Expression of matrix metalloproteinases during vascularization and ossification of normal and impaired avian growth plate

Enzymes of the matrix metalloproteinase (MMP) family regulate angiogenesis and are involved in the endochondral ossification process. Tibial dyschondroplasia (TD) and rickets are 2 disorders associated with impairments in this process, mainly in the vascularization of the avian growth plate. In this paper, we induced TD and rickets and studied the expression patterns of 4 members of the MMP family known to be important for endochondral ossification, MMP-2, 3, 9, and 13, in normal and impaired avian growth plates. The expression of MMP-3, 9, and 13 was reduced in the lesions and lined up parallel to the expulsion of blood vessels, which was extended up to the border of the lesion, but did not penetrate into it. Matrix metallopro-teinase-2 was not expressed in the TD lesion but was overexpressed in the rachitic lesion. We also studied the differentiation stage of the chondrocytes populating the lesions and found that the rachitic lesions were populated with proliferative chondrocytes, whereas the TD lesions were filled with chondrocytes that presented both proliferative and hypertrophic markers. These results suggest that MMP-3, 9, and 13 play a role in the vascularization and ossification processes, whereas MMP-2 is related to chondrocyte differentiation and may be involved in cartilage remodeling in the avian growth plate.     

Effect of diet on longitudinal bone growth and osteochondrosis in swine

Weanling gilts were fed either a 12% or 16% protein diet for 10 weeks. Animals fed the 12% protein diet had reduced body weights and reduced longitudinal bone growth as measured in the distal radial growth plate. There was no difference in the growth plate widths between the two animal groups, but there was a significant reduction in the daily rate of cell production in the proliferative zone of animals fed the 12% protein diet. No effect of diet on the rate of expansion of the epiphysis at the articular-epiphyseal junction of the distal femur or humerus could be detected. All animals in both groups had morphologic cartilage lesions consistent with early changes associated with osteochondrosis (OCD), and there was no difference in the lesion morphology between the dietary groups. Areas of disorderly endochondral ossification in the radial growth plate were associated with perpendicular growth cartilage infractions. Growth plate lesions were characterized by increased widths of the maturing cartilage zone without increased width of the proliferative zone or an increase in the daily rate of cell production. Focal growth plate lesions developed because of a transitory inhibition of cartilage mineralization and resorption. Disorderly foci of endochondral ossification beneath articular cartilage were characterized by an area of chondrocyte necrosis which prevented normal cartilage matrix mineralization. Lamellae of cartilage necrosis were also present within the reserve zone of the articular cartilage. These were associated with abnormalities of the cartilage canal vessels, and chondrocyte necrosis was considered to precede degenerative changes in articular cartilage matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical analysis of matrix metalloproteinase-1, -3, and -13 in naturally occurring cartilaginous tumors of dogs

OBJECTIVE: To determine immunoreactivity of matrix metalloproteinase (MMP)-1, -3, and -13 in cartilaginous tumors of dogs, correlate expression of MMP with histologic grade of tumors and clinical outcome of dogs, and compare MMP immunoreactivity between chondrosarcomas and chondromas. SAMPLE POPULATION: Formalin-fixed, paraffin-embedded tissues obtained from samples of naturally occurring chondrosarcomas (n = 31) and chondromas (8) of dogs that were submitted to our veterinary medical diagnostic laboratory. PROCEDURE: Histologic sections from each sample were stained with H&E and monoclonal antibody to MMP-1, -3, and -13 by use of an avidin-peroxidase immunohistochemical technique. For each section, histologic grade (I, II, or III) and immunohistochemical expression (0, 1, 2, or 3) were evaluated. Clinical outcome was obtained from medical records or interviews with referring veterinarians and scored as a good outcome, moderate outcome, or poor outcome. Correlations among variables and differences between chondrosarcomas and chondromas were analyzed. RESULTS: Samples from chondrosarcomas had significantly higher immunoreactivity of MMP-1 and -13, compared with immunoreactivity in samples from chondromas. In chondrosarcomas, a significant positive correlation (r, 0.386) was found between MMP-1 and -13 immunoreactivities, and a significant negative correlation (r, -0.390) was detected between MMP-3 and -13 immunoreactivities. CONCLUSIONS AND CLINICAL RELEVANCE: A significant increase in expression of collagenases (MMP-1 and -13) in chondrosarcomas, compared with expression in chondromas, suggests that collagenases may play an important role in tumor progression, and possibly metastasis, in chondrosarcomas of dogs.     

肉鸡骨骼发育的调控机制

肉鸡骨骼发育主要是通过软骨内骨化完成的。在软骨内骨化的进程中,生长板软骨细胞经历增殖、肥大、转分化和软骨基质矿化等,最终成骨逐渐取代软骨原基,实现骨骼的线性延长。软骨内成骨是一个复杂精密的过程,由SOX9、RUNX2、MEF2C、OSX、TGF-β、BMP2、FGFs、IHH和PTHrP等多种信号因子和转录因子协调调控,这些调控因子由生长板不同区的软骨细胞表达或特异性的调控软骨细胞的增殖、分化及血管侵入等过程。在家禽养殖中,肉鸡常发腿病且治疗难度大,而有关肉鸡腿病发病机制的研究报道相对较少。本文综述了骨形成过程及具体的分子调控机制,为了解肉鸡腿病的发生以及提供有效治疗方案提供参考。     

Influence of glucosamine on matrix metalloproteinase expression and activity in lipopolysaccharide-stimulated equine chondrocytes

OBJECTIVE: To characterize potential mechanisms of action of glucosamine inhibition of matrix metalloproteinase (MMP) expression and activity in lipopolysaccharide (LPS)-stimulated equine chondrocytes. SAMPLE POPULATION: Chondrocytes cultured from samples of metacarpophalangeal articular cartilage collected from cadaveric limbs of horses. PROCEDURE: The effect of glucosamine on MMP activity in conditioned medium from LPS-stimulated cartilage explants was determined by a colorimetric assay with azocoll substrate. Treatments consisted of negative and positive controls, glucose (50 mM), and glucosamine (50, 25, 6.25, 3, and 1.5 mM). The influence of glucosamine on MMP synthesis was determined in chondrocytes in pellet culture incubated with LPS (20 microg/mL). Concentration of MMP-13 was quantified in spent medium via ELISA; nonspecific MMP activity was determined via azocoll digestion in organomercurial-activated medium. Effects of glucosamine on MMP mRNA concentration in similarly treated chondrocytes were determined by northern blot hybridization with MMP-1, -3, and -13 probes. Statistical analyses were performed with 2-way ANOVA. RESULTS: Glucosamine had no effect on activated MMP activity but inhibited MMP protein expression, as determined by azocoll digestion (glucosamine, 3 to 50 mM) and MMP-13 ELISA (glucosamine, 1.5 to 50 mM). Resting mRNA concentrations for MMP-1, -3, and -13 mRNA were significantly lower in cultures exposed to glucosamine at concentrations of 50 and 25 mM than those of positive controls. CONCLUSIONS AND CLINICAL RELEVANCE: Glucosamine appears capable of pretranslational, and possibly also translational, regulation of MMP expression; data suggest a potential mechanism of action for chondroprotective effects of this aminomonosaccharide.     

Evaluation of collagenase activity, matrix metalloproteinase-8, and matrix metalloproteinase-13 in horses with chronic obstructive pulmonary disease.

OBJECTIVES: To determine collagenase activity and evaluate matrix metalloproteinase (MMP)-8 and MMP-13 in horses with chronic obstructive pulmonary disease (COPD). ANIMALS: 12 horses with COPD and 12 healthy control horses. PROCEDURE: Collagenase activity was determined by use of an assay for degradation of type-I collagen. Western immunoblot analysis was used to identify interstitial collagenases MMP-8 and MMP-13 in tracheal epithelial lining fluid (TELF). Immunocytochemistry and in situ hybridization were used to determine cellular expression of these 2 collagenases in cells in bronchoalveolar lavage fluid (BALF). RESULTS: Collagenase activity was approximately 7 times higher in samples obtained from horses with COPD, compared with control horses. During stabling, horses with COPD had significantly higher collagenase activity than after being maintained on summer pasture, when activity was similar to that of control horses. Immunoreactivity of MMP-8 and MMP-13 was significantly increased in TELF of horses with COPD, compared with healthy horses. In TELF, a positive correlation was detected between immunoreactivity of MMP-8 and MMP-13 and the amount of degradation of type-I collagen. Macrophages and epithelial cells were the major cellular sources of MMP-8 and MMP-13. CONCLUSIONS AND CLINICAL RELEVANCE: Increased collagenase activity in TELF indicates active ongoing disease and, thus, may reflect lung tissue changes in horses with COPD. Measurements of collagenase activity and MMP immunoreactivity may provide additional diagnostic tools to identify the active phase of chronic lung disease.     

肉鸡胫骨软骨发育不良早期钙黏蛋白1(CDH1)差异表达研究

为研究福美双诱发肉鸡胫骨软骨发育不良(Tibial Dyschondroplasia,TD)早期钙黏蛋白1(CDH1)的差异表达,基础日粮中添加福美双,在试验第1、2、6天,对试验鸡进行剖杀,迅速采取胫骨生长板故入4％多聚甲醛溶液于4℃固定,做CDH1免疫组化分析;提取对照组和饲喂福美双组的生长板总RNA,采用Real-time PCR对CDH1 基因进行差异表达验证.结果钙黏蛋白1 (CDH1/E-cadherin)基因在TD生长板表达上调,在对照和TD软骨生长板,其蛋白合成主要在前肥大、肥大区软骨细胞质,增殖区软骨细胞无表达,钙化区软骨细胞表达少,在饲喂福美双第1、2、6天其表达增加与定量PCR变化结果相一致,且在饲喂福美双第2、6天呈明显高表达.结果表明CDH1 参与细胞黏附、血管入侵及调节Wnt/β-cat的信号传递,和其它分子共同调节软骨内骨化.     

Consequences of capital femoral dyschondroplasia in young adult and skeletally mature broilers

Radiographs revealed defective endochondral ossification in proximal femora of 52 lame broilers. Cartilage retention either involved epiphyseal cartilage close to the articular surface or growth plate cartilage (dyschondroplasia). In at least seven birds proximal femoral lesions were the only abnormalities detected which could have caused lameness. In cases of moderate or severe cartilage retention, fracture lines traversed necrotic cartilage to undermine part or all of the femoral head. Evidence of reparative fibrosis was seen with augmentation of metaphyseal trabeculae by woven bone apposition. Foveal blood vessels aided repair by re-establishing a new ossification centre but in many cases attempts at repair were inadequate.     

RETARDATION OF ENDOCHONDRAL OSSIFICATION AT THE DISTAL ULNAR GROWTH PLATE IN DOGS

Bilateral forelimb deformities associated with retained cartilage cores in the distal ulna were diagnosed in 8 immature giant breed dogs. Five of the dogs were autopsied. Histopathological examination of the distal ulnar growth plate revealed a localised central thickening due to the accumulation of hypertrophic chondrocytes which had failed to undergo the usual development to degeneration. Also the intercellular matrix septa of this cartilage were not calcified. Projections of the abnormal cartilage even persisted into the primary spongiosa. These abnormalities in the process of endochondral ossification of the growth plate produced the lesion of retained cartilage core which was responsible for the development of forelimb deformity.     

Assessment of cartilage degradation effects of matrix metalloproteinase-13 in equine cartilage cocultured with synoviocytes

OBJECTIVE: To determine the effects of matrix metalloproteinase (MMP)-13, compared with interleukin (IL)-1alpha, on cartilage matrix molecule gene expression in a coculture system of equine cartilage explants and synoviocytes. SAMPLE POPULATION: Articular cartilage and synovium specimens harvested from femoropatellar joints of 4 horses, aged 3 to 5 years. PROCEDURES: Synoviocytes were isolated and cocultured with cartilage explants. Cultures were treated with human recombinant MMP-13 (1, 25, or 100 ng/mL) or IL-1alpha (0.01, 0.1, 1.0, or 10 ng/mL) for 96 hours, with medium exchange at 48 hours. Cartilage extracts and media were analyzed for glycosaminoglycan (GAG) content, and results were adjusted to cartilage DNA content. Quantitative PCR was performed on mRNA from cartilage (MMP-3, MMP-13, aggrecan, and collagen type IIB [COL2A1]) and synoviocytes (MMP-3 and MMP-13), and results were adjusted to 18S ribosomal subunit mRNA expression. Treatments were performed in triplicate, and the experiment was repeated 4 times. RESULTS: Cultures treated with MMP-13 or IL-1alpha had increased media GAG concentration at 48 and 96 hours. Aggrecan and COL2A1 mRNA expression were increased by application of MMP-13 or IL-1alpha. Gene expression of the catabolic mediator, MMP-3, in cartilage and synoviocytes was increased in cultures treated with MMP-13 or IL-1alpha. Expression of MMP-13 mRNA in cartilage was increased by IL-1alpha, but decreased in synoviocytes by MMP-13 treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the use of recombinant MMP-13 in a coculture system of synoviocytes and cartilage explants for the study of osteoarthritis.     

Hypertrophy and physiological death of equine chondrocytes in vitro

REASON FOR PERFORMING STUDY: Equine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes. OBJECTIVE: To optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo. METHODS: Chondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). The pellets were examined by light and electron microscopy. Total RNA was extracted from the pellets, and quantitative PCR carried out to investigate changes in expression of a number of genes regulating endochondral ossification. RESULTS: Chondrocytes from fetal foals, grown as pellets, underwent hypertrophy and died by a process morphologically similar to that seen in vivo. Chondrocytes from horses age >5 months did not undergo hypertrophy in pellet culture. They formed intramembranous inclusion bodies and the cultures included cells of osteoblastic appearance. Pellets from neonatal foals cultured in FCS resembled pellets from older horses, however pellets grown in HS underwent hypertrophy but contained inclusion bodies. Chondrocytes from fetal foals formed a typical cartilage-like tissue grossly and histologically, and expressed the cartilage markers collagen type II and aggrecan mRNA. Expression of Sox9, collagen type II, Runx2, matrix metalloproteinase-13 and connective tissue growth factor mRNA increased at different times in culture. Expression of fibroblast growth factor receptor-3 and vascular endothelial growth factor mRNA decreased with time in culture. CONCLUSIONS: Freshly isolated cells from fetal growth cartilage cultured as pellets provide optimal conditions for studying hypertrophy and death of equine chondrocytes. POTENTIAL RELEVANCE: This culture system should greatly assist laboratory studies aimed at elucidating the pathogenesis of osteochondrosis.     

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its expected roles in the bovine endometrium during gestation

Extracellular matrix metalloproteinase inducer (EMMPRIN) and its induced matrix metalloproteinases (MMPs) play a crucial role in tissue remodeling during the peri-implantation period. However, the role of EMMPRIN in the bovine placenta is still unclear. We have postulated that EMMPRIN might play a regulatory role in trophoblastic cell functions during gestation by itself or through the regulation of MMP expression. In this study, EMMPRIN mRNA was detected in the bovine placentome and interplacentome throughout gestation, and its expression was significantly higher in the cotyledon during late gestation. In situ hybridization showed that EMMPRIN mRNA was expressed in the caruncular epithelium and the cotyledonary epithelium, including binucleate cells. Western blot analysis detected a band representing a protein of approximately 65 kDa in the caruncular and cotyledonary tissues, and the intensity of its expression was increased in both of these tissues during late gestation. The expression levels of MMP-2 and MMP-14 in the bovine placenta were higher during late gestation, as was observed for EMMPRIN. Therefore, EMMPRIN might regulate trophoblastic cell functions, especially those of binucleate cells, through MMP expression in the bovine placenta.     

Matrix metalloproteinase activity in stifle synovial fluid of cranial cruciate ligament deficient dogs and effect of postoperative doxycycline treatment

This prospective clinical study investigated the activity of matrix metalloproteinases (MMPs) in stifle synovial fluid (SF) of 13 dogs with acute cranial cruciate ligament (CCL) rupture, and the effect of a postoperative doxycycline treatment. MMP-2, 3, 9 and 13 activities were compared with respect to the time of sampling (preoperatively or 1 month after surgical stabilisation) and the type of postoperative adjuvant treatment (doxycycline or not). No significant activity was detected for both MMP-3 and MMP-13. MMP-2 and MMP-9 activities were found to be significantly highly increased in SF of CCL ruptured stifles compared to control stifles of unaffected dogs. No significant effect from surgical stabilisation and postoperative doxycycline treatment on MMP-2 and MMP-9 activities was found, indicating that doxycycline may not be an appropriate postoperative medical treatment after CCL rupture.     

Active expression of matrix metalloproteinase-13 mRNA in the granulation tissue of equine superficial digital flexor tendinitis

The DNA microarray analysis for matrix metalloproteinase (MMP)-related mRNA expression in equine superficial digital flexor tendinitis indicated that mRNA level of MMP-13 was apparently up-regulated in the tendinitis as compared to normal tendon. In situ hybridization also revealed that fibroblastic cells proliferated in the granulation tissue generated in the tendinitis actively expressed MMP-13 mRNA. On the other hand, in normal tendon, a few fibroblastic cells and vascular components lied in the endotenon barely expressed its mRNA, but other cellular components in the tendon bundle were not positively hybridized. As mentioned above, MMP-13 but not other collagenases or gelatinases, may play an important role in tendon injuries in the racehorses.     

Matrix metalloproteinase-9 in laminae of black walnut extract treated horses correlates with neutrophil abundance

We sought to determine whether a correlation exists between neutrophil infiltration and tissue matrix metalloproteinase-9 (MMP-9) content in digital laminae collected during the prodromal and acute phases of laminitis in horses treated with an aqueous black walnut heartwood extract (BWE). Hoof laminar tissue was obtained at the onset of leukopenia and at the onset of clinical signs of lameness from BWE-treated horses and at equivalent times from control horses. Thin sections of laminae were screened for neutrophils by immunohistochemistry with an anti-CD13 monoclonal antibody and extracts of the same tissues were screened for SDS-renaturable and native MMP-9 activities by denaturing and non-denaturing gelatin zymography. Samples were also screened for MMP-2 and MMP-9 gene expression by RT-qPCR. Control laminae were devoid of both MMP-9 and neutrophils, whereas neutrophils and SDS-renaturable MMP-9 activity were detected in laminae from BWE-treated horses and were strongly correlated at the acute stage of the disease at which time laminar MMP-9 gene expression was significantly (15-fold) elevated. In contrast, BWE-treatment did not significantly elevate MMP-2 gene or protein expression in the laminae. Interestingly, MMP-9 that was present in extracts of laminae from BWE-treated horses at both the prodromal and acute stages of the disease was mainly in the zymogen form, suggesting that the accumulation of the MMP did not contribute to pathology during these stages. However, elevated presence of the MMP-9 zymogen in the tissue would predispose it to catastrophic damage should conditions arise that cleave the regulatory propeptide domain.     

Variations on brain microglial gene expression of MMPs, RECK, and TIMPs in inflammatory and non-inflammatory diseases in dogs

Matrix metalloproteinases (MMPs), MMP inhibitors (TIMPs, tissue inhibitors of matrix metalloproteinases), and the membrane-anchored glycoprotein RECK (reversion-inducing cysteine-rich protein with Kazal motifs) contribute to the pathogenesis of many CNS diseases. To assess the potential pathogenetic roles of microglial MMP, TIMP, and RECK generation in extracellular matrix breakdown, opening of the blood brain barrier (BBB) and subsequent recruitment of leukocytes in the CNS, twenty-four dogs suffering from spontaneously occurring different intracranial and extracranial (control group) diseases were examined. Microglia cells were isolated ex vivo by density gradient centrifugation and their expressions of MMP-2, MMP-9, MMP-12, MMP-13, MMP-14, TIMP-1, TIMP-2, and RECK were examined via quantitative real-time polymerase chain reaction (qPCR). Zymography on CNS tissues in selected cases was performed to assess differences at the protein level. Dogs were grouped in different disease categories according to histopathological examinations, in groups with or without inflammatory reactions, and in groups with/without contrast enhancement in advanced diagnostic imaging as a function of BBB breakdown. The results showed a significant up-regulation of MMP-9 in dogs with inflammation in the nervous system compared to dogs with non-inflammatory diseases. An increased expression of MMP-9 might lead to a facilitated invasion of white blood cells. Furthermore, down-regulation of MMP-13 was found in dogs with contrast enhancement. Zymographical data reflected MMP-2 qPCR data. In conclusion, differential expression of MMPs and their inhibitors, but not of RECK, which might crucially influence the pathogenesis of a given disease, could be demonstrated in canine microglia. This reflects a further pathway in the microglial repertoire to respond to various disease conditions in the CNS, a characteristic that might be of particular relevance as a target for specific treatments.     

Matrix metalloprotease-9 activity in the cerebrospinal fluid and spinal injury severity in dogs with intervertebral disc herniation

We investigated whether matrix metalloproteinase (MMP)-9 expression in the cerebrospinal fluid (CSF) of dogs with intervertebral disc herniation (IVDH) is associated with the severity of neurological signs and prognosis. CSF from the cisterna magna (C-CSF) and the lumbar spine (L-CSF) of 34 dogs with IVDH was analyzed using zymography. Activity of MMP-9 in L-CSF was detected in 6 of 34 dogs with IVDH, often for more than 7 days after injury. MMP-9 activity was not detected from any of the C-CSF samples. Of the six cases that were MMP-9 positive, all four cases with grade V that had loss of deep pain were non-ambulatory 6 months after treatment. The remaining two cases with grade III and IV could recover mobility. In dogs with grade V thoracolumbar IVDH, MMP-9 expression in the CSF may indicate severe spinal cord injury with poor prognosis.     

Elevated rate of collagen solubilization and postmortem degradation in muscles of lambs with high growth rates: possible relationship with activity of matrix metalloproteinases

The extracellular matrix, composed mainly of collagen, is considered responsible for the residual toughness of meat. Matrix metalloproteinases (MMP) responsible for the degradation of connective tissue are found in most tissues, but their participation in meat aging has not been tested. We recently showed that skeletal muscle has multiple MMP activities, as well as regulators and tissue inhibitors of metalloproteinases. Here we present the first observations of physiologic and postmortem variation of MMP activities in muscle. Growing lambs were offered two levels of intake: hay + concentrate for lambs with high growth rate (average daily gain > 250 g) and hay only for those with low growth rate (average daily gain < 25 g). At slaughter and at 21 d of postmortem aging of longissimus and semimembranosus muscles, we studied collagen content, collagen solubility, free hydroxyproline (OH-pro), and levels of latent and active forms of a matrix metalloproteinase (MMP-2) by gelatin zymography. Our results demonstrate the presence of an active isoform of MMP-2 in lamb muscle. Its level was higher (+90%, P < 0.01) in lambs that expressed a high growth rate. Activity of MMP-2 was also present at 21 d postmortem, at levels similar to those detected at slaughter. At slaughter and at 21 d, all muscles contained latent MMP-2 and the quantity of proenzyme was greater than that present in the activated form. The levels of free OH-pro in muscles of lambs with high growth rate increased significantly (P < 0.001) over 21 d from 3.75 to 5.08% of total collagen, and this was significantly related to the level of active MMP-2 at slaughter. By contrast, the amount of free OH-pro in muscles of lambs with low growth rate was not different at 21 d (1.63% of total OH-pro) than it had been at slaughter (1.84% of total OH-pro). These results suggest that collagen degradation all the way to free amino acids occurs postmortem in muscle and that there are active MMP simultaneously present that may account for this catabolism. The growth rate of animals at slaughter influences collagen turnover in vivo, as well as postmortem collagen degradation.     

An immunohistochemical study of the role of matrix metalloproteinases and their tissue inhibitors in chronic mitral valvular disease (valvular endocardiosis) in dogs

While the pathogenesis of chronic valvular disease (CVD) in dogs remains unclear, alterations in the activity of specific metalloproteinase enzymes and their inhibitors within the valve stroma are suspected of having a role. This study describes the immunohistochemical distribution pattern of matrix metalloproteinase (MMP) types 2, 9 and 14 and their tissue inhibitors, termed tissue inhibitors of metalloproteinase (TIMP), types 2 and 3, in normal canine mitral valves (MVs) (n=10) and in dogs with mild (n=7), moderate (n=14) and severe (n=9) CVD. In normal MVs, MMP-2 and -14, and TIMP-2 were expressed in isolated stromal cells. Tissue inhibitor of metalloproteinase-3 exhibited moderate intracellular and mild extracellular expression. With increasing severity of CVD, the expression of MMP-2 decreased. The number of stromal cells expressing MMP-14 increased, predominantly in the margins of the nodular lesions. Tissue inhibitor of metalloproteinase-2 and -3 expression increased both intra- and extracellularly. Matrix metalloproteinase-9 was not detected in normal or diseased valves. In conclusion, CVD was characterised by alterations in the distribution and intensity of valvular MMP and TIMP expression, suggesting that depressed catabolism and the accumulation of extracellular matrix components within affected valves contributes to their structural alteration and consequent loss of function.