Effects Of The ACE2-Ang-(1-7)-Mas Axis On Collagen Synthesis In Hepatic Stellate Cells And Its Signal Transduction Mechanism

Background & ObjectivesIt is well known that liver fibrosis is a result of chronic hepatocellular damage due to a variety of liver diseases, and it usually progresses to liver cirrhosis. Liver fibrosis has traditionally been regarded as an irreversible process. However, increasing evidence indicates that even advanced fibrosis may, in fact, be a reversible condition. Yet till now there is no effective treatment for liver fibrosis. Therefore, it is quite important and valuable to investigate underlying mechanisms and to develop more effective preventive and therapeutic interventions for liver fibrosis.Recent reports and our previous studies have shown that intrahepatic renin-angiotensin system (RAS) is closely related with liver fibrosis. Experimental evidence indicates that there is marked upregulation of intrahepatic RAS components in experimental liver injury, which will further lead to oxidative stress, accumulation of inflammatory cells, and finally, development of liver fibrosis. Angiotensin II (Ang II) is the most important effector of this system. It induces the activation and proliferation of hepatic stellate cells (HSC), releases some inflammatory cytokines, growth factors and pro-fibrosis factors, and stimulates extracellular matrix (ECM) production and inhibits its degradation, leading to excessive deposition of ECM in liver, thus liver fibrosis progression occurs. Recent evidence has shown that blocking of RAS may reduce liver damage, alleviate fibrogenesis and relieve portal hypertension.More recently, the newly found ACE2-Ang-(1-7)-Mas axis has aroused great interest among many researchers. Angiotensin-converting enzyme 2 (ACE2) degrades AngⅡand simultaneously produces Ang-(1-7).Ang-(1-7) counterworks the effects of AngⅡby dilation of blood vessels, down-regulation of blood pressure, diuresis, and inhibition of fibroblast proliferation, and other roles. In addition, Ang-(1-7) may also competitively bind to the AngⅡtype 1 receptor (AT1R). Therefore Ang-(1-7) has been considered as an endogenous antagonist of AngⅡ. As the new ACE2-Ang-(1-7)-Mas axis may counter-regulate the classical RAS (ACE-AngⅡ-AT1R axis), it is presumed to have a role in reducing fibrosis, and is expected to be a new potential target for treatment of liver fibrosis.However, the role of the ACE2-Ang-(1-7)-Mas axis in liver fibrosis has been rarely reported. Recent several reports demonstrated that the intrahepatic ACE2-Ang-(1-7)-Mas axis was up-regulated during liver injury, and that blocking of this axis worsened liver damage. However, the direct effects of the ACE2-Ang-(1-7)-Mas axis in hepatic fibrosis and the underlying mechanisms have not been elucidated. Liver fibrosis is characterized by activation of hepatic stellate cells, which are then involved in synthesis of matrix proteins and in regulating matrix degradation. It has been widely believed that HSC are the main cells producing ECM and collagen is the main component of ECM. Recent studies suggest that two newly discovered signaling pathways, Smad and Rho/ROCK pathways, are strongly related with the profibrogenic role of the RAS, and also closely associated with collagen synthesis. In our study, we observe the expression of the ACE2-Ang-(1-7)-Mas axis in animal models and in cultured HSC, and then evaluate the effects of this novel axis on Smad and Rho/ROCK signal transduction pathways, as well as on collagen synthesis in HSC. We hope that these investigations will help to understand the roles and mechanisms of the ACE2-Ang-(1-7)-Mas axis in liver fibrosis. Materials and Methods1. Liver fibrosis was induced in rats by repeated injections of 40% CCl4 (2.5mL/kg, twice per week). The pathological changes of rat livers were evaluated by HE staining. ACE2 immunoreactivity was assessed by immunohistochemical staining, ACE2 protein expression was detected by Western blot, ACE2 and Mas mRNA expression was determined by reverse transcription-polymerase chain reaction (RT-PCR). As an in vitro study, HSC-T6 were treated with AngⅡat concentrations ranging from 0.1 to 10×10-6mol/L. Western blot and RT-PCR were used to measure ACE2 expression and Mas mRNA levels in HSC.2. HSC-LX-2 were treated with AngⅡor Ang-(1-7) alone or together. Some cells were pretreated with Irbesartan (AT1R antagonist), A779 (Mas antagonist), SB203580 (p38 MAPK inhibitor), PD98059 (ERK inhibitor), or Y27632 (ROCK inhibitor) before AngⅡtreatment. The phosphorylated-Smad3 protein expression level was analyzed by Western blot, nuclear translocation of Smad4 was observed by immunofluorescence microscopy, and Smad DNA binding activity was assessed by electrophoretic mobility shift assay (EMSA) using digoxin labeled double-stranded oligonucleotide containing Smad consensus sequence. Western blot and RT-PCR were used to examine RhoA protein and ROCK2 mRNA expression levels, respectively.3. A lentiviral vector encoding a short hairpin RNA specifically targeting human ACE2 was constructed, and the silencing efficiency was determined by real time PCR and Western blot in HSC-LX-2 after transduction with the lentiviral vector. RT-PCR, Western blot and Gene quantification (QuantiGene Plex) were performed to analyze the expression of collagen type 1 (COL1) and connective tissue growth factor (CTGF) in HSC treated with AngⅡ, Ang-(1-7) or after knock-down of ACE2 gene. Inhibitors of p38 MAPK, ERK or ROCK were employed to assess the effects of these signaling pathways on the expression of COL1 and CTGF mRNA in HSC.Results1. The expression of ACE2 and Mas was increased in CCl4-intoxicated rat liver.Hematoxylin-eosin (HE) staining revealed that, in rat livers after subcutaneous injection of CCl4 for 4 weeks, the normal structure of hepatic lobules was destroyed and the regenerative nodules were separated by dense fibrous septa. Immunohisto-chemical staining showed that intrahepatic ACE2 expression was increased and redistribution after 4 weeks of CCl4 exposure. RT-PCR showed that ACE2 mRNA expression levels were mildly increased after 2 weeks of CCl4 administration, and markedly increased at the 4th week (0.509±0.105 vs 0.114±0.044, P=0.000). ACE2 protein expression as determined by Western blotting mirrored the changes observed in ACE2 mRNA levels. In addition, in rats treated with both CCl4 and perindopril, ACE2 expression were significantly increased by 2 weeks as compared with rats receiving CCl4 alone (P<0.01), and showed a further slight increase by week 4. Mas gene expression followed a similar pattern to that seen for ACE2 in all three groups of rats.2. The expression of ACE2 and Mas was increased in AngⅡ-treated HSC.Incubation of HSC with different concentrations of AngⅡfor 24 hr led to a dose-dependent increase in ACE2 and Mas mRNA expression. Although these increases were only slight following treatment with 0.1μmol/L Angll and did not reach statistical significance, they were marked following treatment of HSC with 1μM AngⅡ(1.553±0.090 and 1.536±0.053 fold over control, respectively, both P< 0.01), and increased further at 10μM. Western blot analysis showed similar changes in ACE2 protein. Besides, ACE2 and Mas expression was significantly increased in HSC simultaneously administered with AngⅡand ACEI as compared with cells treated with AngⅡalone (P<0.01).3. Ang-(1-7) inhibited AngⅡ-induced activation of Smad pathway in HSC.The results of Western blot showed that AngⅡinduced phosphorylation of Smad3 (pSmac3) in HSC in a time-dependent manner. The levels of pSmad3 were rapidly increased at 5 min (2.183±0.222 fold of control, P=0.003), and reached with a peak at 15 min, then gradually declined. No change was observed in the levels of non-phosphorylation of Smad3 protein expression. Immunofluorescence assay showed that AngⅡinduced translocation of Smad4 from cytoplasm to nuclear, while gel shift assay (EMSA) showed that Ang II elevated Smad DNA binding activity (9.007±1.007 fold over control, P=0.000). Ang-(1-7) alone showed little impact on the Smad pathway in HSC. However, it inhibited AngⅡ-induced phosphorylation of Smad3 (P<0.01), nuclear translocation of Smad4 and elevation of Smad DNA-binding activity (P<0.05) in HSC. Mas receptor antagonist A779 significantly reversed the inhibiting effect of Ang-(1-7) on AngⅡinduced pSmad3 expression and Smad DNA-binding activity (P<0.01). Irbesartan (AT1R blocker) also potently restrained the activation of Smad pathways induced by AngⅡ. These results indicated that AngⅡactivates the Smad pathway through AT1R, and Ang-(1-7) plays an opposite role in Ang II-induced Smad pathway through Mas receptor. In addition, the levels of AngⅡ-induced pSmad3 were significantly lowered by SB203580 (p38 MAPK inhibitor) (P=0.000), but not by PD98059 (ERK inhibitor) (P=0.575), indicating that Ang II activates Smad signaling pathway at least partly via the p38 MAPK pathway.4. Ang-(1-7) inhibited AngⅡ-induced activation of Rho/ROCK pathway in HSC.The expression levels of RhoA protein and ROCK2 mRNA were significantly increased in HSC after treated with Ang II for 24 hr, as compared with controls (both P=0.000). Irbesartan, Ang-(1-7) and Y27632 (ROCK inhibitor) markedly abrogated the effect of Ang II on RhoA protein and ROCK2 mRNA expression (all P<0.01). A779 reversed the inhibition of Ang-(1-7) on AngⅡ-induced activation of RhoA and ROCK2 (both P<0.01). These results suggested that AngⅡactivates Rho/ROCK pathway through AT1R, and Ang-(1-7) antagonizes this effect of AngⅡthrough Mas receptor.5. ACE2-Ang-(1-7) inhibited AngⅡ-induced collagen synthesis in HSC.A lentiviral vector encoding a shRNA directed against ACE2 was designed and constructed, and then transferred into HSC. The know-down efficiency was near 70% as determined by real-time PCR at the mRNA levels and Western blot at protein levels (0.154±0.018 vs 0.446±0.026, P=0.000). This vector was named as Lenti-SiACE2.The expression of COL1 mRNA and CTGF protein were evaluated by RT-PCR and Western blot respectively, and their mRNA expression levels were further analyzed by QuantiGene Plex assay. The results showed that AngⅡstimulated significant increase of COL1 and CTGF expression levels in HSC as compared with controls (both P<0.01). The expression levels of COL1 and CTGF were little changed in cells transferred with Lenti-SiACE2 (P=1.000), but they were significantly increased following AngⅡstimulation, as compared with normal HSC treated with AngⅡ(both P<0.01). This result indicated that knock-down of ACE2 prolongs AngⅡ-stimulated collagen synthesis. Ang-(1-7) abolished AngⅡ-induced COL1 and CTGF expression (both P<0.01), which was reversed by Mas receptor antagonist A779 (both P<0.01). Besides, QuantiGene assay showed that the degree of inhibition of Ang-(1-7) on AngⅡ-induced upregulation of COL1 and CTGF mRNA was more pronounced than that of SB203580, PD98059 and Y27632 (all P< 0.01, except P<0.05 compared with the CTGF mRNA level in HSC pre-treated with Y27632). These data indicated that collagen synthesis in HSC may involve a variety of pathways, yet it can be dramatically inhibited by Ang-(1-7) through directly against AngⅡ.Conclusions1. In the present study, we observed an increase in hepatic ACE2 and Mas receptor expression with the progression of liver fibrosis induced by CCl4 in rats. Furthermore, in our in vitro experiments, we found that AngⅡupregulated ACE2 and Mas expression in a dose-dependent manner in HSC. The changes in ACE2 and Mas expression indicate that it may be a resistance response to the augmented levels of AngⅡ, and may further represent a negative feedback mechanism involved in maintaining equilibrium in the intrahepatic RAS. Therefore, our results support the previous hypothesis that the ACE2-Ang-(1-7)-Mas axis may play a protective role in liver fibrosis. Besides, our study showed that ACEI is able to upregulate ACE2 expression under conditions of liver injury, both in vivo and in vitro, which may in turn contribute to additional benefits of ACEI on liver fibrosis.2. Our investigation showed that AngⅡactivated the Smad and Rho/ROCK signaling pathways in HSC through interacting with its receptor AT1R, which may be important mechanisms of the pro-fibrosis effect of AngⅡ. Ang-(1-7) inhibited the activation of these two pathways through counteracting with AngⅡ, which may be attributed to its anti-fibrosis function in liver. In addition, p38 MAPK inhibitor reduced the AngⅡ-mediated phosphorylation of Smad3, suggesting that Ang II can directly activate the Smad signaling pathway in a TGF-β-independent manner.3. We also found that AngⅡstimulation significantly increased COL1 and CTGF expression in HSC. Silencing of ACE2 further enhanced AngⅡ-induced overexpression of these two effectors. Ang-(1-7) abated the activation of COL1 and CTGF caused by AngⅡ, and this inhibiting effect was stronger than that of p38 MAPK, ERK 1/2 or Rho/ROCK inhibitors. These results indicated that ACE2 and Ang-(1-7) can suppress collagen synthesis in HSC through antagonizing the effect of Ang II. Therefore, our study provides experimental evidence that the new ACE2-Ang-(1-7)-Mas axis has a protective role in liver, and thus, it can be a promising target for the therapy of liver fibrosis.