Substance P-Mediated Expression of the Pro-Angiogenic Factor CCN1 Modulates the Course of Colitis
2008; Elsevier BV; Volume: 173; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2008.080222
ISSN1525-2191
AutoresHon–Wai Koon, Dezheng Zhao, Hua Xu, Collin Bowe, Alan C. Moss, Mary Pat Moyer, Charalabos Pothoulakis,
Tópico(s)Apelin-related biomedical research
ResumoSubstance P (SP) regulates important intestinal functions, such as mucosal permeability, motility, chloride secretion, and inflammation via the neurokinin-1 receptor (NK-1R). Previous reports showed that vascularization and expression of angiogenic factors are evident in the colonic mucosa of rats with colitis and patients with inflammatory bowel disease. Here we determined whether SP is associated with angiogenesis. Human NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) and mice with dextran sodium sulfate-induced colitis were used. We found that expression of the angiogenic factor CCN1 was increased in the colons of patients with Crohn's disease and ulcerative colitis. Mucosal extracts from inflammatory bowel disease patients induced human intestinal microvascular endothelial cell migration that was inhibited by blockade of CCN1 and its receptor integrin αvβ3. Both the degree of angiogenesis and CCN1 expression were elevated in the colons of mice with dextran sodium sulfate-induced colitis, which was reduced by treatment with the NK-1R antagonist CJ-12255. SP also increased CCN1 expression in NCM460-NK-1R colonocytes. SP exposure to human intestinal microvascular endothelial cells co-cultured with NCM460-NK-1R cells induced angiogenic activity that was inhibited by CCN1 silencing. In addition, intracolonic overexpression of CCN1 induced angiogenesis in mouse colon. Thus, SP mediates angiogenesis via CCN1 during colitis. Substance P (SP) regulates important intestinal functions, such as mucosal permeability, motility, chloride secretion, and inflammation via the neurokinin-1 receptor (NK-1R). Previous reports showed that vascularization and expression of angiogenic factors are evident in the colonic mucosa of rats with colitis and patients with inflammatory bowel disease. Here we determined whether SP is associated with angiogenesis. Human NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) and mice with dextran sodium sulfate-induced colitis were used. We found that expression of the angiogenic factor CCN1 was increased in the colons of patients with Crohn's disease and ulcerative colitis. Mucosal extracts from inflammatory bowel disease patients induced human intestinal microvascular endothelial cell migration that was inhibited by blockade of CCN1 and its receptor integrin αvβ3. Both the degree of angiogenesis and CCN1 expression were elevated in the colons of mice with dextran sodium sulfate-induced colitis, which was reduced by treatment with the NK-1R antagonist CJ-12255. SP also increased CCN1 expression in NCM460-NK-1R colonocytes. SP exposure to human intestinal microvascular endothelial cells co-cultured with NCM460-NK-1R cells induced angiogenic activity that was inhibited by CCN1 silencing. In addition, intracolonic overexpression of CCN1 induced angiogenesis in mouse colon. Thus, SP mediates angiogenesis via CCN1 during colitis. Substance P (SP), an 11-amino acid neuropeptide member of the tachykinin family isolated by Leeman and Chang,1Chang 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 is localized in the central nervous system,2Mantyh PW Neurobiology of substance P and the NK1 receptor.J Clin Psychiatry. 2002; 63: 6-10PubMed Google Scholar sensory neurons,3Maggi CA Capsaicin-sensitive nerves in the gastrointestinal tract.Arch Int Pharmacodyn Ther. 1990; 303: 157-166PubMed Google Scholar enteric nerves4Costa M Furness JB Franco R Llewellyn-Smith I Murphy R Beardsley AM Substance P in nerve tissue in the gut.Ciba Found Symp. 1982; 91: 129-144PubMed Google Scholar and immune cells.5Bost 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 SP binding to its high-affinity neurokinin-1 receptor (NK-1R) modulates important intestinal functions, such as mucosal permeability,6Pothoulakis 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 motility,7Holzer P Holzer-Petsche U Tachykinins in the gut. Part I. Expression, release and motor function.Pharmacol Ther. 1997; 73: 173-217Crossref PubMed Scopus (311) Google Scholar chloride secretion,8Riegler M Castagliuolo I So PT Lotz M Wang C Wlk M Sogukoglu T Cosentini E Bischof G Hamilton G Teleky B Wenzl E Matthews JB Pothoulakis C Effects of substance P on human colonic mucosa in vitro.Am J Physiol. 1999; 276: G1473-G1483PubMed Google Scholar and inflammation, via activation of COX-2, PGE2, and nuclear factor-κB-regulated cytokine genes.9Koon 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, 10Koon 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 SP-NK-1R interaction also induces proliferation of colonic epithelial cells that involves activation of metalloproteinases and transactivation of the epithelial growth factor receptor.11Koon 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 SP also exerts an anti-apoptotic effect in colonic epithelial cells via Akt phosphorylation in vitro and in vivo.12Koon 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 Angiogenesis is a critical component of colonic inflammation. The occurrence of angiogenesis and expression of angiogenic factor have been established in inflammatory bowel disease (IBD) including Crohn's disease (CD) and ulcerative colitis (UC).13Danese S Sans M de la Motte C Graziani C West G Phillips MH Pola R Rutella S Willis J Gasbarrini A Fiocchi C Angiogenesis as a novel component of inflammatory bowel disease pathogenesis.Gastroenterology. 2006; 130: 2060-2073Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar In both CD and UC mucosa, the local microvasculature undergoes intensive inflammation-dependent angiogenesis. Evidence from a sponge angiogenesis model14Fan TP Hu DE Guard S Gresham GA Watling KJ Stimulation of angiogenesis by substance P and interleukin-1 in the rat and its inhibition by NK1 or interleukin-1 receptor antagonists.Br J Pharmacol. 1993; 110: 43-49Crossref PubMed Scopus (133) Google Scholar and synovial inflammation in rats indicates that SP may mediate angiogenesis in nonintestinal tissues.18Brigstock DR Regulation of angiogenesis and endothelial cell function by connective tissue growth factor (CTGF) and cysteine-rich 61 (CYR61).Angiogenesis. 2002; 5: 153-165Crossref PubMed Scopus (272) Google Scholar, 19Moss AC Anton P Savidge T Newman P Cheifetz AS Gay J Paraschos S Winter MW Moyer MP Karalis K Kokkotou E Pothoulakis C Urocortin II mediates pro-inflammatory effects in human colonocytes via corticotropin-releasing hormone receptor 2{alpha}.Gut. 2007; 56: 1210-1217Crossref PubMed Scopus (68) Google Scholar, 20Chang CC Shih JY Jeng YM Su JL Lin BZ Chen ST Chau YP Yang PC Kuo ML Connective tissue growth factor and its role in lung adenocarcinoma invasion and metastasis.J Natl Cancer Inst. 2004; 96: 364-375Crossref PubMed Scopus (161) Google Scholar, 21Vermeulen PB Gasparini G Fox SB Toi M Martin L McCulloch P Pezzella F Viale G Weidner N Harris AL Dirix LY Quantification of angiogenesis in solid human tumours: an international consensus on the methodology and criteria of evaluation.Eur J Cancer. 1996; 32A: 2474-2484Abstract Full Text PDF PubMed Scopus (671) Google Scholar However, whether SP or any other neuropeptide mediates vascular angiogenesis during colonic inflammation is unknown and the mechanisms governing these responses have never been examined. Members of the CCN family, including CCN1 (cysteine-rich 61, Cyr61), CCN2 (connective tissue growth factor, CTGF), CCN3 (nephroblastoma, NOV), CCN4/5/6 (Wnt-inducible secreted protein, WISP-1, -2, and -3), are 30- to 40-kDa cysteine rich proteins15Brigstock DR The CCN family: a new stimulus package.J Endocrinol. 2003; 178: 169-175Crossref PubMed Scopus (445) Google Scholar that stimulate mitosis, adhesion, apoptosis, extracellular matrix, angiogenesis, and tumor growth.16Moussad EE Brigstock DR Connective tissue growth factor: what's in a name?.Mol Genet Metab. 2000; 71: 276-292Crossref PubMed Scopus (444) Google Scholar Recent data indicate that CCN1, -2, and -3 bind to cell surface integrins and thereby activate intracellular signaling, including kinase phosphorylation and gene transcription.15Brigstock DR The CCN family: a new stimulus package.J Endocrinol. 2003; 178: 169-175Crossref PubMed Scopus (445) Google Scholar CCN1 plays a pivotal role in vasculogenesis during embryogenesis because mice lacking CCN1 cannot develop in utero because of vascular defects in the placenta, confirming the importance of this protein in endothelial cell proliferation and angiogenesis.17Mo FE Muntean AG Chen CC Stolz DB Watkins SC Lau LF CYR61 (CCN1) is essential for placental development and vascular integrity.Mol Cell Biol. 2002; 22: 8709-8720Crossref PubMed Scopus (345) Google Scholar, 18Brigstock DR Regulation of angiogenesis and endothelial cell function by connective tissue growth factor (CTGF) and cysteine-rich 61 (CYR61).Angiogenesis. 2002; 5: 153-165Crossref PubMed Scopus (272) Google Scholar However, the expression and role of CCN1 in the intestinal tract has never been investigated. In this study, we found increased CCN1 levels in the colons of UC and CD patients and in the colonic mucosa of mice with experimental colitis. We also demonstrate that intracolonic overexpression of CCN1 induced colonic vascularization in vivo and that SP increased expression of this angiogenic factor in human colonic epithelial cells. Colon samples from UC and CD patients were obtained as previously described.19Moss AC Anton P Savidge T Newman P Cheifetz AS Gay J Paraschos S Winter MW Moyer MP Karalis K Kokkotou E Pothoulakis C Urocortin II mediates pro-inflammatory effects in human colonocytes via corticotropin-releasing hormone receptor 2{alpha}.Gut. 2007; 56: 1210-1217Crossref PubMed Scopus (68) Google Scholar Total RNA was isolated from eight pairs of inflamed colon and matching adjacent normal regions and then converted into cDNA. All patients gave informed consent and the Beth Israel Deaconess Medical Center Institutional Review Board approved the protocol. Total RNA was isolated from colon tissues by Trizol reagent (Invitrogen, Carlsbad, CA) and reverse-transcribed into cDNA using the Superscript III reverse transcription kit (Invitrogen). The PCR reactions were run in the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). The levels of CCN1, TACR1 (NK-1R), TAC1 (SP), and VEGFA, VEGFB, VEGFC, and VEGFD mRNA were determined using their respective real-time primer sets obtained from Applied Biosystems according the manufacturer's instructions. Levels were normalized to equal levels of GAPDH mRNA and results were expressed as fold induction compared to their respective controls. NCM460 cells overexpressing NK-1R (NCM460-NK-1R), previously generated by us,9Koon 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, 10Koon 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, 11Koon 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, 12Koon 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 were cultured in M3D medium (INCELL, San Antonio, TX) containing 10% fetal calf serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). Human intestinal microvascular endothelial cells (HIMECs) were generously provided by Dr. Claudio Fiocchi (Department of Pathobiology, The Cleveland Clinic Foundation, Cleveland, OH)13Danese S Sans M de la Motte C Graziani C West G Phillips MH Pola R Rutella S Willis J Gasbarrini A Fiocchi C Angiogenesis as a novel component of inflammatory bowel disease pathogenesis.Gastroenterology. 2006; 130: 2060-2073Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar and cultured for five to six generations in MCDB131 medium (Sigma, St. Louis, MO) containing 10% fetal bovine serum (Cambrex, East Rutherford, NJ) on fibronectin-coated culture plates (Becton-Dickinson, Franklin Lakes, NJ). Cells were treated with SP or human CCN1 protein (Peprotech Co., Rocky Hill, NJ) as indicated. Male, 8- to 10-week old C57BL6 mice (n = 6 per group) (Charles River Laboratories, Wilmington, MA) were maintained at the animal facility under standard conditions. Animal studies were approved by the institutional animal care and use committee of Beth Israel Deaconess Medical Center. Mice received standard pelleted chow and tap water ad libitum, except the colitis group, which received water containing dextran sodium sulfate (DSS) 5% (w/v), as previously described.9Koon 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, 11Koon 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, 12Koon 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 Groups of mice were also injected intraperitoneally with 200 μl of phosphate-buffered saline (PBS) containing the NK-1R antagonist CJ-12255 (2.5 mg/kg/twice per day) or PBS. After 5 days, mice were sacrificed by carbon dioxide euthanasia. Colon tissues were dissected and homogenized in immunoprecipitation buffer (Santa Cruz Biotechnology, Santa Cruz, CA), and equal amounts of protein (40 μg/lane) were subjected to Western blotting. Some colon tissues were also fixed in formalin and paraffin-embedded for immunohistochemistry. Cells were lysed in 1× lysis buffer (62.5 mmol/L Tris-HCl, 2% sodium dodecyl sulfate, 10% glycerol, 0.01% bromophenol blue, and 1% 2-ME). Equal amounts 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 TBST (50 mmol/L Tris, pH 7.5, 0.15 mol/L NaCl, 0.05% Tween 20), and then incubated with antibodies against phospho-integrin β3, CCN120Chang CC Shih JY Jeng YM Su JL Lin BZ Chen ST Chau YP Yang PC Kuo ML Connective tissue growth factor and its role in lung adenocarcinoma invasion and metastasis.J Natl Cancer Inst. 2004; 96: 364-375Crossref PubMed Scopus (161) Google Scholar (Santa Cruz Biotechnology) and β-actin (Sigma). Horseradish peroxidase-labeled antibody was detected by enhanced chemiluminescence (Pierce, Rockford, IL) and was exposed to X-ray film (Fujifilm; Fisher, Pittsburgh, PA). In some experiments, Western blot bands were quantified by densitometry using Scion Image Analysis software (Frederick, MD, USA). The DNA fragments of the CCN1 promoter (1.1 kb) upstream of the translational start site were cloned by PCR from human colonocyte genomic DNA and after confirming the sequence identity, subcloned into a pGL3 vector (pGL3-CCN1). NCM460-NK-1R cells were seeded in 12-well plates (2 × 106 cells/plate) overnight and transiently transfected with the pGL3-CCN1 construct together with an internal control pRL-TK (Promega, Madison, WI) as previously described.9Koon 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 Transfected cells were serum-starved for 24 hours and then treated with SP for up to 24 hours. The relative promoter activity of CCN1 in equal amounts of cell extracts was measured using a dual luciferase reporter assay system (Promega). NCM460-NK-1R colonocytes were seeded in 12-well plates (2 × 105 cells/well) overnight and transiently transfected with equal amounts of CCN1 or control siRNA (Dharmacon, Chicago, IL) using the X-tremeGene transfection reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions.9Koon 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, 10Koon 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, 12Koon 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 Reduced endogenous CCN1 expression was confirmed by Western blot analyses. Colon tissues were fixed in 4% paraformaldehyde and embedded in paraffin. After incubation with blocking buffer, sections were incubated with a rabbit polyclonal anti-vWF antibody (AB7356; Millipore, Billerica, MA) overnight at 4°C. After washing, sections were incubated with donkey anti-rabbit IgG and slides were stained with an ABC kit for color development (sc-2018, Santa Cruz Biotechnology). Sections were photographed under the microscope and computerized vWF-stained image analysis was performed using the Scion Image Software as previously described.13Danese S Sans M de la Motte C Graziani C West G Phillips MH Pola R Rutella S Willis J Gasbarrini A Fiocchi C Angiogenesis as a novel component of inflammatory bowel disease pathogenesis.Gastroenterology. 2006; 130: 2060-2073Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 21Vermeulen PB Gasparini G Fox SB Toi M Martin L McCulloch P Pezzella F Viale G Weidner N Harris AL Dirix LY Quantification of angiogenesis in solid human tumours: an international consensus on the methodology and criteria of evaluation.Eur J Cancer. 1996; 32A: 2474-2484Abstract Full Text PDF PubMed Scopus (671) Google Scholar, 22Weidner N Current pathologic methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors.Breast Cancer Res Treat. 1995; 36: 169-180Crossref PubMed Scopus (768) Google Scholar Human CCN1 cDNA clone no. TC310465 was obtained from Origene (Rockville, MD). The CCN1-overexpressing construct was mixed with polyethyleneimine-based transfection reagent (in vivo Jet, 201-50G; Polyplus, San Marcos, CA) in a N:P ratio = 5 (polyethyleneimine nitrogen to DNA phosphate ratio). Before induction of colitis, C57/BL6 mice were starved overnight and then anesthetized by pentobarbital sodium (60 mg/kg i.p.), followed by intracolonic injection of CCN1 cDNA/in vivo Jet mixture (100 μg/200 μl/mice) (day 0). Mice were returned to consciousness and provided with 5% DSS in their drinking water or water alone for 5 days ad libitum. A separate group of mice were injected with the same amount of enhanced green fluorescent protein (EGFP)-expressing plasmid. CCN1 transfection was repeated on day 3 to increase transfection efficiency. Colon tissues were embedded in OCT solution. Frozen sections were made, fixed in 10% formalin, and permeabilized using 0.5% Triton X-100. After incubation with blocking buffer, slides were incubated with a rabbit polyclonal anti-CCN1 antibody, anti-green fluorescent protein (GFP) antibody, or 4,6-diamidino-2-phenylindole stain (blue emission signal to stain nuclei) overnight at 4°C. Samples were then washed with PBS and stained by rhodamine (red) or fluorescein isothiocyanate (green)-conjugated secondary antibodies for 1 hour. Slides were then rinsed and mounted with 4,6-diamidino-2-phenylindole mounting solution. Images were analyzed with an Axioskop-2 microscope (Carl Zeiss, Thornwood, NY). HIMECs (5 × 103 cells/well) were seeded in 96-well plates coated with ECMatrix (in vitro angiogenesis kit, no. ECM625; Millipore). In co-culture experiments, NCM460-NK-1R colonocytes were pretransfected with control or CCN1 siRNA as described above and then 1 × 103 cells were added to HIMEC cultures. To block CCN1 activity, anti-CCN1 antibody (Santa Cruz Biotechnology) or rabbit control IgG were added to the cell culture at 20 μg/ml. SP or CCN1 was added shortly after cell seeding and incubated at 37°C for 8 hours. The pattern of angiogenesis (tube formation) was observed under a phase contrast light microscope and the images were recorded.23Madri JA Pratt BM Endothelial cell-matrix interactions: in vitro models of angiogenesis.J Histochem Cytochem. 1986; 34: 85-91Crossref PubMed Scopus (145) Google Scholar Quantitative evaluation of angiogenesis was done in multiple wells of the same group to provide an average scoring of vascular patterns as suggested by the manufacturer in the following: 0, individual separated cells; 1, cells begin to migrate and align; 2, capillary tubes visible but no sprouting; 3, sprouting of capillary tubes visible; 4, closed polygon formation, and 5, complex mesh-like structure development. HIMEC migration was assessed by a standard Transwell migration system using the Chemicon ECM508 assay kit (Chemicon, Temecula, CA).24Goligorsky MS Budzikowski AS Tsukahara H Noiri E Co-operation between endothelin and nitric oxide in promoting endothelial cell migration and angiogenesis.Clin Exp Pharmacol Physiol. 1999; 26: 269-271Crossref PubMed Scopus (93) Google Scholar Briefly, polycarbonate filter inserts (8-μm pore size) serving as upper chambers were seeded with HIMECs (1 × 106 cells/ml in 150 μl of serum-free media) for migration to lower chamber with serum-added media. Filtered (0.22 μm, Millipore) human mucosal extracts from inflamed and unaffected areas of IBD patients (40 μg/ml) were preincubated (30 minutes, 37°C) with 20 μg/ml anti-VEGF antibody, anti-integrin αVβ3 antibody (Upstate Technology, Lake Placid, NY), anti-CCN1 antibody (Santa Cruz Biotechnology), or mouse IgG and then added to the lower chamber. In the case of HIMEC and NCM460-NK-1R co-culture, an additional 1 × 104 CCN1/control siRNA-transfected NCM460-NK-1R colonocytes were added into the lower chamber. SP or CCN1 was added to the lower chamber with anti-CCN1 antibody (20 μg/ml) or anti-VEGF antibody (20 μg/ml) and incubated at 37°C for 16 hours. For control experiments, NCM460-NK-1R cells were seeded onto the upper chamber in the absence of HIMECs. Cells that migrated to the lower chamber were stained and lysed with buffers provided with the kit and cell migration was detected by absorbance at A560. Two hundred μl of Matrigel (BD Biosciences, Bedford, MA) was mixed with 200 μl of PBS containing SP (10−7 mol/L) or CCN1 (1 μmol/L). Colonic mucosa from IBD patients or mouse colon from DSS colitis experiments was homogenized and centrifuged (12,000 × g, 5 minutes, 4°C). Supernatants were collected and aliquots containing 20 μg of protein/ml were mixed with anti-CCN1 or control antibodies (20 μg/ml). The Matrigel mixtures (400 μl) were injected subcutaneously into the laterodorsal abdominal region of 8-week-old male C57/BL6 mice. After 5 days, the mice were sacrificed; and the Matrigel plugs were removed and photographed. The hemoglobin concentration in new blood vessels within the Matrigel was measured by the hemoglobin assay (Sigma) as previously described.25Athanasopoulos AN Schneider D Keiper T Alt V Pendurthi UR Liegibel UM Sommer U Nawroth PP Kasperk C Chavakis T Vascular endothelial growth factor (VEGF)-induced up-regulation of CCN1 in osteoblasts mediates proangiogenic activities in endothelial cells and promotes fracture healing.J Biol Chem. 2007; 282: 26746-26753Crossref PubMed Scopus (83) Google Scholar, 26Norrby K In vivo models of angiogenesis.J Cell Mol Med. 2006; 10: 588-612Crossref PubMed Scopus (232) Google Scholar Results were analyzed using Prism professional statistics software program (Graphpad, San Diego, CA). Analysis of variance was used for intergroup comparisons. Angiogenesis has been recently suggested as an important component of IBD pathophysiology.13Danese S Sans M de la Motte C Graziani C West G Phillips MH Pola R Rutella S Willis J Gasbarrini A Fiocchi C Angiogenesis as a novel component of inflammatory bowel disease pathogenesis.Gastroenterology. 2006; 130: 2060-2073Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar However, there is no evidence to indicate whether the angiogenic factor CCN1 is expressed in human or animal colon and whether its expression is differentially regulated during colitis. We first determined the mRNA levels of various inflammatory and angiogenic mediators in colonic biopsies obtained from inflamed and noninflamed (control) colonic mucosa of IBD patients. As expected,27Goode 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 NK-1R mRNA levels in the inflamed colon of CD and UC patients were significantly elevated by 20- and 10-fold, respectively, compared to the noninvolved colonic sections from the same patients (Figure 1A). SP (TAC1) mRNA levels in the inflamed colon of CD and UC patients were also significantly elevated by 16- and 9-fold, respectively (Figure 1A). CCN1 mRNA levels in inflamed colonic mucosa of CD and UC patients were also significantly increased by 3.9- and 2.8-fold, respectively (Figure 1B). We next examined the importance of CCN1 in angiogenesis associated with IBD using human colonic mucosal extracts from IBD patients to induce migration of HIMECs in Boyden chambers. We found that human colonic mucosal extracts from inflamed IBD colon, but not from noninvolved areas, induced HIMEC migration (Figure 1C). Blockade of CCN1 and its receptor integrin αVβ3 by neutralizing antibodies almost abolished HIMEC migration after neutralization of the known angiogenic factor, VEGF, also inhibited HIMEC migration, but to a lesser degree (Figure 1C). This result suggested the CCN1/αVβ3 system is important in inducing angiogenic function during IBD. Angiogenesis occurs in the colon of animals with experimental colonic inflammation, including DSS-induced colitis.28Danese S Sans M Spencer DM Beck I Donate F Plunkett ML de la Motte C Redline R Shaw DE Levine AD Mazar AP Fiocchi C Angiogenesis blockade as a new therapeutic approach to experimental colitis.Gut. 2007; 56: 855-862Crossref PubMed Scopus (107) Google Scholar Pharmacological antagonism of NK-1R also reduces colonic inflammation during the course of DSS-induced colitis,29Stucchi 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. 2000; 279: G1298-G1306Google Scholar whereas NK-1R expression is also increased in this model.30Reed KL Fruin AB Gower AC Gonzales KD Stucchi AF Andry CD O'Brien M Becker JM NF-kappaB activation precedes increases in mRNA encoding neurokinin-1 receptor, proinflammatory cytokines, and adhesion molecules in dextran sulfate sodium-induced colitis in rats.Dig Dis Sci. 2005; 50: 2366-2378Crossref PubMed Scopus (58) Google Scholar As also shown in Figure 2A, we observed strong CCN1 expression (green) in the colon of DSS-administered mice that was reduced by NK-1 receptor antagonist CJ-12255 treatment (Figure 2A). CCN1 staining was very low in water-treated mice and not affected by CJ-12255 treatment (Figure 2A). Using endothelial-specific vWF staining we found many dilated blood vessels in the colon 5 days after DSS administration, compared to water-treated mice (Figure 2A). To examine whether SP and its receptor are involved in this response we treated animals with the NK-1R antagonist CJ-12255. NK-1R antagonism decreased blood vessel density in colonic tissues of DSS-exposed
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