Substance P-Mediated Expression of the Pro-Angiogenic Factor CCN1 Modulates the Course of Colitis  Hon-Wai Koon, Dezheng Zhao, Hua Xu, Collin Bowe, Alan Moss, Mary P. Moyer, Charalabos Pothoulakis  The American Journal of Pathology  Volume 173, Issue 2, Pages 400-410 (August 2008) DOI: 10.2353/ajpath.2008.080222 Copyright © 2008 American Society for Investigative Pathology Terms and Conditions

Figure 1 CCN1 is overexpressed during colonic inflammation and mediates angiogenesis. Colon tissues of UC and CD patients were used to perform real-time RT-PCR to measure NK-1R and SP gene expression levels (A), CCN1 (B). The change of gene expression was compared to adjacent normal regions and normalized by GAPDH expression. Each group of data represented eight matching pairs of samples from human patients. ***P < 0.001, **P < 0.01; ###P < 0.001, ##P < 0.01, and #P < 0.05 versus respective normal colon. C: Forty μg/ml of human colonic mucosal extracts from inflamed and matching unaffected normal areas with 20 μg/ml of control IgG, anti-VEGF, anti-CCN1, and anti-αVβ3 neutralizing antibodies were added to the lower chamber of a modified Boyden chamber. HIMECs that migrated to the lower chamber were measured as described in Materials and Methods. ***P < 0.001, *P < 0.05 versus IgG-treated IBD group. The American Journal of Pathology 2008 173, 400-410DOI: (10.2353/ajpath.2008.080222) Copyright © 2008 American Society for Investigative Pathology Terms and Conditions

Figure 2 SP mediates angiogenesis and angiogenic factor expression in vivo during DSS colitis. A: Mice were treated with 5% DSS added to their drinking water or water alone for 5 days. The NK-1R receptor antagonist CJ-12255 was administered to block NK-1R. Colon tissues were obtained for 4,6-diamidino-2-phenylindole, CCN1, and vWF staining to visualize CCN1 expression and blood vessels. B: Quantitative analyses of blood vessel number per field were performed using Scion Image Analysis software. CCN1 protein was determined by Western blot analysis (C) and its intensity was quantified by densitometry (D). ***P < 0.001 versus PBS-treated DSS colitis group. Results are representative of four mice per group. The American Journal of Pathology 2008 173, 400-410DOI: (10.2353/ajpath.2008.080222) Copyright © 2008 American Society for Investigative Pathology Terms and Conditions

Figure 3 SP induces CCN1 production from colonocytes. A: NCM460-NK-1R colonocytes were exposed to SP (10−8 mol/L) for up to 24 hours. B: NCM460-NK-1R cells were exposed to SP (0 to 10−7 mol/L) for 4 hours. Equal amounts of protein were fractioned by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the levels of CCN1 in cell lysate (∼45 kDa) and media (∼28 kDa) and β-actin. C and D: NCM460-NK-1R cells were transfected with the CCN1 promoter construct, followed by exposure to different concentrations of SP at the indicated times. Cells were lysed and luciferase assays were performed to determine CCN1 promoter activity. ***P < 0.001, **P < 0.01, *P < 0.05 versus respective control group. The results are representative of three experiments. The American Journal of Pathology 2008 173, 400-410DOI: (10.2353/ajpath.2008.080222) Copyright © 2008 American Society for Investigative Pathology Terms and Conditions

Figure 4 SP stimulates in vitro angiogenesis via CCN1 expression from colonocytes. A: HIMECs cultured with or without NCM460-NK-1R were seeded onto ECMatrix and incubated with SP (10−8 mol/L) or CCN1 (10−9 mol/L) for 8 hours. Angiogenesis patterns were recorded by a camera under ×20 magnification. Before the experiment, NCM460-NK-1R cells were pretransfected with control/CCN1 siRNA. B: Angiogenesis patterns were evaluated and quantified according to the scoring system provided by the manufacturer. C and D: HIMECs were seeded onto the upper compartment and NCM460-NK-1R colonocytes were seeded onto the lower compartment of Boyden chambers and treated with SP/CCN1. Cells migrated to the lower compartment were stained, lysed, and absorbance at A560 was determined. **P < 0.01 versus HIMEC migration alone. ##P < 0.01 versus HIMEC +/− NCM460-NK-1R with control siRNA or IgG. E: NCM460-NK-1R colonocytes were stimulated with SP (10−7 mol/L) for 6 hours. SP-conditioned media were filtered (0.22 μm) and then added to HIMEC cultures pretreated with IgG, anti-CCN1, or anti-αVβ3 (LM609) for 15 minutes. Cells were then lysed and used for detection of phospho-β3 integrin by Western blots. The American Journal of Pathology 2008 173, 400-410DOI: (10.2353/ajpath.2008.080222) Copyright © 2008 American Society for Investigative Pathology Terms and Conditions

Figure 5 CCN1 mediates angiogenesis in vivo. A: Matrigel mixed with SP, CCN1, and mucosal extracts from DSS-treated mice or human IBD patients were isolated 5 days after implantation. B: New blood vessel formation in Matrigel was quantified by the hemoglobin assay as described in the Materials and Methods. Results are representative of three independent experiments. The American Journal of Pathology 2008 173, 400-410DOI: (10.2353/ajpath.2008.080222) Copyright © 2008 American Society for Investigative Pathology Terms and Conditions

Figure 6 CCN1 induces colonic angiogenesis. A: Mice (C57/BL6, n = 4 per group) were transfected intracolonically with CCN1- or EGFP-overexpressing constructs and then treated with 5% DSS in their drinking water or water alone for 5 days. Colon tissues were obtained for 4,6-diamidino-2-phenylindole, GFP, CCN1, and vWF staining to visualize CCN1 expression and blood vessels. B: Quantitative analyses of blood vessel number per field were performed using the Scion image analysis software. Levels of CCN1 and β-actin were determined by Western blot analyses (C) and their signal intensities were quantified by densitometry (D). ***P < 0.001, **P < 0.01 versus EGFP-transfected water-treated group. ##P < 0.01 versus EGFP-transfected DSS-treated group. The American Journal of Pathology 2008 173, 400-410DOI: (10.2353/ajpath.2008.080222) Copyright © 2008 American Society for Investigative Pathology Terms and Conditions