Pulmonary Angiogenesis in a Rat Model of Hepatopulmonary Syndrome Junlan Zhang, Bao Luo, Liping Tang, Yongming Wang, Cecil R. Stockard, Inga Kadish, Thomas Van Groen, William E. Grizzle, Selvarangan Ponnazhagan, Michael B. Fallon  Gastroenterology  Volume 136, Issue 3, Pages 1070-1080 (March 2009) DOI: 10.1053/j.gastro.2008.12.001 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Immunofluorescent localization of factor VIII (FVIII) and pulmonary microvessel counts and von Willebrand factor (vWf) and vascular endothelial cadherin (VE-cadherin) levels in lung after CBDL or TAA administration. Representative images are shown of FVIII, a specific endothelial cell marker (red) with DAPI a nuclear marker (blue) in (A) control and (B) 3-week CBDL animals (original magnification, 40×). (C) Graphical summary of lung microvessel counts after CBDL and TAA administration. (D) Correlation of alveolar-arterial oxygen gradient with lung microvessel count in control and CBDL animals. The linear correlation coefficient was significant for each comparison (r = 0.71, P < .0001). Representative immunoblots and summary of protein levels of vWf (∼130/250 kilodalton; E) and VE-cadherin (130 kilodaltons; F) after CBDL or TAA administration. Values are expressed as means ± SE (n = 5–8 animals for each group). *P < .05 compared with normal control. Gastroenterology 2009 136, 1070-1080DOI: (10.1053/j.gastro.2008.12.001) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Immunohistochemical localization and protein levels of pulmonary proliferating cell nuclear antigen (PCNA) in CBDL and TAA-treated animals. Representative immunohistochemical sections from (A) control and (B) 3-week CBDL lung (original magnification, 40×). (C) Representative immunoblot showing the ∼36-kilodalton PCNA protein band and graphical summary of PCNA protein levels in lung. Values are expressed as means ± SE (n = 5–8 animals for each group). *P < .05 compared with normal controls. Gastroenterology 2009 136, 1070-1080DOI: (10.1053/j.gastro.2008.12.001) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Immunofluorescent localization of pulmonary vascular endothelial growth factor (VEGF)-A and intravascular monocytes (ED1) after CBDL and lung VEGF-A and phospho-vascular endothelial growth factor receptor-2 (p-VEGFR-2) levels in CBDL and TAA-treated animals. Double-label images of VEGF-A (red, left) and ED1 (green, middle) and superimposition (right, with blue DAPI nuclear stain) in control (A–C) and 3-week CBDL animals (D–F, original magnification, 40×). Colocalization of VEGF-A and ED1 in monocytes as well as increased microvascular VEGF-A staining were noted after CBDL. Representative immunoblots and graphical summary of protein levels for (G) VEGF-A (∼21/42 kilodaltons) and (H) p-VEGFR-2 and VEGFR-2 (∼150 or 200 kilodaltons, respectively) are shown. Values are expressed as means ± SE (n = 5–8 animals for each group). *P < .05 compared with normal controls. Gastroenterology 2009 136, 1070-1080DOI: (10.1053/j.gastro.2008.12.001) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Immunofluorescent localization of FVIII, microvessel counts and vWf, PCNA, and ED1 levels and alveolar-arterial oxygen gradients (AaPO2) in pentoxifylline (PTX)-treated 3-week CBDL animals. Representative images of FVIII (red) with DAPI (blue) in (A) 3-week CBDL and (B) PTX-treated 3-week CBDL lung show a reduction in staining after PTX treatment (original magnification, 40×). Graphical summary of (C) lung microvessel counts and representative immunoblots and graphical summaries of (D) vWf, (E) PCNA, and (F) ED1 along with (G) AaPO2 levels in control, CBDL, and PTX-treated CBDL are shown. Values are expressed as means ± SE (n = 5–8 animals for each group). *P < .05 compared with normal control. †P < .05 compared with 3-week CBDL. Gastroenterology 2009 136, 1070-1080DOI: (10.1053/j.gastro.2008.12.001) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Immunofluorescent localization and protein levels of VEGF-A and VEGFR-2 phosphorylation in lung from PTX-treated 3-week CBDL animals. Double-label images of VEGF-A (red), ED1 (green) superimposed with blue DAPI nuclear stain from (A) 3-week CBDL and (B) PTX-treated 3-week CBDL lung showing a reduction in ED1 (intravascular monocytes) and VEGF-A signal after PTX treatment (original magnification, 40×). Representative immunoblots and graphical summaries of protein levels for (C) VEGF-A and (D) p-VEGFR-2 and VEGFR-2 are shown. Values are expressed as means ± SE (n = 5–8 animals for each group). *P < .05 compared with normal controls. †P < .05 compared with 3-week CBDL. Gastroenterology 2009 136, 1070-1080DOI: (10.1053/j.gastro.2008.12.001) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Effects of rAAV E+A administration on the development of lung angiogenesis and HPS in 3-week CBDL animals. Graphical summaries of the reduction in (A) lung microvessel counts and VWF levels, (B) AaPO2 levels, (C) lung VEGF-A and p-VEGFR-2, and (D) lung ED1 (intravascular monocytes) in treated animals relative to rAAV GFP injected CBDL control animals. Values are expressed as means ± SE (n = 5–8 animals for each group). *P < .05 compared with normal controls. †P < .05 compared with 3-week CBDL. Gastroenterology 2009 136, 1070-1080DOI: (10.1053/j.gastro.2008.12.001) Copyright © 2009 AGA Institute Terms and Conditions