Ontogeny of canine myxomatous mitral valve disease; cellular and molecular events over a lifetime
Markby, Greg Robert
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Myxomatous mitral valve disease (MMVD) is the most common cardiac disease in dogs and the second most common cardiac valvular disease in humans. MMVD is particularly prevalent in small breed dogs (such as the Cavalier King Charles Spaniel (CKCS)) but by age 10, almost all dogs will have some form disease developing on the valve. Pathologically, there is a progressive deterioration in the organised structure of the valve extracellular matrix with an accumulation of proteoglycans and glycosaminoglycans, breakdown of collagen fibrils and loss of elastin and basement membrane components. Alongside this, there is an activation of quiescent valvular interstitial cells (VICs) into a myofibrotic phenotype, denuding of endothelial cells and endothelial-to-mesenchymal transition. Despite this knowledge of pathology and the clinical significance of MMVD, there is a lack of understanding of the underlying cellular and molecular mechanisms controlling the disease. This is particularly true of early disease development as the majority of previous studies have focused on comparing normal valves with end-stage disease. This project aimed to address this problem by examining the transcriptomic changes occurring in MMVD in the dog: across the entire pathogenesis of the disease, in regional development of the disease on the valve and in in vitro models of disease. Utilising the Whitney grading system (Grades 0-4), where Grade 4 represents severe disease, valves were collected (5 grades, n=6 per grade) from the entire spectrum of disease and in a mixture of dog breeds. Transcriptomic profiling (Affymetrix GeneChip™ Canine Gene 1.1 Sense Target (ST) Array) was performed and 1002 differentially expressed genes were identified across all grades of disease. Network analysis was used to cluster genes with similar expression profiles and establish trends of progressive up- or down-regulation over the course of MMVD. Gene enrichment analysis highlighted GO terms both associated with these trends and in a grade-specific manner. Pathway analysis established the top canonical pathways, upstream regulators and disease function networks associated with each grade of disease. As a whole, these results indicated dysregulation of metalloproteases (both up- and downregulation) and involvement of immune-related pathways as well as, most importantly, the repeated implication that TGFβ signalling is a controller in disease development. Furthermore, sample-to-sample analysis indicated CKCS, who have an earlier onset of disease than other dogs, had a slightly different transcriptomic profile compared to other valves, with analysis indicating a role for down-regulated calcium signalling and cell contractility in early disease development in this breed. Transcriptomic analysis comparing distinct diseased and normal areas of the same moderately affected valves (n=7) found 289 differentially expressed genes, including hallmarks of disease ACTA2 and 5HTR2B which were upregulated in the diseased sections of the valves. Comparison of the ‘normal’ tissue in this dataset with the whole normal valve dataset and dissection of comparable regions of normal valves and assessment by RT-qPCR indicated that the changes being measured were disease-specific and validated the approach used. Gene enrichment analysis of this dataset also implicated TGFβ1 as the top upstream regulator of disease. To further explore the role of TGFβ1 signalling in MMVD, VICs from normal (n=3) and diseased (n=3) valves were treated with 5ng/mL TGFβ1 and 10 μM SB431542 (TGFβ pathway inhibitor) respectively, with appropriate vehicle controls. Differential gene expression was identified comparing normal VIC to normal VIC TGF-β1-treated (275 genes), diseased VIC to diseased VIC SB431542-treated (236 genes) and diseased VIC to normal VIC (902 genes). Normal VICs transformed to myofibrotic cells in the presence of TGFβ1, with increased αSMA (ACTA2) expression and a 5 fold increase in proteoglycan secretion (p<0.05), consistent with the pathology in vivo. Diseased VICs showed a significant 2 fold increase in TGFβ1 secretion compared to normal VICs, and in the presence of SB431542 reverted to a normal phenotype with a reduction in αSMA expression and 2.8 fold decrease in proteoglycan secretion (p<0.05). To summarise, this study provides insights into the molecular pathogenesis of MMVD from its early development to its end stage consequences and identifies TGFβ1 signalling as a central pathway in disease development.