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D Barritault

Publications and source records attributed to D Barritault.

At least 73 records · Page 4Linked to original sources
PubMed1994

FGFs and their receptors, in vitro and in vivo studies: new FGF receptor in the brain, FGF-1 in muscle, and the use of functional analogues of low-affinity heparin-binding growth factor receptors in tissue repair.

Several heparin-binding growth factors (HBGFs) are thought to play a key role in the natural processes of tissue homeostasis, regeneration or repair. The HBGFs are active upon release from neighbouring inflammatory or circulating cells, as well as upon release from heparan sulfate proteoglycosaminoglycans that are associated with the extracellular matrix (ECM). To better understand the physiological role of these HBGFs, we have focused our effort on studying a subset of HBGFs, namely FGF-1 and FGF-2 and their receptors. We present the purification and characterisation of a new form of heparin-binding FGF receptor from adult bovine brain (Perderiset et al., 1992). This receptor has now been purified to homogeneity. Ligand blot and cross-linking experiments performed with labeled FGF-1 or FGF-2 revealed 80-kd and 130-kd bands. Preliminary sequence information indicates that receptor is different from the receptors, FGFR-1 to -4, but it may be related the cysteine-rich-FGF receptor (CFR). We have previously shown that FGF-1, but not FGF-2, is specifically expressed in myoblastic satellite cells during the proliferating phase preceding myoblast alignment and fusion. We have now transfected primary cultures of rat myoblastic satellite cells with FGF-1 cDNA and expressed this growth factor constitutively. The transfected cells were no longer able to form myotubes. Transfection with antisense FGF-1 induced myotube formation suggesting that endogenous expression of FGF-1 is associated with myoblastic cell differentiation. Numerous studies have concluded that the ECM represents a natural reservoir for various HBGFs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Interaction of basic fibroblast growth factor (FGF-2) with nonresponsive HeLa cells.

Human HeLa adenocarcinoma cells did not respond to basic fibroblast growth factor (bFGF or FGF-2) but did bind the same amount of bFGF as responsive Chinese hamster lung fibroblasts (CCL 39). Heparinase II treatment of HeLa and CCL 39 cells resulted in a decrease of bFGF binding by 96 and 57%, respectively, indicating that heparan sulfate molecules were involved in bFGF binding. On HeLa cells, bFGF bound to a single family of low-affinity sites. Cross-linking experiments of 125I-bFGF to HeLa cells yielded several labeled complexes. Cell-associated 125I-bFGF was internalized in both cell types either by high-affinity receptors and heparitinase-sensitive sites in CCL 39 cells or by heparitinase-sensitive binding sites only in HeLa cells. The binding of bFGF to nonresponsive HeLa cells and its internalization via a family of heparitinase-sensitive binding sites might illustrate other functions of bFGF unrelated to cell proliferation.

Animals↗
PubMed1994

New approaches to tissue regeneration and repair.

Several Heparin Binding Growth Factors (HBGFs) are thought to play a key role in the natural processes of tissue regeneration or repair after being released by neighbouring, inflammatory or circulating cells as well as from extracellular matrix associated heparan sulfate proteoglycosaminoglycans. In order to better understand how the bioavailability of these HBGFs can take part in the regulation of the wound healing processes, we have studied the healing effect of various chemically substituted dextrans (CMDBS) selected for their affinity for HBGFs, alone and in association with HBGFs. The CMDBS was obtained by substitution of methylcarboxylic (CM), benzylamide (B) and benzylamine sulfonate (S) groups in proportion of 83%, 23% and 13% respectively for CMDBS K that we have further used (Mauzac et al., 1985 Biomaterials. 6: 61-63). CMDBS K could 1: potentiate the biological activity of 1 or 2 FGFs, 2: protect 1 and 2 FGFs against thermal or pH inactivation, 3: protect a and b FGFs against proteolytic degradation (Tardieu et al., 1992 J. Cell. Physiol. 150: 194-203). CMDBS K was tested alone in cutaneous and bone wound healing models and for its ability to stabilize FGFs. Rats were punched and skin regeneration was studied by morphometric and histological analysis. The wounds (6 mm diameter) were filled with collagen plaster alone or soaked with CMDBS. CMDBS K in collagen plaster was able to induce a remarkable effect both on the kinetics and on the quality of the restored skin. These results suggest that endogenous growth factors naturally released during the regeneration process could be trapped, protected and released by CMDBS.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

CMDBS, functional analogue of heparin sulfate as a new class of corneal ulcer healing agents.

Soluble dextran polymer derivatives (CMDBSs) are originally synthesized as heparin-like plasma substitutes. Some of them mimic heparin in its interactions and stabilize, protect and facilitate actions of heparin binding growth factors. The wound healing activity of one specific CMDBS was studied in a model of corneal ulcer on the rabbit eye and compared with the activity of basic fibroblast growth factors (bFGF) added alone or in association with CMDBS. Total reepithelization was observed with bFGF + CMDBS, bFGF alone and CMDBS alone after, respectively, 3.8 +/- 0.78, 4.3 +/- 0.67 and 4.4 +/- 0.51 days. All treatments were efficient if compared with eyes treated with saline (p < 0.0001). The grade of significance of the applied treatments was as follows: bFGF + CMDBS > bFGF > CMDBS > saline. Our study pinpoints that some specific CMDBS are as potent agents as bFGF for corneal ulcer healing, and can therefore be proposed for therapeutic use.

Animals↗
PubMed1994

Mitogenic and in vitro angiogenic activity of human recombinant heparin affin regulatory peptide.

We have previously described the purification of a heparin binding growth factor from adult bovine brain named heparin affin regulatory peptide (HARP), which was identical to an uterus derived growth factor named pleiotrophin and to a developmentally regulated neurite promoting factor named heparin-binding growth associated molecule. However, for yet unclear reasons, the mitogenic activity of this purified polypeptide following isolation from animal tissue extracts is a subject of controversy, due to conflicting and irreproducible data when produced by recombinant DNA technologies in E. coli or insect cells. The purified protein was inactive in mitogenic assays but the natural molecule was active in assay of neurite outgrowth. In order to clarify these conflicting results and to obtain a recombinant protein free from other contaminating heparin-binding growth factors, we have cloned human cDNA encoding human HARP, engineered its expression in NIH 3T3 cells and characterised the resulting recombinant polypeptide. Purified recombinant HARP displayed mitogenic activity for capillary endothelial cells with half-maximal stimulation at approximately 1 ng/ml (55 pM) and induced angiogenesis in an in vitro model. Interestingly, while the NH2 terminal sequence of tissue purified HARP was NH2-GKKEKPEKK, the NH2 terminal sequence of the biologically active recombinant protein was NH2-AEAGKKEKPEKK, corresponding to a three amino acid extended form.

3T3 Cells↗
PubMed1994

Mesoglycan and sulodexide act as stabilizers and protectors of fibroblast growth factors (FGFs).

Heparin and heparan sulfate proteoglycans (HSPGs) stabilize FGFs which belong to heparin-binding growth factors (HBGFs) on active conformation. They also strongly potentiate their mitogenic activity on many cell types, and protect them against thermal denaturation and enzymatic degradation. In the present work we have tested two heparin-like substances named mesoglycan and sulodexide obtained from bovine intestinal mucosal extracts. These products are used as heparin, in various of therapeutic fields such as atherosclerosis or antithrombotic therapy. The compositions of mesoglycan and sulodexide are partially known and include chondroitin, dermatan and heparan sulfate. We have shown that mesoglycan and sulodexide potentiated the mitogenic activity of FGF1 and FGF2. The magnitude of this effect was identical with that of heparin used as a control substance but at double concentration. Mesoglycan and sulodexide also exerted stabilizing and protective effects on FGFs for heat denaturation and enzymatic degradation. The suppression of the protective properties after heparinase treatment of mesoglycan and sulodexide indirectly demonstrated the presence of heparan sulfate which was shown to represent about 60% of the commercial products.

Animals↗
PubMed1994

[CMDBS, functional analogs of sulfate heparanes, used as osseous cicatrizing agents].

Several Heparin-Binding Growth Factors (HBGFs) are known to play an important role in bone repair. When osseous tissue is injured, an important increase of protease activities and a massive release of HBGFs occur. The local increase in HBGFs content at the wounded site, produced by a release of this factors from cells implicated in haemostasis and inflammatory reaction and from extracellular matrix associated heparan sulfate proteoglycans (HSPGs), seems to be a crucial step in bone healing. The proteolysis associated with the tissue injury probably limits the growth factors activities at the wound site. In order to define the bone healing potential of molecules that would be able to protect HBGFs against proteolytic activation, we studied the effect of derived dextrans, named carboxymethyl-benzylamide-sulfonated dextrans (CMDBS), behaving as heparan like molecules, in 5 mm in diameter skull trepaned defects in young adult rats. In this model CMDBS induced an important bone regeneration in a dose dependent manner while controls were not repaired. In CMDBS treated animals the defects were repaired and contained a tissue of normal appearance; in several treated animals the sagittal suture, initially removed by the trephination, was restored. This remarkable bone healing potential of CMDBS may result from the capacity to protect the endogenous HBGFs from proteolysis and to modulate their biological activities, in a similar manner to that observed for fibroblast growth factors and HSPGs. CMDBS represent a new form of bone healing agents, which have the advantage of being produced by a controlled chemical synthesis, and of avoiding the use of exogenous growth factors because of their capacity to enhance the bone healing potential of the endogenous growth factors.

Animals↗
PubMed1993

Increased immunodetection of acidic fibroblast growth factor in bladder cancer, detectable in urine.

Acidic fibroblast growth factor is a regulatory peptide involved in cell proliferation, differentiation and motility. We used a polyclonal antiserum raised against purified native bovine acidic fibroblast growth factor, with no cross-reactivity for basic fibroblast growth factor to detect acidic fibroblast growth factor in tissue extracts and urine samples by means of a competitive enzyme immunoassay. Histochemical analysis was also performed on 10 specimens of normal urothelium and 50 of bladder cancer. Acidic fibroblast growth factor immunoreactive material was found in normal urothelium (1.77 +/- 2 ng./gm. tissue) and was increased more than 10-fold in patients with transitional cell carcinoma of the bladder (20.36 +/- 12 ng./gm. tissue). Immunohistochemical analysis localized immunoreactivity in the epithelial compartment of bladder tumors. Acidic fibroblast growth factor was assayed in urine from 579 individuals comprising a control group (114) and patients with benign prostatic hypertrophy (133), carcinoma of the prostate (96) or transitional cell carcinoma of the bladder (236). There was a significant difference in the frequency of urinary acidic fibroblast growth factor detection among the patients with invasive transitional cell carcinoma, the control group (p < 0.001) and the patients with prostatic disease (p < 0.01). The sensitivity was 72% and the specificity was 91%. Furthermore, the frequency of acidic fibroblast growth factor detection by enzyme immunoassay in the urine and the intensity of immunostaining was correlated with the stage of the disease. These data strongly suggest that acidic fibroblast growth factor is a potential marker for bladder tumors that may be of use in the noninvasive followup of patients with bladder cancer. We present a simple and reliable enzyme immunoassay for the detection of acidic fibroblast growth factor in voided urine that might be useful to quantitate this marker.

Biomarkers, Tumor↗
PubMed1992

Growth in serum-free medium of NIH3T3 cells transformed by the EJ-H-ras oncogene: evidence for multiple autocrine growth factors.

Rodent fibroblastic cells transformed by ras oncogenes can grow in serum-free (S-) medium. We have studied clonal lines of mouse NIH3T3 fibroblasts transfected with the EJ-H-ras oncogene, and observed that practically all become independent of exogenous growth and attachment factors shortly after transfection. Moreover, all the clones tested soon form anchorage-independent (AI) colonies in S- medium, and most give rise to spheroids able to grow in suspension. The cell-conditioned S- medium of the transformed (TR) cells stimulates autocrinally the AI and anchored growth of these cells, in the absence of serum, and it contains growth factors related to TGF-alpha (or EGF), PDGF and bFGF, and other uncharacterized factors. Some of these factors are not found, or are found only in very small amounts, in the S- medium of non-transformed NIH3T3 cells, which also stimulates the growth of the TR cells, in the absence of serum. In addition, the TR cells contain 4-6 times more cell-associated bFGF than the non-transformed cells and release more latent TGF-beta activatable by acid treatments. However, no active TGF-beta is secreted by either cell type. Activated TGF-beta and pure TGF-beta 1 stimulate the growth of the anchored TR and NIH3T3 cells, but inhibit the AI growth of the TR cells. Another inhibitor of this growth is also found in the concentrated medium of the NIH3T3 cells.

3T3 Cells↗
PubMed1992

High and low affinity membrane binding sites for fibroblast growth factors in the developing chick brain.

Acidic and basic fibroblast growth factors (aFGF and bFGF), two mitogenic, neurotrophic and angiogenic molecules, are present in the embryonic chick brain but their function remains unclear. In order to approach the biological activity of FGFs during brain development, we have looked for their receptors and studied their regulation through chick brain development. Competitive binding studies realized on brain membranes indicated the presence of two classes of FGF binding sites: high affinity binding sites (dissociation constant, Kd = 100 pM) and low affinity binding sites (Kd = 20 nM). Cross-competition experiments show that these two classes of binding sites both interact with aFGF and bFGF. The number of sites in these two classes of binding sites changes during embryogenesis. On the one hand, the membrane capacity of high affinity sites decreases from E7 (1 +/- 0.2 pmol/mg of protein) to E15 (0.5 +/- 0.2 pmol/mg of protein); on the other hand, the membrane capacity of low affinity sites increases from E15 (25 +/- 4 pmol/mg of protein) to P1 (75 +/- 20 pmol/mg of protein). Cross-linking experiments revealed the presence of two putative receptor forms of molecular masses of about 130 and 95 kDa. These results suggest that the biological activity of aFGF and bFGF during brain embryogenesis could be regulated by the expression of high and low affinity binding sites for these growth factors.

Animals↗
PubMed1992

Derivatized dextrans mimic heparin as stabilizers, potentiators, and protectors of acidic or basic FGF.

Acidic and basic fibroblast growth factors (aFGF and bFGF) belong to a family of structurally related polypeptides characterized by a high affinity for heparin. a and bFGF display mitogenic activity for many cell types. Biological activity is strongly potentiated by heparin which stabilizes their molecular conformation by preventing physicochemical or enzymatic degradation. In our previous study we have shown that a water-soluble derivatized dextran named DDE, containing 82.2% methyl carboxylic acid groups, 6.1% benzylamide, and 5.6% sulfonate with a specific anticoagulant activity equivalent to heparin of 0.5 IU/mg could potentiate the mitogenic activity of aFGF on CCL39 cells. Optimal concentrations for maximal potentiation of 400 micrograms/ml and 20 micrograms/ml were obtained respectively for DDE and heparin. In the present report, we have uncovered the fact that several carboxymethyl benzylamide sulfonate dextrans differing in degree and positioning of the substituent groups can mimic heparin in regard to the protection, stabilization, and potentiating effects with aFGF or bFGF. Our data establishes that the dextran derivatives studied can act as potentiating agents for FGFs. Native dextran (DDA) had no effect. Dextran derivatives can also protect aFGF and bFGF from heat as well as from pH denaturation, and against trypsic and chymotrypsic degradation. The dextran derivative DDI (82% methylcarboxylic acid, 23% benzylamide, 13% sulfonate) was studied in greater detail and exhibited a greater protection for bFGF and a lesser protecting effect for aFGF than heparin. Derivatized dextrans which have very weak anticoagulant activity are of great interest as alternatives to heparin for use as stabilizers, potentiators, protectants, and slow-release matrices for FGFs in pharmaceutical formulations.

Animals↗
PubMed1992

Immunohistochemical detection of acidic fibroblast growth factor in bladder transitional cell carcinoma.

Acidic fibroblast growth factor (aFGF) is a regulatory peptide which, on account of its structural homologies with the products of oncogenes, is involved in cell proliferation, differentiation, and motility. We previously reported the presence of aFGF in the urine of patients with transitional cell carcinoma (TCC). aFGF can also induce the motility of a rat-derived bladder carcinoma cell line (NBTII). This immunohistochemical study used polyclonal rabbit antibodies against acidic and basic FGF and peroxidase detection. Native NBTII nude mice xenografts and aFGF transfected NBTII (NFS14) nude mice xenografts were used as tissue controls for antibody specificity. The samples included 4 normal urothelia and 12 TCC. In addition, cytospins of 4 different tumoral cell lines of human bladder and normal bladder cells were stained. The results showed strong immunostaining in all tumoral urothelium samples using anti-aFGF and a very low amount of staining or none at all in healthy tissues. A primary analysis suggested that the strongest reaction was obtained in high-grade tumors (3 + vs + for lower-grade tumors). Using bFGF antibody, strong immunohistochemical staining was detected on basal membranes and stromal vessels and none in urothelium. These data confirm aFGF expression in the epithelial cell compartment of bladder cancer and the likely involvement of this regulatory peptide in the biology of TCC.

Animals↗
PubMed1992

Purification of a heparin binding FGF receptor (HB-FGFR) from adult bovine brain membranes.

A new form of high affinity fibroblast growth factor receptor has been purified from adult bovine brain membranes. Purification was performed by chromatography on DEAE-Trisacryl and wheat germ agglutinin-agarose followed by FGF-2 affinity chromatography. Affinity labeling of purified fractions with 125I-FGF-2 showed after cross-linking a 170-kDa complex, suggesting the existence of a 150-kDa FGF receptor. No cross-reactivity with anti-FGF receptor 1 (FGFR-1 or flg) or with anti-receptor 2 (FGFR-2 or bek) antibodies could be detected with this partially purified receptor. Heparitinase treatment of the partially purified FGF receptor abolished the formation of the ligand receptor complex. The complex was restored in the presence of heparin in a dose dependent fashion, supporting the idea that heparin-like molecules are needed for proper binding. Further purification of the receptor was achieved by heparin-Sepharose affinity chromatography and yielded a purification of over 320,000-fold. The purified receptor fraction was radiolabeled and loaded on RPLC C4 column. Eluted fractions were analysed by SDS-PAGE. A major 150-kDa band was detected. These data show for the first time a new form of FGF receptor isolated from bovine brain membranes. This purified receptor displays affinity for heparin and was therefore named heparin binding FGF receptor (HB-FGFR). It remains unclear whether the receptor is a proteo-heparin sulfate or whether heparans are strongly associated and therefore are copurified. Large scale preparations are in progress for core protein structure studies.

Animals↗
PubMed1992

Interactions of FGFs with target cells.

Growth factors play a key role in cellular communication, a necessary step for the development of pluricellular organisms. The fibroblast growth factors (FGF) are among these polypeptides and have seven known members: FGF 1 to FGF 7 which are also known as acidic FGF, basic FGF, translation products of oncogenes hst, int 2, FGF 5, FGF 6 and FGF 7 or keratinocyte growth factor (KGF) respectively [1]. The best known and the most abundant in normal adult tissues are acidic and basic FGFs, or FGF 1 and 2 respectively, which have been subjected to extensive studies both in vitro and in vivo. These two factors have almost ubiquitous distribution and a wide spectrum of biological activity including action on cellular proliferation and differentiation, as well as neurotrophic and angiogenic properties [1]. These different activities are induced by triggering specific receptors present at the surface of the target cell. Following this interaction, the FGF-receptor complexes are internalized and activate intracellular pathways. An important effort of investigations has been produced to characterize these receptors and intracellular pathways. It is the purpose of this review to present this work which will focus on FGFs 1 and 2. The existence of two classes of interactions has been reported as early as 1987 [52, 53, 54] suggesting the presence of high and low affinity receptors for FGFs.

Animals↗
PubMed1992

Quantification of angiogenesis due to basic fibroblast growth factor in a modified rabbit corneal model.

The rabbit corneal angiogenesis assay was modified to allow quantification of the neovascular response induced by growth factors. Basic Fibroblast Growth Factor (bFGF) was used for this experiment. bFGF was diluted in phosphate buffer solution (PBS) in such a way that 2 microliter of solution contained concentrations ranging from 17 to 1,200 ng. Each drop was absorbed by a 1 x 2 mm particle of a dried 70% hydratable hydrogel and implanted into a mid-stroma corneal pocket, 2 mm from the limbus. Iodinated bFGF was used to measure growth factor diffusion in the cornea, which was found to be isotropic. Autoradiography showed that bFGF was stored in the cornea at both epithelial and endothelial level. Corneal neovascularization occurred on the second day after implantation and was maximal on the 7th day. At this time the neovascular surface was measured by planimetry on corneal photographs and compared with controls. This method allows precise definition of the dose of any substance to be implanted into the cornea and induces a rapid neovascular response, thus allowing quantitative evaluation of neovascularization within one week. A neovascular response was detectable for doses as low as 35 ng of bFGF and increased proportionally to the dose of bFGF implanted.

Animals↗
PubMed1992

Extracellular matrix covered biomaterials for human endothelial cell growth.

The aim of this study is to optimize conditions for growing endothelial cells on vascular biomaterials. Bovine cornea endothelial cells (BCEC), stimulated by basic Fibroblast Growth Factor (bFGF) secrete an extracellular matrix (ECM) similar to the Descemet membrane produced in vivo by these cells. This ECM, obtained by removing BCEC with an hypotonic shock can be used as a substratum for other endothelial cell growth. Human endothelial cells (HEC) were purified from omentum that was digested with a solution of collagenase-dispase, then filtered through nylon meshes. The cells were further purified by centrifugation onto a Percoll gradient. A comparative study on the attachment and growth of HEC on various coatings (laminin, poly-L-lysine, fibronectin or ECM) indicates that ECM is the most performing substratum. The quality of this endothelium was confirmed by the presence of factor VIII, and MHC class I and the absence of class II antigens.

Animals↗