Ontogenic Transcriptomic Profiling Identifies Signalling Pathways Driving Pathogenesis in Canine Myxomatous Mitral Valve Disease

Presenter Brendan Corcoran
Authors Greg R. Markby1, Kim M. Summers1,2, Vicky E. MacRae1 and Brendan M. Corcoran1,3
Affiliations 1The Roslin Institute, Edinburgh, Scotland. 2Mater Research Institute, Brisbane, Australia. 3The Royal (Dick) School of Veterinary Studies, Edinburgh, Scotland.
Presentation Type Poster


Chronic degenerative diseases (CDGs) are a major welfare concern in canine medicine with myxomatous mitral valve disease (MMVD) being an important example. For some breeds CDGs can have an inherited basis, but often this is a polygenic trait and so understanding the mechanisms that drive disease pathogenesis requires examining molecular events in tissue. Specifically for CDGs this requires examination in both temporal and spatial terms changes in gene and protein expression. In this study we have examined the valvular gene expression at different stages of disease (temporal), different locations (spatial) and in different cell culture models of MMVD.
Transcriptomic profiling (Affymetrix canine 1.1ST microarray), with validation using RT-qPCR for selected genes, was performed on, whole valves from normal and the 4 grades of MMVD (n=6),normal and diseased regions of grade 2 valves (n=7), and cultured (all experiments n=3) normal and diseased valve interstitial cells (VICs), normal cells treated with 5ng/µL TGFβ1 and diseased cells treated with 10µM of the TGFβ pathway inhibitor SB431542. Microarray data were analysed using a range of bioinformatics platforms (Affymetrix Console, IPA, Miru (Biolayout Express)).
Significantly differentially expressed genes (DEG) were identified comparing: 1) normal and the 4 grades of MMVD (1002 genes); 2) diseased and normal tissue within the same valve (315 genes); 3) normal and diseased VICs (1027 genes); 4) normal VICs and normal VICs treated with TGF-β1 (302 genes); 5) diseased VICs and diseased treated with VICs SB431542 (269 genes). Grade-dependent up and down regulated gene clusters were identified, and microarray data were validated by RT-PCR for ACTA2, TAGLN and 5HTR2B. Important GO-terms were found to be associated with myofibroblast differentiation and extracellular matrix homeostasis. In all data sets altered DEGs implicated TGF-β1 as the important up-stream regulator of disease pathogenesis, with minor contributions from TNF and IFNG. 75 DEGs were shared in common between grade 4 whole valve and the diseased sections of the dissected valves. Cultured cell data, in addition to TGFβ1, predicted genes involved in cell cycle and apoptosis as important up-stream regulators.
This study shows how transcriptomic profiling of chronic degenerative disease over an entire lifetime, in tandem with cell culture models, can identify the signalling pathways important in disease pathogenesis. TGFβ1 signalling has been identified as the fundamentally important pathway in MMVD initiation and development, and progression to eventual end-stage valve pathology.