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Published online before print July 3, 2008
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From the Gastrointestinal Neuropeptide Center,* Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts; the Inflammatory Bowel Disease Center, Division of Digestive Diseases, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California; and INCELL Corporation, San Antonio, Texas
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Abstract |
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vβ3. 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.
B-regulated cytokine genes.9,10
SP-NK-1R interaction also induces proliferation of colonic epithelial cells that involves activation of metalloproteinases and transactivation of the epithelial growth factor receptor.11
SP also exerts an anti-apoptotic effect in colonic epithelial cells via Akt phosphorylation in vitro and in vivo.12 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).13 In both CD and UC mucosa, the local microvasculature undergoes intensive inflammation-dependent angiogenesis. Evidence from a sponge angiogenesis model14 and synovial inflammation in rats indicates that SP may mediate angiogenesis in nonintestinal tissues.18-21 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 proteins15 that stimulate mitosis, adhesion, apoptosis, extracellular matrix, angiogenesis, and tumor growth.16 Recent data indicate that CCN1, -2, and -3 bind to cell surface integrins and thereby activate intracellular signaling, including kinase phosphorylation and gene transcription.15 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.17,18 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.
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Materials and Methods |
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Colon samples from UC and CD patients were obtained as previously described.19 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.
Quantitative Real-Time Polymerase Chain Reaction (PCR) Analyses
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.
Cell Cultures
NCM460 cells overexpressing NK-1R (NCM460-NK-1R), previously generated by us,9-12 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)13 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.
Mouse Colon Tissue Preparation
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.9,11,12 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.
Western Blot Analyses
Cells were lysed in 1x 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, CCN120 (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).
Promoter Activity of CCN1 Gene
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 x 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.9 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).
CCN1 Knockdown by siRNA Transfection
NCM460-NK-1R colonocytes were seeded in 12-well plates (2 x 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.9,10,12 Reduced endogenous CCN1 expression was confirmed by Western blot analyses.
Immunohistochemistry of von Willebrand Factor (vWF) for Endothelial Cell Staining
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.13,21,22
In Vivo Transfection of CCN1
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.
Immunofluorescent Staining of CCN1 and EGFP
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).
In Vitro Angiogenesis
HIMECs (5 x 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 x 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.23 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.
Endothelial Cell Migration
HIMEC migration was assessed by a standard Transwell migration system using the Chemicon ECM508 assay kit (Chemicon, Temecula, CA).24
Briefly, polycarbonate filter inserts (8-µm pore size) serving as upper chambers were seeded with HIMECs (1 x 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 Vβ3 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 x 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.
In Vivo Matrigel Plug Angiogenesis Assay
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 x 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.25,26
Statistical Analyses
Results were analyzed using Prism professional statistics software program (Graphpad, San Diego, CA). Analysis of variance was used for intergroup comparisons.
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Results |
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Angiogenesis has been recently suggested as an important component of IBD pathophysiology.13
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,27
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 Vβ3 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/Vβ3 system is important in inducing angiogenic function during IBD.
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Angiogenesis occurs in the colon of animals with experimental colonic inflammation, including DSS-induced colitis.28
Pharmacological antagonism of NK-1R also reduces colonic inflammation during the course of DSS-induced colitis,29
whereas NK-1R expression is also increased in this model.30
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 mice (Figure 2A)
. In contrast, CJ-12255 did not affect vascular patterns in water-treated mice (Figure 2A)
. Image analyses of vWF staining demonstrated that CJ-12255 reduced blood vessel density by 50% (Figure 2B)
. Thus, SP, via NK-1R, mediates angiogenic and inflammatory responses during colitis. Western blot analyses of colonic tissues indicate that CJ-12255 significantly reduced colonic CCN1 induction by DSS (Figure 2, C and D)
, suggesting that SP modulates CCN1 expression in vivo.
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We next examined whether SP-induced CCN1 expression in NCM460-NK-1R colonocytes. We found that SP stimulated CCN1 protein expression in both cell lysates and cell-conditioned media at 2 to 8 hours and then decreased by 24 hours (Figure 3A)
. SP stimulated CCN1 expression in a dose-dependent manner starting from 10–8 mol/L (Figure 3B)
. Consistent with these results, SP also induced a time- and dose-dependent increase of CCN1 promoter activity (Figure 3, C and D)
. Real-time RT-PCR analysis in RNA purified from HIMECs failed to identify CCN1 expression with or without SP (10–7 mol/L) exposure (data not shown).
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Although CCN1 mediates angiogenesis in endothelial cells such as human umbilical vein endothelial cells31
and HIMECs32
via integrin Vβ3 signaling, whether SP can mediate angiogenesis in the intestinal vasculature has not been investigated. We exposed HIMECs cultured on ECMatrix to CCN1 or SP for 8 hours and found that CCN1, but not SP, induced formation of capillary tubes that connected independent HIMEC colonies (Figure 4A)
. Moreover, co-culture of HIMEC and NK-1R-transfected NCM460 cells failed to induce any capillary tube formation (Figure 4A)
. However, stimulation of HIMEC-NCM460-NK-1R co-cultured cells with SP resulted in formation of capillary tubes, which was inhibited by silencing of CCN1 in colonocytes (Figure 4, A and B)
. Successful knockdown of CCN1 in colonocytes is shown in Supplementary Figure S1A at http://ajp.amjpathol.org. These results indicate that SP increases expression of functional CCN1 in colonic epithelial cells, which in turn stimulates angiogenesis in HIMECs.
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Next we determined whether SP or CCN1 affects endothelial cell migration. For this purpose, HIMECs were seeded into the upper compartment of Boyden chambers and the migration of these cells to the lower chamber was examined after SP or CCN1 exposure. The data show that CCN1, but not SP, stimulated HIMEC migration (Figure 4, C and D)
that was inhibited by anti-CCN1, but not anti-VEGF antibody. This suggests CCN1 mediates direct angiogenic effects without involvement of VEGF. Moreover, SP had no migratory effect in NCM460-NK-1R colonocytes (Figure 4C)
. However, co-culture of HIMECs with NCM460-NK-1R accelerated HIMEC migration in response to SP that was blocked by knockdown of CCN1, NK-1R, or anti-CCN1 antibody but not anti-VEGF antibody in colonocytes (Figure 4, C and D)
. CCN1 silencing reduced SP-induced CCN1 protein expression (see Supplementary Figure S1A at http://ajp.amjpathol.org). Also NK-1R silencing reduced NK-1R expression as well as SP-induced CCN1 protein expression (see Supplementary Figure S1B at http://ajp.amjpathol.org). Thus, SP-associated pro-angiogenic responses are mediated via CCN1 released from colonic epithelial cells.
The angiogenic effects of CCN1 are mediated via binding to its cell surface receptor integrin Vβ3.15
However, there is no evidence indicating a role for this integrin in modulating CCN1 activity in HIMECs, whereas the effect of SP in this response has never been examined. To determine the function of CCN1 secreted from SP-stimulated colonocytes, we first stimulated NCM460-NK-1R with SP for 6 hours; conditioned media were then collected and added into HIMEC culture. As shown in Figure 4E
, conditioned media from SP-stimulated NCM460-NK-1R cells induced rapid integrin β3 phosphorylation in HIMECs and this response was attenuated by the Vβ3 neutralizing antibody LM609, and an anti-CCN1 antibody. Thus, SP stimulates colonocytes to release functional CCN1 that binds to integrin Vβ3 and mediates integrin β3 phosphorylation in HIMECs.
CCN1 Mediates Angiogenesis in Vivo
To further characterize the angiogenic effects of SP and CCN1 in vivo, we performed Matrigel angiogenesis assay. Matrigel plugs, containing different angiogenic factors indicated in Figure 5A
, were first implanted into the flank skin space of mice, and after 5 days, plugs were removed and photographed. The data show that, unlike control Matrigel plugs that did not show significant blood vessel formation, Matrigel plugs filled with CCN1, but not SP, induced neovascularization (Figure 5A)
. These results confirmed that SP is not a direct angiogenic factor. Colonic extracts from DSS-treated mice (5% DSS for 5 days) also induced angiogenesis (Figure 5A)
. However, angiogenesis was significantly less evident when inflamed colonic extracts from DSS-exposed mice injected with CJ-12255 were used (Figure 5A)
. Similarly, human colonic mucosal extracts from inflamed, but not noninvolved colon of IBD patients also induced angiogenesis in mice in vivo (Figure 5A)
. In addition, colonic mucosal extracts from DSS-exposed mice and IBD patients induced angiogenesis that was diminished by addition of an anti-CCN1 antibody in the Matrigel. Quantitation of blood vessels infiltrated into the Matrigel measured by hemoglobin assay confirmed this response (Figure 5B)
.
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To directly demonstrate an angiogenic role of CCN1 in the colon, we transfected CCN1 or control, EGFP-overexpressing constructs into mouse colon in vivo followed by treatment with 5% DSS for 5 days. In CCN1-transfected mice, we observed increased CCN1 expression (red), whereas transfection of EGFP into mouse colon resulted in increased colonic expression of GFP (green) (Figure 6A)
, indicating successful transfection of these genes. As shown by endothelial-specific vWF immunohistochemistry (Figure 6A)
, CCN1-transfected, water-treated mice had more blood vessels along the submucosal layer, compared to EGFP-transfected, water-treated mice, indicating that CCN1 induced angiogenesis in the colon (Figure 6, A and B)
. As expected, EGFP-transfected DSS-treated mice showed higher number of vascular networks in the colonic submucosa (Figure 6, A and B)
, and quantification of blood vessels confirmed this response (Figure 6, A and B)
. All CCN1-transfected mice had increased CCN1 protein expression, compared to EGFP-transfected mice (Figure 6, C and D)
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Discussion |
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Although some neuropeptides, such as, ie, neuropeptide Y (NPY) and gastrin,33,34 are associated with angiogenesis-related responses in nonintestinal tissues, there is little evidence that neuropeptides are involved in angiogenesis in the intestinal tract. Most notably, there are no studies to link neuropeptides with angiogenesis in the pathophysiology of intestinal inflammation. SP regulates vascular tone and blood flow and initiates angiogenesis in the cardiovascular system35 and also mediates angiogenesis in synovial inflammation36 and chronic airway inflammation.37 Results presented in this report strongly suggest that SP mediates angiogenesis during colitis, and this event is involved in the pathophysiology of IBD.
Human umbilical vein endothelial cells express NK-1R38 and SP exposure induces human umbilical vein endothelial cell migration, suggesting angiogenic activity.39 SP also induces COX-2 expression40 and neutrophil adhesion41 in these cells. Our results show that HIMECs do not respond to SP directly. Consistent with these results, real-time PCR experiments indicate that HIMECs express very low levels of NK-1R mRNA, whereas Western blot analysis failed to identify NK-1R protein in these cells (data not shown). Along these lines, HIMECs have been previously shown to have different functional properties compared to human umbilical vein endothelial cells.42
Our results indicate that SP stimulates release of CCN1 from colonocytes that could be detected in the conditioned media of these cells (Figure 3)
. A recent report also suggested that, to exert angiogenic activity, CCN1 is released into cell media via protease cleavage, such as tissue plasminogen activator (tPA).45
Interestingly, SP causes a rapid and substantial local release of tPA when injected in human forearm.46
There are some reports indicating functional interactions between CCN1 and VEGF toward angiogenesis.17
CCN1 and VEGF-C knockout mice share similar phenotypes of compromised vascular integrity, leading to embryonic death. VEGF can stimulate CCN1 expression25
whereas CCN1 can induce expression of VEGF-C and thus CCN1 knockout mice have impaired VEGF-C expression.17
Although we did not perform experiments to directly examine the possibility that VEGF-CCN1 interactions are involved in the angiogenic responses in our experimental system, our data presented in Figure 4D
indicate that VEGF might not play a role in SP-induced angiogenesis and endothelial cell migration.
Recent studies suggest that angiogenesis is an important component of colonic inflammation. Several angiogenic factors such as vascular endothelial growth facto, basic fibroblast growth factor, and interleukin-8 are elevated in the colon of IBD patients.13
However, the potential input of angiogenic factors in the development of colitis, both during the acute and the repair or healing phase are not fully understood. Angiogenesis during inflammation will increase the influx of inflammatory cells, help provide nutrients to metabolically active immune cells, and transport cytokines and chemokines in the circulation.45,46
For example, anti-angiogenic therapy with ATN-161, a novel integrin (5)β(1) antagonist, inhibits DSS-induced colitis,28
consistent with the notion for a proinflammatory role of angiogenesis in the development of colonic inflammation. In addition, evidence indicates that temporal expression of VEGF and angiopoietin-1 can accelerate healing during experimental colitis.48
We did not examine whether CCN1 can also mediate mucosal healing after colitis. However, CCN1 has been shown to stimulate proliferation in human skin fibroblasts, and to activate anti-apoptotic pathways in pulmonary epithelial cells.48
In conclusion, we have identified CCN1 as an important angiogenic factor that might promote angiogenesis in experimental colitis and possibly IBD. Our data also present evidence that the neuropeptide SP via its high-affinity NK-1R mediates angiogenesis by inducing CCN1 secretion from colonocytes. These results provide a novel neuropeptide-colonocyte-mucosal endothelial cell paradigm involved in angiogenesis-related intestinal inflammation with putative implications in IBD pathophysiology.
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health (grant DK 47343 to C.P.) and the Crohn’s and Colitis Foundation of America, Inc. (research fellowship award to H.W.K.).
Supplemental material for this article can be found on http://ajp. amjpathol.org.
Accepted for publication May 1, 2008.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
45 kDa) and media (
