ACE2 Therapy Using Adeno-associated Viral Vector Inhibits Liver Fibrosis in Mice

Abstract

Angiotensin converting enzyme 2 (ACE2) which breaks down profibrotic peptide angiotensin II to antifibrotic peptide angiotensin-(1-7) is a potential therapeutic target in liver fibrosis. We therefore investigated the long-term therapeutic effect of recombinant ACE2 using a liver-specific adeno-associated viral genome 2 serotype 8 vector (rAAV2/8-ACE2) with a liver-specific promoter in three murine models of chronic liver disease, including carbon tetrachloride-induced toxic injury, bile duct ligation-induced cholestatic injury, and methionine- and choline-deficient diet-induced steatotic injury. A single injection of rAAV2/8-ACE2 was administered after liver disease has established. Hepatic fibrosis, gene and protein expression, and the mechanisms that rAAV2/8-ACE2 therapy associated reduction in liver fibrosis were analyzed. Compared with control group, rAAV2/8-ACE2 therapy produced rapid and sustained upregulation of hepatic ACE2, resulting in a profound reduction in fibrosis and profibrotic markers in all diseased models. These changes were accompanied by reduction in hepatic angiotensin II levels with concomitant increases in hepatic angiotensin-(1-7) levels, resulting in significant reductions of NADPH oxidase assembly, oxidative stress and ERK1/2 and p38 phosphorylation. Moreover, rAAV2/8-ACE2 therapy normalized increased intrahepatic vascular tone in fibrotic livers. We conclude that rAAV2/8-ACE2 is an effective liver-targeted, long-term therapy for liver fibrosis and its complications without producing unwanted systemic effects.

Figure 1

ACE2 expression in the liver and other major organs of healthy C57BL/6 mice treated with rAAV2/8-ACE2 gene therapy. ACE2 expression was evaluated by (a) qPCR, (b) western blotting, and (c) activity assay. rAAV2/8-ACE2 significantly increased liver specific ACE2 mRNA expression, ACE2 protein and activity compared to mice treated with saline injection. Each bar represents the mean ± SEM profile from n = 5 mice per treatment group. *P < 0.0001 and ^#P < 0.01 were calculated by one-way analysis of variance with Tukey comparison test.

Figure 2

Plasma aminotransferase level (ALT) and hepatic angiotensin peptides levels in the BDL, CCl₄, and MCD models with ACE2 gene therapy. rAAV2/8-ACE2 treated animals displayed significant reduction of (a) plasma alanine aminotransferase (ALT). In addition, ACE2 therapy decreased (b) hepatic Ang II concentrations and (c) increased hepatic Ang-(1–7) concentrations. Each bar represents the mean ± SEM profile from n >10 mice per treatment group. *P < 0.0001, ^#P < 0.01 and ^P < 0.05 and were calculated by one-way analysis of variance with Tukey comparison test.

Figure 3

ACE2 inhibited liver fibrosis and hepatic stellate cell activation. Fibrosis in the BDL, CCl₄, and MCD models was evaluated by morphometric analysis of (a and b) liver sections stained with picrosirius red, by (c) liver hydroxyproline content, and by (d) qPCR for COL1A mRNA, showed a significant reduction of hepatic fibrosis by rAAV2/8-ACE2 therapy. ACE2-treated mice also displayed a significantly decreased (e) positive staining of α-SMA, a marker for activated HSCs as well as (f) the mRNA level. Each bar represents the mean ± SEM profile from n >10 mice per treatment group. *P < 0.0001, ^#P < 0.01, ^P < 0.05, and ^ξP = 0.06 were calculated by one-way analysis of variance with Tukey comparison test.

Figure 4

ACE2 gene therapy reduced profibrotic mediators and proinflammatory cytokines expression. rAAV2/8-ACE2 therapy reduced mRNA levels of profibrotic mediators (a) CTGF, (b) TGF-β1, as well as proinflammatory cytokines (c) MCP-1 and (d) IL-6 in the BDL, CCl₄, and MCD models. Each bar represents the mean ± SEM profile from n >10 mice per treatment group. *P < 0.0001, ^#P < 0.01, ^P < 0.05, and ns = nonsignificant were calculated by one-way analysis of variance with Tukey comparison test.

Figure 5

ACE2 attenuated NADPH oxidase subunit translocation from the membrane to the cytosol. rAAV2/8-ACE2 therapy markedly reduced protein level of NADPH p67^phox protein subunit in the membrane (a and c) but had no effect on cytosolic subunit levels (b and d) in the BDL, CCl₄, and MCD models, suggesting rAAV2/8-ACE2 inhibited membrane translocation of p67^phox subunit. Each bar represents the mean ± SEM profile from n >10 mice per treatment group. *P < 0.0001, ^#P < 0.01, ^P < 0.05, and ns = nonsignificant were calculated by one-way analysis of variance with Tukey comparison test.

Figure 6

ACE2 inhibited hepatic oxidative stress via reduction of lipid peroxidation. Oxidative stress in the BDL, CCl₄, and MCD models was evaluated morphometric analysis of (a and b) liver sections stained with 4-hydroxynonenal, which showed a significant reduction of hepatic oxidative stress by rAAV2/8-ACE2 therapy. Each bar represents the mean ± SEM profile from n > 10 mice per treatment group. *P < 0.0001, ^#P < 0.01, and ^P < 0.05 were calculated by one-way analysis of variance with Tukey comparison test.

Figure 7

ACE2 reduced phosphorylation of MAPKs including (a and c) p38 in the BDL, MCD, and CCl₄ models and (b and c) ERK1/2 in the CCl₄ model. Phosphorylation of MAPKs was evaluated by western blotting in total liver proteins. Each bar represents the mean ± SEM profile from n > 10 mice per treatment group. *P < 0.0001, ^#P < 0.01, and ^P < 0.05 were calculated by one-way analysis of variance (ANOVA) with Tukey comparison test.

Figure 8

ACE2 significantly reduced hepatic vasoconstriction. Perfusion pressure in the liver was evaluated by in situ perfused mouse liver experiment. rAAV2/8-ACE2 gene therapy reduced methoxamine induced vasoconstriction in the livers of BDL mice, thus improving intrahepatic vascular resistance to portal inflow. Each symbol represents the mean ± SEM profile from n = 4 to 5 per treatment group. ^P < 0.05 was calculated by Student's two-tailed, unpaired t-test.