Abstract
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 TGFβ1, 2 and 3, the TGFβ RI kinase inhibitor SB431542 and TGFβ neutralising antibodies, 5HT and the 5HT2RB antagonist LY272015. Cultures were examined by morphology, transcriptomic profiling, protein expression of the cell specific markers αSMA and SM22α (VICs), and CD31 (VECs), deposition of proteoglycans (PG), the PG versican, and the TGFβs themselves. VECs derived from normal valves were CD31+/αSMA-, but those from diseased valves were αSMA+, indicating endothelial-to-mesenchymal (EndoMT) transition had occurred. The TGFβs induced EndoMT in normal VECs, and this was abolished by SB431542, with significant changes in αSMA, CD31 and HAS2 expression (P<0.05). Normal VICs cultured in 10% FBS media were αSMA+ (activated myofibroblast (disease) phenotype), but were αSMA- when grown in 2% FBS. VICs from diseased dogs were αSMA+ in 2% FBS (retention of the activated myofibroblast disease phenotype), with significantly increased TGFβ1 expression (P<0.05) compared to normal cells. Treatment of normal and diseased VICs with the TGFβs significantly increased expression of αSMA, SM22α, versican, the TGFβs themselves, and deposition of PGs (P<0.05), with TGFβ1 being the most potent activator. These effects were either abolished or markedly reduced by SB431542 and a pan-TGFβ neutralizing antibody (P<0.05). SB431542 also markedly reduced αSMA 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 (αSMA), 5HTR2B, TAGLN (SM22α) 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 TGFβ1 and the TGFβ 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.
Highlights
Myxomatous mitral valve degeneration (MMVD) is the single most common acquired cardiovascular disease of the dog, and an important pathological component of a range of valvulopathies in humans, including Barlow’s Disease and Fibroelastic Deficiency, making the dog a potentially useful naturally-occurring large animal model for acquired human mitral valvulopathies [1,2,3,4]
endothelial-to-mesenchymal transition (EndoMT) has been shown to be activated in canine MMVD and a sheep model where valve endothelial cells (VECs) lose expression of CD31 (PECAM1, platelet and endothelial cell adhesion molecule 1) and CDH5, and gain αSMA expression, and transcriptomic data would suggest this occurs in human MMVD [12]. αSMA is a marker for activated myofibroblasts
VECs (n = 4) were grown in CEM and were morphologically normal and αSMA, unlike those grown in conventional high serum DMEM medium, which underwent EndoMT and transformed into activated myofibroblasts
Summary
Myxomatous mitral valve degeneration (MMVD) is the single most common acquired cardiovascular disease of the dog, and an important pathological component of a range of valvulopathies in humans, including Barlow’s Disease and Fibroelastic Deficiency, making the dog a potentially useful naturally-occurring large animal model for acquired human mitral valvulopathies [1,2,3,4]. Loss of mitral valve endothelial cells (VECs), endothelial-to-mesenchymal transition (EndoMT) and transition of normally quiescent valvular interstitial cells (qVICs) into activated myofibroblasts (aVICs) likely contribute to the ECM changes seen [1, 8,9,10,11,12]. Diseased canine valves have increasing numbers of αSMA+ myofibroblasts and TGFβ1 mediates αSMA+ myofibroblast transformation in cultured VICs [11, 20,21,22]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
