Biomedical subjects

D Barritault

Publications and source records attributed to D Barritault.

At least 19 recordsLinked to original sources

PubMed2004

Regulation of the collagen phenotype expression of gamma-irradiated vascular smooth muscle cells by heparan mimetics (RGTA).

Restenosis is characterized by vascular smooth muscle cell (VSMC) proliferation and accumulation of collagen III in a hypertrophic and disorganized extracellular matrix. Restenosis is prevented by antimitotic agents or irradiation but no significant progress has been made to control collagen expression deregulation. Previously, we have shown that a new family of biopolymers named RGTA (heparan mimetics elaborated by grafting on dextran of carboxylate, sulfate, and benzylamide units) stimulate in vivo tissue repair and reduce fibrosis in various models. Using VSMC in vitro (pig aortic VSMC irradiated with a 60Co source and labeled with [3H]Proline), we now show that gamma-irradiation reduced cell survival by 50% and collagen synthesis 6-fold with a major increase in the ratio of collagen III to collagen I biosynthesis taken as a fibrotic index. RGTA added to the cells enhanced their survival up to 80% and reduced collagen III/I ratio back to values found in normal vascular tissues. These results suggest that RGTA combined with gamma-radiation could be an efficient strategy against restenosis.

Animals↗

PubMed2004

Reversal of abnormal collagen production in Crohn's disease intestinal biopsies treated with regenerating agents.

BACKGROUND: Crohn's disease (CD) is characterised by inflammation, muscle layer overgrowth, and collagenous fibrosis of the intestinal tract, with no effective therapy against collagen accumulation. AIMS: We quantified production of collagen in resection specimens from normal and CD patients and investigated the effect of regenerating agents (RGTAs) on collagen production. RGTAs are chemically substituted dextrans engineered to mimic the growth factor protecting effects of heparan sulphates. RGTAs have been shown to enhance tissue repair in various in vivo models and to modulate in vitro collagen phenotype differentially according to their structure. PATIENTS: We studied intestinal biopsies from two groups of CD patients: treated with glucocorticoids (CD-GC group: 10 patients) or not treated (CD group: seven patients), and from seven control patients. METHODS: After 24 hours of ex vivo incubation with (3H) proline, collagen I, III, and V were extracted by pepsin and quantitatively separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Biosynthesis of each collagen type was quantified on radiolabelled isolated collagen. RESULTS: Total intestinal collagen production in CD patients compared with controls was increased up to 3.5-fold overall (p<0.001). In particular, collagen III biosynthesis was enhanced by 6.2-fold (p<0.001) in CD patients. In the CD-GC group, collagen production abnormalities were less marked. RGTAs added to the incubation medium in the CD group decreased total collagen production by 50% and decreased collagen III synthesis by 76%. CONCLUSION: This finding offers a rationale for using RGTAs in the treatment of intestinal fibrosis in CD, thus opening up a potential new therapeutic field for this family of drugs.

Adult↗

PubMed2003

A new approach to treat tissue destruction in periodontitis with chemically modified dextran polymers.

Periodontitis are diseases of the supportive tissues of the teeth provoked by bacteria and characterized by gingival inflammation and bone destruction. We have developed a new strategy to repair tissues by administrating agents (RGTA) that mimic heparan sulfates by protecting selectively some of the growth factors naturally present within the injured tissue and interfering with inflammation. After periodontitis induction in hamsters, the animals were left untreated or received weekly i.m. injections of RGTA1507 at a dose of 100 microg/kg, 400 microg/kg, 1.5 mg/kg, or 15 mg/kg for 4 wk. RGTA treatment significantly reduced gingival tissue inflammation, thickened the pocket epithelium by increasing cell proliferation, and enhanced collagen accumulation in the gingiva. A marked reduction in bone loss was observed, resulting from depression of osteoclasia and robust stimulation of bone formation at the dose of 1.5 mg/kg. RGTA treatment for 8 wk at this dose reversed macroscopic bone loss, sharply contrasting with the extensive bone destruction in the untreated animals. RGTA treatment decreased gelatinase A (MMP-2) and B (MMP-9) pro-forms in gingival tissues. Our data indicate that a 4 wk treatment dose-dependently attenuated gingival and bone manifestations of the disease, whereas a longer treatment restored alveolar bone close to controls. By modulating and coordinating host responses, RGTA has unique therapeutic properties and is a promising candidate for the treatment of human periodontitis.

Animals↗

PubMed2003

Effect of RG1192, a substituted dextran, on inflammatory response.

RG1192 is one of a series of substituted dextran heparin-like polymers that were shown to accelerate tissue repair in various experimental models. Many efforts have been made to investigate the mechanisms by which these compounds act In these experiments, we studied the effect of RG1192 on inflammatory cells. We showed that intraperitoneal RG1192 injection caused a 5-fold increase in peritoneal polymorphonuclear (PMN) cell number at 6 hr, 8-fold increase at 12 hr and 3-fold increase at 24 hr, as compared to controls. Although the circulating white blood cell count did not show any changes, the number of circulating PMN showed a small early increase. Further investigation had been done on the chemotactic effect of RG1192 on PMN in vitro. The results showed that RG1192 alone increased PMN migration at 0.5 mg/ml and 1 mg/ml, without potentiation by additional IL-8.

Animals↗

PubMed2001

The lysine-rich C-terminal tail of heparin affin regulatory peptide is required for mitogenic and tumor formation activities.

Heparin affin regulatory peptide (HARP) is a 18-kDa heparin-binding polypeptide that is highly expressed in developing tissues and in several primary human tumors. It seems to play a key role in cellular growth and differentiation. In vitro, HARP displays mitogenic, angiogenic, and neurite outgrowth activities. It is a secreted protein that is organized in two beta-sheet domains, each domain containing a cluster of basic residues. To assess determinants involved in the biological activities of HARP, C-terminally truncated proteins were produced in Chinese hamster ovary-K1 cells and tested for their mitogenic, tumor formation in nude mice and neurite outgrowth activities. Our data clearly indicate that the residues 111-136 of the lysine-rich C-terminal domain are involved in the mitogenic and tumor formation activities of HARP. Correlatively, no signal transduction was detected using the corresponding mutant, suggesting the absence of HARP binding to its high affinity receptor. However, this C-terminal domain of HARP is not involved in the neurite outgrowth activity. We also demonstrate that HARP signal peptide cleavage could led to two maturated forms that are both but differentially mitogenic.

3T3 Cells↗

PubMed2001

Beneficial use of fibroblast growth factor 2 and RGTA, a new family of heparan mimics, for endothelialization of PET prostheses.

We have studied the endothelialization of polyethylene terephtalate (PET) prostheses coated with collagen by adult human saphenous endothelial cells (EC) under various in vitro conditions. Collagenous PET was impregnated either by Fibroblast Growth Factor 2 (FGF2), heparin, a synthetic heparan sulfate mimic named RGTA 11 (for ReGeneraTing Agent), or combinations of these products. RGTA 11 belongs to a new family of drugs, which have been previously described as stabilizer and protector of heparin binding growth factors (HBGF), and to act in vivo as to stimulate wounded tissue repair. As endothelialization of prosthesis can be obtained in vivo after EC seeding and/or by transanastomotic, as well as by transprosthetic EC migrations, we have designed in vitro models to study the growth of EC seeded on PET, the EC colonization of an acellular area on PET, and the migration of EC from a collagen gel through the prosthesis. The combinations of either RGTA11 or heparin with FGF2 enhanced after a week by 5-fold the growth of seeded EC compared to RGTA or heparin alone and by 3-fold compared to FGF2 alone (p < 0.05). More than 80% of the colonization of an acellular area was achieved within 6 days when FGF2 was combined with RGTA11 or heparin. In contrast, colonization was only of 20% promoted in presence of FGF2 alone and not promoted in the presence of RGTA or heparin alone (p < 0.05). In addition, transprosthetic migration of EC and endothelialization of the luminal side were observed only when gel contained RGTA11 or heparin in combination with FGF2. The present work did strongly indicate that RGTA11 could be used in vivo as to improve endothelialization and should be the focus of continued investigation.

Adult↗

PubMed2001

Heparan sulfate mimetics modulate calpain activity during rat Soleus muscle regeneration.

Skeletal muscle regenerates after injury. Tissue remodelling, which takes place during muscle regeneration, is a complex process involving proteolytic enzymes. It is inferred that micro and milli calpains are involved in the protein turnover and structural adaptation associated with muscle myolysis and reconstruction. Using a whole-crush injured skeletal muscle, we previously have shown that in vivo muscle treatment with synthetic heparan sulfate mimetics, called RGTAs (for ReGeneraTing Agents), greatly accelerates and improves muscle regeneration after crushing. This effect was particularly striking in the case of the slow muscle Soleus that otherwise would be atrophied. Therefore, we used this regeneration model to study milli and micro calpain expressions in the regenerating Soleus muscle and to address the question of a possible effect of RGTAs treatment on calpain levels. Micro and milli calpain contents increased by about five times to culminate at days 7 and 14 after crushing respectively, thus during the phases of fibre reconstruction and reinnervation. After 64 days of regeneration, muscles still displayed higher levels of both calpains than an intact uninjured muscle. Milli calpain detected by immunocytochemistry was shown in the cytoplasm whereas micro calpain was in both nuclei and cytoplasm in small myofibres but appeared almost exclusively in nuclei of more mature fibres. Interestingly, the treatment of muscles with RGTA highly reduced the increase of both milli and micro calpain contents in Soleus regenerating muscles. These results suggest that the improvement of muscle regeneration induced by RGTA may be partly mediated by minimising the consequences of calpain activity.

Animals↗

PubMed2001

Specific RGTA increases collagen V expression by cultured aortic smooth muscle cells via activation and protection of transforming growth factor-beta1.

Regenerating agents (RGTA) are defined as heparan sulfate mimics, which in vivo stimulate tissue repair. RGTA are obtained by controlled grafting of carboxymethyl and sulfate groups on dextran polymers. RGTA are selected in vitro, on their ability to protect heparin binding growth factors such as TGF-beta1 for example, as well as to alter extracellular matrix biosynthesis. We had reported that RGTA were able to modulate smooth muscle cell (SMC) collagen biosynthesis. Here, we demonstrated that a specific RGTA (RG-1503), altered differentially collagen type expression by post-confluent SMC and that this action involves TGF-beta1. RG-1503 decreased, by 50%, collagen I and III biosynthesis and stimulated specifically, by twofold, collagen V biosynthesis. TGF-beta1 stimulated collagen I and V by 1.5- and threefold, respectively. A synergic action for RGTA in association with TGF-beta1 was observed specifically for collagen V expression (eightfold increase). The stimulation of collagen V biosynthesis by RGTA was abolished by TGF-beta1 neutralizing antibodies. These modulations occurred at protein and mRNA levels. RG-1503 did not alter TGF-beta1 mRNA steady state level or total TGF-beta1 protein content (latent+active forms). However, RG-1503 significantly induced an elevated proportion of active TGF-beta1 form, which could result from the selective protection from proteolytic degradation of TGF-beta1 by RG-1503. These data open a rationale for understanding the stimulation of tissue repair induced by RGTA, and also, a new insight for developing drugs adapted to inhibit excess collagen deposition in smooth muscle cells associated vascular disorder, and in fibrotic diseases.

Animals↗

PubMed2001

Heparan mimetic regulates collagen expression and TGF-beta1 distribution in gamma-irradiated human intestinal smooth muscle cells.

Radiation-induced intestinal fibrosis is characterized by collagen accumulation, a process in which TGF-beta1 plays a key role. We analyzed the effects of gamma radiation on collagen expression and TGF-beta1 distribution in human intestinal smooth muscle cells (HISM). We investigated the activity of a carboxymethylated and sulfated dextran (RG-1503), exhibiting antifibrotic properties and promoting in vivo intestinal tissue repair, on irradiated HISM. After (60)Co irradiation (10 Gy), HISM were labeled with [(3)H] proline (+/-RG-1503). Radiolabeled collagen I, III, and V were quantified by SDS-PAGE. TGF-beta1 was quantified by ELISA in culture medium, pericellular and intracellular compartments. Irradiation induced a specific 2.85-fold increase in collagen III production by HISM. Collagen V decreased by 80% 72 h after irradiation. Pericellular TGF-beta1 was increased (up to twofold) in irradiated HISM. RG-1503 added before or after irradiation reversed both mRNA and protein levels of collagen III and V to control values. RG-1503 decreased the amount of TGF-beta1 in the cell layer below the control values. Irradiation of HISM induced the development of a fibrotic phenotype in terms of collagen production and TGF-beta1 distribution. The antifibrotic RG-1503 restored HISM physiological characteristics and may represent a promising therapeutic approach for radiation-induced intestinal fibrosis.

Cells, Cultured↗

PubMed2001

The angiogenic factor heparin affin regulatory peptide (HARP) induces proliferation of human peripheral blood mononuclear cells.

Heparin affin regulatory peptide (HARP) also named pleiotrophin (PTN) is a polypeptide that belongs to a family of heparin-binding molecules. HARP displays mitogenic activity for a wide variety of cells, including fibroblast, endothelial and epithelial cells. This study reports, to our knowledge for the first time that HARP induced the stimulation of triated thymidine incorporation in quiescent human peripheral blood mononuclear cells in a dose-dependant manner, measured after 7 days of culture. Maximal stimulation was observed at picomolar concentration with ED50 corresponding to the half maximum effect at 50 pM. In contrast, midkine (MK), a related heparin-binding growth/differentiation factor, with more than 50% amino acid sequence homology with HARP was ineffective. These results suggest that HARP could be considered as a new cytokine involved inthe growth regulation of cell mediated immunity.

Adult↗

PubMed2000

Human plasmin enzymatic activity is inhibited by chemically modified dextrans.

Some synthetic dextran derivatives that mimic the action of heparin/heparan sulfate were shown to promote in vivo tissue repair when added alone to wounds. These biofunctional mimetics were therefore designated as "regenerating agents" in regard to their in vivo properties. In vitro, these biopolymers were able to protect various heparin-binding growth factors against proteolytic degradation as well as to inhibit the enzymatic activity of neutrophil elastase. In the present work, different dextran derivatives were tested for their capacity to inhibit the enzymatic activity of human plasmin. We show that dextran containing carboxymethyl, sulfate as well as benzylamide groups (RG1192 compound), was the most efficient inhibitor of plasmin amidolytic activity. The inhibition of plasmin by RG1192 can be classified as tight binding hyperbolic noncompetitive. One molecule of RG1192 bound 20 molecules of plasmin with a K(i) of 2.8 x 10(-8) m. Analysis with an optical biosensor confirmed the high affinity of RG1192 for plasmin and revealed that this polymer equally binds plasminogen with a similar affinity (K(d) = 3 x 10(-8) m). Competitive experiments carried out with 6-aminohexanoic acid and kringle proteolytic fragments identified the lysine-binding site domains of plasmin as the RG1192 binding sites. In addition, RG1192 blocked the generation of plasmin from Glu-plasminogen and inhibited the plasmin-mediated proteolysis of fibronectin and laminin. Data from the present in vitro investigation thus indicated that specific dextran derivatives can contribute to the regulation of plasmin activity by impeding the plasmin generation, as a result of their binding to plasminogen and also by directly affecting the catalytic activity of the enzyme.

Dextrans↗

PubMed2000

The endogenous oxindoles 5-hydroxyoxindole and isatin are antiproliferative and proapoptotic.

Oxindole-core synthetic molecules are currently being developed as anticancer drugs that target protein tyrosine kinases associated with growth factor receptors. Oxindole, 5-Hydroxyoxindole, and 2, 3-dioxindole [isatin] are natural molecules found in mammalian body fluids and tissues and we addressed the question of similar properties of endogenous oxindoles. 5-Hydroxyoxindole and isatin, but not oxindole, inhibited N1E-115, BALB/c3T3, BBC, PC12, and HL60 proliferation at submicromolar concentrations. Acute treatment with 5-hydroxyoxindole and isatin reduced the activity of extracellular signal regulated protein kinases (ERKs) by 35% at 100 microM and ERK1 activity was strongly inhibited by 5-Hydroxyoxindole at 10 microM. Survival of PMA-differentiated HL60 and FGF(2)-differentiated PC12 cells was not affected by 5-Hydroxyoxindole and isatin treatment, suggesting that endogenous oxindoles interact with growth factors signaling. The physiological implications of these data and the potential utility of 5-Hydroxyoxindole and isatin as antitumor agents are discussed.

Animals↗

PubMed2000

Increased synthesis of heparin affin regulatory peptide in the perforant path lesioned mouse hippocampal formation.

Heparin affin regulatory peptide (HARP), also known as pleiotrophin or heparin-binding growth-associated molecule, is a developmentally regulated extracellular matrix protein that induces cell proliferation and promotes neurite outgrowth in vitro as well as pre- and postsynaptic developmental differentiation in vivo. Here we have investigated the expression of HARP mRNA and protein in the perforant path lesioned C57B1/6 mouse hippocampal formation from 1 to 35 days after surgery. This type of lesion induces a dense anterograde and terminal axonal degeneration, activation of glial cells, and reactive axonal sprouting within the perforant path zones of the fascia dentata and hippocampus as well as axotomy-induced retrograde neuronal degeneration in the entorhinal cortex. Analysis of sham- and unoperated control mice showed that HARP mRNA is expressed in neurons and white and gray matter glial cells as well as vascular and pial cells throughout the normal, adult brain. Lesioning induced high levels of HARP mRNA in astroglial-like cells in the denervated zones of fascia dentata and hippocampus as soon as day 2 postlesion. This expression reached maximum at day 4, and declined toward normal at day 7-14. Combined HARP in situ hybridization and glial fibrillary acidic protein (GFAP) immunohistochemical staining and double immunofluorescent stainings for GFAP and HARP at day 4 postlesion showed colocalization of HARP mRNA and protein to hypertrophic GFAP-immunopositive astrocytes in the denervated areas. Finally, the axotomized entorhinal layer II neurons, which expressed high levels of HARP mRNA in the normal brain, exhibited a marked decline in hybridization signal after axotomy. The induction of high levels of HARP mRNA and protein in astrocytes in the denervated areas of fascia dentata and hippocampus is of particular interest as astrocytes and astrocyte-derived factors are known to be implicated in axonal growth and regeneration and in rescuing injured neurons.

Animals↗

PubMed2000

New agents for the treatment of infarcted myocardium.

Local delivery of angiogenic growth factors for the treatment of myocardial ischemia has been well documented in various animal models, and clinical trials are now in progress. Our strategy was radically different, based on selective protection of some of the growth factors naturally present within the injured tissue. This protection was obtained by applying a chemically defined substitute for Dextran called RGTA11 (for ReGeneraTing Agent). RGTA is a family of agents, which has properties mimicking those of heparan sulfates toward heparin-binding growth factors (HBGF) and which stimulate tissue repair and protection. Indeed, we have previously shown that RGTA prevents most of the damage resulting from acute skeletal muscle ischemia [FASEB J. (1999) 13, 761-766]. We now show that the same agent can be used for the treatment of myocardial infarction. Acute myocardial infarction was induced in pigs by ligation of the left circumflex artery. One hour later, a single injection of 10 mg of RGTA11 was made in the center of the infarcted area. Three weeks later we observed 1) recovery of 84% of the initial left ventricular ejection fraction (only 55% in saline-treated controls), 2) an almost 50% reduction in the infarct size, 3) a reduction in fibrotic tissue formation, 4) significant preservation of myocytes, and 5) an increase in the number of blood vessels. The treatment of ischemic heart disease with RGTA would have clear advantages over other therapies such as growth factor, gene, or cell transplants, based on a stable, simple, and easy-to-develop chemical product.

Actins↗

PubMed2000

Bioactive functionalized polymer of malic acid for bone repair and muscle regeneration.

A bioactive poly(beta-hydroxyalkanoate) derived from malic acid was prepared and tested on bone repair and muscle regeneration. This functionalized and hydrolyzable polymer was obtained after several steps, the first one being the anionic copolymerization of three malolactonic acid esters. Chemical modifications were carried out on the terpolymer to turn benzyl-protecting groups into carboxyl groups and allyl groups into sulfonate groups. The resulting polymer bore carboxylate, sulfonate, and sec-butyl pendent groups in 65/25/10 molar proportions and were aimed at interacting with heparan binding growth factors. This polymer did not present any toxic effect in cell viability of HepG2 cells, over a large range of concentrations (0.01-0.25 mgl(-1)). Its ability to improve wound healing was tested in vivo and positive results are reported. Furthermore, the bioactivity of this polymer was evaluated using the regeneration model of Extensor digitorum longus (EDL) rat muscle. The study displayed a significant increase in the muscle regeneration and maturation.

Animals↗

PubMed2000

Differential expression of FGF receptors and of myogenic regulatory factors in primary cultures of satellite cells originating from fast (EDL) and slow (Soleus) twitch rat muscles.

In the rat, the fast and slow twitch muscles respectively Extensor digitorum longus (EDL) and Soleus present differential characteristics during regeneration. This suggests that their satellite cells responsible for muscle growth and repair represent distinct cellular populations. We have previously shown that satellite cells dissociated from Soleus and grown in vitro proliferate more readily than those isolated from EDL muscle. Fibroblast growth factors (FGFs) are known as regulators of myoblast proliferation and several studies have revealed a relationship between the response of myoblasts to FGF and the expression of myogenic regulatory factors (MRF) of the MyoD family by myoblasts. Therefore, we presently examined the possibility that the satellite cells isolated from EDL and Soleus muscles differ in the expression of FGF receptors (FGF-R) and of MRF expression. FGF-R1 and -R4 were strongly expressed in proliferating cultures whereas FGF-R2 and R3 were not detected in these cultures. In differentiating cultures, only -R1 was present in EDL satellite cells while FGF-R4 was also still expressed in Soleus cells. Interestingly, the unconventional receptor for FGF called cystein rich FGF receptor (CFR), of yet unknown function, was mainly detected in EDL satellite cell cultures. Soleus and EDL satellite cell cultures also differed in the expression MRFs. These results are consistent with the notion that satellite cells from fast and slow twitch muscles belong to different types of myogenic cells and suggest that satellite cells might play distinct roles in the formation and diversification of fast and slow fibres.

Animals↗

PubMed1999

Glycosaminoglycans promote HARP/PTN dimerization.

Heparin affin regulatory peptide (HARP), also called pleiotrophin (PTN), is a secreted polypeptide which binds to heparin and plays a key role in cellular growth and differentiation. In order to assess the determinants potentially important to its biological activity, we tested the ability of HARP to oligomerize, a process involved in mitogenic activity of the heparin-binding fibroblast growth factor. Using dissuccinimidyl suberate cross-linking experiments and affinity chromatography, we report that human HARP forms noncovalent dimers. Dimerization is dependent on the presence of heparin or other sulfated glycosaminoglycans, as chlorate treatment of cells inhibits this process. In vitro, different glycosaminoglycans, such as dermatan sulfate and chondroitin sulfate-C, also induce a dimer assembly of HARP. The relevance of this process was supported by experiments demonstrating that HARP is secreted as a dimer in conditioned medium of NIH-3T3 cells that overexpressed this growth factor and is also associated to the cell surface or to the extracellular matrix.

3T3 Cells↗

PubMed1999

Functional Rac-1 and Nck signaling networks are required for FGF-2-induced DNA synthesis in MCF-7 cells.

The effects of Fibroblast Growth Factor-2 (FGF-2) on breast cancer cell DNA synthesis are controversial. To elucidate the mechanisms by which FGF-2 stimulates or inhibits DNA synthesis, we analysed FGF-2 signaling pathways in breast cancer MCF-7 and MCF-7 cells overexpressing Ha-Ras (MCF-7ras). We found that FGF-2-induction of DNA synthesis correlates with Ras transient activation, FRS-2 tyrosine phosphorylation and low level of expression of p66Shc. In addition, Nck-associated proteins are highly tyrosine phosphorylated and JNK reaches a higher level of activation when FGF-2 triggers DNA synthesis. Interestingly upon FGF-2 treatment, JNK activation and DNA synthesis are dependent on Rac-1 activity. These results confirm that in MCF-7 cells, induction of DNA synthesis by FGF-2 requires a transient activation of the Ras/MAPK cascade and demonstrates for the first time that intact Rac-1 and Nck signaling networks are required.

Adaptor Proteins, Signal Transducing↗