Oral Administration of Recombinant Adeno-associated Virus-mediated Bone Morphogenetic Protein-7 Suppresses CCl4-induced Hepatic Fibrosis in Mice  Zhi-Ming Hao, Min Cai, Yi-Fei Lv, Yan-Hua Huang, Hong-Hong Li  Molecular Therapy  Volume 20, Issue 11, Pages 2043-2051 (November 2012) DOI: 10.1038/mt.2012.148 Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 The oral administration of AAV–BMP-7 mediates the ectopic expression of BMP-7 in the GI tract and leads to elevated serum BMP-7 concentrations in mice. The in situ β-Gal staining of the intestine 3 days after the second oral administration of virus showed the ectopic expression of LacZ (blue) in the lamina propria and epithelia in mice that received (a) AAV-LacZ but not in mice that received (b) PBS. Likewise, the immunohistochemical staining of the intestine revealed ectopic BMP-7 expression in the mice that received (c) AAV–BMP-7 but not in the mice that received (d) PBS. Bars = 200 µm. (e) The serum BMP-7 concentrations were determined by ELISA 3 days after the second oral dose of 1 × 1010 vg AAV–BMP-7, AAV-LacZ or an equal volume of PBS. The error bars indicate SEM. (f) The mice were orally dosed twice, 3 days apart, with AAV–BMP-7 (1 × 1010 vg for the AAV–BMP-7/CCl4 group), AAV-LacZ (1 × 1010 vg for the AAV-LacZ/CCl4 group), or PBS (for the NC and CCl4 groups). Three days after the second dose, the mice were injected i.p. with CCl4 twice a week for 7 weeks (1 ml/kg BW for the AAV–BMP-7/CCl4, AAV-LacZ/CCl4 and CCl4 groups) or olive oil (for the NC group). The serum BMP-7 concentrations were determined after 7 weeks by ELISA. The error bars indicate SEM. AAV, adeno-associated virus; BMP-7, bone morphogenetic protein-7; CCl4, carbon tetrachloride; ELISA, enzyme-linked immunosorbant assay; i.p., intraperitoneal; IH, immunohistochemistry; NC, normal control; PBS, phosphate-buffered saline; SEM, standard error of the mean; vg, viral genome; β-Gal, β-Galactosidase. Molecular Therapy 2012 20, 2043-2051DOI: (10.1038/mt.2012.148) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 The oral administration of AAV–BMP-7 suppresses hepatic fibrosis in mice, as shown by Masson's trichrome staining, hepatic hydroxyproline content and serum HA concentration. The AAVs were administered, and fibrosis was induced as described the legend to Figure 1f. Representative figures of the Masson's trichrome-stained mouse livers from the (a) NC, (b) AAV–BMP-7/CCl4, (c) AAV-LacZ/CCl4, and (d) CCl4 groups. Bars = 200 µm. (e) The liver hydroxyproline content was determined by a biochemical assay. The hydroxyproline content is expressed as µg/mg wet liver weight. The error bars indicate SEM. (f) The serum hyaluronic acid concentration was determined by ELISA and is expressed as ng/ml serum. The error bars indicate SEM. AAV, adeno-associated virus; BMP-7, bone morphogenetic protein-7; CCl4, carbon tetrachloride; HA, hyaluronic acid; NC, normal control; SEM, standard error of the mean. Molecular Therapy 2012 20, 2043-2051DOI: (10.1038/mt.2012.148) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 The ectopic expression of BMP-7 reduces the expression of α-SMA in the livers of CCl4-treated mice. AAV was administered, and fibrosis was induced as described in the legend to Figure 1f. The fixed liver tissues were sectioned and were immunostained with anti-α-SMA antibody. Representative immunostained liver sections are shown for the (a) NC; (b) AAV–BMP-7/CCl4; (c) AAV-LacZ/CCl4; and (d) CCl4 groups. Bars = 200 µm. (e) The quantitative computer-assisted morphometric analysis of the immunostained activated HSCs (α-SMA–positive cells) in the four groups. The error bars indicate SEM. AAV, adeno-associated virus; BMP-7, bone morphogenetic protein-7; CCl4, carbon tetrachloride; NC, normal control; SEM, standard error of the mean. Molecular Therapy 2012 20, 2043-2051DOI: (10.1038/mt.2012.148) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 The ectopically expressed BMP-7 reduces the expression of hepatic desmin in CCl4-treated mice. The AAVs were administered, and fibrosis was induced as described in the legend to Figure 1f. The fixed liver tissues were sectioned and immunostained with an anti-desmin antibody. The immunostained liver sections are from the (a) NC; (b) AAV–BMP-7/CCl4; (c) AAV-LacZ/CCl4; and (d) CCl4 groups. (e) The immunostaining indicates that some of the desmin-positive cells are hepatocytes. Bars = 200 µm. (f) Quantitative computer-assisted morphometric analysis of the immunostained desmin-positive cells in the four groups. The error bars indicate SEM. AAV, adeno-associated virus; BMP-7, bone morphogenetic protein-7; CCl4, carbon tetrachloride; NC, normal control; SEM, standard error of the mean. Molecular Therapy 2012 20, 2043-2051DOI: (10.1038/mt.2012.148) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 The oral administration of AAV–BMP-7 promotes parenchymal cell proliferation but inhibits mesenchymal cell proliferation in the fibrotic mouse livers. The AAVs were administered, and fibrosis was induced as described in Figure 1f. The fixed liver tissues were sectioned and immunostained with an anti-PCNA antibody. The immunostained liver sections are shown from the (a) NC; (b) AAV–BMP-7/CCl4; (c) AAV-LacZ/CCl4; and (d) CCl4 groups. Bars = 200 µm. The PCNA LIs of the (e) mesenchymal and (f) parenchymal cells. The error bars indicate SEM. AAV, adeno-associated virus; BMP-7, bone morphogenetic protein-7; CCl4, carbon tetrachloride; LI, labeling index; NC, normal control; PCNA, proliferating cell nuclear antigen; SEM, standard error of the mean. Molecular Therapy 2012 20, 2043-2051DOI: (10.1038/mt.2012.148) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions