Pathology, protein expression and signaling in myxomatous mitral valve degeneration: comparison of dogs and humans.

Myxomatous degenerative mitral valve disease (MMVD) is a common heart disease in dogs. Although several morphological similarities occur between human and canine MMVD differences exist. However, in advanced stages the accumulation of proteoglycans is the main finding in both species. The extracellular matrix (ECM) in normal canine and human mitral valves is similar. In MMVD of both species proteoglycans is the major alteration, although specific changes in collagen distribution exists. The valvular expression pattern of matrix metalloproteinases (MMPs) and of their inhibitors (TIMPs) differs, in part, between dogs and humans. The MMPs and TIMPs expression patterns are similar in normal canine and human mitral valves, but they are quite different during degenerative progression. Valve endothelial cells (VEC) and interstitial cells (VIC) are phenotypically transformed in canine and human MMVD. Inflammation is an unlikely cause of valve degeneration in humans and dogs. There are several lines of evidence suggesting that transforming growth factor ß1 (TGF ß1) and serotonin signaling may mediate valve degeneration in humans and dogs. Although human and canine MMVD share structural similarities, there are some differences in ECM changes, enzyme expression and cell transformation, which may reflect a varied pathogenesis of these diseases.

Faecal excretion of intestinal spirochaetes by urban dogs, and their pathogenicity in a chick model of intestinal spirochaetosis.

This study aimed to obtain information about the types of spirochaetes colonising urban dogs in Thailand, and to investigate their pathogenic potential in a day-old chick model of intestinal spirochaetosis. Spirochaetes were isolated from the faeces of six of 47 (12.8%) healthy dogs and 11 of 104 (10.6%) dogs with diarrhoea. Their biochemical properties and 16S ribosomal DNA sequences were analysed. Four isolates were identified as Brachyspira pilosicoli, three resembled "Brachyspira pulli", nine clustered with "Brachyspira canis" and one was similar to Brachyspira intermedia. Canine isolates of B. pilosicoli, "B. canis" and "B. pulli", and control strains of Brachyspira hyodysenteriae, B. pilosicoli and Brachyspira innocens colonised experimentally infected day-old chicks. The chicks did not develop diarrhoea, but were significantly lighter than the non-infected group and those infected with B. innocens after 21 days (P<0.05). Using immunohistochemistry, spirochaetes were observed covering the surface epithelium and in the crypts of chicks in all three groups challenged with the canine isolates. Variable histopathological changes were seen, with the greatest inflammatory cell infiltration into the lamina propria occurring in the group infected with "B. pulli". Canine "B. canis", "B. pulli" and B. pilosicoli isolates may have pathogenic potential.

Serotonin transmembrane transporter is down-regulated in late-stage canine degenerative mitral valve disease.

OBJECTIVE: To compare expression of the serotonin transmembrane transporter (SERT) in normal, early-stage degenerative, and late-stage degenerative canine mitral valve disease. ANIMALS: 24 post-mortem canine mitral valves. METHODS: SERT expression was determined in canine normal (n = 8), early-stage degenerative (n = 8), and late-stage degenerative (n = 8) mitral valves by immunohistochemistry (IHC) and immunoblot (IB) analyses. RESULTS: SERT was expressed in valve interstitial cells of all layers of normal and early-stage degenerative mitral valves based on IHC. SERT was markedly down-regulated in valve interstitial cells, but not valve endothelial cells, of late-stage degenerative mitral valves. SERT expression was significantly decreased in late-stage compared to normal and early-stage degenerative mitral valves based on IB analysis (P < 0.05). CONCLUSIONS: Down-regulation of SERT expression occurs in valve interstitial cells of late-stage, but not early-stage, canine degenerative mitral valves. Down-regulation of SERT could enhance the recently speculated role of serotonin in canine DMVD by decreasing serotonin metabolism and increasing interaction with its receptor. Down-regulation of SERT likely does not play an initiating role in canine DMVD since it does not occur in early-stage disease.

Tryptophan hydroxylase 1 expression is increased in phenotype-altered canine and human degenerative myxomatous mitral valves.

BACKGROUND AND AIM OF THE STUDY: Serotonin is a known mediator of myxomatous pathology in heart valves. Tryptophan hydroxylase 1 (TPH1) is the limiting enzyme for peripheral serotonin synthesis, and its expression by valve interstitial cells (IC) could implicate an autocrine serotonin signaling mechanism in primary degenerative myxomatous mitral valve disease. Thus, the expression of TPH1 in canine and human myxomatous mitral valves was determined, and IC phenotypes expressing TPH1 identified. METHODS: TPH1 expression was determined in canine and human myxomatous and normal mitral valves by immunoblot (IB) and immunofluorescence microscopy (IFM). Co-localization of TPH1 expression with markers of IC phenotype transformation, alpha-smooth muscle actin (a-SMA) and non-muscle embryonic myosin (SMemb) was determined using double-IFM. RESULTS: TPH1 expression by IB was increased (p < 0.05) by three- to five-fold in canine early-stage and late-stage myxomatous valves, and in human surgically excised myxomatous valves compared to canine and human normal control valves, respectively. The number of TPH1 immunopositive cells per x400 field was increased (p < 0.005) in canine (14.9 +/- 1.2) and human (14.9 +/- 2.9) myxomatous valves compared to canine (5.0 +/- 2.4) and human (2.9 +/- 0.6) normal control valves, respectively. Patterns for alpha-SMA and SMemb IC phenotype transformation were distinctly different in myxomatous valves. TPH1 expression was more closely associated with the SMemb IC phenotype in canine and human myxomatous valves. CONCLUSION: An increased expression of TPH1 in canine and human myxomatous mitral valves implicates an autocrine serotonin signaling mechanism in primary degenerative myxomatous mitral valves. TPH1 expression is associated with the SMemb-positive IC phenotype.

Autocrine serotonin and transforming growth factor beta 1 signaling mediates spontaneous myxomatous mitral valve disease.

BACKGROUND AND AIM OF THE STUDY: Although serotonin and serotoninergic drugs are known to cause myxomatous-like valvulopathy, the role of serotonin in spontaneous myxomatous valve disease (MVD) remains unclear. Tryptophan hydroxylase 1 (TPH1) is the limiting enzyme for peripheral serotonin synthesis, and its expression in myxomatous valves could implicate an autocrine serotonin signaling mechanism. Studies in cultured cells demonstrate a close coupling between serotonin and transforming growth factor beta1 (TGFbeta1) signaling. The study aim was to investigate serotonin and TGFbeta1 signaling in spontaneous MVD. METHODS: In canine normal and myxomatous mitral valves, target signaling proteins including TPH1, serotonin 2B receptor (5HT(2B)R), serotonin transmembrane transporter (SERT), total and phosphorylated extracellular signaling-regulated kinase (ERK) 1/2, latent TGFbeta1 and TGFbeta1 receptors I and II, were studied using immunohistochemistry and immunoblot analysis. In human myxomatous valves, TPH1 was determined using immunofluorescence and immunoblot analysis. RESULTS: In canine mitral valves, both 5HT(2B)R and TPH1 were increased in myxomatous valves, whereas SERT, a key protein in serotonin metabolism, was decreased in myxomatous valves. Phosphorylated, but not total, ERK 1/2 was increased in myxomatous valves, consistent with an enhanced active serotonin signaling. The expression of TGFbeta1 receptors I and II, and of latent TGFbeta1, was increased in myxomatous valves. Human myxomatous mitral valves expressed TPH1. CONCLUSION: The expression of TPH1 by canine and human myxomatous valves demonstrates a capacity for local serotonin production. Key signaling protein expression patterns support active serotonin and TGFbeta1 signaling in canine myxomatous valves. These findings implicate an autocrine serotonin and TGFbeta1 mechanism in the pathogenesis of spontaneous MVD.

Differential protein expression between normal, early-stage, and late-stage myxomatous mitral valves from dogs.

Valvular heart disease accounts for over 20 000 deaths and 90 000 hospitalizations yearly in the United States. Myxomatous valve disease (MVD) is the most common disease of the mitral valve in humans and dogs. MVD is pathologically identical in these species and its pathogenesis is poorly understood. The objectives of this study were to (i) develop proteomic methodology suitable for analysis of extracellular matrix-rich heart valve tissues and (ii) survey over- and under-expressed proteins that could provide mechanistic clues into the pathogenesis of MVD. Normal, early-stage, and late-stage myxomatous mitral valves from dogs were studied. A shotgun proteomic analysis was used to quantify differential protein expression. Proteins were classified by function and clustered according to differential expression patterns. More than 300 proteins, with 117 of those being differentially expressed, were identified. Hierarchical sample clustering of differential protein profiles showed that early- and late-stage valves were closely related. This finding suggests that proteome changes occur in early degeneration stages and these persist in late stages, characterizing a diseased proteome that is distinct from normal. Shotgun proteome analysis of matrix-rich canine heart valves is feasible, and should be applicable to human heart valves. This study provides a basis for future investigations into the pathogenesis of MVD.

Interstitial cells from dogs with naturally occurring myxomatous mitral valve disease undergo phenotype transformation.

BACKGROUND AND AIM OF THE STUDY: Myxomatous mitral valve disease is a common naturally occurring heart disease of dogs that is pathologically similar to myxomatous mitral valve disease in humans. It was hypothesized that interstitial cell phenotype transformation recently described in human myxomatous valves might also occur in dogs with myxomatous mitral valves, and correlate with disease severity. METHODS: Normal and early-, intermediate- and late-stage myxomatous canine mitral valves were examined histologically and immunohistochemically for cytoskeletal (vimentin, desmin, smooth muscle alpha-actin, smooth muscle myosin, and non-muscle myosin), collagenolytic (MMP-1, MMP-13), cell surface (CD-31, CD-45, CD-68) and proliferation (Ki-67) proteins. RESULTS: Normal canine mitral valve interstitial cells were positive for vimentin, but negative for alpha-actin, desmin and non-muscle myosin (i.e., fibroblast phenotype). Interstitial cells from myxomatous valves showed progressive positive staining for alpha-actin and desmin, but were negative for smooth muscle myosin (i.e., myofibroblast phenotype). Positive-staining cells first appeared as cellular clusters in the subendocardial region of the lamina atrialis and extended into deeper layers with increasing severity. Interstitial cells from myxomatous valves showed positive staining for non-muscle myosin (i.e., activated mesenchymal cell phenotype). Positive-staining cells increased with disease severity and were dispersed throughout the valve layers. The expression of MMP-1 and MMP-13 increased in myxomatous mitral valves and correlated with disease severity. Interstitial cellularity increased dramatically in degenerative mitral valves, though Ki-67 staining was only mildly increased. CONCLUSION: Two patterns of interstitial cell phenotype transformation were identified in dogs with myxomatous mitral valve disease, and both correlated with disease severity. Myofibroblast transformation characterized by positive staining for alpha-actin and desmin occurred in cellular clusters primarily in the lamina atrialis. Mesenchymal cell activation characterized by positive staining to non-muscle myosin occurred throughout the valve. The dog may be a natural model for studying the cell biology of progressive myxomatous valve disease.

The effects of age and heart rate on tricuspid annular motion velocities in healthy nonsedated cats.

BACKGROUND: Age and heart rate have effects on myocardial velocities as assessed by color tissue Doppler imaging (TDI) in people. A similar phenomenon has been identified when left ventricular velocities are assessed in cats. To date, the effects of age and heart rate on tricuspid annular velocities of cats have not been assessed. OBJECTIVES: This study was designed to determine the relationships between age and heart rate and tricuspid annular velocities in cats as assessed by 2-dimensional (2D) color TDI. ANIMALS: Fifty healthy nonsedated cats with age range from 3 months to 19 years old were studied. METHODS: Tricuspid annular velocities were obtained with 2D color TDI. Effects of age and heart rate on tricuspid annular velocities were evaluated by simple linear regression. The strength of the linear relationship was determined by using coefficient of determination (R2). RESULTS: A significant weak negative relationship was found between age and peak early diastolic annular velocity (E'; R2 = 0.135, P = .018). No significant relationships between age and right ventricular (RV) systolic TDI values were found. Diastolic and systolic TDI parameters were not affected by heart rate with the exception of deceleration rate of early diastolic motion (DR; R2 = 0.100, P = .025). CONCLUSIONS AND CLINICAL IMPORTANCE: Age and heart rate have minimal effects on tricuspid annular velocities. The present study provides reference ranges for tricuspid annular velocities in healthy cats and information for assessing the clinical utility of color TDI for evaluation of RV function in cats.

Immunohistochemical staining of IFN-gamma positive cells in porcine reproductive and respiratory syndrome virus-infected lungs.

Paraffin-embedded lungs were obtained from a previous porcine reproductive and respiratory syndrome virus (PRRSV)-challenged experiment involving three groups: an uninfected control group, a low virulence (LV, Resp PRRSV/Repro)-infected group, and a high virulence (HV, VR-2385)-infected group. Tissues were collected at 3, 7, 10, 14 or 28 days post-inoculation (DPI) (n=5). Lungs were examined to detect IFN-gamma positive cells by immunohistochemical staining using polyclonal antibodies to IFN-gamma. The microscopic lung lesions induced by the HV group were more severe than those in the LV group. A significant increase in number of lymphocytes in the HV group was observed at 10 DPI (24.90+/-9.79%), 14 DPI (22.00+/-11.47%) and 28 DPI (28.95+/-15.11%) (P<0.05). A relative decrease in macrophage numbers was observed and correlated well with the increase in lymphocyte numbers when the disease progressed. IFN-gamma positive cells were demonstrated in both lymphocytes and macrophages, particularly pulmonary alveolar macrophages. A significant increase in IFN-gamma positive cells was found at 7 DPI (15.90+/-13.65%), 10 DPI (46.95+/-13.79%), 14 DPI (10.90+/-5.13%) and 28 DPI (13.40+/-4.89%) in the HV group (P<0.05). The results suggested that the increase in IFN-gamma positive cells in the HV group correlated well with the severity of the lung lesions, which may be because of the presence of PRRSV in the lung.