Myxomatous Degeneration of the Canine Mitral Valve: From Gross Changes to Molecular Events

Myxomatous mitral valve disease (MMVD) is the single most common acquired heart disease of the dog, but is also of emerging importance in human medicine, with some features of the disease shared between both species. There has been increased understanding of this disease in recent years, with most research aiming to elucidate the cellular and molecular events of disease pathogenesis. For gross and histological changes, much of our understanding is based on historical studies and there has been no comprehensive reappraisal of the pathology of MMVD. This paper reviews the gross, histological, ultrastructural, cellular and molecular changes in canine MMVD. (C) 2017 Elsevier Ltd. All rights reserved

Comparative transcriptomic profiling of myxomatous mitral valve disease in the Cavalier King Charles Spaniel

BACKGROUND: Almost all elderly dogs develop myxomatous mitral valve disease by the end of their life, but the cavalier King Charles spaniel (CKCS) has a heightened susceptibility, frequently resulting in death at a young age and suggesting that there is a genetic component to the condition in this breed. Transcriptional profiling can reveal the impact of genetic variation through differences in gene expression levels. The aim of this study was to determine whether expression patterns were different in mitral valves showing myxomatous degeneration from CKCS dogs compared to valves from non-CKCS dogs. RESULTS: Gene expression patterns in three groups of canine valves resulted in distinct separation of normal valves, diseased valves from CKCS and diseased valves from other breeds; the latter were more similar to the normal valves than were the valves from CKCS. Gene expression patterns in diseased valves from CKCS dogs were quite different from those in the valves from other dogs, both affected and normal. Patterns in all diseased valves (from CKCS and other breeds) were also somewhat different from normal non-diseased samples. Analysis of differentially expressed genes showed enrichment in GO terms relating to cardiac development and function and to calcium signalling canonical pathway in the genes down-regulated in the diseased valves from CKCS, compared to normal valves and to diseased valves from other breeds. F2 (prothrombin) (CKCS diseased valves compared to normal) and MEF2C pathway activation (CKCS diseased valves compared to non-CKCS diseased valves) had the strongest association with the gene changes. A large number of genes that were differentially expressed in the CKCS diseased valves compared with normal valves and diseased valves from other breeds were associated with cardiomyocytes including CASQ2, TNNI3 and RYR2. CONCLUSION: Transcriptomic profiling identified gene expression changes in CKCS diseased valves that were not present in age and disease severity-matched non-CKCS valves. These genes are associated with cardiomyocytes, coagulation and extra-cellular matrix remodelling. Identification of genes that vary in the CKCS will allow exploration of genetic variation to understand the aetiology of the disease in this breed, and ultimately development of breeding strategies to eliminate this disease from the breed

Ontogeny of canine myxomatous mitral valve disease; cellular and molecular events over a lifetime

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

Isolation and characterization of primary rat valve interstitial cells: a new model to study aortic valve calcification

Calcific Aortic Valve Disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: α-smooth muscle actin (α-SMA) vimentin and tissue growth factor beta-(TGFβ)1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD

Survival of Activated Myofibroblasts in Canine Myxomatous Mitral Valve Disease and the Role of Apoptosis

Myxomatous mitral valve disease (MMVD) is the single most important acquired cardiovascular disease of the dog. Much is known about the cellular changes and the contribution of activated myofibroblasts (valve interstitial cells (aVICs) to the valve extra-cellular matrix remodelling characteristic of the disease. However, little is known on how aVIC survival might contribute to disease pathogenesis. This study examined the temporal (disease severity-dependent) and spatial distribution of aVICs in MMVD valves, the expression of a range of apoptosis-related genes in cultured VICs from both normal (quiescent VIC (qVIC) and diseased (aVIC) valves, and the differential effects of doxorubicin treatment, as a trigger of apoptosis, on expression of the same genes. Activated myofibroblasts were identified in normal valves at the valve base only (the area closest to the annulus), and then became more numerous and apparent along the valve length as the disease progressed, with evidence of cell survival at the valve base. There were no significant differences in basal gene expression comparing qVICs and aVICs for CASP3, FAS, BID, BAX, BCL2, CASP8, DDIAS, XIAP and BIRC5. After doxorubicin treatment (2mM) for 8hrs there was significant difference (P&lt;0.05) in the expression of BID, BCL2, DDIAS, and CASP8, but when assessed for interactions using a mixed model ANOVA only CASP8 was significantly different because of treatment (P&lt;0.05). These data suggest aVIC survival in MMVD valves may be a consequence of heightened resistance of aVICs to apoptosis, but would require confirmation examining expression of the relevant proteins progression

Expression of calcification and extracellular matrix genes in the cardiovascular system of the healthy domestic sheep (Ovis aries).

The maintenance of a healthy cardiovascular system requires expression of genes that contribute to essential biological activities and repression of those that are associated with functions likely to be detrimental to cardiovascular homeostasis. Vascular calcification is a major disruption to cardiovascular homeostasis, where tissues of the cardiovascular system undergo ectopic calcification and consequent dysfunction, but little is known about the expression of calcification genes in the healthy cardiovascular system. Large animal models are of increasing importance in cardiovascular disease research as they demonstrate more similar cardiovascular features (in terms of anatomy, physiology and size) to humans than do rodent species. We used RNA sequencing results from the sheep, which has been utilized extensively to examine calcification of prosthetic cardiac valves, to explore the transcriptome of the heart and cardiac valves in this large animal, in particular looking at expression of calcification and extracellular matrix genes. We then examined genes implicated in the process of vascular calcification in a wide array of cardiovascular tissues and across multiple developmental stages, using RT-qPCR. Our results demonstrate that there is a balance between genes that promote and those that suppress mineralization during development and across cardiovascular tissues. We show extensive expression of genes encoding proteins involved in formation and maintenance of the extracellular matrix in cardiovascular tissues, and high expression of hematopoietic genes in the cardiac valves. Our analysis will support future research into the functions of implicated genes in the development of valve calcification, and increase the utility of the sheep as a large animal model for understanding ectopic calcification in cardiovascular disease. This study provides a foundation to explore the transcriptome of the developing cardiovascular system and is a valuable resource for the fields of mammalian genomics and cardiovascular research