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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 7, 2004 as doi:10.1096/fj.03-1126fje. |
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CRRET/CNRS FRE 2412, Faculté des Sciences, Université de Paris 12, Créteil Cedex, France; and
* Laboratoire de Pharmacologie et d’Immunologie, Commissariat à l’Energie Atomique, Gif-sur-Yvette Cedex, France
2Correspondence: CRRET/CNRS FRE 2412, Laboratoire CRRET, Université Paris 12 Val de Marne, Avenue du Général de Gaulle, 94010 Créteil cedex, France. E-mail: kern{at}univ-paris12.fr
SPECIFIC AIMS
The aim of this study was to understand the mode of action of a new family of drugs elaborated in our laboratory, named RGTAs (for regenerating agent), in the control of collagen synthesis and prevention of fibrosis during atherosclerosis development. RGTAs are synthetic polymers engineered to mimic the protective and potentiating properties of heparan sulfates toward heparin binding growth factors such as FGF-2 and TGF-ß1. RGTAs enhance the quality and speed of repair of several tissues and exhibit antifibrotic properties. We have investigated the possibility that RGTAs may modulate collagen-type expression via pathways involving FGF-2 or TGF-ß1, depending on the structure of the RGTA molecules.
PRINCIPAL FINDINGS
1. Structurally different RGTAs modulate differentially collagen-type synthesis by vascular smooth muscle cells (SMCs)
Cultured vascular SMCs showing alterations similar to those seen in atherosclerosis are useful for investigating drugs with potential to regulate excessive production of collagens by SMCs. To separate potential influences of RGTAs, heparin, FGF-2, and TGF-ß1 on biosynthesis by SMCs from their influence on cell proliferation, these compounds were incubated with serum-free, postconfluent cell cultures for 24 h, during which time SMC numeration, morphology, and viability remained unaltered in control or treated cells.
We studied two structurally different RGTAs: RG-1503, a carboxylmethylsulfate dextran, and RG-1192, a carboxylmethylsulfate dextran with additional benzylamide substitution.
RG-1192 induced an 80% specific decrease in collagen III expression by SMCs at the maximum efficacy dose of 400 µg/106 cells. Heparin in molar concentrations equivalent to RG-1192 were significantly less effective in diminishing collagen III production (up to 46% decrease) and had no effect on other collagen types. RG-1503 specifically increased by 2-fold collagen V production at the optimum dose of 400 µg/106 cells.
2. RG1192 specifically decreases collagen III synthesis via interaction with FGF-2
FGF-2 specifically decreased collagen III production (up to 45%). Used together, RG1192 and FGF-2 demonstrated a cooperative effect in decreasing collagen III synthesis (Fig. 1
). When heparin was used with FGF-2, no evidence of a comparable cooperative effect was found. The maximum decrease in collagen III production obtained with RG-1192 was abolished by neutralizing FGF-2 antibody, whereas the heparin-induced decrease in collagen III was not significantly altered. All results presented were obtained both at protein and mRNA levels.
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3. RG-1503 specifically increases collagen V production via interaction with TGF-ß1
As an extension of our previous study, we report a strong cooperative interaction between RG-1503 and TGF-ß1 upon stimulation of collagen V production (Fig. 2
). Heparin added to TGF-ß1 under the same conditions induced no further modification in collagen V synthesis. Adding to the specificity of these interactions between a given RGTA and growth factor, no significant cooperation in collagen-type production was found between RG-1192 and TGF-ß1 or between RG-1503 and FGF-2.
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4. RG-1192 increases the proportion of pericellular endogenous FGF-2 in SMCs
At postconfluence, cultured SMCs expressed FGF-2 in a concentration of 321(±47) pg/105 cells. Of the total FGF-2, 86% was intracellular and 16% pericellular. RG-1192 (400 µg/mL for 24 h) significantly increased the proportion of pericellular FGF-2 (up to 34% of total FGF-2).
5. RG-1192 protects FGF-2 activity against proteolysis
Trypsin induced up to an 84% decrease in FGF-2 bioactivity. RG-1192 almost abolished this FGF-2 degradation. We earlier demonstrated a similar protective effect of RG-1503 on TGF-ß1 activity.
6. RG-1192 has affinity to FGF-2
Surface plasmin resonance analysis of RG-1192 binding to FGF-2 indicated that RG-1192 an affinity for FGF-2 similar to that of heparin: 15.7 ± 2.2 nM and 10.6 ± 1.4 nM, respectively.
CONCLUSIONS AND SIGNIFICANCE
Vascular SMCs synthesize a complex extracellular matrix composed mainly of collagen I and III according to a highly regulated process. This process is altered during the development of atherosclerosis, restenosis, and other vascular disorders.
We engineered a family of pharmacological agents, RGTAs, by derivation from dextran via controlled chemical substitutions. These polymers enhanced tissue remodeling in vivo and were devoid of significant anticoagulant activity. In vitro, they inhibited SMC proliferation. Two RGTAs were used: RG-1503, a carboxylmethylsulfate polymer; RG-1192, a carboxylmethylsulfate polymer with added benzylamide groups. RG-1192 preferentially decreases fibrillar collagen III production whereas RG-1503 selectively increases collagen V production. These results suggest that RGTAs with different structures (i.e., presence or absence of hydrophobic benzylamide groups) may exert differential effects on collagen phenotype expression. Further, RGTAs were demonstrated to potentiate or form complexes with heparin binding growth factors such as FGF-2 or TGF-ß1. Among several factors that may influence SMC behavior during the course of atherosclerosis, FGF-2 and TGF-ß1 may play a pivotal role in altering the production of collagen by these cells. These data prompted us to look for mechanisms by which RGTAs may interact with FGF-2 and TGF-ß1 to modulate the types of collagen produced by SMCs. Recently, we demonstrated that RG-1503, which has no benzylamide groups, interacted closely with TGF-ß1 to stimulate collagen V production. For comparison, we extended the earlier study and found that RG-1503 and TGF-ß1 acted cooperatively to increase collagen V production (Fig. 2)
.
We found that RG-1192, which contains benzylamide groups, selectively decreased collagen III biosynthesis by SMCs in a dose-dependent manner. FGF-2 alone also specifically decreased collagen III expression, but this effect was only half that of RG-1192. This specific alteration in collagen III synthesis suggests that distinct regulatory pathways may be involved for collagen I and collagen III in arterial SMCs. Our evaluation of the RG-1192/FGF-2 interaction shows that RG-1192 and FGF-2 in combination display significant synergy in stimulating collagen III expression (Fig. 1)
; no such effect was demonstrated when heparin was added to FGF-2.
On the other hand, FGF-2 antibody inhibited the selective collagen III decrease induced by RG-1192 at protein and the mRNA levels, indicating that endogenous FGF-2 expressed by cultured SMCs possibly mediated the effect of RG-1192 on collagen III expression. In keeping with this, endogenous FGF-2 produced by postconfluent SMCs was confined to pericellular and intracellular compartments. FGF-2 in this location may represent a protected pool of endogenous FGF-2 that interacts with and is protected by RG-1192. In our study, RG-1192 significantly increased the relative proportion of pericellular FGF-2 without changing the total amount of cell layer-associated FGF-2. Pericellular FGF-2, acting through a FGF-2 receptor-mediated pathway, may be more effective than intracellular FGF-2 in regulating collagen synthesis. We found that RG-1192 protected FGF-2 from proteolytic degradation. Thus, just as heparan sulfate proteoglycans can bind to and concentrate cytokines at appropriate cellular sites, thereby enhancing the emission by those cytokines of signals to their receptors, RG-1192 may increase the local accumulation of endogenous FGF-2, which apparently mediates the effect of RG-1192 on collagen III expression.
Using surface plasmin resonance analysis, we found that the binding affinity of FGF-2 for RG-1192 (Kd=21.7 nM) was not very different from that for heparin (Kd=10.6 nM). Our results indicate greater affinity than suggested by published data on the heparin/FGF-2 interaction (Kd=74 nM). Moreover, the affinity of FGF-2 for RG-1192 is quantitatively comparable to the physiological affinity of FGF-2 for heparan sulfate proteoglycans present on the SMC surface (Kd=5–50 nM), further supporting the use of RG-1192 as a functional analog of native heparan sulfates.
The mechanistic results presently reported could be tentatively correlated with in vivo activity of RGTAs on the speed and quality of healing in various tissues (intestine, periodontal tissue, oral mucositis, bone, muscle, or myocardium). During healing, RGTAs seem to limit fibrotic alterations. Collagen III accumulation could be considered a biological marker for the extent of fibrotic disorders in several tissues. As an extension of the in vitro antifibrotic activity of RGTA, we can establish a parallelism between the RGTA-induced decreased of collagen III production by cultured cells and our recent demonstration of the direct diminution of collagen III synthesis in human intestinal tissue from Crohn’s disease patients ex vivo exposed to RGTAs. Concerning the mechanism of the growth factor-RGTA interactions implicated in all these processes, we propose that RGTAs may act as a survival and protective agent through maintenance and protection of the bioavaibility of preexisting and newly synthesized HBGF among those FGF-2 and TGF-ß1. These factors are stored on the heparan sulfate moieties of the extracellular matrix. Heparinase is one of the enzymes first activated after injury and, by cleaving heparan sulfates, releases bound HBGF. RGTAs could bind to the heparan sulfate binding sites of the extracellular matrix that became available after heparanase destruction. Then, RGTAs represent new stable protective sites of binding for HBGF, since RGTAs are less degradable than heparan sulfates by mammalian glycanase.
Taken together (Fig. 3
), these data open up new possibilities for developing drugs such as RGTAs characterized by 1) a more rigorously defined chemical structure than the heterogeneous population of heparin molecules; 2) controlled structural variations associated with marked differences in collagen phenotype modulation as opposed to the modest effect of heparin on collagen III only; 3) significant cooperative activity with FGF-2 (or TGF-ß1, depending on RGTA structure), which is not seen with heparin; 4) none of the adverse effects associated with the anticoagulant activity of heparin; 5) a weak degradability by enzymatic activities.
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Our data provide a rationale for engineering a range of RGTAs, each adapted to treatment of a specific collagen deposition-associated disorder. RG-1192 may be particularly well-suited for inhibiting the overproduction of collagen that occurs in SMC-related vascular disorders. Actually, RG-1192 has been found effective in inhibition of tissue fibrosis affecting other tissues such as myocardial infarcts and ischemic skeletal muscle.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1126fje; doi: 10.1096/fj.03-1126fje
1 These authors contributed equally to this work. 
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