| Autor: |
Aize, Margaux, Roussel, Benoit D., Boileve, Arthur, Lebrun, Alexandre, Saplacan, Vladimir, Manrique, Alain, Guinamard, Romain, Simard, Christophe |
| Zdroj: |
Archives of Cardiovascular Diseases; 2024 Supplement, Vol. 117, pS196-S197, 2p |
| Abstrakt: |
Osteogenic differentiation of valvular interstitial cells (VIC), which share properties with fibroblasts, is a key element in valvular calcification leading to aortic stenosis. TRPM4, a Ca2+-activated non-selective cation channel, is involved in cardiac fibroblasts remodeling, and expressed in human VIC (hVIC). Moreover, TRPM4 protein expression is increased on calcified aortic valves from patients suffering of aortic stenosis. The purpose of this study was to evaluate the participation of TRPM4 in osteogenic differentiation of hVIC. hVIC were isolated by enzymatic digestion from aortic valves obtained from patients undergoing surgical aortic valve replacement. They were maintained 7 or 14 days in standard or pro-calcifying media. TRPM4 expression was repressed by transduction of hVIC by shRNA and inhibition of channel activity was realized with 9-phenanthrol application. Channel expression was followed by Western Blot and immunocytochemistry. Osteogenic differentiation of hVIC was evaluated by measuring hydroxyapatite crystals (Alizarin red staining) and by monitoring osteogenic marker protein expression, BMP2 and Runx2. Viability and cell cycle were studied by flow cytometry. Activation of BMP2 pathway was studied by quantification of SMAD1/5 phosphorylated and SMAD1 total protein expression. TRPM4 protein expression was detected in hVIC by immunocytochemistry and confirmed by Western Blot. When maintained in pro-calcifying media, hVIC developed hydroxyapatite crystals and an increase of TRPM4 expression. Pharmacological inhibition of TRPM4 reduced mineralization by 35% after 14 days of culture. Consistently, repression of TRPM4 by shRNA reduced mineralization by 40% after 14 days of culture. Moreover, pharmacological inhibition as well as shRNA treatment significantly decreased BMP2 and Runx2 expression after 7 and 14 days without affecting cell viability and cell cycle. Finally, activation of BMP2 pathway was reduced with repression of TRPM4 expression or inhibition of its activity after 14 days of culture in pro-calcifying media. Thereby, TRPM4 participates in osteogenic differentiation of hVIC in culture through BMP2 pathway and appears as a potential therapeutic target to slow down this deleterious process. This work was conducted as part of the FHU CARNAVAL project (GSC G4) and with a support of GIP Cancéropôle Nord-Ouest. [ABSTRACT FROM AUTHOR] |
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