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, due to the poor understanding of underlying causes and therefore surgical intervention remains the only effective option. Valve calcification is mediated by valve interstitial cells (VICs) which in response to pathological stimuli abnormally express bone development genes and differentiate into osteoblast-like cells termed osteoVICs. These cells then deposit a calcified matrix that limit cusp movement and leads to stenosis. Despite this, the mechanisms that promote differentiation of osteoVICs are unknown, but our data suggests that Sox9 plays a role. Our data shows that in non-diseased valves Sox9 is highly expressed in the nuclei of VICs where it prevents calcification by functioning as a transcription factor to repress osteogenic gene expression while activating chondrogenic mRNAs that make up healthy valve leaflets. In contrast, we observe reduced Sox9 nuclear localization in several models of calcification including excised valves from human patients and mouse models of CAVD, and in vitro calcification assays, suggesting that Sox9 nuclear export is a common feature of CAVD. Furthermore, our published in vivo work shows that reduced Sox9 function in mice promotes differentiation of osteoVICs and CAVD while attenuating the contribution of chondrogenic matrix proteins indicating a causative role for reduced Sox9 function in CAVD. While our published and preliminary data have identified Sox9 as a key regulator of CAVD, the mechanism underlying its nuclear localization in health and disease are not known, yet identifying this will provide insights for the development of mechanistic-based therapies beyond surgical intervention. In this proposal we demonstrate that in healthy valves, Tgf1 secreted from valve endothelial cells (VECs) molecularly communicates with Sox9 in VICs to retain nuclear localization and prevent calcification via RhoA kinase (ROCK) signaling. Therefore, we hypothesize that: In calcific aortic valve disease, abrogated expression of Tgf1 signaling molecules in VECs prevents ROCK-mediated phosphorylation and nuclear localization of Sox9 in VICs, leading to differentiation of osteoVICs and CAVD. To do this, we will use innovative in vitro, in vitro, genomic and clinical approaches to address the following three specific aims: 1) Determine the mechanism by which Tgf1 mediates Sox9 nuclear localization in healthy and osteogenic valve interstitial cells; 2) VECs isolated from mouse models of CAVD commonly demonstrate altered expression of Tgf signaling molecules associated with dysfunction; and 3) Determine if pharmacological and genetic targeting to prevent Sox9 nuclear export prevents CAVD in vivo. Findings from this proposal will for the first time define a molecular pathway that underlies CAVD and identify mechanistic- based therapies that can be developed to treat affected human patients.

Public Health Relevance

The proposed study is relevant to public health as it addresses the molecular mechanisms that initiate and propagate calcific aortic valve disease, a disease process that is becoming increasing prevalent with an aging population. The planned research has the potential to increase the fundamental understanding of disease pathogenesis and discover new molecular pathways that may lead to novel therapeutic targets to prevent or treat the disease.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Cardiovascular Differentiation and Development Study Section (CDD)
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Evans, Frank
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Nationwide Children's Hospital
United States
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Dutta, Punashi; Lincoln, Joy (2018) Calcific Aortic Valve Disease: a Developmental Biology Perspective. Curr Cardiol Rep 20:21
Hulin, Alexia; Anstine, Lindsey J; Kim, Andrew J et al. (2018) Macrophage Transitions in Heart Valve Development and Myxomatous Valve Disease. Arterioscler Thromb Vasc Biol 38:636-644
Laurent, Frédéric; Girdziusaite, Ausra; Gamart, Julie et al. (2017) HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Development. Cell Rep 19:1602-1613
Lincoln, Joy (2016) The cardiac matrix revolution: Post-translational modification of Scleraxis. J Mol Cell Cardiol 93:106-7
Anstine, Lindsey J; Bobba, Chris; Ghadiali, Samir et al. (2016) Growth and maturation of heart valves leads to changes in endothelial cell distribution, impaired function, decreased metabolism and reduced cell proliferation. J Mol Cell Cardiol 100:72-82
Huk, Danielle J; Austin, Blair F; Horne, Tori E et al. (2016) Valve Endothelial Cell-Derived Tgf?1 Signaling Promotes Nuclear Localization of Sox9 in Interstitial Cells Associated With Attenuated Calcification. Arterioscler Thromb Vasc Biol 36:328-38
Horne, Tori E; VandeKopple, Matthew; Sauls, Kimberly et al. (2015) Dynamic Heterogeneity of the Heart Valve Interstitial Cell Population in Mitral Valve Health and Disease. J Cardiovasc Dev Dis 2:214-232