1. Volume 13, Issue 4, Pages 705-715 (April 2006)
Combined Angiopoietin-1 and vascular endothelial growth factor gene transfer restores cavernous angiogenesis and erectile function in a rat model of hypercholesterolemia 
Ji-Kan Ryu, Chung-Hyun Cho, Hwa-Yean Shin, Sun U. Song, Seung-Min Oh, Minhyung Lee, Shuguang Piao, Jee-Young Han, In-Hoo Kim, Gou Young Koh, Jun-Kyu Suh 
Molecular Therapy 
Volume 13, Issue 4, Pages (April 2006)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
In vivo expression of Ang1 and VEGF gene and protein. (A) Detection of human Ang1- and VEGF-specific mRNA in the corpus cavernosum from hypercholesterolemic rats. PCR was performed with primers specific for human Ang1 and VEGF165. Tissue RNA was extracted from the corpus cavernosum 3, 7, 14, and 21 days after intracavernous administration of ad-Ang1 (1 × 1010 parts/0.1 ml), ad-VEGF165 (1 × 1010 parts/0.1 ml), or a combination of both (1 × 1010 parts, respectively/0.1 ml). Densitometric analyses are presented as the relative ratio of human Ang1 or VEGF165 mRNA to β-actin mRNA. The relative ratio measured 3 days after injection of ad-Ang1 or ad-VEGF165 is arbitrarily presented as 1. Bars represent the mean ± SE from four experiments. (B) Western blot analysis of the protein expression of Ang1 and VEGF in cavernous tissues from hypercholesterolemic rats 3, 7, 14, and 21 days after intracavernous administration of ad-Ang1, ad-VEGF165, or a combination of both. The relative ratio of Ang1 or VEGF to β-actin measured in the control group is arbitrarily presented as 1. Bars represent the mean ± SE from four experiments. (C, D) Immunohistochemical staining of cavernous tissue performed with antibody to Ang1 (C) or VEGF (D) in hypercholesterolemic rats 7 days after intracavernous injection of ad-LacZ (1 × 1010 parts/0.1 ml), ad-Ang1 (1 × 1010 parts/0.1 ml), or ad-VEGF165 (1 × 1010 parts/0.1 ml). 2nd Ab, secondary antibody control; arrowheads, endothelial cells; arrows, smooth muscle cells; asterisks, nerve bundles. Bars indicate 50 μm. (E) Localization of β-galactosidase in the whole penis from hypercholesterolemic rats 3 days after administration of virus vehicle alone or of ad-LacZ (1 × 1010 parts/0.1 ml). Arrows denote positive endothelial cells (E), smooth muscle cells (SM), and nerve bundles (N). Tissues were counterstained with nuclear fast red. Bars indicate 10 μm. (F) β-Galactosidase activity in the corpus cavernosum from hypercholesterolemic rats 3, 7, 14, and 21 days after intracavernous administration of vehicle or ad-LacZ (1 × 1010 parts/0.1 ml). Each bar depicts the mean ± SE for n = 4 animals in each group. *P < 0.01, †P < 0.05 vs control by ANOVA.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Combined gene therapy with Ang1 and VEGF165 completely restores intracavernous pressure (ICP) elicited by electrical stimulation of the cavernous nerve. (A) Representative ICP responses for age-matched control (Am-C) or hypercholesterolemic rats stimulated 14 and 56 days after intracavernous injection of ad-LacZ (1 × 1010 parts/0.1 ml), ad-Ang1 (1 × 1010 parts/0.1 ml), ad-VEGF165 (1 × 1010 parts/0.1 ml), ad-Ang1 + ad-VEGF165 (1 × 1010 parts, respectively/0.1 ml), or cholesterol only (Co-C). The stimulus interval is indicated by a solid bar. SAP, systemic arterial pressure. (B–D) Ratio of mean maximal ICP to mean arterial pressure (MAP), total ICP (area under the curve), and tumescence slope calculated for each group. The slope and area under the curve were calculated from ICP recordings normalized to MAP. Each bar depicts the mean ± SE for n = 12 animals per group. (B) Ratio of ICP to MAP. *P < 0.01 vs ad-LacZ and Co-C groups, †P < 0.05 vs Am-C group, ‡P < 0.01 vs ad-LacZ, ad-Ang1, ad-VEGF165, and Co-C groups by ANOVA. (C) Total ICP (area under the curve). *P < 0.01 vs ad-LacZ and Co-C groups, †P < 0.01 vs ad-LacZ, ad-Ang1, and Co-C groups, ‡P < 0.05 vs ad-VEGF165 group, **P < 0.01 vs ad-LacZ, ad-Ang1, ad-VEGF165, and Co-C groups. (D) Slope. *P < 0.01 vs ad-LacZ and Co-C groups, †P < 0.01 vs ad-LacZ, ad-Ang1, ad-VEGF165, and Co-C groups.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Combined gene therapy with Ang1 and VEGF165 completely restores cavernous blood flow elicited by electrical stimulation of the cavernous nerve. (A) Representative blood flow responses for age-matched control (Am-C) or hypercholesterolemic rats stimulated 14 days after intracavernous injection of ad-LacZ (1 × 1010 parts/0.1 ml), ad-Ang1 (1 × 1010 parts/0.1 ml), ad-VEGF165 (1 × 1010 parts/0.1 ml), ad-Ang1 + ad-VEGF165 (1 × 1010 parts, respectively/0.1 ml), or cholesterol only (Co-C). The stimulus interval is indicated by a solid bar. (B–D) Maximal perfusion unit (PU), total PU, and slope calculated for each group. Each bar depicts the mean ± SE for n = 4 animals per group. *P < 0.05 vs ad-LacZ and Co-C groups by ANOVA.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Quantification of endothelial area, eNOS and nNOS immunoreactivity, and smooth muscle area. (A) Immunohistochemical staining of cavernous tissue performed with antibody to factor VIII, eNOS, nNOS, or α-SMA in age-matched control (Am-C) or hypercholesterolemic rats 14 days after intracavernous injection of ad-LacZ (1 × 1010 parts/0.1 ml), ad-Ang1 (1 × 1010 parts/0.1 ml), ad-VEGF165 (1 × 1010 parts/0.1 ml), ad-Ang1 + ad-VEGF165 (1 × 1010 parts, respectively/0.1 ml), or cholesterol only (Co-C). Bars indicate 200 μm for factor VIII, eNOS, and α-SMA and 50 μm for nNOS. (B–E) Quantitative analysis of endothelium, eNOS- and nNOS-positive area, and smooth muscle content in cavernous tissue was done with an image analyzer. Each bar depicts the mean ± SE for n = 12 animals per group. (B) Endothelial area. *P < 0.05 vs ad-LacZ and Co-C groups, †P < 0.01 vs ad-LacZ, ad-Ang1, and Co-C groups, ‡P < 0.05 vs ad-VEGF165 group by ANOVA. (C) eNOS-positive area. (D) nNOS-positive area. *P < 0.01 vs ad-LacZ and Co-C groups. (E) Smooth muscle area. *P < 0.05, †P < 0.01 vs Am-C group.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Quantification of endothelial area, eNOS and nNOS immunoreactivity, and smooth muscle area. (A) Immunohistochemical staining of cavernous tissue performed with antibody to factor VIII, eNOS, nNOS, or α-SMA in age-matched control (Am-C) or hypercholesterolemic rats 56 days after intracavernous injection of ad-LacZ (1 × 1010 parts/0.1 ml), ad-Ang1 (1 × 1010 parts/0.1 ml), ad-VEGF165 (1 × 1010 parts/0.1 ml), ad-Ang1 + ad-VEGF165 (1 × 1010 parts, respectively/0.1 ml), or cholesterol only (Co-C). Bars indicate 200 μm for factor VIII, eNOS, and α-SMA and 50 μm for nNOS. (B–E) Quantitative analysis of endothelium, eNOS- and nNOS-positive area, and smooth muscle content in cavernous tissue was done with an image analyzer. Each bar depicts the mean ± SE for n = 12 animals per group. (B) Endothelial area. *P < 0.05 vs ad-LacZ and Co-C groups, †P < 0.01 vs ad-LacZ, ad-Ang1, and Co-C groups, ‡P < 0.05 vs ad-VEGF165 group by ANOVA. (C) eNOS-positive area. (D) nNOS-positive area. *P < 0.05 vs Am-C, ad-LacZ, and Co-C groups. (E) Smooth muscle area. *P < 0.01 vs Am-C group.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Western blot analysis demonstrating the relative protein abundance of phospho-eNOS (Ser1177) in age-matched control (Am-C) and hypercholesterolemic rat penis 7, 14, and 56 days after transfection with ad-LacZ (1 × 1010 parts/0.1 ml), ad-Ang1 (1 × 1010 parts/0.1 ml), ad-VEGF165 (1 × 1010 parts/0.1 ml), ad-Ang1 + ad-VEGF165 (1 × 1010 parts, respectively/0.1 ml), or cholesterol only (Co-C). (A) Representative Western immunoblot of phospho-eNOS (Ser1177) and total eNOS in each group. (B) Quantitative analysis of phospho-eNOS (Ser1177). Data are representative of four densitometric values from independent experiments with separate rats for each experiment. Each bar represents the mean ± SE of the ratio of phospho-eNOS/eNOS expressed relative to the control result. *P < 0.05 vs Am-C group, **P < 0.01 vs Am-C group, †P < 0.01 vs Am-C, ad-LacZ, and Co-C groups, ‡P < 0.01 vs Am-C, ad-LacZ, ad-Ang1, ad-VEGF165, and Co-C groups, ***P < 0.01 vs ad-LacZ, ad-Ang1, ad-VEGF165, and Co-C groups by ANOVA.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

8. FIG. 7
Changes in cavernous cGMP concentrations in age-matched control (Am-C) or hypercholesterolemic rats 7, 14, and 56 days after transfection with ad-LacZ (1 × 1010 parts/0.1 ml), ad-Ang1 (1 × 1010 parts/0.1 ml), ad-VEGF165 (1 × 1010 parts/0.1 ml), ad-Ang1 + ad-VEGF165 (1 × 1010 parts, respectively/0.1 ml), or cholesterol only (Co-C). Each bar depicts the mean ± SE for n = 4 animals per group. *P < 0.01 vs Am-C group, †P < 0.01 vs ad-LacZ and Co-C groups, **P < 0.05 vs ad-LacZ and Co-C groups, ‡P < 0.01 vs Am-C, ad-LacZ, ad-Ang1, ad-VEGF165, and Co-C groups by ANOVA.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions