Oral Administration of Recombinant Adeno-associated Virus-mediated Bone Morphogenetic Protein-7 Suppresses CCl4-induced Hepatic Fibrosis in Mice

original article© The American Society of Gene & Cell Therapy

Molecular Therapy vol. 20 no. 11, 2043–2051 nov. 2012 2043

Fibrogenesis and hepatocyte degeneration are the main pathological processes in chronic liver diseases. Trans-forming growth factor-β1 (TGF-β1) is the key profi-brotic cytokine in hepatic fibrosis. Bone morphogenetic protein-7 (BMP-7) is a potent antagonist of TGF-β1 and an antifibrotic factor. In this study, we generated a recombinant adeno-associated virus carrying BMP-7 (AAV–BMP-7) and tested its ability to suppress carbon tetrachloride (CCl4)-induced hepatic fibrosis when orally administered to mice. Our results show that the ectopic expression of BMP-7 in gastrointestinal (GI) mucosa due to the AAV–BMP-7 administration led to the long-term elevation of serum BMP-7 concentrations and resulted in the drastic amelioration of CCl4-induced hepatic fibrosis in BALB/c mice. Immunostaining for α-smooth muscle actin (α-SMA) and desmin demonstrated that AAV–BMP-7 inhibited the activation of hepatic stellate cells (HSCs) in the fibrotic mouse liver. Moreover, the ectopic expression of BMP-7 promoted hepatocyte proliferation, as confirmed by an increase in the amount of proliferat-ing cell nuclear antigen (PCNA)-positive hepatocytes in the mice that received AAV–BMP-7. Our results clearly indicate that BMP-7 is capable of inhibiting hepatic fibrosis and promoting hepatocyte regeneration. We suggest that oral AAV–BMP-7 could be developed into a safe, simple, and effective therapy for hepatic fibrosis.

Received 19 November 2011; accepted 22 February 2012; advance online publication 31 July 2012. doi:10.1038/mt.2012.148

IntroductIonLiver fibrosis is characterized by the accumulation of extracellular matrix (ECM), including fibrillar type I and III collagens, proteo-glycans and glycoproteins. The accumulation of ECM disrupts the hepatic architecture by forming fibrous scars around nodules of regenerating hepatocytes, eventually leading to cirrhosis. During cirrhosis, the excessive deposition of ECM leads to hepatic failure due to the hepatocyte malfunction and portal hypertension caused by hemodynamic alterations. Eliminating the injurious stimulus is

the obvious first choice for interrupting hepatic fibrosis. However, in most cases, the removal of the cause of hepatic fibrosis is quite difficult or even impossible. Moreover, fibrosis can still persist for a long time even after the cause is eliminated. Therefore, specific antifibrotic therapy is essential in the management of chronic liver diseases. Unfortunately, few effective, safe and convenient antifi-brotic approaches are clinically available.1,2

Although the underlying causes of chronic liver diseases vary widely and include chronic viral hepatitis, alcohol abuse and autoimmune disorders, the cellular and molecular mechanisms underlying hepatic fibrosis and cirrhosis are relatively common. Activation of hepatic stellate cells (HSCs) is the central event in hepatic fibrosis. HSCs are sinusoidal constituent cells that play multiple roles in the liver. In the normal liver, HSCs localize in the space of Disse as pericytes surrounding the sinusoids to store vitamin A-containing lipid droplets. When the liver is chroni-cally injured by diseases, elimination of the damaged liver cells causes the release of multiple peptide, lipid and gaseous mediators from the Kupffer cells, infiltrating inflammatory cells, endothelial cells, and hepatocytes, thereby activating HSCs. HSC activation is particularly driven by profibrogenic cytokines and is charac-terized by increased cell proliferation, altered cellular morphol-ogy to a more myofibroblast (MF)-like cell type, and upregulated ECM expression.3,4 Upon HSC activation, enhanced profibro-genic cytokine responses occur due to the excessive production of cytokines, increased expression of cell membrane receptors, and enhanced signal transduction.5 Transforming growth factor-β1 (TGF-β1) is the most potent profibrogenic cytokine. During liver damage, Kupffer cells, infiltrating inflammatory cells, endothe-lial cells, and hepatocytes produce excessive TGF-β1 that acti-vates HSCs. The activated HSCs also produce TGF-β1, thereby stimulating their own collagen gene expression in an autocrine feedback loop. TGF-β1 also upregulates the expression of tissue inhibitors of metalloproteinases, which have antiapoptotic effects on MF-like cells.6–8

Bone morphogenetic protein-7 (BMP-7) (also termed osteo-genic protein-1, OP-1), a 25–40 kDa homodimeric protein belonging to the TGF-β superfamily, was originally identified due to its ability to induce bone formation.9 However, BMP-7 has been

Correspondence: Zhi-Ming Hao, Department of Gastroenterology, The First Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an 710061, China. E-mail: [email protected]

Oral Administration of Recombinant Adeno-associated Virus-mediated Bone Morphogenetic Protein-7 Suppresses CCl4-induced Hepatic Fibrosis in MiceZhi-Ming Hao1, Min Cai1, Yi-Fei Lv1, Yan-Hua Huang1 and Hong-Hong Li2

1Department of Gastroenterology, The First Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, China; 2Department of Pathology, The First Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, China

http://www.nature.com/doifinder/doi:10.1038/mt.2012.148

mailto:[email protected]

2044 www.moleculartherapy.org vol. 20 no. 11 nov. 2012

© The American Society of Gene & Cell TherapyOral AAV–BMP-7 Suppresses Hepatic Fibrosis in Mice

identified as a multifunctional cytokine capable of regulating the proliferation and differentiation of multiple cell types.9,10 Recent studies have shown that BMP-7 inhibits fibrosis in the kidney,11–15 lung,16,17 liver,18,19 heart,20 peritonium,21,22 oral submucosa tissue,23 and colonic wall24 by antagonizing TGF-β1 signal transduction, which suggests that BMP-7 could be developed into a therapeutic agent for treating organ fibrosis.

In this study, we demonstrated that the oral administration of a recombinant adeno-associated virus carrying BMP-7 (AAV–BMP-7) mediates the long-term ectopic expression of BMP-7 in the gastrointestinal (GI) mucosa, elevates the circulating BMP-7 concentration, and significantly inhibits carbon tetrachloride (CCl4)-induced hepatic fibrosis in mice. Additionally, our results show that AAV–BMP-7 promotes hepatocyte proliferation in the fibrotic liver. This study suggests that oral AAV–BMP-7 could be developed into an efficient, safe, and convenient therapy for treat-ing hepatic fibrosis.

resultsthe oral administration of AAV–BMP-7 efficiently mediates the ectopic expression of BMP-7 in the GI tract and increases serum BMP-7 concentrations in miceTo test whether orally administered AAV–BMP-7 could inhibit hepatic fibrosis in mice, we generated adeno-associated vectors containing BMP-7 (AAV–BMP-7) or LacZ (AAV-LacZ) under the transcriptional control of the ubiquitous cytomegalovirus promoter. We verified the ectopic expression of BMP-7 and LacZ in the GI tract after the oragastric administration of 1 × 1010 viral genomes of AAV–BMP-7 or AAV-LacZ, respectively, in BALB/c mice. To reinforce the viral transduction efficiency, the AAVs were re-administered arbitrarily 3 days later. Three days after the second administration of AAV-LacZ and AAV–BMP-7, in situ β-Galactosidase staining and BMP-7 immunostaining revealed that LacZ (Figure 1a,b) and BMP-7 (Figure 1c,d), respectively, were expressed in the lamina propria and in the epithelia of the stomach and intestines, with the small intestine displaying the strongest staining. To confirm the expression of the secreted BMP-7 and the elevation of the circulating BMP-7 levels after the oral administration of AAV–BMP-7, the serum BMP-7 concen-tration was determined by sandwich enzyme-linked immuno-sorbant assay (ELISA). Figure 1e shows that the serum BMP-7 concentration in the AAV–BMP-7 mice sharply increased 3 days following the second dose of oral AAV compared with that in both the AAV-LacZ and phosphate-buffered saline (PBS)-control mice (P < 0.001). To determine the duration of the AAV-mediated ectopic expression in the GI tract following oral administration, in situ β-Galactosidase staining and BMP-7 immunostaining of the GI tract, and the ELISA determination of the serum BMP-7 concentration following 7 weeks of CCl4 injection were per-formed. The ectopic expression of LacZ and BMP-7 due to the AAVs persisted in the GI mucosa, although the staining intensity was weaker than that obtained before the induction of fibrosis (data not shown). The ELISA results showed that the mice which received repeated CCl4 injections had significantly higher serum BMP-7 concentrations than the normal control mice (P < 0.001), whereas the AAV–BMP-7/CCl4 mice had significantly higher

serum BMP-7 levels than the mice from either the AAV-LacZ/CCl4 or CCl4 groups (P < 0.001) (Figure 1f). These results col-lectively indicate that orally administered recombinant type 2 AAV can resist destruction by gastric acid and intestinal fluids can infect GI mucosa cells and can efficiently mediate the long-term expression of exogenous genes.

oral AAV–BMP-7 inhibits ccl4-induced hepatic fibrosis in miceNext, we investigated the protective effect of oral AAV–BMP-7 against CCl4-induced hepatic fibrosis in mice. During the 7 weeks of CCl4 injections, five mice (one in the AAV–BMP-7/CCl4 group and two each in the AAV-LacZ/CCl4 and CCl4 groups) died due to liver failure. The remaining mice were sacrificed, and hepatic fibro-sis was evaluated by hematoxylin and eosin and Masson’s trichrome staining. The mice in the AAV-LacZ/CCl4 and CCl4 groups exhib-ited obvious and uniform hepatic fibrosis after 7 weeks of repeated CCl4 injections, with most of the mice (12/13) showing stage 4 or above fibrosis in both groups (P > 0.05) (Figure 2c,d and Table 1). The severity of the hepatic fibrosis was significantly milder in the AAV–BMP-7/CCl4 group than that in either the AAV-LacZ/CCl4 or CCl4 groups (P < 0.05), with most of the mice (12/14) showing stage 2 or 3 fibrosis (Figure 2b and Table 1).

The hydroxyproline content, a commonly accepted indicator of fibrous tissue, was determined in the liver tissue. As in the his-tological grading, the liver hydroxyproline content of the AAV–BMP-7/CCl4 group was significantly lower than that of either the AAV-LacZ/CCl4 or CCl4 groups (P < 0.001), and those of the latter two groups were similar (P > 0.05) (Figure 2e).

In this study, we also determined the concentration of serum hyaluronic acid, a useful marker of fibrosis, by sandwich ELISA. As shown in Figure 2f, the serum hyaluronan concen-tration mirrored the histological fibrosis score and the liver hydroxyproline content. These results collectively demonstrate that oral AAV–BMP-7 may inhibit CCl4-induced hepatic fibro-sis in mice.

ectopic BMP-7 expression suppresses Hsc activation in the ccl4-induced fibrotic liversBecause the activation of HSCs is the key event in hepatic fibro-sis, the expression of α-smooth muscle actin (α-SMA), a typical marker of activated HSCs, was assessed by immunohistochemistry to evaluate the effect of orally administered AAV–BMP-7 on HSC activation during hepatic fibrosis. In normal mice, α-SMA–positive staining was confined to the smooth muscle cells lining the portal and central veins and large arteries in the liver (Figure 3a). Mice subjected to 7 weeks of CCl4 injections showed a drastic increase in the number of α-SMA–positive cells observed throughout the fibrotic septa (Figure 4b,c,d). As expected, the computer-assisted semiquantitative analysis of the α-SMA–positive staining showed that the AAV–BMP-7/CCl4 mice (Figure 4b) had significantly smaller α-SMA–positive areas than either the AAV-lacZ/CCl4 (Figure 4c) or CCl4 (Figure 4d) groups (P < 0.001), whereas no significant differences in the α-SMA–positive staining were found between the two latter groups (P > 0.05) (Figure 4e).

Desmin is also regarded as a specific marker of activated HSCs, and it was detected by immunohistochemistry in this

Molecular Therapy vol. 20 no. 11 nov. 2012 2045


study. In the NC mice, positive immunostaining for desmin was restricted to the portal areas, central veins, and large arteries within the liver, similar to the distribution patterns of α-SMA (Figure 4a). After 7 weeks of CCl4 injection, there was a marked increase in the amount of desmin-positive cells (Figure 4b,c,d). Interestingly, the expression pattern of desmin in the fibrotic livers was different from that of α-SMA. α-SMA was strictly found in the core of fibrous septa, whereas desmin was located at the rim of fibrous septa and in neighboring hepatocytes (Figure 4e), as described by Ballardini et al.25 and Cassiman et al.26 Computer-assisted semiquantitative analysis (Figure 4f) showed that the desmin-positive area in the AAV–BMP-7/CCl4

livers (Figure 4b) was significantly lower than that of the AAV-LacZ/CCl4 (Figure 4c) or CCl4 (Figure 4d) livers (P < 0.001).

ectopic BMP-7 expression promotes parenchymal cell proliferation, whereas it inhibits mesenchymal cell proliferation in fibrotic liversIn addition to verifying that BMP-7 inhibited hepatic fibrosis, we also examined its influence on parenchymal and mesenchymal cell proliferation by measuring proliferating cell nuclear antigen (PCNA)-positive cells. The mesenchymal PCNA labeling indices (PCNA LIs) were higher in the CCl4 (Figure 5d) and AAV-LacZ/CCl4 (Figure 5c) mice than in the NC mice (Figure 5a), whereas

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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 immuno-histochemical 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, immu-nohistochemistry; NC, normal control; PBS, phosphate-buffered saline; SEM, standard error of the mean; vg, viral genome; β-Gal, β-Galactosidase.



the PCNA LIs in the AAV–BMP-7/CCl4 mice (Figure 5b) were significantly lower than that in the CCl4 or AAV-LacZ/CCl4 mice (P < 0.001) (Figure 5e). This result indicates that the proliferation of HSCs/MFs is suppressed by BMP-7 because HSCs/MFs are the main mesenchymal cell type in fibrotic livers. However, the PCNA LIs in the parenchymal cells (predominantly hepatocytes) were higher in the CCl4 (Figure 5d) and AAV-LacZ/CCl4 (Figure 5c) mice than in the NC mice (Figure 5a) (P < 0.001), possibly reflect-ing a complementary tissue regeneration after liver damage. The PCNA LIs of the parenchymal cells were significantly higher in

the livers of the AAV–BMP-7/CCl4 mice (Figure 5b) than in either the CCl4 or AAV-LacZ/CCl4 mice (P < 0.001) (Figure 5f), indicating that BMP-7 promoted the proliferation and regenera-tion of hepatocytes.

dIscussIonIn this study, we demonstrated that the oral administration of AAV–BMP-7 mediates long-term ectopic expression of BMP-7 in the GI tract, elevates circulating BMP-7 levels, and significantly alleviates CCl4-induced hepatic fibrosis in mice. The effects of

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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.



BMP-7 on hepatic fibrosis have been reported previously18,19,27 with some discrepancies. Our data are consistent with those reported by Kinoshita et al., who reported that the adenovirus-mediated ectopic expression of BMP-7 suppressed TAA-induced

hepatic fibrosis in rats,18 and the results from Zeisberg et al., who showed that administering recombinant human BMP-7 (300 μg/kg) intraperitoneally every other day alleviated CCl4-induced hepatic fibrosis in mice.19 However, Tacke et al. found that a BMP-7-containing adenovirus resulted in the accelerated proliferation, elevated α-SMA expression, and upregulated type I collagen syn-thesis in telomerase reverse transcriptase-transformed primary human HSCs.27 Additionally, other studies reported a discrepancy between the in vitro and in vivo activation of HSCs by a soluble TGF-β receptor, as measured by α-SMA expression.28 Based on the above results, BMP-7 may activate HSCs in a context- dependent manner.

MFs are the primary ECM-producing cell type during fibro-sis. However, the origin of the MFs in fibrotic livers is still under debate. Although HSCs are considered to be the principal source of MFs in hepatic fibrosis, recent studies have provided substan-tial evidence that hepatocytes undergo epithelial-to-mesenchymal transition in the presence of TGF-β1 and contribute to the accu-mulation of activated MFs in the fibrotic mouse livers, whereas the presence of BMP-7 inhibits this transition.19,29 Although both α-SMA and desmin have been used as specific markers for HSCs/MFs, some studies have demonstrated that these two makers represent different HSC/MF phenotypes. Positive α-SMA stain-ing indicates differentiated MFs, whereas positive desmin stain-ing represents intermediately differentiated cells.25,26 In this study, we found many desmin-positive cells near the fibrous septa that exhibited a hepatocyte phenotype after repeated CCl4 injection, possibly indicating that the hepatocytic epithelial-to-mesenchymal transition contributes to MF accumulation in the CCl4-induced fibrotic livers. Moreover, a decrease in the α-SMA- and desmin-positive areas due to orally administered AAV–BMP-7 supports previous results that suggest that BMP-7 inhibits HSC activation and epithelial-to-mesenchymal transition in fibrotic livers.

Under normal physiological conditions, BMP-7 is predomi-nantly produced by the kidney and bone and has a circulating blood concentration ranging between 100 and 300 pg/ml.30 Neither humans nor rodents produce hepatic BMP-7, but hepatocytes and HSCs express BMP-7 receptors.30,31 At physiological levels, BMP-7 may promote liver regeneration and regulate mesenchymal regen-eration and ECM production in adults.31 Hepatocytes in chroni-cally inflamed livers restore the expression of BMP-7, leading to an elevated BMP-7 concentration in the bloodstream,27,32 as dem-onstrated by this study. However, the role of the elevated endoge-nous BMP-7 in fibrotic livers has not yet been elucidated. The fact that exogenous BMP-7 suppresses experimental hepatic fibrosis, as demonstrated in this and other studies,18,19 suggests that BMP-7 may exert antifibrotic functions in a dose-dependent manner. The upregulation of BMP-7 in the fibrotic liver may reflect an adap-tive reaction to liver damage and fibrosis. However, the increased expression of endogenous BMP-7 may not be sufficient to neutral-ize the profibrogenic effects of TGF-β1 in fibrotic livers. Only a drastic increase in BMP-7 levels, as in our experiment using AAV-mediated ectopic expression, can significantly alleviate hepatic fibrosis.

Apart from its antifibrogenic properties, this study demon-strated that BMP-7 also promotes hepatocyte regeneration after repeated CCl4 injections. This finding was not unexpected because

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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 tetrachlo-ride; NC, normal control; SEM, standard error of the mean.

table 1 Grading of hepatic fibrosis in various groups

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AAV–BMP-7/CCl4† 14 0 0 7 5 2 0 0

AAV-LacZ/CCl4‡ 13 0 0 1 0 5 6 1

CCl4* 13 0 0 0 1 6 6 0

Abbreviations: AAV, adeno-associated virus; BMP-7, bone morphogenetic protein-7; CCl4, carbon tetrachloride; N, number of mice; NC, normal control.†P < 0.05 as compared to the AAV-LacZ/CCl4 and CCl4 groups. ‡P > 0.05 as compared to the CCl4 group by the Mann–Whitney U-test.



BMP-7 induces liver bud sprouting from the central foregut endo-derm, which eventually gives rise to hepatocytes during embryo development.10,33 Our results are also consistent with a previous study by Sugimoto et al. showing that recombinant BMP-7 infusion leads to accelerated liver regeneration, as indicated by improved liver function, whereas endogenous BMP-7 neutralized with a spe-cific antibody significantly impaired liver regeneration after partial hepatectomies in mice.31 The chronic loss of functional hepatocytes is an important pathological alteration in chronic liver diseases that eventually leads to liver failure. Promoting hepatocyte regeneration by BMP-7 will help restore liver structure and function.

AAV is one of the most promising vectors in gene therapy because it has a broad host range, transduces both dividing and

nondividing cells, has a long-term expression profile, shows no toxic side-effects and lacks the antigenicity observed in other vec-tors, which allows for repeated applications. The AAV-mediated ectopic protein expression persists for at least 6 months, mak-ing AAVs a suitable vector for gene therapy in chronic diseases. For these reasons, AAV has become an increasingly common vector for gene therapy in human clinical trials. As of the sum-mer of 2010, 75 AAV-based gene therapy trials were registered at the Journal of Gene Medicine Clinical Trial site.34 In addition to the advantages mentioned above, several studies have shown that AAVs are resistant to extreme pH and solvent conditions, can transduce the GI mucosa of rats and mice, and can efficiently mediate exogenous gene expression.35–43 After oral administration,

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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 indi-cates that some of the desmin-positive cells are hepatocytes. Bars = 200 μm. (f) Quantitative computer-assisted morphometric analysis of the immu-nostained 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.



AAV mediates exogenous gene expression primarily in the lam-ina propria and the epithelia lining the stomach and the small intestine.35–43 The AAV-mediated expression of foreign genes in the GI tract is potentially therapeutically useful due to the ben-efits not only for diseases of the GI system but also extra-GI dis-orders. Ectopic proteins expressed in the well-vascularized GI mucosa first go to the liver via the portal blood flow, resulting in the delivery of sustained high levels of the foreign proteins to the liver. Therefore, peroral administration is the preferred route of administration of the AAVs that express secretory proteins in the treatment of chronic liver diseases, such as hepatic fibrosis. Orally administered AAVs can simplify the complicated, laborious and

costly viral vector purification procedure. Our results consis-tently show that oral AAV–BMP-7 mediates the ectopic expres-sion of BMP-7 in the GI tract, leading to the long-term elevation of BMP-7 serum concentrations and the significant alleviation of CCl4-induced hepatic fibrosis. Although AAVs are fairly stable in the GI tract, their stability and survival are adversely affected by gastric acid and intestinal enzymes, which influence the survival and efficiency of gene transfer. In this study, we arbitrarily chose to administer two doses of AAV over an interval of three days, but we still need to investigate if this repeated protocol increases the efficiency of the gene transfer as compared with a single dose protocol.

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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.



In conclusion, our results indicate that orally administered AAV–BMP-7 shows excellent antifibrotic effects in the liver. These results suggest that oral AAV–BMP-7 could be developed into a safe, simple, and efficient therapy for treating hepatic fibrosis because of the preferential localization of AAVs in the human gut.44

MAterIAls And MetHodsPreparation of AAVs. AAVs (type 2) were prepared using the AAV Helper-Free System (Strategene, La Jolla, CA) according to the manufacturer’s instructions. Briefly, the CDS of hBMP-7 (kindly provided by Dr Xiang-Hui Huang, 2nd Hospital, School of Medicine, Xi’an Jiaotong University) was subcloned into pAAV-MCS between the BamHI and SalI sites to yield pAAV-hBMP-7. pAAV–BMP-7, or pAAV-LacZ were cotransfected with pHelper and pAAV-RC into AAV-293 cells by a calcium phosphate-based protocol. The cells were collected 72 hours later, and the viral particles were harvested. The viruses were designated as AAV–BMP-7 and AAV-LacZ, respectively. The viral particle titers were estimated by both dot–blot hybridization and quantitative real-time PCR and were expressed as the number of viral genomes/ml.

Animal protocol. Specific pathogen-free, 6-week-old male BALB/c mice were purchased from the Experimental Animal Center, School of Medicine, Xi’an Jiaotong University. All the animals received human care as detailed in the experimental protocol that was approved by the Committee of Laboratory Animal Research according to institutional guidelines. The mice were allowed free access to water and laboratory chow diet and were housed for one week before experiments. First, 15 mice were used to verify the AAV-mediated expression of BMP-7 and LacZ in the GI tract. The mice were divided into three equal groups: AAV–BMP-7, AAV-LacZ, and PBS control. All mice were deprived of food for 12 hours and were then given AAV–BMP-7 (1×1010 viral genomes in 0.5 ml), AAV-LacZ (1×1010 viral genomes in 0.5 ml), or an equal volume of PBS oragastrically followed by 4 hours of fasting. Three days later, the oral protocol was repeated as described above. The mice were sacrificed 3 days after the second dose. Blood samples were collected to determine the serum BMP-7 concentra-tions. The stomach and intestines were fixed with 4% paraformaldehyde for the immunohistochemical detection of BMP-7 or were cryosectioned for the histochemical examination of LacZ.

In the fibrosis experiment, 55 mice were assigned to four groups: normal controls (n = 10), AAV–BMP-7/CCl4 (n = 15), AAV-LacZ/CCl4 (n = 15), and CCl4 (n = 15). The mice in the AAV–BMP-7/CCl4 and AAV-LacZ/CCl4 groups were given the appropriate AAV oragastrically twice at an interval of 3 days, as in the above experiment. The mice in the other two groups were given an equal volume of PBS. The mice in the AAV–BMP-7/CCl4, AAV-LacZ/CCl4, and CCl4 groups were injected intraperitoneally with CCl4 (1 ml/kg BW dissolved in olive oil; final concentration of 20%) twice a week, for 7 weeks, starting 3 days after the second AAV dose. The mice in the NC group were given an equal volume of olive oil. Following 7 weeks of injections, the mice were sacrificed. Blood samples were collected for detecting the BMP-7 and hyaluronic acid concentrations by ELISA. The left lobe of the liver was fixed in 4% paraformaldehyde for the histological examinations. The right lobe was snap-frozen in liquid nitrogen and was stored at –70 °C for the quantification of hydroxyproline content.

In situ β-gal staining. Cryosectioned, 5-μm thick sections of the stomach and intestines were fixed with 0.2% glutaraldehyde for 15 minutes. Freshly prepared β-Galactosidase staining solution (1 mg/ml X-gal, 5 mmol/l potassium ferrocyanide, 5 mmol/l potassium ferrocyanide, 150 mmol/l NaCl, 2 mmol/l MgCl2, and 40 mmol/l citrate, titrated to pH 6.0 with NaH2PO4) was added to the sections. The sections were stained for 14 hours at 37 °C.

ELISA of serum BMP-7 and HA. The serum BMP-7 and HA levels were determined by sandwich ELISA using commercially available ELISA kits

(BMP-7 ELISA kit; R&D Systems, Minneapolis, MN. Hyaluronic acid ELISA kit, K-1200; Echelon Biosciences, Salt Lake City, UT) according to the manufacturer’s instructions. Duplicate analyses were carried out for each sample, and the results were averaged.

Histological evaluation. Formalin-fixed liver tissues were paraffin- embedded in an automated tissue processor. Five-micrometer liver sec-tions were stained with hematoxylin and eosin staining and Masson’s trichrome staining to assess the architectural alternations and hepatic col-lagen deposition (fibrosis).

The degree of fibrosis was evaluated semiquantitatively by the Ishak system.45 Fibrosis was graded as follows: stage 0—no fibrosis; stage 1—fibrous expansion of some portal areas, with or without short fibrous septa; stage 2—fibrous expansion of most portal areas, with or without short fibrous septa; stage 3—fibrous expansion of most portal areas with occasional portal to portal bridging; stage 4—fibrous expansion of portal areas with marked bridging (portal to portal or portal to central); stage 5—marked bridging (portal to portal and/or portal to central) with occasional nodules (incomplete cirrhosis); and stage 6—probable or definite cirrhosis. The fibrosis scoring was evaluated by two independent pathologists.

Immunohistochemistry. Immunohistochemistry was performed using the Histostain-Plus SP kit. Briefly, the paraffin-embedded sections were depar-affinized with xylene and were rehydrated by gradient ethanol immersion. The endogenous peroxidase activity was quenched by 0.3% (vol/vol) hydro-gen peroxide in methanol for 20 minutes, followed by three 5-minute washes with PBS. The sections were then blocked with 10% (vol/vol) normal goat serum in PBS for 1 hour followed by overnight incubation at 4 °C with the primary antibodies (mouse anti-BMP-7 monoclonal antibody, MAB4350; Millipore, Billerica, MA; 1:200 dilution; mouse anti-α-SMA monoclonal antibody, MS-113-PO clone 1A4; Lab Vision, Fremont, CA, 1:800 dilution; rabbit anti-desmin polyclonal antibody, Cat No. RB-9014-P0, Lab Vision, 1:400 dilution; or mouse anti-PCNA monoclonal antibody, ms-106-po, Lab Vision, 1:400 dilution) in PBS containing 3% (wt/vol) bovine serum albumin. The negative controls were performed by replacing the primary antibodies with preimmune mouse or rabbit serum. After washing in PBS with 0.02% (vol/vol) Tween-20 (PBST) three times for 5 minutes, the sec-tions were treated with biotinylated secondary antibodies for 20 minutes at room temperature followed by three 5-minute washes with PBST. The spec-imens were then incubated with Streptavidin-HRP for 20 minutes at room temperature followed by repeated washes as described above. HRP was processed using diaminobenzidine (DAB) at room temperature for 5 min-utes. After counterstaining with hematoxylin for 30 seconds, the sections were rinsed with tap water, immediately dehydrated by sequential immer-sion in gradient ethanol and xylene and mounted with Permount and cover slips. The images were obtained with a light microscope (Olympus BX51; Olympus, Tokyo, Japan) equipped with a DP70 digital camera.

Computer-assisted semiquantitative analysis was used to evaluate the areas of the positive α-SMA and desmin staining. All the images were quantified using Image-ProPlus version 4.5, a commercially available software package from Media Cybernetics (Silver Spring, MD). The imaging of the tissue sections was performed using an automated Image-Pro Plus macro calibrated for each microscope objective. The data for both the α-SMA and desmin staining were expressed as the mean percentage of the positively stained area out of the total tissue section area.

The parenchymal and non-mesenchymal cells were blindly counted (1,000 cells analyzed in 10 randomly chosen fields centered on a centrilobular vein at 400× enlargement) for PCNA expression, which was represented as the PCNA LI.

Hepatic hydroxyproline content. The total hydroxyproline content in the liver was determined as described previously.46 Briefly, the liver specimens were weighed, and an ~100 mg frozen sample was hydrolyzed in 1 ml 2 N



sodium hydroxide by autoclaving at 120 °C for 20 minutes. Chloramine-T (450 μl) was added to the lysate, mixed gently, and the oxidation was allowed to proceed for 25 minutes at room temperature. Finally, 500 μl of Ehrlich’s aldehyde reagent ([5 g p-dimethylaminobenzaldehyde dissolved in n-propanol/perchloric acid (2:1 vol/vol) in 100 ml)] was added to each sample. The samples were mixed gently, and the chromophore was devel-oped by incubating the samples at 65 °C for 20 minutes. The absorbance was measured at 550 nm. The hydroxyproline content was expressed as μg/mg wet liver.

Statistical analysis. The quantitative data are expressed as the means ± SEM. To assess the statistical significance of the intergroup differences of the quantitative data, Bonferroni’s multiple comparison tests were per-formed after one-way analysis of variance, followed by Bartlett’s tests to determine the homology of variance. The nonparametric data were ana-lyzed by the Mann–Whitney U-test. P < 0.05 was considered to be statisti-cally significant.

AcKnoWledGMentsThis work was supported by a grant from the National Foundation of Natural Sciences, China (No. 30871144). We thank Dr Xiang-Hui Huang, 2nd Hospital, School of Medicine, Xi’an Jiaotong University, for providing the CDS of hBMP-7.

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Oral Administration of Recombinant Adeno-associated Virus-mediated Bone Morphogenetic Protein-7 Suppresses CCl4-induced Hepatic Fibrosis in Mice