Effects And Mechanisms Of Carvedilol On Improving The Structural Factors Of Intrahepatic Vascular Resistance And Reducing Portal Venous Pressure In Liver Cirrhosis

Background and Aims Liver cirrhosis is the advanced stage of liver fibrosis and a common pathological process of chronic liver diseases(CLDs)with high morbidity and mortality.As the most common and severe complication in patients with liver cirrhosis,portal hypertension(PHT)results in serious clinical consequences including variceal bleeding,ascites and hepatic encephalopathy.In patients with liver cirrhosis,PHT and its complications are the leading causes of death and requirement for liver transplantation.Pharmacological therapies that could halt or reverse the progression liver cirrhosis are of great importance for improving the life quality and expectancy in cirrhotic patients.Recently,great advances have been made in the pathophysiology of cirrhotic PHT.Two factors are involved in the pathogenesis of PHT:one is the initial determinant that is the increased intrahepatic vascular resistance(IHVR),the other is the remaining determinant that is the increased portal venous blood flow.The increased IHVR mainly results from structural factors and functional factors.Liver fibrosis,regenerative nodules formation and hepatic sinusoidal remodeling are important structural factors responsible for the increased of IHVR in liver cirrhosis.The most remarkable characteristic in hepatic sinusoidal remodeling has been referred to as "hepatic sinusoidal capillarization".The most obvious feature of liver cirrhosis is the excessive deposition of the extracellular matrix(ECM).On the one hand,the formation of scars,the regeneration of nodules and the proliferation of fibrous connective tissue could lead to the architectural distortion and the compression of hepatic venous systems that increase IHVR.On the other hand,the excessive deposition of ECM in the subendothelium of LSECs results in the formation of basement membranes under the liver sinusoidal endothelial cells(LESCs),termed"hepatic sinusoidal capillarization",thus promoting the development of liver cirrhosis and PHT.Various types of cells and cytokines are involved in the pathogenesis and progression of liver fibrosis,cirrhosis and PHT.Hepatic stellate cells(HSCs)are the most important effector cells.Following liver injuries,the quiescent HSCs undergo transdifferentiation into a myofibroblastic phenotype.Activated HSCs are characterized by proliferation,migrate to the damaged region,increased production of ECM proteins and secretion of profibrogenic factors.LESCs secrete ECM proteins and profibrogenic cytokines including fibronectin(FN),transforming growth factorβ1(TGF-β1)and platelet-derived growth factor(PDGF),which are involved in HSCs activation and hepatic sinusoidal capillarization.They play important roles in the progression of liver fibrosis,cirrhosis and PHT.The TGF-β1 signaling pathway is one of the most important pathways in the progression of liver fibrosis,and TGF-β1 is the most profibrogenic cytokine in the live that promotes the synthesis of ECM proteins in both HSCs and LESCs.The TGF-pl signaling pathways include Smad-dependent pathway which is associated with Smads famity members,and Smad-independent pathways including phosphatidylinositol-3-kinase/protein kinase B(PI3K/AKT)pathway,extracellular signal-regulated kinase(ERK)pathway,ect.Carvedilol is a novel nonselective beta-blocker(NSBB)with selective α1 adrenoreceptor antagonist activity,and is used widely in the cardiovascular fields.Carvedilol has anti-proliferative,anti-inflammatory,antioxidant and anti-angiogenic properties.Thus,carvedilol has more advantages than traditional beta-blockers.NSBBs are the first-line pharmacological therapy for the phrophylaxis of variceal bleeding due to their effective reduction of portal venous pressure.Studies have shown that carvedilol is more effective than traditional NSBBs(propranolol and nadolol)in reducing hepatic venous pressure gradient(HVPG).The effect of carvedilol on reducing portal venous pressure has mainly based on decreasing the increased portal inflow that is the maintenance factor of PHT.No structural factors of the increased IHVR that is the initial determinant of PHT has been found to be involved in the effect of carvedilol on reducing portal venous pressure.It has been found that carvedilol could improve myocardial fibrosis and remodeling,as well as renal fibrosis and vascular remodeling.Therefore,we speculate that carvedilol may improve the structural factors that account for the increased IHVR,thereby reducing the portal venous pressureIn the present study,the rat model of liver cirrhosis induced by carbon tetrachloride(CCl4)was used to explore the therapeutic effects of carvedilol.Animal experiments proved that carvedilol could inhibit HSCs activation and reduce the deposition of ECM,improve the intrahepatic architectural distortion,liver fibrosis and hepatic sinusoidal capillarization,and reduce the portal venous pressure.The improvements of the structural factors that account for the increased IHVR are involved in the effect of carvedilol on reducing portal venous pressure.Human HSC cell line LX-2 cells,primary LSECs and human umbilical vein endothelial cells(HUVECs)were used in vitro to explore the mechanism of carvedilol on improving the structural factors that account for the increased IHVR.Cellular experiments showed that carvedilol could inhibit the proliferation,activation and invasion of HSCs,and inhibit the synthesis of ECM proteins in HSCs,LSECs and HUVECs by suppressing the TGF-pl/Smads pathway.These results revealed that carvedilol inhibited HSCs proliferation,activation and invasion of,and supressed the synthesis of ECM proteins in HSCs and LSECs,thereby improving the structural factors that responsible for the increased IHVR.At present,carvedilol is mainly used for the prophylaxis of variceal bleeding due to its reduction of portal venous pressure in patients with decompensated liver cirrhosis.We believe that carvedilol could act on the structural factors of the initial determinant of PHT(the increased IHVR),thus reducing the portal venous pressure.Hence,the clinical application of carvedilol should not be limited in the decompensated stage of liver cirrhosis.It is of great importance that patients with CLDs may benefit more from the application of carvedilol in the early stage of CLDs.Part 1 Carvedilol improves the structural factors of intrahepatic vascular resistance and reduces portal venous pressure in cirrhotic ratsObjective To investigate the therapeutic effects of carvedilol on cirrhotic rats induced by CCI4.Methods Male Wistar rats were randomly divided into three groups:the control group,the liver cirrhosis group and the carvedilol-treated group.The rats in the control group received an intraperitoneal injection of olive oil twice a week.The rats in the liver cirrhosis group received an intraperitoneal injection of CC14 twice a week.The rats in the carvedilol-treated group received an intraperitoneal injection of CCl4 twice a week and a concurrent intragastrie administration of carvedilol daily.After 9 weeks,the body weight of all the rats were weighed and recorded,then the portal vein pressure was measured under anesthesia.Blood samples were collected and the serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and albumin(ALB)were measured.Liver specimens were collected,then Hematoxylin-Eosin(HE)staining and Sirius Red(S-R)staing were used for the histological examination and ECM deposition evaluation in the liver tissues,respectively.Immunohistochemistry(IHC)and Western blot were used to detect the indexes of liver fibrosis including the expression of α-smooth muscle actin(α-SMA)and FN.Transmission electron microscope(TEM)was used to observe the deposition of ECM in the subendothelium of LSECs.Results1.The body weight of cirrhotic rats was significantly lower than that of the control group.Carvedilol aggravated the CCl4-induced weight loss of the rats.2.The serum levels of ALT and AST were increased,whereas the serum level of ALB was decreased in cirrhotic rats.Carvedilol inhibited the CCl4-induced elevation of serum ALT and AST,but did not improve the CCl4-induced decrease of serum ALB in the rats3.The portal vein pressure was increased significantly in cirrhotic rats.Carvedilol reduced the CCl4-induced increase of portal vein pressure in the rats.4.The architecture of livers was distorted in cirrhotic rats,which could be improved by carvedilol.5.The excessive ECM deposition was observed in the livers of cirrhotic rats.C arvedilol significantly decreased the CCl4-induced ECM deposition in the crats.6.HSCs were activatedin the livers of cirrhotic rats,which could be suppressed by carvedilol.7.The excessive deposition of ECM was observed in the subendothelium of LSECs in the livers of cirrhotic rats,which resulted in the formation of continuous basement membrane.Carvedilol obviously attenuated the CCl4-induced ECM deposition in the subendothelium of LSECs and improved the hepatic sinusoidal capillarization in the livers.Conclusion Carvedilol inhibited HSCs activation and ECM synthesis,improved the architectural distortions,liver fibrosis and hepatic sinusoidal capillarization in the livers of cirrhotic rats,thus improving the structural factors that responsible for the increased IHVR and reducing the portal venous pressure in cirrhotic rats.Part 2 Carvedilol inhibits the proliferation,invasion,activation and the synthesis of extracellular matrix proteins in hepatic stellate cellsObjectiveTo investigate the effects of carvedilol on the proliferation,invasion,activation and the synthesis of ECM proteins in HSCs.Methods1.Human HSCs cell line LX-2 cells were cultured in vitro.2.CCK-8 was used to detect the effect of carvedilol on the proliferation of HSCs.3.Transwell assay was used to investigate the effect of carvedilol on the invasive ability of HSCs.4.Immunofluorescence(IF)was used to detect the protein expression of FN in the cytoplasm of HSCs.5.Western blot was used to assay the effect of carvedilol on th eactivation and synthesis of ECM proteins in HSCs,and explore the molecular mechanisms.Results1.Carvedilol inhibited the proliferation of HSCs.2.Carvedilol inhibited the invasion of HSCs.3.Carvedilol inhibitedHSCs activation.4.TGF-β1 stimulated the synthesis of ECM proteins in HSCs.Carvedilol inhibited the elevated expression of ECM proteins induced by TGF-β1.5.TGF-β1 upregulated the phosphorylation levels of Smad2 and Smad3 in HSCs,without changing the expression of total Smad4 and phosphorylated AKT and ERK.6.Carvedilol inhibited the up-regulation of phosphorylated Smad2 and Smad3 induced by TGF-β1.The application of SIS3(pSmad3 specific inhibitor)confirmed that carvedilol inhibited the up-regulation of ECM proteins induced by TGF-pl through inhibition of TGF-β1/Smads pathway.Conclusion Carvedilol inhibited HSCs proliferation,invasion,activation,and the synthesis of ECM proteins by inhibiting the TGF-pl/Smads pathway,thus improving the improving the structural factors that responsible for the increased IHVR in cirrhotic rats.Part 3 Carvedilol inhibits the synthesis of fibronectin in liver sinusoidal endothelial cellsObjective To investigate the effects of carvedilol on the synthesis of fibronectin in LSECs.Methods1.HUVECs and primary LSECs were cultured in vitro.The endothelial cell line HUVECs were used to explore the conditions of cellular experiments,and the effects of carvedilol were verified in LSECs2.IF was used to detect the FN protein expression in the cytoplasm of LSECs and HUVECs.3.Quantitative real-time polymerase chain reaction(qTR-PCR)was used to investigate the effects of carvedilol on the mRNA expression of FN in LSECs and HUVECs induced by TGF-β1.4.Western blot was used to investigate the effect of carvedilol on the protein expression of FN inLSECs and HUVECs induced by TGF-β1 and explore its molecular mechanisms.5.Inhibitors were used to explore the signaling pathway by which carvedilol inhibited the TGF-β1-inducedFN protein expressionin LSECs and HUVECs.Results1.IF confirmed the protein expression of FN in the cytoplasm of LSECs and HUVECs.2.TGF-β1 up-regulated the mRNA and protein expression of FNinLSECs and HUVECs.3.Carvedilol inhibited the up-regulation of FN mRNA and protein expressions induced by TGF-β1 in LSECs and HUVECs.4.TGF-β1 upregulated the phosphorylation levels of Smad2 and Smad3 in LSECs,while it upregulated the phosphorylation levels of ERK,Smad2 and Smad3 in HUVECs.5.Carvedilol inhibited the up-regulation of phosphorylated Smad2 and Smad3 induced by TGF-β1 in LSECs,and inhibited the up-regulation of phosphorylated ERK,Smad2 and Smad3 induced by TGF-β1 in HUVECs.6.The application of SIS3 and PD98059 confirmed that carvedilol inhibited the TGF-β1-induced up-regulation of FN protein expression in LSECs and HUVECs through suppressing TGF-β1/Smads pathway.Conclusion Carvedilol inhibited the TGF-β1-induced up-regulation of FN protein expression in LSECs through the suppression of TGF-β1/Smads pathway,thus alleviating liver fibrois and hepatic sinusoidal capillarization,thus improving improving the structural factors that responsible for the increased IHVR in cirrhotic rats.