Dataset Information

Evaluation of canine 2D cell cultures as models of myxomatous mitral valve degeneration.

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 TGF?1, 2 and 3, the TGF? RI kinase inhibitor SB431542 and TGF? neutralising antibodies, 5HT and the 5HT2RB antagonist LY272015. Cultures were examined by morphology, transcriptomic profiling, protein expression of the cell specific markers ?SMA and SM22? (VICs), and CD31 (VECs), deposition of proteoglycans (PG), the PG versican, and the TGF?s themselves. VECs derived from normal valves were CD31+/?SMA-, but those from diseased valves were ?SMA+, indicating endothelial-to-mesenchymal (EndoMT) transition had occurred. The TGF?s induced EndoMT in normal VECs, and this was abolished by SB431542, with significant changes in ?SMA, CD31 and HAS2 expression (P<0.05). Normal VICs cultured in 10% FBS media were ?SMA+ (activated myofibroblast (disease) phenotype), but were ?SMA- when grown in 2% FBS. VICs from diseased dogs were ?SMA+ in 2% FBS (retention of the activated myofibroblast disease phenotype), with significantly increased TGF?1 expression (P<0.05) compared to normal cells. Treatment of normal and diseased VICs with the TGF?s significantly increased expression of ?SMA, SM22?, versican, the TGF?s themselves, and deposition of PGs (P<0.05), with TGF?1 being the most potent activator. These effects were either abolished or markedly reduced by SB431542 and a pan-TGF? neutralizing antibody (P<0.05). SB431542 also markedly reduced ?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 (?SMA), 5HTR2B, TAGLN (SM22?) 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?1 and the TGF? 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.

SUBMITTER: Tan K

PROVIDER: S-EPMC6695117 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Evaluation of canine 2D cell cultures as models of myxomatous mitral valve degeneration.

Tan Karen K Markby Greg G Muirhead Rhona R Blake Rachel R Bergeron Lisa L Fici Greg G Summers Kim K Macrae Vicky V Corcoran Brendan B

PloS one 20190815 8

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, p ...[more]

PMID: 31415646

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Project description:Myxomatous mitral valve disease (MMVD) is the most common acquired canine cardiovascular disease and shares many similarities with human mitral valvulopathies. While transcriptomic datasets are available for the end-stage disease in both species, there is no information on how gene expression changes as the disease progresses, such that it cannot be stated with certainty if the changes seen in end-stage disease are casual or consequential. In contrast to humans, the disease in dogs can be more readily examined as it progresses, and this allows an opportunity for insight into disease pathogenesis relevant to both species. The aim of this study was to identify changes in valve gene expression as canine MMVD advances over an entire life-time, from normal (grade 0) to severely affected (grade 4), and differences in gene expression comparing normal and disease areas of the same valve. Transcriptomic profiling identified 1002 differentially expressed genes (DEGs) across all four disease grades when compared with normal valves with the greatest number of DEGs in grade 3 (673) and grade 4 (507). DEGs were associated with a large number of gene families, including genes encoding cytoskeletal filaments, peptidases, extra-cellular matrix (ECM) proteins, chemokines and integrins. Gene enrichment analysis identified significant grade-dependent changes in gene clustering, with clusters trending both up and down as disease progressed. Significant grade-dependent changes in hallmark disease gene expression intensity were identified, including ACTA2, HTR2B, MMP12, and CDKN2A. Gene Ontology terms were dominated by terms for ECM and inflammation with TGF?1, TNF, IFGN identified as the top up-stream regulators in both whole and dissected diseased valve samples. These data show that while disease progression in MMVD is associated with increasing numbers of DEGs, TGF? appears to be the dominant signaling pathway controlling pathogenesis irrespective of disease severity.

Project description:Myxomatous mitral valve disease (MMVD) is functionally and histologically identical to mitral valve prolapse (MVP) in humans. Currently, there are no medical treatments that can delay the progression of this valvular disease or associated cardiac remodelling. Therefore, there is a need to understand the molecular pathology associated with MMVD and MVP better, and thus identify potential therapeutic targets. Circulating exosomes contain small RNA, including miRNA, which reflect cell physiology and pathology. This study explored the association between circulating exosomal miRNA (ex-miRNA) content and MMVD, heart failure due to MMVD (MMVD-CHF) and ageing, which is strongly associated with MMVD. Ex-miRNA was isolated from old normal/healthy dogs (n = 6), young normal dogs (n = 7), dogs with MMVD (n = 7) and dogs with MMVD-CHF (n = 7). Separately, total plasma miRNA was isolated from normal dogs (n = 8), dogs with MMVD (n = 8) and dogs with MMVD-CHF (n = 11). Using reverse transcription quantitative polymerase chain reaction, exosomal miR-181c (p = 0.003) and miR-495 (p = 0.0001) significantly increased in dogs with MMVD-CHF compared to the other three groups. Exosomal miR-9 (p = 0.002) increased in dogs with MMVD and MMVD-CHF compared to age-matched (old) normal dogs. Exosomal miR-599 (p = 0.002) decreased in dogs with MMVD compared to old normal dogs. In total plasma, 58 miRNA were deemed significantly different (p < 0.04) between normal dogs, dogs with MMVD and dogs with MMVD-CHF. However, in contrast to ex-miRNA, none of the miRNA in total plasma remained statistically significant if the false discovery rate was <15%. Changes in ex-miRNA are observed in dogs as they age (miR-9, miR-495 and miR-599), develop MMVD (miR-9 and miR-599) and progress from MMVD to CHF (miR-181c and miR-495). Ex-miRNA expression-level changes appear to be more specific to disease states than total plasma miRNA. RESPONSIBLE EDITOR Elena Aikawa, Harvard Medical School, USA.

Dataset Information

Evaluation of canine 2D cell cultures as models of myxomatous mitral valve degeneration.

Publications

Evaluation of canine 2D cell cultures as models of myxomatous mitral valve degeneration.

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