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)