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1.  Active synovial matrix metalloproteinase-2 is associated with radiographic erosions in patients with early synovitis 
Arthritis Research  2000;2(2):145-153.
Serum and synovial tissue expression of the matrix metalloproteinase (MMP)-2 and -9 and their molecular regulators, MMP-14 and TIMP-2 was examined in 28 patients with inflammatory early synovitis and 4 healthy volunteers and correlated with the presence of erosions in the patients. Immunohistological staining of MMP-2, MMP-14 and TIMP-2 localized to corresponding areas in the synovial lining layer and was almost absent in normal synovium. Patients with radiographic erosions had significantly higher levels of active MMP-2 than patients with no erosions, suggesting that activated MMP-2 levels in synovial tissue may be a marker for a more aggressive synovial lesion.
Introduction:
In cancer the gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] have been shown to be associated with tissue invasion and metastatic disease. In patients with inflammatory arthritis the gelatinases are expressed in the synovial membrane, and have been implicated in synovial tissue invasion into adjacent cartilage and bone. It is hypothesized that an imbalance between the activators and inhibitors of the gelatinases results in higher levels of activity, enhanced local proteolysis, and bone erosion.
Objectives:
To determine whether the expression and activity levels of MMP-2 and MMP-9, and their regulators MMP-14 and tissue inhibitor of metalloproteinase (TIMP), are associated with early erosion formation in patients with synovitis of recent onset.
Patients and method:
A subset of 66 patients was selected from a larger early synovitis cohort on the basis of tissue availability for the study of synovial tissue and serum gelatinase expression. Patients with peripheral joint synovitis of less than 1 years' duration were evaluated clinically and serologically on four visits over a period of 12 months. At the initial visit, patients underwent a synovial tissue biopsy of one swollen joint, and patients had radiographic evaluation of hands and feet initially and at 1year. Serum MMP-1, MMP-2, MMP-9, MMP-14, and TIMP-1 and TIMP-2 levels were determined, and synovial tissue was examined by immunohistology for the expression of MMP-2 and MMP-9, and their molecular regulators. Gelatinolytic activity for MMP-2 and MMP-9 was quantified using a sensitive, tissue-based gel zymography technique. Four healthy individuals underwent closed synovial biopsy and their synovial tissues were similarly analyzed.
Results:
Of the 66 patients studied, 45 fulfilled American College of Rheumatology criteria for rheumatoid arthritis (RA), with 32 (71%) being rheumatoid factor positive. Of the 21 non-RA patients, seven had a spondylarthropathy and 14 had undifferentiated arthritis. Radiographically, 12 of the RA patients had erosions at multiple sites by 1 year, whereas none of the non-RA patients had developed erosive disease of this extent. In the tissue, latent MMP-2 was widely expressed in the synovial lining layer and in areas of stromal proliferation in the sublining layer and stroma, whereas MMP-9 was expressed more sparsely and focally. MMP-14, TIMP-2, and MMP-2 were all detected in similar areas of the lining layer on consecutive histologic sections. Tissue expression of MMP-14, the activator for pro-MMP-2, was significantly higher in RA than in non-RA patients (8.4 ± 5 versus 3.7 ± 4 cells/high-power field; P = 0.009). In contrast, the expression of TIMP-2, an inhibitor of MMP-2, was lower in the RA than in the non-RA samples (25 ± 12 versus 39 ± 9 cells/high-power field; P = 0.01). Synovial tissue expressions of MMP-2, MMP-14, and TIMP-2 were virtually undetectable in normal synovial tissue samples. The synovial tissue samples of patients with erosive disease had significantly higher levels of active MMP-2 than did those of patients without erosions (Fig. 1). Tissue expression of MMP-2 and MMP-9, however, did not correlate with the serum levels of these enzymes.
With the exception of serum MMP-2, which was not elevated over normal, serum levels of all of the other MMPs and TIMPs were elevated to varying degrees, and were not predictive of erosive disease. Interestingly, MMP-1 and C-reactive protein, both of which were associated with the presence of erosions, were positively correlated with each other (r = 0.42; P < 0.001).
Discussion:
MMP-2 and MMP-9 are thought to play an important role in the evolution of joint erosions in patients with an inflammatory arthritis. Most studies have concentrated on the contribution of MMP-9 to the synovitis, because synovial fluid and serum MMP-9 levels are markedly increased in inflammatory arthropathies. Previously reported serum levels of MMP-9 have varied widely. In the present sample of patients with synovitis of recent onset, serum MMP-9 levels were elevated in only 21%. Moreover, these elevations were not specific for RA, the tissue expression of MMP-9 was focal, and the levels of MMP-9 activity were not well correlated with early erosions. Although serum MMP-2 levels were not of prognostic value, high synovial tissue levels of MMP-2 activity were significantly correlated with the presence of early erosions. This may reflect augmented activation of MMP-2 by the relatively high levels of MMP-14 and low levels of TIMP-2 seen in these tissues. We were able to localize the components of this trimolecular complex to the synovial lining layer in consecutive tissue sections, a finding that is consistent with their colocalization.
In conclusion, we have provided evidence that active MMP-2 complexes are detectable in the inflamed RA synovium and may be involved in the development of early bony erosions. These results suggest that strategies to inhibit the activation of MMP-2 may have the potential for retarding or preventing early erosions in patients with inflammatory arthritis.
PMCID: PMC17808  PMID: 11062605
early synovitis; erosion; metalloproteinase; matrix metalloproteinase-2; rheumatoid arthritis
2.  Effects of induced mast cell activation on prostaglandin E and metalloproteinase production by rheumatoid synovial tissue in vitro 
OBJECTIVE—To determine whether induced mast cell activation/degranulation in rheumatoid synovial explants modulates the production of prostaglandin E (PGE2), and the matrix metalloproteinases (MMPs) collagenase 1, gelatinase A, and stromelysin 1.
METHODS—Synovial explant cultures were treated either with rabbit IgG anti-human IgE as a mast cell (MC) secretagogue or with non-immune rabbit IgG as controls. After 20 hours conditioned medium was assayed for the release of MC tryptase, PGE2, collagenase 1, gelatinase A, and stromelysin 1 using radioimmunoassay, enzyme linked immunosorbent assay, western blot, and zymogram techniques; tissue explants were examined immunohistologically for the relative distributions of MC tryptase, collagenase 1, and stromelysin 1.
RESULTS—Over a 20 hour incubation period the MC secretagogue treated explants showed a significant increase in the quantities of released tryptase and PGE2 compared with controls. By contrast, the three MMPs showed variable values between experiments in response to MC activation; no reproducible trend of either an increased or decreased production of each MMP over control values was evident. Each MMP initially appeared as an inactive precursor form; collagenase 1 and stromelysin 1 were more effectively processed to active forms in the MC activated cultures. Immunolocalisation studies of MC activated explants showed that areas of extracellular tryptase were commonly associated with the local production of both collagenase 1 and stromelysin 1.
CONCLUSION—MC degranulation induced artificially in rheumatoid synovial explant cultures consistently resulted in an increased production of PGE2 but had variable effects on the quantification of released collagenase 1, gelatinase A, and stromelysin 1. Such observations support the concept that activated synovial MCs within their native environment stimulate the production of non-MC derived PGE2 and may contribute to the regulation and processing of specific MMPs; both aspects represent important components of the inflammatory and degradative processes of the rheumatoid lesion.

 Keywords: mast cells; matrix metalloproteinases; prostaglandin E2; rheumatoid synovium
PMCID: PMC1752465  PMID: 9536819
3.  Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. 
Journal of Clinical Investigation  1994;94(6):2493-2503.
Dysregulated extracellular matrix (ECM) metabolism may contribute to vascular remodeling during the development and complication of human atherosclerotic lesions. We investigated the expression of matrix metalloproteinases (MMPs), a family of enzymes that degrade ECM components in human atherosclerotic plaques (n = 30) and in uninvolved arterial specimens (n = 11). We studied members of all three MMP classes (interstitial collagenase, MMP-1; gelatinases, MMP-2 and MMP-9; and stromelysin, MMP-3) and their endogenous inhibitors (TIMPs 1 and 2) by immunocytochemistry, zymography, and immunoprecipitation. Normal arteries stained uniformly for 72-kD gelatinase and TIMPs. In contrast, plaques' shoulders and regions of foam cell accumulation displayed locally increased expression of 92-kD gelatinase, stromelysin, and interstitial collagenase. However, the mere presence of MMP does not establish their catalytic capacity, as the zymogens lack activity, and TIMPs may block activated MMPs. All plaque extracts contained activated forms of gelatinases determined zymographically and by degradation of 3H-collagen type IV. To test directly whether atheromata actually contain active matrix-degrading enzymes in situ, we devised a method which allows the detection and microscopic localization of MMP enzymatic activity directly in tissue sections. In situ zymography revealed gelatinolytic and caseinolytic activity in frozen sections of atherosclerotic but not of uninvolved arterial tissues. The MMP inhibitors, EDTA and 1,10-phenanthroline, as well as recombinant TIMP-1, reduced these activities which colocalized with regions of increased immunoreactive MMP expression, i.e., the shoulders, core, and microvasculature of the plaques. Focal overexpression of activated MMP may promote destabilization and complication of atherosclerotic plaques and provide novel targets for therapeutic intervention.
Images
PMCID: PMC330083  PMID: 7989608
4.  Matrix Metalloproteinases and their Inhibitors in Vascular Remodeling and Vascular Disease 
Biochemical pharmacology  2007;75(2):346-359.
Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade various components of the extracellular matrix (ECM). Members of the MMP family include collagenases, gelatinases, stromelysins, matrilysins and membrane-type MMPs. ProMMPs are cleaved into active forms that promote degradation of ECM proteins. Also, recent evidence suggests direct or indirect effects of MMPs on ion channels in the endothelium and vascular smooth muscle, and on other mechanisms of vascular relaxation/contraction. Endogenous tissue inhibitors of metalloproteinases (TIMPs) reduce excessive proteolytic ECM degradation by MMPs. The balance between MMPs and TIMPs plays a major role in vascular remodeling, angiogenesis, and the uterine and systemic vasodilation during normal pregnancy. An imbalance in the MMPs/TIMPs activity ratio may underlie the pathogenesis of vascular diseases such as abdominal aortic aneurysm, varicose veins, hypertension and preeclampsia. Downregulation of MMPs using genetic manipulations of endogenous TIMPs, or synthetic pharmacological inhibitors such as BB-94 (Batimastat) and doxycycline, and Ro-28-2653, a more specific inhibitor of gelatinases and membrane type 1-MMP, could be beneficial in reducing the MMP-mediated vascular dysfunction and the progressive vessel wall damage associated with vascular disease.
doi:10.1016/j.bcp.2007.07.004
PMCID: PMC2254136  PMID: 17678629
MMP; TIMP; blood vessels; extracellular matrix; aneurysm; varicose veins
5.  Determinants of extracellular matrix remodelling are differentially expressed in paediatric and adult dilated cardiomyopathy 
European Journal of Heart Failure  2010;13(3):271-277.
Aims
The left ventricular phenotype of idiopathic dilated cardiomyopathy (DCM) can appear similar in paediatric and adult patients. However, the aetiology of paediatric DCM is usually idiopathic, and often leads an aggressive clinical course. A structural underpinning of DCM is extracellular matrix changes, which are determined by a balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). This study tested the hypothesis that different MMP/TIMP profiles occur in paediatric and adult DCM patients.
Methods and results
Left ventricular samples from paediatric (age 9 ± 5 years; n = 10) and adult (age 62 ± 3 years; n = 20) DCM (at time of transplant) were subjected to an MMP/TIMP multiplex array and immunoassay in order to measure the MMP subclasses; collagenases (MMP-8, -13), gelatinases (MMP-2, -9), stromelysin/matrilysin (MMP-3, -7), membrane type (MT1-MMP), as well as for the four known TIMPs. MMP-8 and -9 levels increased by over 150% (P < 0.05), whereas MMP-3 and -7 levels decreased by over 30% (P < 0.05) in paediatric DCM when compared with adult DCM. TIMP-1 and -2 levels increased two-fold (P < 0.05), but TIMP-3 fell by 41% (P < 0.05) in paediatric DCM. Myocardial levels of specific interleukins (IL-1beta, IL-2, IL-8) were increased by approximately 50% in paediatric DCM.
Conclusions
These unique findings demonstrated that a specific MMP/TIMP profile occurs in paediatric DCM when compared with adult DCM, and that local cytokine induction may contribute to this process. These distinct differences in the determinants of myocardial matrix structure and function may contribute to the natural history of DCM in children.
doi:10.1093/eurjhf/hfq184
PMCID: PMC3041467  PMID: 21147820
Cardiomyopathy; Extracellular matrix; Matrix metalloproteinase; Paediatrics
6.  Matrix metalloproteinase protein expression profiles cannot distinguish between normal and early osteoarthritic synovial fluid 
Background
Osteoarthritis (OA) and Rheumatoid arthritis (RA) are diseases which result in the degeneration of the joint surface articular cartilage. Matrix Metalloproteinases (MMPs) are enzymes that aid in the natural remodelling of tissues throughout the body including cartilage. However, some MMPs have been implicated in the progression of OA and RA as their expression levels and activation states can change dramatically with the onset of disease. Yet, it remains unknown if normal and arthritic joints demonstrate unique MMPs expression profiles, and if so, can the MMP expression profile be used to identify patients with early OA. In this study, the synovial fluid protein expression levels for MMPs 1, 2, 3, 7, 8, 9, 12 & 13, as well as those for the Tissue Inhibitors of MMPs (TIMPs) 1, 2, 3, & 4 were examined in highly characterized normal knee joints, and knee joints with clinically diagnosed OA (early and advanced) or RA. The purpose of this study was to determine if normal, OA, and RA patients exhibit unique expression profiles for a sub-set of MMPs, and if early OA patients have a unique MMP expression profile that could be used as an early diagnostic marker.
Methods
Synovial fluid was aspirated from stringently characterized normal knee joints, and in joints diagnosed with either OA (early and advanced) or RA. Multiplexing technology was employed to quantify protein expression levels for 8 MMPs and 4 TIMPs in the synovial fluid of 12 patients with early OA, 17 patients diagnosed with advanced OA, 15 with RA and 25 normal knee joints. Principle component analysis (PCA) was used to reveal which MMPs were most influential in the distinction between treatment groups. K – means clustering was used to verify the visual grouping of subjects via PCA.
Results
Significant differences in the expression levels of MMPs and TIMPs were observed between normal and arthritic synovial fluids (with the exception of MMP 12). PCA demonstrated that MMPs 2, 8 & 9 can be used to effectively separate individuals diagnosed with advanced arthritis from early osteoarthritic and normal individuals, however, these MMP profiles do not separate early OA from normal synovial fluid. An apparent separation between advanced OA and RA subjects was also revealed through PCA. K-means clustering verified the presence of 3 clusters: normal joints clustered with early OA, and separate clusters of advanced OA or RA.
Conclusions
This study demonstrates that unique MMP and TIMP expression profiles are present within normal, advanced OA and RA synovial fluid. These MMP profiles can be used to distinguish advanced OA & RA synovial fluid from early OA & normal synovial fluid, and even between synovial fluid samples from OA and RA joints. Although this methodology cannot be used for the diagnosis of early OA, high throughput multiplex technology of MMPs and TIMPs in synovial fluid may prove useful in determining the severity of the disease state, and/or quantifying the response of individuals to disease interventions.
doi:10.1186/1471-2474-13-126
PMCID: PMC3532375  PMID: 22824140
7.  Matrix Metalloproteinase Inhibitors as Investigative Tools in the Pathogenesis and Management of Vascular Disease 
Exs  2012;103:209-279.
Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade various components of the extracellular matrix (ECM). MMPs could also regulate the activity of several non-ECM bioactive substrates, and consequently affect different cellular functions. Members of the MMPs family include collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs and others. Pro-MMPs are cleaved into active MMPs, which in turn act on various substrates in the ECM and on the cell surface. MMPs play an important role in the regulation of numerous physiological processes including vascular remodeling and angiogenesis. MMPs may also be involved in vascular diseases such as hypertension, atherosclerosis, aortic aneurysm, and varicose veins. MMPs also play a role in the hemodynamic and vascular changes associated with pregnancy and preeclampsia. The role of MMPs is commonly assessed by measuring their gene expression, protein amount, and proteolyic activity using gel zymography. Because there are no specific activators of MMPs, MMP inhibitors are often used to investigate the role of MMPs in different physiologic processes and in the pathogenesis of specific diseases. MMP inhibitors include endogenous tissue inhibitors (TIMPs) and pharmacological inhibitors such as zinc chelators, doxycycline and marimastat. MMP inhibitors have been evaluated as diagnostic and therapeutic tools in cancer, autoimmune and cardiovascular disease. Although several MMP inhibitors have been synthesized and tested both experimentally and clinically, only on MMP inhibitor, i.e. doxycycline, is currently approved by the Food and Drug Administration. This is mainly due to the undesirable side effects of MMP inhibitors especially on the musculoskeletal system. While most experimental and clinical trials of MMP inhibitors have not demonstrated significant benefits, some trials still showed promising results. With the advent of new genetic and pharmacological tools, disease-specific MMP inhibitors with fewer undesirable effects are being developed and could be useful in the management of vascular disease.
PMCID: PMC3367802  PMID: 22642194
TIMP; endothelium; vascular smooth muscle; extracellular matrix; angiogenesis; atherosclerosis; hypertension; aneurysm; varicose veins; pregnancy; preeclampsia
8.  Matrix metalloproteinases and their natural inhibitors in fibrovascular membranes of proliferative diabetic retinopathy 
The British Journal of Ophthalmology  2000;84(10):1091-1096.
AIM—To examine epiretinal membranes of proliferative diabetic retinopathy (PDR) for the presence of selective matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs), in order to determine whether neovascularisation and fibrosis, characteristic of this complication of diabetes mellitus, are associated with specific anomalies of MMP or TIMP expression.
METHODS—The presence of selected MMPs and TIMPs was investigated in 24 fibrovascular epiretinal membranes of PDR, and the findings compared with that observed in 21 avascular epiretinal membranes of proliferative vitreoretinopathy (PVR) and five normal retinas. Specimens were examined for deposition of interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), and three tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2, and TIMP-3).
RESULTS—The results showed that unlike normal retina, which constitutively expresses MMP-1 and TIMP-2, a large proportion of PDR membranes (> 62%) stained for MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, and TIMP-3. There were no differences in the expression of these molecules when compared with PVR membranes. A characteristic staining for MMP-9 was observed within the perivascular matrix of PDR membranes, and there was a significant increase in TIMP-2 expression by PDR membranes (p= 0.036) when compared with PVR membranes.
CONCLUSIONS—The findings that MMPs involved in degradation of fibrovascular tissue matrix, as well as TIMP-1 and TIMP-2, are found in a large proportion of PDR membranes, and that their expression does not differ from that of PVR membranes, suggest the existence of common pathways of extracellular matrix degradation in pathological processes leading to retinal neovascularisation and fibrosis.


doi:10.1136/bjo.84.10.1091
PMCID: PMC1723275  PMID: 11004090
9.  Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis 
Annals of the Rheumatic Diseases  2000;59(6):455-461.
OBJECTIVE—Matrix metalloproteinases (MMPs) are expressed in joint tissues of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The objective of this study was to define the steady state levels of seven different MMPs and two tissue inhibitors of metalloproteinases (TIMPs) as well as the potential metalloproteinase activity in the synovial fluid (SF) to provide more insight into the role of MMPs in cartilage destruction in RA and OA.
METHODS—Levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, TIMP-1, and TIMP-2 in SF aspirated from knee joints of 97 patients with RA and 103 patients with OA were measured by the corresponding one step sandwich enzyme immunoassays. Proteolytic activity of MMPs in these SFs was examined in an assay using [3H]carboxymethylated transferrin substrate in the presence of inhibitors of serine and cysteine proteinases after activation with p-aminophenylmercuric acetate (APMA). Destruction of RA knee joints was radiographically evaluated.
RESULTS—Levels of MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 were significantly higher in RA SF than in OA SF. MMP-7 and MMP-13 were detectable in more than 45% of RA SFs and in less than 20% of OA SFs, respectively. Among the MMPs examined, MMP-3 levels were extremely high compared with those of other MMPs. Direct correlations were seen between the levels of MMP-1 and MMP-3 and between those of MMP-8 and MMP-9 in RA SF. Although the levels of MMP-1 and MMP-3 increased even in the early stage of RA, those of MMP-8 and MMP-9 were low in the early stage and increased with the progression of RA. Molar ratios of the total amounts of the MMPs to those of the TIMPs were 5.2-fold higher in patients with RA than in OA, which was significant. APMA-activated metalloproteinase activity in SF showed a similar result, and a direct correlation was seen between the molar ratios and the activity in RA SF.
CONCLUSIONS—Our results show that high levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and TIMP-1 are present in RA SF and suggest that once these MMPs are fully activated, they have an imbalance against TIMPs, which may contribute to the cartilage destruction in RA.


doi:10.1136/ard.59.6.455
PMCID: PMC1753174  PMID: 10834863
10.  Determination of interstitial collagenase (MMP-1) in patients with rheumatoid arthritis. 
Annals of the Rheumatic Diseases  1995;54(12):970-975.
OBJECTIVES--To investigate whether interstitial collagenase (MMP-1) concentration in synovial fluid can be useful as a marker for disease activity in rheumatoid arthritis (RA), to determine the main route by which collagenase degrades the matrix of articular cartilage, and to investigate if an imbalance between metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMP) is responsible for the activity of MMPs in RA. METHODS--Collagenase concentrations were measured in synovial fluid and paired serum samples using a specific sandwich enzyme linked immunosorbent assay. Collagenase activities were also assayed in synovial fluid samples. Synovial tissues obtained from the same patient were examined by immunohistochemical staining and the numbers of cells expressing collagenase were counted. RESULTS--Collagenase concentrations in synovial fluid did not correlate with C reactive protein and collagenase levels in serum, but did correlate positively with the degree of synovial inflammation, and increased with increasing numbers of cells identified as expressing collagenase in synovial tissue. Collagenase activities did not correlate with TIMP-1 concentrations, but did correlate strongly with the ratios of collagenase concentration to TIMP-1 (r = 0.73). CONCLUSION--The collagenase concentration in synovial fluid cannot be used as a marker for systemic disease activity, but can be used as a marker for the degree of synovial inflammation in the joint from which the sample is aspirated. In advanced RA, most of the collagenase is probably produced in synovial lining cells and released into synovial fluid, where it degrades the matrix of articular cartilage. An imbalance between MMP and TIMP may be of importance in the degradation of extracellular matrix of articular cartilage in RA.
Images
PMCID: PMC1010062  PMID: 8546529
11.  Expression and functional properties of antibodies to tissue inhibitors of metalloproteinases (TIMPs) in rheumatoid arthritis 
Arthritis Research & Therapy  2005;7(5):R1014-R1022.
Tissue inhibitors of matrix metalloproteinases (TIMPs) regulate the breakdown of extracellular matrix components and play an important role in tissue remodelling and growth, in both physiological and pathological conditions. We studied the autoimmune response to TIMPs in patients with rheumatoid arthritis (RA). Eighty-nine paired blood and synovial fluid samples from patients with RA were assessed for their reactivity with recombinant tissue inhibitors of metalloproteinases (TIMPs) 1 to 4 by an ELISA and were compared with blood from 62 healthy controls and 21 synovial fluid samples from patients with degenerative joint diseases. Presence of antibodies was established as the absorbance of the sample more than 2 standard deviations above the mean of the controls. In addition, immunoglobulin G (IgG) from blood samples of RA patients possessing TIMP antibodies was isolated on protein A–sepharose and tested for the in vitro ability to neutralize TIMP-2-dependent effects on metalloproteinase 9 (MMP9). Anti-TIMP antibodies were found in 56% of RA samples but in only 5% of the controls (P < 0.005). RA patients had high frequencies of antibodies against all TIMPs except TIMP-3. TIMP-2 antibodies were most frequently found (33%), being significantly more prevalent (P = 0.024) in patients with nonerosive than erosive RA. TIMP-1 antibodies were significantly more often found in synovial fluid samples than in the matched blood samples (P < 0.025). Importantly, the IgG fraction containing TIMP antibodies down-regulated the TIMP-2 inhibitory effect, thereby supporting MMP9 activity in vitro. In the present study, we show that RA patients frequently develop autoimmune response to TIMPs that may act as a functionally significant regulator of MMP activity and thereby of joint destruction.
doi:10.1186/ar1771
PMCID: PMC1257425  PMID: 16207317
12.  Upregulation and differential expression of matrilysin (MMP-7) and metalloelastase (MMP-12) and their inhibitors TIMP-1 and TIMP-3 in Barrett's oesophageal adenocarcinoma 
British Journal of Cancer  2001;85(3):383-392.
Oesophageal adenocarcinoma is believed to arise from metaplastic mucosa in the distal oesophagus, a condition also known as Barrett's oesophagus (BE). BE develops as a result of injury caused by refluxing gastric and duodenal contents and is associated with increased risk of malignant transformation. Matrix metalloproteinases (MMPs) have been implicated in all aspects of tumour progression; tumour growth, basement membrane degradation, invasion and metastatic spread. Using in situ hybridization, we investigated the expression patterns of collagenases-1 and -3, stromelysin-2, matrilysin, metalloelastase and TIMPs-1 and -3 in BE, adenocarcinoma and lymph-node metastases. Matrilysin was expressed abundantly in 12/15 tumours and in 4/6 lymph-node metastases and its expression correlated with the histological aggressiveness of tumour. Matrilysin and metalloelastase were upregulated already in BE. Stromelysin-2 and collagenase-3 expression was detected only in a few tumours. Collagenase-1 was expressed by cancer and stromal cells in 9/15 tumours. Tumour-infiltrating macrophages expressed metalloelastase in 13/15 cancers. TIMPs-1 and -3 were expressed in 12/15 and 11/15 tumours, respectively. Laminin-5 and tenascin were abundantly expressed at the invasive front of poorly differentiated tumours, but not in BE. Our results indicate that matrilysin is the principal MMP expressed by tumour cells in oesophageal adenocarcinoma, and further studies are needed to investigate whether matrilysin or tenascin-C could be used as a predictive marker for progression of BE to cancer. © 2001 Cancer Research Campaign http://www.bjcancer.com
doi:10.1054/bjoc.2001.1929
PMCID: PMC2364078  PMID: 11487270
collagenase; in situ hybridization; laminin-5; tenascin-C
13.  Activities of matrix metalloproteinases and tissue inhibitor of metalloproteinase-2 in idiopathic hemotympanum and otitis media with effusion 
Acta oto-laryngologica  2008;128(2):144-150.
Conclusion
The expression profile of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) was specific to the type of middle ear effusion. Further studies are necessary for elucidating its correlation with the sequelae of otitis media with effusion (OME) and idiopathic hemotympanum.
Objectives
We aimed to investigate the relative activities of gelatinases (MMP-2 and 9), stromelysin-1 (MMP-3), matrilysin-1 (MMP-7) as well as measuring TIMP-2 levels in the serous and mucous effusions of OME and hemorrhagic effusion of the idiopathic hemotympanum.
Method
Middle ear effusions were collected from patients with OME and idiopathic hemotympanum, and were classified as mucoid, serous or hemorrhagic. MMP activity in the effusion samples was examined by gelatin and casein zymography. Levels of TIMP-2 were measured by ELISA. Human temporal bones sections, with and without otitis media (OM), were examined histologically.
Results
One case showed tympanic membrane thinning in the OM group, but none in the control group. While MMP-2 was present in all effusions, the active form of MMP-2 was found only in mucous effusions. MMP-3 and MMP-7 activity was detected only in the mucous effusions. MMP-9 exhibited activity in all effusions, with the highest levels in mucous effusions. TIMP-2 levels were markedly elevated in serous effusions.
doi:10.1080/00016480701477610
PMCID: PMC2577605  PMID: 17851959
Matrix metalloproteinase; Tissue inhibitor of metalloproteinase; Otitis media with effusion; Idiopathic hemotympanum
14.  Collagenolytic and gelatinolytic matrix metalloproteinases and their inhibitors in basal cell carcinoma of skin: comparison with normal skin 
British Journal of Cancer  2000;82(3):657-665.
Tissue from 54 histologically-identified basal cell carcinomas of the skin was obtained at surgery and assayed using a combination of functional and immunochemical procedures for matrix metalloproteinases (MMPs) with collagenolytic activity and for MMPs with gelatinolytic activity. Collagenolytic enzymes included MMP-1 (interstitial collagenase), MMP-8 (neutrophil collagenase) and MMP-13 (collagenase-3). Gelatinolytic enzymes included MMP-2 (72-kDa gelatinase A/type IV collagenase) and MMP-9 (92-kDa gelatinase B/type IV collagenase). Inhibitors of MMP activity including tissue inhibitor of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2) were also assessed. All three collagenases and both gelatinases were detected immunochemically. MMP-1 appeared to be responsible for most of the functional collagenolytic activity while gelatinolytic activity reflected both MMP-2 and MMP-9. MMP inhibitor activity was also present, and appeared, based on immunochemical procedures, to reflect the presence of TIMP-1 but not TIMP-2. As a group, tumours identified as having aggressive-growth histologic patterns were not distinguishable from basal cell carcinomas with less aggressive-growth histologic patterns. In normal skin, the same MMPs were detected by immunochemical means. However, only low to undetectable levels of collagenolytic and gelatinolytic activities were present. In contrast, MMP inhibitor activity was comparable to that seen in tumour tissue. In previous studies we have shown that exposure of normal skin to epidermal growth factor in organ culture induces MMP up-regulation and activation. This treatment concomitantly induces stromal invasion by the epithelium (Varani et al (1995) Am J Pathol146: 210–217; Zeigler et al (1996 b) Invasion Metastasis16: 11–18). Taken together with these previous data, the present findings allow us to conclude that the same profile of MMP/MMP inhibitors that is associated with stromal invasion in the organ culture model is expressed endogenously in basal cell carcinomas of skin. © 2000 Cancer Research Campaign
doi:10.1054/bjoc.1999.0978
PMCID: PMC2363319  PMID: 10682680
interstitial collagenase; collagenase-3; tissue inhibitoral metalloproteinase invasion; fibroblast; epithelial cells; endothelial cells
15.  Stromelysin-1 Regulates Adipogenesis during Mammary Gland Involution 
The Journal of Cell Biology  2001;152(4):693-703.
The matrix metalloproteinase MMP-3/stromelysin-1 (Str1) is highly expressed during mammary gland involution induced by weaning. During involution, programmed cell death of the secretory epithelium takes place concomitant with the repopulation of the mammary fat pad with adipocytes. In this study, we have used a genetic approach to determine the role of Str1 during mammary involution. Although Str1 has been shown to induce unscheduled apoptosis when expressed ectopically during late pregnancy (Alexander, C.M., E.W. Howard, M.J. Bissell, and Z. Werb. 1996. J. Cell Biol. 135:1669–1677), we found that during post-lactational involution, mammary glands from transgenic mice that overexpress the tissue inhibitor of metalloproteinases, TIMP-1 (TO), or mice carrying a targeted mutation in Str1 showed accelerated differentiation and hypertrophy of adipocytes, while epithelial apoptosis was unaffected. These data suggest that matrix metalloproteinases (MMPs) do not induce unscheduled epithelial cell death after weaning, but instead alter the stromal microenvironment. We used adipogenic 3T3-L1 cells as a cell culture model to test the function of MMPs during adipocyte differentiation. Fibroblastic 3T3-L1 progenitor cells expressed very low levels of MMPs or TIMPs. The transcription of a number of MMP and TIMP mRNAs [Str1, MT1-MMP, (MMP-14) collagenase-3 (MMP-13), gelatinase A (MMP-2), and TIMP-1, -2 and -3] was induced in committed preadipocytes, but only differentiated adipocytes expressed an activated MMP, gelatinase A. The addition of MMP inhibitors (GM 6001 and TIMP-1) dramatically accelerated the accumulation of lipid during differentiation. We conclude that MMPs, especially Str1, determine the rate of adipocyte differentiation during involutive mammary gland remodeling.
PMCID: PMC2195781  PMID: 11266461
transgenic mouse; mammary involution; MMP-3; TIMP-1; 3T3-L1 adipocytes
16.  Co-culture of human breast adenocarcinoma MCF-7 cells and human dermal fibroblasts enhances the production of matrix metalloproteinases 1, 2 and 3 in fibroblasts. 
British Journal of Cancer  1995;71(5):1039-1045.
No measurable amounts of matrix metalloproteinases (MMPs) were produced by human breast adenocarcinoma cell lines MCF-7 and BT-20 in culture. When MCF-7 cells were co-cultured with human dermal fibroblasts enhanced production of precursors of MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1) and tissue inhibitor of metalloproteinase type 1 (TIMP-1) was observed. Immunohistochemical studies indicated that these pro-MMPs originated primarily from the fibroblasts, suggesting that MCF-7 cells have a stimulatory effect on stromal cells to produce at least three pro-MMPs and TIMP-1. BT-20 cells also enhanced the production of pro-MMP-2 and TIMP-1 in the dermal fibroblasts, but not of pro-MMP-1 and pro-MMP-3. Normal mammary epithelial cells promoted only TIMP-1 production. To investigate further the stimulatory factors from MCF-7 cells, the conditioned medium and the cell membrane were prepared and examined. The cell membrane fraction enhanced the production of pro-MMP-1 and -3 and TIMP-1, but not of pro-MMP-2. The conditioned medium, on the other hand, augmented the production of all four proteins in the fibroblasts. These observations suggest that breast adenocarcinoma MCF-7 cells in culture produce both soluble and membrane-bound factor(s) which stimulate the production of pro-MMPs and TIMP-1 in neighbouring stromal cells, but the factor(s) released into the medium and that associated with cell membranes are probably different. Such communication between the normal and malignant cell types may, in part, assist the cancer cells to invade and metastasise.
Images
PMCID: PMC2033797  PMID: 7734296
17.  Upregulation of matrix metalloproteinases in a model of T cell mediated tissue injury in the gut: analysis by gene array and in situ hybridisation 
Gut  2002;51(4):540-547.
Background and aim: Matrix metalloproteinases (MMPs) have been implicated in tissue remodelling and ulceration in inflammatory bowel disease and coeliac disease. Studies to date have concluded that stromelysin 1 is functionally involved in mucosal degradation. However, there are many other MMPs whose function in the gut is currently unknown. This work had two aims: firstly, to use gene array technology to measure changes in MMP and tissue inhibitor of metalloproteinase (TIMP) expression in a model of T cell mediated injury in the gut, and secondly, to correlate data from gene arrays with that generated by in situ hybridisation.
Methods: T cells in explants of human fetal gut were activated with pokeweed mitogen or anti-CD3 plus interleukin 12. Gene array analysis and in situ hybridisation were performed to investigate changes in MMP gene expression.
Results: Both gene array analysis and in situ hybridisation indicated marked upregulation of stromelysin 2 and macrophage metalloelastase expression in the explants associated with mucosal destruction. The arrays also confirmed our previous observation that interstitial collagenase (MMP-1), stromelysin 1 (MMP-3), and gelatinase B (MMP-9) are upregulated but there was no change in MMP-2, -7, -8, -9, -11, -13, -14–17, or -19. Following T cell activation, transcripts for TIMPs were reduced.
Conclusions: These results show that there is differential upregulation of MMPs during T cell responses in the gut and suggest that further studies on the role of stromelysin 2 and macrophage metalloelastase may show that they have a functional role. In addition, the increase in MMPs and reduction in TIMPs suggest that the protease/antiprotease balance in the mucosa may determine the extent of mucosal degradation.
PMCID: PMC1773375  PMID: 12235077
collagenase; matrilysin; metalloelastase; tissue inhibitor of metalloproteinase; stromelysin
18.  Analysis of 16 different matrix metalloproteinases (MMP-1 to MMP-20) in the synovial membrane: different profiles in trauma and rheumatoid arthritis 
Annals of the Rheumatic Diseases  1999;58(11):691-697.
OBJECTIVE—To define the pattern of mRNA expression of all human matrix metalloproteinases (MMPs) described to date in rheumatoid arthritis (RA) and traumatic synovial membrane, in order to differentiate between a physiological tissue remodelling pattern and that associated with inflammatory tissue destruction.
METHODS—Analysis of SwissProt protein and EMBL/GenBank nucleotide sequence banks, protein sequence alignment, reverse transcriptase-polymerase chain reaction and nucleotide sequencing were used.
RESULTS—MMP-2 (gelatinase A), MMP-3 (stromelysin-1), MMP-11 (stromelysin-3) and MMP-19 were constitutively expressed. MMP-1 (fibroblast type collagenase), MMP-9 (gelatinase B) and MMP-14 (MT1-MMP) were expressed in all RA, but only in 55-80% of trauma samples. MMP-13 (collagenase-3) and MMP-15 (MT2-MMP) were expressed exclusively in RA (80-90% of the samples). MMP-20 (enamelysin) was absent and MMP-8 (collagenase-2) was rarely found in RA or trauma. All other MMPs (-7, -10, -12, -16, -17) had an intermediate pattern of expression.
CONCLUSIONS—Some MMPs without interstitial collagenase activity seem to have a constitutive pattern of expression and probably participate in physiological synovial tissue remodelling. Some MMPs are exclusively associated to RA synovitis, for example, MMP-13, which preferentially degrades type II collagen and aggrecan, and MMP-15, which activates proMMP-2 and proMMP-13 and is involved in tumour necrosis factor α processing. This clear cut rheumatoid/inflammatory MMP profile, more complex than has been previously appreciated, may facilitate inflammatory tissue destruction in RA.


PMCID: PMC1752794  PMID: 10531073
19.  Hypoxia upregulates angiogenesis and synovial cell migration in rheumatoid arthritis 
Introduction
Rheumatoid arthritis (RA) is characterised by invasion of cartilage, bone and tendon by inflamed synovium. Previous studies in our laboratory have shown that hypoxia is a feature of RA synovitis. In the present study, we investigated the consequences of hypoxia on angiogenesis and synovial fibroblast migration in RA.
Methods
Synovial tissue was harvested from RA patients, and synovial membrane cells were cultured under conditions either of hypoxia (1% oxygen) or normoxia (21% oxygen). Protein levels of matrix metalloproteinases (MMPs) and angiogenic factors were measured, while RNA was extracted for PCR quantification of MMPs/tissue inhibitors of MMP (TIMPs) and angiogenic factors. Migration of RA synovial fibroblasts through collagen, and the effect of RA synovial cell supernatants in an in vitro angiogenesis assay, were utilised to determine the functional relevance of changes in mRNA/protein.
Results
We observed upregulation under hypoxic conditions of MMPs responsible for collagen breakdown, specifically collagenase MMP-8, and the gelatinases MMP-2 and MMP-9, at both mRNA and protein levels. Increased MT1-MMP mRNA was also observed, but no effect on TIMP-1 or TIMP-2 was detected. RA fibroblast migration across collagen was significantly increased under hypoxic conditions, and was dependent on MMP activity. Furthermore, expression of angiogenic stimuli, such as vascular endothelial growth factor (VEGF), and VEGF/placental growth factor heterodimer, was also increased. Crucially, we show for the first time that hypoxia increased the angiogenic drive of RA cells, as demonstrated by enhanced blood vessel formation in an in vitro angiogenesis assay.
Conclusions
Hypoxia may be responsible for rendering RA synovial lining proangiogenic and proinvasive, thus leading to the debilitating features characteristic of RA.
doi:10.1186/ar2689
PMCID: PMC2714109  PMID: 19426483
20.  Patterns of matrix metalloproteinase expression in cycling endometrium imply differential functions and regulation by steroid hormones. 
Journal of Clinical Investigation  1994;94(3):946-953.
Matrix metalloproteinases are a highly regulated family of enzymes, that together can degrade most components of the extracellular matrix. These proteins are active in normal and pathological processes involving tissue remodeling; however, their sites of synthesis and specific roles are poorly understood. Using in situ hybridization, we determined cellular distributions of matrix metalloproteinases and tissue inhibitor of metalloproteinase-1, an inhibitor of matrix metalloproteinases, in endometrium during the reproductive cycle. The mRNAs for all the metalloproteinases were detected in menstrual endometrium, but with different tissue distributions. The mRNA for matrilysin was localized to epithelium, while the others were detected in stromal cells. Only the transcripts for the 72-kD gelatinase and tissue inhibitor of metalloproteinases-1 were detected throughout the cycle. Transcripts for stromelysin-2 and the 92-kD gelatinase were only detected in late secretory and menstrual endometrium, while those for matrilysin, the 72-kD gelatinase, and stromelysin-3 were also consistently detected in proliferative endometrium. These data indicate that matrix metalloproteinases are expressed in cell-type, tissue, and reproductive cycle-specific patterns, consistent with regulation by steroid hormones, and with specific roles in the complex tissue growth and remodeling processes occurring in the endometrium during the reproductive cycle.
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PMCID: PMC295134  PMID: 8083380
21.  Expression of metalloproteinases and their inhibitors in primary pulmonary carcinomas. 
British Journal of Cancer  1992;66(6):1188-1194.
Nine primary pulmonary carcinomas, one metastatic carcinoma, and two malignant pleural mesotheliomas have been analysed for the expression at the mRNA level of metalloproteinases (MPs) and tissue inhibitors of MPs (TIMPs). In situ hybridisation showed TIMP-1 and TIMP-2 transcripts predominantly over tumour stroma and gelatinases evenly distributed over both stromal and tumour cells. While both TIMP-1 and TIMP-2 were expressed in non-neoplastic lungs (NNL) as well as in carcinomas, stromelysin 3 (ST3), 92 kDa gelatinase and interstitial collagenase were expressed only by carcinomas. Expression of these MPs by carcinomas was independent of histologic type and such tumour features as fibrosis or necrosis. The consistent expression of ST3 by all of the carcinomas examined and absence of its expression in NNL indicates that ST3 production is likely associated with the malignant phenotype. However, since 92 kDa gelatinase and interstitial collagenase transcripts were found in some but not all tumour samples, their expression is not a uniform feature of pulmonary carcinomas. The possible prognostic significance of the expression of the latter two enzymes by carcinomas remains to be established.
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PMCID: PMC1978055  PMID: 1457364
22.  Synovial procollagenase activation by human mast cell tryptase dependence upon matrix metalloproteinase 3 activation. 
Journal of Clinical Investigation  1989;84(5):1657-1662.
Mast cells have been implicated in the pathogenesis of the matrix degradation observed in the cartilaginous and osseous structures of the rheumatoid joint. We previously reported that human mast cell tryptase, a 134-kD granule-associated neutral protease, is present in rheumatoid synovium and can activate collagenase in crude culture medium in vitro. the present study attempts to depict the precise mechanism of this activation. To express full activation of latent collagenase, matrix metalloproteinase 3 (MMP-3) or stromelysin, can be activated by tryptase in a time and dose-dependent manner. Tryptase was not capable of generating active collagenase in the crude media from cultured rheumatoid synoviocytes depleted of proMMP-3 by immunoadsorption. In addition, the function of the tissue inhibitor of metalloproteinases (TIMP) was not altered by tryptase, and SDS-PAGE analysis revealed no degradation of TIMP by tryptase. The tryptase dependent activation of synoviocyte procollagenase thereby appears to be entirely dependent upon its ability to activate proMMP-3.
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PMCID: PMC304033  PMID: 2553780
23.  MMP- And TIMP-Secretion by Human Cutaneous Keratinocytes and Fibroblasts – Impact of Coculture and Hydration 
Epithelial-mesenchymal interactions are important in wound healing and scarring, but are difficult to study in vitro. We have previously reported on an in vitro keratinocyte-fibroblast coculture system exploring these interactions and found that coculture modifies the levels of cytokines they secrete. The same coculture model was used to study changes in MMP- and TIMP-activity. We hypothesized that the previously shown decrease of collagen is partly due to increased MMPs.
Adult human cutaneous keratinocytes and fibroblasts were cocultured under serum-free conditions. Keratinocytes were either kept at the air-liquid-interface or hydrated. The conditioned medium was submitted to a multiplex sandwich enzyme-linked immunosorbent assay including gelatinases, collagenases, stromelysins, and tissue inhibitors of metalloproteinases. Collagen content was determined by western blot. Zymography depicted the gelatinases in conditioned media. For confirmation of the coculture results fibroblasts were treated with conditioned media from keratinocyte monocultures as well.
MMP-1, MMP-9, and MMP-10 were mainly secreted by keratinocytes, whereas MMP-2, TIMP-1 and -2 by fibroblasts. MMP-13 was secreted by both cell types at comparable levels. Collagenases, gelatinases, MMP-3, and TIMPs increased significantly in cocultures compared to monocultures. Hydration of keratinocytes revealed a significant increase of MMP-3 and MMP-2, and a decrease of TIMP-2.
Paracrine interactions between keratinocytes and fibroblasts modify strongly MMPs and TIMPs, whereas hydration of keratinocytes had a smaller impact in this context. The observed changes may be in part responsible for reduced collagen in coculture conditioned media. The present coculture experiments reemphasize the role of epidermis in controlling scarring.
doi:10.1016/j.bjps.2010.03.051
PMCID: PMC2939938  PMID: 20542748
MMP-3; MMP-2; TIMP-2; MMP-1; hydration; hypertrophic scar
24.  Matrix Metalloproteinases as Potential Targets in the Venous Dilation Associated with Varicose Veins 
Current drug targets  2013;14(3):287-324.
Varicose veins (VVs) are a common venous disease of the lower extremity characterized by incompetent valves, venous reflux, and dilated and tortuous veins. If untreated, VVs could lead to venous thrombosis, thrombophlebitis and chronic venous leg ulcers. Various genetic, hormonal and environmental factors may lead to structural changes in the vein valves and make them incompetent, leading to venous reflux, increased venous pressure and vein wall dilation. Prolonged increases in venous pressure and vein wall tension are thought to increase the expression/activity of matrix metalloproteinases (MMPs). Members of the MMPs family include collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs and others. MMPs are known to degrade various components of the extracellular matrix (ECM). MMPs may also affect the endothelium and vascular smooth muscle, causing changes in the vein relaxation and contraction mechanisms. ECs injury also triggers leukocyte infiltration, activation and inflammation, which lead to further vein wall damage. The vein wall dilation and valve dysfunction, and the MMP activation and superimposed inflammation and fibrosis would lead to progressive venous dilation and VVs formation. Surgical ablation is an effective treatment for VVs, but may be associated with high recurrence rate, and other less invasive approaches that target the cause of the disease are needed. MMP inhibitors including endogenous tissue inhibitors (TIMPs) and pharmacological inhibitors such as zinc chelators, doxycycline, batimastat and marimastat, have been used as diagnostic and therapeutic tools in cancer, autoimmune and cardiovascular disease. However, MMP inhibitors may have side effects especially on the musculoskeletal system. With the advent of new genetic and pharmacological tools, specific MMP inhibitors with fewer undesirable effects could be useful to retard the progression and prevent the recurrence of VVs.
PMCID: PMC3584231  PMID: 23316963
MMP; endothelium; vascular smooth muscle; extracellular matrix; chronic venous insufficiency disease; TIMP
25.  Rat pancreatic stellate cells secrete matrix metalloproteinases: implications for extracellular matrix turnover 
Gut  2003;52(2):275-282.
Background: Pancreatic fibrosis is a characteristic feature of chronic pancreatic injury and is thought to result from a change in the balance between synthesis and degradation of extracellular matrix (ECM) proteins. Recent studies suggest that activated pancreatic stellate cells (PSCs) play a central role in pancreatic fibrogenesis via increased synthesis of ECM proteins. However, the role of these cells in ECM protein degradation has not been fully elucidated.
Aims: To determine: (i) whether PSCs secrete matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) and, if so (ii) whether MMP and TIMP secretion by PSCs is altered in response to known PSC activating factors such as tumour necrosis factor α (TNF-α), transforming growth factor β1 (TGF-β1), interleukin 6 (IL-6), ethanol, and acetaldehyde.
Methods: Cultured rat PSCs (n=3–5 separate cell preparations) were incubated at 37°C for 24 hours with serum free culture medium containing TNF-α (5–25 U/ml), TGF-β1 (0.5–1 ng/ml), IL-6 (0.001–10 ng/ml), ethanol (10–50 mM), or acetaldehyde (150–200 μM), or no additions (controls). Medium from control cells was examined for the presence of MMPs by zymography using a 10% polyacrylamide-0.1% gelatin gel. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine gene expression of MMP9 and the tissue inhibitors of metalloproteinases TIMP1 and TIMP2. Western blotting was used to identify a specific MMP, MMP2 (a gelatinase that digests basement membrane collagen and the dominant MMP observed on zymography) and a specific TIMP, TIMP2. Reverse zymography was used to examine functional TIMPs in PSC secretions. The effect of TNF-α, TGF-β1, and IL-6 on MMP2 secretion was assessed by densitometry of western blots. The effect of ethanol and acetaldehyde on MMP2 and TIMP2 secretion was also assessed by this method.
Results: Zymography revealed that PSCs secrete a number of MMPs including proteinases with molecular weights consistent with MMP2, MMP9, and MMP13. RT-PCR demonstrated the presence of mRNA for metalloproteinase inhibitors TIMP1 and TIMP2 in PSCs while reverse zymography revealed the presence of functional TIMP2 in PSC secretions. MMP2 secretion by PSCs was significantly increased by TGF-β1 and IL-6, but was not affected by TNF-α. Ethanol and acetaldehyde induced secretion of both MMP2 and TIMP2 by PSCs.
Conclusions: Pancreatic stellate cells have the capacity to synthesise a number of matrix metalloproteinases, including MMP2, MMP9, and MMP13 and their inhibitors TIMP1 and TIMP2. MMP2 secretion by PSCs is significantly increased on exposure to the proinflammatory cytokines TGF-β1 and IL-6. Both ethanol and its metabolite acetaldehyde increase MMP2 as well as TIMP2 secretion by PSCs.
Implication: The role of pancreatic stellate cells in extracellular matrix formation and fibrogenesis may be related to their capacity to regulate the degradation as well as the synthesis of extracellular matrix proteins.
PMCID: PMC1774949  PMID: 12524413

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