The Regulation Mechanism Of ACE2-Ang-(1-7)-Mas Axis On Hepatic Stellate Cell Contraction And Portal Hypertension

Background and ObjectiveIt is well known that various kinds of chronic liver disease may develop hepatic cirrhosis from hepatic fibrosis, while portal hypertension is a common and serious complication of cirrhotic liver disease. Hepatic stellate cells (HSCs) are major cells of extracellular matrix, activated HSCs play key roles in the hepatic fibrosis as well as portal hypertension. The activated-HSCs may transdifferentiate to myofibroblast-like and gain contractility. One hand, contracted HSCs induce the diminution of sinus hepaticus diameter which increase the intrahepatic vascular resistance. On the other hand, they cause hepatic cicatricial contraction, and further increase intrahepatic vascular resistance which result in portal hypertension finally. Therefore, the regulatory mechanism for the contraction of HSC has become an important factor of investigation to portal hypertension.Current studies have demonstrated that Ca2+-independent pathways are mainly involved in HSC contraction. RhoA/ROCK pathway which is one of the Ca2+-independent pathways has been focus study recently. The proteins of Rho family regulate cells contraction and movement mainly through actin regulation and cytoskeleton combination. Nevertheless, RhoA/ROCK pathway has down-regulation effect on NO/PKG pathway. Activated RhoA/ROCK pathway promoted HSC contraction by inhibition of eNOS activity which increased intrahepatic vascular resistance and leaded to portal hypertension.Intrahepatic renin-angiotensin-aldosterone system (RAAS) has been recognized as one of important fields in liver fibrosis research. In liver, upregulation of ACE-Angll-ATIR axis induced HSC activation, aggravated liver injury, advanced liver fibrosis and leaded to portal hypertension. ACE2-Ang-(1-7)-Mas axis which was new composition of RAAS has significant inhibitory effect on ACE-AngⅡ-AT1R axis and protect organs like heart, lung, kidney and liver from injury or fibrosis. Ang-(1-7) antagonized AngⅡ activity by competively binding to AT1R. Besides after binding with Mas receptor, Ang-(1-7) facilitated NO synthsis and resulted in vasodilation.Therefore, the aim of our study is to investigate the effect of ACE2-Ang-(1-7)-Mas axis on HSC contraction induced by AngⅡ through RhoA/ROCK and NO/PKG pathway in vitro;the effect of Ang-(1-7) on the liver cirrhosis and portal hypertension.Methods and materials1. Effects of ACE2-Ang-(1-7)-Mas axis on AngⅡ induced HSC contraction HSC-T6cell line was used in vitro study. The change of cell morphology was observed under electron microscopy; HSC contraction was evaluated by type I rat tail collagen; TRITC-phalloidin staining filaments actin (F-actin) in cells displayed its distribution and dynamics change.2. The regulation mechanism of ACE2-Ang-(1-7)-Mas axis on HSC contraction induced by AngⅡ in vitroAfter HSC-T6cells were pretreated by AT1R inhibitor (10nM Irbesartan), Mas receptor inhibitor (10nM A779), ROCK-2inhibitor (10nM Y27632) for1hour, AngⅡ (lnM) with Ang-(1-7)(lnM), AngⅡ (lnM)+Ang-(1-7)(lnM) with A779(10nM), AngⅡ(lnM) with Y27632(10nM), AngⅡ(lnM) with Irb (10nM) were co-treated for15min, and then RhoA GTP active protein was extracted by Pull-down GST in different groups, and the levels of actived RhoA GTP were detected by Western blotting. Membrane proteins were extracted from different groups; and the expressions of Ras, RhoA protein were examined by Western blottings. Levels of P-moesin/moesin and P-MLC/MLC proteins in RhoA/ROCK signaling pathway were measured using Western blottings in different groups. P-VASP/VASP and P-eNOS/eNOS proteins in NO/PKG pathway were evaluated by Western blot. Celluar RNA was extracted from different groups; fluorescence quantitative polymerase chain reaction (Quantitative Real-time PCR, Q-PCR) detected the expression of CTGF mRNA levels. Lenti-ACE2and ACE2siRNA were transfected into HSC-T6cells respectively and different proteins in either RhoA/ROCK or NO/PKG pathway assessed by Western Bloting.3. The regulation mechanism of Ang-(1-7) on portal hypertension in vivoISHAK and METAVIR fibrosis score were evaluated by HE staining, hydroxyproline (Hyp) content and Masson staining was detected in liver tissue among sham operation model (Sham)、double bile duct ligation rat model (BDL) and the treatment model (BDL+Ang-(1-7)). The protein levels in RhoA/ROCK and NO/PKG signaling pathway were measured by Western blotting and immunohistochemistry. Rho GEF, RhoA GTP and ROCK-2(Rho kinase) mRNA expressions were examined by Q-PCR. The hepatic portal vein blood flow velocity and the hepatic portal vein inside diameter were detected by Color Doppler Flow Imaging (CDFI); Portal pressure and intrahepatic resistance were evaluated using in situ liver perfusion method.Results1. ACE2-Ang-(1-7)-Mas axis inhibits HSC-T6contraction induced by AngⅡThe cells extension and volume increment stimulated by AngⅡ could be inhibited by Irb or Ang-(1-7) under the scanning electron microscope; Ang-(1-7), Irb and Y27632markedly inhibited the number of F-action increase staining with FITC-phalloidine observed by laser confocal microscopy; Rat tail collagen type I showed that Angll induced HSC-T6cells contraction and this effect could be inhibited by Ang-(1-7), Irb and Y27632. 2. The regulation mechanism of ACE2-Ang-(1-7)-Mas axis on inhibiting HSC-T6contraction induced by Angll in vitroAt the protein level, ACE2-Ang-(1-7)-Mas axis could significantly inhibit AngⅡ induced RhoA GTP protein activity (P=0.001), moesin protein phosphorylation (P=0.000), MLC phosphorylation and a-SMA levels (P=0.000). Y27632(RhoA kinase inhibitor) could also significantly reduce the RhoA GTP protein activity (P=0.000) and moesin protein phosphorylation (P=0.000). At the gene expression level, CTGF mRNA (P=0.000) were significantly increased after treated with AngⅡ but attenuated by Ang-(1-7)(P<0.01). AngI+Ang-(1-7), AngⅡ+Irb and AngⅡ+Y27632group were significantly reduced the expression levels of CTGF mRNA (all P<0.001). In addition, after lenti-ACE2was transfered into HSC-T6, RhoA/ROCK activity was markedly inhibited while NO/PKG pathway was upregulated. In contrast, ACE2siRNA transfected HSCs had the opposite effect.3. The regulatory mechanism of Ang-(1-7) on lowering portal hypertension in vivoMETAVIR&ISHAK fibrosis score demenstrated that BDL group was significantly higher than that of Sham group (P=0.000), and Ang-(1-7) treatment group significantly reduced compared with BDL group (P=0.000), but both higher than the Sham group. The contents of liver tissue Hydroxyproline (Hyp) and type I collagen in different rat groups showed that they were significantly highest in BDL group (P=0.000), and BDL+Ang-(1-7) group significantly reduced the levels of liver tissue Hyp (P=0.000) and type I collagen (P=0.000) comparing with BDL group. Immunohistochemistry found that that only a small part of the liver cells expressed P-moesin and a-SMA proteins. After rats were subjected to bile duct ligation for4weeks, hepatic P-moesin and a-SMA protein expressions were significantly increased. While Ang-(1-7) treatment group decreased the expressions compared with BDL group including the decreased number and staining positive cells signal intensity. At the protein level, in BDL group the expressions of RhoA, Ras, P-moesin and a-SMA on RhoA/ROCK signaling pathway were significantly increased (both P<0.01); while treatment with Ang-(1-7) decreased those protein levels and all were statistically significant (both P<0.01) except for RhoA and Ras. On NO/PKG pathway, the expressions of P-eNOS and P-VASP were significantly increased in BDL+Ang-(1-7) group (both P=0.000). At the gene level, on RhoA/ROCK signaling pathway the expressions of Rho GEF, RhoA and ROCK-2mRNA in BDL group were significantly increased (all P=0.000). On NO/PKG pathway, the expression of eNOS mRNA in BDL+Ang-(1-7) group was also significantly increased (P=0.000).The change of hemodynamic index was:CDFI detected that the visible surface of the liver was uneven, jagged or wavy in BDL group compared with that of in Sham group; the performance of the hepatic liver echo was showed light point thickening, uneven distribution or network-like hyperechoic separation in BDL rat groups. Moreover, Hepatic vein was squeezed thinner or uneven thickness. The result of hemodynamics in different rats groups showed that hepatic portal vein blood flow velocity and the hepatic portal vein inside diameter in BDL rats were significantly increase (P=0.000) compared with Sham group. Meanwhile, using in situ liver perfusion method extrahepatic portal vein pressure and intrahepatic vascular resistance were significantly lower in BDL+Ang-(1-7) groups than BDL groups (P=0.000).Conclusion1. ACE2-Ang-(1-7)-Mas axis inhibits AngⅡ induced HSC-T6contraction.2. ACE2-Ang-(1-7)-Mas axis inhibits AngⅡ induced HSC-T6contraction through inhibition of RhoA/ROCK activity and upregulation NO/PKG pathway.3. Ang-(1-7) lowers portal hypertension by improvement of liver fibrosis and inhibition of intrahepatic vasoconstriction via down-regulating ROCK-2activity and activating NO/PKG pathway.