A fine structural modification of glycosaminoglycans is correlated with the progression of muscle regeneration after ischaemia: towards a matrix-based therapy?

Critical limb ischaemia often leads to amputation of the limb and potential mortality. Moreover, there are still significant problems with current therapeutic treatments, according to poor revascularisation of degenerated tissue probably due to modifications within the microenvironment. This study is focused on the changes of structure and bioactivity of glycosaminoglycans (GAGs), especially heparan sulphate (HS) and chondroitin sulphate (CS) in rat Extensor Digitorum Longus (EDL) muscle after ischaemia. Male Wistar rats were subjected to ischaemic-injury by ligation of the neurovascular trunk accompanying EDL-tendon. After 4, 8, 15, 21, 60 and 90 d, the rats were sacrificed and the muscles were collected and submitted to histological, biochemical and gene expression assays. We demonstrated that ischaemia induced modification of expression of enzymes involved in GAG biosynthesis which correlated with significant changes in HS and CS structural features such as size and sulphation pattern. These major structural changes are associated to modifications of GAG abilities to bind growth factors and to modulate cell activity. Moreover, a CS hallmark of injury is maintained as well after the regeneration process. Finally, we showed the relevance of the role of this glycanic matrix remodelling, since a GAG mimetic treatment accelerated muscle repair after ischaemia.

RGTA-based matrix therapy in severe experimental corneal lesions: safety and efficacy studies.

Corneal alteration potentially leading to ulceration remains a major health concern in ocular surface diseases. A treatment that would improve both the quality and speed of healing and control the inflammation would be of great interest. Regenerating agents (RGTAs) have been shown to stimulate wound healing and modulate undesired fibrosis in various in vivo systems. We investigated the effects of RGTA-OTR4120(®) in a rabbit corneal model in order to assess its potential use in ocular surface diseases. First, we assessed its safety for 7 and 28 days using the Draize test criteria in healthy rabbit eyes; then, we investigated the effect of a single dose (50µl, 5µg) in an alkali-burned cornea model. Daily follow-up of clinical signs of healing was scored, and histology was performed at D7. RGTA was well tolerated; no signs of ocular irritation were observed. In the corneal alkali-burn model, non-RGTA-treated eyes showed inflammatory clinical signs, and histology confirmed a loss of superficial corneal layers with epithelial disorganization, neovascularization and infiltration of inflammatory cells. When compared to NaCl control, RGTA treatment appeared effective in reducing clinical signs of inflammation, enhancing re-epithelialization, and improving histological patterns: edema, fibrosis, neovascularization and inflammation. Three to four layers of epithelial cells were already organized, stroma was virtually unvascularized and keratocytes well implanted in parallel collagen fibers with an overall reorganization similar to normal cornea. RGTA appears to be a promising agent for controlling ocular surface inflammation and promoting corneal healing and was well tolerated. This study offers preclinical information and supports the findings of other (compassionate or pilot) studies conducted in patients with various ocular surface diseases.

[Basement of matrix therapy in regenerative medicine by RGTA(®): From fundamental to plastic surgery]. / Les bases de la thérapie matricielle en médecine régénérative par les RGTA(®) : du fondamental à la chirurgie plastique.

Cells present continuous renewal, permitting permanent regeneration which is called tissue homeostasis. The signaling protein, known as growth factors, cytokines, interleukins and chemokines, but also the extracellular matrix play a key role in the cellular communication. All processes are deregulated after tissue injury, inducing scars. By reconstituting the extracellular matrix, it is possible to avoid the development of scar and to favorize the regeneration of the injured tissue. Glycosaminoglycans, and particularly heparan sulfates, by participating to the extracellular matrix structure, are implicated in cellular communication. This article describes how, by creating heparan sulfate mimetic or Regenerating Agent (RGTA), a French academic team has demonstrated that mammals have the ability to regenerate, by restoring the proper cellular micro-environment. After a first clinical development in two severe and chronic pathologies (corneal and skin ulcers), we show now the potential of these agents in plastic and reconstructive surgery, to regulate fibrosis and to enhance speed and quality of tissue healing.

Insights on a new path of pre-mitochondrial apoptosis regulation by a glycosaminoglycan mimetic.

Lysosomal cathepsins have recently been reported to play crucial roles in the regulation of the mitochondrial death cascade by an unclear mechanism leading to mitochondrial membrane permeabilization. Glycosaminoglycans (GAG) are a family of ionic polysaccharides present at the lysosomal compartment and shown to inhibit lysosomal cathepsin activities. The implication of this family of polysaccharides in the regulation of the pre-mitochondrial death cascade has still not been considered. Here, we demonstrate in a model of skin fibroblasts submitted to oxidative stress that a GAG-mimetic protects the lysosome from membrane disruption, reduces intracellular ROS levels, and inhibits mitochondrial membrane potential collapse, cytochrome c release and caspases-9 and -3 activations without affecting the extrinsic pathway of apoptosis. Heparan sulfate and chondroitin sulfate, but not heparin, showed also protecting effects when assessing key points of the intrinsic pathway of apoptosis. We suggest the existence of molecular links between endogenous GAGs and the regulation of apoptosis.

Matrix therapy in regenerative medicine, a new approach to chronic wound healing.

Nonhealing wounds remain a major health problem whose treatment is challenging and costly. Treatments based on cells or growth factors are still not very effective. We developed an entirely novel strategy consisting in treatment of the wound-tissue matrix with biopolymers engineered to mimic heparan sulfates called OTR4120. This compound was dextran polymer with sulfated and carboxymethyl groupments. After binding to matrix proteins, the heparan-sulfate-mimicking polymer protects the microenvironment, maintaining the normal production of signals and growth factors needed for healing to occur. Here, we show that a specific biopolymer accelerates ulcer closure and improves re-epithelialization and dermal-matrix-component remodeling. OTR4120 treatment was associated with faster maturation of epidermal structures, most notably regarding the number of epithelial-cell layers, and with an appearance that more closely resembled normal skin. Treatment had also a main effect on collagen I and III expression. Necrotic skin ulcers induced in mice with doxorubicin recovered normal collagen levels and organization, with no evidence of fibrosis. Thus, appropriate polymer-based matrix therapy is a valid and simple alternative to regenerative medicine.

[Pilot study of a new matrix therapy agent (RGTA OTR4120) in treatment-resistant corneal ulcers and corneal dystrophy]. / Etude pilote d'un nouvel agent de thérapie matricielle (RGTA OTR4120) dans les ulcères de cornée et les dystrophies cornéennes rebelles.

AIMS: This study's objective was to evaluate the tolerance and safety of a new ophthalmic solution based on ReGeneraTing agent (RGTA) technology in a pilot noncontrolled exploration on compassion use for corneal ulcers and severe chronic dystrophies resistant to the usual treatments. RATIONALE: RGTAs are large biopolymers engineered to replace heparan sulfates specifically bound to matrix proteins and growth factors destroyed after a lesion has occurred. The RGTA-bound proteins are protected from proteolysis and this allows the extracellular matrix microenvironment to restore its original proper organization. The initial endogenous signals needed for tissues to regenerate are back on the restored matrix. They are expected to trigger the natural onset of events, signaling cells to migrate and multiply with the cascades and equilibrium found in tissue homeostasis. RGTA-induced matrix therapy is a possible alternative to cell or gene therapy in regenerative medicine. In a rabbit preclinical model of alkali-induced severe corneal ulcers, a single instillation of RGTA ophthalmic solution was found sufficient to enhance speed and quality of healing, restoring an almost normal corneal histology after only 1 week. These data prompted us to initiate this study. PATIENTS AND METHODS: Eleven eyes from ten patients were included in this study. All patients had severe dystrophic cornea or painful corneal ulcers rated over 50 on the VAS pain scale ranging from 0 to 100 and had undergone unsuccessful treatments. The RGTA ophthalmic solution was administered by the investigator during each weekly consultation as a single drop over 1 month. Tolerance and efficacy were judged on subjective criteria based on pain evaluation and functional inconvenience as well as on objective clinical criteria through a complete ophthalmic examination at days 3, 7, 14, 21, 28 and after 2 and 3 months from the beginning of the treatment. RESULTS: The study was conducted to completion for all patients included at the beginning. Tolerance was excellent both locally and generally: no uneasiness during instillation, no worsening of the initial pathology, no occurrence of ocular inflammation or increase in ocular pressure, and no general side effects were observed. In addition, we observed a noticeable analgesic effect, increasing with time and instillations, but pain reappeared in the majority of cases as treatment ended. The mean visual analog scale pain score was 72.73 +/- 7.86, it decreased significantly with the first drops of treatment. After 1 month, the mean visual analog scale pain score was 32+/-15.49, then it increased after the end of the treatment, confirming the link between the effects observed and the treatment. Efficacy on keratitis was moderate but with an overall tendency toward improvement. The initial Oxford Score was 3.37 +/- 1.06. After 1 month, it decreased significantly to 1.57 +/- 0.97 and then it rose again after the end of the treatment. As for corneal ulcers, of the five cases included, four healed during the protocol. Two reversed when the treatment stopped, two healed without reversion at the last follow-up visit. The last case was characterized by stem cell deficiency and no improvement was noted. It is important to keep in mind that these ulcers were all resistant to usual therapies. CONCLUSION: This RGTA ophthalmic solution is the first matrix therapy product in ophthalmology. The RGTA OTR4120 was used in treating chronic and severe corneal dystrophies as well as corneal ulcers resistant to usual treatments. It was very well tolerated with no side effects. It significantly reduced pain and favored corneal healing in almost all corneal ulcers. Weekly instillation of a single drop seems insufficient and these very promising data need to be confirmed on a larger population in a controlled trial with more adapted dosages. Based on these preliminary data, a RGTA-based matrix therapy product may be a very innovative solution to unresolved pain and corneal surface healing problems.

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.

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.

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.

Kinetic study of early regenerative effects of RGTA11, a heparan sulfate mimetic, in rat craniotomy defects.

We previously reported that RGTA, a synthetic heparan sulfate mimetic, induces almost complete closure of craniotomy defects one month after surgery in adult rats. RGTA-treated wounds showed features suggesting unusual cell and matrix interactions reminiscent of developmental events. As healing success or failure is determined shortly after wounding, we examined early events in RGTA-treated wounds. Collagen plasters soaked in a solution of RGTA11 (1.5 Microg per piece) or saline (control) were implanted in rat craniotomy defects. Seven control and seven treated rats were killed daily from days 1 to 7 after surgery. The lesions and adjacent tissues were sampled and processed for morphometry. A layer of type III collagen along the dura mater (DM) thickened up to day 5 in RGTA-treated wounds (p < 0.05 vs day 1), but became thinner in control wounds. Alkaline phosphatase-positive osteoprogenitor cells were detected on day 1 in this layer. Their number increased, and they migrated toward the mid-sagittal sinus and to connective tissue adjacent to the sinus, where they aggregated and differentiated into osteoblasts, forming bone nodules on day 6. These features were not seen in control wounds. Angiogenesis was significantly enhanced in RGTA-treated wounds, especially near the sinus. In vitro, bovine bone endothelial (BBE) cell proliferation was inhibited by RGTA11 in a concentration-dependent manner. In contrast, RGTA11 strongly enhanced the effect of fibroblast growth factor-2 on BBE cell proliferation. These results show that RGTA11, possibly by interacting with heparin-binding growth factors, elicits vascular reactions accompanying the recruitment of a large pool of committed osteoprogenitors from the DM. The DM and the sinus appear to be important centers of organization for craniotomy defect healing. RGTA probably creates an environment that starts a program of directing healing towards bone formation and defect closure.

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.

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.

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.

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.

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.

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.

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.

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.