RGTA OTR4120, a heparan sulfate mimetic, is a possible long-term active agent to heal burned skin.

Burn-related skin fibrosis leads to loss of tissue function and hypertrophic scar formation with damaging consequences for the patient. There is therefore a great need for an efficient agent to treat burned skin. We report that ReGeneraTing Agent (RGTA) reduces burn-induced skin alteration. The tissue-regenerating effect of RGTA OTR4120 was evaluated after 1-6 days and after 10 months in a rat skin burn model. This effect was also examined in vitro using fibroblasts isolated from control and 6-day-old burned skins. We measured production of dermal collagen I, III, and V and activities of metalloproteinases 2 and 9 (MMP-2 and MMP-9). Ratio of collagen III over collagen I production increased 6 days after the burn, because of a decrease in collagen I production. After 10 months, ratio of collagen III over collagen I in burn sites was still increased compared with control skin, because of an increase in collagen III production. Both abnormalities were corrected by OTR4120. OTR4120 increased pro- and active MMP-2 and MMP-9, compared with healthy and burned controls and therefore accelerated remodeling. Similar data were obtained with cultured fibroblasts from healthy and burned skins. OTR4120 enhanced healing in short- and long-term after burns, reducing the formation of fibrotic tissue, and then represents a potential agent to improve burned skin healing.

Periodontitis destructions are restored by synthetic glycosaminoglycan mimetic.

Periodontitis are bacterium-driven inflammatory diseases that destroy tooth-supporting tissues whose complete restoration is not currently possible. RGTA, a new class of agents, have this capacity in an animal model. Periodontitis was induced in hamsters and, starting 8 weeks later, injected RG1503, a glycosaminoglycan synthesized from a 40 kDa dextran behaving like a heparan sulfate mimetic (1.5 mg kg(-1) w(-1)) or saline for 8 weeks. The three periodontium compartments were evaluated by immunohistochemistry and morphometry. The gingival extracellular matrix disorganized by inflammation was restoring under treatment. The collagen network was repaired and resumed its previous organization. Fibrillin-1 expression was restored so that the elastic network rebuilt at a distance from the pocket and began to reconstruct near the pocket. Apoptotic cell numbers were decreased in the pocket epithelium, and more so in the infiltrated connective tissue. The continuity and the thickness of the basement membrane were restored and testified normalization of epithelium connective tissue interaction. The amount of alveolar bone increased around the first molar, and the interradicular bone was rebuilt. The root cementum was thickened and the number of proliferating cells in the periodontal ligament was increased close to the cementum. RG1503 treatment induces potent anabolic reactions in the extracellular matrices of the different tissues of the periodontium and recruitment of progenitors. In particular, the cell proliferation close to the root surface suggests the reformation of a functional attachment apparatus. These results demonstrate that RG1503 reverses the degenerative changes induced by inflammation and favors the conditions of a regenerative process. Thus, RGTA, a known matrix component mimetic and protector, may be considered as a new therapeutic tool to regenerate the tissues destroyed by periodontitis.

[Regenerating agents (RGTAs): a new therapeutic approach]. / Les agents de régénération (ou RGTAs): une nouvelle approche thérapeutique.

RGTAs, or ReGeneraTing Agents constitute a new class of medicinal substance that enhance both speed and quality of tissue healing and leading in some case to a real tissue regenerating process. RGTAs consist of chemically engineered polymers adapted to interact with and protect against proteolytic degradation of cellular signaling proteins known as growth factors, cytokines, interleukins, colony stimulating factors, chemokines, neurotrophic factors etc. Indeed almost all these proteins of cellular communication are naturally stored in the extra cellular matrix interacting specifically with the heparan sulfates or HS. After tissue injury of any cause, cells die liberating glycanases and proteases inducing first HS degradation then liberation of the cytokines which in turn are susceptible to degradation as they are no longer protected. By replacing the natural HS, RGTAs will protect cytokines from proteolyses as they are liberated from the matrix compartment matter in the wound. This spatio-temporal selective protection of cytokines results in a preservation of the natural endogenous signaling of a tissue and is reflected by spectacular tissue regeneration or by a very greatly improved tissue repair. These observations indicate that mammals have an unexpected ability to regenerate and that RGTA helps to reveal this capacity. The aim of OTR3 is to develop RGTA into a drug to treat specific tissue lesions.

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.

Functional, cellular and molecular aspects of skeletal muscle recovery after injury induced by snake venom from Notechis scutatus scutatus.

We have analysed the rate and ultimate extent of muscle functional recovery after snake venom-induced myotoxicity, as well as the relationships between functional, biochemical and structural indices of recovery. We also compared the effects of various injuries leading to muscle necrosis, loss of innervation/vasculature and/or precursors of muscle cells (pmc). We found that several parameters of rat soleus muscle such as maximal isometric force, slow myosin heavy chain, and citrate synthase, were fully and rapidly restored within 6 weeks after treatment with snake Notechis scutatus venom (im, 2 microg/muscle). In contrast, some muscle contractile properties (degree of tetanic fusion, fatigue resistance...) were not fully recovered even by 12 weeks after venom treatment. However, when compared to other injuries, recovery 3 weeks after venom treatment, was better than that observed after severing the terminal nerve and accompanying vessels and after cryodamage known to kill pmc. In conclusion, our studies demonstrate that-contrary to what is commonly believed -- muscle treated by myotoxic agent does not recover rapidly and fully. However, the degree or rate of muscle recovery after snake venom treatment was much better when compared to other types of injury. In addition, histological and biochemical parameters cannot be used as such to easily predict functional recovery following injury.

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.

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.

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.

Upregulation of the angiogenic factor heparin affin regulatory peptide by progesterone in rat uterus.

Heparin affin regulatory peptide (HARP), also named pleiotropin, is a secreted polypeptide that belongs to a new family of heparin-binding growth/differentiation factors. In this study, we investigated the expression and distribution of HARP mRNA and protein in rat uterus. Semi-quantitative reverse transcriptase PCR experiments showed variations in HARP mRNA levels throughout the estrous cycle, with a maximum during diestrus, pointing to hormonal regulation of HARP mRNA expression. Uterine expression of HARP mRNA was studied in ovariectomized animals treated with 17 beta-estradiol, progesterone alone or progesterone and RU486. In these experiments, progesterone upregulated HARP mRNA expression. Induction was observed 6 h after progesterone injection and was inhibited by RU486 treatment. In contrast, after 17 beta-estradiol injection, a slight decrease in HARP mRNA expression was observed. In situ hybridization studies with digoxigenin-labeled DNA probe revealed that HARP mRNA was present in smooth muscle cells of both myometrium and blood vessels and also in endothelial cells from endometrium. Immunohistochemical studies showed that HARP expression was not limited to cells that expressed HARP mRNA, but also occurred in both the luminal and glandular epithelium even though its transcript was never detected. We conclude that HARP may mediate the effects of progesterone on the homeostasis and vascularization of uterine tissue.

Signaling mechanisms of basic fibroblast growth factor in arterial cells from genetically hypertensive rat.

The mechanisms of vascular structural alterations in hypertension were studied in cultured adventitial fibroblasts isolated from aortas of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Basic fibroblast growth factor (bFGF)-, epidermal growth factor (EGF)-, or platelet-derived growth factor (PDGF)-induced DNA synthesis and phospholipase C activity were estimated by determining 3H-thymidine incorporation and 3H-inositol phosphate production, respectively. The role of protein tyrosine kinases was assessed by stimulating the cells in the presence of tyrphostin, a protein tyrosine kinase inhibitor. Both the mitogenic potency of bFGF, EGF, and PDGF and the phospholipase C activity elicited by these factors were increased markedly in SHR (v WKY) fibroblasts. SHR fibroblasts were significantly less sensitive to tyrphostin inhibition of bFGF-induced 3H-thymidine incorporation than WKY fibroblasts, whereas when the cells were stimulated with EGF, PDGF, or 5% serum, SHR and WKY fibroblasts were equally sensitive to tyrphostin inhibition. At doses that abolished bFGF-induced 3H-thymidine incorporation, tyrphostin did not affect bFGF-induced 3H-inositol phosphate production. These results indicate that in aortic fibroblasts phospholipase C activation is not sufficient for bFGF-induced DNA synthesis. They suggest that tyrosine kinase activation is a necessary step in the transduction of bFGF mitogenic signal and plays an important role in the enhanced DNA synthesis exhibited by SHR (v WKY) cells. Therefore, one may envisage that bFGF contributes, through paracrine/autocrine mechanisms, to the vascular smooth muscle hyperplasia/hypertrophy in SHR.

Developmental changes of acidic fibroblast growth factor (aFGF) transcription and expression in mouse brain.

In order to increase our knowledge of the in vivo role of acidic fibroblast growth factor (aFGF) in the central nervous system, we have examined aFGF levels during mouse brain development. Using a specific polyclonal antibody raised against aFGF, we measured levels of aFGF-immunoreactive material (IRMaFGF) in extract of total mouse brain taken at different days of development. We found that the level of measurable IRMaFGF remained low and without significant variation during fetal brain development (0.2 ng/mg of extracted proteins). During the first 11 days postnatal (P0 to P11), IRMaFGF increased from 0.5 to 1.5 ng/mg. Between P11 and P14 IRMaFGF levels went up more rapidly, reaching 5 ng/mg. From P30 to adulthood a constant value of 2.5 ng/mg was measured, aFGF content in the different brain extracts was further characterized by its affinity for heparin-Sepharose, its elution at 1 M NaCl from this column and its capacity to induce thymidine incorporation in quiescent fibroblasts. These results were confirmed at the mRNA level. Northern blot analyses of poly A+ mRNA from brains with a specific riboprobe for bovine aFGF, revealed a major 4.5-Kb transcript and a minor 2.7-Kb transcript detectable only in postnatal brains. A similar pattern to that observed for IRMaFGF was seen with these mRNA transcripts, indicating that these aFGFmRNA are translated in the mouse brain. Our results suggest that aFGF may act in the postnatal phases of brain maturation.

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.

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.

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)

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.

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.

[CMDBS, functional analogs of sulfate heparanes, used as osseous cicatrizing agents]. / Les CMDBS, analogues fonctionnels des héparanes sulfates, utilisés comme agents de la cicatrisation osseuse.

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.