Influence of heparin(s) on the interleukin-1-beta-induced expression of collagenase, stromelysin-1, and tissue inhibitor of metalloproteinase-1 in human gingival fibroblasts.

Here, we describe the influence of heparin(s) on the interleukin-1-beta (IL-1beta)-induced expression of collagenase (matrix metalloproteinase-1, MMP-1), stromelysin-1 (matrix metalloproteinase-3, MMP-3) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in human gingival fibroblasts (HGF). Amounts of secreted enzymes and inhibitors as well as their mRNA steady-state levels increased significantly following supplementation of HGF culture medium with 2 ng/mL of IL-1 beta1. Addition of heparin to cell culture medium 1 hour following IL-1beta decreased MMP and TIMP-1 expression in a dose-dependent manner. The inhibitory effect of heparin was significant at a concentration as low as 1 microg/mL. These findings could be reproduced with a low Mr heparin fragment devoid of anticoagulant activity. Heparin and fragments might therefore reduce the excessive proteolytic capacity of the gingival fibroblast during inflammation and could be useful as pharmacological agent(s) in gingivitis and periodontitis.

Collagen zymography as a sensitive and specific technique for the determination of subpicogram levels of interstitial collagenase.

We describe the use of casting native collagen type I in SDS-polyacrylamide gel (collagen zymography) for the determination of interstitial collagenase. As with gelatin, the incorporation of collagen in the gels reduced protein migration and the need for making corrections for an accurate Mr evaluation. This method proved to be very sensitive: 0.1 pg of APMA-activated procollagenase could be detected, and specific levels of active gelatinase or stromelysin lower than 5 ng were inactive under our experimental conditions. It was used to demonstrate the increased expression of collagenase following treatment of human gingival fibroblasts with interleukin-1 beta; the amounts of enzyme quantified by either collagen zymography or immunodot blot assay are comparable.

Increased expression of gelatinases A and B by skin explants from patients with anetoderma.

The extent of alterations to the elastic fibre network in lesional skin areas of three patients with anetoderma was assessed by quantitative image analysis of tissue sections and compared with morphometric parameters from unaffected sites of the same individuals. In the anetodermic skins pre-elastic fibres were undetectable or extremely rare: the volume fraction (Vv%) occupied by these pre-elastic fibres was 0-0.3%, while in unaffected skins the Vv% occupied by pre-elastic fibres was 0.5-0.8%. A nearly complete absence of dermal elastic fibres in lesional skins from the three patients was evidenced (Vv% = 0.2-0.3%). Organ cultures were performed using explants from skin with or without anetodermic lesions to quantify the expressions of elastase-type proteinases. All tissues from anetodermic lesions expressed proforms of gelatinases A and B and the activated form of gelatinase A; their levels increased with the culture time. In comparison, enzymatic activities on oligopeptide substrates specific for leucocyte elastase and fibroblast plasma membrane-associated metalloelastase were not detected in the conditioned media of any explants at any time of culture from 1 to 5 days. Increased production of progelatinases A and B and activation of progelatinase A could be mainly responsible for the degradation of skin elastic fibres demonstrated in anetodermic skins.

Influence of successive and combined ultraviolet A and B irradiations on matrix metalloelastases produced by human dermal fibroblasts in culture.

To study the cumulative influence of UV irradiations on skin matrix alterations, human skin fibroblasts were irradiated successively three-fold, at 24 h intervals, with UVA (3x5J/cm2), UVB (3x8mJ/cm2), UVA plus UVB (3x5J/cm2 and 3x8mJ/cm2) and the levels of 92 kDa gelatinase (pro-MMP9), 72 kDa gelatinase (pro-MMP2) and plasma-membrane elastase type protease were determined, following subsequent 24-h culture in 10% serum-containing medium. UV irradiations had only minor influence (1.4-fold increase for UVB) on secreted levels of pro-MMP2 and decreased the amount of plasma membrane elastase produced by cells. It did however, for UVA and UVB alone, induce a significant increase of 66 kDa activated MMP2 production: 2.5- and 1.7-fold respectively. Such enhancement was not observed when combined irradiations were administered. UV exposure possessed a much higher influence on pro-MMP9 secretion by dermal fibroblast enhancing enzyme levels by 2.5-, 6.5- and 5-fold for UVA, UVB and UVA+UVB, respectively.

Influence of interleukin 1 beta on tenascin distribution in human normal and osteoarthritic cartilage: a quantitative immunohistochemical study.

OBJECTIVE: To determine the influence of IL-1 beta on the presence and the distribution of tenascin in matrix of human normal and osteoarthritic cartilage explants. METHODS: Cartilage was grown in organotypic culture with or without IL-1 beta (10 ng ml1). Tenascin antigen was detected on cryopreserved cartilage sections by immunohistochemical techniques with a monoclonal antibody directed against all tenascin isoforms (BC-4), and then quantified by video imaging densitometry. RESULTS: Tenascin was present in normal cartilage explants and increased in osteoarthritic cartilage explants. Treatment of normal and osteoarthritic cartilage explants with IL-1 beta (10 ng ml-1) induced an increase in tenascin content, which was particularly high in normal cartilage and predominated in the superficial layers of damaged cartilage. There was no obvious correlation between proteoglycan loss and presence of tenascin. CONCLUSIONS: In human normal and osteoarthritic cartilage explants, the presence and the distribution of tenascin are influenced by IL-1 beta.

Presence of ED-A containing fibronectin in human articular cartilage from patients with osteoarthritis and rheumatoid arthritis.

OBJECTIVE: To determine whether normal (fetal and adult) and osteoarthritic (OA) and rheumatoid arthritic (RA) cartilage express a specific isoform of fibronectin, the extra domain A (ED-A) containing fibronectin (EDA+Fn). METHODS: Presence of fibronectin (EDA+Fn and native molecule) in cartilage matrix was studied using immunohistochemical assays with specific monoclonal antibodies. Fibronectins were identified by Western blots, in synovial fluids (SF) and cartilage extracts. RESULTS: EDA+Fn was either moderately present in the surface zone or undetectable in normal cartilage, while it was increased in OA cartilage surface. In one OA cartilage sample, EDA+Fn was localized in the matrix distant from the cartilage surface (patches of staining) and its presence was confirmed by immunoblotting. In RA cartilage EDA+Fn was present in the pericellular areas of the different layers. By Western blots, the presence of EDA+Fn was confirmed in OA SF (2/3) and RA SF (3/3) (with different patterns of fragmentation). CONCLUSION: EDA+Fn generally accumulates in the surface zone of OA cartilage, where it may play a role in extracellular matrix remodelling. Its presence was more abundant in SF and in cartilage from patients with RA.

Proteoglycan-degrading activity associated with the 40 kDa collagen-binding fragment of fibronectin.

The osteoarthritis (OA) process is characterized by the progressive destruction of articular cartilage. There is a loss of cartilage proteoglycan content and disorganization of the collagen network, as well as an increase in other non-collagenous protein such as fibronectin (Fn). Increased proteolytic activity may lead to the degradation of native Fn and generation of Fn proteolytic fragments. Among them, the 45 kDa collagen-binding Fn fragment can be autoactivated in vitro into a 40 kDa fragment. This 40 kDa fragment induces an average of 30% of proteoglycan release per day from human OA cartilage explants and can degrade proteoglycan using dead cartilage sections. Proteoglycan-degrading activity related to the 40 kDa Fn fragment was decreased up to 66% by fetal calf serum (10%), but was not prevented by protein synthesis inhibitors (cycloheximide or actinomycin D). The action of this 40 kDa Fn fragment was greater on OA than on normal cartilage. This study suggests that enzymatic activity induced by the 40 kDa collagen-binding fragment of Fn might be involved in cartilage matrix turnover.

Elastase activity in cartilage extracts and synovial fluids from subjects with osteoarthritis or rheumatoid arthritis: the prominent role of metalloproteinases.

OBJECTIVE: Polymorphonuclear leukocyte (PMN) elastase is able to degrade the extra-cellular matrix components of cartilage. However, in vitro several proteinases can degrade elastin. The purpose of this study was to evaluate the role of the serine proteinases and metalloproteinases in the elastase activity measured in cartilage extracts from patients with osteoarthritis (OA), as well as in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and OA. METHODS: Elastase activity was determined using synthetic low molecular weight substrates and radiolabelled insoluble elastin. Aminophenyl mercuric acetate was used to activate the prometalloproteinases. RESULTS: Elastase activity, measured using synthetic substrates, was higher in BA SF (0.76 + 0.03 microU/ml, n = 12) than in OA SF (0.14 + 0.04 microU/ml, n = 12) (p < 0.001). This activity was inhibited by metal chelating agents: 86% inhibition in OA and 75% inhibition in RA. However, in RA SF the inhibitor of serine proteinase (PMSF) also induced a 40% inhibition. Elastase activity, measured using radiolabelled elastin, in OA SF and RA SF samples and in OA cartilage extract was very low, but increased following activation by mercurial agents. Again this activity was inhibited by metal chelating agents. CONCLUSIONS: Taken together these results indicate that elastase activity (measured by standard methods) in OA and RA SF is mainly due to metalloenzymes.

Increased expression of the Ed-B-containing fibronectin (an embryonic isoform of fibronectin) in human osteoarthritic cartilage.

Fibronectin is non-collagenous protein which accumulates in osteoarthritic cartilage. The presence of fibronectin and its specific isoform containing the B sequence, Ed-B fibronectin (B.Fn), was studied in normal and osteoarthritic human cartilage using immunohistochemical and biochemical assays, with a specific monoclonal antibody. Results showed substantial amounts of B.Fn in osteoarthritic cartilage, especially in the superficial and middle layers. Western blot analysis confirmed the presence of B.Fn with a molecular mass of 220 and 55 kDa. In contrast, in normal cartilage, expression of B.Fn was extremely low. In conclusion, the expression of a specific isoform of fibronectin during the osteoarthritic process suggests that this isoform might have specific functions in extracellular matrix remodelling.

Tenascin distribution in articular cartilage from normal subjects and from patients with osteoarthritis and rheumatoid arthritis.

OBJECTIVE: To determine whether tenascin is present in normal and diseased human cartilage. METHODS: Immunohistochemical and biochemical assays with a monoclonal antibody against all tenascin isoforms (BC-4) were used. RESULTS: Cartilage samples from osteoarthritis and rheumatoid arthritis patients contained increased amounts of tenascin compared with the levels in normal cartilage. Human fetal cartilage was also found to contain tenascin. In normal cartilage explants treated with interleukin-1 beta, tenascin was present in pericellular areas of all layers. Immunolocalization studies revealed that tenascin was most abundant in the superficial layers of osteoarthritic cartilage. Western blot analysis performed from dissociative extracts of diseased cartilage confirmed the presence of subunits of the native molecule. CONCLUSION: Tenascin is increased in arthritic cartilage and is weakly expressed in normal cartilage.

Biosynthesis and distribution of fibronectin in normal and osteoarthritic human cartilage.

The biosynthesis of fibronectin was determined in explants from normal and osteoarthritic human cartilage after metabolic labeling and immunoprecipitation. Each sample of osteoarthritic cartilage was divided into three regions taken at different distances from the eburnated bone area. Only full depth cartilage samples were taken into consideration. We could detect a low level of fibronectin biosynthesis in normal cartilage. In osteoarthritic cartilage increased synthesis of fibronectin was demonstrated, the most important in the region close to the eburnated area. Increased synthesis, although to a lower extent, was also demonstrated in the two other regions at increasing distances from the eburnated areas. Immuno-histological examinations performed on tissue samples and similar studies on articular chondrocyte cell cultures confirmed the accumulation of newly synthesized fibronectin in pathological conditions.

[Study of the effect of procyanidole oligomers on cultured mesenchymatous cells. III. Size and form of cells and nuclei. Quantitative morphologic study]. / Etude de l'action des oligomères procyanidoliques sur des cellules mésenchymateuses en culture. III. Taille et forme des cellules et de leurs noyaux. Etude par morphologie quantitative.

The effect of procyanidole oligomers (PCOs) on the morphology of cultured human skin fibroblasts (FB) and swine aorta smooth muscle cells (SMC) was studied. Exposure to PCOs induced dose-dependent changes in the size, shape and arrangement of cultured fibroblasts. Smooth muscle cells did not exhibit similar changes. These findings demonstrate that procyanidole oligomers interact with fibroblasts membrane and cytoskeletal constituents. With smooth muscle cells the main site of action of procyanidole oligomers may be the basement membrane surrounding the cells, which is lacking in fibroblasts. Thus, in addition to their action on extracellular matrix constituents, i.e., collagen and elastin fibers, procyanidole oligomers affect structural components of cells, i.e., the cell membrane and cytoskeleton. This twofold action of procyanidole oligomers on mesenchymatous cells and their extracellular matrix may be a significant component of the pharmacologic action of procyanidole oligomers.

[The effect of procyanidolic oligomers on vascular permeability. A study using quantitative morphology]. / Action des oligomères procyanidoliques sur la perméabilité de la paroi vasculaire. Etude par morphologie quantitative.

In normal conditions vascular permeability is precisely regulated by mechanisms which involve among others the macromolecules of extracellular matrix of the vascular wall. Permeability for a given substance will vary according to the anatomical localisation of the vessel determining also its structure and composition. In some pathological conditions, such as inflammation or diabetes, permeability can be abnormally increased. Increased permeability can be reproduced by i.v. collagenase injection. This permeability increase can be quantified by image analysis using appropriate tracers such as FITC-dextrans or horse-radish peroxidase, on histological sections from control and collagenase treated rats, pretreated or not with procyanidolic oligomers (PCO). We studied cerebral capillaries, aorta and cardiac muscle capillaries. It could be shown that previous treatment of animals with procyanidolic oligomers prevented the permeability increase produced by collagenase injection.

[The effect of procyanidolic oligomers on mesenchymal cells in culture. II--Attachment of elastic fibers to the cells]. / Etude de l'action des oligomères procyanidoliques sur des cellules mésenchymateuses en culture. II--L'attachement des fibres élastiques aux cellules.

Tissues are composed of cells and extracellular matrix (EM). Adhesion of cells to extracellular matrix is mediated by membrane-bound glycoproteins such as fibronectin, laminin and others. Elastonectin was shown recently to be involved in the mediation of interactions between elastic fibers and cells such as human skin fibroblasts (FB) and smooth muscle cells (SMC) from the media of the aorta. A strong interaction between fibers and cells is important for the maintenance of the quality of the vascular wall. We studied the action of procyanidolic oligomers (PCO) on the attachment of fibroblasts from human skin and smooth muscle cells from porcin aorta to elastic fibers. A dose-dependent increase of cell-fiber interaction could be demonstrated with both cell-types. Elastonectin is located on the cell membrane as well as an elastolytic serine-protease exhibiting an age- and pathology-dependent increase in activity. This will result in a degradation of elastic lamellae, the detachment of cells from elastic fibers and a weakening of the vascular wall. The activity of procyanidolic oligomers increasing the resistance of elastic fibers to degradation by elastases and enhancing the interaction between fibers and cells can be considered as favouring the maintenance of the normal functional state of the vascular wall.

[Effect of procyanidolic oligomers on cultured mesenchymal cells. I. Effect on attachment, proliferation and detachment of cells]. / Etude de l'action des oligomères procyanidoliques sur des cellules mésenchymateuses en culture. I. Effet sur l'attachement, la prolifération et le détachement des cellules.

The effect of procyanidolic oligomers (OPC) was studied on mesenchymal cells in culture: human skin fibroblasts (FB) and porcine aorta smooth muscle cells (CML). In presence of OPC part of the freshly seeded FB did not attach. There was no significant effect on the attachment of CML. Proliferation of FB was also slowed down in a dose-dependent manner. Proliferation of CML-s was also decreased, but much less than for FB-s. The detachment of the cells was also studied by adding trypsin to previously attached cells. Detachment of FB-s was strongly inhibited in presence of OPC in a dose-dependent manner. Much less effect was seen on CML. It appears therefore that OPC may interact with some components of the FB cell membrane and modify the attachment, proliferation and detachment of these cells. The only cell kinetic parameter significantly influenced by OPC for CML was their rate of proliferation. This may be due to the different constitution of the CML cell surface as compared to the FB cell surface.

Immunolocalisation of extracellular matrix macromolecules in the rat spinal cord.

Extracellular matrix macromolecules are involved in many aspects of cell biology. The knowledge concerning the presence, the distribution and the role of these macromolecules in the central nervous system has not yet received sufficient attention. In the present work we studied by indirect immunohistochemical methods the localisation of five extracellular matrix macromolecules in the spinal cord of rats: three collagens: type I, type III, and type IV, and two structural glycoproteins: laminin and fibronectin. We found that all five macromolecules are present in the spinal cord of normal animals. They are localised exclusively in the connective type tissues: the meningeal sheets and the vascular walls. Only type I and type III collagens and fibronectin could be demonstrated around the epithelial cells of the ependyma.

Effect of chromocarb diethylamine on the permeability of the blood-brain barrier.

Intravenously injected collagenase, detectable in brain microvessels by immunological methods, partially degrades the constituents of the vascular wall and so increases the permeability of the blood-brain barrier (BBB). Intravenous administration of collagenase is a model for diseases in which the concentration of endogenous proteases is increased. Peroral treatment of rats with chromocarb diethylamine (CD) significantly reduced the degradation of the vascular wall by intravenous collagenase, as demonstrated by a lesser permeability increase of the BBB, a shorter recovery time, lower hydroxyproline levels in the cerebrospinal fluid and a lesser decrease of the collagen content of the brain capillary basal lamina.

[Cross antigenicity between human arterial tissue and various germs]. / Antigénicité croisée entre le tissue artériel humain et divers germes.

Cross antigenicity was demonstrated between human arterial tissue and enterobacteriaceae, some streptococcus strains or some viruses, using the indirect immunoenzymatic test. Absorption of antigerm antisera by the glycoproteins of either the human serum or aorta suggested that a glycoprotein or some fragment of it acted as a target-antigen or target-epitope for the investigated antibodies and that these antibodies might attack human arterial tissue.

[Experimental immune arteriosclerosis in the rabbit. Immunogenic and pathogenic properties of glycoproteins and elastins in arterial tissue]. / Artériosclérose expérimentale immune du lapin. Pouvoir immunogène et pathogène des glycoprotéines et élastines du tissu artériel.

Rabbits immunized with kappa elastin produced arteriosclerosis and antibodies that bound to target-structures (elastic fiber sheaths, endothelial and smooth muscle cells). These antibodies were cytotoxic for cultured rabbit or rat arterial smooth muscle cells. Absorption of the antielastin antiserum with pig aorta or human serum glycoproteins inhibited its binding to target-structures and suppressed its in vitro cytotoxicity. These data are discussed.

[Possible intervention of immune mechanisms in the genesis and course of human atherosclerosis]. / Possible intervention de mécanismes immuns dans la genèse et l'evolution de l'athérosclérose humaine.

Aortas, coronary and carotid arteries from 31 patients who died of myocardial or cerebral infarction were examined by direct immunoenzymatic tests (using peroxidase-labelled anti human IgG sheep Fab or anti human complement sheep IgG) and compared to those of 9 patients who died of non atherosclerotic diseases. Immunoglobulins and complement bound to all atherosclerotic lesions, all elastic fiber alterations, all lipid infiltration in patients who died of atherosclerosis, as well as in patients who died of various other causes. Binding was generally more intensive in patients who died of atherosclerosis and in arteries irrigating infarcted areas. These data are discussed.