The role of Sox9 in heart valve maintenance and disease

of a dissertation at the University of Miami. Dissertation supervised by Professor Joy Lincoln. No. of pages in text. (110) Heart valve disease results in over 23,000 deaths annually in the United States, with calcific aortic valve disease (CAVD) being the most prevalent (Go et al., 2014). To date, there are no pharmacological therapies to prevent or slow the progression of CAVD, and the only effective treatment involves surgical intervention (Beckmann et al., 2010). Mature aortic valve cusps are composed of a stratified trilaminar extracellular matrix (ECM) that provides the necessary biomechanical properties for function throughout life. It has been previously shown that several ECM proteins within these layers share molecular similarities with cartilage including the expression of type II collagen (Col2a1) and cartilage link protein (CLP) (Lincoln and Yutzey, 2011). The valvular connective tissue is interspersed with valve interstitial cells (VICs) and overlayed by a monolayer of valve endothelial cells (VECs) that seem to be important in sensing changes in the hemodynamic environment and communicating with the underlying VICs (Bosse et al., 2013; Butcher and Nerem, 2006; Gould et al., 2014; Richards et al., 2013; Walker et al., 2004). In contrast to healthy valves, calcified valves are characterized by a loss of ECM stratification and VEC disruption(Hinton et al., 2006; Lincoln and Yutzey, 2011; New and Aikawa, 2011; O'Brien, 2006). In addition, CAVD pathogenesis involves phenotypic changes in the VICs associated with increased expression of osteogenic genes including Spp1 and Runx2, and deposition of a mineralized bone-like matrix that leads to valve stiffening and incomplete opening (Hinton et al., 2006; Lincoln and Yutzey, 2011). Despite the clinical significance, the mechanisms the cartilage-like ECM and prevent CAVD in healthy valves are poorly understood. It has been previously shown that the SRY-transcription factor Sox9 is expressed in the valves beginning in early valvulogenesis and maintained throughout life, and is essential for ECM stratification and preventing calcification of adult valves (Akiyama et al., 2004; Lincoln et al., 2006; Lincoln et al., 2007). This is largely achieved by Sox9-mediated transcriptional activation of cartilaginous genes including Col2a1 and CLP (Lincoln et al., 2007; Peacock et al., 2010). Simultaneously, Sox9 prevents calcification by repressing osteogenic gene programs, although this mechanism of Sox9 action is poorly understood. In this current work, we aim to increase our understanding of the role of Sox9 in valve maintenance and disease by examining both upstream regulators and downstream targets of Sox9 in the valve, as well as mechanisms of regulation of Sox9 in calcification processes. To better define the repressive activity of Sox9 in preventing ectopic bone formation in the valve, we identify and characterize a novel target of Sox9 transcriptional regulation, the osteogenic transcription factor Spp1. Additionally, we identify a new risk factor for development of CAVD through investigations into upstream regulation of Sox9 by retinoic acid. Finally, we investigate the mechanisms of heart valve maintenance and prevention of CAVD by investigating molecular communications between the VICs and VECs to control the VIC phenotype and prevent progression to disease through Sox9 dependent mechanisms. Through these studies we further our understanding of the role of Sox9 in valve maintenance and the pathogenesis of CAVD, and highlight new potential targets for mechanism-based therapeutic