Volume 122, Issue 2, Pages 458-468 (February 2002) Esophageal ulceration activates keratinocyte growth factor and its receptor in rats: Implications for ulcer healing  Dolgor Baatar, Hirofumi Kawanaka, Imre L. Szabo, Rama Pai, Michael K. Jones, Seigo Kitano, Andrzej S. Tarnawski  Gastroenterology  Volume 122, Issue 2, Pages 458-468 (February 2002) DOI: 10.1053/gast.2002.31004 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Macroscopic appearance of acetic acid–induced esophageal ulcers. Esophagus was dissected and opened longitudinally. (A) At 3 days, ulcer appears as an oval-shaped mucosal defect with clearly defined margins (arrows) and necrotic tissue (NT) present at the ulcer base. (B) At 9 days, ulcer was re-epithelialized and a mucosal scar was formed (arrow). Scale is marked in millimeters. Gastroenterology 2002 122, 458-468DOI: (10.1053/gast.2002.31004) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Ulcer healing dynamics. Ulcer area was measured 3, 6, and 9 days after ulcer induction by a computerized video analysis of the ulcer images. Values are mean ± SEM. For each column, n = 6. Gastroenterology 2002 122, 458-468DOI: (10.1053/gast.2002.31004) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Photomicrographs of esophageal ulcer (H&E stain) at (A) 1 day, (B) 3 days, (C) 6 days, and (D) 9 days after ulcer induction. NT, necrotic tissue; E, epithelium; SM, submucosa; MP, muscularis propria; GT, granulation tissue. Open arrows indicate muscularis mucosae, and closed arrows indicate basal zone (bar = 500 μm). Gastroenterology 2002 122, 458-468DOI: (10.1053/gast.2002.31004) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 RT-PCR analysis of KGF and KGF-R mRNA expression in ulcerated esophageal tissue (UL) versus nonulcerated esophageal tissue from sham-operated (SO) rats 3 and 9 days after ulcer induction or sham operation. (A) RT-PCR products obtained with use of specific primers for KGF (570 base pairs), KGF-R (342 base pairs), and β-actin. (B) Quantitative data for KGF mRNA expression. (C) Quantitative data for KGF-R mRNA expression. Data were obtained by computerized analysis of amplified PCR products. Each signal was normalized against the corresponding β-actin signal, and the results are expressed as KGF/β-actin and KGF-R/β-actin. Values are means ± SEM. For each column, n = 6. Gastroenterology 2002 122, 458-468DOI: (10.1053/gast.2002.31004) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 Western blot analysis of KGF, KGF-R, and phosphorylated KGF-R (pKGF-R) protein expression in ulcerated esophageal tissue (UL) versus nonulcerated esophageal tissue from sham-operated (SO) rats 3 and 9 days after ulcer induction or sham operation. KGF was immunoprecipitated before blotting as described in Materials and Methods. (A) Immunoblotting with specific antibodies detected the specific bands for KGF, KGF-R, and pKGF-R. (B) Quantitative data for KGF protein expression. (C) Quantitative data for KGF-R protein expression. (D) Quantitative data for pKGF-R protein expression. Data were obtained by a computerized video analysis of the Western blots. Values are expressed in intensity units and represent means ± SEM. For each column, n = 6. Gastroenterology 2002 122, 458-468DOI: (10.1053/gast.2002.31004) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Photomicrographs of esophageal tissue showing immunofluorescence staining for KGF 6 days after ulcer induction or sham operation. (A) KGF expression in nonulcerated esophagus from sham-operated rats. (B) KGF expression in the esophageal tissue adjacent to the ulcer. E, epithelium; LP, lamina propria; SM, submucosa; MP, muscularis propria. Arrows indicate muscularis mucosae (bars = 200 μm). Gastroenterology 2002 122, 458-468DOI: (10.1053/gast.2002.31004) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Photomicrographs of esophageal tissue showing immunofluorescence staining for KGF-R 6 days after ulcer induction or sham operation. (A) KGF-R expression in nonulcerated esophageal epithelium from sham-operated rats. (B) KGF-R expression in the epithelium of the ulcer margin. (C) KGF-R expression at the ulcer margin. (D) KGF-R expression in the epithelium distant from the ulcer (microscopic field next to that in C). BZ, basal zone; E, epithelium; NT, necrotic tissue; GT, granulation tissue (bars: A and B, 50 μm; C and D, 200 μm). Gastroenterology 2002 122, 458-468DOI: (10.1053/gast.2002.31004) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Effects of exogenous KGF injection on ulcer healing. Ulcer area was measured by a computerized video analysis of the ulcer images before (3 days after ulcer induction) and 3 days after injection of recombinant human KGF (1 mg/kg body wt) or its vehicle (VHC) into the submucosa around the ulcer (6 days after ulcer induction). Values are means ± SEM. For each column, n = 6. Gastroenterology 2002 122, 458-468DOI: (10.1053/gast.2002.31004) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 Photomicrographs of rat esophageal epithelium 1 cm distant from ulcer and epithelium of the ulcer margin immunostained for PCNA. Rats were treated with local injections of either recombinant human KGF (1 mg/kg body wt) or its vehicle. (A) Epithelium distant from the ulcer in rats treated with vehicle. (B) Epithelium distant from the ulcer in rats treated with KGF. (C) Epithelium of the ulcer margin in rats treated with vehicle. (D) Epithelium of the ulcer margin in rats treated with KGF. PCNA expression is present in the cell nuclei (dark staining) (bars = 100 μm). Gastroenterology 2002 122, 458-468DOI: (10.1053/gast.2002.31004) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 10 Quantitative data for PCNA expression in esophageal epithelium distant from the ulcer (distant) and epithelium of the ulcer margin (margin) in rats treated with human recombinant KGF (1 mg/kg body wt) or its vehicle (VHC). The results are expressed as the percentage of positively stained cells in a total number of cells. LI, labeling index. *P < 0.05 vs. distant of the same treatment group. Values are means ± SEM. For each column, n = 6. Gastroenterology 2002 122, 458-468DOI: (10.1053/gast.2002.31004) Copyright © 2002 American Gastroenterological Association Terms and Conditions