Project Summary Valvular heart disease (VHD), which is most commonly associated with mitral valve prolapse (MVP), causes severe regurgitation leading to sudden cardiac arrest or sudden cardiac death. Mitral valve endothelial cells (ECs) are crucial participants in establishing mitral valve structures and are essential for the maintenance of mitral valve integrity and function. However, the mechanisms by which mitral valve ECs govern structural changes in the mitral valve during the progression of MVP have yet to be adequately characterized. The long- term goal of this proposal is to elucidate the fundamental processes that regulate EC function in the integrity and function of the mitral valve and to understand how the disruption of these mechanisms leads to MVP. FOXC1 and FOXC2 are closely related members of the FOX transcription factor family and have numerous essential roles in cardiovascular development, health, and disease. There is some evidence that human FOXC1 mutations are associated with mitral valve abnormalities, including mitral regurgitation. We have previously shown that in mice, global homozygous knockout mutations of Foxc1 and/or Foxc2 are associated with vascular anomalies; however, the mutations also lead to embryonic or perinatal lethality, so attempts to determine how the two Foxc genes function in adult animals have generally been inconclusive. To overcome this limitation, we generated a line of mice carrying tamoxifen-inducible, EC-specific, compound Foxc1;Foxc2 mutations (i.e., EC- Foxc-DKO mice), and the parent award is aimed at elucidating the role of Foxc1/Foxc2 in vascular repair and intestinal regeneration after injury by directly regulating the expression of CXCL12 and R-spondin 3 paracrine factors in intestinal ECs. During the course of performing the proposed studies for the parent award using the Foxc-DKO mice, we also obtained the results from preliminary investigations with these adult mutants, which indicate that the Foxc1/c2 mutations in the adult impair the structure and integrity of the mitral valve of the heart. Thus, our central hypothesis is that the transcriptional activity of Foxc1/c2 in mitral valve ECs contributes to mitral valve integrity and fucntion. We will test our central hypothesis by pursuing the two Specific Aims: (1) To determine whether Foxc1 and Foxc2 are required in mitral valve ECs for proper development of the mitral valve and (2) To determine the mechanisms by which EC expression of Foxc1 and Foxc2 regulates the integrity and function of the mitral valve. In summary, the experiments described in this proposal will provide crucial information about how Foxc1/c2 deficiency in mitral valve EC populations contributes to mitral valve defects such as MPV. Furthermore, since impairments in the mitral valve contribute to various cardiac disorders, our findings may have important implications for other pathologic conditions associated with VHD such as heart failure.