PROJECT SUMMARY Heart valve disease results in over 23,000 annual deaths in the United States with calcific aortic valve disease (CAVD) being the most prevalent. There is no pharmacological treatment to prevent or reverse CAVD and therefore surgical intervention remains the only effective option which comes with insuperable complications and no guarantee of long-term success. In healthy individuals, valve leaflets maintain unidirectional blood flow by sustaining a highly organized extracellular matrix (ECM) structure. The major resident cell type responsible for ECM homeostasis is the valve interstitial cell (VIC) that resembles a fibroblast in healthy individuals. In CAVD, VICs transition towards an osteoblast-like cells and this is associated with perturbations in ECM organization including calcific nodule formation on the aortic surface leading to stenosis. Despite this, the signaling pathways underlying osteogenic changes in valve structures are not known and therefore the development of pharmacological therapies to attenuate or reverse the process have been stalled. KPT-330 is a CRM1-dependent nuclear export inhibitor currently in Phase III clinical trials to treat cancer. We show that KPT-330 is sufficient to prevent CAVD in mice (Klotho-/-), as well as prevent, attenuate and rescue calcific nodule formation in human and porcine aortic VICs in vitro. The mechanism underlying the beneficial effect of KPT-330 in CAVD is not known, but our proteomic analysis suggests that it inhibits nuclear export of the CRM1-dependent transcription factor, NFAT5 to reduce calcification. The goal of this proposal is to further test the therapeutic potential of KPT-330 in the treatment of CAVD and delineate the mechanisms underlying its function. Work by us and others has shown that human CAVD is associated with reduced Sox9 and increased Wnt signaling. Interestingly, studies in chondrocytes and intestinal cells have shown that Sox9 and Wnt are commonly regulated by NFAT5, albeit in opposing directions. We have preliminary data to suggest that KPT-330 enhances crosstalk between these signaling pathways and will therefore test the overall hypothesis that: KPT-330 is a novel, therapeutic drug that treats CAVD by preventing nuclear export of NFAT5 and inhibiting osteogenic markers by increasing Sox9 and repressing Wnt. To test this we will address the following three specific aims: 1) Determine the therapeutic window of KPT-330 administration in the treatment of CAVD in vivo;; 2) Delineate the mechanism of KPT-330 mediated treatment of CAVD through NFAT5 signaling;; and 3) Determine if NFAT5 loss of function is sufficient to cause CAVD in vivo. Upon completion we will have determined the therapeutic potent...