Volume 138, Issue 7, Pages 2457-2467.e5 (June 2010) Corticotropin-Releasing Hormone Family of Peptides Regulates Intestinal Angiogenesis  Eunok Im, Sang Hoon Rhee, Yong Seek Park, Claudio Fiocchi, Yvette Taché, Charalabos Pothoulakis  Gastroenterology  Volume 138, Issue 7, Pages 2457-2467.e5 (June 2010) DOI: 10.1053/j.gastro.2010.02.055 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Dextran sodium sulfate (DSS)–induced colitis is reduced in corticotropin-releasing hormone receptor (CRHR)1−/− mice but increased in CRHR2−/− mice compared with littermate controls. Mice were fed with 4% DSS for 14 days. (A, C) Difference in the survival is shown by the Kaplan–Meier plot. The log-rank test indicates there are significant survival differences between CRHR1−/− and CRHR1+/+ mice (n = 15 mice per genotype, P = .001), as well as between CRHR2−/− and CRHR2+/+ mice (n = 15 mice per genotype, P < .0001). (B, D) Body weight data are mean ± standard deviation (CRHR1: n = 12–15 mice per genotype; CRHR2: n = 6–11 mice per genotype). *P < .05 vs controls. Two-way analysis of variance results show a significant genotype-treatment interaction; P < .0001 (B, D). Gastroenterology 2010 138, 2457-2467.e5DOI: (10.1053/j.gastro.2010.02.055) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Histological damage and inflammatory cytokine production induced by dextran sodium sulfate (DSS) are reduced in corticotropin-releasing hormone receptor (CRHR)1−/− mice but increased in CRHR2−/− mice. (A, B) Representative images of H&E-stained sections from the mice fed with 4% DSS for 7 days indicate reduced tissue damage in CRHR1−/− mice, but increased tissue damage in CRHR2−/− mice compared with controls. Original magnification: 50× in the upper panel and 200× in the lower panel. Scale bar = 50 μm. (C) Ulcers, leukocyte infiltration, and edema are decreased in CRHR1−/− mice, but increased in CRHR2−/− mice with colitis compared with controls. Data are mean ± standard deviation (n = 8 mice per genotype). *P < .05 vs controls. (D–F) Enzyme-linked immunosorbent assay was performed to measure mouse keratinocyte-derived chemokine (KC) (D), interleukin (IL)-6 (E), and tumor necrosis factor–α (TNFα) (F) levels in tissues from DSS-treated CRHR1−/−, CRHR2−/−, and control mice. Data are mean ± standard deviation (n = 3–7 mice per genotype). *P < .05 vs controls. Gastroenterology 2010 138, 2457-2467.e5DOI: (10.1053/j.gastro.2010.02.055) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 The corticotropin-releasing hormone receptor (CRHR)1 or CRHR2 antagonist attenuates or worsens dextran sodium sulfate (DSS)–induced colitis, respectively. (A, B) DSS-induced mortality and body weight loss are reduced in mice (CD1) with a CRHR1-specific antagonist, antalarmin (Antal, 20 mg/kg) but increased in mice with a CRHR2-specific antagonist, astressin 2B (Ast2B, 30 μg/kg) compared with the vehicle control. (A) Difference in the survival is shown by Kaplan–Meier plot. The log-rank test followed by the multiple-comparison Bonferroni method shows that there are significant survival differences between water versus DSS, water versus DSS plus Ast2B, DSS versus DSS plus Antal, and DSS versus DSS plus Ast2B treated mice (n = 4–10 mice per genotype, P < .05). (B) Body weight data are mean ± standard deviation (SD) (n = 4–10 mice per genotype). (C) Ulcers, leukocyte infiltration, and edema are reduced by Antal, but increased by Ast2B in mice with colitis. Data are mean ± SD (n = 4–8 mice per genotype). (D–F) Enzyme-linked immunosorbent assay was performed to measure mouse keratinocyte-derived chemokine (KC) (D), interleukin (IL)-6 (E), and tumor necrosis factor–α (TNFα) (F) levels in the colon from DSS-treated mice with Antal or Ast2B or vehicle for 7 days. Data are mean ± SD (n = 4–7 mice per genotype). The results of 1-way analysis of variance followed by a Newman–Keuls post-hoc test show a significant difference compared to DSS-alone–treated mice; *P < .05 (B–D). Gastroenterology 2010 138, 2457-2467.e5DOI: (10.1053/j.gastro.2010.02.055) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Increased versus decreased vascular endothelial growth factor (VEGF)-A levels during colitis in corticotropin-releasing hormone receptor (CRHR)2−/− versus CRHR1−/− mice. VEGFR2 blockade reduces colitis in CRHR2−/− mice. (A, B) Enzyme-linked immunosorbent assay was performed to measure VEGF-A levels in the colon from dextran sodium sulfate (DSS)–treated (for 7 days) CRHR1−/−, CRHR2−/−, and control mice. Data are mean ± standard deviation (SD) (n = 6–7 mice per genotype). *P < .05 vs controls. (C, D) DSS-induced colitis is reduced in CRHR2−/− mice injected intraperitoneally with Ki8751 (Ki, 10 mg/kg) compared with the vehicle control. Mice were fed with 4% DSS for 14 days. (C) Difference in the survival is shown by Kaplan–Meier plot. The log-rank test indicates there are significant survival differences between CRHR2−/− mice with Ki and CRHR2−/− mice (n = 19 mice per genotype, P < .001). (D) Body weight data are mean ± SD (n = 15 mice per genotype). *P < .05 vs CRHR2−/− mice. (E, with quantification in F) Immunohistochemistry on CD31 demonstrates that microvascular density is reduced in CRHR2−/− mice with Ki. H&E staining also shows reduced tissue damage in the Ki-treated mice compared with vehicle-treated mice. Scale bar = 50 μm. NC, negative control. Gastroenterology 2010 138, 2457-2467.e5DOI: (10.1053/j.gastro.2010.02.055) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Deletion of corticotropin-releasing hormone receptor (CRHR)1 impairs the vessel outgrowth from aortic explants, whereas deletion of CRHR2 enhances it. (A) Aortic explants from CRHR1−/−, CRHR2−/−, and control mice were embedded in Matrigel and cultured for up to 14 days with mouse vascular endothelial growth factor (VEGF) (25 ng/mL). Representative images from 3 independent experiments are shown. Scale bar = 200 μm. (B) Average vessel length on samples pooled from 3 independent experiments is shown as mean ± standard deviation (n = 3 mice per genotype). *P < .005 vs wild-type littermates. Gastroenterology 2010 138, 2457-2467.e5DOI: (10.1053/j.gastro.2010.02.055) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Corticotropin-releasing hormone (CRH) augments tube formation, but urocortin (Ucn) III inhibits it. (A) Quantitative real-time polymerase chain reaction analyses reveal that human intestinal microvascular endothelial cells (HIMECs) express both corticotropin-releasing hormone receptor (CRHR)1 and CRHR2, but not CRH or Ucn III. The complementary DNA from human NCM460 colonocytes was used as a calibrator. ND, not detected. (B, with quantification in C) CRH increases spontaneous tube formation but Ucn III decreases it. The HIMECs were subjected to a tube formation assay and then medium containing CRH (10 nM) or Ucn III (10 nM) or vehicle was added for 5 hours. Representative photos from 5 independent experiments are shown. Scale bar = 100 μm. *P < .05 vs vehicle. (D) CRH or Ucn III–induced tube responses are diminished in the presence of antalarmin (1 nM) or astressin 2B (100 nM). The antagonists were added for 30 minutes prior to the addition of CRH or Ucn III or vehicle. *P < .05 vs CRH. #P < .05 vs Ucn III. (E) The result of XTT assays indicates that CRH increases cell proliferation but Ucn III decreases it. Data are mean ± standard deviation (SD). *P < .05 vs vehicle. (F) The results of wound injury assays (24 hours) indicate that CRH enhances wound closure but Ucn III delays it. Representative photos of 3 independent experiments are shown. Scale bar = 100 μm. *P < .05 vs vehicle. Data are mean ± SD. The results of 1-way analysis of variance followed by a Newman–Keuls post-hoc test show a significant difference compared to vehicle (C, E, F). Gastroenterology 2010 138, 2457-2467.e5DOI: (10.1053/j.gastro.2010.02.055) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Corticotropin-releasing hormone (CRH) stimulates the phosphatidylinositol 3 kinase (PI3K) pathway, whereas urocortin (Ucn) III inhibits it in human intestinal microvascular endothelial cells (HIMECs). (A, with quantification in B) Western blot analysis shows concentration-dependent increase in phospho-Akt by CRH (10 nM, 15 minutes), but decrease by Ucn III (10 nM, 15 minutes). (C) Inhibition of PI3K activity by LY294002 (LY) diminishes tube formation by CRH. The HIMECs were subjected to a Matrigel tube formation assay and then medium containing LY294002 (5 μM) was added for 30 minutes prior to the addition of CRH (10 nM) or vehicle for 5 hours. *P < .05 vs CRH. (D) Synthetic lipid substrate of PI3K rescued the inhibition of tube response by Ucn III. HIMECs were subjected to a tube assay. A mixture of synthetic lipids (PI45P or PI3P) and histone was added simultaneously with Ucn III (10 nM). *P < .05 vs Ucn III plus PI3P. Data are mean ± standard deviation. The results of 1-way analysis of variance followed by a Newman–Keuls post-hoc test show a significant difference (B, C). Gastroenterology 2010 138, 2457-2467.e5DOI: (10.1053/j.gastro.2010.02.055) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 (A, B) Genetic deletion of corticotropin-releasing hormone receptor (CRHR)1 or CRHR2 does not induce any changes in natural weight gains. Body weight data are mean ± standard deviation (SD) (n = 4 mice per genotype). There is no significant difference compared with wild-type littermates. In addition, we did not find any difference in basal colonic architecture. (C, D) The basal expression levels of cytokines were not different between knockout mice and wild type littermates. Data are mean ± SD (n = 3 mice per genotype). There is no significant difference compared with wild type littermates. Gastroenterology 2010 138, 2457-2467.e5DOI: (10.1053/j.gastro.2010.02.055) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 (A) Genetic deletion of corticotropin-releasing hormone receptor (CRHR)1 or CRHR2 does not show any difference in the basal expression level of vascular endothelial growth factor (VEGF)–A compared with controls. Data are mean ± standard deviation (SD) (n = 3 mice per genotype). (B) Microvascular density (CD31) is reduced in CRHR1−/− mice but increased in CRHR2−/− mice compared with controls. H&E staining shows reduced tissue damage in CRHR1−/− mice but increased tissue damage in CRHR2−/− mice compared with controls. (C–E) Colitis is reduced in CRHR2+/+ mice injected intraperitoneally with Ki8751 (Ki, 10 mg/kg) compared with CRHR2+/+ mice injected with vehicle. (C) Difference in the survival is shown by Kaplan–Meier plot. The log-rank test indicates there are significant survival differences between CRHR2+/+ mice with Ki and CRHR2+/+ mice (n = 9 mice per genotype, P < .05). (D) Body weight data are mean ± SD (n = 9 mice per genotype). *P < .05 vs CRHR2+/+ mice. (E) Ki reduces microvascular density (CD31) in CRHR2−/− mice. H&E staining also shows reduced tissue damage in the Ki-treated mice compared with vehicle-treated mice. Scale bar = 50 μm. NC, negative control. Gastroenterology 2010 138, 2457-2467.e5DOI: (10.1053/j.gastro.2010.02.055) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 3 Time-lapse images of human intestinal microvascular endothelial cells (HIMEC) tube formation. HIMECs were subjected to Matrigel tube formation assays, and then culture medium containing corticotropin-releasing hormone (CRH) (10 nM) was added. Time-lapse images are captured by ZEISS observer D1 microscope every 30 minutes over the course of 5.5 hours and serial images are presented. Gastroenterology 2010 138, 2457-2467.e5DOI: (10.1053/j.gastro.2010.02.055) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 4 (A, B) Production of pro-angiogenic factors in human intestinal microvascular endothelial cells (HIMECs) is not affected by corticotrophin-releasing hormone (CRH) or urocortin (Ucn) III stimulation. Enzyme-linked immunosorbent assay was performed to measure human fibroblast growth factor (A) and interleukin-8 (IL-8) (B) levels in HIMECs treated with CRH (10 nM for IL-8) or Ucn III (10 nM of IL-8) for 8 hours. Data are mean ± standard deviation. Differences are not statistically significant. Gastroenterology 2010 138, 2457-2467.e5DOI: (10.1053/j.gastro.2010.02.055) Copyright © 2010 AGA Institute Terms and Conditions