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Biomedical subjects

D Barritault

Publications and source records attributed to D Barritault.

At least 55 records · Page 3Linked to original sources
PubMed1997

Cellular distribution of the angiogenic factor heparin affin regulatory peptide (HARP) mRNA and protein in the human mammary gland.

The heparin affin regulatory peptide (HARP) growth factor, also known as pleiotrophin, is a developmentally regulated protein that displays biological functions during cell growth and differentiation. To study the physiological role of this protein, we investigated the cellular distribution of HARP mRNA and protein in the resting human mammary gland. In situ hybridization histochemistry revealed that HARP mRNA was localized in alveolar myoepithelial cells, whereas alveolar epithelial cells were negative. In the stroma, HARP mRNA was localized in endothelial cells and smooth muscle cells of blood vessels. Interestingly, HARP protein and mRNA were not always co-localized. HARP protein immunocytochemistry staining was observed in an area including both alveolar myoepithelial and epithelial cells, although epithelial cells do not express HARP transcript. In contrast, the distribution of HARP protein is parallel to that of HARP mRNA in endothelial and vascular smooth muscle cells. In the light of these results, the putative role of HARP in controlling the proliferation and/or differentiation of the different mammary cell types is proposed and discussed.

Blotting, Western↗
PubMed1997

Transmural endothelialization of vascular prostheses is regulated in vitro by Fibroblast Growth Factor 2 and heparan-like molecule.

Endothelialization of vascular prostheses may result from transmural migration of endothelial cells. Angiogenesis is controlled by growth factors like Fibroblast Growth Factor 2 (FGF2) and regulators like heparan-like molecules. To that end, we used heparan-like molecules named RGTA for ReGeneraTing Agent. The RGTA11 used was a chemically derived dextran obtained by successive substitutions with carboxymethyl, benzylamide, and benzylamide sulfonate groups on glucose residues. This agent was further selected for its ability to bind, stabilize and protect FGF2. We defined firstly the angiogenic capability of FGF2 in combination with RGTA11 on bovine aortic endothelial cells (BAEC) cultured on collagen I gels. Secondly, the role of FGF2 and RGTA11 in transmural endothelialization was assessed in a three-dimensional in vitro model using a polyethylene terephtalate prosthesis included in collagen gel. BAEC seeded on the external face can migrate to the luminal face of the prosthesis. Microscopic and histological evaluations were performed at 4 and 7 days. Results showed that the addition of RGTA11 alone did not promote angiogenesis while FGF2 alone did. However, RGTA11 combined with FGF2 produced a significant acceleration in angiogenesis compared to FGF2 alone. This combination magnifies and enhances the angiogenic processes leading to endothelialization of luminal face through transmural cellular migration. Our data demonstrates that in vitro transmural endothelialization of porous vascular prostheses by BAEC cultured on collagen I gels is upregulated by RGTA11 combined with FGF2.

Animals↗
PubMed1996

Heparin-like polymers derived from dextran enhance colonic anastomosis resistance to leakage.

A new tissue repair agent, RGTA11, is described for its ability to enhance colonic anastomosis repair and resistance to leakage. RGTA11 is a dextran derivative containing 110% carboxymethyl groups, 2.6% carboxymethyl benzylamide groups, and 36.6% carboxymethyl benzylamide sulfonate groups. RGTA11 was deemed efficient to protect the heparin-binding growth factors FGF2 against trypsin digestion. By this property RGTA11 mimicked heparin or heparan sulfate. We have also found that RGTA11 protected TGF beta 1 against trypsin digestion while heparin did not. RGTA11 was then tested in an in vivo wound-healing model of colonic anastomosis. Our results indicate that after 48 h, RGTA11- or RGTA11/FGF-2-treated animals presented a resistance of the anastomosis to leakage which was increased twofold (p < 0.05) over untreated controls. After 96 h and until day 7 there was no more difference with control animals. Our results suggest that RGTA11 presents potential clinical interest by preventing earlier leakage of colonic anastomosis.

Anastomosis, Surgical↗
PubMed1996

Visualization of several binding sites for basic fibroblast growth factor (FGF-2) on fibroblasts by photoaffinity labeling: evidence for intracellular complexes.

The internalization of basic fibroblast growth factor (FGF-2) was studied in Chinese hamster lung fibroblasts (CCL39). Recombinant FGF-2 was derivatized with a photoactivable agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), iodinated, and used to visualize intracellular FGF-2-affinity-labeled molecules after internalization at 37 degrees C. Iodinated HSAB-FGF-2 maintained the properties of natural FGF-2 such as affinity for heparin, binding to Bek and Fig receptors, interaction with high- and low-affinity binding sites, and reinitiating of DNA synthesis in CCL39 cells. Affinity-labeling experiments at 4 degrees C with 125I-HSAB-FGF-2 led to the detection of several FGF-cell surface complexes with apparent molecular mass of 80, 100, 125, 150, 170-180, 220, 260, and about 320 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two specific bands at 80 and 130-160 kDa were obtained using the homobifunctional cross-linking reagent, disuccinimidyl suberate. When the cells, preincubated with 125I-HSAB-FGF-2 at 4 degrees C and then washed, were shifted to 37 degrees C, irradiation of the internalized labeled FGF-2 led to detection of a similar but fainted profile with one major specific band at 80 kDa. Heparitinase II treatment of the cells reduced binding of 125I-HSAB-FGF-2 to its cell surface sites by 80% and internalization by 55%, indicating the involvement of heparan sulfate proteoglycans in these processes. Among the heparitinase-sensitive bands was the 80-kDa complex.

Affinity Labels↗
PubMed1996

FGF protection and inhibition of human neutrophil elastase by carboxymethyl benzylamide sulfonate dextran derivatives.

Several derivatized dextrans (DxD) containing defined percentage of carboxymethyl, carboxymethyl benzylamide and carboxymethyl benzylamide sulfonate groups have been shown to stimulate tissue repair in various in vivo models including skin, bone, muscle and cornea. These selected DxD were also shown to mimic heparin or heparan sulfate by their ability to interact with, stabilise and protect the heparin-binding growth factor of the fibroblast growth factor family against trypsin digestion (Tardieu et al., J. Cell. Physiol. 1992; 150: 94). The wound healing action of these DxD was explained by postulating that the endogenously released heparin-binding growth factors could be protected within the wound. To further understand the action of these DxD on tissue repair, we have studied their effect on the human neutrophil elastase (HNE) activity, one of the proteases involved in wound repair. These DxD inhibited HNE in an hyperbolic non-competitive manner. Extent of HNE inhibition by DxD increased with their molecular weight and benzylamide sulfonate substitution levels. One DxD, RGT11, was the best inhibitor (Ki 40 pM) and efficiently inhibited FGF-2 proteolysis by HNE, restoring its growth-promoting activity towards human skin fibroblasts. The data contribute to a better understanding of the wound-healing property and anti-inflammatory activity of these polymers.

Cell Division↗
PubMed1996

Growth factors in skeletal muscle regeneration.

Adult skeletal muscles are able to regenerate after injury. This process is due to the activation of quiescent muscle precursor cells, also called satellite cells, which proliferate and differentiate to form new myotubes. In this regeneration process, several growth factors which come from the muscle and/or from the motor nerve and inflammatory cells have been shown to play key roles. However, most of our knowledge comes from in vitro studies, where, during myogenesis, proliferation of satellite cells is regulated by FGFs, TGF beta s, PDGF, IGF-I and II, while differentiation appears to be promoted mainly by IGFs. During regeneration in vivo, most of these factors have been shown to operate and interact. Other factors also appear to condition the regeneration process, such as LIF, which acts predominantly as a proliferative factor; and HARP/PTN/HB-GAM and other neurotrophic factors, which may be necessary for the formation of new neuromuscular junctions. TGF beta has a major influence on the reorganisation of the extracellular matrix. This review presents a critical summary of the known effects of growth factors on skeletal muscle regeneration.

Animals↗
PubMed1996

Modulation of cranial bone healing with a heparin-like dextran derivative.

Substituted dextran polymers have been shown to bind growth factors and protect them from enzymatic degradation. Using this information, other researchers have been able to use these substituted dextrans to enhance the healing of bone in an environment where bone would otherwise not regenerate. We used substituted dextran polymers to evaluate their ability to accelerate the healing of cranial bone in a rabbit model. We were able to document a more rapid rate of healing and demonstrate micrographic evidence to support that conclusion. Possible mechanisms are postulated.

Analysis of Variance↗
PubMed1996

New concepts in tissue repair: skin as an example.

Several heparin-binding growth factors (HBGFs) are thought to play a key role in natural processes of tissue regeneration or repair after release from inflammatory or circulating cells and extracellular matrix-associated heparan sulfate proteoglycosaminoglycans. To clarify how the bioavailability of these HBGFs can help regulate wound-healing processes, we studied the healing effect of various chemically substituted dextrans (RGTA) selected for their affinity for HBGFs. One member of the RGTA family, RGTA11, obtained by substitution of carboxymethyl (CM), benzylamide (B) and benzylamide sulfonate (S) groups in a proportion of 110% (CM), 2.6% (B) and 36.5% (S) respectively was used in these studies. RGTA11 may potentiate the biological activity of fibroblast growth factors 1 and 2 and protect them against heat or pH inactivation and proteolytic degradation. RGTA11 was tested in a rat punch-biopsy skin-healing model for its ability to enhance wound repair. Wounds were filled with collagen plaster alone or soaked with RGTA, and skin regeneration was studied by histological analysis. In collagen plaster, RGTA11 affected both the kinetics and quality of restored skin. It seems likely that endogenous growth factors naturally released during the regeneration process are trapped and protected against natural proteases, thereby preserving their ability to stimulate tissue repair. Since most known growth factors have a nearly ubiquitous distribution and blind to heparin, our hypothesis was verified by studying the ability of RGTA to induce repair in damaged tissue. We demonstrated the RGTA could stimulate wound repair in various models, including bone, muscle, nerve, cornea and colonic anastomosis. The data presented here concern wound-healing in a deep skin model and suggest that heparan-like biopolymers constitute a new family of tissue-repair agents with a wide variety of potential uses. The efficiency of this approach in cases in which impaired healing is associated with a pathology, as in diabetes, remains to be determined.

Animals↗
PubMed1995

Influence of a dextran derivative on myosin heavy chain expression during rat skeletal muscle regeneration.

We recently described dextran derivatives (RGTA) which stimulate tissue repair in several in vivo models. One of them, RGTA11, has been shown to accelerate crush-induced regeneration and reinnervation of rat EDL and Soleus muscles. In this study we wanted to know if RGTA11 alters the pattern of myosin heavy chain expression during regeneration. In both EDL and Soleus muscles, RGTA11, injected at the moment of the crush, was found to accelerate the shift from neonatal to adult myosin heavy chain isoforms within 2 weeks. The proportion of slow fibers increased considerably, especially in the Soleus where RGTA11 induced a precocious and permanent expression of slow myosin isoform, thus confirming that a more efficient innervation had occurred in the presence of RGTA11. These results illustrate the interesting potential pharmacological use of such dextran derivatives in neuromuscular disease.

Animals↗
PubMed1995

Effect of heparin on bovine epithelial lens cell proliferation induced by heparin affin regulatory peptide.

HARP (heparin affin regulatory peptide) is an 18 kDa heparin binding protein, also known as HB-GAM or pleiotrophin (PTN) which has been primarily isolated from brain and uterus, and displays neurite outgrowth, angiogenic and mitogenic activities. Previously, we have expressed the human cDNA encoding human HARP in NIH 3T3 cells. Purified recombinant HARP displayed mitogenic activity for endothelial cells. Its NH2-terminal sequence indicates that the HARP molecule possesses a three amino acid extension from the signal peptide more than the NH2-terminal described. For HB-GAM or PTN, these three amino acids may be essential for the stability and the mitogenic activity of this growth factor. In an attempt to further study the mode of action of this growth factor, we have investigated the mitogenic effect of HARP on various cell types. In contrast to FGF-2, HARP failed to induce stimulation of DNA synthesis on a CCL39 cell line. However, we found that in quiescent bovine epithelial lens (BEL) cells, the stimulation of DNA synthesis induced by HARP is dose-dependent (EC50: 2.5 ng/ml) and maximal stimulation is as potent as that induced by FGF-2 (EC50: 25 pg/ml). Interestingly, when BEL cells were allowed to quiesce in the presence of serum, the stimulation induced by HARP is considerably less potent. In this highly responsive cell system, heparin could potentiate the mitogenic activity of HARP at very low doses (0.1-1 microgram/ml) and inhibit this activity at concentrations of 10 micrograms/ml. In contrast to its protective effect on FGF-1 and -2, heparin was unable to preserve HARP from tryptic and chymotryptic degradations.

Animals↗
PubMed1995

Production and purification of active FGF2 via recombinant fusion protein.

Basic fibroblast growth factor (FGF2) is involved in both cell proliferation and differentiation processes. Heparin may interfere in the stability and biological activities of FGFs. However, it is difficult to obtain FGF preparation without traces of heparin since heparin affinity chromatographies are routinely used to prepare this growth factor. We have therefore devised a means of production of active recombinant FGF2 devoid of heparin traces. The bovine FGF2 gene was inserted into the pMAL-c prokaryotic expression vector and the recombinant protein was synthesised as a fusion product between the maltose binding protein (MBP) and FGF2. Purification of the FGF2 fusion protein was performed by an amylose affinity chromatography. Yields were similar to those obtained by using a traditional heparin affinity column purification procedure. The fusion protein (MBP-FGF2) and the cleaved-off FGF2 were tested for some of their biological properties and compared to recombinant FGF2 purified by heparin affinity chromatography. Mitogenic activity on Chinese hamster lung fibroblasts (CCL39) and neurite outgrowth on pheochromocytoma culture cells (PC12) were used as biological assays. The cleaved-off FGF2 was as active as commercially available recombinant FGF2 (ED50 at 0.16 and 0.04 nM respectively). However MBP-FGF2 was less active (ED50 at 0.9 nM) in both tests.

Amylose↗
PubMed1995

Inhibition by dextran derivatives of FGF-2 plasmin-mediated degradation.

In previous work we have shown that some new regenerating agents (RGTAs), molecules which correspond to some dextran derivatives (DxD) containing defined amounts of carboxymethyl (CM), benzylamide (B) and benzylamide sulfonate (BS) groups, were able to stimulate tissue repair when applied at the site of injury. Based on in vitro studies showing that these DxD could interact and protect heparin binding growth factors (HBGFs), we postulated that DxD could also act in vivo by protecting endogenously released HBGFs against protease degradation. We now present data demonstrating that human plasmin (HP1), one of the known proteases involved in extracellular matrix remodelling and in the local activation of some growth factors is specifically inhibited by some specific DxD. The most efficient compounds for inhibiting the amidolytic activity were substituted by all functions with IC50 at 0.26 microM for RGTA11 (a DxD obtained from a 40,000 Da dextran containing 110% of CM, 2.5% of B and 36.5% of BS units and with IC50 at 1.1 microM for RGTA10 (derived from 10,000 Da dextran and containing 110% of CM, 0% of B and 27.3% of BS). Compounds which were substituted with only one or two functions were less effective. The degradation of FGF-2 by HP1 was analyzed by SDS-PAGE and by measuring its residual growth promoting activity using a bioassay on human skin fibroblasts. In this assay, RGTA11 at a concentration of 1 microM could inhibit by 80-100% FGF-2 degradation induced by HP1 treatment. In conclusion, the inhibitory activity of some DxD towards HP1 as well as the ability of these DxD to protect FGF-2 against this proteinase could partially explain its beneficial influence on extracellular matrix remodelling following tissue injury.

Adult↗
PubMed1995

Biochemical and mitogenic properties of the heparin-binding growth factor HARP.

Heparin affin regulatory peptide (HARP), also called Pleiotrophin (PTN), is a polypeptide that displays a high affinity for heparin and that shares approximately 50% sequence homology with Midkine (MK). According to this structural homology, these two molecules constitute a new family of heparin-binding proteins. The biological properties of HARP and MK remain largely a subject of debate. Both proteins have been described as neurite outgrowth promoting agents whereas until recently the mitogenic activity has been controversial. The aim of this review is to summarize the information on HARP with special focus on the recent data relating to its mitogenic properties.

Amino Acid Sequence↗
PubMed1995

Heparan-like molecules induce the repair of skull defects.

Heparin-binding growth factors (HBGFs) are known to stimulate bone repair when applied to bone lesions. Nevertheless, successful treatments are obtained with high protein doses since HBGFs are rapidly degraded in situ by multiple proteolytic activities associated with the inflammatory period of tissue healing. Like heparin or heparan sulfates, heparan-like molecules, named carboxymethyl-benzylamide-sulfonated dextrans (CMDBS), are known to potentiate fibroblast growth factor activities by stabilizing them against pH, thermal or proteolytic denaturations, and by enhancing their binding with cell surface receptors. We have postulated that CMDBS stimulate in vivo bone healing by interacting with endogenous HBGFs, spontaneously released in the wounded site. The effect of CMDBS on bone repair was studied in a skull defect model in rats by computer-assisted radio-morphometry and histomorphometry. Single application of CMDBS in a collagen vehicle to skull defects induced a dose-dependent increase in bone defect closure and new bone formation after 35 days. Complete bony bridging occurred in defects treated with 3 micrograms CMDBS, whereas bone formation was not observed in vehicle-treated defects which contained only dense fibrous connective tissue between the defect margins. These results indicate that heparan-like molecules, such as CMDBS, are able to induce bone regeneration of skull defects. This action is possibly mediated by potentiation of endogenous growth factor activities and/or by neutralization of proteolytic activities.

Animals↗
PubMed1995

[Acceleration of the regeneration of skeletal muscles in adult rats by dextran derivatives].

Dextran derivatives were obtained by controlled successive substitutions of carboxymethyl, carbomethyl-benzylamide and carboxymethyl-benzylamide sulfonate groups on glucose residues. Among these derivatives, RGT11 was selected since it mimicked some properties of heparin or heparan sulfate to stabilise and protect heparin binding growth factors such as FGFs and TGF-beta. Furthermore RGT11 inhibited plasmine and leukocyte elastase. In previous works, we have explained the healing effects of RGT obtained in skin or bone repair models by these protecting and inhibiting properties. We now present the results obtained after a single injection of RGT11 in a regenerating crushed extensor digitorum longus (EDL) muscle. After 8 days, RGT11 injected muscles contained 10 times the number of fibers than controlled injected muscles. Fibers were organized in bundles of normal size. Histological analysis indicated that regeneration was comparable to that observed after 3 weeks without RGT. Hence in vivo, RGT11 could act by protecting the heparin binding growth factors involved in the natural process of muscle regeneration and therefore favour their actions. This family of polymers should offer a new pharmaceutical potential for treating muscle atrophy and destruction and is worth studying further.

Animals↗
PubMed1995

Fibroblast growth factors and their specific binding sites in normal urothelium and transitional cell carcinoma (TCC) of the human bladder.

In order to further investigate the involvement of FGF1 and FGF2 in the transitional cell carcinoma of the bladder (TCC), we compared the expression of FGF1, FGF2 and their membrane binding sites by biochemical and immunological methods in both human normal urothelium and transitional cell carcinoma of the bladder. Using a specific enzyme immunoassay (EIA), we observed that neoplastic tissue exhibited a lower FGF2 content than normal tissue. FGF1 and FGF2 Western-blot analysis revealed in both types of tissues a major FGF1 form with a low molecular weight (14 kDa) and a single FGF2 form (21 kDa). Immunohistological studies showed that FGF1 was specifically located in normal or transformed urothelial cells, while FGF2 was associated with bladder stroma. Binding analysis in tissue membrane preparations revealed a dramatic drop in both high and low affinity binding sites for FGF2 in TCC bladder in comparison to normal bladder. FGF2 cross-linking experiments illustrated a qualitative modification of FGF2 binding complexes in tumors. These data suggest that invasive bladder carcinoma is associated with modifications of FGF1 and FGF2 tissue levels and alterations in the characteristics of the FGF2 membrane binding sites.

Binding Sites↗
PubMed1995

[Effects of substituted dextran on reinnervation of a skeletal muscle in adult rats during regeneration].

RGTA11 is a chemically substituted dextran that mimics some of the properties of heparin or heparan sulphates towards heparin binding growth factors as well as inhibits some heparin binding proteases. In vivo RGTA11 has been shown to enhance muscle regeneration after crush. We now present evidence that RGTA11 can alos favour reinnervation of fast (EDL) as well as slow (soleus) crushed muscles. Both types of muscles were injected with RGTA11 after crushing and nerve cutting. In EDL muscles, after 16 days, motor end plates were more rapidly reformed and choline acetyl-transferase activity was 2 fold higher than in controls. In soleus muscles, after 16 days, motor end plates were reformed at normal size while controls were on average 30% smaller, the 16S form of acetyl-cholinesterase and choline acetyl-transferase activity were twice those of non injected regenerating controls. In conclusion, RGTA11 favours axonal growth and synaptic differentiation, allowing a more rapid reinnervation and maturation of the regenerated fibers. RGTA may present a new family of drugs against neuromuscular degenerescence.

Acetylcholinesterase↗