Probing glycosaminoglycan spectral signatures in live cells and their conditioned media by Raman microspectroscopy.

Spectroscopic markers characteristic of reference glycosaminoglycan molecules were identified previously based on their vibrational signatures. Infrared spectral signatures of glycosaminoglycans in fixed cells were also recently demonstrated but probing live cells still remains challenging. Raman microspectroscopy is potentially interesting to perform studies under physiological conditions. The aim of the present work was to identify the Raman spectral signatures of GAGs in fixed and live cells and in their conditioned media. Biochemical and Raman analyses were performed on five cell types: chondrocytes, dermal fibroblasts, melanoma (SK-MEL-28), wild type CHO, and glycosaminoglycan-defective mutant CHO-745 cells. The biochemical assay of sulfated GAGs in conditioned media was only possible for chondrocytes, dermal fibroblasts, and wild type CHO due to the detection limit of the test. In contrast, Raman microspectroscopy allowed probing total glycosaminoglycan content in conditioned media, fixed and live cells and the data were analysed by principal component analysis. Our results showed that the Raman technique is sensitive enough to identify spectral markers of glycosaminoglycans that were useful to characterise the conditioned media of the five cell types. The results were confirmed at the single cell level on both live and fixed cells with a good differentiation between the cell types. Furthermore, the principal component loadings revealed prominent glycosaminoglycan-related spectral information. Raman microspectroscopy allows monitoring of the glycosaminoglycan profiles of single live cells and could therefore be developed for cell screening purposes and holds promise for identifying glycosaminoglycan signatures as a marker of cancer progression in tissues.

Elastin peptides regulate HT-1080 fibrosarcoma cell migration and invasion through an Hsp90-dependent mechanism.

BACKGROUND: The elastin-derived peptides (EDPs) exert protumoural activities by potentiating the secretion of matrix metalloproteinases (MMP) and the plasminogen-plasmin activating system. In the present paper, we studied heat-shock protein 90 (Hsp90) involvement in this mechanism. METHODS: HT-1080 fibrosarcoma cell migration and invasion were studied in artificial wound assay and modified Boyden chamber assay, respectively. Heat-shock protein 90 was studied by western blot and immunofluorescence. Matrix metalloproteinase-2 and urokinase plasminogen activator (uPA) were studied by gelatin ± plasminogen zymography and immunofluorescence. Heat-shock protein 90 partners were studied by immunoprecipitation. Messenger RNA expression was studied using real-time PCR. Small interfering RNAs were used to confirm the essential role of Hsp90. RESULTS: We showed that kappa-elastin and VGVAPG elastin hexapeptide stimulated Hsp90, pro-MMP-2 and uPA secretion within 6 h, whereas AGVPGLGVG and GRKRK peptides had no effect. No increase of mRNA level was observed. Heat-shock protein 90-specific inhibitors inhibit EDP-stimulated HT-1080 cell-invasive capacity and restrained EDP-stimulated pro-MMP-2 and uPA secretions. The inhibitory effect was reproduced by using Hsp90-blocking antibody or Hsp90 knockdown by siRNA. Heat-shock protein 90 interacted with and stabilised uPA and pro-MMP-2 in conditioned culture media of HT-1080 fibrosarcoma cells. CONCLUSIONS: Taken together, our results demonstrate that EDPs exert protumoural activities through an Hsp90-dependent mechanism involving pro-MMP-2 and uPA.

Extracellular matrix and wound healing.

Extracellular matrix has been known for a long time as an architectural support for the tissues. Many recent data, however, have shown that extracellular matrix macromolecules (collagens, elastin, glycosaminoglycans, proteoglycans and connective tissue glycoproteins) are able to regulate many important cell functions, such as proliferation, migration, protein synthesis or degradation, apoptosis, etc., making them able to play an important role in the wound repair process. Not only the intact macromolecules but some of their specific domains, that we called "Matrikines", are also able to regulate many cell activities. In this article, we will summarize main findings showing the effects of extracellular matrix macromolecules and matrikines on connective tissue and epithelial cells, particularly in skin, and their potential implication in the wound healing process. These examples show that extracellular matrix macromolecules or some of their specific domains may play a major role in wound healing. Better knowledge of these interactions may suggest new therapeutic targets in wound healing defects.

Analytical methods for measuring collagen XIX in human cell cultures, tissue extracts, and biological fluids.

Type XIX collagen is a minor collagen associated with basement membranes in vascular, neuronal, mesenchymal, and epithelial tissues. We demonstrated that the NC1, C-terminal, domain of collagen XIX inhibits the migration capacities of tumor cells and exerts a strong inhibition of tumor growth. Other basement membrane collagens or derived fragments were measured in biological fluids such as blood and urine of patients and appeared to be useful for diagnosis, prognosis, or treatment monitoring. The aim of this study was to develop and validate methods to measure collagen XIX and its fragments in human cell cultures, tissue extracts, and human biological fluids. For that purpose, we developed real-time PCR, Western blot, and competitive enzyme-linked immunosorbent assays. We demonstrated that the methods developed in this paper are specific for collagen XIX. We showed that it is expressed in human cell cultures, tissue extracts, and various biological fluids. These methods may be used in various human tissue extracts and biological fluids such as serum, amniotic fluid, cord blood, and many other fluids. Collagen XIX or its fragments could constitute new biomarkers for human diseases as well as for diagnosis and/or prognosis.

Celebration of the 50th anniversary of the foundation of the French society for connective tissue research. Its short history in the frame of the origin and development of this discipline.

The science of connective tissues has (at least) a double origin. Collagen, their major constituent was first studied in conjunction with the leather industry. Acid mucopolysaccharides (now glycosaminoglycans) were characterised by (bio)-chemists interested in glycoconjugates. They joined mainly hospital-based rheumatology departments. Later started the study of elastin with the discovery of elastases and of connective tissue-born (structural) glycoproteins. Besides rhumatologists and leather-chemists mainly pathologists became involved in this type of research, followed closely by ophthalmology research. The first important meetings of these diverse specialists were organised under the auspices of NATO, first in Saint-Andrew's in GB in 1964 and a few years later (1969) in Santa Margareta, Italy. With the discovery of fibronectin, a "structural glycoprotein", started the study of cell-matrix interactions, reinforced by the identification of cell-receptors mediating them and the "cross-talk" between cells and matrix constituents. The first initiative to organise societies for this rapidly growing discipline was that of Ward Pigman in New York in 1961, restricted however to glycol-conjugates. Next year, in 1962 was founded the first European Connective Tissue Society in Paris: the "Club français du tissu conjonctif", which played a crucial role in the establishment of schools, laboratories, national and international meetings in the major cities of France: Paris, Lyon, Reims, Caen,Toulouse. A second European society was born in Great Britain, and at a joint meeting with the French society at the Paris Pasteur Institute, was founded in 1967 by these societies the Federation of European Connective Tissue Societies (FECTS). Their meetings, organised every second year, drained a wide attendance from all over the world. An increasing number of young scientists joined since then this branch of biomedical discipline with several international journals devoted to connective tissue research, to matrix biology. The increasing number and quality of the young generation of scientists engaged in research related to the extracellular matrix or better Biomatrix and cell-matrix interactions is a further guarantee for the continued interest in this crucial field of science at the interface of basic and medically oriented research.

Decreased expression of GPC1 in human skin keratinocytes and epidermis during ageing.

BACKGROUND: Glypicans (GPCs) are heparan sulfate cell membrane proteoglycans containing glycosylphosphatidylinositol (GPI) anchor. They play important role in cell behavior by activating/presenting numerous growth factors and cytokines. OBJECTIVES: The expression of GPCs was investigated in primary culture of skin keratinocytes sampled from healthy donors of different age. MATERIALS AND METHODS: Primary keratinocytes from healthy female donors aged from 20 to 89 years old (n = 30) were either isolated from breast or abdominal skin samples (n = 27) or purchased (n = 3). GPCs expression was examined by qPCR, immunohistochemistry and western blot. Its role in proliferation induced by fibroblast growth factor 2 (FGF2) was also studied. RESULTS: Glypican 1 (GPC1) was the major expressed GPC in human keratinocytes. Its expression was up to two orders of magnitude higher than other GPCs and was significantly decreased with the age of the donors. It was localized at the cell surface and associated with intracellular granules. In skin sections, GPC1 was mainly localized in basal layer of epidermis. Shedding of GPCs decreased the proliferative effect of FGF2, confirming their role of modulator of growth factor effects on keratinocytes. These results established GPC1 as an important player in epidermis biology and skin ageing.

17Beta-oestradiol up-regulates the expression of a functional UDP-glucose dehydrogenase in articular chondrocytes: comparison with effects of cytokines and growth factors.

OBJECTIVES: To investigate the mechanisms by which cytokines and 17beta-oestradiol (17beta-E2) modulate gene expression and activity of uridine diphosphoglucose dehydrogenase (UGDH), a key enzyme of GAG synthesis in articular chondrocytes. METHODS: Rabbit articular chondrocytes (RAC) from 3-week-old animals were incubated for 24 h with TGF-beta, insulin like growth factor-I (IGF-I), IL-1beta, IL-6 and 17beta-E2. GAG synthesis was measured by [35S]-sulphate labelling and the expression of the UGDH gene was estimated by both real-time polymerase chain reaction and western blotting, whereas the enzyme activity was assayed by a spectrophotometric procedure. In addition, the transcriptional activity of several UGDH gene promoter constructs was determined in RAC transiently transfected with wild-type or deleted human oestrogen receptor-alpha gene (hER alpha66 or hER alpha46, respectively). RESULTS: 17Beta-E2 and its receptor hER alpha66 enhanced GAG neosynthesis in rabbit articular chondrocytes, as did TGF-beta1 whereas IL-1beta decreased this synthesis. 17Beta-E2 was found to exert positive regulatory effects at mRNA, protein and UGDH activity levels. In addition, the receptor hER alpha66, but not hER alpha46, increased the transcriptional activity of the UGDH gene. In contrast, no clear correlation between transcription, translation and activity of the UGDH was found under the effects of the cytokines studied. However, TGF-beta enhanced the enzyme activity, whereas IL-1beta, IL-6 and IGF-I were without significant effect. CONCLUSIONS: 17Beta-E2 enhanced GAG synthesis in chondrocytes via up-regulation of the UGDH gene expression and enzyme activity. These data provide insights into the molecular mechanisms involved in the regulation of the UGDH gene and offer new approaches to investigate its potential alteration in joint diseases.

In vivo stimulation of connective tissue accumulation by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+ in rat experimental wounds.

The tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+ (GHK-Cu) was first described as a growth factor for differentiated cells. Recent in vitro data showed that it possesses several properties of a potential activator of wound repair. We investigated the effects of GHK-Cu in vivo, using the wound chamber model described previously (Schilling, J.A., W. Joel, and M.T. Shurley, 1959. Surgery [St. Louis]. 46:702-710). Stainless steel wire mesh cylinders were implanted subcutaneously on the back of rats. The animals were divided into groups that received sequential injections into the wound chamber of either saline (control group) or various concentrations of GHK-Cu. At the end of the experiments, rats were killed, wound chambers were collected, and their content was analyzed for dry weight, total proteins, collagen, DNA, elastin, glycosaminoglycans, and specific mRNAs for collagens and TGF beta. In the GHK-Cu-injected wound chambers, a concentration-dependent increase of dry weight, DNA, total protein, collagen, and glycosaminoglycan contents was found. The stimulation of collagen synthesis was twice that of noncollagen proteins. Type I and type III collagen mRNAs were increased but not TGF beta mRNAs. An increase of the relative amount of dermatan sulfate was also found. A control tripeptide, L-glutamyl-L-histidyl-L-proline, had no significant effect. These results demonstrate that GHK-Cu is able to increase extracellular matrix accumulation in wounds in vivo.

A specific sequence of the noncollagenous domain of the alpha3(IV) chain of type IV collagen inhibits expression and activation of matrix metalloproteinases by tumor cells.

The invasive properties of melanoma cells correlate with the expression of matrix metalloproteinases (MMPs) and their physiological modulators (tissue inhibitors of metalloproteinase and membrane-type MMPs) and with that of the alphaVbeta3 integrin. We investigated the effect of anterior lens capsule type IV collagen and of the alpha3(IV) collagen chain on the invasive properties of various tumor cell lines (HT-144 melanoma cells, HT-1080 fibrosarcoma cells). We demonstrated that anterior lens capsule type IV collagen or specifically the synthetic peptide alpha3(IV) 185-203 inhibited both the migration of melanoma or fibrosarcoma cells as well as the activation of membrane-bound MMP-2 by decreasing the expressions of MT1-MMP and the beta3 integrin subunit.

[Role of matrikins in melanoma progression]. / Rôle des matrikines dans la progression du mélanome.

Expression of melanoma invasiveness, ultimately leading to the formation of metastases, requires that cancer cells break through the successive skin barriers (dermo-epidermal junction, dermis) constituted of various extracellular matrix constituents. In order to facilitate their progression, melanoma cells express, in concert with stromal cells, a group of proteolytic systems which degrade this extracellular structures. However, proteolysis of basement membrane, collagen or elastic fibers can uncover cryptic sites or/and liberate matrix fragments whose properties appeared distinct from their intact macromolecule counterparts. Those fragments, called matrikines, are able to empede or to accelerate melanoma progression ex vivo and in vivo. Non-collagenous domains of basement membrane collagens, which behave like potent "matstatins", are seen as potential pharmacological agents in melanoma.

A glucose-containing fraction extracted from the young erythrocyte membrane is capable of transferring glucose to hemoglobin in vitro.

Red blood cell (RBC) membranes are rich in a glycoconjugate that is extractable in chloroform/methanol solutions (2/1, v/v) and contains several hexoses, such as glucose. Old and young RBC are separated and their respective glycoconjugates are prepared. HbA0 is purified by column chromatography and incubated with solutions of this conjugate. After 24-h incubation, Hb is dialyzed and the amount of glycosylated Hb is measured by a method of column chromatography adapted from Trivelli. A very significant amount of HBAlc is formed when young RBC extracts are incubated: 3.6% of total Hb becomes HBAlc with the extracts, versus 3.2% with free glucose, and only 2.5% for controls. No increase in HbAlc is obtained when extracts of old RBC are incubated. Another difference between the action of the glycoconjugate and free glucose is that the former induces the increase of only the HBAlc fraction, whereas glucose induces the increase of all the minor Hb fractions. The evaluation of glucose contained in the conjugate before and after the glycosylation reaction demonstrates that it is due to an exchange of glucose units from the conjugate to Hb. The reaction is stereospecifically inhibited by p-nitrophenyl-beta-D-glucoside. The nature of the formed HbAlc is demonstrated by isoelectric focusing. A slight increase of HbAlc observed in the incubated controls may be due to an internal migration of some residues of glucose primitively bound to lysyl residues in an unstable form and also to some degree of denaturation during the incubation.

Matrikines in the regulation of extracellular matrix degradation.

The term "matrikines" was coined for designating peptides liberated by partial proteolysis of extracellular matrix macromolecules, which are able to regulate cell activities. Among these peptides, some of them may modulate proliferation, migration, protease production, or apoptosis. In this review, we summarize the activity of matrikines derived from elastin and interstitial or basement membrane collagens on the regulation of matrix metalloproteinases expression and/or activation, and on the plasminogen/plasmin system. Due to their activity, matrikines may play a significant role in physiological or pathological processes such as wound healing or tumor invasion.

[A very curious electrophoresis]. / Une bien curieuse électrophorèse.

Preanalytical steps may be the source of many errors. In this paper, we report the case of an exploration of cerebrospinal fluid (CSF) proteins in which the pre-analytical step was defective. Discordance in the results of the CSF protein level measurement, associated with an aberrant electrophoresis led us to suspect a preanalytical interference. After investigating the preanalytical treatment of the sample, we suspected a possible interference by previous formaldehyde treatment, which was confirmed by several tests performed in our laboratory. These data point out the importance of preanalytical steps for the quality of results.

Gamma-interferon inhibits extracellular matrix synthesis and remodeling in collagen lattice cultures of normal and scleroderma skin fibroblasts.

Three-dimensional collagen lattice cultures of fibroblasts mimic the in vivo situation better than monolayer cultures. Here, skin fibroblasts from scleroderma patients and healthy controls were cultivated in collagen lattices, and the effects of recombinant human gamma-interferon (IFN-gamma) on these cultures investigated. IFN-gamma inhibited collagen lattice retraction in a dose-dependent way at concentrations ranging from 10 to 10,000 U/ml. This effect was independent of any alteration to the cell proliferation within the lattices. The inhibition was of the same order of magnitude in normal and pathological fibroblasts. The synthesis of collagen and non-collagen proteins, particularly fibronectin, was increased in scleroderma cultures. It was inhibited in both normal and scleroderma fibroblasts by IFN-gamma, with a maximal effect at the concentration 1000 U/ml, but the inhibition of protein synthesis was far more intense in scleroderma than in normal cells. In situ hybridization, Northern blot and dot blot analyses showed that mRNA coding for pro alpha 1(I) collagen was decreased in IFN-gamma-treated cells, indicating an effect at the pretranslational level. IFN-gamma also inhibited glycosaminoglycan synthesis, but in scleroderma cells only. This study shows that IFN-gamma regulates cell behavior in three-dimensional collagen matrices: (i) it decreases protein and specifically glycosaminoglycan synthesis in scleroderma fibroblasts, (ii) it modulates the interactions between cells and matrix that lead to the retraction of the lattice. Whereas collagen synthesis is largely decreased in lattice cultures like in vivo, it remains increased in the case of scleroderma compared to normal fibroblasts and may be down-regulated by IFN-gamma. Similar conclusions may be drawn for fibronectin and glycosaminoglycans. The inhibitory effect of IFN-gamma on the retraction capacity of fibroblasts and on their ability to synthesize increased amounts of extracellular matrix macromolecules may be of potential interest for therapeutic use of IFN-gamma in scleroderma patients.

Expression of glycosaminoglycans and small proteoglycans in wounds: modulation by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu(2+).

Glycyl-histidyl-lysine-Cu(2+) is a tripeptide-copper complex previously shown to be an activator of wound healing. We have investigated the effects of glycyl-histidyl-lysine-Cu(2+) on the synthesis of glycosaminoglycans and small proteoglycans in a model of rat experimental wounds and in rat dermal fibroblast cultures. Repeated injections of glycyl-histidyl-lysine-Cu(2+) (2 mg per injection) stimulated the wound tissue production, as appreciated by dry weight and total protein measurements. This stimulation was accompanied by an increased production of type I collagen and glycosaminoglycans (assessed, respectively, by hydroxyproline and uronic acid contents of the chamber). Electrophoretic analysis of wound tissue glycosaminoglycans showed an accumulation of chondroitin sulfate and dermatan sulfate in control wound chambers, whereas the proportion of hyaluronic acid decreased with time. The accumulation of chondroitin sulfate and dermatan sulfate was enhanced by glycyl-histidyl-lysine-Cu(2+) treatment. The expression of two small proteoglycans of the dermis, decorin and biglycan, was analyzed by northern blot. The biglycan mRNA steady-state level in the chamber was maximal at day 12, whereas the decorin mRNA increased progressively until the end of the experiment (day 22). Glycyl-histidyl-lysine-Cu(2+) treatment increased the mRNA level of decorin and decreased those of biglycan. In dermal fibroblast cultures, the stimulation of decorin expression by glycyl-histidyl-lysine-Cu(2+) was also found. In contrast, biglycan expression was not modified. These results show that the expression of different proteoglycans in wound tissue are regulated in a different manner during wound healing. The glycyl-histidyl-lysine-Cu(2+) complex is able to modulate the expression of the extracellular matrix macromolecules differently during the wound repair process.

Antagonistic effects of interferon-gamma and interleukin-4 on fibroblast cultures.

A major characteristic of scleroderma (SSc) fibroblasts is an increased biosynthesis of extracellular matrix macromolecules that could be linked to impaired regulation by cytokines. We investigated the effects of two cytokines from T lymphocytes, interleukin-4 (IL-4) and interferon-gamma (IFN-gamma), on normal and scleroderma fibroblast cultures. In both types of fibroblasts, IL-4 strongly stimulated collagen synthesis, whereas IFN-gamma was a potent inhibitor. The effects of these cytokines were localized at the pre-translational level, and both mRNA steady-state level and protein synthesis were equally affected. SSc fibroblasts responded to IL-4 and IFN-gamma as well as normal fibroblasts. When fibroblasts were incubated with combinations of both cytokines, IFN-gamma completely suppressed the stimulation of collagen gene expression induced by IL-4. Northern blot and western blot analyses demonstrated that IFN-gamma induced a rapid and strong decrease in the expression of the IL-4 receptor-alpha by fibroblasts. This effect might explain the antagonistic effects of IFN-gamma on the IL-4-dependent enhancement of collagen synthesis. Thus, our data suggest that the alteration of collagen production in scleroderma fibroblasts does not depend on an altered sensitivity of these cells to stimulatory or inhibitory cytokines but is more likely the consequence of an imbalance in the local production of autocrine or paracrine regulatory factors.

Expression and activation of matrix metalloproteinases in wounds: modulation by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+.

We investigated the expression and activation of matrix metalloproteinases in a model of experimental wounds in rats, and their modulation by glycyl-L-histidyl-L-lysine-Cu(II), a potent activator of wound repair. Wound chambers were inserted under the skin of Sprague-Dawley rats and received serial injections of either 2 mg glycyl-L-histidyl-L-lysine-Cu(II) or the same volume of saline. The wound fluid and the neosynthetized connective tissue deposited in the chambers were collected and analyzed for matrix metalloproteinase expression and/or activity. Interstitial collagenase increased progressively in the wound fluid throughout the experiment. Glycyl-L-histidyl-L-lysine-Cu(II) treatment did not alter its activity. Matrix metalloproteinase-9 (gelatinase B) and matrix metalloproteinase-2 (gelatinase A) were the two main gelatinolytic activities expressed during the healing process. Pro-matrix metalloproteinase (pro-form of matrix metalloproteinase)-9 was strongly expressed during the early stages of wound healing (day 3). In the wound fluid, it decreased rapidly and disappeared after day 18, whereas in the wound tissue, matrix metalloproteinase-9 expression persisted in the glycyl-L-histidyl-L-lysine-Cu(II) injected chamber until day 22. Pro-matrix metalloproteinase-2 was expressed at low levels at the beginning of the healing process, increased progressively until day 7, then decreased until day 18. Activated matrix metalloproteinase-2 was present in wound fluid and wound tissue. It increased until day 12, then decreased progressively. Glycyl-L-histidyl-L-lysine-Cu(II) injections increased pro-matrix metalloproteinase-2 and activated matrix metalloproteinase-2 during the later stages of healing (days 18 and/or 22). These results demonstrate that various types of matrix metalloproteinases are selectively expressed or activated at the various periods of wound healing. Glycyl-L-histidyl-L-lysine-Cu(II) is able to modulate their expression and might significantly alter wound remodeling.

Alteration of matrix macromolecule synthesis by fibroblasts from a patient with pachydermoperiostosis.

Pachydermoperiostosis (primary hypertrophic osteoarthropathy) is a very rare genetic disease characterized by pachydermia, periostosis, arthralgia, and finger clubbing. Its pathophysiology is still unclear, but previous studies have reported connective tissue hypertrophy in the skin of these patients. We investigated the synthesis of collagen, fibronectin, and proteoglycans by fibroblasts from affected and unaffected skin from one patient with pachydermoperiostosis and four normal donors. We found that collagen synthesis was largely decreased in fibroblasts from the diseased skin, whereas the synthesis of the small dermatan-sulfate-containing proteoglycan decorin strongly increased. Fibroblasts from the unaffected skin of the patient exhibited syntheses of these macromolecules similar to control fibroblasts from healthy donors. Northern blot and dot blot analyses showed decreased pro alpha 1 (I) collagen in patient's affected and unaffected skin fibroblasts whereas increased decorin mRNA levels were found in fibroblasts from the patient's affected skin. No change in cell proliferation was observed. These data demonstrate an alteration of fibroblast biosynthetic activity in the skin lesions of pachydermoperiostosis, which may be responsible, at least in part, for the patient's phenotype.

Inhibition of collagen production in scleroderma fibroblast cultures by a connective tissue glycoprotein extracted from normal dermis.

It was shown in a previous paper that a connective tissue glycoprotein (CTGP) extracted from normal rabbit dermis was able to inhibit total protein and collagen syntheses by normal dermis fibroblast cultures. In the present study, the effects of CTGP on scleroderma fibroblasts were investigated. [14C]Proline incorporation into total proteins of the supernatant was not significantly different from that found in controls. By contrast, the amount of collagen, expressed as percentage of total secreted protein, was far higher in scleroderma cultures than in normal ones (14.4% +/- 6.0% vs 4.6% +/- 0.9%). Addition of CTGP to the medium induced a concentration-dependent inhibition of [14C]proline incorporation into proteins from both control and scleroderma cells. In control cultures, no significant decrease of the percentage of collagen was observed, but over 60 micrograms/ml, both cytotoxic effects and inhibition of protein synthesis occurred. In scleroderma cultures, the inhibition was twice as effective on collagen as on noncollagen protein synthesis. The inhibition of collagen secretion was not related either to changes in collagen hydroxylation or to the intracellular catabolism of newly synthesized procollagen.

The rate of fibrinolysis is increased by free retraction of human gingival fibroblast populated fibrin lattices.

We previously demonstrated that human gingival fibroblasts (HGF), but not their dermal counterparts, when seeded in retracting fibrin lattices induced intense fibrinolysis that was observed at the earliest stages of contraction and led to complete matrix degradation by day 7 of culture. Our aim was to examine the influence of mechanical forces in such fibrinolytic processes. HGF were seeded in retracting (R) e.g. free floating or non retracting (NR) e.g. anchored fibrin lattices (FL). Cultures were analysed from day 1-12 by phase contrast microscopy and scanning electron microscopy (s.e.m.). Levels of fibrin degradation products (FDP) and tissue plasminogen activator (tPA) accumulating in culture media were quantified by ELISA. Urokinase (uPA) and gelatinase A (MMP2) were identified by zymographic techniques. At the s.e.m. level, vacuolization around some HGF was noticed at the earliest stages of culture for RFL and complete degradation of lattices occurred at day 7. Formation of lysed matrix cavity was far less intense in NRFL even after 12 days of culture. FDP amounts at day 4 of culture were equal to 79 +/- 14 and 8.5 +/- 0.6 micrograms/10(5) cells for RFL and NRFL, respectively; tPA levels were equal to 5.8 +/- 0.6 (RFL) and 2.1 +/- 0.3 ng/10(5) cells (NRFL) and differences were still evident at day 7. The kinetics of tPA production were identical in either retracting fibrin or collagen lattices. On the contrary, uPA and proMMP2 productions were similar in RFL and NRFL. Isometric forces, but not the matrix support, were responsible for accelerated tPA production and fibrinolysis in HGF populated lattices.