The utility of cells cultured from the mitral valve as models of myxomatous diseases needs to be properly validated. In this study valve interstitial cells (VICs) and valve endothelial cells (VECs) were cultured from normal and diseased canine mitral valves in 2% (v/v) or 10% FBS media, in the presence of TGF beta 1, 2 and 3, the TGF beta RI kinase inhibitor SB431542 and TGF beta neutralising antibodies, 5HT and the 5HT2RB antagonist LY272015. Cultures were examined by morphology, transcriptomic profiling, protein expression of the cell specific markers alpha SMA and SM22 alpha (VICs), and CD31 (VECs), deposition of proteoglycans (PG), the PG versican, and the TGF beta s themselves. VECs derived from normal valves were CD31 +/alpha SMA-, but those from diseased valves were alpha SMA+, indicating endothelial-to-mesenchymal (EndoMT) transition had occurred. The TGF beta s induced EndoMT in normal VECs, and this was abolished by SB431542, with significant changes in alpha SMA, CD31 and HAS2 expression (P<0.05). Normal VICs cultured in 10% FBS media were alpha SMA+ (activated myofibroblast (disease) phenotype), but were alpha SMA- when grown in 2% FBS. VICs from diseased dogs were alpha SMA+ in 2% FBS (retention of the activated myofibroblast disease phenotype), with significantly increased TGF beta 1 expression (P<0.05) compared to normal cells. Treatment of normal and diseased VICs with the TGF beta s significantly increased expression of alpha SMA, SM22 alpha, versican, the TGF beta s themselves, and deposition of PGs (P<0.05), with TGF beta 1 being the most potent activator. These effects were either abolished or markedly reduced by SB431542 and a pan-TGF beta neutralizing antibody (P<0.05). SB431542 also markedly reduced alpha SMA expression in VICs from diseased valves, but 5HT and LY272015 had no effect on VIC phenotype. Transcriptomic profiling identified clear differences in gene expression for the different conditions and treatments that partially matched that seen in native diseased valve tissue, including changes in expression of ACTA2 (alpha SMA), 5HTR2B, TAGLN (SM22 alpha) and MYH10 (SMemb), gene ontology terms and canonical signalling pathways. Normal and diseased VICs and normal VECs from canine mitral valves can be successfully grown in culture with retention of phenotype, which can be manipulated using TGF beta 1 and the TGF beta RI kinase inhibitor SB431542. This optimized cell system can now be used to model MMVD to elucidate disease mechanisms and identify key regulators of disease progression.
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