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• W2149705807 abstract "Substance P (SP) and the neurokinin-1 receptor (NK-1R) are involved in the development of colitis and mucosal healing after colonic inflammation. We studied whether SP modulates colonic fibrosis by using a chronic model of trinitrobenzenesulfonic acid (TNBS)-induced colitis in wild-type (WT) and NK-1R-deficient (NK-1R KD) mice. We found increased mRNA expression levels of collagen, vimentin, and the fibrogenic factors transforming growth factor β1 and insulin-like growth factor 1 in the chronically inflamed colons of WT mice treated with repeated intracolonic TNBS administrations. Fibrosis in TNBS-treated mice was also evident immunohistochemically by collagen deposition in the colon. Treatment of TNBS-exposed WT mice with the NK-1R antagonist CJ-12255 reduced colonic inflammation, colonic fibrosis, fibroblast accumulation, and expression levels of the fibrogenic factors. NK-1R knockout mice chronically exposed to TNBS had similar colonic inflammation compared with WT, but reduced colonic fibrosis, fibroblast accumulation, and expression levels of fibrogenic factors. Immunohistochemical staining also showed co-localization of NK-1R with fibroblasts in inflamed colons of mice and in colonic mucosa of patients with Crohn's disease. Exposure of human colonic CCD-18Co fibroblasts to SP (10 nmol/L) increased cell migration. SP stimulated collagen synthesis in CCD-18Co fibroblasts in the presence of transforming growth factor β1 and insulin-like growth factor 1, and this effect was reduced by Akt inhibition. Thus, SP, via NK-1R, promotes intestinal fibrogenesis after chronic colitis by stimulating fibrotic responses in fibroblasts. Substance P (SP) and the neurokinin-1 receptor (NK-1R) are involved in the development of colitis and mucosal healing after colonic inflammation. We studied whether SP modulates colonic fibrosis by using a chronic model of trinitrobenzenesulfonic acid (TNBS)-induced colitis in wild-type (WT) and NK-1R-deficient (NK-1R KD) mice. We found increased mRNA expression levels of collagen, vimentin, and the fibrogenic factors transforming growth factor β1 and insulin-like growth factor 1 in the chronically inflamed colons of WT mice treated with repeated intracolonic TNBS administrations. Fibrosis in TNBS-treated mice was also evident immunohistochemically by collagen deposition in the colon. Treatment of TNBS-exposed WT mice with the NK-1R antagonist CJ-12255 reduced colonic inflammation, colonic fibrosis, fibroblast accumulation, and expression levels of the fibrogenic factors. NK-1R knockout mice chronically exposed to TNBS had similar colonic inflammation compared with WT, but reduced colonic fibrosis, fibroblast accumulation, and expression levels of fibrogenic factors. Immunohistochemical staining also showed co-localization of NK-1R with fibroblasts in inflamed colons of mice and in colonic mucosa of patients with Crohn's disease. Exposure of human colonic CCD-18Co fibroblasts to SP (10 nmol/L) increased cell migration. SP stimulated collagen synthesis in CCD-18Co fibroblasts in the presence of transforming growth factor β1 and insulin-like growth factor 1, and this effect was reduced by Akt inhibition. Thus, SP, via NK-1R, promotes intestinal fibrogenesis after chronic colitis by stimulating fibrotic responses in fibroblasts. Inflammatory bowel diseases, including Crohn's disease (CD) and ulcerative colitis, are chronic relapsing immune disorders that significantly impact health-related quality of life.1Shih DQ Targan SR Insights into IBD Pathogenesis.Curr Gastroenterol Rep. 2009; 11: 473-480Crossref PubMed Scopus (67) Google Scholar Intestinal fibrosis is presented as excessive accumulation of fibrotic tissue in inflamed areas of the small intestine and colon of both ulcerative colitis and CD patients.2Rieder F Fiocchi C Intestinal fibrosis in IBD: a dynamic, multifactorial process.Nat Rev Gastroenterol Hepatol. 2009; 6: 228-235Crossref PubMed Scopus (248) Google Scholar As a result, patients with CD often develop transmural luminal narrowing and form strictures caused by excessive extracellular matrix deposition.3Fiocchi C Inflammatory bowel disease: etiology and pathogenesis.Gastroenterology. 1998; 115: 182-205Abstract Full Text Full Text PDF PubMed Scopus (1864) Google Scholar Although the mechanisms involved in extracellular matrix deposition are complex and not well understood, it is well accepted that this process is driven by increased expression of collagen and other pro-fibrogenic factors such as transforming growth factor β1 (TGF-β1) and insulin-like growth factor 1 (IGF-1).4Di Sabatino A Jackson CL Pickard KM Buckley M Rovedatti L Leakey NA Picariello L Cazzola P Monteleone G Tonelli F Corazza GR MacDonald TT Pender SL Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn's disease strictures.Gut. 2009; 58: 777-789Crossref PubMed Scopus (169) Google Scholar, 5Fichtner-Feigl S Young CA Kitani A Geissler EK Schlitt HJ Strober W IL-13 signaling via IL-13R alpha2 induces major downstream fibrogenic factors mediating fibrosis in chronic TNBS colitis.Gastroenterology. 2008; 135: 2003-2013Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 6Simmons JG Pucilowska JB Keku TO Lund PK IGF-I and TGF-beta1 have distinct effects on phenotype and proliferation of intestinal fibroblasts.Am J Physiol Gastrointest Liver Physiol. 2002; 283: G809-G818PubMed Google Scholar Substance P (SP) is an 11-amino acid neuropeptide belonging to the tachykinin family.7Chang MM Leeman SE Isolation of a sialogogic peptide from bovine hypothalamic tissue and its characterization as substance P.J Biol Chem. 1970; 245: 4784-4790Abstract Full Text PDF PubMed Google Scholar SP is expressed in the central nervous system,8Mantyh PW Neurobiology of substance P and the NK1 receptor.J Clin Psychiatry. 2002; 63: 6-10PubMed Google Scholar enteric nerves,9Costa M Furness JB Franco R Llewellyn-Smith I Murphy R Beardsley AM Substance P in nerve tissue in the gut.Ciba Found Symp. 1982; : 129-144PubMed Google Scholar sensory neurons,10Maggi CA Capsaicin-sensitive nerves in the gastrointestinal tract.Arch Int Pharmacodyn Ther. 1990; 303: 157-166PubMed Google Scholar immune cell types, such as macrophages11Bost KL Quantification of macrophage-derived substance P receptor mRNA using competitive polymerase chain reaction.Adv Exp Med Biol. 1995; 373: 219-223Crossref PubMed Scopus (17) Google Scholar, 12Castagliuolo I Keates AC Qiu B Kelly CP Nikulasson S Leeman SE Pothoulakis C Increased substance P responses in dorsal root ganglia and intestinal macrophages during Clostridium difficile toxin A enteritis in rats.Proc Natl Acad Sci USA. 1997; 94: 4788-4793Crossref PubMed Scopus (143) Google Scholar and T-lymphocytes,13Blum A Setiawan T Hang L Stoyanoff K Weinstock JV Interleukin-12 (IL-12) and IL-23 induction of substance p synthesis in murine T cells and macrophages is subject to IL-10 and transforming growth factor beta regulation.Infect Immun. 2008; 76: 3651-3656Crossref PubMed Scopus (25) Google Scholar as well as in colonic epithelial cells.14Koon HW Zhao D Zhan Y Simeonidis S Moyer MP Pothoulakis C Substance P-stimulated interleukin-8 expression in human colonic epithelial cells involves protein kinase Cdelta activation.J Pharmacol Exp Ther. 2005; 314: 1393-1400Crossref PubMed Scopus (59) Google Scholar, 15Koon HW Zhao D Zhan Y Rhee SH Moyer MP Pothoulakis C Substance P stimulates cyclooxygenase-2 and prostaglandin E2 expression through JAK-STAT activation in human colonic epithelial cells.J Immunol. 2006; 176: 5050-5059PubMed Google Scholar, 16Simeonidis S Castagliuolo I Pan A Liu J Wang CC Mykoniatis A Pasha A Valenick L Sougioultzis S Zhao D Pothoulakis C Regulation of the NK-1 receptor gene expression in human macrophage cells via an NF-kappa B site on its promoter.Proc Natl Acad Sci USA. 2003; 100: 2957-2962Crossref PubMed Scopus (80) Google Scholar In the intestine SP binding to its high affinity receptor neurokinin-1 (NK-1) modulates several important intestinal functions, including colonic inflammation.17Koon HW Pothoulakis C Immunomodulatory properties of substance P: the gastrointestinal system as a model.Ann NY Acad Sci. 2006; 1088: 23-40Crossref PubMed Scopus (110) Google Scholar Pharmacological antagonism of NK-1 receptor (NK-1R) or genetic deletion of this receptor results in reduced intestinal inflammation in the acute model of Clostridium difficile toxin A-induced enteritis or the acute phase of 2,4,6-trinitrobenzensulfonic acid (TNBS)-induced colitis.18Pothoulakis C Castagliuolo I LaMont JT Jaffer A O'Keane JC Snider RM Leeman SE CP-96,345, a substance P antagonist, inhibits rat intestinal responses to Clostridium difficile toxin A but not cholera toxin.Proc Natl Acad Sci USA. 1994; 91: 947-951Crossref PubMed Scopus (255) Google Scholar, 19Mantyh CR Pappas TN Lapp JA Washington MK Neville LM Ghilardi JR Rogers SD Mantyh PW Vigna SR Substance P activation of enteric neurons in response to intraluminal Clostridium difficile toxin A in the rat ileum.Gastroenterology. 1996; 111: 1272-1280Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 20Stucchi AF Shofer S Leeman S Materne O Beer E McClung J Shebani K Moore F O'Brien M Becker JM NK-1 antagonist reduces colonic inflammation and oxidative stress in dextran sulfate-induced colitis in rats.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G1298-G1306PubMed Google Scholar, 21Castagliuolo I Riegler M Pasha A Nikulasson S Lu B Gerard C Gerard NP Pothoulakis C Neurokinin-1 (NK-1) receptor is required in Clostridium difficile-induced enteritis.J Clin Invest. 1998; 101: 1547-1550Crossref PubMed Scopus (160) Google Scholar In contrast, NK-1R deficient (knockout [KO]) mice exposed to TNBS or sodium dextran sulfate,22Castagliuolo I Morteau O Keates AC Valenick L Wang CC Zacks J Lu B Gerard NP Pothoulakis C Protective effects of neurokinin-1 receptor during colitis in mice: role of the epidermal growth factor receptor.Br J Pharmacol. 2002; 136: 271-279Crossref PubMed Scopus (54) Google Scholar or mice treated with the NK-1R receptor antagonist CJ-12255 during the healing phase of sodium dextran sulfate-induced colitis23Koon HW Zhao D Zhan Y Moyer MP Pothoulakis C Substance P mediates antiapoptotic responses in human colonocytes by Akt activation.Proc Natl Acad Sci USA. 2007; 104: 2013-2018Crossref PubMed Scopus (91) Google Scholar developed more severe colitis than control mice suggesting that NK-1R is also involved in mucosal healing during chronic colitis. However, there are no studies examining whether SP, or any other neuropeptide, is involved in colonic fibrosis. Based on the ability of SP and NK-1R to modulate colonic inflammation and fibrosis through its interaction with fibroblasts,22Castagliuolo I Morteau O Keates AC Valenick L Wang CC Zacks J Lu B Gerard NP Pothoulakis C Protective effects of neurokinin-1 receptor during colitis in mice: role of the epidermal growth factor receptor.Br J Pharmacol. 2002; 136: 271-279Crossref PubMed Scopus (54) Google Scholar, 24Pucilowska JB Williams KL Lund PK Fibrogenesis. IV. Fibrosis and inflammatory bowel disease: cellular mediators and animal models.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G653-G659PubMed Google Scholar we hypothesized that SP/NK-1R interactions participate in colitis-associated fibrosis. To address our hypothesis, we used NK-1R KO and wild-type (WT) mice injected with the specific nonpeptide NK-1R antagonist CJ 12255 and the murine model of chronic TNBS-induced colitis associated with intestinal fibrosis.25Lawrance IC Wu F Leite AZ Willis J West GA Fiocchi C Chakravarti S A murine model of chronic inflammation-induced intestinal fibrosis down-regulated by antisense NF-kappa B.Gastroenterology. 2003; 125: 1750-1761Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar Our results indicate that WT mice treated with the NK-1R antagonist as well as NK-1R KO mice have reduced colonic fibrosis, fibroblast accumulation, and expression of fibrogenic factors in the colonic mucosa. NK-1R was co-localized with IGF-1 in colonic mucosa of patients with CD and TNBS-treated mice. Furthermore, in vitro experiments with human colonic CCD-18Co fibroblasts showed that SP stimulated fibroblast migration and, in the presence of TGF-β1 and IGF-1, increased collagen synthesis in an Akt-dependent manner. Total RNA was isolated by Trizol Reagent (Invitrogen, Carlsbad, CA) and reverse-transcribed into cDNA by using a Superscript III reverse transcription kit (Invitrogen). Quantitative PCR reactions were run in an ABI Prism 7700 Sequence Detection System as we previously described.26Koon HW Zhao D Xu H Bowe C Moss A Moyer MP Pothoulakis C Substance P-mediated expression of the pro-angiogenic factor CCN1 modulates the course of colitis.Am J Pathol. 2008; 173: 400-410Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar The levels of mRNA were determined by using cataloged primers (Applied Biosystems, Foster City, CA) for human (COL1A2 Hs01028956_m1) and mouse (Col1a2 Mm01309565_m1; vimentin Mm00449208_ m1; Igf-1 Mm00439561-m1; and Tgf-β1 Mm00441724_m1). Expression of these genes was normalized to expression of GAPDH/18S mRNA (human, 18S Hs99999901_s1; mouse, GAPDH Mm99999915_g1) and the results were expressed as relative fold differences. NK-1R KO mice were generated by targeted disruption of the NK-1R gene in embryonic stem cells as we previously described.27Bozic CR Lu B Hopken UE Gerard C Gerard NP Neurogenic amplification of immune complex inflammation.Science. 1996; 273: 1722-1725Crossref PubMed Scopus (296) Google Scholar Aged matched NK-1R KO and WT littermates (10- to 12-week-old) were maintained at the animal research facility of the University of California at Los Angeles. All animal studies were approved by the institutional animal care and use committee of the University of California, Los Angeles. Mice received standard pelleted chow and tap water ad libitum. Chronic TNBS colitis was induced as previously reported.25Lawrance IC Wu F Leite AZ Willis J West GA Fiocchi C Chakravarti S A murine model of chronic inflammation-induced intestinal fibrosis down-regulated by antisense NF-kappa B.Gastroenterology. 2003; 125: 1750-1761Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar Briefly, mice were injected intracolonically with a 50-μl of 30% ethanol containing a TNBS solution or 30% ethanol weekly for 5 weeks as illustrated in Figure 1A. Some groups were injected intraperitoneally with the specific nonpeptide NK-1R antagonist CJ-12255 (Pfizer, New York, NY; 5 mg/kg in 100 μl PBS, every 3 days during the last 4 weeks of the 8-week experimental period). Other groups were injected intraperitoneally with the same volume of PBS. Mice were then returned to their cages and sacrificed at week 8 by carbon dioxide euthanasia. Colon tissues of mice were sectioned and stained with H&E. Microphotographs at 200× magnification were recorded at multiple locations of colons and analyzed by two pathologists in a blinded manner who scored the specimens at a 0 to 12 scale for the following chronic colitis parameters: mucosal transformation, round cell infiltrates in the lamina propria mucosae, goblet cell death, submucosa fibrosis, and granuloma formation as previously established.28Kruschewski M Foitzik T Perez-Canto A Hubotter A Buhr HJ Changes of colonic mucosal microcirculation and histology in two colitis models: an experimental study using intravital microscopy and a new histological scoring system.Dig Dis Sci. 2001; 46: 2336-2343Crossref PubMed Scopus (76) Google Scholar Zero = no inflammation; 1 to 4 = mild inflammation; 5 to 8 = moderate inflammation; and 9 to 12 = severe inflammation. Human colonic tissue frozen sections were purchased from Cytomyx, Inc. (Rockville, MD). Colonic tissues were embedded in OCT solution, and frozen sections were made and fixed and permeabilized by using acetone. After incubation with blocking buffer, slides were incubated with a rabbit polyclonal rabbit anti-NK-1R antibody (sc-15323) or goat anti-vimentin antibody (sc-7557; Santa Cruz biotechnology, Santa Cruz, CA; all at 1:50 dilution) overnight at 4°C. Samples were then washed with PBS and stained by Texas Red (red)-conjugated and fluorescein isothiocyanate (green)-conjugated secondary antibodies (1:200 dilution) for 1 hour. Slides were then rinsed and mounted with 4′,6-diamidino-2-phenylindole (DAPI; blue emission signal to stain nuclei) mounting solution. Images were analyzed with a Zeiss (Thornwood, NY) AX10 microscope at 200× magnification. Colon tissues were fixed in 4% paraformaldehyde and embedded in paraffin. After incubation with blocking buffer, sections were incubated with a goat polyclonal anti-vimentin antibody (sc-7557; Santa Cruz; 1:50 dilution) overnight at 4°C. After washing, sections were incubated with donkey anti-goat IgG, and slides were stained with an avidin-biotin complex kit for color development (Santa Cruz; sc-2018). Images were analyzed with a Zeiss AX10 microscope at magnification of 200×. Staining of collagen deposition of deparaffinized tissue sections was performed by a Masson Trichrome Staining kit (HT-10) and Bouin solution (HT-10132; Sigma, St. Louis, MO) following the manufacturer's instructions. Human CCD-18Co colonic fibroblasts were cultured in minimal essential medium Eagle's medium (ATCC, Manassas, VA) containing 10% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). The levels of mouse IGF-1 and TGF-β1 levels were measured by the enzyme-linked immunosorbent assay kits (IGF-1 DY291 and TGF-β1 DY791; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Cell migration assays of CCD-18Co fibroblasts were performed by using a modified Boyden chamber approach (number ECM508; Millipore, Billerica, MA) as we previously described.26Koon HW Zhao D Xu H Bowe C Moss A Moyer MP Pothoulakis C Substance P-mediated expression of the pro-angiogenic factor CCN1 modulates the course of colitis.Am J Pathol. 2008; 173: 400-410Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 29Koon HW Kim YS Xu H Kumar A Zhao D Karagiannides I Dobner PR Pothoulakis C Neurotensin induces IL-6 secretion in mouse preadipocytes and adipose tissues during 2,4,6,-trinitrobenzensulphonic acid-induced colitis.Proc Natl Acad Sci USA. 2009; 106: 8766-8771Crossref PubMed Scopus (56) Google Scholar Briefly, 0.1% Trifluoroacetic acid (TFA), SP (number 05-23-0600; Calbiochem, Gibbstown, NJ), IGF-1 (number 407240; Calbiochem), or TGF-β1 (number 616455; Calbiochem) was added into the lower chamber. CCD-18Co fibroblasts (2.5 × 104 cells) were seeded in the upper chamber and incubated for 6 hours at 37°C. Fibroblasts that had migrated through the membrane were stained according to the manufacturer's protocol, and migrated cells were determined by absorbance at 650 nm. CCD-18Co fibroblasts, seeded on 96-well plates (106 cells/plate), were pretreated with 1 μmol/L CJ-12255 or vehicle for 30 minutes and then exposed to SP (10 nmol/L) or vehicle (0.01% TFA). After 24 hours, 20 μl of CellTiter AQeuous One solution (MTS tetrazolium compound, G3580; Promega, Madison, WI) was added, cells were incubated at 37°C for 1 hour, and absorbance at 490 nm (indicating cell viability) was measured on a 96-well plate reader as we described.30Koon HW Zhao D Na X Moyer MP Pothoulakis C Metalloproteinases and transforming growth factor-alpha mediate substance P-induced mitogen-activated protein kinase activation and proliferation in human colonocytes.J Biol Chem. 2004; 279: 45519-45527Crossref PubMed Scopus (101) Google Scholar Cells were lysed in 1× blue loading buffer (Cell Signaling, Danvers, MA). Equal amount of cell extracts were fractioned by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and proteins were transferred onto nitrocellulose membranes (400 mA for 2 hours at 4°C; Bio-Rad, Hercules, CA). Membranes were blocked in 5% nonfat milk in Tris-buffered saline Tween 20 (TBST; 50 mmol/L Tris, pH 7.5, 0.15 M NaCl, and 0.05% Tween 20), and then incubated with antibodies against phospho-Akt (ser473) number 4060, β-actin number 4967, β-tubulin number 2146 (Cell Signaling), NK-1R (sc-15323), or collagen 1A2 (sc-28655). Horseradish peroxidase-labeled antibodies were detected by chemiluminescence (Fisher Scientific, Pittsburgh, PA), and signals were captured by using the luminescent image analyzer LAS4000 (Fujifilm, Tokyo, Japan). Results were expressed as mean ± SEM and analyzed by using the Prism professional statistics software program (Graphpad, San Diego, CA). Student's t-tests with Mann-Whitney post tests were used for intergroup comparisons. Colonic fibrosis is characterized by collagen deposition and overexpression of fibrogenic factors.2Rieder F Fiocchi C Intestinal fibrosis in IBD: a dynamic, multifactorial process.Nat Rev Gastroenterol Hepatol. 2009; 6: 228-235Crossref PubMed Scopus (248) Google Scholar, 31Burke JP Mulsow JJ O'Keane C Docherty NG Watson RW O'Connell PR Fibrogenesis in Crohn's disease.Am J Gastroenterol. 2007; 102: 439-448Crossref PubMed Scopus (225) Google Scholar To investigate the role of NK-1R in the development of fibrosis, we used a murine model of chronic colitis that develops colonic fibrosis25Lawrance IC Wu F Leite AZ Willis J West GA Fiocchi C Chakravarti S A murine model of chronic inflammation-induced intestinal fibrosis down-regulated by antisense NF-kappa B.Gastroenterology. 2003; 125: 1750-1761Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar in NK-1R KO mice as well as WT mice treated with the NK-1R receptor antagonist CJ-12255. Details of this model are included in Materials and Methods and illustrated in Figure 1A. As shown in Figures 1C and 2A, compared with vehicle administration, WT mice treated with multiple TNBS cycles develop substantial fibrosis in the mucosa and submucosa. In contrast, CJ-12255 treated, TNBS-exposed mice have less mucosal and submucosal fibrosis (Figure 1C and 2A) and overall better mucosal tissue integrity. Quantification of these histological differences shown in Figure 1B as “histological score” show mild to moderate levels of colitis (histological score 4 to 5) after TNBS administration and a ∼60% reduction of colitis after CJ-12255 administration (P < 0.001). Notably, histology (Figure 1C) and histological scores (Figure 1B) of ethanol (vehicle)-treated mice are low and not affected by CJ-12255 treatment. Moreover, NK-1R KO mice treated with multiple TNBS cycles develop similar histological tissue damage and histological score as TNBS-treated WT mice (Figure 1, B and C). Similar to WT TNBS-treated mice, colonic sections from NK-1R KO mice exposed to TNBS show signs of chronic inflammation including mucosal transformation with altered cryptal architecture, immune cell infiltration, and occasional formation of granulomas (Figure 1, B and C). In contrast, NK-1R KO mice have substantially less colonic fibrosis than TNBS-exposed WT littermates (Figure 2A). These results indicate that NK-1R is involved in colonic fibrosis in murine model of chronic colitis. Collagen is secreted by fibroblasts and fibroblast-like cells32Lawrance IC Maxwell L Doe W Inflammation location, but not type, determines the increase in TGF-beta1 and IGF-1 expression and collagen deposition in IBD intestine.Inflamm Bowel Dis. 2001; 7: 16-26Crossref PubMed Scopus (106) Google Scholar and can be identified immunohistochemically by the fibroblast marker vimentin.33Motomura Y Khan WI El-Sharkawy RT Verma-Gandhu M Verdu EF Gauldie J Collins SM Induction of a fibrogenic response in mouse colon by overexpression of monocyte chemoattractant protein 1.Gut. 2006; 55: 662-670Crossref PubMed Scopus (52) Google Scholar Masson Trichrome staining in the mucosa and submucosa of WT mice after chronic TNBS treatment showed increased collagen formation (Figure 2A). Compared with controls, we observed an increased number of vimentin-positive cells in the colonic mucosa and submucosa of WT TNBS-treated mice (Figures 2B and 3A, arrows) in areas with increased collagen deposition (Figure 2A), suggesting that colonic fibroblasts mediate increased collagen secretion. Treating TNBS-exposed wild-type or NK-1R KO mice with CJ-12255 reduced collagen deposition (Figure 2A) and fibroblast infiltration (Figure 2B). This is correlated with a reduction of vimentin-positive cells in TNBS-exposed colonic tissues that are treated with CJ-12255 (Figure 3B). NK-1R KO mice exposed to TNBS also have reduced vimentin-positive cells compared with WT (Figure 3B). The number of fibroblasts among ethanol-treated groups is low and not affected by CJ-12255 treatment or NK-1R deficiency (Figure 3B). Consistent with prior observations,25Lawrance IC Wu F Leite AZ Willis J West GA Fiocchi C Chakravarti S A murine model of chronic inflammation-induced intestinal fibrosis down-regulated by antisense NF-kappa B.Gastroenterology. 2003; 125: 1750-1761Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar WT mice treated with multiple TNBS cycles demonstrate high mRNA levels of collagen and vimentin, compared with ethanol control (Figure 3, C and D). NK-1R antagonism by CJ-12255 significantly lowers collagen and vimentin mRNA levels (Figure 3, C and D). Moreover, NK-1R KO mice have also lower collagen and vimentin mRNA after TNBS exposure (Figure 3, C and D), directly demonstrating an essential role for NK-1R in colitis-associated fibrogenesis. Increased colonic mRNA expression of fibrogenic factors TGF-β1 and IGF-1 in the colons of patients with CD has been reported previously.32Lawrance IC Maxwell L Doe W Inflammation location, but not type, determines the increase in TGF-beta1 and IGF-1 expression and collagen deposition in IBD intestine.Inflamm Bowel Dis. 2001; 7: 16-26Crossref PubMed Scopus (106) Google Scholar, 34Zimmermann EM Li L Hou YT Mohapatra NK Pucilowska JB Insulin-like growth factor I and insulin-like growth factor binding protein 5 in Crohn's disease.Am J Physiol Gastrointest Liver Physiol. 2001; 280: G1022-G1029PubMed Google Scholar Consistent with these results, our experiments show increased TGF-β1 and IGF-1 colonic protein levels in inflamed colons of TNBS-treated mice (Figure 3, E and F). However, colonic levels of both TGF-β1 and IGF-1 are significantly lower in TNBS-exposed NK-1R KO mice and in WT mice treated with CJ-12255 (Figure 3, E and F). NK-1R is broadly expressed in the colons of patients with CD (Figure 4A), in line with previous reports.26Koon HW Zhao D Xu H Bowe C Moss A Moyer MP Pothoulakis C Substance P-mediated expression of the pro-angiogenic factor CCN1 modulates the course of colitis.Am J Pathol. 2008; 173: 400-410Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 35Goode T O'Connell J Anton P Wong H Reeve J O'Sullivan GC Collins JK Shanahan F Neurokinin-1 receptor expression in inflammatory bowel disease: molecular quantitation and localisation.Gut. 2000; 47: 387-396Crossref PubMed Scopus (119) Google Scholar Double immunofluorescence staining show increased expression of vimentin (fibroblast marker) positive cells and colocalization of many of these cells with NK-1R expressing cells (Figure 4A). Similarly, we found strong widespread NK-1R staining in the colon of TNBS-treated mice, which is reduced after CJ-12255 administration (Figure 4B). Several vimentin positive cells also express NK-1R in the colonic mucosa of TNBS-exposed mice (Figure 4B). Thus, colonic fibroblasts in human and mouse inflamed colon express NK-1R. To mediate colonic fibrosis, fibroblasts migrate to sites of inflammation where they deposit collagen. To determine the role of SP in fibroblast migration, cultured human colonic CCD-18Co fibroblasts were seeded in the upper chamber of modified Boyden chambers. Addition of SP to the lower, but not upper, chamber induces significant fibroblast migration similar to that seen in response to TGF-β1 and IGF-1 serving as positive controls (Figure 5A). We next tested whether SP induces proliferation in CCD-18Co cells. Addition of SP (10 nmol/L) alone does not increase cell proliferation, measured by the MTS assay (Figure 5B). SP also fail to stimulate CCD-18Co cell proliferation even in the presence of TGF-β1 (1 ng/ml) and IGF-1 (10 ng/ml; Figure 5B). Moreover, SP (10 nmol/L), either alone or in the presence of TGF-β1 and IGF-1, does not affect TGF-β1 or IGF-1 mRNA expression (Figure 5, C and D). To determine whether SP affects fibrogenesis in vitro, we exposed human colonic CCD-18Co fibroblasts to SP in the presence or absence of TGF-β1 and IGF-1. Incubation with SP alone for 48 hours does not induce collagen COL1A2 protein expression in CCD-18Co cells (data not shown). Exposure of fibroblasts to IGF-1 and TGF-β1 does not increase expression of protein bands representing COL1A2 (Figure 6A). In the presence of TGF-β1 and IGF-1, however, SP increases COL1A2 protein expression in a dose-dependent manner (Figure 6A). Similarly, SP in combination with TGF-β1 and IGF-1, but not alone, induces COL1A2 mRNA expression (Figure 6B). In contrast, SP does not influence collagen COL1A2 mRNA expression when incubated with either TGF-β1 alone or IGF-1 alone (Figure 6B). TGF-β1 alone, serving as positive control, moderately increases collagen COL1A2 mRNA levels that is further augmented by IGF-1 co-exposure in CCD-18Co fibroblasts, consistent with prior observations6Simmons JG Pucilowska JB Keku TO Lund PK IGF-I and TGF-beta1 have distinct effects on phenotype and proliferation of intestinal fibroblasts.Am J Physiol Gastrointest Liver Physiol. 2002; 283: G809-G818PubMed Google Scholar(Figure 6B). IGF-1 induces Akt phosphorylation in lung fibroblasts,36Choi JE Lee SS Sunde DA Huizar I Haugk KL Thannickal VJ Vittal R Plymate SR Schnapp LM Insulin-like growth factor-I receptor blockade improves outcome in m" @default.
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• W2149705807 date "2010-11-01" @default.
• W2149705807 modified "2023-10-15" @default.
• W2149705807 title "Substance P Modulates Colitis-Asscociated Fibrosis" @default.
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