Concentration of collagen, aggrecan and cartilage oligomeric matrix protein (COMP) in synovial fluid from equine middle carpal joints.

The aim of the present investigation was to study the metabolic activity of the third carpal bone and the release of COMP, aggrecan and collagen type II molecules in the synovial fluid as a result of injury. Cartilage oligomeric matrix protein (COMP), aggrecan and collagen type II or fragments of these molecules released to the synovial fluid and serum (COMP) were quantified in samples from 73 left equine middle carpal joints from 2 breeds with different activity profiles (52 Standardbred trotters [STB] and 21 Swedish Warmblood riding horses [SWH]) and different articular cartilage lesions. Synovial and serum samples were analysed using inhibition ELISA for COMP and aggrecan. An ELISA that combines features of both the competitive and capture ELISAs was used for collagen type II. COMP and aggrecan concentrations decreased in synovial fluid from the joints with moderate lesions of STB compared with the normal joints; COMP from 16.6 to 12.0 microg/ml and aggrecan from 93.0 to 68.1 microg/ml. In serum, COMP concentrations were also lowered in the STB with moderate lesions compared with the normal joints, while in the SWH, the COMP concentration in synovial fluids from joints with moderate lesions was somewhat increased at 19.6 microg/ml compared with the normal joints (17.6 microg/ml). The ratio between aggrecan/COMP in the synovial fluid from joints with moderate lesions was higher in the STB (6.2) than in the SWH (3.4). The level of collagen type II in synovial fluid was higher in the SWH (8.8 microg/ml) than the STB (1.6 microg/ml), but there was no correlation between joint damage and collagen concentrations in synovial fluids (10.0 and 1.8 microg/ml in joints with moderate lesions from SWH and STB, respectively). A marked difference in COMP synthesised upon metabolic labelling between the normal and osteoarthritic cartilage was seen and the synthesis of COMP in the articular cartilage of the third carpal bone with moderate articular lesions (from an STB) was lower than in the joint with mild lesions. This difference between breeds may reflect different load characters, in release of macromolecules in osteoarthritic and normal joints. This a novel finding that should be considered in studies of equine traumatic arthritis.

Evaluation of the effects of swainsonine, captopril, tangeretin and nobiletin on the biological behaviour of brain tumour cells in vitro.

Although intrinsic tumours of the brain seldom metastasize to distant sites, their diffuse, infiltrative-invasive growth within the brain generally precludes successful surgical and adjuvant therapy. Hence, attention has now focused on novel therapeutic approaches to combat brain tumours that include the use of anti-invasive and anti-proliferative agents. The effect of four anti-invasive agents, swainsonine (a locoweed alkaloid), captopril (an anti-hypertensive drug), tangeretin and nobiletin (both citrus flavonoids), were investigated on various parameters of brain tumour invasion such as matrix metalloproteinase (MMP) secretion, migration, invasion and adhesion. A standard cytotoxicity assay was used to optimize working concentrations of the drugs on seven human brain tumour-derived cell lines of various histological type and grade of malignancy. A qualitative assessment by gelatin zymography revealed that the effect of these agents varied between the seven cell lines such that the low grade pilocytic astrocytoma was unaffected by three of the agents. In contrast, downregulation of the two gelatinases, MMP-2 and MMP-9 was seen in the grade 3 astrocytoma irrespective of which agent was used. Generally, swainsonine was the least effective whereas the citrus flavonoids, particularly nobiletin, showed the greatest downregulation of secretion of the MMPs. Furthermore, captopril and nobiletin were most efficient at inhibiting invasion, migration and adhesion in four representative cell lines (an ependymoma, a grade II oligoastrocytoma, an anaplastic astrocytoma and a glioblastoma multiforme). Yet again, the effects of the four agents varied between the four cell lines. Nobiletin was, nevertheless, the most effective agent used in these assays. In conclusion, the differential effects seen on the various parameters studied by these putative anti-invasive agents may be the result of interference with MMPs and other mechanisms underlying the invasive phenotype. From these pilot studies, it is possible that these agents, especially the citrus flavonoids, could be of future therapeutic value. However, further work is needed to validate this in a larger study.

The effects of exogenous growth factors on matrix metalloproteinase secretion by human brain tumour cells.

Matrix metalloproteinases (MMPs) are a growing family of zinc-dependent endopeptidases that are capable of degrading various components of the extracellular matrix. These enzymes have been implicated in a variety of physiological and pathological conditions including embryogenesis and tumour invasion. The synthesis of many MMPs is thought to be regulated by growth factors, cytokines and hormones. In this study, we investigated the effects of five exogenous growth factors known to be expressed by gliomas [epidermal growth factor (EGF), basic growth factor (bFGF), transforming growth factor beta (TGF-beta1,2) and vascular endothelial growth factor (VEGF)].on MMP-2 and MMP-9 expression in an ependymoma, two grade III astrocytomas, a grade III oligoastrocytoma and a benign meningioma. Zymogram analysis revealed that the effects of the growth factors depended upon the cell lines used in the study. Growth factors generally up-regulated MMP-2 and MMP-9 expression in the gliomas but were least effective in the meningioma; the effect being most prominent with TGF-beta1 and TGF-beta2 in all the cell lines. It is hypothesized that paracrine growth factor interplay may be crucial in the regulation of MMP expression by glioma invasion of the normal brain.

Primary structure of the helical domain of porcine collagen X.

The entire primary structure of the collagen X helical region is presented, including identification of the extensive post-ribosomal modifications by amino acid sequencing and mass spectrometry. As in collagen I, a single residue of 3-hydroxyproline was identified, but for collagen X this was located near the N-terminal end of the helix. Lysine residues in collagen X are extensively hydroxylated/glycosylated: at least 11 sites were localized and shown to be fully glycosylated, exclusively as glucosyl-galactosyl derivatives. The lysine-derived crosslinks, dihydroxylysinonorleucine and hydroxylysinonorleucine, were shown to be present in a 3:2 molar ratio primarily within the C-terminal portion of the helix.

Comparative analysis of matrix metalloproteinases by immunocytochemistry, immunohistochemistry and zymography in human primary brain tumours.

Matrix metalloproteinases (MMPs) are a growing family of zinc-dependent endopeptidases which are characterised by their ability to degrade various extracellular matrix (ECM) components. The family includes collagenases, gelatinases, stromelysins, metalloelastase and membrane type metalloproteinases. Consistent with their proteolytic activities, MMPs have been implicated in a variety of physiological and pathological conditions, such as normal embryogenesis, tissue morphogenesis and are thought to play a role in facilitating tumour cell invasion of the normal brain. In this comparative study, we have used zymography, immunohistochemical and immunocytochemical techniques to demonstrate the expression of gelatinase-A and B (MMP-2 and 9, respectively) and membrane type metalloproteinase (MMP-14) in 8 intrinsic human primary brain tumours of various histological type and grade. Zymography results showed that MMP-2 was the most prominent proteolytic enzyme in all the cell lines studied (with one exception), while MMP-9 was only faintly expressed. However, the corresponding paraffin sections showed no expression of either MMP-2, 9 or 14 within the tumour cells, positivity being confined to haematogenous cells and the vascular endothelium. Fluorescence immunocytochemical studies, using monoclonal antibodies to MMP-2, 9 and 14, showed granular cytoplasmic reactivity in vitro. In addition, there was strong focal positivity at the cell membrane with MMP-14 in some high grade tumours suggesting that MMPs are produced at the leading edge of the cell by individual subpopulations of invading glia, in small quantities and on demand in vivo. It can be concluded that local microenvironmental conditions in vitro appear to stimulate such MMP activity.

An inverse correlation between expression of NCAM-A and the matrix-metalloproteinases gelatinase-A and gelatinase-B in human glioma cells in vitro.

Matrix metalloproteinases (MMPs) are an homologous family of proteolytic enzymes capable of degrading components of the extracellular matrix (ECM) and thereby facilitating the invasion of tumour cells into normal tissues. The neural cell adhesion molecules (NCAMs) of neuronal and glial cells provide a Ca2+-independent mechanism for cell-cell and cell-ECM adhesion. NCAMs are downregulated to promote cell disaggregation during cell migration in the developing nervous system whereas MMPs facilitate migration. Recent studies have shown downregulation of MMP secretion in rat glioma cells transfected with an NCAM cDNA, implying an inverse correlation between NCAM and MMP expression. The purpose of this study was to establish whether such a correlation could be demonstrated in a panel of nine human glioma cell-lines, one metastatic carcinoma and one foetal astrocyte derived cell line. The secretion of two MMPs, 72 kDa gelatinase (MMP-2 or gelatinase-A) and 92 kDa gelatinase (MMP-9 or gelatinase-B), was investigated using SDS-PAGE zymography; NCAM-A was assayed by an immunochemiluminescent assay following SDS-PAGE of whole-cell extracts. An inverse correlation was found between the expression of NCAM-A and that of both MMPs studied although the patterns of expression showed no obvious correlation with histological type or grade of the parent tumours. Our results suggest that downregulation of NCAM-A may contribute to tumour invasiveness by promoting both cell disaggregation and protease secretion.

Deer antler does not represent a typical endochondral growth system: immunoidentification of collagen type X but little collagen type II in growing antler tissue.

The collagen isotypes present at early (6 week) and late (5 month) stages of growing deer antler were isolated and identified. Pepsin-digested collagens were separated by differential salt fractionation, SDS-PAGE and Western blotting and subsequently identified by immunostaining. Cyanogen bromide digestion of antler tissue was used to establish a collagen type-specific pattern of peptides, and these were also identified by immunoblotting. Collagen type I was found to be the major collagen in both early- and late-stage antler. Collagen type II was present in the young antler in small amounts but was not confined to the soft "cartilaginous" tip of the antler. Collagen type XI was found in the pepsin digest of the young antler, but collagen type IX was not present at either stage of antler growth. Collagen type X was found in the young antler in all fractions studied. Microscopic study showed that the deer antler did not possess a discrete growth plate as found in endochondral bone growth. Unequivocal immunolocalization of the different collagen types in the antler were unsuccessful. These results show that, despite the presence in the antler of many cartilage collagens, growth does not occur through a simple endochondral process.

Preparation of biotinylated, affinity-purified antibodies for enzyme-linked immunoassays using blotting membrane as an antigen support.

A method combining the affinity purification and biotinylation of antibodies has been developed utilizing the high-capacity binding of blotting membranes and glutaraldehyde treatment to immobilize antigen. Following reaction of the antisera with the membrane-bound antigen, the biotinylation of the antibodies is performed in situ, before release of the IgG-biotin complex with low pH buffer. The antibody is able to recognize antigen in subsequent reactions in enzyme-linked immunosorbent assays, and preliminary results have shown that the antibody can also be used in immunocytochemical localization of antigen in frozen sections. Biotinylation in situ may protect the variable region of the IgG compared to antibodies biotinylated in solution, thus increasing their antigen recognition. The method has the advantage of being suitable for multiple use of the antigen-coated membrane, making it particularly attractive where only small amounts of antigen may be available.

Identification of lysine-derived crosslinks in porcine collagen type X from growth plate and newly mineralized bone.

Intact collagen type X cannot readily be extracted from the growth plate. Both the use of pepsin to release this molecule from tissue and the relative solubility of collagen type X following treatment of chick embryos with beta-aminopropionitrile (Chen et al., 1992) suggest that the insolubility may by brought about by the formation of lysine-derived crosslinks. By immunocytochemical labelling using antibodies specific for collagen type X, we have shown that this collagen type persists in the cartilaginous spicules present in metaphyseal bone and appears to be colocalized with collagen type II. The combined concentration of the reducible bifunctional crosslinks, dihydroxylysinonorleucine and monohydroxylysinonorleucine, in collagen type X isolated from the premineralized and newly mineralized growth plate was about 0.6 residues/ molecule, a level which might explain the relative intractability of collagen type X. Pyridinoline and deoxypyridinoline were present in very small amounts in collagen type X; this suggests that, unlike the situation in other types of collagen, few of the bifunctional crosslinks undergo maturation to pyridinium compounds. Although it is clear that collagen type X contains lysinederived crosslinks, work is in progress to establish which molecule also participates in the formation of these crosslinks.

Collagen type X: a component of the surface of normal human, pig, and rat articular cartilage.

Collagen type X, a protein generally associated with hypertrophic chondrocytes of avian and mammalian growth plate during endochondral growth of long bones, has been previously shown to be present during disruption of normal metabolic status of articular cartilage during osteoarthritis. We have demonstrated that collagen type X is present as a component of normal articular cartilage in adult human, growing pig and new-born rat. The protein was immunolocalized in these tissues at the surface of the articular cartilage and for human tissue there was some staining of chondrocytes adjacent to the "tidemark' zone. The presence of collagen type X was confirmed by isolating the protein from these tissues and its identification by SDS-PAGE and immunodetection with antibodies specific for collagen type X.

The effect of exogenous gangliosides on matrix metalloproteinase secretion by human glioma cells in vitro.

Matrix metalloproteinases (MMPs) are zinc-dependent peptidases and are amongst those enzymes responsible for extracellular matrix (ECM) degradation during tumour-cell migration. Gangliosides are a family of acidic membrane glycolipids thought to play a role during cell development, differentiation and oncogenic transformation. In this descriptive study, we investigated the effects of six exogenous gangliosides (GM1, GM3, GD1a, GD1b, GD3 and GT1b) on the secretion of MMP-2 (72 kDa gelatinase or gelatinase-A) and MMP-9 (92 kDa gelatinase or gelatinase-B). Cell-conditioned media from eight human glioma-derived cell-lines served as the source of MMPs and were investigated using SDS-PAGE zymography. Six of the cell lines showed upregulation of secretion of both enzymes by all six gangliosides. Of the remaining two cell lines, one showed inhibition of MMP secretion by all gangliosides and the other had a small but differential response to the range of gangliosides investigated. These results suggest that gangliosides may stimulate glioma cell invasiveness by promoting MMP expression.

The influence of sequential, in vitro passage on secretion of matrix metalloproteinases by human brain tumour cells.

Matrix metalloproteinases (MMP) are a family of zinc-dependent enzymes which degrade various components of the extracellular matrix (ECM) and play an important role in facilitating tumour cell invasion of the normal brain. The family includes the gelatinases, stromelysins and collagenases. Preliminary studies have shown that there is a differential expression four metalloproteinases in human brain tumour cell lines derived from neoplasms of various histological types and grades of malignancy. Morphological and antigenic changes in human glioma-derived cell lines over many serial in vitro passages have been reported in earlier studies. When established cell lines are maintained in culture over a long period, it is possible that the secretion of enzymes such as metalloproteinases may differ according to the passage level examined. This report presents a study on the secretion of four matrix metalloproteinases - interstitial collagenase (MMP-), 72-kDa and 92-kDa gelatinases (MMP-2 and MMP-9 respectively), and stromelysin (MMP-3) - in three human brain tumour-derived cell lines at sequentially increasing passage numbers, ranging from passage 2 to passage 50; foetal astrocytes were used as a positive control. Reverse zymography and substrate degradation analysis were employed to demonstrate the presence of these enzymes in cell- conditioned culture medium. Aminophenyl mercuric acetate (APMA) was used to activate latent zymogen. Results demonstrate that there is no definite pattern of change in the levels of enzyme secretion common to all cell lines studied. Instead, the fluctuations in APMA- activated metalloproteinase activity in serial passage seems to vary considerably depending on the cell line and the type of enzyme studied. The variation in metalloproteinase expression observed on serial passage may be due to in vitro selection processes or karyotype evolution where the transcription of either the enzyme and/or its inhibitor may be affected. Thus an imbalance of the two products could be occurring in serial passage. Ideally, experiments requiring the measurement of relative enzyme activities should use cultures as near to the biopsy stage as possible, i.e. very low passages, to avoid artifacts that may arise on prolonged culturing.

Leptomeningeal tissue: a barrier against brain tumor cell invasion.

BACKGROUND: Primary brain tumors are characterized by an extensive infiltrative growth into the surrounding brain tissue. This process is confined to the central nervous system, and tumor cell metastasis to other organs is rare. However, other tumors of non-neural origin may frequently metastasize to the central nervous system. PURPOSE: The purpose of the present study was to examine the invasive behavior of different glioma cells into tissues of neural (brain aggregates) as well as non-neural origin (leptomeningeal tissue). Using the same target tissues, the invasive characteristics of two neural metastatic tumors (one malignant melanoma and one small-cell lung carcinoma) were also studied. This direct comparison of the invasive behavior between tumors of neural and non-neural origin provides valuable information regarding the mechanisms of glioma cell dissemination in the central nervous system. METHODS: The in vitro invasive behavior of human tumors of the central nervous system into human leptomeningeal tissue as well as into normal rat brain tissue was studied. For this purpose, a co-culture system consisting of tumor biopsy specimens, human leptomeningeal cell aggregates, and brain cell aggregates was established. Three glioblastomas, one oligodendroglioma, one meningioma, one small-cell lung carcinoma, and one malignant melanoma were studied. RESULTS: In co-cultures of gliomas and leptomeningeal cell aggregates, a well-defined border between the two tissues was observed. The brain cell aggregates, in contrast, were consistently invaded by the glioma cells. The brain metastases showed a different invasion pattern. The metastatic cells invaded and progressively destroyed leptomeningeal cell aggregates, whereas they did not invade the brain cell aggregates. Upon confrontation of the leptomeningeal tissue with the meningioma, a fusion of the two tissues was observed. Immunostaining of the leptomeningeal tissue showed a strong expression of the basement membrane components fibronectin, collagen type IV, and laminin with no expression of glial fibrillary acidic protein, neuron-specific enolase, or S-100 protein. CONCLUSIONS: The present study indicates that there may be important biologic differences between the invasive behavior of gliomas and non-neuroepithelial tumors. Our co-culture experiments suggest that leptomeningeal cells and associated acellular components may constitute a barrier against glioma cell invasion. However, this barrier may not be functional for metastatic tumors to the brain. The presence of glioma cells within the leptomeninges should not necessarily be taken as evidence of aggressive growth or as an indicator of malignancy.

Laminin: a potential inhibitor of rat glioma cell invasion in vitro.

The lack of metastatic behaviour of primary glioma is poorly understood. A possible natural barrier accounting for this phenomenon may be the proteins of the extracellular matrix which are found in the basement membranes of the blood vascular system. This hypothesis is reinforced by the finding that glioma invasion in vitro using a syngeneic model system results in a lack of invasion of areas of target tissue which contain extracellular matrix proteins. The study was extended by examining the effect of the incorporation of these proteins during the formation of fetal rat brain cell aggregates and glioma spheroids and on the invasion of aggregates by tumour spheroids. Laminin was shown to reduce the size of the aggregates and spheroids during their formation while fibronectin and type IV collagen had no effect. Laminin also prevented the invasion of the tumour spheroid into the target aggregate and appeared to inhibit migration of glioma cells on laminin coated tissue culture plastic.

Turnover rates of different collagen types measured by isotope ratio mass spectrometry.

The rates of collagen turnover in different tissues have been estimated in growing rats previously exposed to gaseous 18O2. The abundance of the stable isotope was measured using isotope ratio mass spectrometry following combustion of isolated collagen-derived hydroxyproline. Using this method, problems of label reutilization associated with radiolabelling methods are avoided. In general the results confirm the slow turnover rates with half-lives of total collagen in skin, muscle and gut of 74, 45 and 244 d, respectively. The use of cyanogen bromide digests of whole tissues followed by isolation of collagen type-specific peptides has allowed the comparison of turnover rates of collagen types I and III, indicating that collagen type III is turned over more rapidly than type I.

Invasion of brain tissue by primary glioma: evidence for the involvement of urokinase-type plasminogen activator as an activator of type IV collagenase.

The immunocharacterization of a metalloproteinase isolated from rat glioma cell conditioned medium is described and confirms that the enzyme is identical to type IV collagenase. Free, active plasminogen activator (PA) and PA-PAI complexes were identified as being secreted by the same cells. Using affinity-purified metalloproteinase we demonstrate that the enzyme can be partially activated by u-PA but not by plasmin in vitro. On the basis of these findings and previous published work we propose a scheme for the proteolytic degradation of normal brain tissue during tumour invasion.

A metalloproteinase, capable of destroying cultured brain tissue isolated from rat glioma cells.

A continuous rat glioma cell line, BT5C, which causes severe invasion and tissue destruction when cocultured with fetal rat brain aggregates, secreted into the culture medium a metalloproteinase in a hMr latent (86000) and an lMr active (76000) isoform. Purified preparations of the enzyme were added to cultures of fetal rat brain aggregates, which were then examined by light and by scanning electron microscopy. After 48h of enzyme exposure, destruction of the neural tissue was observed. This was morphologically similar to the tissue destruction seen when BT5C spheroids were cocultured with brain aggregates. The protease had no effect on the morphology of BT 5C tumour spheroids. The report suggests a possible role of metalloproteinases in tissue degradation during brain tumour invasion.

Isolation and characterization of a metalloproteinase secreted by rat glioma cells in serum-free culture.

The isolation of a metalloproteinase secreted by a rat glioma cell line (BT5C) in serum-free media is described. After affinity purification, the activity was present as a double band with Mr 86000 and 76000 both of which required CaCl2 for activity. The enzyme was able to degrade gelatin but not casein. It was unable to degrade native types I, III, IV and V collagens but their denatured counterparts were degraded. Using a radiolabel release assay the enzyme was inhibited by EDTA, 1:10 phenanthroline and TIMP confirming that it belongs to the family of metalloproteinases. Its activity was not affected by either serine or cysteine protease inhibitors. The proteinase was activated by APMA but was unaffected by trypsin treatment.

A radiolabel-release microwell assay for proteolytic enzymes present in cell culture media.

A modified method for the measurement of proteolytic enzyme activity in cell culture-conditioned media has been developed. Using the release of 3H-labeled peptides from 3H-labeled gelatin the method is performed in microwell plates. The substrate is insolubilized and attached to the wells by glutaraldehyde treatment, thus eliminating the need for a precipitation step at the end of the assay. The assay is sensitive, reproducible, and convenient for small sample volumes. The effect of different protease inhibitors on activity can be assessed rapidly allowing an early characterization of the enzyme. It can also be adapted to microplate spectrophotometric analysis by staining residual substrate with Coomassie blue.