Volume 135, Issue 5, Pages 1729-1738 (November 2008) Hepatic Stellate Cells Secrete Angiopoietin 1 That Induces Angiogenesis in Liver Fibrosis  Kojiro Taura, Samuele De Minicis, Ekihiro Seki, Etsuro Hatano, Keiko Iwaisako, Christoph H. Osterreicher, Yuzo Kodama, Kouichi Miura, Iwao Ikai, Shinji Uemoto, David A. Brenner  Gastroenterology  Volume 135, Issue 5, Pages 1729-1738 (November 2008) DOI: 10.1053/j.gastro.2008.07.065 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Liver fibrosis is associated with increased vascularity and increase in angiopoietin 1 expression in human liver. Nontumorous portions of the liver were obtained from patients undergoing partial liver resection. Quantitative real time RT-PCR was performed to measure mRNA expression of collagen α1(I) (A), CD31 (B), and angiopoietin 1 (D). Fibrotic stage was classified into 3 categories; z0, normal liver (n = 19); z1, mild to moderate fibrosis (n = 18); and z2, cirrhosis (n = 17), according to the Liver Cancer Study Group of Japan.16 Boxes indicate 25th, 50th, and 75th percentiles; bars indicate 10th and 90th percentiles; circles indicate values outside 10th and 90th percentiles. Ribosomal 18S was used as an internal control. The values are ratios to the mean value in z0. *Significant difference (P < .05) by the Mann–Whitney U test. mRNA levels were correlated between CD31 and collagen α1(I) (C), angiopoietin 1 and CD31 (E), and angiopoietin 1 and collagen α1(I) (F). Gastroenterology 2008 135, 1729-1738DOI: (10.1053/j.gastro.2008.07.065) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Angiogenesis accompanies liver fibrosis induced by CCl4 in mice. Liver fibrosis was induced in BALB/c mice by injection with 0.5 μl/g of CCl4 twice a week. Development of liver fibrosis was assessed by Sirius red staining (A, left panels) and angiogenesis was evaluated by immunostaining for CD31 (A, right panels) and von Willebrand factor (B). (C) Percentage of CD31-positive pixels. mRNA expression of the liver was evaluated for CD31 (D), VEGFR2 (E), and Tie2 (F). Ribosomal 18S was used as an internal control. The values are ratios to the mean value of time 0 (pretreatment with CCl4); n = 5 at each time point. Bars indicate standard errors. Gastroenterology 2008 135, 1729-1738DOI: (10.1053/j.gastro.2008.07.065) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Angiopoietin 1 mRNA increases during liver fibrosis induced by CCl4 in mice. mRNA expression in CCl4-treated liver was evaluated for VEGFA (A), angiopoietin 1 (B), and angiopoietin 2 (C). The values are ratios to the mean value of time 0 (pretreatment with CCl4). Ribosomal 18S was used as an internal control; n = 5 at each time point. Bars indicate standard errors. (D) Protein was extracted from the liver. Tie2 was immunoprecipitated, electrophoresed, transblotted, and probed with an anti-phosphotyrosine antibody. The densities of the bands were analyzed by a densitometer and the intensity of phosphotyrosine was corrected by that of Tie2. The indicated values are percentage against time 0 (pretreatment with CCl4). Gastroenterology 2008 135, 1729-1738DOI: (10.1053/j.gastro.2008.07.065) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 HSCs are the primary source of angiopoietin 1 in the liver. The 4 major liver cell fractions were obtained as described in Materials and Methods. Each fraction was evaluated for the expression of VEGFA mRNA (A), angiopoietin 1 mRNA (B), and angiopoietin 2 mRNA (C). Ribosomal 18S was used as an internal control. Average of 3 independent isolations is shown. The values are ratio to the expression in hepatocytes. Bars indicate standard errors. HEP, hepatocyte; KC, Kupffer cell; EC, endothelial cell; HSC, hepatic stellate cell. (D) Double immunofluorescence for angiopoietin 1 (green) and desmin (red) was performed. Untreated and CCl4-induced fibrotic liver tissues were fixed in 4% formalin, embedded in OCT compound, and sectioned at 5 μm thickness. An anti-angiopoietin 1 antibody (R&D Systems) and an anti-desmin antibody (LAB VISION) were used. Gastroenterology 2008 135, 1729-1738DOI: (10.1053/j.gastro.2008.07.065) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Activation of HSCs increases angiopoietin 1 expression. HSCs were isolated from untreated mouse liver and plated on uncoated plastic plates to induce activation. RNA was isolated at 20 hours after plating for quiescent HSCs and 5 days for culture-activated HSCs. Quantitative real-time RT-PCR was performed to evaluate mRNA expression of angiopoietin 1 (A) and VEGFA (B). Ribosomal 18S was used as an internal control; n = 7 for each group. The values are ratios to the expression in quiescent HSCs. Bars indicate standard errors and asterisk indicates statistically significant difference by the Student t test. (C) Hepatocytes and HSCs were separately isolated from mice and plated at a density of 5 × 105/well on 6-well plates. Culture media were replaced every 2 days and concentration of angiopoietin 1 in the collected culture media was determined by ELISA. (D) Immunofluorescence for angiopoietin 1 in cultured HSCs. HSCs were isolated and fixed at 20 hours after plating for quiescent HSCs and 5 days for culture-activated HSCs with 4% formalin. After permeabilization with 0.05% Triton X-100, the cells were incubated with a primary antibody for angiopoietin 1 (R&D Systems), followed by an Alexa-Fluor 488 conjugated secondary antibody and nuclear staining with Hoechst 33342. Gastroenterology 2008 135, 1729-1738DOI: (10.1053/j.gastro.2008.07.065) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 TNF-α–stimulated angiopoietin 1 expression is NF-κB–dependent in HSCs. (A) HSCs were isolated from rats, allowed to activate in culture for 7 days, and then incubated with 5 ng/ml TGFβ-1, 200 ng/ml leptin, or 30 ng/ml murine TNF-α. RNA was extracted 24 hours after stimulation. (B) hTERT-HSCs were stimulated with 5 ng/ml TGFβ-1, 200 ng/ml leptin, or 10 ng/ml human TNF-α and RNA was extracted 24 hours after stimulation. (C) hTERT-HSCs were stimulated with 10 ng/ml human TNF-α and RNA was extracted before and 8, 24, and 48 hours after stimulation. (D) hTERT-HSCs were infected with either adenovirus-expressing mutant I-κB (Ad IκBsr) or GFP as a control at 800 moi, stimulated with 10 ng/ml human TNF-α 24 hours after infection, and RNA was extracted 24 hours later. (E) hTERT-HSCs were stimulated with TNF-α with or without proteosome inhibitor MG-132 (5 mmol/l), I-κB kinase inhibitor PS1145 (10 mmol/l). RNA was extracted 24 hours after stimulation. Quantitative real-time RT-PCR was performed to measure mRNA expression of angiopoietin 1 with ribosomal 18S as an internal control. The values are ratio to the expression in unstimulated cell. Gastroenterology 2008 135, 1729-1738DOI: (10.1053/j.gastro.2008.07.065) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Adenovirus expressing soluble Tie2 (AdsTie2) inhibits angiogenesis accompanying liver fibrosis. BALB/c mice were injected intravenously with either AdsTie2 or Adempty at 5 × 108 plaque forming unit/body. (A) Expression of sTie2 in the serum and the liver was evaluated by Western blotting with a primary antibody for Tie2 that recognizes the extracellular domain of Tie2 (Cell Signaling). Lane 1, no injection; lane 2, 3 days after Adempty; lane 3, 3 days after AdsTie2. (B) Liver fibrosis was induced by intraperitoneal injection with 0.5 μl/g CCl4 twice a week starting at 2 days after adenovirus injection. Control mice received corn oil (Oil) injection. Mice were killed 2 days after the 4th injection of CCl4 and evaluated for angiogenesis in the liver by immunohistochemistry for CD31. (C) CD31-positive pixels were quantified (n = 5). *Significant difference by the Student t test. E, Adempty; S, AdsTie2. Gastroenterology 2008 135, 1729-1738DOI: (10.1053/j.gastro.2008.07.065) Copyright © 2008 AGA Institute Terms and Conditions

Figure 8 Adenovirus expressing soluble Tie2 (AdsTie2) inhibits liver fibrosis induced by CCl4. BALB/c mice were injected intravenously with either AdsTie2 or Adempty at 5 × 108 plaque forming unit/body and liver fibrosis was induced by injection with CCl4 twice a week starting at 2 days after adenovirus injection (n = 5). Control mice received corn oil injection (Oil). The mice were killed 2 days after the 4th injection of CCl4. (A) Liver fibrosis was evaluated by Sirius red staining. (B) Quantification of Sirius red positive area. (C) Collagen deposition was quantified by measuring hydroxyproline content in the liver. The values are difference from the respective controls (Oil-injected mice). (D) The expression of α-smooth muscle antibody (SMA) was evaluated by immunofluorescence. (E) Quantification of α-SMA–positive area. *Significant difference by the Student t test. E, Adempty; S, AdsTie2. Gastroenterology 2008 135, 1729-1738DOI: (10.1053/j.gastro.2008.07.065) Copyright © 2008 AGA Institute Terms and Conditions