Insulin-like growth factor I and insulin-like growth factor binding protein 5 in Crohn's disease

E. M. Zimmermann1, L. Li1, Y. T. Hou1, N. K. Mohapatra2, and J. B. Pucilowska2

1 Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109-0589; and 2 Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, North Carolina 27599


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Insulin-like growth factor (IGF)-I and its binding protein IGF binding protein 5 (IGFBP-5) were highly expressed in inflamed and fibrotic intestine in experimental Crohn's disease. IGF-I induced proliferation and increased collagen synthesis by smooth muscle cells and fibroblasts/myofibroblasts in vitro. Here we studied IGF-I and IGFBP-5 in Crohn's disease tissue. Tissue was collected from patients undergoing intestinal resection for Crohn's disease. IGF-I and IGFBP-5 mRNAs were quantitated by RNase protection assay and Northern blot analysis, respectively. In situ hybridization was performed to localize mRNA expression, and Western immunoblot was performed to quantitate protein expression. IGF-I and IGFBP-5 mRNAs were increased in inflamed/fibrotic intestine compared with normal-appearing intestine. IGF-I mRNA was expressed in multiple cell types in the lamina propria and fibroblast-like cells of the submucosa and muscularis externa. IGFBP-5 mRNA was highly expressed in smooth muscle of the muscularis mucosae and muscularis externa as well as fibroblast-like cells throughout the bowel wall. Tissue IGFBP-5 protein correlated with collagen type I (r = 0.82). These findings are consistent with a mechanism whereby IGF-I acts on smooth muscle and fibroblasts/myofibroblasts to increase collagen synthesis and cellular proliferation; its effects may be modulated by locally expressed IGFBP-5.

inflammatory bowel disease; fibrosis; smooth muscle cells; inflammation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INSULIN-LIKE GROWTH FACTOR (IGF)-I is a mitogen for a variety of cell types and promotes cellular differentiation in many tissues, including the intestine (17, 23). IGF-I has potent fibrogenic actions. For example, IGF-I induced proliferation of fibroblasts, myofibroblasts, and smooth muscle cells and increased collagen synthesis by those cells (17, 18, 36). In the peptidoglycan-polysaccharide (PG-PS)-induced rat model of Crohn's disease, IGF-I mRNA was increased in inflamed intestinal tissue and was highly expressed in cells in regions of intense fibrosis (37). Similarly, in a model of colitis induced by intercolonic instillation of trinitrobenzene- sulfonic acid, IGF-I expression was increased in intestinal mucosa and in smooth muscle, especially in areas of transmural inflammation and smooth muscle disorganization (31). These data are consistent with a role for IGF-I in promoting tissue fibrosis in the setting of intestinal inflammation.

IGF-I can bind to a family of secreted proteins known as IGF binding proteins (IGFBPs) (16, 27). IGFBPs have conserved structures that are distinct from the IGF receptor. Circulating binding proteins act as carriers that prolong the plasma half-life of IGF-I and limit IGF-I's insulin-like endocrine actions (16). The IGFBPs are expressed in a wide range of peripheral tissues. Unique patterns of expression suggest important roles during growth and development, during the estrous cycle, during bone growth and remodeling, and during alterations in nutritional status (16). IGFBPs can enhance or inhibit IGF actions on target cells depending on the binding protein and system under study (3, 16). IGF-independent actions of IGFBPs have also been documented (7, 27, 30).

One of the IGFBPs, IGFBP-5, may be important in the pathogenesis of intestinal fibrosis in inflammatory bowel disease (IBD). In the PG-PS rat model of IBD, IGFBP-5 mRNA was highly expressed in intestinal smooth muscle cells. In vitro, IGFBP-5 enhanced the mitogenic actions of IGF-I on cultured fibroblasts and smooth muscle cells by associating with extracellular matrix (ECM) near the target cell (5, 16, 34, 29). ECM-associated IGFBP-5 may act by accumulating IGF-I near its receptor by modulating the interaction of IGF-I with its receptor or by protecting it from proteolysis (17). In cultured intestinal smooth muscle cells, IGF-I stimulated IGFBP-5 and collagen synthesis (19, 36). Our hypothesis is that IGF-I is induced during the process of chronic intestinal inflammation and acts on intestinal smooth muscle to stimulate proliferation and to increase collagen and IGFBP-5 synthesis. IGFBP-5, in turn, is secreted from smooth muscle cells, associates with the ECM, and enhances the actions of IGF-I on those cells, thereby acting in a positive feedback loop. The result is increased smooth muscle layer thickness and increased tissue collagen deposition, leading to luminal narrowing and loss of intestinal wall compliance.

The aim of this study was to quantitate IGF-I and IGFBP-5 mRNA expression in inflamed and strictured regions of surgically resected specimens from Crohn's disease patients and to compare expression with that in normal-appearing tissue from the same resected specimen. Consistent with our hypothesis, we found that IGF-I and IGFBP-5 mRNAs were increased in inflamed and strictured segments of tissue compared with normal-appearing tissue. IGF-I mRNA was most highly expressed in fibroblast-like cells in the submucosa and muscularis externa. IGFBP-5 mRNA was expressed primarily in smooth muscle of the muscularis mucosae and muscularis externa, as well as in fibroblast-like cells in the lamina propria and submucosa.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue collection. All tissues were collected at the time of intestinal resection for Crohn's disease. Use of excess surgical tissue was approved by the Institutional Review Board at the University of Michigan and obtained through the Tissue Procurement Core of the University of Michigan Comprehensive Cancer Center. The tissue was visually inspected, and a portion of tissue that was visually normal or less inflamed and a portion of inflamed or strictured intestine were obtained from the same specimen. An ~2-cm2 piece of tissue was removed from each region. A small corner of each piece was fixed in formalin and processed for routine histology. The remaining tissues were processed for RNA extraction or in situ hybridization. Small intestine was studied in all patients; colon was also available in two of the patients. Tissues for Northern blot analysis and RNase protection assay were snap frozen in liquid nitrogen and stored at -80°C until processed for RNA extraction. Tissues for in situ hybridization were oriented in Tissue-Tek optimal cutting temperature compound (Miles, Elkhart, IN) and frozen in isopentane at -50°C. Histology was determined on hematoxylin and eosin- and trichrome-stained sections and graded in a blinded fashion from 0 to 2 for normal histology, mild abnormality, or severe abnormality, respectively, in each of four categories: surface epithelial damage, lamina propria inflammation, thickness of the muscularis externa, and fibrosis. All tissue labeled "normal" or "less inflamed" had histological scores of <= 4; all tissues labeled "inflamed/fibrotic" had histological scores of >= 6.

RNA extraction. Total RNA was extracted from 1 g of human intestine by the guanidine isothiocyanate-cesium chloride method and enriched for poly(A+) RNA by oligo(dt) cellulose chromatography (1, 37). RNA concentrations of the final preparations were calculated on the basis of optical absorbency at 260 nm. Aliquots of the RNA preparations were analyzed on 0.8% agarose gels (GIBCO BRL, Gaithersburg, MD) to ensure integrity before further analysis.

Protein extraction. Total protein was extracted from small pieces of human intestine by using radioimmunoprecipitation buffer (RIPA) containing 20 mM Tris, 0.16 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 µg/ml leupeptin, 0.1 trypsin inhibitory units aprotinin/ml, and 1 mM Na3VO4 (pH 7.2) (21). Tissues were sonicated for 20 s, and RIPA-insoluble components were removed by centrifugation for 10 min at 14,000 rpm. A portion of the sample was reserved for protein quantitation by DC protein assay (Bio-Rad, San Francisco, CA).

Northern blot analysis. The human IGFBP-5 cDNA was isolated from the pHBP5-501/pBluescript SK+ plasmid (kindly provided by Shunichi Shimasaki, Scripps Research Institute, La Jolla, CA) by using appropriate restriction enzymes. The cDNA was purified from a 1% agarose gel by using QIAquick gel extraction kit (Qiagen, Santa Clarita, CA) and was radiolabeled with [32P]dCTP (Amersham, Arlington Heights, IL) by using a random priming kit (Boehringer Mannheim, Indianapolis, IN). RNA was electrophoresed on 1% agarose gels (GIBCO BRL) with 6% formaldehyde (Sigma Chemical, St. Louis, MO). The gels were soaked in H2O to decrease the formaldehyde concentration, stained with ethidium bromide for 30 min, and then destained for 3 h. The presence of sharp bands corresponding to the 18S and 28S ribosomal RNAs was confirmed by ultraviolet illumination. RNA was transferred overnight to Nytran (Schleicher & Schuell, Keene, NH) by using capillary action, and the blots were baked at 80°C. Blots were prehybridized for 3 h and then hybridized overnight in solution containing 50% formamide, 5× standard sodium citrate (SSC), 150 µg/ml salmon sperm DNA, 32P-labeled cDNA probe, and a buffer containing 250 mM Tris (pH 7.5), 0.5% SDS, 1% polyvinylpyrrolidone, 1% Ficoll, 25 mM EDTA, and 1% BSA. Membranes were washed in increasingly stringent SSC washes, with the most stringent being 0.5× SSC and 0.1% SDS at 45°C for 30 min. Membranes were exposed to radiographic film overnight at -80°C with intensifying screens. The autoradiogram was digitized by flatbed scanning and was imported for densitometric analysis into NIH Image (National Institutes of Health, Bethesda, MD). The relative densitometric value for each band was adjusted for minor variations in loading by using the corresponding signal for blots probed with a 32P-labeled cDNA probe for glyceraldehyde-3-phosphate dehydrogenase [American Type Culture Collection (ATCC), Rockville, MD].

Immunoblot analysis. Proteins (50 µg/lane for bowel samples) were electrophoresed on 12% polyacrylamide gels (for IGFBP-5) or 8% polyacrylamide gels (for collagen type I) with 0.1% SDS, then transferred to nitrocellulose (21, 36) (0.2 µm; Schleicher & Schuell). Blots were incubated for 3 h with primary antibody: rabbit anti-human IGFBP-5 (1:500 dilution; Upstate Biotechnology, Lake Placid, NY) or rabbit anti-collagen type I (1:2,000 dilution; Rockland Immunochemicals, Gilbertsville, PA). Blots were washed and then exposed to the secondary antibody: goat anti-rabbit IgG (1:10,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h. Blots were washed, and specific antibody was detected by an enhanced chemiluminescence detection system (Amersham) and exposed to X-ray film for 1-5 min. Human type I collagen purified from human placenta (0.5 µg/lane; Rockland Immunochemicals) was used as a positive control in collagen immunoblot studies. Molecular weights of proteins were estimated by comparison with prestained molecular weight standards (Amersham). The autoradiogram was digitized by flatbed scanning and was imported for densitometric analysis into NIH Image.

To validate quantitation of collagen type I using the Western immunoblot technique, rat intestinal tissue was studied by using immunoblot analysis as described above and a standard hydroxyproline technique (20). Frozen samples of intestine from rats with PG-PS-induced chronic enterocolitis, an experimental model of Crohn's disease that induces marked chronic inflammation with prominent fibrosis (36), were divided and studied in a blinded fashion. The hydroxyproline assay was performed as previously described (20). Briefly, 100- to 120-mg wet wt sections of intestine (ileum or cecum) were homogenized in 0.5 M acetic acid and dried in a SpeedVac for determination of dry weight. The dried tissue was then suspended in 6 N HCl and hydrolyzed at 120°C for 7.5 h. Triplicate 25-µl and 50-µl portions of each hydrolysate were dried in the SpeedVac, resuspended in citrate-acetate buffer (pH 6.0), and oxidized by chloramine T. After addition of p-dimethylaminobenzaldehyde, the absorbance at 550 nm was measured. The hyrdoxyproline content was calculated from a standard curve of purified hydroxy-L-proline (Sigma). Western immunoblot autoradiograms were digitized and analyzed by densitometry as above. There was a significant correlation between collagen measured by the two techniques (µg hydroxyproline/mg tissue vs. densitometric value/mg protein; n = 8; r = 0.7; P = 0.025).

In situ hybridization. The plasmid containing the human IGFBP-5 cDNA was linearized using appropriate restriction enzymes. Sense and antisense 35S-labeled probes were generated by using the T3 and T7 DNA-dependent RNA polymerases (GIBCO BRL), respectively, in a standard in vitro transcription protocol (36).

In situ hybridization was performed on 10-µm-thick sections of human intestine by using methods previously described (37). Briefly, sections were fixed in 4% neutral buffered formaldehyde for 30 min, washed in 0.1 M PBS, and treated with proteinase K (1 µg/µl) for 10 min. Slides were exposed to triethyl ammonium and acetic anhydride (Sigma) for 10 min, washed, and dehydrated through graded alcohols. Sections were exposed to standard hybridization buffer containing 75% formamide (GIBCO BRL) and 1-2 × 106 cpm radiolabeled probe for 18 h at 55°C. After hybridization, coverslips were removed and the slides were treated with RNase A (200 µg/ml; Sigma) for 30 min, then washed in increasingly stringent SSC buffers, the most stringent being 0.5× SSC for 1 h at 55°C. Slides were dehydrated and exposed to X-ray film for 24 h, then dipped in radiographic emulsion and maintained at 4°C for 2 wk. Slides were developed and then viewed and photographed under light- and darkfield illumination using a Zeiss Axiophot microscope (Carl Zeiss, Thornwood, NJ). Negative controls performed included slides exposed to RNase A for 60 min before hybridization with an antisense probe and slides hybridized with a sense probe. Background labeling was observed in all negative control sections and was defined as a density of exposed silver grains over an area of the slide with no tissue section that was nearly indistinguishable from the density of exposed grains over the tissue section (36). In situ hybridization was considered qualitative.

RNase protection assay. A Pst I-BamH I fragment (420 bp) of human IGF-I cDNA (hIGF-I; ATCC) was subcloned into pBluescript SK (Stratagene, La Jolla, CA). The plasmid was linearized with BsaH I (New England Biolabs, Beverly, MA) and transcribed using the T3 RNA polymerase and [32P]UTP to generate a 300-bp antisense RNA probe. The resultant probe was purified after electrophoresis on a 5% acrylamide gel containing 8 M urea. This probe consisted of 247 nt complementary to IGF-I Ea mRNA (144 nt from exon 4 and 103 nt from exon 6) and 53 nt derived from the vector. An antisense riboprobe specific for human cyclophilin, used to equate loading among lanes, was generated from pTRI-cyclophilin (Ambion, Austin, TX) at a full length of 165 nt. The size of the protected band (103 nt) was estimated from the 32P-labeled DNA marker [pBR 322 Msp I digest (New England Biolabs, Beverly MA)].

The RPA II kit (Ambion) was used according to the manufacturer's instructions. Briefly, 10 µg of poly(A+) RNA was hybridized with 250,000 cpm of 32P-labeled hIGF-1 and cyclophilin cRNA probes at 45°C overnight in 20 µl of buffer (80% formamide, 100 mM sodium citrate, pH 6.4, 300 mM sodium acetate, pH 6.4, 1 mM EDTA). Samples were digested with a mixture of RNase A (5 U/ml) and RNase T1 (100 U/ml) for 45 min at 37°C. After inactivation of the RNases, the RNA was pelleted and resuspended in loading buffer containing 80% formamide and then electrophoresed on 5% polyacrylamide gels containing 8 M urea. The gel was dried and subjected to autoradiography. The autoradiogram was digitized by flatbed scanning and was imported for densitometric analysis into NIH Image. The relative densitometric value for each IGF-I band was adjusted for minor variations in loading by using the corresponding signal for cyclophilin.

Statistical analysis. Bands from autoradiograms were digitized by flatbed scanning and quantitated by densitometry by using NIH Image software. Densitometric values for each sample were normalized to control for minor variations in sample loading as described above. Samples were handled as paired samples throughout analysis. mRNA or protein abundance was expressed as percent abundance in normal-appearing tissue. Comparisons were made between groups of paired samples by the Wilcoxon's signed rank test. Correlation between variables was calculated using the Pearson correlation coefficient, and a P value for the correlation was determined by the usual t-distribution-based test. Data were considered significant if P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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IGF-I mRNA was increased in inflamed intestine compared with normal-appearing tissue from the same patient, as determined by RNase protection assay (Fig. 1; 4.4 ± 0.5-fold; n = 5 pairs; P = 0.04). Northern blot analysis was used to determine the abundance of IGFBP-5 mRNA. There was a 3.6 ± 0.7-fold increase in IGFBP-5 mRNA in inflamed intestine compared with normal-appearing tissue (Fig. 2; n = 10 pairs; P = 0.005). IGFBP-5 protein (31 kDa) was increased in inflamed intestine compared with normal-appearing intestine by Western immunoblot (Fig. 3A). A smaller 25-kDa band, possibly representing an IGFBP-5 proteolytic fragment, was observed only in samples from inflamed intestine (Fig. 3A; see DISCUSSION). As expected, collagen type I appeared as a strong band at ~110 kDa (Fig. 3A) and as a faint band at 95 kDa in the bowel samples and in the positive control (human collagen type I purified from human placenta). Collagen type I was increased in inflamed intestine compared with normal-appearing tissue, and the abundance of tissue collagen type I correlated with IGFBP-5 levels (Fig. 3; n = 12; r = 0.82; P = 0.001).


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Fig. 1.   Insulin-like growth factor (IGF)-I mRNA is increased in inflamed intestine as determined by RNase protection assay. RNA was hybridized with radiolabeled RNA probes specific for human IGF-I and cyclophilin. After digestion with RNase, protected fragments were separated on 5% polyacrylamide/8 M urea gels. IGF-I mRNA was increased in inflamed/strictured intestine compared with normal-appearing tissue (4.4 ± 0.5-fold ; n = 5 pairs; P = 0.04). Representative gel is shown in A; quantitation of gels is shown in B. Lane 1, mol wt marker; lane 2, probes alone; lane 3, probes + RNase; lane 4, liver RNA (5 µg; positive control); lanes 5, 7, and 9, RNA from normal-appearing intestine from 3 Crohn's patients; lanes 6, 8, and 10, RNA from inflamed intestine from the same 3 Crohn's patients. The error bar in B represents SE of the mean increase in IGF-I mRNA abundance in inflamed/fibrotic intestine compared with controls. Exposure time was 16 h. *Significant difference from "normal" (P < 0.05).



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Fig. 2.   IGF binding protein (IGFBP)-5 mRNA is increased in inflamed intestine as determined by Northern blot analysis. Northern blot analysis of IGFBP-5 mRNA in intestine from 4 different patients is shown. Twenty micrograms of total RNA in each lane were used from paired samples of normal-appearing and inflamed intestine. After electrophoresis and transfer, the blot was hybridized with 32P-labeled probes for human IGFBP-5 and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Messages for human IGFBP-5 and GAPDH were 6.0 and 1.2 kb, respectively. IGFBP-5 mRNA was increased in inflamed/strictured intestine compared with normal-appearing tissue from the same patient (3.6 ± 0.7-fold ; n = 10 pairs; P = 0.005). Representative gel is shown in A; quantitation of gels is shown in B. The error bar in B represents SE of the mean increase in IGFBP-5 mRNA abundance in inflamed/fibrotic intestine compared with controls. Exposure time was 16 h. *Significant difference from "normal" (P < 0.05).



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Fig. 3.   IGFBP-5 and collagen type I are increased in inflamed intestine as determined by Western immunoblot. Protein was extracted from paired samples of normal-appearing and inflamed intestine from the same patient. Samples were size separated on polyacrylamide gels using SDS and transferred to nitrocellulose. Immunoblot procedure was performed as described in METHODS by using primary antibodies for IGFBP-5 or collagen type I. Representative gels are shown in A, and correlation between densitometric values for IGFBP-5 and collagen type I in the same sample are shown in B.

In situ hybridization using IGF-I-specific riboprobes was performed on paired samples from six patients with Crohn's disease. In the mucosa, hybridization was observed in lamina propria; no signal was observed in epithelial cells. Most normal-appearing tissue demonstrated a barely detectable or weak diffuse signal in multiple cells within the mucosa and submucosa (Fig. 4, A and B) and a slightly stronger hybridization in matched inflamed/fibrotic tissue (Fig. 4, C and D). Some cells expressing IGF-I mRNA in the lamina propria appeared to be immune cells with round nuclei and scant cytoplasm, whereas others were fibroblast-like mesenchymal cells with spindle-shaped nuclei and irregular cytoplasm. In the submucosa, cells expressing IGF-I mRNA were fibroblast-like. There was significant variability in level of intensity of IGF-I mRNA expression in the lamina propria and submucosa between patients.


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Fig. 4.   In situ hybridization localizing IGF-I mRNA expression. In situ hybridization was performed on frozen sections of normal-appearing and inflamed intestine from patients with Crohn's disease. Representative sections are shown. Lightfield and corresponding darkfield photomicrographs are shown using the antisense riboprobe: A and B: section from normal-appearing intestine. Mucosa is indicated by {. C and D: section from inflamed/strictured intestine from the same patient. Mucosa is indicated by {, and submucosa is indicated by [. E and F: section of muscularis externa (ME) from normal-appearing intestine. MEL, longitudinal muscle layer; MEC, circular muscle layer. G and H: section of thickened muscularis externa from inflamed/strictured intestine. I and J: section of muscularis externa from inflamed/strictured intestine. S, serosa. Sections were exposed to emulsion for 10 days. Magnification, ×10 on film.

The strongest signal with the IGF-I probe was seen in fibroblast-like mesenchymal cells in the muscularis externa of both normal-appearing and inflamed/strictured bowel. In normal-appearing bowel, there was expression of IGF-I mRNA in the muscularis externa, especially at the margins of the circular and longitudinal smooth muscle layers (Fig. 4, E and F) and in septae traversing the smooth muscle layers. In inflamed/fibrotic tissue, strong hybridization was observed in the muscularis externa and adjacent submucosa and serosa in bands surrounding disrupted smooth muscle (Fig. 4, G-J). IGF-I was also expressed in smooth muscle cells of the inflamed/fibrotic tissue, but the signal was not as strong as that seen in the septae. As seen in the rat IBD model (40), IGF-I mRNA was not detectable in inflammatory cells within granulomas (not shown). Sections of tissue from Crohn's disease patients hybridized with the sense IGF-I probe demonstrated only background labeling.

In situ hybridization with the antisense riboprobe specific for IGFBP-5 was also performed on paired samples of normal-appearing and inflamed/strictured intestine. The IGFBP-5 signal was intense in the muscularis mucosae of normal-appearing and inflamed intestine, although in some sections the muscularis mucosae was not well seen, possibly due to disruption by inflammatory cells (Fig. 5, A and B). In inflamed tissue, there was strong hybridization in fibroblast-like cells in the lamina propria and submucosa (Fig. 5, C and D). In addition, there was IGFBP-5 mRNA expression in blood vessel walls within the submucosa (Fig. 5, C and D). There was no clear-cut expression of IGFBP-5 mRNA in the epithelium, although in some sections there appeared to be expression in the crypt epithelium (Fig. 5, C and D). This is a common site for nonspecific hybridization and was likely an artifact.


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Fig. 5.   In situ hybridization localizing IGFBP-5 mRNA expression. In situ hybridization was performed on frozen sections of normal-appearing and inflamed/strictured intestine from patients with Crohn's disease. Lightfield and corresponding darkfield photomicrographs are shown by using the antisense and sense IGFBP-5 riboprobes. A and B: section from normal-appearing intestine. Mucosa is indicated by {; arrow points to muscularis mucosae. C and D: section from inflamed/strictured intestine. Mucosa is indicated by {; arrow indicates expression in submucosal blood vessels. E and F: section of muscularis externa (ME) from normal-appearing intestine. MEL, longitudinal muscle layer; MEC, circular muscle layer. Arrow indicates myenteric plexus. G and H: section of muscularis externa from inflamed/strictured intestine. I and J: section of inflamed/strictured intestine hybridized with the sense probe demonstrating background labeling. Mucosa is indicated by {. Sections were exposed to emulsion for 10 days. Magnification, ×10 on film.

The bowel region with strongest IGFBP-5 signal was the muscularis externa (Fig. 5, E-H). Expression was observed in smooth muscle cells of both the circular and longitudinal muscle layers. A less intense signal was observed in unidentified cells within the myenteric plexus in some samples (see Fig. 5E). The hybridization signal was much more intense in muscularis externa of inflamed/fibrotic intestine compared with normal-appearing intestine (Fig. 5, G and H). Sections of tissue from Crohn's disease patients hybridized with the sense IGFBP-5 probe demonstrated only background labeling (Fig. 5, I and J).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Patients with Crohn's disease develop intestinal fibrosis that often leads to luminal narrowing and loss of wall compliance and contributes to symptoms of abdominal pain and diarrhea. Despite new and effective therapies to treat intestinal inflammation, intestinal fibrosis often progresses to critical luminal narrowing, necessitating surgical resection. Morphological studies of strictured ileum from patients with Crohn's disease suggest that smooth muscle cells are major sites of collagen synthesis. Fibroblasts and myofibroblasts also synthesize collagen and other ECM components (4, 11). Regulation of collagen synthesis in inflamed intestine is poorly understood. Several fibrogenic growth factors, including transforming growth factor-beta (TGF-beta ) and IGF-I, have been shown to stimulate collagen synthesis by intestinal cells. In human intestinal smooth muscle cells, TGF-beta caused a 58% increase in collagen synthesis over noncollagen protein production without a mitogenic effect (10). TGF-beta increased collagen type III synthesis by human fibroblasts obtained from strictured intestines from Crohn's disease patients (28). IGF-I increased collagen synthesis and synthesis of IGFBP-5 by rat intestinal smooth muscle cells (36). Interestingly, TGF-beta enhanced the effects of IGF-I on cell proliferation and differentiation in other systems (2, 9). Therefore, it is tempting to speculate that IGF-I and TGF-beta act synergistically to stimulate collagen synthesis by intestinal cells.

Our study showed that IGF-I and IGFBP-5 mRNAs were increased in inflamed intestine compared with normal-appearing tissue from Crohn's disease patients. IGF-I mRNA was expressed in multiple cell types in the mucosa, submucosa, and muscularis externa, consistent with widespread low-level expression previously observed in normal tissue (32). The strongest IGF-I signal was seen in fibroblast-like mesenchymal cells in septae between smooth muscle cells in the muscularis externa and in adjacent submucosa and serosa. In contrast, the strongest IGFBP-5 signal was observed in smooth muscle cells of the muscularis mucosae and muscularis externae. Findings there are consistent with the sites of IGF-I and IGFBP-5 expression in the PG-PS rat model of Crohn's disease (37) and are consistent with a paracrine interaction between cells expressing IGF-I and IGFBP-5.

In vitro, IGF-I increases IGFBP-5 and collagen synthesis by intestinal smooth muscle cells. Emerging data suggest that IGFBP-5 that is secreted by fibroblasts and smooth muscle cells associates with the ECM and enhances the fibrogenic actions of IGF-I, possibly by accumulating IGF-I near its target cell (5, 16, 34). The observation that IGFBP-5 enhances IGF-I actions is a well-recognized in vitro phenomenon, but in vivo evidence to support this hypothesis is limited (17). The localization of IGF-I and IGFBP-5 in adjacent cell populations in and around thickened and fibrotic smooth muscle is strong in vivo support for in vitro observations.

In many in vitro systems, IGFBP-5 is rapidly degraded by specific proteases, and IGFBP-5 is detected in conditioned media lacking protease inhibitors as a 21-kDa proteolytic fragment instead of the 31-kDa intact protein (13, 25, 35). It is postulated that proteolysis may be one mechanism controlling IGFBP-5 abundance and, therefore, an important factor in modulating IGF-I actions. In cultured intestinal smooth muscle, IGFBP-5 was detected in high quantities in conditioned media as the intact protein without evidence of proteolysis, even in the absence of protease inhibitors (36). In this study of human tissue, IGFBP-5 appeared as an intact 31-kDa protein in all tissue; a second faint 25-kDa band was seen in some inflamed bowel samples, likely representing a product of the specific IGFBP-5 protease. This may indicate active IGFBP-5 proteolysis in inflamed tissue but not in the absence of inflammation. This awaits further study.

IGF-I is mitogenic for many cell types, including intestinal smooth muscle (16, 18). IGF-I functions as a progression factor and is a key regulator mediating progression of cells through the cell cycle (16). Quiescent cells in G0 progress to G1 in the presence of competence factors that include growth factors such as platelet-derived growth factor and basic fibroblast growth factor. Treatment of cells with a competence factor and IGF-I allows progression through G1 and continuation through the cell cycle, resulting in DNA synthesis and cell proliferation. In vitro, IGF-I stimulated proliferation of human intestinal smooth muscle (18). In vivo, exogenous IGF-I increases thickness of the muscularis externa in rat intestine (33). IGF-I induced in the course of chronic inflammation may contribute to smooth muscle proliferation, leading to luminal narrowing. Our study showed that IGF-I was expressed widely in inflamed tissue including in smooth muscle cells and fibroblast-like cells near smooth muscle cells. IGF-I may act in an autocrine and/or paracrine manner to increase proliferation of intestinal smooth muscle cells.

IGF-I has potent effects on promoting cellular differentiation. IGF-I increased expression of myf-5 and myogenin, transcription factors important in the differentiation of fibroblasts to myoblasts (8, 24). In vitro, IGF-I stimulated differentiation of myoblasts, and the effect was modulated by IGFBP-5 (6, 14). In addition, IGF-I receptor knockout mice had impaired muscle development and died after birth (22). Evidence suggests that in inflamed intestine contractile smooth muscle cells proliferate and begin synthesizing collagen. In addition, colonic subepithelial fibroblasts differentiate into myofibroblasts and synthesize collagen (15). Processes that mediate the switch from "resting" cells to proliferating, collagen-synthesizing cells are unclear. Similar mechanisms likely mediate changes that occur in hepatic stellate cells (Ito cells) during the development of cirrhosis (12) and changes in pulmonary fibroblasts/myofibroblasts in the pathogenesis of pulmonary fibrosis (26). The factors that regulate these processes are likely key to the development of fibrosis in many fibrotic diseases, including Crohn's disease. IGF-I may promote fibrosis in Crohn's disease by modulating cellular differentiation, inducing cellular proliferation, and increasing collagen synthesis.


    ACKNOWLEDGEMENTS

We thank Jane Kimball for secretarial assistance and Julie Bedore for editorial assistance.


    FOOTNOTES

This work used resources of the Michigan Gut Peptide Center at the University of Michigan and the Center for Gastrointestinal Biology and Disease at the University of North Carolina.

This work was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grants R29-DK-49628 (E. M. Zimmermann) and KO8-DK-02402 (J. B. Pucilowska).

Present address of Y. T. Hou: Dept. of Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China 200031.

Address for reprint requests and other correspondence: E. Zimmermann, 4410 Kresge III, Univ. of Michigan, Ann Arbor, MI 48109-0589 (E-mail: ezimmer{at}umich.edu).

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.

Received 20 October 1999; accepted in final form 18 December 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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