Biologically active oligosaccharides related to glycosaminoglycans are accumulating increased attention because of their therapeutic potential and for their value in mechanistic studies. Heparan mimetics (HMs) are a family of dextran based polymer known to mimic the properties of glycosaminoglycans, and particularly those of heparan sulfates, as to interact with heparin binding proteins. HMs have shown to stimulate tissue repair in various animal models. Here, we use different methods to depolymerize HMs in order to produce a library of related oligosaccharides and study their biological activities. Since HMs were resistant to endoglycanases activities, depolymerization was achieved by chemical approaches. In vitro biological studies showed that HM oligosaccharides can differentially potentiate FGF-2 mitogenic and antithrombotic activities. In vivo, a selected oligosaccharide (H-dp12) showed to be able to regenerate tissue almost as well as the related polymeric product. The very low anticoagulant activity and high biological activity of low mass oligosaccharides give to these products a new therapeutic potential.

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.

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.

We have engineered polymers called ReGeneraTing Agents (RGTAs), which mimic the protecting and potentiating properties of heparan sulfates toward heparin-binding growth factors (HBGF). RGTAs have been shown to optimize cell growth and regulate collagen production in vitro. Here, we studied relationships between RGTA structure and collagen-type expression in aortic smooth muscle cells by using two RGTAs, the carboxylmethylsulfate dextran RG-1503 and the carboxylmethylsulfate dextran with added benzylamide RG-1192. RG-1192 specifically induced a fivefold decrease in collagen III synthesis. This effect was abolished by FGF-2 neutralizing antibody. RG-1192 and FGF-2 acted synergistically to decrease collagen III. RG-1192 was more effective than heparin in this process. RG-1192 increased the pericellular localization of FGF-2 and protected FGF-2 from proteolysis. Surface plasmon resonance analysis indicated a Kd of 15.7 nM for the RG-1192/FGF-2 interaction (10.6 nM for the heparin/FGF-2 interaction). The structurally different RG-1503 (without benzylamide) did not interact with FGF-2 and worked synergistically with TGF-beta1 to specifically induce a twofold increase in collagen V. RGTAs with different structures exert different modulating effects on the collagen phenotype. Selection of appropriate RGTAs, which had been shown to enhance in vivo tissue repair, may provide a mean of correcting collagen abnormalities in vascular disorders and more generally in fibrotic diseases.

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.

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.

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-β1 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-β1. RG-1503 decreased, by 50%, collagen I and III biosynthesis and stimulated specifically, by twofold, collagen V biosynthesis. TGF-β1 stimulated collagen I and V by 1.5- and threefold, respectively. A synergic action for RGTA in association with TGF-β1 was observed specifically for collagen V expression (eightfold increase). The stimulation of collagen V biosynthesis by RGTA was abolished by TGF-β1 neutralizing antibodies. These modulations occurred at protein and mRNA levels. RG-1503 did not alter TGF-β1 mRNA steady state level or total TGF-β1 protein content (latent+active forms). However, RG-1503 significantly induced an elevated proportion of active TGF-β1 form, which could result from the selective protection from proteolytic degradation of TGF-β1 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.

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.

Gingival fibroblasts are particularly involved in the physiologic maintenance and repair of periodontium. During these processes, cell proliferation and synthesis of a collagen-rich gingival matrix should be controlled. A dextran derivative, namely, carboxy methyl dextran benzylamide sulfonate (CMDBS), considered to be a functional analog of heparin, was previously described to regulate proliferation of different types of cells and independently to modulate the expression of collagen biosynthesis. In this report, we demonstrate that CMDBS and heparin inhibited gingival fibroblast proliferation. We then analyzed collagen biosynthesis by measuring the incorporation of the radiolabeled [3H]proline precursor into collagen by postconfluent gingival fibroblasts. Our results showed CMDBS did not alter total collagen synthesis; it induced the preferential accumulation of newly synthesized collagen into the pericellular matrix; and it decreased the expression of type III collagen, particularly in the cell layer. Taken together, our results suggest that by inhibiting cell proliferation, CMDBS could induce the synthesis of an extracellular collagenous matrix which forms a network between gingival fibroblasts.

We developed regenerating agents (RGTAs) corresponding to polysaccharides derived from dextran and containing defined amounts of carboxymethyl (CM), carboxymethyl sulfate (CMS), carboxymethyl benzylamide (CMB), or carboxymethyl benzylamide sulfate (CMBS) groups with varying degrees of substitution. These compounds mimicked some effects of heparin on smooth muscle cell (SMC) proliferation and promoted in vivo tissue remodeling. We demonstrated that only RGTAs containing both CM and sulfate groups decreased SMC proliferation, in correlation with increased sulfation level. This effect was amplified by the presence of benzylamide. Independent of this activity on cell proliferation (i.e., with postconfluent cells), RGTAs modulated collagen biosynthesis by SMCs. On the one hand, CMBS more than CMS RGTAs induced a decrease of collagen III synthesis at the level of mRNA steady state and protein production. On the other hand, CMS to a greater extent than CMBS RGTAs increased both collagen V mRNA and protein production. In addition, only benzylamide-containing RGTAs increased accumulation of collagen I and III in the cell layer. In conclusion, RGTA bioactivities required the presence of CM functions, increased with the sulfation level, and varied with benzylamide substitution. RGTAs that modulate cell proliferation and collagen biosynthesis by differential mechanisms may represent potential antifibrotic agents.

Extracellular matrix macromolecules such as collagen and fibronectin are progressively altered during aging and age-related diseases like diabetes. We investigated the effect of high-glucose concentration (mimicking diabetic conditions) and the influence of in vitro cell aging [comparing 4th-passage fibroblasts (P4) to 15th-passage fibroblasts (P15)] on collagen and fibronectin synthesis. Fibroblasts were incubated at postconfluency with radiolabeled precursors, [3H] proline for collagen, [35S] methionine for fibronectin. We report that in control conditions (5 mM glucose) collagen III production increased with in vitro cell aging. High glucose concentrations (10 and 15 mM) increased specifically collagen III synthesis both at the mRNA and protein levels, without alteration of collagen I production in P4 and P15 cells. Fibronectin synthesis was also increased both during in vitro cell aging and in high glucose-treated P4 fibroblasts. Taken together, these data suggest similarities between changes of phenotypic expression of collagen and fibronectin induced by in vitro cell aging and conditions imitating diabetes.

Numerous studies on interactions between insoluble polymers and cell membrane receptors indicated modulation of cellular proliferation and cell phenotype by these polymers considered as biospecific. We synthesized several biospecific polymers in order to investigate the interactions between polymers and intracellular receptors as estrogen receptors considered as tumoral indicator of breast cancer. Biospecific polymers were obtained by random substitutions of crosslinked polystyrene beads with suitable chemical groups (sulfonate and amino acid sulfamides). These polymers were used as microcarriers for culture of MCF7 cells, a cellular model of human breast cancer. Quantification of MCF7 cell estrogen receptors was determined by radioligand binding assay for different days of cellular proliferation. The data obtained with MCF7 cells cultured on biospecific polymers show an inverse relationship between polymer induced inhibition of cell proliferation and polymer induced increase of estrogen receptors. Similar inverse relationship was obtained with MCF7 cell cultured on standard polystyrene tissue culture plates. The various interaction between insoluble polymers and MCF7 cells could be related to the proportion and the nature of the substitutive chemical groups. These biospecific polymers could presents sites of interaction with cell membrane receptors leading to modulation of cell biological activity. The different insoluble polymers were used as preliminary models: a practical application could be a methodology of cellular selection using soluble biospecific polymers (for example chemically modified dextrans).

The biosynthesis of basement membrane components in Engelberth Holm Swarm-bearing mice with or without streptozotocin-induced diabetes and the effect of low-molecular-weight heparin derivatives (CY222, Sanofi Recherche/Institut Choay) on the relative rates of these synthetic activities were studied. In diabetic mice, the laminin mRNA level increased, whereas type IV collagen mRNA decreased. In vivo treatment with heparin fragments decreased the mRNA level of laminin to control values without altering the mRNA level of collagen IV. Biosynthetic studies with radiolabeled precursors ([3H]-proline for collagen and [35S]-methionine for laminin) confirmed these results. Laminin protein biosynthesis increased in diabetic mice. Treatment with CY222 corrected this alteration. Our results suggested an increased labeling of polymeric forms of collagen IV in diabetic mice. In addition, we showed that biosynthesis of acid-extractable collagen IV decreased in diabetic mice and that CY222 treatment corrected this disturbance. These experiments suggest that low-molecular-weight heparin fragments CY222 can modulate the biosynthesis of extracellular matrix macromolecules altered in diabetic animals by different pathways, including pretranslational and posttranslational steps.

Production of various components of the extracellular matrix (ECM) modulates biological functions of the vascular tissue. This process is generally amplified in pathologic states as atherosclerosis. Atheroma originates from smooth muscle cells (SMC) which have migrated and proliferated in the vascular intima. In this study we investigated protein synthesis, collagen synthesis, and types I, III, and V collagen distribution by SMC in the presence of three families of watersoluble polysaccharides, heparin, fucans, and derivatized dextrans. We observed that fucan and derivatized dextran were able, as was heparin, to inhibit rat aortic SMC growth in culture. We then analyzed collagen modulation by measuring the incorporation of the radiolabeled precursor (3H)-proline into vascular SMC. Our results showed uncoupling of the antiproliferative capacity with collagen biosynthesis. However, fucan, the most antiproliferative polysaccharide, was also the most active in inhibiting protein and collagen synthesis. In addition, compounds that decreased total collagen synthesis preferentially increased the proportion of cell-associated collagen. Interestingly, only the antiproliferative polysaccharides inhibited significantly type V collagen biosynthesis. These new biomaterials appear to be valuable tools to study and control extracellular-matrix interactions with cells from the vascular walls.

The effect of specifically derivatized dextrans, with or without antiproliferative activity on smooth muscle cells (SMC), was investigated on type I and type III collagen biosyntheses and mRNA levels in post-confluent SMC cultures. Our results indicate that dextran derivatives decreased total protein and collagen syntheses independently of their antiproliferative activities. However, the most substituted dextran, the one exhibiting the strongest antiproliferative activity towards SMC, was the most active in modulating type III collagen expression. In addition, only the two dextran derivatives bearing benzylamide groups inhibited collagen excretion.

The effectiveness of the two proteolytic enzymes, pepsin and proctase (isolated from Aspergillus niger), was investigated in order to compare their relative efficiencies for the solubilization of collagen from bovine skin. Some of the molecular properties of gelatins derived from these collagen preparations were also studied. Pepsin and proctase solubilized collagen with similar yields (75% and 76% of total collagen as calculated from hydroxyproline). The relative amounts of collagen extracts converted to gelatin were also comparable (32% and 39%, respectively). However, proctase-extracted collagen exhibited a decrease of high-molecular-weight components compared to pepsin-prepared collagen. Furthermore, gelatin obtained from proctase-extracted collagen showed significant proportions of molecular species smaller than collagen alpha-chains. Type III to type I collagen ratios were also analyzed in both preparations. The results indicate an increase of the proportion of type III collagen in proctase extract as compared to pepsin extract. Rheological properties of gelatin obtained by proctase solubilization exhibited a very significant decrease of gel strength, viscosity, and turbidity parameters as compared to pepsin-derived gelatin. These parallel biochemical and biophysical data indicate that proctase-prepared gelatin is markedly altered compared with pepsin-derived gelatin.

We demonstrated that the ratio of type III to type I collagen increases during chronological aging. This increase became even more significant in hairless mice receiving chronic ultra-violet B irradiation. We investigated the regulation of these processes at the transcriptional level by extraction and quantification of mRNAs coding for fibrillar collagens. Total RNA from non-irradiated or irradiated mice was analysed by RNA-DNA hybridization on slot-blots. We observed changes in the transcription rate of these specific genes during aging and induced "photoaging". During chronological aging, we found a decrease of mRNA coding for type I collagen without any alteration in the amount of mRNA coding for type III collagen. In ultraviolet B-treated mouse skin, we found a significant increase of mRNA coding for type III collagen with no change in the amount of mRNA coding for type I collagen. These data are in agreement with the results obtained at the translational level. Comparison between chronological aging and induced "photoaging" at the transcriptional level shows evidence for a separate regulation of the expression of the genes coding for these two procollagens and shows also differences in the modifications of collagen gene expression between chronological aging and photoaging.

Annals of the New York Academy of SciencesVolume 673, Issue 1 p. 16-22 Biomarkers of Connective Tissue Aging: Biosynthesis of Fibronectin, Collagen Type III, and Elastase J. LABAT-ROBERT, J. LABAT-ROBERT Laboratoire de Biologie du Tissu Conjonctif URA CNRS 1460 Faculté de Médecine Université Paris XII 94010 Créteil, FranceSearch for more papers by this authorP. KERN, P. KERN Laboratoire de Biologie du Tissu Conjonctif URA CNRS 1460 Faculté de Médecine Université Paris XII 94010 Créteil, FranceSearch for more papers by this authorL. ROBERT, L. ROBERT Laboratoire de Biologie du Tissu Conjonctif URA CNRS 1460 Faculté de Médecine Université Paris XII 94010 Créteil, FranceSearch for more papers by this author J. LABAT-ROBERT, J. LABAT-ROBERT Laboratoire de Biologie du Tissu Conjonctif URA CNRS 1460 Faculté de Médecine Université Paris XII 94010 Créteil, FranceSearch for more papers by this authorP. KERN, P. KERN Laboratoire de Biologie du Tissu Conjonctif URA CNRS 1460 Faculté de Médecine Université Paris XII 94010 Créteil, FranceSearch for more papers by this authorL. ROBERT, L. ROBERT Laboratoire de Biologie du Tissu Conjonctif URA CNRS 1460 Faculté de Médecine Université Paris XII 94010 Créteil, FranceSearch for more papers by this author First published: December 1992 https://doi.org/10.1111/j.1749-6632.1992.tb27431.xCitations: 29AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume673, Issue1Physiopathological Processes of Aging: Towards a Multicausal InterpretationDecember 1992Pages 16-22 RelatedInformation