被破坏或增强。间充质细胞将被检测成骨细胞特异性基因表达的激活或抑制，在某些情况下还会有钙沉积。Notchl信号与先前涉及心血管钙化的三个分子通路之间的相互作用，即Runx2，BMP2和Wnt，将被研究。 (摘要结束)

DESCRIPTION (provided by applicant): Aortic valve calcification/stenosis is the third leading cause of adult heart disease and the most common form of acquired valvular disease in developed countries. The risk factors most closely linked to calcific aortic stenosis are the presence of a bicuspid aortic valve, increased age, male gender, smoking, hypertension, and elevated lipoproteins. Examination of human calcified aortic valve tissue has shown that several pathologic pathways are involved. However, the molecular mechanisms involved in valvular calcification have not been elucidated. Recently, the Srivastava lab reported two human families in which mutations in NOTCH1 were associated with early calcification of the aortic valve and bicuspid aortic valves. Notchl is a part of a highly conserved signaling pathway involved in many cellular events, including cell fate decisions and cell differentiation. This study demonstrated that Hrt2, a transcriptional represser that mediates the Notchl signal, could regulate the activity of Runx2, a central regulator of osteoblast cell fate determination in vitro. Our preliminary data show that Notchl heterozygous mice develop increased aortic valve calcification as compared to wild-type age matched littermates. I hypothesize that Notchl signaling represses pro-osteogenic pathways in aortic valve mesenchyme and that Notchl disruption may sensitize the valve to known risk factors that promote calcification. Specific Aim 1: To determine if disrupting Notchl signaling causes calcification of the aortic valve by activation of osteogenic pathways in mice. Notchl signaling will be conditionally disrupted in aortic valve tissue valves prenatally or postnatally. The aortic valves will be examined for functional stenosis, calcification, and expression of genes involved in bone formation. In addition, Notchl-deficient mice will be crossed with a mouse model of hyperlipidemia to examine the contribution of this risk factor to Notchl-mediated aortic valve calcification. Specific Aim 2: To elucidate the calcific pathways mediated by Notchl in mesenchymal and endocardial cells. Notchl expression will be disrupted or enhanced in cultured mesenchymal and endocardial cells under various conditions. Mesenchymal cells will be examined for activation or repression of osteoblast-specific gene expression and in some cases deposition of calcium. The interactions between Notchl signaling and three molecular pathways previously implicated in cardiovascular calcification, namely Runx2, Bmp2, and Wnt, will be examined. (End of Abstract)