TGF-ß phospho antibody array identifies altered SMAD2, PI3K/AKT/SMAD, and RAC signaling contribute to the pathogenesis of myxomatous mitral valve disease.

Background: TGFß signaling appears to contribute to the pathogenesis of myxomatous mitral valve disease (MMVD) in both dogs and humans. However, little is known about the extent of the downstream signaling changes that will then affect cell phenotype and function in both species. Objective: Identify changes in downstream signals in the TGFß pathway in canine MMVD and examine the effects of antagonism of one significant signal (SMAD2 was selected). Materials and methods: Canine cultures of normal quiescent valve interstitial cells (qVICs) and disease-derived activated myofibroblasts (aVICs) (n = 6) were examined for TGFß signaling protein expression using a commercial antibody array. Significant changes were confirmed, and additional proteins of interest downstream in the TGFß signaling pathway and markers of cell phenotype were examined (PRAS40, S6K, elF4E IRS-1, αSMA, and VIM), using protein immunoblotting. RT-PCR examined expression of gene markers of VIC activation (ACTA2, TAGLN, and MYH10; encoding the proteins αSMA, SM22, and Smemb, respectively). Attenuation of pSMAD2 in aVICs was examined using a combination of RNA interference technology (siRNA) and the SMAD7 (antagonizes SMAD2) agonist asiaticoside. Results: The antibody array identified significant changes (P < 0.05) in 19 proteins, of which six were phosphorylated (p). There was increased expression of pSMAD2 and pRAC1 and decreased expression of pmTOR, pERK1/2, and pAKT1. Expression of pPRAS40 and pIRS-1 was increased, as was the mTOR downstream transcription factor pS6K, with increased expression of peIF4E in aVICs, indicating negative feedback control of the PI3K/AKT/mTOR pathway. SMAD2 antagonism by siRNA and the SMAD7 agonist asiaticoside decreased detection of pSMAD by at least 50%, significantly decreased expression of the aVIC gene markers ACTA2, TAGLN, and MYH10, and pαSMA, pAKT2, and pERK1, but had no effect on pS6K, pERK2, or pVIM expression in aVICs. SMAD2 antagonism transitioned diseased aVICs to normal qVICs, while maintaining a mesenchymal phenotype (VIM+) while concurrently affecting non-canonical TGFß signaling. Conclusion: MMVD is associated with changes in both the canonical and non-canonical TGFß signaling pathway. Antagonism of SMAD2 transitions diseased-activated myofibroblasts back to a normal phenotype, providing data that will inform studies on developing novel therapeutics to treat MMVD in dogs and humans.

TGF-ß-induced PI3K/AKT/mTOR pathway controls myofibroblast differentiation and secretory phenotype of valvular interstitial cells through the modulation of cellular senescence in a naturally occurring in vitro canine model of myxomatous mitral valve disease.

PI3K/AKT/mTOR signalling contributes to several cardiovascular disorders. The aim of this study was to examine the PI3K/AKT/mTOR pathway in myxomatous mitral valve disease (MMVD). Double-immunofluorescence examined expression of PI3K and TGF-ß1 in canine valves. Valve interstitial cells (VICs) from healthy or MMVD dogs were isolated and characterized. Healthy quiescent VICs (qVICs) were treated with TGF-ß1 and SC-79 to induce activated myofibroblast phenotypes (aVICs). Diseased valve-derived aVICs were treated with PI3K antagonists and expression of RPS6KB1 (encoding p70 S6K) was modulated using siRNA and gene overexpression. SA-ß-gal and TUNEL staining were used to identify cell senescence and apoptosis, and qPCR and ELISA to examine for senescence-associated secretory phenotype. Protein immunoblotting was used to examine expression of phosphorylated and total proteins. TGF-ß1 and PI3K are highly expressed in mitral valve tissues. Activation of PI3K/AKT/mTOR and increased expression of TGF-ß are found in aVICs. TGF-ß transitions qVICs to aVICs by upregulation of PI3K/AKT/mTOR. Antagonism of PI3K/AKT/mTOR reverses aVIC myofibroblast transition by inhibiting senescence and promoting autophagy. Upregulation of mTOR/S6K induces transformation of senescent aVICs, with reduced capacity for apoptosis and autophagy. Selective knockdown of p70 S6K reverses cell transition by attenuating cell senescence, inhibiting apoptosis and improving autophagy. TGF-ß-induced PI3K/AKT/mTOR signalling contributes to MMVD pathogenesis and plays crucial roles in the regulation of myofibroblast differentiation, apoptosis, autophagy and senescence in MMVD.

Metformin protects against vascular calcification through the selective degradation of Runx2 by the p62 autophagy receptor.

Vascular calcification is associated with aging, type 2 diabetes, and atherosclerosis, and increases the risk of cardiovascular morbidity and mortality. It is an active, highly regulated process that resembles physiological bone formation. It has previously been established that pharmacological doses of metformin alleviate arterial calcification through adenosine monophosphate-activated protein kinase (AMPK)-activated autophagy, however the specific pathway remains elusive. In the present study we hypothesized that metformin protects against arterial calcification through the direct autophagic degradation of runt-related transcription factor 2 (Runx2). Calcification was blunted in vascular smooth muscle cells (VSMCs) by metformin in a dose-dependent manner (0.5-1.5 mM) compared to control cells (p < 0.01). VSMCs cultured under high-phosphate (Pi) conditions in the presence of metformin (1 mM) showed a significant increase in LC3 puncta following bafilomycin-A1 (Baf-A; 5 nM) treatment compared to control cells (p < 0.001). Furthermore, reduced expression of Runx2 was observed in the nuclei of metformin-treated calcifying VSMCs (p < 0.0001). Evaluation of the functional role of autophagy through Atg3 knockdown in VSMCs showed aggravated Pi-induced calcification (p < 0.0001), failure to induce autophagy (punctate LC3) (p < 0.001) and increased nuclear Runx2 expression (p < 0.0001) in VSMCs cultured under high Pi conditions in the presence of metformin (1 mM). Mechanistic studies employing three-way coimmunoprecipitation with Runx2, p62, and LC3 revealed that p62 binds to both LC3 and Runx2 upon metformin treatment in VSMCs. Furthermore, immunoblotting with LC3 revealed that Runx2 specifically binds with p62 and LC3-II in metformin-treated calcified VSMCs. Lastly, we investigated the importance of the autophagy pathway in vascular calcification in a clinical setting. Ex vivo clinical analyses of calcified diabetic lower limb artery tissues highlighted a negative association between Runx2 and LC3 in the vascular calcification process. These studies suggest that exploitation of metformin and its analogues may represent a novel therapeutic strategy for clinical intervention through the induction of AMPK/Autophagy Related 3 (Atg3)-dependent autophagy and the subsequent p62-mediated autophagic degradation of Runx2.

The Role of Transforming Growth Factor-ß Signaling in Myxomatous Mitral Valve Degeneration.

Mitral valve prolapse (MVP) due to myxomatous degeneration is one of the most important chronic degenerative cardiovascular diseases in people and dogs. It is a common cause of heart failure leading to significant morbidity and mortality in both species. Human MVP is usually classified into primary or non-syndromic, including Barlow's Disease (BD), fibro-elastic deficiency (FED) and Filamin-A mutation, and secondary or syndromic forms (typically familial), such as Marfan syndrome (MFS), Ehlers-Danlos syndrome, and Loeys-Dietz syndrome. Despite different etiologies the diseased valves share pathological features consistent with myxomatous degeneration. To reflect this common pathology the condition is often called myxomatous mitral valve degeneration (disease) (MMVD) and this term is universally used to describe the analogous condition in the dog. MMVD in both species is characterized by leaflet thickening and deformity, disorganized extracellular matrix, increased transformation of the quiescent valve interstitial cell (qVICs) to an activated state (aVICs), also known as activated myofibroblasts. Significant alterations in these cellular activities contribute to the initiation and progression of MMVD due to the increased expression of transforming growth factor-ß (TGF-ß) superfamily cytokines and the dysregulation of the TGF-ß signaling pathways. Further understanding the molecular mechanisms of MMVD is needed to identify pharmacological manipulation strategies of the signaling pathway that might regulate VIC differentiation and so control the disease onset and development. This review briefly summarizes current understanding of the histopathology, cellular activities, molecular mechanisms and pathogenesis of MMVD in dogs and humans, and in more detail reviews the evidence for the role of TGF-ß.

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.

Disease Severity-Associated Gene Expression in Canine Myxomatous Mitral Valve Disease Is Dominated by TGFß Signaling.

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 TGFß1, 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, TGFß appears to be the dominant signaling pathway controlling pathogenesis irrespective of disease severity.

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 (2 mM) for 8 h there was significant difference (P < .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 < .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.

Exploiting novel valve interstitial cell lines to study calcific aortic valve disease.

Calcific aortic valve disease (CAVD) involves progressive valve leaflet thickening and severe calcification, impairing leaflet motion. The in vitro calcification of primary rat, human, porcine and bovine aortic valve interstitial cells (VICs) is commonly employed to investigate CAVD mechanisms. However, to date, no published studies have utilised cell lines to investigate this process. The present study has therefore generated and evaluated the calcification potential of immortalized cell lines derived from sheep and rat VICs. Immortalised sheep (SAVIC) and rat (RAVIC) cell lines were produced by transduction with a recombinant lentivirus encoding the Simian virus (SV40) large and small T antigens (sheep), or large T antigen only (rat), which expressed markers of VICs (vimentin and α­smooth muscle actin). Calcification was induced in the presence of calcium (Ca; 2.7 mM) in SAVICs (1.9 fold; P<0.001) and RAVICs (4.6 fold; P<0.01). Furthermore, a synergistic effect of calcium and phosphate was observed (2.7 mM Ca/2.0 mM Pi) on VIC calcification in the two cell lines (P<0.001). Analysis of SAVICs revealed significant increases in the mRNA expression of two key genes associated with vascular calcification in cells cultured under calcifying conditions, runt related transcription factor­2 (RUNX2;1.3 fold; P<0.05 in 4.5 mM Ca) and sodium­dependent phosphate transporter­1 (PiT1; 1.2 fold; P<0.05 in 5.4 mM Ca). A concomitant decrease in the expression of the calcification inhibitor matrix Gla protein (MGP) was noted at 3.6 mM Ca (1.3 fold; P<0.01). Assessment of RAVICs revealed alterations in Runx2, Pit1 and Mgp mRNA expression levels (P<0.01). Furthermore, a significant reduction in calcification was observed in SAVICs following treatment with established calcification inhibitors, pyrophosphate (1.8 fold; P<0.01) and etidronate (3.2 fold; P<0.01). Overall, the present study demonstrated that the use of immortalised sheep and rat VIC cell lines is a convenient and cost effective system to investigate CAVD in vitro, and will make a useful contribution to increasing current understanding of the pathophysiological process.

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: alpha-smooth muscle actin (α-SMA) vimentin and tissue growth factor beta (TGFß) 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.

Comparative Transcriptomic Profiling and Gene Expression for Myxomatous Mitral Valve Disease in the Dog and Human.

Myxomatous mitral valve disease is the single most important mitral valve disease in both dogs and humans. In the case of the dog it is ubiquitous, such that all aged dogs will have some evidence of the disease, and for humans it is known as Barlow's disease and affects up to 3% of the population, with an expected increase in prevalence as the population ages. Disease in the two species show many similarities and while both have the classic myxomatous degeneration only in humans is there extensive fibrosis. This dual pathology of the human disease markedly affects the valve transcriptome and the difference between the dog and human is dominated by changes in genes associated with fibrosis. This review will briefly examine the comparative valve pathology and then, in more detail, the transcriptomic profiling and gene expression reported so far for both species.

Further characterization of computed tomographic and clinical features for staging and prognosis of idiopathic pulmonary fibrosis in West Highland white terriers.

Idiopathic pulmonary fibrosis is an interstitial lung disease of unknown etiology resulting in progressive interstitial fibrosis, with a known predilection in West Highland white terriers. In humans, computed tomography (CT) is a standard method for providing diagnostic and prognostic information, and plays a major role in the idiopathic pulmonary fibrosis staging process. Objectives of this retrospective, analytical, cross-sectional study were to establish descriptive criteria for reporting CT findings and test correlations among CT, clinical findings and survival time in West Highland white terriers with idiopathic pulmonary fibrosis. Inclusion criteria for affected West Highland white terriers were a diagnosis of idiopathic pulmonary fibrosis and available CT, bronchoscopy, bronchoalveolar lavage, echocardiography, and routine blood analysis findings. Clinically normal West Highland white terriers were recruited for the control group. Survival times were recorded for affected dogs. The main CT lung pattern and clinical data were blindly and separately graded as mild, moderate, or severe. Twenty-one West Highland white terriers with idiopathic pulmonary fibrosis and 11 control West Highland white terriers were included. The severity of pulmonary CT findings was positively correlated with severity of clinical signs (ρ = 0.48, P = 0.029) and negatively associated with survival time after diagnosis (ρ = -0.56, P = 0.025). Affected dogs had higher lung attenuation (median: -563 Hounsfield Units (HU)) than control dogs (median: -761 HU), (P < 0.001). The most common CT characteristics were ground-glass pattern (16/21) considered as a mild degree of severity, and focal reticular and mosaic ground-glass patterns (10/21) considered as a moderate degree of severity. Findings supported the use of thoracic CT as a method for characterizing idiopathic pulmonary fibrosis in West Highland white terriers and providing prognostic information for owners.

Developmental pathways and endothelial to mesenchymal transition in canine myxomatous mitral valve disease.

Epithelial to mesenchymal transition (EMT) and the cardiovascular equivalent, endothelial to mesenchymal transition (EndoMT), contribute to a range of chronic degenerative diseases and cancer metastasis. Chronic valvulopathies exhibit some features of EndoMT and activation of developmental signalling pathways, such as osteogenesis and chondrogenesis, expression of cell differentiation markers, basement membrane damage and endothelial transformation. The aim of the present study was to investigate the potential role of developmental mechanisms in canine myxomatous mitral valve disease (MMVD) using a combination of transcriptomic array technology, RT-PCR and immunohistochemistry. There was significant differential expression for genes typically associated with valvulogenesis and EndoMT, including markers of inflammation (IL6, IL18 and TLR4), basement membrane disarray (NID1, LAMA2 and CTSS), mesenchymal and endothelial cell differentiation (MYH11 and TAGLN) and EndoMT (ACTA2, SNAI1, CTNNB1, HAS2, CDH5, and NOTCH1), with fold changes from +15.35 (ACTA2) to -5.52 (LAMA2). These changes in gene expression were confirmed using RT-PCR, except for HAS2. In silico analysis identified important gene networks and canonical pathways in MMVD that have associations with development and organogenesis, including inflammation, valve morphogenesis and EMT, as well as components of the basement membrane and extra-cellular matrix. Immunohistochemistry identified changes in the expression of hyaluronic acid synthase (Has2), Snai1, α-smooth muscle actin (α-SMA) and VE-cadherin (CDH5), and co-expression of Has2 with α-SMA. These research findings strongly suggest involvement of developmental signalling pathways and mechanisms, including EndoMT, in the pathogenesis of canine MMVD.

An official American thoracic society workshop report: comparative pathobiology of fibrosing lung disorders in humans and domestic animals.

BACKGROUND: The clinical outcome of idiopathic pulmonary fibrosis (IPF) is poor, with a 50% survival rate at 3 years. Furthermore, current treatments provide little amelioration of symptoms. Despite significant advances in understanding the clinical features and pathobiology of IPF, further advances have been hampered by a lack of suitable animal models of the disease. Interestingly, spontaneously occurring disorders with a similarity to IPF have been recognized in the dog, cat, horse, and donkey. These disorders share clinical and pathologic features with human IPF and are emerging diseases of veterinary importance. PURPOSE: To improve awareness about these disorders in domestic animals and stimulate interactions between disciplines, and to facilitate the elucidation of mechanisms of fibrosing lung disorders using a comparative natural-occurrence disease model approach. METHODS: A 1-day meeting joined physicians, veterinarians, pathologists, researchers, and advocacy experts to discuss information available in this area. A review of the literature was conducted, and an executive committee discussed the findings and prepared a summary statement during subsequent meetings. RESULTS: Clinical, diagnostic, and treatment opportunities were identified, and common areas of interest where collaborative efforts could accelerate discovery regarding etiological factors, methods for early detection, determinants of disease progression, and novel therapies were defined. CONCLUSIONS: Comparing fibrosing lung disorders in humans and domestic animals will allow for a better understanding of the similarities and differences among species and may offer novel insights into the underlying mechanisms of spontaneously occurring fibrotic lung diseases.

Characterisation and differentiation potential of bone marrow derived canine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have potential for use in regenerative therapeutics, since they are capable of multi-lineage differentiation. In this study, primary canine MSCs (cMSCs) were isolated from bone marrow aspirates and characterised using marker expression and morphology. cMSCs expressed CD44 and STRO-1, but not CD34 or CD45. Morphologically, cMSCs were similar to previously described MSCs and were capable of chondrocyte differentiation towards articular type cartilage, characterised by increased collagen type II vs. collagen type I expression and expression of Sox-9. cMSCs demonstrated no significant alterations in marker profiles and failed to differentiate into cardiomyocytes in response to a cardiac differentiation protocol or when co-cultured with canine cardiac stem cells. The study indicated that cMSCs can be derived readily from bone marrow and are capable of differentiation into articular cartilage, but appear to have limited ability to differentiate into cardiomyocytes using current protocols.

Characterisation and cardiac directed differentiation of canine adult cardiac stem cells.

This study describes the isolation and characterisation of adult canine cardiac stem cells, and explores their ability to differentiate into cardiac myocytes. Direct comparisons are also made with available human data. Atrial cardiac explants were taken from dogs post-mortem and cultured to isolate adult stem cells. Cells were able to survive successive passages in serum-free media, were able to form cardiospheres, and under controlled culture conditions were capable of clonal expansion, demonstrating their ability for self-renewal. Characterisation of these cells demonstrated the following marker profile: c-kit, GATA 4 and flk-1 positive; cardiac troponin T and NKx2.5 low. Cardiac lineage directed differentiation was performed based on the published literature. Gene expression studies demonstrated that cardiac directed differentiation was partially achieved, with up-regulation of cardiac troponin T and NKx2.5, and down-regulation of c-kit and endothelial lineage markers. However the cells did not express the ryanodine receptor or ß(1)-adrenergic receptors and did not contract spontaneously.

Cardiac specific gene expression changes in long term culture of murine mesenchymal stem cells.

Murine MSCs are a readily available source of adult stem cells enabling extensive in vitro study of this cell population. MSCs have been described as multipotent, and have been proven capable of differentiation into several connective tissue types. Furthermore some studies have suggested an ability to differentiate into non-connective tissue cell types such as the cardiomyocyte. The aim of this study was to differentiate murine MSCs toward cardiac lineage with the commonly used method of culture with 5' Azacytidine. Critically, baseline analysis of gene expression of passage four MSCs demonstrated expression of key cardiac markers including cardiac troponin T and I, and the ryanodine receptor. Furthermore, expression analysis of these genes changed with time in culture and passage number. However, there was no significant alteration when cells were subjected to a differentiation protocol. This study therefore highlights the importance of analyzing baseline cells extensively, and indicates the limitations in extrapolating data for comparison between species. Furthermore this data brings into question the efficacy of cardiac differentiation using MSCs.

Structural and cellular changes in canine myxomatous mitral valve disease: an image analysis study.

BACKGROUND AND AIM OF THE STUDY: Myxomatous mitral valve disease (MMVD) is the single most common cardiac disease of the dog, and bears close similarities to chronic degenerative mitral valve disease in humans. However, limited quantitative data are available on cellular and morphological changes in both species. The study aim was to use an image analysis system to examine various morphological changes associated with MMVD, and in particular to measure changes in cell numbers in overtly myxomatous areas of the distal portion of the valve. METHODS: Mitral valve complexes were collected from normal dogs and dogs with varying severity of myxomatous mitral valve disease (veterinary Whitney grades 1-4; a measure of disease severity and age-related disease progression in the dog). An image analysis technique (ImageJ; National Institutes of Health, USA) was used to measure valve leaflet length, thickness, connective tissue content and density, glycosaminoglycan (GAG) content, cell number and shape in normal and myxomatous areas of diseased valves. RESULTS: There was a change in the valve leaflet anterior/posterior length ratio in the diseased valves, suggestive of valve lengthening. Distinct and statistically significant (p < 0.01) changes occurred in the valve thickness ratio for both anterior and posterior leaflets as the disease progressed, and the posterior leaflet thickness ratios were consistently higher than for the anterior leaflets. There was a statistically significant decrease in cell numbers in overtly myxomatous areas of the distal portion of affected valves compared to similar locations in normal valves, but there was no difference between the different grades of disease. The majority of cells in both diseased and normal valves had a circularity score typical of a spindle (elongated) shape. Connective tissue derangement was clearly seen in the myxomatous areas, and this was associated with a significant reduction in connective tissue density. The reduction in connective tissue density was associated with advancing disease severity (age). There was an increase in GAG expression with disease severity, as shown by the level of Alcian blue staining, but this could not be quantified with ImageJ. CONCLUSION: Mitral valve myxomatous degeneration in the dog is associated with lengthening and thickening of valve leaflets, a loss of connective tissue, and a decrease in cell numbers in selected myxomatous areas, but no change in cell circularity. Some of these changes were age- (disease severity-) related.

Evaluation of innervation of the mitral valves and the effects of myxomatous degeneration in dogs.

OBJECTIVE: To map aspects of the innervation of the mitral valve complex and determine any association with the development or progression of myxomatous mitral valve disease (MMVD) in dogs. SAMPLE POPULATION: Septal mitral valve leaflets from 11 dogs aged 6 months to > 10 years. PROCEDURES: Expression of protein gene product 9.5 (general neuronal marker), tyrosine hydroxylase (adrenergic innervation marker), vasoactive intestinal peptide (parasympathetic innervation marker), and calcitonin gene-related peptide (sensory innervation marker) was assessed by use of a standard immunohistochemical technique. Innervation was assessed qualitatively and semiquantitatively. Differences between valvular zones and between groups were analyzed statistically. RESULTS: MMVD was present in leaflets of all dogs > or = 5 years of age. Innervation was confirmed in all leaflets but was markedly reduced in leaflets of dogs > 10 years of age. Innervation was most dense at the base of valves and mainly associated with the epimysial, perimysial, and endomysial layers of the muscle and blood vessels within the valve. Innervation was reduced within the middle zone of the valve and lacking at the free edge. Innervation was not identified at the tip of the leaflet, the free edge, or the chordae. Nerve fibers were mostly sympathetic, with the remainder being parasympathetic or sensory. Existence of MMVD did not alter the pattern or density of innervation. CONCLUSIONS AND CLINICAL RELEVANCE: Mitral valve leaflets in the study dogs were innervated, with most of the nerve fibers associated with the myocardium in the valve base. Development of MMVD appeared to precede the reduction of innervation associated with advancing age.

Molecular changes in fibrillar collagen in myxomatous mitral valve disease.

INTRODUCTION: Myxomatous mitral valve disease (MMVD) is the single most common acquired cardiac disease of dogs and is a disease of significant veterinary importance. It also bears close similarities to mitral valve prolapse in humans and therefore is a disease of emerging comparative interest. We have previously mapped the structure of collagen fibrils in valve leaflets using synchrotron X-rays and have demonstrated changes in collagen structure associated with the regions of disease. METHODS: Differential scanning calorimetry (DSC),biochemical assay of collagen content, high-performance liquid chromatography (HPLC), and neutron diffraction were combined with further analysis of our previous X-ray data to elucidate molecular changes in fibrillar collagen in mild to moderately affected MMVD dogs. RESULTS: Comparing diseases and adjacent grossly uninvolved areas in the same leaflets, there was a 20% reduction in collagen fibrils, but only a 10% depletion of collagen content. The enthalpy of collagen denaturation was reduced in affected areas. Chromatography showed a 25% decrease in mature nonreducible covalent cross-links in the affected samples, and neutron diffraction data showed fewer reducible immature covalent cross-links in grossly uninvolved tissue samples. CONCLUSIONS: Mild to moderate MMVD in the dog is associated with a marginal decline in collagen content in overtly diseased areas of valves, but more importantly is associated with an increase in immature collagen content. These changes will contribute to the mechanical dysfunction of the leaflet, and this study provides important information on the structure-mechanical alterations associated with this disease. The data suggests MMVD involves a dyscollagenesis process in the development of valve pathology.

Pulsed-wave Doppler tissue imaging velocities in normal geriatric cats and geriatric cats with primary or systemic diseases linked to specific cardiomyopathies in humans, and the influence of age and heart rate upon these velocities.

Pulsed-wave Doppler tissue imaging (pw-DTI) techniques allow the non-invasive assessment of myocardial dynamics. pw-DTI has demonstrated regional and global diastolic impairment in various forms of human and feline cardiomyopathy. We hypothesise that in geriatric cats with systemic diseases that have been linked to specific cardiomyopathies in human beings, the myocardial velocity profile will be altered when compared to either normal or hypertrophic cardiomyopathy (HCM) cats; and that both age and heart rate have a significant affect upon pw-DTI velocities. The aims of this study were to determine whether the feline M-mode or myocardial velocity profile is altered in geriatric cats with disease states that have been linked to specific cardiomyopathies in humans when compared to normal geriatric cats or geriatric cats with HCM and to determine whether age or heart rate has a significant effect upon pw-DTI velocities within these groups of cats. Sixty-six cats aged 8 years or above were included in the study, and were divided as follows: Unaffected (n=8), basilar septal bulge (BSB) (17), HCM (14), hyperthyroid (HiT(4)) (12) and chronic renal failure (CRF) (15). Systolic blood pressure was normal in all the cats. pw-DTI systolic (S'), early (E') and late diastolic (A') velocities were assessed from standardised sites within the myocardium, and the relationships between these and disease group, age and heart rate were then assessed. In cats with HCM, the E' velocity was decreased at various sites. Conversely, the HiT(4) cats demonstrated increased S' velocities. The only site at which the age of the cat was significantly related to myocardial velocities was the S' velocity from the apical mid-septum. There were also significant positive relationships between heart rate and the magnitude of myocardial S', E' and A' velocities of radial motion and S' and A' velocities of longitudinal motion. pw-DTI detected diastolic dysfunction in untreated cats with HCM and increased systolic function in HiT(4) cats. The age of the cat was of little significance, whereas heart rate significantly influenced myocardial velocity profiles.