INFLAMMATORY BOWEL DISEASE Reversal of abnormal collagen production in Crohn's disease intestinal biopsies treated with regenerating agentsC Alexakis, J P Caruelle, A Sezeur, J Cosnes, J P Gendre, H Mosnier, L Beaugerie, D Gallot,M Malafosse, D Barritault, P Kern. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gut 2004;53:85–90 Background: Crohn's disease (CD) is characterised by inflammation, muscle layer overgrowth, andcollagenous 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 andinvestigated the effect of regenerating agents (RGTAs) on collagen production. RGTAs are chemicallysubstituted dextrans engineered to mimic the growth factor protecting effects of heparan sulphates. RGTAshave been shown to enhance tissue repair in various in vivo models and to modulate in vitro collagen See end of article for authors' affiliations 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.
Correspondence to: Professor D Barritault, Methods: After 24 hours of ex vivo incubation with (3H) proline, collagen I, III, and V were extracted by CRRET-CNRS FRE 2412, pepsin and quantitatively separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
Faculte´ des Sciences de Biosynthesis of each collagen type was quantified on radiolabelled isolated collagen.
Cre´teil, Universite´ Paris 12, Results: Total intestinal collagen production in CD patients compared with controls was increased up to Avenue du Ge´ne´ral de Gaulle, 94010 Cre´teil 3.5-fold overall (p,0.001). In particular, collagen III biosynthesis was enhanced by 6.2-fold (p,0.001) in cedex, France; barritault@ 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 Accepted for publication 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.
the gastrointestinal tract characterised by transmural stimulate tissue repair and protection in vivo, which we granulomatous inflammation and thickening of the called ReGeneraTing Agents or RGTAs. These polymers are bowel wall, with muscle layer overgrowth and collagenous obtained by chemical substitutions of dextran (see fig 1 for fibrosis. Excess of fibrillar collagens occurs in all bowel wall detailed structures). In several experimental models, they layers.1 2 Recent findings suggest that increased collagen promote healing of various tissues, including muscle,12 production may be pivotal in the mechanism leading to bone,13 skin,14 and intestine.15 They also induce extracellular intestinal fibrosis, stricture development, and intestinal matrix remodelling by decreasing collagen production,16 17 obstruction.3 4 Yet no medications effective against intestinal and collagen III production in particular, relative to other collagen accumulation are available, and surgery remains the collagen types.18 Because this effect on collagen synthesis main treatment for fibrosis related morbidity in CD.1 may be associated with an antifibrotic activity, we studied the Quantitative and qualitative variations in collagen pheno- effects of RGTAs on collagen overproduction by intestinal type have been documented in CD patients with conflicting tissue affected by CD.
The situation is further complicated in the intestine by the MATERIALS AND METHODS heterogeneity of the cell population capable of producing collagens. Mesenchymal intestinal cells producing collagens Intestinal tissue was obtained from three groups of patients include fibroblasts, smooth muscle cells, and myofibro- who required surgical intestinal resection (medical details on blasts.3 9 Our first objective was to investigate collagen patients and precise tissue specimen origins are indicated in production in specimens of entire intestinal tissue fragments table 1). For CD patients, behaviour status was indicated rather than in isolated cells. We quantitated the ex vivo according to the Vienna classification.19 The CD group production of collagens by intestinal tissue biopsies collected included patients with CD who were not receiving glucocor- surgically from CD patients treated or not with glucocorti- ticoids (CD group); the CD-GC group comprised patients with coids. Control biopsies were macroscopically normal intest- CD who were receiving glucocorticoids at a mean dose of inal tissue fragments from patients with distal intestinal approximately 30 mg prednisone/day. Tissue specimens from Evidence that collagen, and collagen III in particular, overproduction is related to fibrosis8 has prompted a search Abbreviations: RGTA, regenerating agent; CD, Crohn's disease; CD- for components capable of selectively regulating the produc- GC, Crohn's disease treated with glucocorticoids; TGF-b1, transforming tion of specific collagen types.10 We have demonstrated growth factor b1; FGF-2, fibroblast growth factor 2; Mr, molecular previously that low molecular weight heparin specifically weight; ds, degree of substitution; SDS-PAGE, sodium dodecyl sulphate- decreased collagen III production.11 Recently, we developed a polyacrylamide gel electrophoresis Alexakis, Caruelle, Sezeur, et al indicated by infrared spectroscopy, with the appearance of two absorption bands at 1250 and 1025/cm, respectively.
For RG1192, the carboxymethyl dextran was dissolved in H2O/EtOH, and carboxylate functions were activated with N- ethoxy-carbonyl-2-ethoxy-1,2- dihydroquinoline for 30 min- utes at pH 3.5 and room temperature. This was followed by overnight incubation with 2 eq of free benzylamide. The product was precipitated and washed with methanol and dried under a vacuum. The infrared spectrum confirmed the presence of benzylamide with the appearance of a new absorption band at 1750/cm, corresponding to the carbonyl bond of the amide. RG1192 was synthesised from the derivatised carboxymethyl-benzylamide dextran by O-sul- phonation. The presence of sulphate groups was indicated byinfrared spectroscopy, with the appearance of two absorptionbands at 1250 and 1025/cm, respectively.
Chemical characterisation of RG1503 and RG1192 was based on the degree of substitution (ds) of each individual group per glucosidic unit (table 2). A ds value of 3 indicatesmaximum substitution as one glucosidic unit contains three reactive OH groups at the C2, C3, and C4 positions. Each ds value was determined by acidimetric titration and elementary analysis and confirmed by 1H nuclear magnetic resonance.
The distribution of the three reactive OH groups is reported in table 2. Average molecular weights of the RGTA molecules were estimated by high performance size exclusion chroma- tography, as described previously.20 These polymers did not show significant anticoagulant activity (less than 5 IU/mg compared with 173 IU/mg for heparin).
Tissue processing Freshly resected tissue was placed in phosphate buffered saline and immediately transported to the laboratory for processing. In order to tentatively maintain uniformity in the sampling of tissue specimens, a standardised procedure wasused to place each tissue specimen (approximately 1 g wet Figure 1 Schematic structure of dextran derivatives. Polymers were weight) on a cutting board and to slice it longitudinally into elaborated from T40 dextran by chemical substitutions, as described in three pieces of similar macroscopic aspect and of approxi- materials and methods. Dextrans were substituted by carboxy- methylation followed by O-sulphonation (RG1503) or by carboxy- mately equal size.
methylation followed by amidation with benzylamine and O-sulphonation (RG1192). The different percentages indicated were Intestinal collagen production calculated from the degree of substitution (ds) relative to the position of One of the three parts of each specimen was incubated in each group in a glucosidic unit, as reported in table 2. For an easy 3 ml of standard medium (Dulbecco's modified Eagle's representation, the substituted glucosidic units A, B, C, and D were arranged in an arbitrary combination. Their respective proportions (%) minimal essential medium) supplemented with 100 IU/ml within each polymer were calculated according to the nature of the penicillin, 100 mg/ml streptomycin, and 2 mM glutamine.
group at the C2 position. R is the proportion (%) of each substituted The two other parts were incubated in 3 ml of standard group at the C3 plus C4 positions.
medium with RG1503 (100 mg/ml) or RG1192 (100 mg/ml),respectively. Then, tissue specimens were labelled for24 hours patients in the CD and CD-GC groups were obtained from Amersham, France) plus ascorbic acid (50 mg/ml). At the totally diseased areas with no portion of macroscopically end of the labelling period, the tissue was washed extensively normal tissue. In the control group, intestinal specimens with distilled water until no radioactivity was detected in the were uninvolved areas of bowel removed from patients with washing solutions.
distal intestinal cancer (at least 10 cm away from thetumour), with normal gross and histological features and Collagen extraction, identification, and quantitation no signs of inflammation.
Each tissue specimen (approximately 300 mg wet weight)was finely minced and homogenised. A small aliquot of homogenised tissue (10–15 mg) was hydrolysed in 6 M HCl The water soluble dextran derivatives RG1503 and RG1192 at 105˚C for 24 hours, for determination of total protein (see fig 1 for detailed structures) were prepared from T40 production, total collagen content, and total collagen dextran (average molecular weight (Mr) 37 000; Pharmacia, production. Total protein production and total collagen Paris, France) as previously described.18 20 21 Briefly, for production were determined by quantitation of (3H) proline RG1503, carboxymethyl dextran was synthesised from and hydroxy (3H) proline by high performance liquid dextran T40 by carboxymethylation of OH residues with chromatography.22 23 Total collagen content was determined monochloracetic acid treatment. The presence of carboxy- by colorimetric hydroxyproline assay.
methyl groups was confirmed by infrared spectroscopy, Then, all of the remaining minced tissue was digested with which showed an absorption band at 1650/cm. Then, pepsin, as previously described.22 24 Briefly, the tissue was RG1503 was obtained from carboxymethyl dextran by dispersed in 0.5 M acetic acid containing pepsin (with a O-sulphonation. The presence of sulphate groups was collagen to pepsin ratio of 10:1) for 24 hours at 4˚C under RGTAs regulate collagen production in Crohn's disease Table 1 Intestinal tissue specimens included in the study Surgical procedure Right colon cancer Right hemicolectomy Right colon adenocarcinoma Right hemicolectomy Small intestine carcinoma Right colon cancer Right colon adenocarcinoma Right hemicolectomy Right colon cancer Right hemicolectomy Colorectal cancer Ileocaecal resection CD, bowel obstruction Ileocaecal resection CD, bowel obstruction Ileocaecal resection CD, ileosigmoid fistula+bowel obstruction Ileocaecal resection CD+bowel obstruction Resection of middle portion of small intestine CD+bowel obstruction Ileocecal resection CD+bowel obstruction Resection of small intestine CD, ileosigmoid fistula Ileocaecal resection CD, right and transverse colon+bowel Ileocolonic resection CD, bowel obstruction Right ileocolonic resection CD, bowel obstruction Ileocecal resection CD, right and left colon Ileocolonic resection CD, perineal lesions+bowel obstruction Ileocecal resection CD, bowel obstruction Ileocecal resection CD, bowel obstruction Ileal and ileocaecal resection CD, bowel obstruction Ileocaecal resection CD, bowel obstruction The control group is defined in materials and methods; CD group, patients with Crohn's disease not treated with glucocorticoids; CD-GC group, patients withCrohn's disease treated with glucocorticoids.
B, behaviour status (non-stricturing non-penetrating (B1), stricturing (B2), penetrating (B3)) according to the Vienna classification.19T, tissue origin of the portion of specimen studied (I, ileal; O, ileocaecal; C, colonic).
constant shaking. This procedure was repeated twice. The carried out in triplicate on each tissue specimen with or insoluble material was hydrolysed in 6 M HCl at 105˚C for without RGTA treatment. Differences between means in the 24 hours, for determination of residual insoluble collagen two groups were evaluated using the Student's unpaired t (hydroxyproline). In all specimens, the amount of insoluble test. A p value ,0.05 was considered statistically significant.
collagen was less than 10–15% of the total intestinal collagen.
Production of pepsin soluble collagen types was deter- mined by sodium dodecyl sulphate-polyacrylamide gel The collagen content of intestinal tissue from the CD, CD-GC, revealed by Coomassie blue staining and identified by and control groups was determined by quantitation of comparison with standard collagen types (I, III, and V).
hydroxyproline per unit of tissue wet weight. Total collagen Separation of collagen III was achieved by delayed reduc- content was significantly higher in the CD and CD-GC groups tion.25 The relative proportions of radioactivity incorporated than in the control group (362 (44) (p,0.001) and 287 (32) in collagens I, III, and V were quantified by excision of each (p,0.02), respectively, versus 183 (20) mg collagen/10 g individual collagen band followed by hydrolysis of the band tissue wet weight). Brief exposure (24 hours) of intestinal in 6 M HCl at 105˚C for 24 hours and by determination of tissue to the RGTAs had no significant effect on total collagen hydroxy (3H) proline in the hydrolysate.22 content in any of the three groups.
Statistical analysis Collagen production For all parameters reported in the text and tables, results are Each intestinal specimen was cut into three pieces: one was expressed as the mean (SD) of independent determinations left untreated, one was treated with RG1503, and one was Table 2 Chemical characterisation of the regenerating agents (RGTA) Position of groups expressed as ds Polymer RG1192 (average Mr 140 000)0.31 Polymer RG1503 (average Mr 62 900)0.26 Chemical characterisation of each RGTA polymer is detailed in materials and methods.
*ds, degree of substitution of an individual group in one glucosidic unit (SD of ds values were less than 5%, withn = 3).
CM, CH2COONa; Su, SO3Na; 1CMB, CH2CONHCH2C6H5; H, non-reacted hydroxyl groups; **C3+C4,global substitution at the C3+C4 positions calculated for each group as the difference between the total ds value andthe ds value at the C2 position.
Alexakis, Caruelle, Sezeur, et al Table 3 Effect of regenerating agents RG1503 and RG1192 on total collagen productionin the bowel of patients with Crohn's disease (CD) Protein production Collagen production (102 dpm hydroxy (total dpm/mg proline) (3H) pro/mg collagen) CD-GC group+RG1503 CD-GC group+RG1192 Control, CD, and CD-GC groups as described in table 1.
RGTA treatment of normal intestinal tissue (control group) did not significantly change total collagen production(data not shown).
*p,0.05, **p,0.01, ***p,0.001 compared with the control group.
p,0.05 between RGTA treatment and no RGTA treatment of specimens from the CD group.
p,0.05 between RGTA treatment and no RGTA treatment of specimens from the CD-GC group.
treated with RG1192. After 24 hours of labelling with (3H) control values and also reduced production of collagens I and proline, total collagen production by the entire tissue V. RG1192 decreased collagen III and collagen V production fragment was determined (table 3). In the control group but had no effect on collagen I production. Treatment of CD- biopsies, RGTA treatment had no significant effect (data not GC biopsies with RG1503 restored collagen I and III shown). There were no significant differences in total protein production to control values but induced a smaller decrease production between the three groups. Total collagen produc- in collagen V production; RG1192 reduced collagen III and tion showed a marked increase in the CD group (by up to 3.5- collagen V production to control values but did not modify fold) and a smaller increase in the CD-GC group (by up to collagen I production.
2.1-fold). Treatment with RG1503 significantly decreased We compared the relative percentages of production of total collagen production in the CD and CD-GC groups, with each collagen type in each group (table 4). Without RGTA a return to control values in the CD-GC group. RG1192 had treatment, the proportion of collagen III in the CD group was no significant effect on total collagen production in the CD selectively increased compared with the control group.
and CD-GC groups. Collagen in each tissue specimen was solubilised by pepsin treatment which produced similar decreased the percentage of collagen III production to control yields in all three groups (the amount of residual insoluble values. Note that the relative proportion of collagen V collagen was less than 10–15% of total intestinal collagen).
production was higher with RG1503. In the CD-GC group, Fibrillar collagen types were separated and quantified by the proportion of collagen III production without RGTA SDS-PAGE. Table 4 shows that production of collagen types I, treatment was not significantly increased compared with III, and V exhibited large increases in the CD group and control values. However, both RG1503 and RG1192 selec- smaller increases in the CD-GC group. Treatment of CD tively decreased the proportion of collagen III production in biopsies with RG1503 decreased collagen III production to Table 4 Effect of regenerating agents RG1503 and RG1192 on production of collagentypes and on the relative production of collagen types in intestinal fragments from patientswith Crohn's disease (CD) Collagen production (102 dpm hydroxy (3H) proline/mg collagen)(collagen type production as % of total collagen production) % of total collagen % of total collagen % of total collagen % of total collagen % of total collagen CD-GC group+RG1503 % of total collagen CD-GC group+RG1192 % of total collagen Control, CD, and CD-GC groups as described in table 1.
RGTA treatment of normal intestinal tissue (control group) did not significantly change collagen type production(data not shown). The percentage of collagen type production compared with total collagen production wascalculated from absolute values.
*p,0.05, **p,0.01, ***p,0.001 compared with the control group.
p,0.05, p,0.01, p,0.001 between RGTA treatment and no RGTA treatment of specimens from the CDgroup.
p,0.05, p,0.01, p,0.001 between RGTA treatment and no RGTA treatment of specimens from the CD-GC group.
RGTAs regulate collagen production in Crohn's disease the effect of RG1192 on the CD collagen phenotype could be CD is a severe intestinal disorder characterised by a chronic mediated by potentiation of the specific FGF-2 induced and unpredictable course with flares of acute inflammation decrease in collagen III production.34 separated by remissions.26 The acute inflammation induces In addition, both RG1503 and RG1192 have been shown to fibrosis with massive accumulation of a collagenous extra- inhibit the proliferation of various mesenchymal cells,17 32 35 cellular matrix. Onset of remission occurs when healing and and thus RGTA may have beneficial effects on the abnormal repair mechanisms become operative. Thus antifibrotic drugs cellular proliferation seen in CD.
that enhance these mechanisms may hold therapeutic Although the duration of tissue exposure to RGTA was extremely short in our study, the results suggest that these The goals of the present study were to improve the polymers may hold promise for correcting the collagen characterisation of collagenous fibrosis in human intestinal production abnormalities seen in CD, thus providing a non- tissue from CD patients and to investigate the effect on this invasive alternative to surgery. Regular heparin and low tissue of two antifibrotic compounds of the RGTA family, molecular weight heparin have shown some efficacy in which promotes repair and remodelling of several tissues,12–14 treating the bowel inflammation seen in CD. However, including the intestine.15 27 treatment duration was kept short because of the risk of Our results indicate that alterations in collagen production bleeding and the rationale for using heparin is unclear.
associated with CD were not related to an overall alteration in RGTAs are heparan sulphates mimetic agents devoid of protein production, which was not significantly modified anticoagulant activity. Thus our findings indicate that further clinical research is warranted.
Intestinal collagen production was increased 3.5-fold in biopsies from CD patients who were not taking glucocorticoid therapy. Our study indicates an important increase in This work was supported by the CNRS, the ‘‘Ministe re de collagen III production, which reached 6.2-fold (table 4) l'Enseignement Supe ´rieur'', and by the association Naturalia et versus 2.9-fold and 4.8-fold for collagen I and collagen V, Biologia. CA was a recipient of a grant from ‘‘Association Franc¸ois respectively. Increases in collagen III and also in collagen I production have been reported in fibrotic processes affecting various tissues,8 including the intestine.7 28 Authors' affiliations CD patients who were taking glucocorticoid therapy (CD- C Alexakis, J P Caruelle, D Barritault, P Kern, CRRET/CNRS FRE 2412, GC group) had milder alterations in collagen production than Universite´ Paris-12, 94010 Cre´teil Cedex, France patients in the CD group. The ability of glucocorticoids to A Sezeur, H Mosnier, Chirurgie Digestive et Colioscopique, Hoˆpital des decrease collagen production has been widely documented.29–31 Diaconesses, 75571 Paris Cedex 12, France However, in addition to exerting desirable effects on J Cosnes, J P Gendre, L Beaugerie, Gastroente´rologie, Hoˆpital Rothschild, 75571 Paris Cedex 12, France inhibit wound healing and tissue repair.
D Gallot, M Malafosse, Chirurgie Ge´ne´rale, Hoˆpital Bichat, 75018 In the present study, very brief ex vivo exposure (24 hours) to RGTA partially or completely corrected collagen productionabnormalities in intestinal tissue from CD patients. In particular, RG1503 reduced the increase in collagen III 1 Becker JM. Surgical therapy for ulcerative colitis and Crohn's disease.
production in the CD group from 6.2- to 1.5-fold of control Gastroenterol Clin North Am 1999;28:371–90.
2 Graham MF, Willey A, Adams J, et al. Corticosteroids increase procollagen levels. It should be noted that RG1503 had an overzealous gene expression, synthesis and secretion by human intestinal smooth muscle inhibitory effect on the percentage of collagen III in the CD- cells. Gastroenterology 1999;109:1454–61.
GC group, indicating that lower doses should be used in 3 Pucilowska JB, Williams KL, Lund PK, et al. Fibrosis and inflammatory bowel function of the present response of intestinal tissue.
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