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(The FASEB Journal. 2001;15:1546-1554.)
© 2001 FASEB

Heparan mimetic regulates collagen expression and TGF-ß1 distribution in gamma-irradiated human intestinal smooth muscle cells

CATHERINE ALEXAKIS, AHMED GUETTOUFI, PATRICIA MESTRIES, CARINE STRUP*, DENIS MATHÉ*, CHRISTEL BARBAUD, DENIS BARRITAULT1, JEAN-PIERRE CARUELLE and PATRICK KERN

CRRET/CNRS UPRESA 7053, Faculté des Sciences de Créteil, Université PARIS-12, France; and
* Institut de Protection et de Sûreté Nucléaire, Département de Protection de la Santé de l’Homme et de Dosimétrie, Section Autonome de Radiobiologie Appliquée à la Médecine, IPSN, BP no. 6, F-92265 Fontenay aux Roses Cedex, France

1Correspondence: CRRET-CNRS UPRESA 7053, Faculté des Sciences de Créteil, Université Paris 12, Avenue du Général de Gaulle, 94010 Créteil cedex, France. E-mail: Barritault{at}univ-paris12.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Radiation-induced intestinal fibrosis is characterized by collagen accumulation, a process in which TGF-ß1 plays a key role. We analyzed the effects of gamma radiation on collagen expression and TGF-ß1 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 60Co irradiation (10 Gy), HISM were labeled with [3H] proline (±RG-1503). Radiolabeled collagen I, III, and V were quantified by SDS-PAGE. TGF-ß1 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-ß1 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-ß1 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-ß1 distribution. The antifibrotic RG-1503 restored HISM physiological characteristics and may represent a promising therapeutic approach for radiation-induced intestinal fibrosis.—Alexakis, C., Guettoufi, A., Mestries, P., Strup, C., Mathé, D., Barbaud, C., Barritault, D., Caruelle, J.-P., Kern, P. Heparan mimetic regulates collagen expression and TGF-ß1 distribution in gamma-irradiated human intestinal smooth muscle cells.


Key Words: gamma radiation • intestinal fibrosis • collagen • transforming growth factor ß1


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
FIBROSIS IS ASSOCIATED with radiotherapy and accidental irradiation of several organs (1) . This fibrotic response has been characterized by an increased accumulation of extracellular matrix, particularly collagen, in the irradiated tissue (2 , 3) . Some data have emphasized the particularly marked increase in collagen III during radio-induced fibrosis (4 5 6) . This collagenous accumulation can impair organ functions with potentially life-threatening consequences. Several observations have indicated that the transforming growth factor ß1 (TGF-ß1) is a key modulator of cellular events leading to radiation-induced fibrosis (1 , 7 8 9) . The mechanisms leading to fibrosis vary between tissues and differ in the intestine in several important ways from those in other organs (10) . For example, intestinal collagen is produced mainly by smooth muscle cells stimulated by TGF-ß1 (11) . During intestinal fibrosis, overgrowth of the muscularis mucosa and muscularis propria occurs (12) ; and within these hypertrophied muscularis layers, the predominant smooth muscle cells could develop a fibrogenic phenotype (13) . After acute injury, intestinal normal healing or intestinal fibrosis occurs via transformation of pluripotent mesenchymal cell type into a fibrogenic, smooth muscle, cell-like phenotype. This transformation is transient during normal healing but may lead to fibrosis when the fibrogenic phenotype is maintained (13) . TGF-ß1 stimulates collagen production by human intestinal smooth muscle cells (11) . In addition, TGF-ß1 expression remains elevated in intestinal smooth muscle cells in regions of chronic fibrosis and TGF-ß1 appears to be the predominant isoform involved in the mechanisms of radiation enteropathy rather than TGF-ß2 and TGF-ß3 (14) . Intestinal radiation injury is associated with sustained increase in TGF-ß1 immunoreactivity in regions of injury (15) . Evidence of relationships between TGF-ß1, collagen overproduction, and fibrosis have led to the search for antifibrotic components with the ability to selectively regulate biosynthesis of specific collagen type (16) . We have demonstrated that heparin-related glycosaminoglycans are able to regulate collagen biosynthesis (17) .

Recently we developed a family of polymers engineered to mimic the stabilizing and protecting properties of heparan sulfates toward heparin binding growth factors (HBGFs) and that, in vivo, stimulates tissue repair and protection. Hereafter named RGTA for regenerating agents, these polymers are believed to enhance the bioavailability of HBGFs in vivo. Indeed, RGTA are able to promote tissue healing in several experimental models including muscle (18) , bone (19) , skin (20) , and intestinal tissue (21) . RGTA interact with heparin binding growth factors such as fibroblast growth factors as well as TGF-ß1 (21) . In vitro, these polymers mimic heparin antiproliferative activity on aortic smooth muscle cells but are devoid of heparin-associated anticoagulant properties (22 , 23) . They also induce extracellular matrix remodeling by interfering with collagen expression (22 , 23) . We recently demonstrated that one RGTA, RG 1503 (see Fig. 1 for detailed structure), specifically decreased the proportion of collagen III synthesized by these cells (24) . This property, which could be of use as a potential antifibrotic activity, prompted us to study the radiation-induced alteration of collagen expression in the gut and the role of TGF-ß1 in this process. We therefore developed an in vitro model of cell irradiation using human intestinal smooth muscle cells (HISM) as these cells are involved in the evolution of intestinal postradiation fibrosis.



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  		Figure 1. Schematic structure of RG-1503. RG-1503 polymer was elaborated as described in Materials and Methods. T40 dextran was substituted by carboxymethylation, followed by O-sulfonation. The different percentages indicated in the figure were calculated from the degree of substitution (d.s.) relative to the position of each group in glucosidic unit as reported in Table 1 . The substituted glucosidic units A–C were arranged in an arbitrary combination. Their respective proportions (%) were calculated according to the nature of the group linked to the C2 position. In addition, R represents the proportion (%) of each substituted group in the global C3 plus C4 positions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Chemicals for cell culture, media, and sera were from Life Technologies (Cerg Pontoise, France). All other reagents were from Merck (Darmstadt, Germany) or Sigma (St. Louis, MO). TGF-ß1 was from R&D Systems (Abingdon, Oxon, UK).

RG-1503 preparation
The water-soluble dextran derivative RG-1503 (see Fig. 1 for detailed structure) was prepared from T40 dextran (average Mr 37000; Pharmacia, Paris, France) as described previously (24 , 25) . Carboxymethyl dextran was synthesized from dextran T40 by carboxymethylation of OH residues with monochloracetic acid treatment. RG-1503 was then obtained from carboxymethylated dextran by O-sulfonation. The presence of carboxymethyl and sulfate groups was confirmed by infrared spectroscopy. The chemical characterization of RG-1503 was based on the degree of substitution (d.s.) of each individual group per glucosidic unit (Table 1 ). A d.s. value of 3 represents the maximum of substitution, since one glucosidic unit contains three reactive OH groups on C2, C3, and C4 positions. Each d.s. value was determined by acidimetric titration and elementary analysis. All these values were confirmed by 1H-NMR. Average molecular weight of RG-1503 was estimated by high-performance size exclusion chromatography (25) . This polymer did not show any significant anticoagulant activity (less than 5 IU/mg compared with 173 IU/mg for heparin).


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  		Table 1. Chemical characterization of RG-1503a

Cell culture
Human intestinal smooth muscle cells (HISM), passage frozen P13, were obtained from American Type Culture Collection (ATTC, Rockville, MD) and were isolated from the muscularis propria of normal human jejunum by digestion with collagenase (26) . The cells were maintained and passaged in Dulbecco’s modified Eagle’s minimal essential medium supplemented with 10% fetal calf serum (FCS), 1% penicillin, and streptomycin at 37°C in humidified 7% CO2/95°C air atmosphere. Viability of the cells in the presence of RG-1503 was tested by determination of lactic dehydrogenase (EC1.1.1.27).

Experimental groups and cell irradiation
HISM were used at postconfluence in all experiments in order to minimize the effect on cell proliferation that could be induced by RG-1503 (24) . In groups pretreated with RG-1503, the polymer (400 µg/ml/106 cells) was added 48 h before irradiation. In groups post-treated with RG-1503, the polymer was added immediately after irradiation. Postconfluent HISM maintained with 10% FCS were irradiated with a 60Co source (IPSN France) for 10 min at a dose rate of 1 Gy/min. Collagen biosynthesis measurement (over a period of 24 h) and TGF-ß1 content determination (collected over a period of 24 h) were performed either immediately or 24 or 72 h after irradiation in each experimental group. The following abbreviations were used to identify experimental groups: A (control); B (nontreated and irradiated); C (post-treated); D (irradiated and post-treated); E (pretreated); F (pretreated and irradiated).

Total protein and collagen biosynthesis
Measurement of protein and collagen biosynthesis (performed during a 24 h period) was started 1) immediately, 2) 24 h, and 3) 72 h after cell irradiation. Labeling of proteins and collagen was performed on postconfluent HISM maintained in 10% FCS throughout the process. As described previously (22 , 24) , cells were incubated for 24 h in culture medium containing [5-3H] proline (925 kBq/ml, 1.1TBq/mM; Amersham, Paris, France), ascorbic acid (50 µg/ml), ±RG-1503 (400 µg/ml/106 cells) optimum dose as previously determined (22) . At the end of labeling, the medium and cell layer were dialyzed against distilled water at 4°C. For measurement of total protein biosynthesis, the radioactivity contained in aliquot of dialyzed medium plus cell layer was determined by liquid scintillation counting and the results are expressed as total [3H] dpm per cell. For determination of total collagen biosynthesis, an aliquot of dialyzed medium plus the corresponding cell layer was hydrolyzed in 6M HCl for 24 h at 105°C. Radiolabeled hydroxyproline as a specific marker of collagen was then separated and quantified (4) . For determination of individual collagen types, medium and cell dialysates were digested with pepsin and the biosynthesis of pepsin-soluble collagen types was determined by SDS-PAGE (17) . The electrophoresis was performed in the presence of standard collagen types (I, III, and V) and the collagen bands were revealed by Coomassie blue staining. Separation of collagen III was achieved by delayed reduction (4) . The relative proportions of radioactivity incorporated in collagen I, III, and V were quantified by excision of each individual collagen band, followed by hydrolysis of the band in 6M HCl at 105°C for 24 h and determination of hydroxy[3H]proline in hydrolysate (17) .

RNA analysis
Total RNA was extracted from cells by guanidium isothiocyanate (27) and analyzed by Northern blot (22) . Northern blots were prehybridized and hybridized as described previously (24) with appropriate [32P]-labeled probes at 42°C. The autoradiograms were quantified by laser densitometric scanning. The following cDNA probes were used: Hf677 for human alpha1(I) collagen (28) ; Hf934 for human alpha1(III) collagen (29) ; HT168 for human alpha1(V) collagen (30) , all from ATCC; and GAPDH (glyceraldehyde-3-phosphate-dehydrogenase), which was provided by Dr. Asselot-Chapel (CEA France). The quantification of each mRNA was obtained from independent hybridization carried out in triplicate on four different HISM cultures.

Immunological determination of total TGF-ß1 protein in cell medium
Cell culture supernatants were collected over 24 h from postconfluent HISM cultured under the same experimental conditions as for collagen biosynthesis (without radiolabeling). Total TGF-ß1 (latent+active forms) was determined after acidification by sandwich ELISA (‘Quantikine’ assay; R&D). The amount of TGF-ß1 present in FCS added to the culture medium was subtracted from all experimental values. Addition of RG-1503 alone did not interfere with the detection of TGF-ß1.

Compartmentalization of TGF-ß1 in cell layers
For determination of TGF-ß1 compartmentalization in cell layers, we separated the pericellular TGF-ß1 extracted by a limited trypsin treatment without cell lysis from the remaining intracellular TGF-ß1 obtained after cell lysis (31) . The medium of cultured HISM was removed and the cell layer was washed exhaustively with PBS until no TGF-ß1 could be detected in the washes. The washed cell layer was trypsinized (0.05% trypsin, 0.02% EDTA in PBS). After addition of soybean trypsin inhibitor, the cells were centrifuged. The supernatant containing the pericellular TGF-ß1 was measured by specific sandwich ELISA. Intracellular TGF-ß1 was obtained from trypsinized cells that were first washed with PBS, then lysed with 2 M NaCl, 1% triton X100, pH 5.5. The lysate was centrifuged and the intracellular TGF-ß1 was analyzed as described above. As a control, total cell-associated TGF-ß1 was measured on direct lysate of the cell layer without trypsin treatment.

Number of experimental determinations and statistical analysis
For all parameters described here, the results are expressed as the mean (±SD) of independent determinations carried out in triplicate on four different HISM cultures. Difference between the means of two groups was evaluated with Student’s paired t test; significance was defined as P < 0.05 and less.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Under all experimental conditions, cell cultures were confluent in order to avoid the effect of treatments on cell proliferation. During the experimental period (up to 96 h after irradiation), no statistically significant alteration in cell number or cell viability could be detected in any group (less than ±15% different from control group). Preliminary experiments showed that T40 dextran (400 µg/ml) or carboxymethyl dextran (400 µg/ml) had no significant activity on collagen biosynthesis by HISM (data not shown).

Effect of irradiation and RG-1503 on protein and collagen biosynthesis by HISM
Total protein synthesis, expressed as total [3H] dpm per cell, ranged from 19.3 ± 2.8 (for 0 to 24 h measurement) to 17.1 ± 2.5 (for 24 to 48 h measurement) and 15.2 ± 2.1 (for 72 to 96 h measurement). This time-dependent decrease of protein biosynthesis was not significant. These values were not significantly changed by irradiation or after RG-1503 treatment of HISM (data not shown). Total collagen biosynthesis was increased in irradiated cells for up to 48 h after irradiation (Table 2 ). This augmentation was completely abolished by addition of RG-1503 before or after irradiation. All RG-1503-treated groups exhibited a decrease in total collagen biosynthesis correlated with duration of RG-1503 treatment. HISM collagen type expression was also altered during the first 48 h after irradiation (Fig. 2 ). In irradiated cells, collagen III synthesis was significantly increased by up to threefold 24 h after irradiation and then decreased with time. RG-1503 treatment of irradiated HISM down-regulated the altered collagen III biosynthesis to control values within 24 h whenever RG-1503 was added before or after irradiation. With time, RG-1503 decreased collagen III production to below control values (Fig. 2) . Collagen I biosynthesis showed no significant differences between any groups. Irradiated HISM synthesized less collagen V than control cells only after 72 to 96 h (Fig. 3 ). RG-1503 largely increased collagen V production during the first 48 h treatment in all groups. Between 72 and 96 h, RG-1503 restored the radiation-induced decrease in collagen V to the control level.


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  		Table 2. Effect of RG-1503 on total collagen biosynthesis by control and irradiated HISM cellsa



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  		Figure 2. Effect of irradiation and RG-1503 on collagen I and collagen III biosynthesis by HISM. Postconfluent control or irradiated cells (as defined in Materials and Methods) were labeled with [3H] proline in the presence or absence of RG-1503 at 400 µg/ml/106 cells. Collagen biosynthesis measurements (for a 24 h period) were performed immediately (0 to 24 h), 24 h (24 to 48 h), or 72 h (72 to 96 h) after irradiation in each experimental group. Pepsin-resistant radiolabeled collagen types I and III from medium plus cell layer were separated on SDS-PAGE as described in Materials and Methods. Radioactivity incorporated in each collagen type is expressed as hydroxy [3H] proline dpm/104 cells. A (control); B (nontreated and irradiated); C (post-treated); D (irradiated and post-treated); E (pretreated); F (pretreated and irradiated). Number of determination and statistical analysis are described in Materials and Methods. *Significant difference (P<0.05 and less) compared with control group; °significant difference (P<0.05 and less) between D (or F) groups and B group.



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  		Figure 3. Effect of irradiation and RG-1503 on collagen V biosynthesis by HISM. Postconfluent control or irradiated HISM (as defined in Materials and Methods) were labeled with [3H] proline in the presence or absence of RG-1503 and collagen biosynthesis measurements (for a 24 h period) were performed immediately (0 to 24 h), 24 h (24 to 48 h), or 72 h (72 to 96 h) after irradiation in each experimental group as described in Fig. 2 . Radioactivity incorporated in collagen V is expressed as hydroxy [3H] proline dpm/104 cells. A (control); B (nontreated and irradiated); C (post-treated); D (irradiated and post-treated); E (pretreated); F (pretreated and irradiated). Number of determination and statistical analysis are described in Materials and Methods. *Significant difference (P<0.05 and less) compared with control group; °significant difference (P<0.05 and less) between D (or F) groups and B group.

Effect of RG-1503 and TGF-ß1 on collagen mRNA levels
To clarify the mechanism by which RG-1503 regulate collagen metabolism in irradiated HISM, we measured steady-state collagen mRNA levels by Northern blot in all experimental groups. We illustrate only the most significant results that could be related to variations in collagen protein synthesis reported in Figs. 2 and 3 . However, all densitometric analysis concerning Northern blot are reported in Table 3 . Figure 4 , part 1) shows a significant increase of collagen III mRNA in irradiated HISM after 24 h. This alteration was abolished by treatment of irradiated cells with RG-1503. After 72 to 96 h (Fig. 4 , part 2), irradiated HISM produced less collagen V mRNA than control cells and RG-1503 largely restored the collagen V mRNA level to the control value. With GAPDH probe taken as control, no significant variation was observed in any group or at any time after irradiation (for illustration, see Fig. 4 , part 3: 72 to 96 h after irradiation).


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  		Table 3. Effect of RG-1503 on collagen mRNA expressed by control and irradiated HISM cellsa



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  		Figure 4. Effect of irradiation and RG-1503 on collagen III, collagen V, and GAPDH mRNA expressed by HISM. Total RNA was extracted from postconfluent control or irradiated HISM and incubated between 0 and 24 h after irradiation (part 1) or between 72 and 96 h (parts 2 and 3) in the presence or absence of RG-1503 (as defined in Materials and Methods). After Northern blot hybridization, collagen III mRNA (parat 1), collagen V mRNA (part 2) and GAPDH mRNA (part 3) were detected with appropriate [32P]-labeled probes as described in Materials and Methods, using 1) COL3A1 probe, 2) COL5A1 probe, and 3) GAPDH probe. A (control); B (nontreated and irradiated); C (post-treated); D (irradiated and post-treated); E (pretreated); F (pretreated and irradiated).

Effect of irradiation and RG-1503 on TGF-ß1 distribution in cultured HISM
The amount of TGF-ß1 present in FCS added to the culture medium was subtracted from all experimental values. TGF-ß1 (latent+active forms) present in HISM culture medium was not significantly altered by irradiation or by RG-1503 (all the values centered around 1100 pg/ml, with no significant between-group variation). However, irradiation induced a 1.5- to 2-fold increase in total TGF-ß1 in the cell layer (Fig. 5 ). RG-1503 reduced both baseline and irradiated cell TGF-ß1 levels. When we examined TGF-ß1 compartmentalization in the HISM cell layer (Fig. 5) , we found that the sum of pericellular plus intracellular TGF-ß1 was in the same range as the value obtained by the direct determination of total cell-associated TGF-ß1 taken as a control of compartmentalization yield (data not shown). We also demonstrated that the increase of TGF-ß1 in the cell layer during the postradiation period was essentially localized to the pericellular domain. RG-1503 treatment of the cells decreased both pericellular and intracellular TGF-ß1 whenever the polymer was added before or after irradiation.



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  		Figure 5. Effect of irradiation and RG-1503 on pericellular and intracellular TGF-ß1 content in HISM cells. Cell layers from postconfluent cells, treated as described in Materials and Methods, were exhaustively washed with PBS. Pericellular and intracellular compartments were separated as described in Materials and Methods. TGF-ß1 was determined in each compartment by sandwich ELISA. A (control); B (nontreated and irradiated); C (post-treated); D (irradiated and post-treated); E (pretreated); F (pretreated and irradiated). Number of determination and statistical analysis are described in Materials and Methods. *Significant difference (P<0.05 and less) compared with control group; °significant (P<0.05 and less) difference between D (or F) groups and B group.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The development of intestinal fibrosis is the main dose-limiting factor during radiotherapy for abdominal cancer. Chronic radiation enteropathy is characterized by progressive accumulation of collagen in the deeper layer of the bowel wall leading to severe long-term morbidity. Hence, abdominal radiotherapy results in permanent impairment of gastrointestinal function in one-half of the patients and important chronic toxicity (32 , 33) , with 5 to 10% of patients developing more severe complications (34) . In intestinal tissue, collagens are produced largely by smooth muscle cells, and this production is positively regulated by the fibrogenic cytokine TGF-ß1 (11 , 13) . A central role in the radiation-induced intestinal collagenous fibrosis has been attributed to TGF-ß1 (35) , and intense TGF-ß1 mRNA expression has been described in smooth muscle cells in area of intestinal injury (14) . To study these mechanisms, we irradiated cultured HISM and investigated in this model the biosynthesis of fibrillar collagens and the distribution of TGF-ß1.

We report that irradiation of HISM specifically increases collagen III biosynthesis during the first 48 h. Similar stimulation of collagen III production was reported in early responses to irradiation in various tissues (2 , 4 , 6) . Moreover, in fibrosis in general, the first steps of the fibrotic process are often characterized by alteration of the collagen III to collagen I ratio (36) . The radiation-induced collagen III overproduction by HISM is correlated with a significant increase in immunodetectable TGF-ß1 in the pericellular domain of the irradiated cells during the same period. The immunoassay used to measure TGF-ß1 content was performed after acidification, which induced activation of the latent form of TGF-ß1. Therefore, this assay measures both the latent and active forms and does not allow the determination of either form separately. It should be emphasized that this TGF-ß1 augmentation is localized to the irradiated cell environment and does not affect TGF-ß1 content measured in cell culture media. In fact, cell-associated TGF-ß1 appeared more directly implicated in the regulation of cell metabolism, especially collagen biosynthesis, than culture medium TGF-ß1. For example, in radiation enteropathy, smooth muscle cells exhibited a large increase in cell-associated TGF-ß1 immunoreactivity compared with control cells (14) . The absence of significant alteration of TGF-ß1 measured in the medium 24 h after HISM irradiation was also described for cultured human skin cells irradiated in similar experimental conditions (8) . These results argue for the importance of the localized alteration of TGF-ß1 concentration. Taken together, these data favor our cellular approach, which examines the relationships between collagen biosynthesis and pericellular TGF-ß1 content rather than total (medium plus cellular) TGF-ß1 in the development of radiation-induced fibrosis.

To regulate this radiation-induced fibrosis, we tested a heparan mimetic, RG-1503 (24 , 25) , on this model which was shown to decrease the biosynthesis of collagen III by smooth muscle cells in vitro (21 , 24) and could therefore present antifibrotic properties. As for heparin or highly sulfated heparan sulfate and other polyanionic polymers (37 38 39) , RG-1503 binds to TGF-ß1 and interferes with its biological activities (21 , 40) . In irradiated-HISM, RG-1503 restored collagen III production to the control level or even below. During the same period, RG-1503 dramatically decreased the amount of cell-associated TGF-ß1 immunoreactivity without significant alteration of the total cell medium TGF-ß1 content. Heparin (41) or heparin fragments (17) have been reported to decrease collagen III biosynthesis, but no study implicating TGF-ß1 had been performed. Our experiments demonstrate that RG-1503 modulates specifically both TGF-ß1 distribution and collagen III expression. This result could suggest at least two hypotheses. 1) RG-1503 could act by competing with the binding of TGF-ß1 to its low-affinity, nonsignaling RIII receptor or beta glycan, which is heparan sulfate proteoglycan (16) . 2) RG-1503 could interfere with TGF-ß1 and mask its immunoreactive moieties. However, we found that RG-1503 does not alter the immunodetection of TGF-ß1 in ELISA. The first hypothesis could then explain the mechanism by which RG-1503 modulate the bioactivity of TGF-ß1 toward collagen III expression. Even though collagen I and collagen III have a TGF-ß1 binding site in their promoter region, it has been shown that expression of collagen I and collagen III could be modulated independently by differential transcriptional mechanisms (42) . As an example, a high-glucose medium that induced an overexpression of TGF-ß1 by mesangial cells (43) caused a specific increase in collagen III biosynthesis without alteration of collagen I biosynthesis (44) . Taken together, these data could be in favor of the differential specific effect of RG-1503 on collagen III not seen on collagen I, which could be via a TGF-ß1-related mechanism. This remains to be investigated at the promoter level

Another collagen isotype disturbance was observed in irradiated HISM, but occurring only at 72 to 96 h postirradiation. The production of the minor fibrillar collagen V was significantly decreased. It is known that collagen V causes reduction of the diameter of heterotypic collagenous fibrils composed in association with the major collagen I and collagen III (45) . Inversely, this radiation-induced diminution of collagen V could promote an amplification of collagen fibril diameter, part of the fibrotic process. As reported previously for aortic smooth muscle cells (24) , RG-1503 specifically increased HISM collagen V biosynthesis, which resumed its original level before irradiation. TGF-ß1 has been described in other cells (46 , 47) to up-regulate collagen V more drastically than collagen I and collagen III. The complex interactions of RG-1503 with cell-associated TGF-ß1 described in this report for HISM might conceivably involve differential regulations of collagen types by mechanisms that remain to be elucidated. Finally, if RG-1503 was not the only RGTA or heparin-related compounds reducing the level of collagen III synthesis (17 , 24 , 41) , RG-1503 was chosen as the only RGTA that could also enhance collagen V synthesis and thus could correct the complex effects of irradiation on collagen phenotype expressed by HISM.

We first demonstrated that all these modulations of collagen types induced by radiation are regulated by RG-1503 at the protein level and are mirrored by comparable variations at mRNA level. These results could suggest that RG-1503 may exert control on collagen mRNA transcription via pathway involving cellular localization and distribution of TGF-ß1.

It is noteworthy that RG-1503 actively influences radiation-induced alterations in synthesis of collagens and distribution of TGF-ß1 whenever it is added before or immediately after irradiation. Restoration by RG-1503 of a normal collagen phenotype expression by irradiated HISM suggests that this finding could be useful in the development of preventive and/or curative therapeutic treatments for radiation-induced fibrosis and more generally for digestive fibrosis. RG-1503 would represent a new class of agent acting directly on collagen biosynthesis alterations associated with the tissular fibrotic response, as opposed to other agents acting on the inflammatory response.


   ACKNOWLEDGMENTS
 
This work was supported by the ‘Ministère de l’Enseignement Supérieur’, the CNRS, the ‘Association de Recherche sur le Cancer’, and the Association Naturalia et Biologia. P.M. was the recipient of an ATER position from the IUT-Créteil, France

Received for publication December 18, 2000. Revision received March 27, 2001.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Burger, A., Löffler, H., Bamberg, M., Rodemann, H. P. (1998) Molecular and cellular basis of radiation fibrosis. Int. J. Radiat. Biol. 73,401-408[Medline]
  2. Followill, D. S., Travis, E. L. (1995) Differential expression of collagen types I and III in consequential and primary fibrosis in irradiated mouse colon. Radiat. Res. 144,318-328[Medline]
  3. Rodeman, H. P., Binder, A., Burger, A., Guven, N., Loffler, H., Bamberg, M. (1996) The underlying cellular mechanism of fibrosis. Kidney Int. Suppl. 54,S32-S36[Medline]
  4. El Nabout, R., Martin, M., Remy, J., Kern, P., Robert, L., Lafuma, C. (1989) Collagen synthesis and deposition in cultured fibroblasts from subcutaneous radiation-induced fibrosis. Modification as a function of cell aging. Matrix 9,411-420[Medline]
  5. Remy, J., Wegrowski, J., Crechet, F., Martin, M., Daburon, F. (1991) Long-term over-production of collagen in radiation-induced fibrosis. Radiat. Res. 125,14-19[Medline]
  6. Ehrhart, A. J., Segarini, P., Tsang, M. L. S., Carrol, A. G., Barcellos-Hoff, M. H. (1997) Latent transforming growth factor ß1 activation in situ: quantitative and functional evidence after low-dose irradiation. FASEB J 11,991-1002[Abstract]
  7. Randall, K., Coggle, J. E. (1995) Expression of transforming growth factor beta 1 in mouse skin during the acute phase of radiation damage. Int. J. Radiat. Biol. 68,301-309[Medline]
  8. Martin, M., Vozenin, M. C., Gault, N., Crechet, F., Pfarr, C. M., Lefaix, J. L. (1997) Co-activation of AP-1 and TGF-ß1 gene expression in the stress response of normal skin cells to ionizing radiation. Oncogene 15,981-989[Medline]
  9. Herskind, C., Bamberg, M., Rodemann, H. P. (1998) The role of cytokines in the development of normal-tissue reactions after radiotherapy. Strahlenther. Onkol. 174(Suppl. III),12-15
  10. Hauer-Jensen, M., Richter, K. K., Wang, J., Abe, E., Sung, C. C., Hardin, J. W. (1998) Changes in transforming growth factor ß1 gene expression and immunoreactivity levels during development of chronic radiation enteropathy. Radiat. Res. 150,673-680[Medline]
  11. Graham, M. F., Bryson, G. R., Diegelmann, R. F. (1990) Transforming growth factor ß1 selectively augments collagen synthesis by Human Intestinal Smooth Muscle cells in culture. Gastroenterology 99,447-453[Medline]
  12. Becker, J. M. (1999) Surgical therapy for ulcerative colitis and Crohn’s disease. Gastroenterol. Clin. N. Am. 28,371-390[Medline]
  13. Pucilowska, J. B., Williams, K. L., Lund, P. K. (2000) Fibrogenesis; IV. Fibrosis and inflammatory bowel disease: cellular mediators and animal models. Am. J. Physiol. 279,G653-G659[Abstract/Free Full Text]
  14. Wang, J., Zheng, H., Sung, C. C., Richter, K. K., Hauer-Jensen, M. (1998) Cellular sources of transforming growth factor ß isoforms in early and chronic radiation enteropathy. Am. J. Pathol. 153,1531-1540[Abstract/Free Full Text]
  15. Landberg, C. W., Hauer-Jensen, M., Sung, C.H., Kane, C. J. M. (1994) Expression of fibrogenic cytokines in rat small intestine after fractionated irradiation. Radiother. Oncol. 32,29-36[Medline]
  16. O’Kane, S., Ferguson, M. W. J. (1997) Transforming growth factor ßs and wound healing. Int. J. Biochem. Cell Biol. 29,63-78[Medline]
  17. Asselot-Chapel, C., Combacou, L., Labat-Robert, J., Kern, P. (1995) Expression of fibronectin and collagen genes in smooth muscle cells: modulation by low-molecular weight heparin fragments and serum. Biochem. Pharmacol. 49,653-659[Medline]
  18. Aamiri, A., Butler-Browne, G. S., Martelly, I., Barritault, D., Gautron, J. (1995) Influence of a dextran derivative on myosin heavy chain expression during rat skeletal muscle regeneration. Neurosci. Lett. 201,243-246[Medline]
  19. Lafont, J., Baroukh, B., Berdal, A., Colombier, M. L., Barritault, D., Caruelle, J. P., Saffar, J. L. (1998) RGTA-11, a new healing agent, triggers developmental events during healing of craniotomy defects in adult rats. Growth Factors 16,23-38[Medline]
  20. Meddahi, A., Blanquaert, F., Saffar, J. L., Colombier, M. L., Caruelle, J. P., Josefonvicz, J., Barritault, D. (1994) New approaches to tissue regeneration and repair. Pathol. Res. Commun. 190,923-928
  21. Meddahi, A., Benoit, A., Ayoub, N., Sézeur, A., Barritault, D. (1996) Heparin-like polymers derived from dextran enhance colonic anastomosis resistance to leakage. J. Biomed. Mater. Res. 31,293-297[Medline]
  22. Benazzoug, Y., Logeart, D., Labat-Robert, J., Robert, L., Jozefonvicz, J., Kern, P. (1995) Derivatized dextrans modulate collagen synthesis in aortic smooth muscle cells. Biochem. Pharmacol. 49,847-853[Medline]
  23. Logeart, D., Letourneur, D., Jozefonvicz, J., Kern, P. (1996) Collagen synthesis by vascular smooth muscle cells in the presence of antiproliferative polysaccharides. J. Biomed Mater. Res. 30,501-508[Medline]
  24. Mestries, P., Borchiellini, C., Barbaud, C., Duchesnay, A., Escartin, Q., Barritault, D., Caruelle, J. P., Kern, P. (1998) Chemically modified dextrans modulate expression of collagen phenotype by cultured smooth muscle cells in relation with degree of carboxymethyl, benzylamide and sulfation substitutions. J. Biomed. Mater. Res. 42,286-294[Medline]
  25. Ledoux, D., Escartin, Q., Papy, D., Sagot, M. A., Cao, Y., Barritault, D., Courtois, J., Caruelle, J. P. (2000) Human plasmin enzymatic activity is inhibited by chemically modified dextrans. J. Biol. Chem. 275,29383-29390[Abstract/Free Full Text]
  26. Graham, M. F., Diegelmann, R. F., Elson, C. O., Bitar, K. N., Ehrlich, H. P. (1984) Isolation and culture of human intestinal smooth muscle cells. P. Soc. Exp. Biol. Med. 176,503-507[Abstract]
  27. Chromczynski, P., Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162,156-159[Medline]
  28. Chu, M. L., Myers, J. C., Bernard, M. J., Ding, S. F., Ramirez, F. (1982) Cloning and characterization of five overlapping cDNAs specific for the human pro-alpha 1(I) collagen. Nucleic. Acids Res. 10,5925-5934[Abstract/Free Full Text]
  29. Chu, M. L., Weil, D., de Wet, W., Bernard, M., Sippola, M., Ramirez, F. (1985) Isolation of cDNA and genomic clones encoding human pro-alpha 1(III) collagen. Partial characterization of the 3' end region of the gene. J. Biol. Chem. 260,4357-4363[Abstract/Free Full Text]
  30. Adams, M. D., Kerlavage, A. R., Fleischman, R. D., Fuldner, R. A., Bult, C. J., Lee, N. H., Kirkness, E. F., Weinstock, K. G., Gocayne, J. D., White, O., et al (1995) Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence. Nature (London) 377,3-174[Medline]
  31. Schmidt, A., Skaletz-Rorowski, A., Breithardt, G., Buddecke, E. (1995) Growth status-dependent changes of bFGF compartmentalization and heparan sulfate structure in arterial smooth muscle cells. Eur. J. Cell Biol. 67,130-134[Medline]
  32. Yeoh, E., Horowitz, M., Russo, A., Muecke, T., Ahmad, A., Robb, T., Chaterton, B. (1993) A retrospective study of pelvic irradiation for carcinoma of the cervix of gastrointestinal function. Int. J. Radiat. Oncol. Phys. 24,229-237
  33. Eifel, P. J., Levenback, C., Wharton, J. T., Oswald, M. J. (1995) Time course and incidence of late complications in patients treated with radiation therapy for FIGO stage IB carcinoma of the uterine cervix. Int. J. Radiat. Oncol. Biol. Phys. 32,1289-1300[Medline]
  34. Coia, L. R., Meyerson, R. J., Tepper, J. R. (1995) Late effects of radiation therapy on the gastrointestinal tract. Int. J. Radiat. Oncol. Biol. Phys. 31,1213-1236[Medline]
  35. Richter, K. K., Landberg, C. W., Sung, C. C., Hauer-Jensen, M. (1997) Increased transforming growth factor ß (TGFß) immunoreactivity is independently associated with chronic injury in both consequential and primary radiation enteropathy. Int. J. Radiat. Oncol. Biol. Phys. 39,187-195[Medline]
  36. Stallmach, A., Schuppan, D., Riese, H. H., Matthes, H., Riecken, E.O. (1992) Increased collagen type III synthesis by fibroblasts isolated from strictures of patients with Crohn’s disease. Gastroenterology 102,1920-1929[Medline]
  37. McCaffrey, T. A., Falcone, D. J., Du, B. (1992) Transforming growth factor-ß1 is a heparin-binding protein: identification of putative heparin-binding regions and isolation of heparins with varying affinity for TGF-ß1. J. Cell. Physiol. 152,430-440[Medline]
  38. McCaffrey, T. A., Falcone, D. J., Vicente, D., Du, B., Consigli, S., Borth, W. (1994) Protection of transforming growth factor-ß1 activity by heparin and fucoidan. J. Cell. Physiol. 159,51-59[Medline]
  39. Lyon, M., Rushton, G., Gallagher, J. T. (1997) The interaction of the transforming growth factor-betas with heparin/heparan sulfate is isoform specific. J. Biol. Chem. 272,18000-18006[Abstract/Free Full Text]
  40. Blanquaert, F., Barritault, D., Caruelle, J. P. (1999) Effects of heparan-like polymers associated with growth factors on osteoblast proliferation and phenotype expression. J. Biomed. Mater. Res. 44,63-72[Medline]
  41. Majack, R. A., Bornstein, P. (1985) Heparin regulate the collagen phenotype of smooth muscle cells: induced synthesis of an Mr 60.000 collagen. J. Cell Biol. 100,613-619[Abstract/Free Full Text]
  42. DeCrombrugghe, B., Karsenty, G., Maity, S., Vuorio, T., Rossi, P., Ruteshouser, E. C., McKinney, S. H., Lozano, G. (1990) Transcriptional mechanisms controlling types I and III collagen genes. Ann. N.Y. Acad. Sci. 580,88-96[Medline]
  43. Ceol, M., Gambaro, G., Sauer, U., Baggio, B., Anglani, F., Forino, M., Facchin, S., Bordin, L., Weigert, C., Nerlich, A., Schleicher, E. D. (2000) Glycosaminoglycan therapy prevents TGF-ß1 overexpression and pathological changes in renal tissue of long-term diabetic rats. J. Am. Soc. Nephrol. 11,2324-2336[Abstract/Free Full Text]
  44. Benazzoug, Y., Borchiellini, C., Labat-Robert, J., Robert, L., Kern, P. (1998) Effect of high-glucose concentrations on the expression of collagens and fibronectin by fibroblasts in culture. Exp. Gerontol. 33,445-455[Medline]
  45. Birk, D. E., Fitch, J. M., Barbiaz, J. B., Doane, K. J., Linsenmayer, T. F. (1990) Collagen fibrillogenesis in vitro: interaction of types I and V collagen regulates fibril diameter. J. Cell Sci. 95,649-657[Abstract/Free Full Text]
  46. Lawrence, L., Hartmann, D. J., Sonenshein, G. E. (1994) Transforming growth factor ß1 stimulates type V collagen expression in bovine vascular smooth muscle cells. J. Biol. Chem. 269,9603-9609[Abstract/Free Full Text]
  47. Parekh, T. V., Wang, X. W., Makri-Werzen, D. M., Greenspan, D. S., Newman, M. J. (1998) Type V collagen is an epithelial cell cycle inhibitor that induced by and mimics the effects of transforming growth factor ß1. Cell Growth Differ 9,423-433[Abstract]



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