Michele Tardieu

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.

Biological and binding studies of acidic fibroblast growth factor in the presence of substituted dextran.

Heparin has been shown to interact with acidic fibroblast growth factor (aFGF) and to potentiate the biological activity of aFGF on fibroblastic cells. Water-soluble dextran substituted with methyl carboxylic benzylamine and sulfonate groups has been shown to mimic the effect of heparin in its anticoagulant and anticomplement activity. We have studied the effect of a dextran derivative named E (DDE), which had an anticoagulant activity equivalent to 0.5 IU heparin/mg, on the mitogenic activity of aFGF on Chinese hamster fibroblasts (CCL39). DDE interacts with aFGF in a comparable manner to heparin. We have shown that 20 micrograms of heparin or 400 micrograms of DDE added to 1 ml of culture medium has no effect on cell proliferation alone but potentiates the mitogenic activity of aFGF ten fold if aFGF is added at doses corresponding to half maximum stimulation (ED50). We have also studied the effect of various concentrations of heparin and DDE on the binding of 125I-aFGF on bovine brain membranes. Interestingly, the binding of 125I-aFGF increased three-fold as the concentration of heparin was increased up to 0.2 microgram/ml. At 1 microgram/ml of heparin, the amount of bound 125I-aFGF is comparable to that obtained in the absence of heparin. At higher concentrations, heparin displaces bound 125I-aFGF, and a 50% displacement is seen with 20 micrograms/ml of heparin. In the presence of DDE, no increase in 125I-aFGF binding is seen and a displacement is obtained with increasing doses. A possible explanation of these results may be the existence of specific receptors to heparin on the cellular membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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 RGTA 11 on bovine aortic endothelial cells (BAEC) cultured on collagen I gels. Secondly, the role of FGF2 and RGTA 11 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 RGTA 11 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 RGTA 11 combined with FGF2.

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.

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.