Biomedical subjects

R M Hembry

Publications and source records attributed to R M Hembry.

At least 37 records · Page 2Linked to original sources

PubMed1996

Production of collagenase by human osteoblasts and osteoclasts in vivo.

Studies in some animal species have demonstrated the production of metalloproteinases by bone cells, suggesting that they may play a role in bone modeling and remodeling. The aim of the present study was to investigate the expression of collagenase in human bone in situ, using heterotopic and osteophytic bone. Immunohistochemistry was performed on chilled sections of bone, using well characterized polyclonal antibodies to human collagenase. The heterotopic and osteophytic bone exhibited high turnover and both bone modeling and remodeling were evident. Collagenase expression by osteoblasts was demonstrated in cells synthesising matrix and in lining cells; the strongest signal was seen in areas of de novo matrix formation, where bridges of woven bone were being formed between areas of mineralized bone. Collagenase was also present in some osteoclasts associated with eroded bone surfaces and in some mononuclear cells that were present in resorption cavities and in the bone marrow. Our results provide the first demonstration, in situ, of collagenase in human bone and suggest that it may play a role in human bone modeling and remodeling. Production of collagenase by active osteoblasts and lining cells suggest that it may be involved both in matrix formation and activation of bone remodeling. The presence of collagenase in osteoclasts provides further evidence that metalloproteinases may play a role in bone resorption.

Acid Phosphatase↗

PubMed1996

Development of recombinant adenoviruses that drive high level expression of the human metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 and -2 genes: characterization of their infection into rabbit smooth muscle cells and human MCF-7 adenocarcinoma cells.

Remodelling of the extracellular matrix resulting from increased secretion of metalloproteinase enzymes (MMPs) is implicated in many pathological conditions, including rheumatoid arthritis, restenosis following balloon angioplasty, atherosclerosis and cancer cell invasion and metastasis. Clear definition of the normal and pathological function of individual MMPs will benefit from approaches that use gene transfer to produce increases in MMP levels that mimic those observed in pathological conditions. Similarly, gene transfer methods leading to controlled increases in levels of the tissue inhibitor of metalloproteinases (TIMPs) will help to define the function of MMPs both in vitro and in vivo. Gene transfer of TIMPs may also have therapeutic potential in pathological conditions where inhibition of MMP activity may be beneficial. We have used the adenovirus serotype 5 vector system to generate replication-deficient recombinant adenoviruses capable of expressing the MMP-9, TIMP-1 or -2 genes. High level expression is driven by the cytomegalovirus major immediate early promoter (CMV IEP). Efficient and selective over-production of each recombinant protein was shown by immunofluorescence in either rabbit smooth muscle cells (SMC) or human MCF-7 adenocarcinoma cells. High level secretion directly dependent on the multiplicity of infection (MOI) was observed for each functional transgene by gelatin zymography. Using a quantitative ELISA assay, levels of recombinant TIMP-1 were detected when SMC were infected with as low as three plaque forming units (pfu) of virus per cell in vitro. A linear increase in TIMP-1 secretion was observed up to 1000 pfu/cell of virus (0.75 ng/10(4) cells/24 h at 3 pfu/cell to 1243 ng/10(4) cells/24h at 1000 pfu/cell). Similar levels of secretion of MMP-9 and TIMP-2 were observed by Western blot analysis using the same MOI of adenovirus. Thus, recombinant adenoviruses are an efficient and flexible system for high level expression of MMPs and TIMPs and will be useful tools in the study of matrix remodelling in vivo and in vitro.

Adenocarcinoma↗

PubMed1996

Matrix metalloproteinases in the formation of human synovial joint cavities.

Matrix metalloproteinases (MMPs) have been implicated in tissue remodelling in growth and development. A histochemical study of human fetal limbs was undertaken to assess the presence, and consequently the possible role, of MMPs and their inhibitor TIMP-1 (tissue inhibitor of metalloproteinases-1) in synovial joint cavity formation. Cryostat sections of fetal limbs from 7 to 14 wk gestation were stained with specific antibodies to collagenase (MMP-1), gelantinases A (MMP-2) and B (MMP-9), stromelysin (MMP-3) and TIMP-1. Immunoreactive (IR) MMP-1, MMP-2 and MMP-3 were seen chiefly in chondrocytes, but in all cases in zones distant from the joint line before cavity formation. IR-MMP-1 and MMP-2 were also localised both in synovium and on the articular surfaces of joints after cavity formation. In addition IR-MMP-2 was seen in a "collar' of perichondrium alongside the hypertrophic zone of chondrocytes and weakly in bone marrow spaces. IR-MMP-9 was seen in neutrophil leucocytes and in bone marrow spaces. IR-TIMP-1 was generally distributed in connective tissue cells. No IR-MMP (1, 2,3 or 9) was seen along potential joint lines before or at the time of cavity formation, nor was there aspecific decrease in IR-TIMP-1 at this site. These findings confirm a role for metalloproteinases in developmental processes such as cartilage remodelling and bone marrow space formation. MMP-1 and MMP-2 may be involved in the remodelling of developing synovial tissue and the articular surfaces subsequent to cavity formation. However, we have failed to find evidence to indicate that the loss of tissue strength at the joint line which allows synovial joint cavity formation relates to high local levels of MMPS.

Cartilage, Articular↗

PubMed1995

Matrix metalloproteinases and TIMP-1 localization at sites of osteogenesis in the craniofacial region of the rabbit embryo.

BACKGROUND: The matrix metalloproteinases (MMPs) are a family of closely related enzymes, the principal members being the collagenases, gelatinases, and stromelysins. They are synthesized and secreted by connective tissue cells and are capable of degrading all the components of connective tissue matrices at physiological pH. METHODS: Patterns of synthesis and distribution of MMPs and their inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1), are documented in the craniofacial region at sites of bone formation during both intramembranous (e.g., calvaria, maxilla, and mandible) and endochondral ossification (e.g., cartilaginous cranial base and synchondroses) using indirect immunolocalization. RESULTS: MMPs and TIMP-1 were detected both as bright intracellular accumulations, indicating active synthesis, and as diffuse matrix-bound extracellular deposits. Gelatinase-A had an extensive distribution in osteogenic tissues and was detected both in cells of the periosteum and spongiosum and as extracellular deposits in the osteoid layer of newly formed bone. In addition, gelatinase-AB synthesis was detected in osteoclasts. All regions of the early cartilaginous cranial base produced MMPs and TIMP-1, and synthesis continued in the established synchondrosis. MMPs and TIMP-1 were also documented in early tooth germs and in Meckel's cartilage. CONCLUSIONS: These data document a prominent role for MMPs, and in particular gelatinase-A, in mediating matrix degradation during osteogenesis. Their detection in tooth germs and Meckel's cartilage further indicates a role for MMPs and TIMP-1 in matrix turnover during morphogenesis.

Animals↗

PubMed1995

The synthesis of collagenase, gelatinase-A (72 kDa) and -B (95 kDa), and TIMP-1 and -2 by human osteoblasts from normal and arthritic bone.

Bone resorption is a complex multistep process that involves removal of both the organic and mineral constituents of bone matrix by proteolytic enzymes synthesized by osteoblasts and osteoclasts. To further understand the role of matrix metalloproteinases (MMPs) and their specific inhibitors TIMPs (tissue inhibitor of metalloproteinases) in this process, human osteoblasts were obtained by sequential enzymatic digestion from samples of bone from normal donors and patients with various forms of arthritis; first passage cells were used in all experiments and cultured on a type I collagen substratum. Collagenase was detected by an ELISA in supernatants from unstimulated osteoblasts (range 12-730 ng/mL), although the levels did not appear to bear any relationship to the age or clinical status of the patient; treatment with parathyroid hormone (PTH; 2 units/mL) and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3, 10 ng/mL] had no added effect, but mononuclear cell conditioned medium (MCM; 5% v/v) and interleukin-1 alpha (IL-1 alpha; 1 ng/mL) both stimulated collagenase synthesis, in the case of MCM by two orders of magnitude. TIMP-1 was detected in unstimulated cultures by an ELISA (range 320-590 ng/mL), the mean level being three-fold greater than for collagenase and was stimulated by 1,25(OH)2D3 and MCM treatment. Degradation studies showed that, over a 120 h culture period, one third of the collagen substratum was degraded by unstimulated cells. PTH and 1,25(OH)2D3 had no effect on this endogenous level of lysis, but addition of MCM and IL-1 alpha resulted in a significant increase in collagen degradation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1995

The distribution of matrix metalloproteinases and tissue inhibitor of metalloproteinases in colorectal cancer.

Studies suggest that the interplay between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of metalloproteinases (TIMPs) is an important mediator of tumour invasion and metastasis. Using immunohistochemistry, 40 specimens of colorectal cancer were examined for the presence of TIMP-1 and the MMPs, stromelysin, gelatinases A and B and interstitial collagenase. Neither enzyme nor TIMP-1 was detected in histologically normal mucosa. Within malignant tissue, stromelysin and gelatinase A were conspicuously absent in tumor cells but were immunolocalized to the extracellular matrix and for gelatinase A also to peritumoural fibroblast-like cells. Gelatinase B was confined to polymorphonuclear leucocytes. Interstitial collagenase was not identified. TIMP-1 was present in only three of the 40 tumours within the malignant stroma. These observations suggest that the mesenchymal elements of colorectal carcinomas, by acting as a source of MMPs and TIMPs, may modulate tumour invasion.

Collagenases↗

PubMed1995

The distribution of matrix metalloproteinases and tissue inhibitor of metalloproteinases in colorectal cancer.

Studies suggest that the interplay between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of metalloproteinases (TIMPs), is an important mediator of tumour invasion and metastasis. Using immunohistochemistry, 40 specimens of colorectal cancer were examined for the presence of TIMP-1 and the MMPs, stromelysin, gelatinases A and B and interstitial collagenase. Neither enzyme nor TIMP-1 was detected in histologically normal mucosa. Within malignant tissue, stromelysin and gelatinase A were conspicuously absent in tumour cells but were immunolocalized to the extracellular matrix and for gelatinase A also to peritumoural fibroblast-like cells. Gelatinase B was confined to polymorphonuclear leucocytes. Interstitial collagenase was not identified. TIMP-1 was present in only three of the 40 tumours within the malignant stroma. These observations suggest that the mesenchymal elements of colorectal carcinomas, by acting as a source of MMPs and TIMPs, may modulate tumour invasion.

Collagenases↗

PubMed1995

Stromelysin, gelatinase A and TIMP-1 in prosthetic interface tissue: a role for macrophages in tissue remodelling.

Aseptic loosening of prosthetic components is the most important long-term complication of total joint replacement. To investigate the underlying destructive mechanisms, periprosthetic tissues from both well-fixed and loosened sites from six patients, undergoing surgery for aseptic loosening of knee or hip prostheses, were analysed in detail by immunohistochemical methods for the presence of matrix metalloproteinases and tissue inhibitor of metalloproteinases-1 (TIMP-1). The tissues contained small numbers of cells positive for either collagenase, stromelysin, gelatinase A or TIMP-1; these were randomly distributed, neither specifically next to the bone interface nor to wear particles, and the number of positive cells did not correlate with macroscopic observations at operation. Gelatinase A was co-localized in cells with prolyl-4-hydroxylase, an enzyme involved in collagen synthesis. The predominant cell type in these tissues was shown to be the macrophage by the use of cell marker antibodies. Dual localization was not technically possible but the results strongly suggest that monocyte/macrophages were the primary source of gelatinase A and TIMP-1. Stromelysin was immunolocalized on connective tissue matrix in four patients, and gelatinase A in one patient, and were also observed in tissues in which there was no evidence of cellular synthesis of these enzymes. This suggests that secretion had taken place previously, resulting in enzyme bound to matrix for some time. Taken together, these data indicate that localized focal connective tissue remodelling occurs in periprosthetic tissues from both well fixed and loosened sites.

Adult↗

PubMed1995

Immunolocalisation studies on six matrix metalloproteinases and their inhibitors, TIMP-1 and TIMP-2, in synovia from patients with osteo- and rheumatoid arthritis.

OBJECTIVE: To assess the likely importance of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) in the arthritic process. METHODS: Synovial samples from seven joints with rheumatoid arthritis and three osteoarthritic joints were analysed by indirect immunofluorescence microscopy. Using specific human antisera, we documented the frequencies and distributions of collagenase, stromelysins 1 and 2, matrilysin, gelatinases A and B, TIMP-1, and TIMP-2. RESULTS: Stromelysin 1 was found in all synovia, bound to extracellular matrix, within cells, or both, indicating stromelysin synthesis. Matrilysin was present in only one active inflammatory synovium, and focal synthesis of collagenase and gelatinase A was seen in four synovia. Stromelysin 2 and TIMP-2 were not observed, but TIMP-1 synthesis was seen in five synovia, and in two active synovia the distribution of TIMP-1 positive cells was more widespread than that of MMPs. CONCLUSIONS: The presence of stromelysin 1 in all synovia clearly implicates this enzyme in joint damage. Collagenase, gelatinase A and matrilysin may also have a role in rheumatoid arthritis, but are not significant in osteoarthritis. However, marked regional variations were found in the synthesis of these MMPs, indicating not only that these diseases are episodic but that control of enzyme synthesis is focal. Only TIMP-1 may be considered an inhibitory factor.

Adult↗

PubMed1994

Rabbit calvarial osteoblasts in culture constitutively synthesize progelatinase-A, and TIMP-1 and TIMP-2.

Defining the TIMPs (tissue inhibitors of metalloproteinases) produced by non-human bone cells is important because animal models and cells are used extensively for research on bone resorption, and because TIMP-2 can bind to and prevent activation of progelatinase-A. By zymography we show that rabbit calvarial osteoblasts in culture constitutively synthesize TIMP-2 in addition to TIMP-1 and progelatinase-A. Previously we found that human osteoblasts produced only low levels of TIMP-2 under any conditions. We also show that stimulated rabbit osteoblasts can degrade a type I collagen film even though both TIMPs are being synthesized.

Animals↗

PubMed1994

Metalloproteinase production by rabbit articular cartilage: comparison of the effects of interleukin-1 alpha in vitro and in vivo.

To assess the effects of interleukin-1 on intact To assess the effects of interleukin-1 on intact articular cartilage in vitro, explants from young and adult rabbits were cultured with interleukin-1 and the distributions of the matrix metalloproteinases and tissue inhibitor of metalloproteinases (TIMP-1) were investigated by indirect immunofluorescence microscopy. One to 2-week-old cartilage chondrocytes synthesized collagenase in response to pure or crude interleukin-1 (monocyte conditioned medium), with subarticular cells most responsive. Collagenase synthesis was not stimulated in adult articular chondrocytes when explants were treated with either pure or crude interleukin-1. Stromelysin, gelatinase and TIMP-1 could not be demonstrated within any zone of the cartilage, indicating that their synthesis was not stimulated by either pure or crude interleukin-1. The addition of fibroblast growth factors, either alone or in combination with interleukin-1, did not modify these responses. These results contrast markedly with observations on cultured chondrocyte monolayers, where interleukin-1 treatment induces near co-ordinate expression of metalloproteinases. To assess the effects of interleukin-1 in vivo, it was injected into adult rabbit knee joint spaces and the articular cartilage subsequently analysed for evidence of altered metalloproteinase production by immunocytochemistry. No significant increase in metalloproteinase or TIMP-1 synthesis by chondrocytes was detected, although the cartilage matrix showed a marked loss of toluidine blue metachromasia. We conclude that metalloproteinases are not involved in the rapid loss of proteoglycan from cartilage matrix in these situations.

Animals↗

PubMed1994

Immunolocalization of matrix metalloproteinases and TIMP-1 (tissue inhibitor of metalloproteinases) in human gingival tissues from periodontitis patients.

The matrix metalloproteinases (MMPs) collagenase, gelatinase A (72 kDa gelatinase), stromelysin, and their specific inhibitor TIMP-1 (tissue inhibitor of metalloproteinases), were immunolocalized using specific polyclonal antisera in gingival tissues from 21 patients with chronic inflammatory periodontal disease. Monoclonal antibodies against macrophages (Leu-M5), B cells (Leu-14), helper T cells (OKT4), suppressor T cells (OKT8) and the HLA-DR epitope were also used to identify leukocyte subsets. MMPs were observed in connective tissues at sites that histologically showed signs of remodelling. The number and distribution of positive cells varied widely, however, not only between individual biopsy specimens, but also within the same specimen. The same was true for the composition and distribution of the inflammatory cell infiltrate. Moreover, although there was a positive correlation between the number of MMP-producing cells and the severity of inflammation in some specimens, for others with comparable leukocyte subset scoring the number was reduced and sometimes absent altogether. Cells secreting MMPs were fibroblasts, macrophages and epithelial cells. It was not possible to determine unequivocally whether a MMP-positive cell within the connective tissue was a fibroblast or a macrophage, since the antisera recognise both fibroblast and macrophage MMPs and the different fixation requirements for MMPs (4% paraformaldehyde) and Leu-M5 (acetone) precluded co-localization on the same section. TIMP-1 was immunolocalized within connective tissue cells at sites of tissue remodelling. Our results support the hypothesis that tissue-derived MMPs may be involved in tissue remodelling in periodontal disease and conclusively demonstrate that epithelial cells may be involved as well as connective tissue cells.

Antibodies, Monoclonal↗

PubMed1994

Distribution of the matrix metalloproteinases stromelysin, gelatinases A and B, and collagenase in Crohn's disease and normal intestine.

AIMS: To investigate the role of the matrix metalloproteinases (MMPs) in the connective tissue changes seen in the intestine in Crohn's disease. METHODS: Indirect immunofluorescence microscopy using specific antibodies to the MMPs (collagenase, gelatinase A and B, and stromelysin) were used to assess the distribution of these enzymes in normal and diseased intestine. RESULTS: In normal intestine the matrix metalloproteinases were confined to a few isolated inflammatory cells, but in Crohn's disease, the inflammatory infiltrate was associated with increased numbers of polymorphonuclear leucocytes which stained positive for gelatinase B. Stromelysin was also detected extracellularly on the connective tissue matrix in regions of smooth muscle cell proliferation and mucosal degradation. Interestingly, in ulcerative colitis, another inflammatory bowel disease, stromelysin was localised in the lamina propria in regions of mucosal loss. CONCLUSIONS: The increased numbers of inflammatory cells containing gelatinase B, and the localisation of extracellular stromelysin in regions of fibrosis and mucosal degradation, suggest that these enzymes have a role in the pathological changes seen in Crohn's disease. In cases of ulcerative colitis stromelysin was also detected on the lamina propria in regions of mucosal loss, and seems to be associated with the connective tissue changes that precede mucosal loss.

Colitis, Ulcerative↗

PubMed1994

Connective tissue degradation in health and periodontal disease and the roles of matrix metalloproteinases and their natural inhibitors.

Connective tissue remodeling is essential for normal growth and development, and many diseases have long been associated with the breakdown of the collagenous matrix of bone, cartilage, and related tissues. Recent work has established that members of the family of matrix metalloproteinases (MMPs) are key enzymes in matrix degradation. They function at neutral pH and can digest synergistically all the matrix macromolecules. Biochemical and cloning studies indicate that there are three major groups, collagenases, gelatinases, and stromelysins. Naturally occurring inhibitors, TIMPs (Tissue Inhibitors of MetalloProteinases), are important controlling factors in the actions of MMPs, and tissue destruction in disease processes often correlates with an imbalance of MMPs over TIMPs. The major inhibitor is TIMP-1 (or TIMP), a 30-kDa glycoprotein that is synthesized by most cells. The expression of MMPs and TIMPs by cells is regulated by many cytokines (particularly interleukin-1, IL-1), growth factors, and hormones, some of which are specific to cell type and others that are ubiquitous (e.g., transforming growth factor beta, TGF-beta). One way in which pathogenic organisms might mediate tissue degradation in periodontal diseases is through the ability of cell wall antigens to stimulate cytokine production by circulating mononuclear cells. These would then induce MMP synthesis by resident gingival cells, thereby initiating degradative events. Direct in vivo evidence for the source of collagenase and other MMPs in periodontal tissues is limited. By using specific polyclonal antibodies and indirect immunofluorescence, we could demonstrate the presence of collagenase, stromelysin-1, gelatinase A, and TIMP in human gingival biopsy specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Connective Tissue↗

PubMed1994

The effects of selective inhibitors of matrix metalloproteinases (MMPs) on bone resorption and the identification of MMPs and TIMP-1 in isolated osteoclasts.

We have compared the effects of a general matrix metalloproteinase (MMP) inhibitor (CT435) with those of a concentration-dependent specific gelatinase inhibitor (CT543; Ki < 20 nM) on bone resorption in vitro. The test systems consisted of measuring: (i) the release of 45Ca2+ from prelabelled mouse calvarial explants; (ii) the release of 45Ca2+ from prelabelled osteoid-free calvarial explants co-cultured with purified chicken osteoclasts; and (iii) lacunar resorption by isolated rat osteoclasts cultured on ivory slices. Both CT435 and CT543 dose-dependently inhibited the release of 45Ca2+ from neonatal calvarial bones stimulated by either parathyroid hormone or 1,25-dihydroxyvitamin D3. Moreover, CT543 produced a 40% inhibition at a concentration (10(-8) M) selective for the inhibition of human gelatinases A and B. CT435 (10(-5) M) and CT543 (10(-5) M) partially inhibited the release of 45Ca2+ from osteoid-free calvarial explants by chicken osteoclasts with a maximum of approximately 25% for unstimulated cultures, and approximately 36% for cultures stimulated by interleukin-1 alpha (IL-1 alpha; 10(-10) M). Neither inhibitor prevented lacunar resorption on ivory by unstimulated rat osteoclasts, but the compounds produced a partial reduction in both the number and total surface area of lacunae in IL-1 alpha-stimulated cultures, with maximal action at 10(-5) M. Neither of the inhibitors affected protein or DNA synthesis, nor the IL-1 alpha-stimulated secretion of the lysosomal enzyme beta-glucuronidase. Immunocytochemistry demonstrated that isolated rabbit osteoclasts constitutively expressed gelatinase A and synthesized gelatinase B, collagenase and stromelysin, as well as the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) following IL-1 alpha stimulation. These experiments have shown that in addition to collagenase, gelatinases A and B are likely to play a significant role in bone resorption. They further suggest that MMPs produced by osteoclasts are released into the sub-osteoclastic resorption zone where they participate in bone collagen degradation.

Animals↗

PubMed1994

Regional and temporal changes in the synthesis of matrix metalloproteinases and TIMP-1 during development of the rabbit mandibular condyle.

Connective tissues synthesise and secrete a family of matrix metalloproteinases (MMPs; collagenases, gelatinases and stromelysins) capable of degrading all the components of connective tissue matrices at physiological pH. We document the patterns of synthesis and distribution of MMPs and the tissue inhibitor of metalloproteinases-1 (TIMP-1) within the developing rabbit mandibular condyle using immunofluorescence microscopy. MMPs and TIMP-1 were detected both as bright intracellular accumulations within Golgi vesicles and also as diffuse matrix-bound extracellular deposits. Cells in the articular zone, proliferative zone, condylar cartilage and bone of the mandibular ramus were shown to produce all 3 classes of MMPs and TIMP-1 with the exception of stromelysin, which was not synthesised by cells of the bone of spongiosum. Temporal synthesis of MMPs and TIMP-1 within these regions varied during the period 18 d postcoitum to 14 d postnatum. Our results document unique patterns of MMP and TIMP-1 synthesis during embryonic and early postnatal development of condylar cartilage and support the concept that cells synthesise and secrete MMPs and TIMP-1 before undergoing proliferation and hypertrophy. A comparison of these results with data in the rabbit growth plate show many similarities, but some differences exist that probably reflect differences in the modes of growth of the 2 cartilages.

Animals↗

PubMed1993

Rabbit models of arthritis: immunolocalization of matrix metalloproteinases and tissue inhibitor of metalloproteinase in synovium and cartilage.

The distribution of the matrix metalloproteinases, collagenase, stromelysin, gelatinases A and B, and the tissue inhibitor of metalloproteinases in cartilage and synovium removed from rabbits up to 27 days after induction of two models of arthritis was investigated by immunolocalization. Following intra-articular injection of poly-D-lysine/hyaluronic acid coacervate, collagenase and stromelysin were found bound to cartilage matrix, but there was little increase in chondrocyte synthesis of these enzymes. The synovium underwent a complex wound healing response involving invagination and encapsulation of the coacervate and inflammatory cell debris, during which all four metalloproteinases and tissue inhibitor of metalloproteinase could be immunolocalized. The second model, intra-articular injection of ovalbumin into sensitized rabbits, caused considerable chondrocyte necrosis; collagenase was found bound to cartilage matrix on day 13, although again there was little evidence of synthesis by chondrocytes. Inflammatory cell infiltration of meniscoid synovia took place initially, followed by fibrosis involving macrophagelike cells secreting gelatinase A. In both models there was rapid loss of glycosaminoglycan metachromasia from the cartilage matrix. These results are discussed in relation to current knowledge of metalloproteinase involvement in the chronic rheumatoid synovial pannus erosion of cartilage in humans. The data suggest that there are considerable differences between rheumatoid arthritis and these models, and their use must therefore be carefully defined.

Animals↗

PubMed1992

Secretion of 72 kDa type IV collagenase/gelatinase by cultured human lipocytes. Analysis of gene expression, protein synthesis and proteinase activity.

The matrix metalloproteinases play an important role in matrix degradation, but there is limited information about this family of enzymes in either normal or diseased human liver. In this study, we have examined the synthesis of a 72 kDa type IV collagenase/gelatinase by human hepatic lipocytes in primary culture. Hepatic lipocytes were isolated from wedges of normal human donor liver by Pronase/collagenase perfusion, purified by density-gradient centrifugation, and established in primary culture on uncoated plastic. By Northern-blot analysis, the total RNA extracted from cultured human lipocytes was found to contain 3.4 kb mRNA for the 72 kDa type IV collagenase/gelatinase. Low levels of expression of this mRNA were observed in freshly isolated lipocytes but expression increased with the duration of lipocyte culture. Using anti-human 72 kDa type IV collagenase/gelatinase IgG, synthesized enzyme was immunolocalized to monensin-treated human lipocyte cultures. De novo synthesis and secretion of 72 kDa type IV collagenase/gelatinase were confirmed by immunoprecipitation of radiolabelled enzyme from medium obtained from [35S]methionine-treated cells. Activity of the secreted enzyme was demonstrated by gelatin-zymography and by degradation of soluble, radiolabelled [14C]gelatin. The enzyme was released both in active and latent pro-enzyme forms and its inhibition profile was that of a metalloproteinase. These studies indicate that cultured human hepatic lipocytes express the gene for the 72 kDa type IV collagenase/gelatinase, and secrete this enzyme, particularly in prolonged primary culture. As this enzyme exhibits degradative activity against basement membrane collagen, its release by activated hepatic lipocytes in the space of Disse could lead to disruption of the normal subendothelial liver matrix. It is suggested that this enzyme may have an important role in human liver injury and fibrosis.

Adipose Tissue↗