[Hyaluronic acid and extracellular matrix: a primitive molecule?]. / Acide hyaluronique et matrice extracellulaire : une molécule primitive ?

Hyaluronic acid, or hyaluronan, is a polymer made of the repetition of a unique disaccharidic unit, D-glucuronic acid and D-N-acetylglucosamine, that can reach a molecular mass of 10(7) daltons. This primitive polymer has emerged as a remarkable extracellular matrix component by its viscoelastic properties, its hygroscopic capacities and the diversity of cell processes it controls. Identified in all vertebrate tissues, more than 50% of acid hyaluronic of the organism is present in skin. Having no protein core, its synthesis is performed through a unique process, depending on enzymatic activity of hyaluronan synthases acting at the internal face of the plasmatic membrane and extruding the nascent polymer to the extracellular medium. This polymer constitutes a scaffold on which a large number of sulfated proteoglycans, up to one hundred, can be linked. These supramolecular structures of considerable size are able to entrap large amounts of water and ions to provide tissues with hydration and turgescence. Hyaluronic acid is recognized by cell membrane receptors, notably CD44 which is the best known. Interaction of hyaluronic acid with its receptors triggers several intracellular signaling pathways regulating proliferation, migration and differentiation. Cell response is largely influenced by the size of the polymer and by that of the fragments generated upon degradation by hyaluronidases or free radicals. Hyaluronic acid is metabolically very active, as, for example, its half-life in skin is less than one day. Detected in epidermis where it could play a role in the control of proliferation and differentiation of basal cells, it is however prominent in dermis in association with versican. The remarkable physicochemical properties of hyaluronic acid as well as the diversity of biological processes it controls largely surpass the primitive character of this polymer.

RGDS and DGEA-induced [Ca2+]i signalling in human dermal fibroblasts.

A pulse of short peptides, RGDS and DGEA in the millimolar range, immediately elicits in normal human fibroblasts a transient increase of intracellular Ca2+ ([Ca2+]i). In the present study, we show that this [Ca2+]i occurs in an increasing number of cells as a function of peptides concentration. It is specific of each peptide and inhibited at saturating concentration of the peptide in the culture medium. The [Ca2+]i transient depends on signalling pathways slightly different for DGEA and RGDS involving tyrosine kinase(s) and phosphatase(s), phospholipase C, production of inositol-trisphosphate and release of Ca2+ from the cellular stores. GFOGER, the classical collagen binding peptide of alpha1- alpha2- and alpha11-beta1 integrins, in triple helical or denatured form, does not produce any Ca2+ signal. The [Ca2+]i signalling induced by RGDS and DGEA is inhibited by antibodies against beta1 integrin subunit while that mediated by RGDS is also inhibited by antibodies against the alpha3 integrin. Delay in the acquisition of responsiveness is observed during cell adhesion and spreading on a coat of fibronectin for RGDS or collagen for DGEA or on a coat of the specific integrin-inhibiting antibodies but not by seeding cells on GFOGER or laminin-5. This delay is suppressed specifically by collagenase acting on the collagen coat or trypsin on the fibronectin coat. Our results suggest that free integrins and associated focal complexes generate a Ca2+ signal upon recognition of DGEA and RGDS by different cellular pathways.

Apoptosis in v-myc-transfected MSU-1.1 fibroblasts is induced by cell-matrix contact and differs from that of normal dermal fibroblasts.

In order to characterize a fibroblast cell line representing normal human skin fibroblasts in three-dimensional cultures, we compared the fibroblast line MSU-1.1, derived from human foreskin and immortalized by v-myc, to primary human dermal fibroblasts (NDF). Our results demonstrate that in contrast to NDF, all MSU-1.1 fibroblasts die within 3-4 d when cultured within three-dimensional contractile collagen matrices. Also, in contrast to NDF. MSU-1.1 cells die markedly in anchored collagen gels as well. Death is due to apoptosis and is attenuated by addition of antibodies against collagen-recognizing receptors alpha1beta1 and alpha2beta1. Apoptosis of NDF in collagen lattices was repressed by an inhibitor of caspase-1, which was ineffective on apoptosis of MSU-1.1. Further, apoptosis by MSU-1.l fibroblasts was also observed in anchored, i.e., restrained collagen lattices, an environment that supports proliferation of NDF.

Topically applied vitamin C enhances the mRNA level of collagens I and III, their processing enzymes and tissue inhibitor of matrix metalloproteinase 1 in the human dermis.

Ascorbic acid (vitamin C) is a cofactor required for the function of several hydroxylases and monooxygenases. It is not synthesized in humans and some other animal species and has to be provided by diet or pharmacologic means. Its absence is responsible for scurvy, a condition related in its initial phases to a defective synthesis of collagen by the reduced function of prolylhydroxylase and production of collagen polypeptides lacking hydroxyproline, therefore, they are unable to assemble into stable triple-helical collagen molecules. In fibroblast cultures, vitamin C also stimulates collagen production by increasing the steady-state level of mRNA of collagen types I and III through enhanced transcription and prolonged half-life of the transcripts. The aim of the experimental work has been to evaluate the effect on dermal cells of a preparation of vitamin C topically applied on one side vs placebo on the other side of the dorsal face of the upper forearm of postmenopausal women. Biopsies were collected on both sides and the level of mRNA measured by non competitive reverse transcription-polymerase chain reaction made quantitative by the simultaneous transcription and amplification of synthetic RNA used as internal standards. The mRNA of collagen type I and type III were increased to a similar extent by vitamin C and that of three post-translational enzymes, the carboxy- and amino-procollagen proteinases and lysyloxidase similarly increased. The mRNA of decorin was also stimulated, but elastin, and fibrillin 1 and 2 were not modified by the vitamin. The expression of matrix metalloproteinases 1, 2, and 9 was not significantly changed, but an increased level of tissue inhibitor of matrix metalloproteinase 1 mRNA was observed without modification of tissue inhibitor of matrix metalloproteinase 2 mRNA. The stimulating activity of topical vitamin C was most conspicuous in the women with the lowest dietary intake of the vitamin and unrelated to the level of actinic damage. The results indicate that the functional activity of the dermal cells is not maximal in postmenopausal women and can be increased.

Down-regulation of vascular endothelial growth factor by tissue inhibitor of metalloproteinase-2: effect on in vivo mammary tumor growth and angiogenesis.

The tissue inhibitor of metalloproteinases-2 (TIMP-2) has at least two independent functions, i.e., regulation of matrix metalloproteinases and growth promoting activity. We investigated the effects of TIMP-2 overexpression, induced by retroviral mediated gene transfer, on the in vivo development of mammary tumors in syngeneic mice inoculated with EF43.fgf-4 cells. The EF43.fgf-4 cells established by stably infecting the normal mouse mammary EF43 cells with a retroviral expression vector for the fgf-4 oncogene, are highly tumorigenic and overproduce vascular endothelial growth factor (VEGF). Despite a promotion of the in vitro growth rate of EF43.fgf-4 cells overexpressing timp-2, the in vivo tumor growth was delayed. At day 17 post-cell injection, the volume of tumor derived from TIMP-2-overexpressing cells was reduced by 80% as compared with that obtained with control cells. Overexpression of TIMP-2 was associated with a down-regulation of VEGF expression in vitro and in vivo, a reduction of vessel size, density, and blood supply in the induced tumors. In addition, TIMP-2 completely inhibited the angiogenic activity of EF43.fgf-4 cell-conditioned medium in vitro using a rat aortic ring model. Our findings suggest that overexpression of TIMP-2 delays growth and angiogenesis of mammary carcinoma in vivo and that down-regulation of VEGF expression may play an important role in this TIMP-2-mediated antitumoral and antiangiogenic effects. Finally the in vivo delivery of TIMP-2, as assessed by i.v. injection of recombinant adenoviruses vectors, significantly reduced the growth of the EF43.fgf-4-induced tumors. This effect of TIMP-2 was shown to be equally comparable with that of angiostatin, a known potent inhibitor of angiogenesis.

Comparative caustic and biological activity of trichloroacetic and glycolic acids on keratinocytes and fibroblasts in vitro.

OBJECTIVES: Beside their causticity, the biological mechanism by which trichloroacetic acid (TCA) and glycolic acid (GA), two agents extensively used for chemical peeling, might act remains unknown. The purpose of this study was to examine in vitro the effect of TCA and GA on human keratinocytes and the influence of the released epithelial mediators on collagen and matrix metalloproteinases (MMPs) production by human dermal fibroblasts. METHOD: Cultured keratinocytes were treated by TCA and GA at 10 mg/ml brought to pH 3, 5 and 7, and the conditioned media neutralized to pH 7 were added to human normal skin fibroblasts. RESULTS: TCA was cytotoxic for keratinocytes at each tested pH. The conditioned medium depressed protein and collagen synthesis and the expression of MMPs when added to fibroblasts as did also TCA when added directly to fibroblasts. GA was not cytotoxic for keratinocytes at neutral pH and the conditioned medium obtained at each pH applied to fibroblasts did not alter protein, collagen nor MMPs production while causing an elevated secretion of IL-6. CONCLUSION: TCA exerts a toxic effect on keratinocytes and fibroblasts while GA does not alter the metabolism of fibroblasts but induces the secretion of IL-6.

Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis are caused by mutations in the procollagen I N-proteinase gene.

Ehlers-Danlos syndrome (EDS) type VIIC is a recessively inherited connective-tissue disorder, characterized by extreme skin fragility, characteristic facies, joint laxity, droopy skin, umbilical hernia, and blue sclera. Like the animal model dermatosparaxis, EDS type VIIC results from the absence of activity of procollagen I N-proteinase (pNPI), the enzyme that excises the N-propeptide of type I and type II procollagens. The pNPI enzyme is a metalloproteinase containing properdin repeats and a cysteine-rich domain with similarities to the disintegrin domain of reprolysins. We used bovine cDNA to isolate human pNPI. The human enzyme exists in two forms: a long version similar to the bovine enzyme and a short version that contains the Zn++-binding catalytic site but lacks the entire C-terminal domain in which the properdin repeats are located. We have identified the mutations that cause EDS type VIIC in the six known affected human individuals and also in one strain of dermatosparactic calf. Five of the individuals with EDS type VIIC were homozygous for a C-->T transition that results in a premature termination codon, Q225X. Four of these five patients were homozygous at three downstream polymorphic sites. The sixth patient was homozygous for a different transition that results in a premature termination codon, W795X. In the dermatosparactic calf, the mutation is a 17-bp deletion that changes the reading frame of the message. These data provide direct evidence that EDS type VIIC and dermatosparaxis result from mutations in the pNPI gene.

Gelatinases in drug-induced toxic epidermal necrolysis.

BACKGROUND: The matrix metalloproteinases (MMPs) MMP2 and MMP9 play a significant role in epidermal detachment, inflammation and re-epithelialization. We have evaluated their activity in toxic epidermal necrolysis (TEN). DESIGN: The level and pattern of activity of MMP2 and MMP9 were investigated by measuring the degradation of 3H-labelled gelatin and by zymography in blister fluid from six TEN patients and compared the results with three other blistering conditions: bullous pemphigoid (n = 6), second-degree burn (n = 13) or suction blister (n = 3). RESULTS: A higher amount of MMP2 was found in TEN blister fluid with the constant presence of a significantly larger proportion of the activated forms of MMP2, a particular feature of TEN, than the other blistering diseases studied. CONCLUSION: This study emphasizes the potential role of MMP2 in the specific inflammatory reaction and reparation process in TEN skin.

An interleukin-1 loop is induced in human skin fibroblasts upon stress relaxation in a three-dimensional collagen gel but is not involved in the up-regulation of matrix metalloproteinase 1.

The integrin-mediated stress relaxation as it occurs in a retracting three-dimensional collagen gel (RCG) is accompanied by a large up-regulation of the interstitial collagenase, matrix metalloproteinase 1 ((MMP-1), EC 3.4.24.7), regulated notably by interleukin-1 (IL-1), phorbol esters, and cytoskeleton-disrupting drugs as cytochalasin D (CD). The repression of MMP-1 up-regulation in RCG by cycloheximide suggested the participation in the regulation process of a de novo synthesized intermediary component. We demonstrate here that culture of human skin fibroblasts in RCG or in CD- and 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated monolayers resulted in the activation of an IL-1 autocrine feedback loop that was switched off by the naturally occurring IL-1 receptor antagonist (IL-1RA), a blocker of the common IL-1 receptor. The IL-1RA did not suppress the MMP-1 up-regulation induced in RCG nor in CD-treated cells, indicating that the up-regulation of MMP-1 and the IL-1 autocrine loop occurred in an independent way, while the TPA-induced MMP-1 expression was suppressed by the receptor antagonist. The RCG- as well as the TPA-, IL-1-, and CD-induced up-regulation of both MMP-1 and IL-1 was totally suppressed by protein tyrosine kinases inhibitors. In contrast bisindoylmaleimide, at a concentration (5 microM) that inhibits the TPA-induced protein kinase C activity, suppressed the CD-induced MMP-1 expression but did not or barely altered that induced in RCG or by IL-1. None of the other tested inhibitors of a variety of signaling pathways including those used by integrins was able to suppress the RCG or CD-induced MMP-1. These results point to a potent regulation of MMP-1 by mechanical stress relaxation, a process depending on de novo protein synthesis and occurring independently of the activation of an IL-1 autocrine feedback loop.

Angiogenesis by fibroblast growth factor 4 is mediated through an autocrine up-regulation of vascular endothelial growth factor expression.

The infection of normal mouse mammary EF43 cells by a retroviral vector carrying either Fgf-3 (EF43.Fgf-3) or Fgf-4 (EF43.Fgf-4) cDNA resulted in the transformation of cells displaying different tumorigenic potentials in nude mice (A. Hajitou and C-M. Calberg-Bacq, Int. J. Cancer, 63: 702-709, 1995). EF43.Fgf-4 produced rapidly developing tumors at all sites of inoculation, whereas EF43.Fgf-3 produced slowly growing tumors only in the mammary fat pad. Cells infected with the vector carrying the selection gene alone (EF43.C) were not tumorigenic. The angiogenic properties of these cells were tested in an in vitro angiogenesis model using human umbilical vein endothelial cells (HUVECs) cultured at the surface of a type I collagen gel and their capacity to form tube-like structures on invasion of the gel. Only the conditioned medium (CM) of EF43.Fgf-4 induced an angiogenic morphotype in HUVECs. In parallel, the mRNA expression of matrix metalloproteinase 1 and c-ETS-1 was increased in the HUVECs displaying a differentiated phenotype, whereas the tissue inhibitor of matrix metalloproteinase 1 mRNA level was decreased. Recombinant human fibroblast growth factor 4 (FGF-4) did not induce an angiogenic phenotype in HUVECs by itself. By Western blot analysis, a high expression of vascular endothelial growth factor (VEGF) was detected in the EF43.Fgf-4 CM. This result was confirmed by Northern blot analysis of total RNA extracted from the three cell types; the steady-state level of VEGF mRNA was low and equivalent in EF43.C and EF43.Fgf-3, whereas it was strongly increased in EF43.Fgf-4. Culturing EF43 cells carrying only the selection gene with increasing concentrations of recombinant human FGF-4 resulted in a dose-dependent stimulation of VEGF. The induction of the angiogenic morphotype and the parallel modulations of the biosynthetic phenotype in HUVECs were completely suppressed by adding a neutralizing antibody directed against VEGF to EF43.Fgf-4 CM. Furthermore, inhibition of protein kinase C by bisindoylmaleimide suppressed the angiogenic phenotype induced by the CM of EF43.Fgf-4. Our results point to an indirect angiogenic activity of FGF-4 through the autocrine induction of VEGF secretion by EF43.Fgf-4 cells, an original signaling pathway that might be significant in tumor progression and metastasis.

Activated forms of MMP2 and MMP9 in abdominal aortic aneurysms.

PURPOSE: This consistent observation of a reduction of the elastin concentration in abdominal aortic aneurysms (AAAs) has led us to investigate in AAA specimens two metalloproteinases that display elastase activity, MMP2 (gelatinase A/72kDa) and MMP9 (gelatinase B/92 kDa). METHODS: Samples of full-thickness aortic wall, adherent thrombus, and serum were collected in 10 patients with AAAs. Samples of normal aortic wall and serum were taken from 6 age-matched control patients. Quantitative gelatin-zymography and gelatinolytic soluble assays after acetyl-phenyl mercuric acid activation were performed on serum and tissue extracts, and the results were expressed in units on a comparative wet-weight basis. Histologic analysis was performed in parallel to score the inflammatory infiltrate. RESULTS: The luminal and parietal parts of the thrombus contained, respectively, 20- and 10-fold more gelantinolytic activity than the serum. The predominate form was MMP9. Although the total gelatinolytic activity was in the same range both in AAAs and in normal walls, a significantly higher proportion of MMP9 was found in the aneurysmal aortic walls. Furthermore, a significant proportion of MMP9 was under its processed active form, which was never observed in normal samples. A significantly higher proportion of MMP2 was also present as processed active form in AAA wall. This latter parameter positively correlated with the inflammatory score. CONCLUSIONS: The presence of activated MMP9 and MMP2 might contribute to the degradation of the extracellular matrix proteins that occurs during the development of aneurysms.

Modifications of the extracellular matrix of aneurysmal abdominal aortas as a function of their size.

Collagen and elastin are the main extracellular matrix proteins providing the aortic wall with adequate mechanical properties and resistance for proper function. Our study aimed at investigating the relationship between the elastin concentration of the wall of normal and aneurysmal abdominal aortas (AAA), the collagen concentration, and its extractability, as a function of their size. Infrarenal aortas were collected from 30 patients undergoing operative repair of abdominal aortic aneurysm. Age-matched control samples were obtained from eight autopsies of individuals without vascular disease. Samples were divided into five groups according to the aortic diameter: control group (group N, n = 8); < 50 mm (group I, n = 6; between 50-75 mm (group II, n = 10); > 75 mm (group III, n = 7); and ruptured (group IV, n = 7). The collagen concentration in samples from group I was similar to the controls. An increased collagen concentration was observed in group II and remained at the same level in the largest and ruptured aneurysms. Extractability of collagen was found to be increased in group III and was even higher in group IV. A highly significant reduction in elastin concentration was observed in group I and there was progressive reduction with increasing diameter and rupture. A significant correlation could be established between aortic diameter, increased collagen extractability and decreased elastin content.

Modulation of expression of endogenous collagenase and collagen genes by electroporation: possible involvement of Ca2+ and protein kinase C.

We have investigated the effect of electroporation on the expression of collagen alpha 1(I), collagenase, c-fos and c-jun genes in human dermal fibroblasts (HDF), human smooth muscle cells (HSMC) and HeLa cells. Collagenase and collagen mRNA levels were respectively increased and decreased in a voltage-dependent manner in HDF harvested 2 days after a sham electroporation. These effects were still observed 10 days after electroporation. Similar effects occurred in electroporated HSMC. Neither collagen nor collagenase mRNAs were detected in control or electroporated HeLa cells. c-fos and c-jun mRNA levels were also increased in electroporated HDF, HSMC and HeLa cells harvested 1 h after plating. This suggests that factor AP1 (fos/jun) could mediate the up-regulation of collagenase expression in electroporated HDF and HSMC. When electroporation of HDF was performed in the presence of H7, an inhibitor of protein kinase C, no increase in collagenase mRNA level was observed, suggesting that protein kinase C might be involved in the transduction of the effect. All the effects reported were also suppressed when cells were electroporated in a medium containing EGTA, suggesting that Ca2+ might mediate the transduction of this effect.

Effect of cell-cell and cell-matrix interactions on the response of fibroblasts to epidermal growth factor in vitro. Expression of collagen type I, collagenase, stromelysin and tissue inhibitor of metalloproteinases.

Investigations of the effect of epidermal growth factor (EGF) on the expression of four genes involved in the turnover of the extracellular matrix, collagen type I, collagenase, stromelysin and tissue inhibitor of metalloproteinases (TIMP) were performed on four strains of skin fibroblasts in vitro. Addition of EGF to subconfluent cultures for increasing periods of time up to 5 days induced an inhibition of procollagen alpha 1(I) mRNA and a strong stimulation of collagenase (100-fold) and stromelysin (1000-fold) mRNAs, whereas the mRNA of TIMP was increased to a lesser extent (5-fold). After a 40 h pulse with EGF, these effects persisted for 24-48 h after withdrawal of the growth factor and slowly diminished thereafter to attain control values after several days. By culturing fibroblasts for increasing periods of time, different levels of confluence were obtained allowing for the deposition of an extracellular biomatrix. The steady-state level of collagenase and stromelysin mRNAs were profoundly depressed in confluent as against non-confluent cultures, whereas no major change for TIMP and procollagen alpha 1(I) mRNAs was observed. Upon treatment of these cultures with EGF for 48h, the steady-state level of collagenase, stromelysin and TIMP increased, whereas procollagen alpha 1(I) mRNA was slightly reduced. These modifications were, at least in part, dependent upon a regulation of the transcription rate, as suggested from run-off experiments. Similar states of confluence were obtained by seeding cells at increasing densities in short-term cultures in which cell-cell contact predominated. In such culture conditions, the collagenase and stromelysin mRNAs were enhanced in high as compared to low density cultures. The response to EGF was progressively decreased for collagenase, stromelysin and, to a lesser extent, TIMP mRNAs at most densities and a complete lack of response to EGF at the highest cell density was observed. Under all culture conditions the modulation of collagenase mRNA was paralleled by similar modifications of enzyme activity. These results emphasize the importance of the cell-cell contacts and cell-matrix interactions in the expression of the genes coding for metalloproteinases or their inhibitor and their modulation by growth factors.

Evidence for a relationship between Ehlers-Danlos type VII C in humans and bovine dermatosparaxis.

Ehlers-Danlos (ED) syndrome type VII is characterized by the accumulation of collagen precursors in connective tissues. ED VII A and B are caused by mutations in the genes of alpha 1 and alpha 2 collagen I which result in the disruption of the cleavage site of procollagen I N-proteinase. The existence of ED VII C in humans has been hypothesized on the basis of a disorder in cattle and sheep related to the absence of the enzyme. We now present evidence for the existence of this disease in humans, characterized by skin fragility, altered polymers seen as hieroglyphic pictures with electron microscopy, accumulation of p-N-alpha 1 and p-N-alpha 2 collagen type I in the dermis and absence of processing of the p-N-I polypeptides in fibroblast cultures.

Pretranslational regulation of extracellular matrix macromolecules and collagenase expression in fibroblasts by mechanical forces.

In vivo, the extracellular matrix modulates the phenotype of the connective tissue cells both through its biochemical composition and the transfer of mechanical information. In this study, the mechanical effect was investigated in collagen gels populated by skin fibroblasts maintained under tension (bound lattices (BL)) compared with free retracting lattices (FL) and monolayer on plastic. The overall proteins and collagen synthesis of human skin fibroblasts, investigated by isotopic labeling, were decreased respectively by a factor of about 20 and 40 in FL compared with monolayers and increased by a factor of 4 and 6 in BL versus FL. As assayed by the degradation of [3H]collagen type I by trypsin-activated medium conditioned by fibroblasts under the three models of culture, collagenase activity was inversely regulated and increased in lattices when compared with monolayer culture. It was four times higher in FL than in BL. The steady-state level of mRNA coding for procollagen types I, III, and VI polypeptides, fibronectin, elastin, beta-actin, and procollagenase was determined by cDNA hybridization. The mRNA coding for beta-actin as well as for the various extracellular matrix macromolecules were increased in BL when compared with FL while the level of procollagenase mRNA was lower. These data demonstrate the existence of a modulation of the function of the fibroblasts performed by mechanical forces. This regulation operates, at least in part, at a pretranslational level.

Invasion of reconstituted basement membrane matrix is not correlated to the malignant metastatic cell phenotype.

Interactions between tumor cells and basement membranes represent a critical step in the progression of neoplasia and in the metastatic process. Reconstituted basement membrane matrix, matrigel, has been recently used with the aim of developing an in vitro assay of tumor cell invasiveness. We have extended these studies by comparing the invasiveness of a large series of normal and malignant epithelial and mesenchymal cells of human and animal origin cultured on matrigel. Normal cells (fibroblasts, glomerular mesangial cells, keratinocytes), human fibrosarcoma cells (HT1080), and reticular sarcoma cells (M5076) clearly established invasive capabilities in the matrix. However, all the other tested cell lines, malignant or virally transformed cells invasive in vivo (MCF7, T47D, SA52, SW613, MO4, A431, BeWo), as well as normal nontransformed cells (MOH22) were incapable of penetration. The morphological features of matrigel invasion by normal fibroblasts and HT1080 cells are described at the light and electron microscope levels. The extent of degradation of a radiolabeled matrigel is minimal and similar in several cell lines reported to be noninvasive or invasive in vivo. Our data suggest that matrigel does not provide a universal model to correlate the invasiveness of cells in vivo and in vitro.

Treatment of alcoholic hepatitis with colchicine. Results of a randomized double blind trial.

A randomized double-blind trial of colchicine vs placebo was conducted in 67 patients with histologically proven alcoholic hepatitis, 33 of whom had cirrhosis. Patients with hepatic encephalopathy, ascites, protracted prothrombin time, severe thrombocytopenia, hepatocellular carcinoma, evident lack of discipline or refusal to participate in the trial were not included. Thirty-three patients received colchicine (1 mg/day) and 34 received placebo for 6 months. Blood parameters including N-terminal peptide of type III procollagen were assessed in the serum, and a percutaneous liver biopsy was performed at the start of the trial and after 3 and 6 months. Alcoholic hepatitis and fibrosis scores were established for each biopsy specimen. Twenty-eight percent of patients were lost to follow-up at 3 months, and fifty-two percent at 6 months. One patient died of liver failure. Fifty-eight percent of patients were abstaining from alcohol at 3 months and fifty percent at 6 months. No significant effect of treatment was noted. Nevertheless, improvement in alcoholic hepatitis core at 3 months was more important in the colchicine group than in the placebo group. No side-effects were noted except transient diarrhea. Our results suggest that colchicine has no important effect on the course of alcoholic hepatitis. A trial including of at least 260 patients might be necessary for the observed alcoholic hepatitis score difference at 3 months, favoring colchicine, to be statistically significant.