Treatment with XAV-939 prevents in vitro Calcification and induces SOX9 Expression in Human Aortic Valvular Interstitial Cells
Claudia Dittfeld1, Gabriel Reimann1, Alice Mieting1, Anett Jannasch1, Katrin Plötze1, Gerald Steiner2, Klaus Matschke1, Sems Malte Tugtekin1.
1Technische Universität Dresden, Faculty of Medicine Carl Gustav Carus, Department of Cardiac Surgery, Herzzentrum Dresden, Dresden, Germany, 2Technische Universität Dresden, Faculty of Medicine Carl Gustav Carus, Clinical Sensoring and Monitoring, Dresden, Germany.
OBJECTIVE: Aortic valve (AV) stenosis is accompanied and mainly results from tissue calcification. The development of a substance based treatment strategy to prevent stenosis is a challenge. Wnt/β-catenin signaling is involved in early heart valve formation and calcific AV disease and β-catenin inactivity induces SOX9 driven chondrogenesis. The tankyrase-inhibitor XAV-939 stabilizes Axin and therewith β-catenin destruction complex. Dephosphorylated nuclear β-catenin promotes transcription leading to osteogenesis. Aim of the study was to develop a setup to induce in vitro calcification of aortic valvular interstitial cells (VIC), to investigate the anti-calcific effect of XAV-939 and the expression of Wnt signaling proteins and SOX9.
METHODS: In vitro calcification of human(h) VICs, isolated from native, surgical replaced patient AV was induced by ADGM (50µM ascorbic-acid-phosphate, 10 nM dexamethasone, 10mM β-glycerophospate) incubation for ≤33 days. Total calcium amount was photometrically detected and related to protein content. Infrared spectroscopic imaging was performed on cryosections to confirm calcification. Cultures were additionally incubated with 1µM XAV-939. Expression of alkaline phosphatase, β-catenin and SOX9 was quantified in Western Blots.
RESULTS: 58 % (11/19) of individual hVIC preparations exhibit calcium accumulation in ADGM. Thereby an increase of calcium amount to 3.3±2.9 mol/kg protein was detected (basic level 0.2±0.1 mol/kg protein). The induction of calcification showed a high biological variability and required 14-33 days culturing in ADGM. IR images of an induced culture reveal calcification, indicated by strong absorption bands of carbonate groups comparable to calcified human AV tissue sections. Simultaneous XAV-939 treatment of hVICs in ADGM prevents calcium accumulation (0.3±0.1 mol/kg protein). Alkaline phosphatase expression solely detected in induced ADGM cultures was significantly reduced after XAV-939 treatment. After induction of calcification significant higher β-catenin levels of low molecular weight probably dephosphorylated were expressed. These bands were not detected after XAV-939 treatment. Sox9 expression was significantly induced (4.0±0.7 times) in hVICs XAV-939 treated but lower after additional ADGM incubation.
CONCLUSIONS: Incubation of XAV-939 in ADGM prevents the induction of calcification in hVICs in vitro, leads to different expression patterns of β-catenin and elevated SOX9 expression. It is hypothesized that Axin-stabilization and modulation of Wnt/ β-catenin signaling via XAV-939 reduces AV calcification.
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