Journal of Veterinary Cardiology (2012)

-, -e-

www.elsevier.com/locate/jvc

Pathology, protein expression and signaling in myxomatous mitral valve degeneration: Comparison of dogs and humans Heike Aupperle, DVM, PhD habil

a,

*, Sirilak Disatian, DVM, MS, PhD

b

a

Institute of Veterinary-Pathology, University Leipzig; moved to: LABOKLIN GmbH; Co. KG, Steubenstr. 4, 97688 Bad Kissingen, Germany b Department of Veterinary Medicine, Faculty of Veterinary Science, Chulalongkorn University, Henry Dunant Rd. Pathumwan, Bangkok 10330, Thailand Received 12 May 2011; received in revised form 25 December 2011; accepted 2 January 2012

KEYWORDS Canine; Collagen; Human; Matrix metalloproteinase; Mitral valve disease; Serotonin; Valve interstitial cells

Abstract 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 b1 (TGF b1) 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. ª 2012 Elsevier B.V. All rights reserved.

* Corresponding author. E-mail address: [email protected] (H. Aupperle). 1760-2734/$ - see front matter ª 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jvc.2012.01.005

Please cite this article in press as: Aupperle H, Disatian S, Pathology, protein expression and signaling in myxomatous mitral valve degeneration: Comparison of dogs and humans, Journal of Veterinary Cardiology (2012), doi:10.1016/j.jvc.2012.01.005

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H. Aupperle, S. Disatian Abbreviations CKCS MMVD MMP MVP TGF TIMP VEC VIC

Cavalier King Charles Spaniel Myxomatous mitral valve disease Matrix metalloproteinase Mitral valve prolapse Transforming growth factor Tissue inhibitor of metalloproteinase Valve endothelial cell Valve interstitial cell

Introduction Myxomatous mitral valve disease (MMVD) is a common acquired cardiac disease in older small and medium-sized dogs.1,2 The mitral valve is comprised of two leaflets (anterior or septal and posterior or mural leaflets). Normal valve leaflets are grossly thin and transparent (Fig. 1A).1 The myxomatous valves are macroscopically characterized by nodular thickening of the valve leaflets (Fig. 1B).3 The chordae tendineae are elongated.4e6 Myxomatous mitral valve disease develops mainly in dogs older than seven years of age.1 Males are more susceptible to develop the disease than females.7 The etiology of canine MMVD is still unclear. Hemodynamic, endogenous and genetic factors have been discussed.1,7,8 In dogs, breeds predisposed to connective tissue disorders including intervertebral disc disease, tracheal collapse and cruciate ligament rupture are also affected with MMVD.9 Such breeds include Dachshund, Cocker spaniel, Poodle, Schnauzer, Chihuahua and Terriers.1 This disease is considered to be inherited in some breeds including Cavalier King Charles Spaniel (CKCS) and Dachshund.10e13 Myxomatous mitral valve disease in CKCS occurs at an early age and may be caused by a polygenic inheritance.12 Clinically, the degeneration of the valves and corresponding chordae tendineae causes an inappropriate coaptation of the leaflets leading to valve regurgitation and finally resulting in left sided congestive heart failure.1,2,14e17 In man, a morphologically similar disease (Fig. 1C) has been described.18 Human MMVD causes prolapse or billowing of mitral valve leaflets into the left atrium during systole. The prevalence of MMVD in the human population is associated with age.19 Mitral valve prolapse, a risk factor for development of MMVD later in life, is more prevalent in females than males.20 Patients with mitral valve prolapse tend to have low body mass index, but the reason for this phenomenon is uncertain.21 The etiology of human MMVD is

Figure 1 Gross findings in mitral valves (A) Normal mitral valve from a 3-year-old dog. The leaflets are thin and regular and the chordae tendineae are uniform. (B) Marked chronic valve disease from 8-year-old dog: The leaflets of the mitral valve are thickened, white in colour and deformed with the free edges rolled upward. There is evidence of chordal rupture (C) Human surgically removed diseased valve from 59-year-old male. The valve is thickened with irregular surface and hemorrhage.

unknown. The risk factor of mitral valve prolapse may be inherited as an autosomal dominant or a polygenic abnormality.22,23 It often displays familial transmission.24 Connective tissue disorders

Please cite this article in press as: Aupperle H, Disatian S, Pathology, protein expression and signaling in myxomatous mitral valve degeneration: Comparison of dogs and humans, Journal of Veterinary Cardiology (2012), doi:10.1016/j.jvc.2012.01.005

Mitral valve disease protein expression and signaling including Ehlers-Danlos-Syndrome, Osteogenesis imperfecta, Marfan syndrome and SticklerSyndrome are strongly associated with MVD in humans.24,25 Normal canine and human mitral valves have three well-defined layers (Fig. 2A): (1) The atrialis layer is mainly composed of elastic fibers. (2) The spongiosa layer is rich in glycosaminoglycans. (3) The fibrosa layer predominantly consists of densely-packed collagen bundles.3,24,26 The pathology of myxomatous valves in humans and in dogs includes increased cellularity, valve structure disorganization as well as transdifferentiation of valve endothelial (VEC) and valve interstitial cells (VIC).1,5,15,27e30 In canine degenerative valves, the changes occur primarily in the atrialis layer by an accumulation of transformed VIC.30e33 In advanced stages the major structural change, glycosaminoglycan infiltration, appears in the spongiosa layer in both human and canine MMVD (Fig. 2B and C).30e32 The degeneration is most pronounced at the free margins of the leaflets. When disease progresses, the amount of

3 glycosaminoglycans increases and invades into the other layers.33 The elastic fibers become fragmented and split. The collagen bundles are disorientated by glycosaminoglycan infiltration. The collagen fibrils also appear disrupted. Similar changes occur in the chordae tendineae.34,35 In humans, the posterior leaflet is more often and more severely affected than the anterior leaflet.36,37 In contrast, the anterior leaflet is more frequently involved in canine MMVD (Table 1).38 In human myxomatous valves, linear thrombus may be present at the base of the leaflet next to the left atrial wall.25 In dogs, platelets adhering to the subendothelial collagen have been detected in endothelial lesions, but marked thrombus formation has not been reported.14,28 It remains to be determined whether abnormalities of platelet function and morphology11,39 or abnormalities in coagulation factors such as plasminogen, are responsible for this finding.28 The occurrence of stroke secondary to emboli has been reported in humans affected with MMVD. However, some studies suggested that this phenomenon might be

Figure 2 Histomorphology of normal and degenerated mitral valves (A) (magnification 4x) Normal mitral valves show well differentiated layer organization with atrialis (A), spongiosa (S) and fibrosa (F). Normal mitral valve from a 3 years old dog (HE stain). Canine chronic disease valve (B) (magnification 2x) and human diseased mitral valve (C) (magnification 2x) appear with nodular valve structure disorganization (HE stain).

Please cite this article in press as: Aupperle H, Disatian S, Pathology, protein expression and signaling in myxomatous mitral valve degeneration: Comparison of dogs and humans, Journal of Veterinary Cardiology (2012), doi:10.1016/j.jvc.2012.01.005

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H. Aupperle, S. Disatian Table 1

Comparison of characteristics in canine and human valvular degeneration. Dog

Age Predilection Pathology

Man

Older dogs1,40 - Small to medium-sized breeds2 - Beginning in the endothelium28 and in the atrialis layer 29,31,33 - Accumulation of glycosaminoglycans in the spongiosa layer and fragmentation of collagen bundles and elastic fibers30,31 - Anterior leaflet is more affected by degeneration.38

a coincidence. The occurrence of thromboembolism has not been reported in canine MMVD.

Extracellular matrix In heart valves, the extracellular matrix (ECM) is produced by endothelial and valve interstitial cells.45,46 ECM is a complex dynamic physical and chemical network consisting of several components including collagen, elastin, fibronectin, laminin, proteoglycans and glucosamines.47 ECM is not a static structure, but is a dynamic network constantly being remodelled. The balance between synthesis and degradation maintains a normal structure and homestasis of the valves.48,49 About 14 types of collagen have been described.50 In heart valves of several species, collagen types I, III, IV, V and VI have been identified.31,46 Collagen I and III are mainly produced by fibroblasts. Collagen IV and VI are synthesized by endothelium, fibroblasts and cardiomyocytes51,52 and are part of basement membranes.53e55 Collagen VI is secreted as a tetramer and aggregates extracellularly.56 It is very resistant against proteolytic digestion and is not detectable by most analytical methods.54,57 Aupperle et al.31 described collagen VI for the first time in normal adult canine heart valves and valvular degeneration. Comparable data in humans is not available. Biochemical analyses of collagen VI in canine mitral valves quote 472 ! 49 mg/g collagen content as normal.8 More detailed biochemical analyses of human mitral valves are available: water content in normal mitral valves is 83%. The collagen, elastin and proteoglycan contents of normal human valves are approximately 60%, 10% and 30% of the dry weight, respectively.33,45,54 Elastin and fibronectin are produced from smooth muscle cells, fibroblasts and endothelium.58e60 Laminin is part of the basement membrane of endothelium and cardiomyocytes.61,62 The primary

Age association19 - Inherited connective tissue disorders24,25 - No study of structural changes associated with disease progression - Accumulation of glycosminaglycans in the spongiosa layer and fragmentation of collagen bundles and elastic fibers32,37,41,42 - Posterior leaflet is more prone to the degeneration and to ruptured chordae tendineae.43,44

role of laminin is the mediation of interactions between cells and the ECM, but laminin can also bind to glycosaminoglycans.62 Proteoglycans (also known as mucopolysaccharides) are the main component of the ECM63 and are involved in degenerating processes of elastic fibers.33 In general, the specific distribution pattern of ECM components in the mitral valve reflects the mechanical forces during systole and diastole: elasticity of the atrialis layer, stability of the fibrosa layer and flexibility of the spongiosa layer.64 Immunohistochemical analyses of the normal canine mitral valve showed that the subendothelial basement membrane consists of moderate amounts of laminin and fibronectin, and small amounts of collagen I, III, IV, VI and heparan sulphate. The atrialis layer is mainly composed of elastic fibers, collagen I, III and VI. The spongiosa layer contains proteoglycans and collagen VI and the fibrosa layer consists of collagen I, III, and collagen VI (Fig. 3A).31 These findings are in accordance to published data in normal human mitral valves.46,65 In human MMVD, the amount of collagen I is mildly decreased and collagen III increased (normal: 74% collagen I, 24% collagen III; MMVD: 67% collagen I, 31% collagen III).45 The study from Hadian et al.66 showed a 10% reduction in total collagen and a 20% reduction in fibrillar collagen content in the myxomatous areas of canine valves with mild to moderate MMVD. Unstable, immature and abnormally crosslinked collagen molecules were identified.66,67 Furthermore, an accumulation of proteoglycans, lipids and hexosamines was detected in canine MMVD.8,68,69 Immunohistochemical studies in mild canine MMVD showed an irregularly split basement membrane. The ECM components, collagen VI and laminin were mildly increased and spread into the atrialis layer. Collagen I and III were no longer arranged in their typical layers, but formed a loose

Please cite this article in press as: Aupperle H, Disatian S, Pathology, protein expression and signaling in myxomatous mitral valve degeneration: Comparison of dogs and humans, Journal of Veterinary Cardiology (2012), doi:10.1016/j.jvc.2012.01.005

Mitral valve disease protein expression and signaling

5 markedly increased (Fig. 3B). Laminin was not only associated with the basement membranes, but also mildly deposited multifocally on the periphery of the myxomatous nodules.31 To the best of the authors’ knowledge, comparable investigations in human MMVD have not been published. However, various congenital connective tissue disorders (Ehlers-Danlos-Syndrome, Osteogenesis imperfecta, Stickler-Syndrome, MarfanSyndrome) have been reported to correlate to MMVD in humans.24,25,70,71 In summary, the ECM in normal canine and human mitral valves is similar. In dogs the early lesions appear in the atrialis layer. In man there are no studies of early alterations. However, in advanced stages, the accumulation of proteoglycans is the main finding in both species (Table 1).

MMPs/TIMPs

Figure 3 Collagen VI immunohistochemistry (A) (magnification 2x) Normal heart valve from 1-year-old Schnauzer: Collagen VI formed a thin subendothelial layer. Expression was mild in atrialis (A) and spongiosa (S), but marked in fibrosa (F). (B) (magnification 1x) Severe chronic valve disease from a 9-year-old Chihuahua: Collagen VI was moderately positive in the subendothelial layer. Furthermore it appeared moderately in the periphery and mildly in the centre of the myxomatous nodules.

and fine fibrillary network in the atrialis and spongiosa layer, resulting in a less intense staining reaction.31 It may be speculated that the atrial deposition of laminin, fibronectin and collagen IV, act as an intensified diffusion barrier, presumably causing malnutrition of underlying tissues. In advanced canine MMVD, nodular lesions were characterized by peripheral fibronectin accumulations.31 The intense immunohistochemical expression of fibronectin is in accordance to microarray gene expression analyses performed by Oyama and Chittur,16 who reported a 3.7 fold increase of fibronectin expression in canine MMVD in comparison to healthy controls. Collagen I was diffusely present throughout the nodular lesions, while collagen III was moderately positive mainly in the center of the nodules. Collagen VI was

The ECM is not static, as it is constantly being remodelled and in equilibrium by the cellular synthesis of ECM components and their breakdown by different enzymes.48 Matrix metalloproteinases (MMPs) appear to play an important role in physiological processes and in the pathogenesis of various diseases affecting the ECM.72,73 MMPs are divided into six groups: (1) collagenase (MMP-1, MMP-8, MMP-13, MMP-18), (2) gelatinase A and B (MMP-2, MMP-9), (3) stromelysin 1 and 2 (MMP-3, MMP-10), (4) stromelysin 3 (MMP-11), (5) metalloelastase (MMP-12), (6) membrane-bound MMPs (MMP-14, MMP-15, MMP-16, MMP-18).72e74 In cardiovascular tissues, predominantly MMP-1, -2, -3, -9, -13 and "14 play a role.55,75,76 MMPs are Ca2þ and Zn2þ dependent proteases that are produced and secreted as inactive zymogens pro-MMPs. Several mediators may induce transcription, translation, secretion or activation of MMPs, for example, angiotensin II, endothelin-1, catecholamines, norepinephrine, TNF-a and interleukine-1b. Oxidative stress77 or mechanical stress66 may also alter MMPs and tissue inhibitor of metalloproteinase (TIMPs) expression. Serine proteases as trypsin, plasmin and urokinase may also activate MMPs.72,73,77 MMPs are inhibited by specific tissue inhibitors TIMPs72e74 Four types of TIMPs, TIMP-1 to TIMP-4, have been described which are all expressed in the myocardium.78 They are mostly expressed by the same cells which also produce the MMPs.79 The balance of MMPs and TIMPs appears to be vital for the regulation and homeostasis of the ECM. The expression of MMPs and TIMPs has been investigated in normal human embryonal, adult,80

Please cite this article in press as: Aupperle H, Disatian S, Pathology, protein expression and signaling in myxomatous mitral valve degeneration: Comparison of dogs and humans, Journal of Veterinary Cardiology (2012), doi:10.1016/j.jvc.2012.01.005

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H. Aupperle, S. Disatian

degenerative canine mitral valves30,81e83 as well as in normal48 and altered human heart valves.32,79,84 Immunohistochemical studies in normal canine mitral valves showed: MMP-1, MMP-2, MMP-14 (Fig. 4A) and TIMP-2 were mildly expressed in individual interstitial cells and TIMP-3 (Fig. 5A) expression was intense in numerous interstitial cells in all layers.31,83 In contrast, MMP-2, was not seen in normal valves in the study from Obayashi et al. (2011).83 MMP-9 was not identified in VIC, but was extensively expressed in cardiomyocytes.31,83 However, mRNA of MMP-9 was detectable in normal canine mitral valves.16,82 The normal findings in dogs partially contrast to the immunohistochemical studies in normal mitral valves in humans (Table 2), which showed an intense expression of MMP-1, TIMP-1, and TIMP-2, but no or little expression of MMP-2, MMP-3,

MMP-9 and TIMP-3 in VIC.48 MMP-14 expression was not detected in normal or diseased human mitral valves.84 With increasing severity of canine MMVD (Table 3) the immunohistochemical expression of MMP-2, and MMP-9 decreased, but MMP-14 (Fig. 4B), TIMP-2 and TIMP-3 (Fig. 5B) expression increased markedly.81 In contrast, Obayachi et al83 found increasing expression of MMP-1 and MMP-3 but negative labeling for MMP-2 and MMP-9 in MMVD. There was a correlation between the elevation in mRNA encoding MMP-14, TIMP-2 and -3 in MMVD, and the expression of these proteins within similarly affected mitral valves.82 The expression of MMP-1 and MMP-13 progressively increased with advancing disease severity.30 Furthermore, the concentration of mRNA encoding MMP-14 and TIMP-3 was significantly elevated, and there was additional transcription of genes encoding MMP-1, TIMP-2 and -4 which were not transcribed in normal mitral valves.82

Figure 4 MMP-14 immunohistochemistry: (A) (magnification 6x) Normal canine mitral valve from a 2-year-old Labrador: Expression of MMP-14 in endothelial cells and in individual interstitial cells of the atrialis (A) and spongiosa (S) but not in fibrosa layer (F). (B) (magnification 6x) 10-year-old German Shepherd: Expression of MMP-14 in numerous interstitial cells in myxomatous nodules.

Figure 5 TIMP-3 immunohistochemistry: (A) (magnification 4x) Normal canine mitral valve from a 2-year-old Labrador: Expression of TIMP-3 in endothelial cells and in numerous interstitial cells within the atrialis (A) and spongiosa (S) layers but not within the fibrosa (F). (B) (magnification 20x) 11-year-old Staffordshire terrier: Expression of TIMP-3 in numerous interstitial cells and in the extracellular space of myxomatous nodules.

Please cite this article in press as: Aupperle H, Disatian S, Pathology, protein expression and signaling in myxomatous mitral valve degeneration: Comparison of dogs and humans, Journal of Veterinary Cardiology (2012), doi:10.1016/j.jvc.2012.01.005

Mitral valve disease protein expression and signaling Table 2

7

Expression patterns of MMPs and TIMPs in normal mitral valves in dogs and humans. Normal MV

Aupperle et al. 200981 Aupperle et al. 200982 Disiatan et al. 200830 Obayashi et al. 201183 Oyama & Chittur 200616 Rabkin et al. 200132 Dreger et al. 200248 Togashi et al. 200785

Dog IHC Dog PCR (mRNA) Dog IHC Dog IHC Dog MR (mRNA) Human IHC Human IHC Human IHC

MMP "1

MMP "2

e

þ

e

þ

MMP "3

MMP "9 e

þ

þ e

MMP "13

MMP "14

(þ)

TIMP "1

TIMP "2

TIMP "3

TIMP "4

e

þ

e

þþ

þþ

þ

þ

þ

þ

þ

þþ

e

þ e

e

e

þ

þ

þ

þ

þ

þ

þ

þ

e

e

þ

þ

þ

e

IHC: imunhistochemistry, PCR: polymerase-chain reaction, MMP: matrix metalloproteinases, mRNA: messenger ribonucleic acid, TIMP: tissue inhibitor of metalloproteinases, MR: microarray analyses, MV: mitral valve. e: negative; (þ):minimal expression, þ: mild expression, þþ: moderate expression.

The results of the studies in canine MMVD contrast with findings in myxomatous human mitral valves in which there is increased expression of genes encoding MMP-1, -2, -9 and -13 compared with normal valves.32,84,85 Furthermore, the elevated MMPs expression was identified immunohistochemically in the VIC.32,85 The expression of TIMPs was not investigated in these studies. Interestingly, a significant promoter polymorphism

Table 3

of the MMP-3 gene was identified correlating to the severity of MMVD in humans.86 Elevated expression of MMP-2, TIMP-1 and TIMP-2 was seen in endocarditis, and in some cases of human degenerative valve disease.32,84,87 In summary, the MMPs and TIMPs expression patterns are similar in normal canine and human mitral valves, but they are quite different during degenerative progression. In canine MMVD, the

Expression patterns of MMPs and TIMPs in degenerated mitral valves in dogs and humans. Species/ Method

Aupperle et al. 200981 Aupperle et al. 200982 Disiatan et al. 200830 Obayashi et al. 201183 Oyama & Chittur 200616 Rabkin et al. 200132 Togashi et al. 200785

Dog IHC Dog PCR Dog IHC Dog IHC Dog MR Human IHC Human IHC

MMP "1

MMP "2

[

±

Ø

Y

MMP "3

MMP "9 Ø

[

MMP "14 [

TIMP "1

[

±

[ [

MMP "13

TIMP "2

TIMP "3

TIMP "4

[

[

[

[

[

Ø

[ Ø

[

Ø

±

±

±

[

[

[

[

[

[

±

Y

IHC: immunohistochemistry, PCR: polymerase-chain reaction, MMP: matrix metalloproteinases, mRNA: messenger ribonucleic acid, TIMP: tissue inhibitor of metalloproteinases, MR: microarray analyses, MV: mitral valve. Compared to the normal mitral valve: ! not changed, Y diminished, [ elevated expression intensity, Ø neither detectable in normal nor in degenerated valves.

Please cite this article in press as: Aupperle H, Disatian S, Pathology, protein expression and signaling in myxomatous mitral valve degeneration: Comparison of dogs and humans, Journal of Veterinary Cardiology (2012), doi:10.1016/j.jvc.2012.01.005

8

H. Aupperle, S. Disatian

expression of TIMPs is the main finding. In contrast, elevated MMP expression is characteristic in human MMVD. These differing results in normal and myxomatous heart valves in dogs and humans probably reflect different pathogenic pathways of degenerative valve disease, although a progressive course of the disease is seen in both species.

Cells Two major cell types, VIC and VEC, exist within the valve. These cells have a major function to maintain valve homeostasis in normal conditions and participate in valve repair as well as remodeling in diseased valves. Both VIC and VEC have the ability to produce ECM, chemokines and catabolic enzymes such as MMPs and their tissue inhibitors (TIMPs). These cells have unique characteristics which are different from those described in interstitial and endothelial cells from other tissue types.88 Furthermore, it has been suggested that VIC isolated from aortic, pulmonary, mitral and tricuspid valves express some molecular components that are different from each other indicating that VIC in different types of valves may have a specific functional role.89

Valve endothelial cells The VEC layer comprises of a single cell lining on the surface of both leaflet sides. Vascular endothelial cells function as a barrier between the leaflets and the blood. Damaged or denuded areas of the endothelial layer have been identified in dogs and humans with MMVD.28,90,91 The damaged area was mostly seen in the distal part of the leaflets. The endothelial cells in affected valves were swollen, separated or had loose contact to each other.92 Calcium crystals were occasionally found in the ulcerated areas of the endothelial layer of affected valves in humans,91 but not in dogs. In canine myxomatous valve tissues, VEC appeared with prominent nuclear bulges and became activated by transdifferentiating into a-actin positive cells or myofibroblasts.29,30,33 The endothelial lining looked irregular and not smooth like in normal valves (Fig. 6A and B). An increased NADPH-diaphorase activity and subsequent augmented nitric oxide synthase expression has been found on endothelial cells in areas of myxomatous changes of canine mitral valves.15 A higher endothelin receptor presentation was also seen in endothelial layers of myxomatous valves in dogs.93

Figure 6 CD31 immunohistochemistry (A) (magnification 20x) The normal canine mitral valve is covered by a layer of flat inactive endothelial cells, which intensively express CD31, a marker of endothelial cells. (B) (magnification 20x) In degenerated canine valves the endothelial cells are activated and proliferated and irregularly arranged.

In humans, the valve endothelium from normal and diseased valves is phenotypically different. An increased expression of adhesion molecules including VCAM-1, ICAM-1 and E-selectin was found in surgically removed diseased valves.94 In summary, changes of VEC morphology and phenotype were seen in both human and canine degenerative valves. Several kinds of markers and proteins were shown to be up-regulated. However, roles of each phenotype and mechanisms mediated phenotypic changes are still unclear.

Valve interstitial cells Valve interstitial cell is a major cell type in the heart valve and scatters throughout in all three valve layers. Valve interstitial cells are thought to have a role in maintaining normal valve structure

Please cite this article in press as: Aupperle H, Disatian S, Pathology, protein expression and signaling in myxomatous mitral valve degeneration: Comparison of dogs and humans, Journal of Veterinary Cardiology (2012), doi:10.1016/j.jvc.2012.01.005

Mitral valve disease protein expression and signaling and function. An increased number of VIC was seen in human and canine degenerative valves.30,32,95 Most VIC accumulated in the atrialis layer.30,96 Several researchers suggested that an increased cellularity may be due to the proliferative process. However, a proliferative marker, Ki-67 antigen was not elevated in canine myxomatous valves.30 In addition, by using a computerized imaging tool, the number of cells in myxomatous areas was not different in various degrees of the disease suggesting a constant number of cells within the canine diseased valves during the disease progression.97 A similar image was seen in human myxomatous valves. However, image analysis has not been studied yet. In diseased valves, VIC undergo phenotypic transformation. Some VIC turn into an aactin positive phenotype.30,32 In both human and canine degenerative valves, these a-actin positive cells or myofibroblasts mostly accumulated in the area underneath the endothelium particularly on the atrial side of the leaflets (Fig. 7A and B).98 Several researchers believe that myofibroblasts or a-actin positive VIC may be a major phenotype that mediates degeneration. A study on human

9 myxomatous valve disease suggested that a-actin positive VIC might be an activated VIC phenotype which play a role in producing catabolic enzymes and ECM.32 However, a co-localization study in dog tissues showed that the expression of MMP-1 and MMP-13 was not restricted to a-actin positive cells.30 In agreement with an immunohistochemical study, an electron microscope study in canine valve tissues suggested that myofibroblast VIC may not have a primary function in production of ECM.29 Secondary to smooth muscle-like structure within these cells, it has been suggested that myofibroblasts may have contractile properties and act as a supporter to maintain valve tone in diseased canine valves. This considers that a-actin VIC or myofibroblasts in human and canine diseased valves may have different functions in the degenerative process. Several studies performed in human tissues suggested that the myofibroblast might be a precursor that implicates the alteration in valve structure. However, several lines of evidence in canine studies implied that transformation of VIC into myofibroblast phenotype might be a later consequence of the diseased process. The nonmuscle embryonic heavy chain myosin, a marker of mesenchymal cell activation was expressed in human and canine diseased valves in a similar pattern of distribution.30,32 This VIC phenotype was suggested to be a marker of cell activation in close correlation with pathologic changes within the valves and displayed a similar pattern of distribution with MMP.30 An increased expression of CD34, a hematopoietic stromal cell marker involved in stromal cells repair was reported in human mitral valve disease.99 This marker has not been studied yet in canine valves. In humans, another phenotype of VIC called osteoblastic VIC has been suggested.100 This phenotype has been thought to be involved in forming calcific nodules and cartilaginous structures in human degenerative valves.98,100e102 The expression of osteoblastic VIC has not been reported in canine myxomatous valves. However, a chondroblastic activity has recently been described.103 In conclusion, the phenotypic transformation of VIC in humans tends to present itself in a similar manner as in canine MMVD. However, the activity of these transformed VIC may be different in humans and dogs.

Inflammatory cells Figure 7 a-actin immunohistochemistry Positive cells are accumulated mainly in the atrialis layer of canine (A) (magnification 10x) and human MMVD (B) (magnification 10x).

It has been suggested that inflammation is not involved in the degenerative process of human MMVD since the inflammatory cells including macrophages and lymphocytes were negligible in

Please cite this article in press as: Aupperle H, Disatian S, Pathology, protein expression and signaling in myxomatous mitral valve degeneration: Comparison of dogs and humans, Journal of Veterinary Cardiology (2012), doi:10.1016/j.jvc.2012.01.005

10 myxomatous valves.32,91,98 In canine valves, mast cells were slightly increased in the perimeter of pathologic areas of diseased valves suggesting a potential role of mast cells in this disease. Interestingly, the human myxomatous valve is prone to develop bacterial endocarditis.104,105 However, this condition is rare in dogs.105 An appearance of mast cells may have a protective role in preventing secondary infection in canine myxomatous valves. Macrophage numbers also increase in canine diseased valves. However, evidence of direct involvement does not exist since the macrophage population mostly accumulated in the disease-free basal zone.69 The infiltration of other inflammatory cells was not detected in diseased valves.1,16,30 However, several inflammatory cytokine genes were up-regulated in affected canine valves. It has been suggested that valve endothelium might be a major source of these inflammatory mediators.16 In human valves, chronic inflammation and neovascularization may be seen as postinflammatory changes.106,107 The histologic appearance of these valves showed layer structure destruction with diffuse thick-walled vessels and inflammatory round cell infiltration. However, the vascularization was hardly seen in canine myxomatous valves. In summary, an inflammatory process is unlikely to be a cause of degeneration in human and canine valves. A post-inflammation may occur in humans101 but not in canine MMVD.30 Small amounts of mast cells were seen in canine myxomatous valves, but it is unclear if they play a role in in the disease process.

The expression of signaling proteins In affected canine valves, the a-actin positive VIC and VEC strongly expressed transforming growth factor (TGF) b1 and b3.33,83 The results of the Aupperle et al33 indicated an increased expression of TGF-b1 and TGF-b3 and weak expression of TGFb2 in canine diseased valves. In contrast, Obayashi et al.83 described an expression of TGF-b3 and of TbR-II in degenerated mitral valves and unchanged expression of TGF-b1 and TGF-b2 in clinically normal dogs and dogs with MMVD. These differences may have been attributable to the use of antibodies obtained from different manufacturers, different fixation times, or the immunohistochemistry methods that were used. An increased expression of TGF-b1 was seen in human myxomatous valves in a similar manner to those described in canine valves.108 Not only TGFb, but also their receptors are up-regulated in

H. Aupperle, S. Disatian affected canine valves.109 In addition, localized production of TGF-b1 has been reported in canine myxomatous valves.33,109 The up-regulation of serotonin 5HT2B receptor mRNA and proteins has been demonstrated in canine degenerative valves.16,109 Tryptophan hydroxylase 1 (TPH1), a rate limiting enzyme for serotonin production was found up-regulated in diseased human and canine valves.109 Interestingly, the expression pattern of TPH 1 is similar to nonmuscle embryonic heavy chain myosin VIC phenotype in canine and human myxomatous valves.96 The expression of the serotonin transporter, a major structure involved in cellular serotonin uptake, was decreased in late stage canine MMVD.110 Thus, there is evidence suggesting that TGF-b1 and serotonin signaling may be related to the pathogenesis of human and canine MMVD. The roles of signaling pathways involved in myxomatous degeneration are the subject of ongoing studies.

Conclusion Human and canine MMVD share several similarities. The structure of the degenerative valves is almost identical in both species. However, there are some differences in details such as the distribution, the type and the amount of ECM. The pathogenesis of MMVD is unclear. In human valves, ECM abnormalities secondary to inherited diseases may be the cause. The alteration of ECM homeostasis i.e. increased production and/or increased degradation by MMPs has been proposed as the cause of canine and human MMVD. The transformation of cells within the canine and human diseased valves has been found, but it is not clear whether this is the cause or result of the degeneration. Inflammation is unlikely to be the cause of MMVD in both species. Finally, changes in serotonin and TGF-b1 signaling proteins have been discussed. It is uncertain what the exact roles of these proteins are in the pathogenesis of MMVD. Further studies are needed to clarify in more details about the similarity and difference between human and canine MMVD.

Conflicts of interest The authors have no conflict of interest.

References 1. Buchanan JW. Chronic valvular disease (endocardiosis) in dogs. Adv Vet Sci Comp Med 1977;21:75e108.

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Mitral valve disease protein expression and signaling 2. Ha ¨ggstro ¨m J, Pedersen HD, Kvart C. New insights into degenerative mitral valve disease in dogs. Vet Clin North Am Small Anim Pract 2004;34:1209e1226. 3. Trautwein G, Brass W, Kersten U, ernst E, Schneider P, Schulz LC, Amtsberg G, Bisping W, Kirchhoff H, Schole J. ¨tiologie und Pathogenese der EndoUntersuchungen zur A kardiose und Endokarditis des Hundes. Dtsch Tiera ¨rztl Wochenschr 1973;80:507e511. 4. Liu S, Tilley LP. Malformation of the mitral valve complex. J Am Vet Med Assoc 1975;167:465e471. 5. Kogure K. Pathology of chronic mitral valvular disease in the dog. Jpn J Vet Sci 1980;42:323e325. 6. Jacobs GJ, Calvert CA, Mahaffey MB, Hall DG. Echocardiographic detection of flail left atrioventricular valve cusp from ruptured chordae tendineae in 4 dogs. J Vet Intern Med 1995;9:341e346. 7. Robinson WF, Maxie MG. Endocardiosis in dogs. In: Jubb KVF, Kennedy PC, Palmer N, editors, The pathology of domestic animals, vol 3. San Diego, USA: Academic press; 1993. p. 23e24. ¨tiologie und Pathogenese 8. Schole J. Untersuchungen zur A der Endokardiose und Endokarditis des Hundes. IV. Biochemische Untersuchungen. Dtsch Tiera ¨rztl Wochenschr 1973;80:472e473. 9. Ha ¨ggstro ¨m J. Chronic valvular disease in Cavalier King Charles Spaniels. Epidemiology, inheritance and pathophysiology. Thesis, Swedish University of Agricultural Sciences, Uppsala, Sweden, 1996. 10. Swift S. The problem of inherited diseases. 5: valvular disease in Cavalier King Charles Spaniels. Vet Clin North Am Small Anim Pract 1996;37:505e506. 11. Olsen LH, Kristensen A, Ha ¨ggstro ¨m J, Jensen AL, Klitgaard B, Hansson H, Pedersen HD. Increased platelet aggregation response in Cavalier King Charles Spaniel with mitral valve prolapse. J Vet Intern Med 2001;15:209e216. 12. Hyun C. Mitral valve prolapse in Cavalier King Charles Spaniel: a review and case study. J Vet Sci 2005;6:67e73. 13. Olsen LH, Fredholm M, Pederson HD. Epidemiology and inheritance of mitral valve prolapse in Dachshunds. J Vet Intern Med 1999;13:448e456. 14. Ernst E, Schneider P, Trautwein G. Die Endokardiose der Atrioventrikularklappen des Hundes IV: Elektronenmikroskopische Untersuchungen. Zentralbl Vet Med A 1974;21:400e416. 15. Olsen LH, Mortensen K, Martinussen T, Larsson LI, Baandrup U, Pedersen HD. Increased NADPH-diaphorase activity in canine myxomatous mitral valve leaflets. J Comp Pathol 2003;129:120e130. 16. Oyama MA. Quantification of mitral regurgitation volume and effective orifice area using the proximal isovelocity surface area (PISA) method. Proc 1st Int Canine Valv Dis Symp 2004. pp. 43e47. 17. Bonagura, JE, Schober, KE. Can ventricular function be assessed by echocardiography in chronic canine mitral valve disease? 2009;50(suppl 1):12e24. 18. Shah PM. Current concepts in mitral valve prolapse e diagnosis and management. J Cardiol 2010;56:125e133. 19. Iung B, Vahanian A. Epidemiology of valvular heart disease in the adult. Nat Rev Cardiol 2011;8:162e172. 20. Avierinos J-F, Inamo J, Grigioni F, Gersh B, Shub CS, Enriquez-Sarano M. Sex differences in the morphology and outcomes of mitral valve prolapse: a cohort study. Ann Intern Med 2008;149:787e795. 21. Movahed MR, Hepner AD. Mitral valvular prolapse is significantly associated with low body mass index in addition to mitral and tricuspid regurgitation. Cardiol Young 2007;17:172e174.

11 22. Jacquet-Davis. Mitral valve prolapse. Prim Care Update Ob/Gyns 1995;2:1e5. 23. Wilcken DEL. Genes, gender and geometry and prolapsing mitral valve. Aust NZ J Med 1992;22:556e561. 24. Deveroux RB, Kramer-Fox R, Kligfield P. Mitral valve prolapse: causes, clinical manifestations and management. Ann Intern Med 1989;111:305e317. 25. Boudoulas H, Kolibash AJ, Baker P, King BD, Wooley CF. Mitral valve prolapse and the mitral valve prolapse syndrome: a diagnostic classification and pathogenesis of symptoms. Am Heart J 1989;118:797e818. 26. Sell SU, Scully RE. Aging changes in the aortic und mitral valves: histological and histochemical studies with observations on calcific aortic stenosis and calcification of the mitral annulus. Am J Pathol 1965;46:345e365. 27. Schneider P, Ernst E, Trautwein G. Enzyme histocytochemical studies of atrioventricular heart valves in endocardiosis in the dog. Vet Pathol 1973;10:281e294. 28. Corcoran BM, Black A, McEwan J, French A, Smith P, Devine C. Identification of surface morphologic changes in the mitral valve leaflets and chordae tendineae of dogs with myxomatous degeneration. Am J Vet Res 2004;65:198e206. 29. Black A, French AT, Dukes-McEwan J, Corcoran BM. Ultrastructural morphologic evaluation of the phenotype of valvular interstitial cells in dogs with myxomatous degeneration of the mitral valve. Am J Vet Res 2005;66: 1408e1414. 30. Disatian S, Ehrhart III EJ, Zimmerman S, Orton EC. Interstitial cells from dogs with naturally-occurring myxomatous mitral valve disease undergo phenotype transformation. J Heart Valve Dis 2008;17(4):402e412. 31. Aupperle H, Ma ¨rz I, Thielebein J, Kiefer B, Kappe A, Schoon HA. Immunohistochemical characterization of the extracellular matrix in normal mitral valves and in chronic valve disease (endocardiosis) in dogs. Res Vet Sci 2009;87: 277e283. 32. Rabkin E, Aikawa M, Stone JR, Fukumoto Y, Libby P, Schoen FJ. Activated interstitial myofibroblasts express catabolic enzymes and mediate matrix remodelling in myxomatous heart valves. Circulation 2001;104:2525e2532. 33. Tamura K, Fukuda Y, Ishizaki M, Masuda Y, Yamanaka N, Ferrans FJ. Abnormalities in elastic fibres and other connective-tissue components of floppy mitral valve. Am Heart J 1995;129:1149e1158. 34. Akhtar S, Meek KM, James VJ. Ultrastructure abnormalities in proteoglycans, collagen fibrils, and elastic fibers in normal and myxomatous mitral valve chordae tendineae. Cardiovasc Pathol 1999;8:191e201. 35. Akhtar S, Meek KM, James VJ. Immunolocalization of elastin, collagen type I and type III, fibronectin, and vitronectin in extracellular matrix components of normal and myxomatous mitral heart valve chordae tendineae. Cardiovasc Pathol 1999;8(4):203e211. 36. Lester WM. Myxomatous mitral valve disease and related entities: the role of matrix in valvular heart disease. Cardiovasc Pathol 1995;4:257e264. 37. Davies MJ, Moore BP, Braimbridge MV. The floppy mitral valve eStudy of incidence, pathology, and complications, in surgical, necropsy, and forensic material. Br Heart J 1978;40:468e481. 38. Terzo E, Marcello MD, McAllister H, Glazier B, Lo Coco D, Locatelli C, Palermo V, Brambilla PG. Echocardiographic assessment of 537 dogs with mitral valve prolapse and leaflet involvement. Vet Radiol Ultrasound 2009;50:416e422. 39. Tanaka R, Yamane Y. Platelet aggregation in dogs with mitral valve regurgitation. Am J Vet Res 2000;61: 1248e1251.

Please cite this article in press as: Aupperle H, Disatian S, Pathology, protein expression and signaling in myxomatous mitral valve degeneration: Comparison of dogs and humans, Journal of Veterinary Cardiology (2012), doi:10.1016/j.jvc.2012.01.005

12 40. Pedersen HD, Ha ¨ggstro ¨m J. Mitral valve prolapsed in the dog: a model of mitral valve prolapse in man. Cardiovasc Res 2000;47:234e243. 41. Agozzino L, Falco A, de Vivo F, de Vincentiis C, de Luca L, Esposito S, Cotrufo M. Surgical pathology of the mitral valve: gross and histological study of 1288 surgically excised valves. Int J Cardiol 1992;37:79e89. 42. Pellerin D, Brecker S, Veyrat C. Degenerative mitral valve disease with emphasis on mitral vlave prolapse. Heart 2002;88(Suppl. 4):iv20eiv28. 43. Prunotto M, Caimmi PP, Bongiovanni M. Cellular pathology of mitral valve prolapse. Cardiovasc Pathol 2010;19: e113ee117. 44. Roberts WC, McIntosh CL, Wallace RB. Mechanisms of severe mitral regurgitation in mitral valve prolapse determined from analysis of operatively excised valves. Am Heart J 1987;113:1316e1323. 45. Cole WG, Chan D, Hickey AJ, Wilcken DEL. Collagen composition of normal and myxomatous human mitral valves. Biochem J 1984;219:451e460. 46. Latif N, Sarathchandra P, Taylor PM, Antoniw J, Yacoub MH. Localization and pattern of expression of extracellular matrix components in human heart valves. J Heart Valve Dis 2005;14:218e227. 47. Pelouch V, Dixon IMC, Golfman L, Beamish RE, Dhalla NS. Role of extracellular matrix proteins in heart function. Mol Cell Biochem 1994;129:101e120. 48. Dreger S, Taylor PM, Allen SP, Youb MH. Profile and localization of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in human heart valves. J Heart Valve Dis 2002;11:875e880. 49. Krieg T, LeRoy EC. Diseases of the extracellular matrix. J Mol Med 1998;76:224e225. 50. Souza de RR. Aging of myocardial collagen. Biogerontology 2002;3:325e335. 51. Eghbali M, Weber KT. Collagen and the myocardium: fibrillary structure, biosynthesis and degradation in relation to hypertrophy and its regression. Mol Cell Biochem 1990;96:1e14. 52. Mollnau H, Mu ¨nkel B, Schaper J. Collagen VI in the extracellular matrix of normal and failing human myocardium. Herz 1995;20:89e94. 53. Sanes JR, Engvall E, Butkowski R, Hunter DD. Molecular heterogeneity of basal laminae: isoforms of laminin and collagen IV at the neuromuscular junction and elsewhere. J Cell Biol 1990;111:1685e1699. 54. Bashey RI, Martinez-Hernandez A, Jimenez SA. Isolation, characterization, and localization of cardiac collagen VI. Circ Res 1992;70:1006e1017. 55. Graham HK, Horn M, Trafford AW. Extracellular matrix profiles in the progression to heart failure. Acta Physiol (Oxf) 2008;194:3e21. 56. Engvall E, Hessle H, Klier G. Molecular assembly, secretion, and matrix deposition of type VI collagen. J Cell Biol 1986;102:703e710. 57. Tru ¨eb B, Schreier T, Bruckner P, Winterhalter KH. Type VI collagen represents a major fraction of connective tissue collagens. Eur J Biochem 1987;166:699e703. 58. Arribas SM, Hinek A, Gonzalez MC. Elastic fibres and vascular structure in hypertension. Pharmacol Ther 2006; 111:771e791. 59. Shekonin BV, Domogatsky SP, Idelson GL, Koteliansky VE. Participance of fibronectin and various collagen types in the formation of fibrous extracellular matrix in cardiosclerosis. J Mol Cell Cardiol 1988;20:501e508. 60. Speiser B, Weihrauch D, Riess CF, Schaper J. The extracellular matrix in human cardiac tissue part II:

H. Aupperle, S. Disatian

61. 62.

63.

64.

65.

66.

67.

68.

69.

70.

71. 72.

73.

74.

75.

76.

77.

78.

79.

vimentin, laminin, and fibronectin. Cardioscience 1992; 3:41e49. Martin GR, Timpl R. Laminin and other basement membrane components. Ann Rev Cell Biol 1987;3:57e85. Talts JF, Andac Z, Gohring W, Branaccio A, Timpl R. Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins. EMBO J 1999;18:863e870. Stern R. Association between cancer and “acid mucopolysaccharides”: an old concept comes of age, finally. Sem Cancer Biol 2008;18:238e243. Kunzelman KS, Cochran RP, Murphree SS, Ring WS, Verrier ED, Eberhart RC. Differential collagen distribution in the mitral valve and its influence on biochemical behaviour. J Heart Valve Dis 1993;2:236e244. Nasuti JF, Zhang PJ, Feldman MD, Pasha T, Kuhrana JS, Gorman JH, Gorman RC, Narula J, Narula N. Fibrillin and other matrix proteins in mitral valve prolapse syndrome. Ann Thorac Surg 2004;77:532e536. Hadian M, Corcoran BM, Bradshaw JP. Molecular changes in fibrillar collagen in myxomatous mitral valve disease. Cardiovasc Pathol 2010;19:c141ec148. Hadian M, corcoran BM, Han RI, Grossmann G, Bradshaw JP. Collagen Organization in canine myxomatous mitral valve disease: an X-ray diffraction study. Biophysic J 2007;93: 2472e2476. Sokkar SM, Trautwein G. Die Endokardiose der Atrioventrikularklappen des Hundes. I. Morphologische und histochemische Untersuchungen. Zentralbl Vet Med A 1970;17: 757e759. Han RI, Black A, Culshaw GJ, French AT, Else RW, Corcoran BM. Distribution of myofibroblasts, smooth muscle like cells, macrophages, and mast cells in mitral valve leaflets of dogs with myxomatous mitral valve disease. Am J Vet Res 2008;69:763e769. Glesby MJ, Pyeritz RE. Association of mitral valve prolapse and systemic abnormalities of connective tissue. A phenotypic continuum. J Am Med Assoc 1989;262:523e528. Milewicz DM, Urban Z, Boyd C. Genetic disorders of the elastic fiber system. Matrix Biol 2000;19:471e480. Li YY, McTiernan CF, Feldman AM. Interplay of matrix metalloproteinases, tissue inhibitors of metalloproteinases and their regulators in cardiac matrix remodeling. Cardiovasc Res 2000;46:214e224. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 2003;92:827e839. Dollery CM, McEwan JR, Henney AM. Matrix metalloproteinases and cardiovascular disease. Circ Res 1995; 77:863e868. Mann DL, Spinale FG. Activation of Matrix metalloproteinases in the failing human heart. Circulation 1998; 98:1699e1702. Kassiri Z, Khokha R. Myocardial extracellular matrix and its regulation by metalloproteinases and their inhibitors. Thromb Haemost 2005;93:212e219. Deschamps AM, Spinale G. Pathways of matrix metalloproteinase induction in heart failure: bioactive molecules and transcriptional regulation. Cardiovasc Res 2006; 69:666e676. Li YY, Feldman AM, Sun Y, McTiernan CF. Differential expression of tissue inhibitors of metalloproteinases in the failing human heart. Circulation 1998;98:1728e1734. Fondard O, Detaint D, Jung B, Chouquex C, AdleBiassette H, Jarraya M, Hvass U, Couetil JP, Henin D, Michel JB, Vahanian A, Jacob MP. Extracellular matrix remodelling in human aortic valve disease: the role of

Please cite this article in press as: Aupperle H, Disatian S, Pathology, protein expression and signaling in myxomatous mitral valve degeneration: Comparison of dogs and humans, Journal of Veterinary Cardiology (2012), doi:10.1016/j.jvc.2012.01.005

Mitral valve disease protein expression and signaling

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90. 91.

92.

93.

94.

matrix metalloproteinases and their tissue inhibitors. Eur Heart J 2005;26:1333e1341. Aikawa E, Whittaker P, Farber M, Mendelson K, Padera RF, Aikawa M, Schoen FJ. Human semilunar cardiac valve remodelling by activated cells from fetus to adult: implications for postnatal adaptation, pathology and tissue engineering. Circulation 2006;113:1344e1352. Aupperle H, Thielebein J, Kiefer B, Ma ¨rz I, Dinges G, Schoon HA. An immunohistochemical study of the role of matrix metalloproteinases and their tissue inhibitors in chronic mitral valvular disease (valvular endocardiosis) in dogs. Vet J 2009;180:88e94. Aupperle H, Thielebein J, Kiefer B, Ma ¨rz I, Dinges G, Schoon HA, Schubert A. Expression of genes encoding matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in normal and diseased canine mitral valves. J Comp Pathol 2009;140:271e277. Obayashi K, Miyagawa-Tomita S, Matsumoto H, Koyama H, Nakanishi T, Hirose H. Effects of transforming growth factor-beta3 and matrix metalloproteinase-3 on the pathogenesis of chronic mitral valvular disease in dogs. Am J Vet Res 2011;72:194e202. Soini Y, Satta J, Ma ¨a ¨tta ¨ M, Autio-Harmainen H. Expression of MMP2, MMP9, MTI-MMP, TIMP1, and TIMP2 mRNA in valvular lesions of the heart. J Pathol 2001;194:225e231. Togashi M, Tamura K, Nitta T, Ishizaki M, Sugisaki Y, Fukuda Y. Role of matrix metalloproteinases and their tissue inhibitor of metalloproteinases in myxomatous change of cardiac floppy valves. Pathol Int 2007;57:251e259. Oceandy D, Ysoff R, Baudoin FM, Neyses L, Ray SG. Promoter polymorphism of the matrix metalloproteinase 3 gene is associated with regurgitation and left ventricular remodelling in mitral valve prolapse patients. Eur J Heart Fail 2007;9:1010e1017. Koullias GJ, Korkoli DP, Ravichandran P, Psyrri A, Hatzara I, Elefteriades JA. Tissue microarray detection of matrix metalloproteinases in diseased tricuspid and bicuspid aortic valves with or without pathology of the ascending aorta. Eur J Cardiothorac Surg 2004;26:1098e1103. Taylor PM, Batten P, Brand NJ, Thomas PS, Yacoub MH. The cardiac valve interstitial cell. Int J Biochem Cell Biol 2003; 35:113e118. Roy A, Brand NJ, Yacoub MH. Molecular characterization of interstitial cells isolated from human heart valves. J Heart Valve Dis 2000;9:459e464. Leask RL, Jain N, Butany J. Endothelium and valvular diseases of the heart. Microsc Res Tech 2003;60:129e137. Stein PD, Wang CH, Riddle JM, Sabbah HN, Magilligan Jr DJ, Hawkins ET. Scanning electron microscopy of operative excised severely regurgitant floppy mitral valves. Am J Cardiol 1989;64:392e394. Mirzaie M, Meyer T, Schwarz P, Lofti S, Rastan A, Schondube F. Ultrastructural alterations in acquired aortic and mitral valve disease as revealed by scanning and transmission electron microscopical investigations. Ann Thorac Cardiovasc Surg 2002;8(1):24e30. Mow T, Pedersen HD. Increased endothelin-receptor density in myxomatous canine mitral valve leaflets. J Cardiovasc Pathol 1999;34:254e260. Muller AM, Cronen C, Kupferwasser LI, Oelert H, Muller KM, Krikpatrick CJ. Expression of endothelial cell adhesion

13

95.

96.

97.

98.

99.

100.

101.

102.

103.

104.

105. 106. 107.

108.

109.

110.

molecules on heart valves: up-regulation in degeneration as well as acute endocarditis. J Pathol 2000;36:2257e2262. Radermecker MA, Limet R, Lapiere CM, Nusgens B. Increased mRNA expression of decorin in the prolapsing posterior leaflet of the mitral valve. Interact Cardiovasc Thorac Surg 2003;2(3):389e394. Disatian S, Lacerda C, Orton EC. Tryptophan hydroxylase 1 expression is increased in phenotype-altered canine and human degenerative myxomatous mitral valves. J Heart Valve Dis 2010;19(1):71e78. Han RI, Black A, Culshaw G, French AT, Corcoran BM. Structural and cellular changes in canine myxomatous mitral valve disease: an image analysis study. J Heart Valve Dis 2010;19:60e70. Lars W, Steffensen T, Mats B. Role of inflammation in nonrheumatic, regurgitant heart valve disease. A comparative, descriptive study regarding apolipoproteins and inflammatory cells in nonrheumatic heart valve disease. Cardiovasc Pathol 2007;16:171e178. Barth PJ, Koster H, Moosdorf R. CD34þ fibrocytes in normal mitral valves and myxomatous mitral valve degeneration. Pathol Res Pract 2005;201:301e304. Liu AC, Joag VR, Gotlieb AI. The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology. Am J Pathol 2007;171(5):1407e1417. Caira FC, Stock SR, Gleason TG, McGee EC, Huang J, Bonow RO, Spelsberg TC, McCathy PM, Rachimtoola SH, Rajamannan NM. Human degenerative valve disease is associated with up-regulation of low density lipoprotein receptor-related protein 5 receptor mediated bone formation. J Am Coll Cardiol 2006;47(8):1707e1712. Mohler ER, Gannon F, Reynolds C, Zimmerman R, Keene MG, Kaplan FS. Bone formation and inflammation in cardiac valves. Circulation 2001;103:1522e1528. MacLea HB, Lacerda C, Kisiday JD, Orton EC. Canine and Human Degenerative mitral valve disease mimics chondrogenesis. 2011; 25:632e676 (research abstract). MacMahon SW, Roberts JK, Kramer-Fox R, Zucker DM, Roberts RB, Devereux RB. Mitral valve prolapse and infective endocarditis. Am Heart J 1987;113: 1291e1298. Pomerance A, Whitney J. Heart valve changes common to man and comparative study. Cardiovasc Res 1970;4:61e66. Vienot VP. Pathology of inflammatory native valvular heart disease. Cardiovasc Pathol 2006;15:243e251. Tomaru T, Uchida Y, Mohri N, Mori W, Furuse A, Asano K. Postinflammatory mitral and aortic valve prolapsed: a clinical and pathological study. Circulation 1987;114(1):63e69. Park JH, Youn HJ, Yoon JS, Park CS, Oh SS, Chung WB, Chung JW, Choi YS, Lee DH, Oh YS, Chung WS, Hong SJ, Lee YS, Sim SB, Lee SH. The expression of transforming growth factor b1 and a-smooth muscle actin is increased in the human myxomatous valve. Korean J Pathol 2009;43:152e156. Disatian S, Orton EC. Autocrine serotonin and transforming growth factor beta 1 signaling mediates spontaneous myxomatous mitral valve disease. J Heart Valve Dis 2009; 19(1):71e78. Scruggs SM, Disatian S, Orton EC. Serotonin transmembrane transporter is down-regulated in late-stage canine degenerative mitral valve disease. J Vet Cardiol 2010;12(3):163e169.

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