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Am J Physiol Gastrointest Liver Physiol 287: G875-G885, 2004. First published June 3, 2004; doi:10.1152/ajpgi.00088.2004
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INFLAMMATION/IMMUNITY/MEDIATORS

Global gene expression profiles reveal an increase in mRNA levels of collagens, MMPs, and TIMPs in late radiation enteritis

Carine Strup-Perrot,1,2 Denis Mathé,1,2 Christine Linard,2 Dominique Violot,1 Fabien Milliat,1,2 Agnès François,1,2 Jean Bourhis,1,3 and Marie-Catherine Vozenin-Brotons1,2

1Laboratoire UPRES EA 27-10 "Radiosensibilité des tumeurs et tissus sains," Institut Gustave Roussy/Institut de Radioprotection et de Sûreté Nucléaire, 94805 Villejuif; 2Laboratoire d'étude des pathologies radio-induites, Service de Radioprotection Radiobiologie et Epidémiologie, Direction de la Radioprotection de l’Homme, Institut de Radioprotection et de Sûreté Nucléaire, 92265 Fontenay-aux-Roses; and 3Radiation Oncology Department, Institut Gustave Roussy, 94805 Villejuif, France

Submitted 25 February 2004 ; accepted in final form 1 June 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Radiation enteritis, a common complication of radiation therapy for abdominal and pelvic cancers, is characterized by severe transmural fibrosis associated with mesenchymal cell activation, tissue disorganization, and deposition of fibrillar collagen. To investigate the mechanisms involved in this pathological accumulation of extracellular matrix, we studied gene expression of matrix components along with that of genes involved in matrix remodeling, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Hybrid selection on high-density cDNA array, real-time RT-PCR, gelatin zymography and imunohistochemistry were used to characterize the mRNA expression profile, activity, and tissue location of extracellular matrix-related genes in radiation enteritis compared with healthy ileum. cDNA array analysis revealed a strong induction of genes coding for collagens I, III, IV, VI, and VIII, SPARC, and tenascin-C, extracellular-matrix degrading enzymes (MMP-1, -2, -3, -14, -18+19), and metalloproteinase inhibitors (TIMP-1, -2, plasminogen activator inhibitor-1) in radiation enteritis. This increase was correlated with the degree of infiltration of the mucosa by inflammatory cells, and the presence of differentiated mesenchymal cells in the submucosa and muscularis propria. Despite the fact that expression of collagens, MMPs, and TIMPs simultaneously increase, quantification of net collagen deposition shows an overall accumulation of collagen. Our results indicate that late radiation enteritis tissues are subjected to active process of fibrogenesis as well as fibrolysis, with a balance toward fibrogenesis. This demonstrates that established fibrotic tissue is not scarred fixed tissue but is subjected to a dynamic remodeling process.

fibrosis; radiation therapy; ileum; cDNA array; extracellular matrix

PELVIC RADIATION THERAPY IS frequently associated with normal intestinal tissue toxicity, which may result in the development of progressive fibrosis. During fibrogenesis, the compliant relationship between the mucosa and the submucosa is lost, which contributes to stricture formation, subsequent intestinal obstruction, and ultimate organ failure. The main feature of tissue fibrosis is excessive accumulation of abnormal and cross-linked collagen mainly composed of fibrillar and immature ECM components (8). The precise mechanisms underlying the dramatic deposition of connective tissue observed in tissue fibrosis remain unclear. However, failure to maintain homeostasis of the ECM and upsetting the balance between synthesis and degradation of ECM components may play an important role.

The synthesis of ECM components is regulated at the transcriptional, posttranscriptional, translational, and posttranslational levels. Collagen lysis is regulated by the balance between the activity of matrix metalloproteinases (MMPs) and that of their tissue inhibitors (TIMPs). MMPs consist of a family of at least 25 zinc-dependent proteases (3, 19, 40). The latter are classified according to their substrate specificity and structural features into gelatinases (MMP-2, -9), stromelysins (MMP-3, -7, -10, -11), elastases (MMP-12), collagenases (MMP-1, -8, -13, -18), and membrane-type MMPs (MMP-14, -15, -16, -17). MMP activity is tightly controlled both at the transcriptional and the translational levels (19). Analysis of the control elements of the promoter region of MMP genes revealed common arrangements of the transcription factor binding sites. This specific promoter structure is thought to be required for the control of the tissue-specific expression of MMPs and to involve a functional cooperation between transcription factors of the AP-1 and Ets family (5). Most MMPs are secreted as zymogens and require proteolytic activation. In vivo activation of pro-MMPs is mostly mediated through the plasminogen-plasmin cascade and by MMPs themselves (18, 30). Another type of MMP activation, which has been reported for MMP-2, is through the membrane-type MMP-1 (MMP-14) (28). This process may be associated to fibrogenesis as MMP-2 degrades basement membrane type IV collagen (4), which is thought to facilitate the deposition of fibril-forming collagen. The third level of control of MMP activity is ensured by TIMPs, which are known to inhibit active MMPs at a stoichiometric ratio of 1:1 (38). Four subtypes of TIMPs (TIMP-1 to -4) have been identified so far (2). Whereas TIMP-1 inhibits a broad range of MMPs, TIMP-2 seems to specifically inhibit MMP-2.

The control of the ECM turnover during the wound healing process and fibrosis depends on a sharp balance between ECM synthesis and degradation, and involves cooperation among three groups of genes: ECM components, proteases, and protease inhibitors. Recent global approaches, such as gene array analysis, allow an overall and integrated view of the regulation of ECM remodeling. This approach has been successfully applied to intestinal inflammatory and fibrotic disorders (34). In experimental models of T-cell-mediated intestinal injury, overexpression of MMPs (MMP-1, -3, -9) was found to be associated with a decrease in TIMP expression (26), whereas concomitant overexpression of MMPs (MMP-1, -2, -3, -14) and TIMP-1 was observed in inflamed mucosa of inflammatory bowel disease (IBD) samples (34). Overexpression of MMPs, however, globally exceeded that of TIMP-1, which led to a net increase in proteolytic activity in the inflamed mucosa. There are very little data on MMPs in relation to intestinal fibrosis and even less in relation to radiation-induced fibrosis, with most studies focusing on the relationship between MMPs and mucosal ulceration (11). However, conflicting theories have been proposed. The excessive accumulation of collagen may be the consequence of increased ECM synthesis associated with decreased ECM degradation. In skin radiation-induced fibrosis, Lafuma et al. (10) reported decreased activity of gelatinases associated with increased TIMP activity. Zhao et al. (42) likely reported that the increased expression of plasminogen activator inhibitor-1 (PAI-1) after exposure to ionizing radiation led to decreased ECM degradation and to collagen accumulation.

This study aimed at investigating the balance between fibrogenesis and fibrolysis during intestinal radiation-induced fibrosis. cDNA array analysis is a global approach that enabled us to simultaneously quantitate mRNA expression of ECM components, MMPs, TIMPs, and PAI-1 in bowel biopsies from patients with radiation enteritis. Changes in expression levels of MMPs and TIMPs were confirmed by real-time RT-PCR, and immunolocalization was used to characterize the cell types involved in the control of ECM remodeling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue sampling. Twenty-two patients treated by surgery for intestinal occlusion caused by delayed radiation-induced enteritis entered the study. The patients characteristics are shown in Table 1. Histological and immunohistological studies were performed in 22 patients and tissue samples from six patients were frozen for subsequent mRNA and gelatin zymography studies. In most cases, the severity of the affliction did not allow resection of healthy intestine. Healthy ileum samples obtained from six patients without radiation enteritis, who underwent colon surgery, were used as controls. These control samples were free of malignancy and showed regular histology after hematoxylin and eosin staining. Collagen deposition was detected by Sirius Red staining. Procurement of tissue samples received prior approval from our institution's Ethics Committee and was performed according to the French Medical Research Council guidelines.


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  		Table 1. Characteristics of the patient population

 
Gene array analysis. Total RNA was extracted from frozen tissue by the method of Chomczynski as already described (35), and quantified by absorption spectrometry. RNA was treated with RNase-free DNase (0.5 U/µl) to remove contaminating genomic DNA. RNA integrity was checked and PolyA RNA was purified from 20 µg of total RNA using the RNA Atlas Pur kit (Clontech, Ozyme, St. Quentin en Yveline, France). Radiolabeled cDNA was prepared according to Clontech's instructions and hybridized with the Cell Interaction and Atlas Human 1.2 arrays. A list of all the genes included in these two arrays as well as their functions can be found at www.clontech.com/atlas and is deposited into the Gene Expression Omnibus database (www.ncbi.nih.gov/geo) under GEO accession nos. GPL127 and GPL135. Phosphorimager intensifying screens were exposed to membranes and mRNA expression levels were determined by scanning the screen with a phosphorimager (Raytest; Fuji, Courbevoie, France). Analysis of differential mRNA expression was carried out by using the Atlas Image 1.5 software, and data were normalized with selected housekeeping genes (HPRT, GAPDH, TUBA1, RPL13A, 40S ribosomal protein S9) as already described (35). Signal intensities had to be significantly above background (i.e., 50% or more) to be considered. Only changes in the expression level greater than twofold the average control level were considered significant.

Determination of net collagen deposition in radiation enteritis. We used the method recently proposed by Sandler et al. (27) to assess net collagen deposition. Briefly, relative change in MMP activity in radiation enteritis was expressed as the ratio of the fold change in MMP mRNA to TIMP mRNA expression. The fold change in collagen mRNA in radiation enteritis was divided by this relative MMP activity: fold change in collagen/(fold change in MMP/fold change in TIMP). Values thus obtained reflected a tendency toward collagen deposition relative to steady state when >1 and toward matrix degradation when <1. Example: Type III collagen is a substrate for MMP-1, -3, and -14. MMP-1 and -3 are both inhibited by TIMP-1, and MMP-14 is inhibited by TIMP-2. The fold change of COL31A1 mRNA in radiation enteritis is 3.9; the fold changes of MMP-1, -3, and -14 mRNA in radiation enteritis are, respectively, 11.7, 19.8, and 2.3; the fold changes of TIMP-1 and -2 mRNA in radiation enteritis are, respectively, 5.4 and 2.5. Relative expressions of MMP-1/TIMP-1, MMP-3/TIMP-1, and MMP-14/TIMP-2 were, respectively, 2.1, 3.6, and 0.92. Collagen type 3 alpha 1 (COL3A1)/MMP-1:TIMP-1 = 1.8, COL3A1/MMP-3:TIMP-1 = 1.08, and COL3A1/MMP-14:TIMP-2 = 4.2.

Confirmation of differential gene expression. Two micrograms of total RNA were reverse transcribed with SuperScript II reverse transcriptase (Invitrogen, Cergy Pontoise, France) using random hexamers. Primers were generated with the Primer Express software (Applied Biosystems, Courtaboeuf, France) and were purchased from Invitrogen: collagen type I alpha 2 (COL1A2), 5'-CGCGGACTTTGTTGCTGCTTG-3' (Forward); 5'-GGAAACCTTGAGGGCCTGGG-3' (Reverse); MMP-2, 5'-CGCTCAGATCCGTGGTGAG-3' (Forward); 5'-TTGTCACGTGGCGTCACAG-3' (Reverse); MMP-3, 5'-CAAGCCCAGGTGTGGAGTTC-3' (Forward); 5'-GGGTTTTGCTCCACTTCGG-3' (Reverse); MMP-14, 5'-TGGACACGGAGAATTTTGTGC-3' (Forward); 5'-ACCCCCATAAAGTTGCTGGAT-3' (Reverse); TIMP-1, 5'-CACCCACAGACGGCCTTCT-3' (Forward); 5'- CTTCTGGTGTCCGCACGAA-3' (Reverse); TIMP-2, 5'-TGACTTCATCGTGCCCTGGG-3' (Forward); 5'-CTGGACCAGTCGAAACCCTTGG-3'(Reverse). Optimized PCR used the ABI PRISM 7700 detection system in the presence of 135 nM specific forward and reverse primers for COL1A2, MMP-2 and -3, TIMP-2, and 45 nM specific forward and reverse primers for MMP-14 and TIMP-1. Both water and genomic DNA controls were included to ensure specificity. The purity of each PCR product was checked by analyzing the amplification plot and dissociation curves. Relative mRNA quantitation was performed by using the comparative {Delta}{Delta}CT method. Relative quantification in radiation enteritis = 2–{Delta}{Delta}CT, where {Delta}{Delta}CT is defined as the difference between the mean {Delta}CT(radiation enteritis) and the mean {Delta}CT(healthy bowel), and {Delta}CT, the difference between the mean CT(COL1A2, MMPs, TIMPs) and CT(18S)-18S was used as endogenous control. Each sample was monitored for fluorescent dyes, and signals were regarded as significant if the fluorescence intensity exceeded 10-fold of the standard deviation of the baseline fluorescence, defined as threshold cycles (CT). CT were selected in the line in which all samples were in logarithmic phase.

Gelatin zymography. Frozen tissue samples were crushed to powder in liquid nitrogen, homogenized in a 50 mM Tris·HCl buffer (pH 7.6), containing 150 mM NaCl, 10 mM CaCl2, 1% Triton X-100, and protease inhibitors (Sigma-Aldrich, St. Quentin Fallavier, France). Supernatants were collected, and protein concentration was determined by using the Lowry method. Gelatinase activity was assessed as follows: 1 mg/ml type A gelatin from porcine skin (Sigma-Aldrich) was copolymerized in 8% SDS-polyacrylamide gel, and was used as substrate. The samples were diluted 1:1 in sample buffer consisting of 62.5 mM Tris·HCl (pH 6.8), 10% glycerol, 2% SDS, and 0.05% bromophenol blue. Four micrograms of each protein sample were separated by electrophoresis at a constant voltage of 100 V for 1–2 h at 4°C. The gel was then washed twice in 2.5% Triton X-100 and incubated overnight at 37°C in a buffer containing 50 mM Tris·HCl (pH 7.8), 5 mM CaCl2·2H2O, 50 mM NaCl, 0.01% Brij 35, and 0.02% NaN3. Gels were stained with 0.5% Coomassie blue in 25% isopropanol and 10% acetic acid for 60 min, and destained in a mixture containing 10% methanol and 10% acetic acid until the stacking gel was destained. Bands of gelatin lysis appear as clear zones countering a blue background. Densitometric analyses were performed by using an image analyzer (Biocom, Les Ulis, France) interfaced with the Phoretix image analysis software (Nonlinear Dynamics, Newcastle upon Tyne, UK).

Immunostaining. Four-micrometer-thick acid formaldehyde alcohol or Bouin-fixed paraffin-embedded sections were used to immunolocalize MMP-2 (1:150, 42–5D11), MMP-3 (1:75, SL-1 IIIC4), MMP-9 (1:3,000, 56–2A4), MMP-14 (1:5,000, 113–5B7), TIMP-1 (1:100, 102D1), and TIMP-2 (1:1,500, 67–4H11). Antibodies were purchased from Chemicon (Euromedex, Mundolscheim, France). These antibodies were described to recognize pro- and active forms of MMP without crossreacting with other MMPs and TIMPs. After dewaxing and rehydration, endogenous peroxidase activity was eliminated with 3% hydrogen peroxide in PBS. MMP-3 and TIMP-1 epitopes were unmasked in 10 mM citrate buffer (pH 6.0). To inhibit nonspecific staining, slides were incubated 10 min at room temperature with serum-free DAKO (Trappes, France) Protein Block and incubated overnight at 4°C with the primary antibody, diluted in DAKO Antibody Diluent. Slides were then rinsed in Tris·HCl/NaCl/Tween 20 (50 mM, 0.3 M, 0.1%, respectively). The primary antibody was detected by using the EnVision+ anti-mouse horseradish peroxidase (DAKO) revealed by Vector NovaRED substrate kit (BioValley) and counterstained with IMeyer’s hemalun. Known positive cases and negative controls (omission of the primary antibody and irrelevant mouse IgG1 incubation) were included in each run and were shown to be positive and negative, respectively. A semiquantitative analysis of MMP-2, -3, -9, and -14, and TIMP-1 and 2 was performed by using the following scoring system. Intensities of staining in epithelium, lamina propria, submucosa, vessels, muscularis propria, and serosa were assigned a score, where – represents no staining; +, weak staining; ++, mild staining; and +++, strong staining. Total score was the mean value obtained for each bowel layer.

Statistical analysis. For real-time RT-PCR and zymography, statistical differences between means of control group and radiation enteritis group were evaluated by using Student's t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Histopathological study. Examination of hematoxylin and eosin-stained and Sirius Red-stained sections (Fig. 1, A–D) revealed common histological features in all radiation enteritis samples. Severe fibrosis affected the whole intestinal wall; transmural collagen accumulation was observed in the mucosa, submucosa, and muscularis propria. Real-time RT-PCR analysis showed that COL1A2 mRNA level increased in radiation enteritis (Fig. 1E).



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  		Fig. 1. Collagen staining by Sirius red in the mucosa (A) and the submucosa (B) of a healthy bowel (HB) sample and in the mucosa (C) and the submucosa (D) of a radiation enteritis sample. Gene expression of collagen type I alpha 2 chain (COL1A2), determined by real-time RT-PCR, was measured in radiation enteritis (RE) samples (n = 6) and compared with the expression in HB samples (n = 6). Values were normalized to 18S RNA and are means ± SE (*P < 0.05). UA, arbitrary unit.

 
Gene array analysis. A "normal" composite membrane, which included mRNAs expressed in all six control samples was generated and compared with membranes established for each radiation enteritis sample. Genes were selected when their expression level was altered by more than twofold compared with controls.

Levels of mRNA coding for the fibrillar collagen type I alpha 2 (COL1A2) and COL3A1 were increased by 3.8- and 5-fold in radiation enteritis (Fig. 2A; notice that collagen type I alpha 1 was not spotted on the array). Furthermore, the collagen I-to-collagen III ratio increased from 1.3 in control samples to 1.8 in radiation enteritis. The level of mRNA coding for the microfibrillar type VI alpha 3 collagen was increased by threefold, whereas mRNA coding for the alpha 1 and alpha 2 chains were, respectively, increased by 1.8 and 1.7 and were not included in the Fig. 2A, because they did not reach the cut-off value. The level of mRNA coding for the stromal component tenascin-C was also increased by threefold in radiation enteritis (Fig. 2A). These observations are consistent with the development of tensile ECM, which characterizes late radiation-induced fibrosis. Compositions of vascular and basement membrane ECM were also found to be altered in radiation enteritis. We observed two- and sixfold increases in type VIII alpha 1 collagen and Sparc mRNA levels, respectively. Collagen type IV alpha 2 (COL4A2) hybridization signal was below the background signal in healthy bowel sample, but was detectable in radiation enteritis samples (notice that COL4A3 and COL4A6 were also slightly increased but did not reach the cut-off value). Composition of basement membrane cDNA array analysis further showed that the expression level of interstitial collagenases (MMP-1 and MMP-18+19), gelatinase (MMP-2), membrane-type MMP (MMP-14), and stromelysin (MMP-3) increased by 2- to 19-fold in radiation enteritis (Fig. 2B), whereas hybridization signal of the macrophage-specific metalloelastase (MMP-12) was below the background signal in radiation enteritis but was detectable in healthy bowel samples. Both MMP-9 and -7 were not found to be differentially expressed. Levels of MMP inhibitors, TIMP-1, TIMP-2, and PAI-1 increased by five-, two-, and threefold in radiation enteritis, respectively (Fig. 2B).



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  		Fig. 2. Gene array analysis of extracellular components (A), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs), and plasminogen activator inhibitor-1 (PAI-1) genes (B) in HB samples (n = 6) and RE samples (n = 6). Hybridization intensities were obtained by using the Atlas Image 1.5 software, converted into ratios, and adjusted for background and housekeeping genes expression (Gene X intensity background)/(average intensity for all 6 housekeeping gene backgrounds). Y-axis includes name and accession no. of genes.

 
Because the balance among collagen synthesis, collagen degradation by MMPs, and inhibition of MMPs by TIMPs regulates collagen deposition, we assessed net collagen deposition in radiation enteritis samples using the method developed by Sandler et al. (27). It is assumed that no net collagen deposition or degradation occurs in control samples. Thus the collagen/MMP:TIMP value was set to 1 for controls and used as a reference. Each collagen mRNA expression value was divided by the fold change value for the relevant MMP and TIMP (Fig. 3) and values above 1 are in favor of net collagen deposition. Results thus obtained suggest that net collagen deposition occurs in radiation enteritis despite the increase in MMP mRNA. Both the Sandler method (27)and Sirius Red spectrophotometric collagen assay (36) revealed a similar trend to net collagen deposition.



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  		Fig. 3. Collagen-to-MMP-to-TIMP ratio in RE. Relative expression of MMP was calculated by determining the fold change of the MMP mRNA expression relative to its relevant inhibitor TIMP (i.e., fold change of MMP-1, -3, and -19 mRNA expression was divided by fold change TIMP-1 mRNA expression). Fold change of MMP-2 and -14 mRNA expression was divided by fold change TIMP-2 mRNA expression. The fold change for collagen mRNA was then divided by the fold change value for the relevant MMP/TIMP to yield the fold change in the ratio of collagen deposition to collagen degradation compared with the steady state.

 
Gelatinase expression. Real-time RT-PCR analysis confirmed that MMP-2 and -14 (Fig. 4I) mRNA level increased in radiation enteritis. As regards MMPs, protein levels are thought to be well correlated with mRNA expression. Thus to investigate whether mRNA levels correlated with MMP activity, gelatin zymography was performed on whole tissue extracts. We found a strong MMP-2 activity (Fig. 4II) in radiation enteritis samples. Furthermore, despite the fact that MMP-9 mRNA induction did not reach the twofold cut-off value in the cDNA array analysis, we observed an increase in MMP-9 activity in zymography experiments although statistically nonsignificant due to interindividual variability (Fig. 4II), whereas real-time RT-PCR showed a fivefold increase of MMP-9 mRNA level (Fig. 4I).



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  		Fig. 4. I: gelatinases and MMP-14 mRNA levels in RE vs. HB. Gene expression of MMP-2, -9, and -14 determined by real-time RT-PCR was measured in RE samples (n = 6) and compared with the expression in HB samples (n = 6). Values were normalized to 18S RNA and are means ± SE (*P < 0.05). II: study of gelatinase activities in RE vs. HB by zymography. Lane STD shows MMP-2 and -9 standards. Gelatin zymography showed basal MMP-2 and -9 activities in HB specimens (n = 6; lanes 1–6) and a statistically significant increase of MMP-2 activity (*P < 0.05) in RE samples (n = 6; lanes A–F), whereas increased MMP-9 activity in RE samples was not found to be statistically significant due to heterogeneity between samples (n = 6; lanes A–F).

 
Cellular localization of MMP-2, -9, and -14 was assessed by immunostaining. Representative immunohistochemical staining patterns in control and radiation enteritis samples are shown in Fig. 5. A strong increase in MMP-2 staining was found in each layer of the bowel in radiation enteritis (Fig. 5, C–F). In the mucosa, MMP-2 was detected at the apical end of epithelial cells, in {alpha}-sm actin positive subepithelial myofibroblasts, and inflammatory cells. Activated fibroblasts and leucocytes infiltrating the submucosa, as well as smooth muscle cells of the muscularis propria were also stained. Because MMP-14 is involved in the proteolytic activation of pro-MMP-2, and MMP-14 mRNA levels were found increased in radiation enteritis, we sought to determine the cell type involved in MMP-14 expression by immunolocalization (Fig. 5, J–O). A gradient of expression was observed along the crypt-villus axis: epithelial cells of the crypt were negative, whereas differentiated epithelial cells were strongly positive. MMP-14 was detected in the same cell types as those found positive for MMP-2 (i.e., subepithelial myofibroblasts, leucocytes, submucosal fibrosis myofibroblasts). Endothelial cells, however, appeared to be MMP-2 and -14 negative. An increased MMP-9 mRNA level was found in radiation enteritis, as shown by real-time RT-PCR experiments, and immunostaining provided evidence that MMP-9 protein was mainly expressed in leucocytes. MMP-9 staining was thus directly related to the degree of infiltration by inflammatory cells, which increased in radiation enteritis (Fig. 5, H–I).



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  		Fig. 5. Gelatinase A (MMP-2), gelatinase B (MMP-9), and membrane-type 1 matrix metalloproteinase (MMP-14) immunostaining in HB and RE specimens. Low MMP-2 immunostaining was seen in the mucosa (A; magnification, x400) and the submucosa (B; magnification, x400) of HB samples. In RE samples, MMP-2 immunostaining increased in mucosal epithelial and inflammatory cells (arrow) (C; magnification, x100). High magnification bright-field image (D; magnification, x400) showed MMP-2 positive staining at the apex of epithelial cells (arrow) and in subepithelial myofibroblasts (*). In the submucosa, increased MMP-2 immunostaining was found in fibrosis myofibroblasts (arrow) (E; magnification, x400) and in infiltrated leucocytes (arrow) (F; magnification, x400). Low MMP-9 immunostaining was seen in the mucosa (G; magnification, x400) and the submucosa (data not shown) of HB samples. In RE samples, MMP-9 immunostaining increased in mucosal (H; magnification, x400) and submucosal inflammatory cells (I; magnification, x400). Low MMP-14 immunostaining was seen in the mucosal inflammatory cells (J; magnification, x400) and the fibroblasts of the submucosa (K; magnification, x400) of HB samples. In RE samples, MMP-14 gradient of expression was observed along the crypt-villus axis in the epithelium (L; magnification, x400) and subepithelial myofibroblasts (arrow) were MMP-14 positive (M; magnification, x400). Submucosal activated fibroblasts (arrow) (N; magnification, x400) and infiltrated leucocytes (arrow) (O; magnification, x400) stained positively for MMP-14.

 
Stromelysin expression. In our experimental conditions (10 ng of cDNA), MMP-3 mRNA was only detected in radiation enteritis samples, thus confirming the induction of MMP-3 mRNA observed by cDNA array analysis. MMP-3 immunostaining showed very few positive cells (mostly leucocytes) in the mucosa of control samples, whereas strong positive staining was observed in the mucosa and the submucosa of radiation enteritis samples. Epithelial cells and mucosal macrophages were hugely labeled, as well as submucosal inflammatory cells, fibrosis myofibroblasts, and endothelial cells (Fig. 6).



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  		Fig. 6. Stromelysin-1 (MMP-3) immunostaining in HB and RE specimens. Low MMP-3 immunostaining was seen in the mucosa (A; magnification, x400), and no MMP-3 staining was seen in the submucosa of HB samples. In RE samples, MMP-3 immunostaining increased in epithelial cells (B; magnification, x400). High-magnification brightfield image showed MMP-3 positive staining in mucosal (C; magnification, x400) and submucosal (D; magnification x400) leucocytes. Endothelial cells (arrow) stained positively for MMP-3 (E; magnification, x400).

 
TIMP expression. Real-time RT-PCR analysis confirmed that TIMP-1 and -2 (Fig. 7, I and II) mRNA level increased in radiation enteritis. Detection of TIMP-1 was restricted to the mucosa in control and radiation enteritis samples. In control samples, however, very few TIMP-1 positive cells were found, whereas the mucosa in radiation enteritis samples was heavily stained (Fig. 7III). Control samples showed sparse staining for TIMP-2, whereas radiation enteritis samples were highly positive for TIMP-2. Transparietal inflammatory cells and fibrosis myofibroblasts of the submucosa and muscularis propria were strongly immunoreactive for TIMP-2 (Fig. 7IV). All immunohistochemical data are summarized in Table 2.



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  		Fig. 7. Tissue inhibitors of metalloproteinase TIMP-1 and -2 mRNA expression and immunolocalization in HB and RE specimens. I and III: gene expression of TIMP-1 and TIMP-2 determined by real-time RT-PCR, was measured in RE samples (n = 6) and compared with the expression in HB samples (n = 6). Values were normalized to 18S RNA and are means ± SE (*P < 0.05). II: immunolocalization of TIMP-1 in RE vs. HB. Both in normal and RE samples, TIMP-1 immunostaining was restricted to the mucosa. Low TIMP-1 immunostaining was seen in the epithelium (A; magnification, x400) of HB samples. In RE samples, TIMP-1 immunostaining increased in epithelial (B and C; magnification, x400) and inflammatory cells (D; magnification, x400). IV: immunolocalization of TIMP-2 in RE vs. HB. Low TIMP-2 immunostaining was seen in the mucosa (E; magnification, x400) and submucosa of HB samples. In RE samples, TIMP-2 immunostaining increased in leucocytes (arrow) of the mucosa (F; magnification, x400) and of the submucosa. In the submucosa, increased TIMP-2 immunostaining was found in activated fibroblasts (arrow) (G; magnification, x400).

 

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  		Table 2. Semiquantitative analysis of MMP-2, -3, -9, -14 and TIMP-1 and -2 in radiation enteritis versus healthy bowel

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To gain further insight into the biological function of various MMPs, their inhibitors, and their involvement in the excessive ECM deposition occurring in chronic radiation fibrosis after radiotherapy in humans, their expression patterns were studied by gene array analysis and immunohistochemistry. Classically, radiation fibrosis has been considered a chronic and progressive process in which normal tissue is replaced by fixed and irreversible fibrotic tissue. This view has however, been challenged, because fibrosis has recently been defined as a dynamic process resembling chronic wound healing. In this study, a marked upregulation of collagen and enzymes involved in ECM remodeling was observed in late radiation enteritis, which concurs with this new definition of radiation-induced fibrosis and is reflective of a continuous repair process.

Collagen is the predominant protein of the intestine's connective tissue. It is secreted by intestinal mesenchymal cells (subepithelial myofibroblasts, smooth muscle cells) located in the lamina propria and in the muscles (14). Fibrillar collagens (type I and III) are found in the lamina propria, the submucosa, and muscles, whereas type IV collagen is found in the basement membrane and the lamina propria (8). Colocalization of collagen deposition and myofibroblasts was demonstrated in one of our previous studies (36). In the present study, we observed a marked increase in type I, III, and IV collagen RNA transcripts in strictured ileum resected from patients with radiation enteritis. Moreover, we observed increase in the noncollagenous stromal component tenascin-C, which is produced in immature and newly formed granulation tissue and promotes migration, proliferation, and activation of matrix-producing cells, such as fibroblasts and smooth muscle cells (reviewed in Ref. 12). Induction of type I collagen, normalized for changes in the expression levels of MMPs and TIMPs, is greater than that of type III collagen. This observation is consistent with previous findings showing accumulation of type I collagen in late phases of radiation fibrosis (25). This abnormal deposition of type I/III collagen that consists of large and tensile collagen fibrils, leads to intestinal stenoses and ensuing obstructive symptomatology. Furthermore, abnormal ECM deposition may affect the ability of cells to express and maintain their differentiated phenotype. Besides having a structural role, molecules of the ECM are now known to have functional roles, such as storage of growth factors and transmission of differentiation signals to cells (1, 31). Furthermore, MMP activation may lead to the release of growth factors and cytokines from the ECM. For instance, release and activation of TGF-{beta}1 may affect the fibrogenic process (15, 39). Consequently, the nature of the cell microenvironment should not be solely seen as a consequence of tissue fibrosis, but also as a mean to ensure cellular activation responsible for the maintenance of the fibrotic process.

Collagen accumulation in radiation fibrosis has been thought to be associated with a decrease in MMP activity and increased TIMP levels. Our results show an induction of each member of the MMP family, i.e., gelatinases, stromelysin, collagenases, and membrane-type MMPs, in late radiation enteritis. The concomitant induction of MMP inhibitors (TIMP-1, TIMP-2, and PAI-1) counterbalances this induction of MMPs, leading to a net collagen deposition. Because MMPs act locally, studies on whole-tissue lysates provide only partial information about ECM remodeling in late radiation enteritis. We therefore performed immunohistochemical studies on 22 radiation enteritis samples to investigate regulation of ECM metabolism in each bowel layer.

Intense ECM remodeling seems to particularly affect intestinal mucosa in radiation enteritis. We report a coordinated expression of MMP-2, -3, -9, and -14, proportional to the extent of inflammatory cell infiltration. Molecular characterization of inflammatory cell subtypes involved in MMP overexpression in radiation enteritis has not been done so far. However, it seems reasonable to assume that collagen synthesis in mesenchymal cells is stimulated by secretory products of inflammatory cells (14) and that MMP/TIMP secretion may be partly achieved by leucocytes themselves (13, 29). IL-1{beta} can be cited among the inflammatory mediators that control MMP synthesis (21). In a previous study (35), IL-1{beta} mRNA was found to be markedly increased in radiation enteritis samples, suggesting that it may be involved in ECM remodeling in the late phase of radiation fibrosis. Monitoring the T-helper 2 response in radiation enteritis may also be relevant, because T-helper 2 response has been shown to be involved in pneumonitis and subsequent pulmonary fibrosis after thoracic radiotherapy (37) and is known to control ECM remodeling in pathogen-induced pulmonary inflammation (27). Upregulation and colocalization of MMP-2 and -14 in activated mesenchymal cells has already been described in various wound healing models in skin (20), liver (32), and gut (34). Furthermore, in accordance with our findings, increased MMP-2 expression in human subepithelial myofibroblasts within irradiated rectum (9) and derived from IBD biopsies (16) has been demonstrated. Contrary to Hovdenak's findings (9), we report strong MMP-2 and -14 staining in epithelial cells with a crypt-villus gradient of expression for MMP-14. The difference between our findings and Hovdenak's may account for the difference in the anatomical location of the biopsy (ileum vs. colon) and the timing at which resection was performed (several months vs. 2 wk after radiation therapy). Furthermore, colocalization of MMP-3 and -14 has already been described (22) in activated fibroblast-like cells from IBD biopsies, whereas in radiation enteritis samples, MMP-3 and -14 were found to be expressed in intestinal epithelial cells and inflammatory cells in the lamina propria. This increased expression of MMP-2, -3, and -14 provides new insight into the differentiation of intestinal epithelial cells in radiation enteritis. Increased MMP-2 and 3 expression has already been described in migrating keratinocytes during cutaneous wound healing and in monostratified epithelia, increased MMP-2 expression has been reported in lung (41) and mouse colon (23). Furthermore, MMP-2 and -14 are known to process laminin and to regulate migration of lung carcinoma-derived epithelial cells (A549) (24). The present observations suggest that remodeling of the basement membrane occurs in late radiation enteritis and could lead to epithelial anoïkis and chronic ulceration. In addition, production of MMPs by epithelial cells may directly affect differentiation and proliferation of the subepithelial myofibroblasts into fibrosis-activated cells. The significance of MMP-14 gradient of expression along the crypt-villus axis is unknown, but increased MMP-2 activity could be required for accelerated migration of enterocytes toward the top of the villi and to desquamation of differentiated enterocytes in radiation enteritis. In conclusion, we propose that MMP-2, -3, and -14 could be useful markers of epithelium activation and could mediate fibrogenic activation signals from epithelial cells to subepithelial myofibroblasts, as recently described by Xu et al. (41) in pulmonary fibrosis.

Although the most dramatic intestinal collagen deposition occurs in the submucosa and muscles, submucosal and muscular ECM remodeling during intestinal fibrosis has been poorly investigated. In accordance with findings from Matthes and Graham on Crohn's disease (14), in vitro studies performed in our laboratory (17) demonstrated that activated smooth muscle cells derived from the muscularis propria of the bowel of patients with radiation enteritis secretes twofold more type I collagen than their normal counterparts. The present study brings some new insight concerning ECM remodeling in deep intestinal layers, because it shows an increased MMP-2, -3, and -14 staining in fibrosis myofibroblasts within the submucosa and the muscles. ECM turnover depends on the MMPs-to-TIMPs ratio. In this study, mRNA analysis showed that TIMP mRNA levels were higher than the MMPs mRNA level in radiation enteritis, which resulted in inhibition of degradation. Immunohistochemistry brought additional clues to the understanding of the ECM remodeling balance in radiation enteritis, because it enabled us to observe an increased TIMP-1 and -2 staining in the mucosa, whereas only TIMP-2 staining was observed in the deeper layers. Classically, the balance between MMPs and TIMPs has been thought to regulate proteolytic activities; however, in addition to its inhibitory activity, TIMP-2 can associate with pro-MMP-2 and activate its proteolytic activity (7). Furthermore, TIMPs can play additional functional roles, such as that of anti-apoptotic factors that may be particularly relevant in a fibrotic context. TIMP-2 protects melanoma cells from apoptosis (33), and recently, TIMP-1 has been shown to inhibit apoptosis in activated hepatic stellate cells in established liver fibrosis (18). In this particular model, the anti-apoptotic effect of TIMP-1 was dependent on the prevention of matrix degradation through inhibition of MMPs. In normal conditions, resorption of granulation tissue occurs through apoptosis of myofibroblasts (6), whereas in fibrosis, persistence of myofibroblasts leads to organ failure. The observed TIMP-1 staining in subepithelial myofibroblasts and TIMP-2 staining in submucosal fibrosis myofibroblasts suggest that TIMP-1 and -2 may mediate persistence of myofibroblastic differentiation in late radiation enteritis.

In conclusion, despite the difficulty in assessing the ECM remodeling process in vivo, these observations nonetheless provide the first evidence of active ECM remodeling in late radiation enteritis. These findings further reinforce the concept that fibrotic tissue is dynamic and undergoes constant renewal, thus opening interesting perspectives for the development of antifibrotic interventional therapies.


   ACKNOWLEDGMENTS
 
We thank Dr. J. Aigueperse (Direction de la Radioprotection de l’Homme, Institut de Radioprotection et de Sûreté Nucléaire, 92265 Fontenay-aux-Roses, France), Dr. J. C. Sabourin (Pathology Department; Institut Gustave Roussy, 94805 Villejuif, France), and Dr. P. Lasser (Surgery Department, Institut Gustave Roussy, 94805 Villejuif, France) for support and scientific advice.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. C. Vozenin-Brotons. Laboratoire UPRES EA 27-10 "Radiosensibilité des tumeurs et tissus sains," PR1, 39, rue Camille Desmoulins, 94805 Villejuif CEDEX (E-mail: vozenin{at}igr.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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