The Mechanism And Clinical Study Of Intrahepatic Angiogenesis In Cirrhosis With Portal Hypertension

BackgroundLiver cirrhosis is a pathological disease due to various chronic liver injuries,which is the most severe pathological stage of all chronic liver diseases.If the injury factors persist,cirrhosis could further lead to severe clinical complications such as portal hypertension,esophageal varices(EV)and ascites.Portal hypertension and its complications are one of the leading causes of death in patients with end-stage liver disease,but the treatment is limited at present.To investigate the pathogenesis of portal hypertension for precisely treatment of cirrhosis with therapeutic target,and to explore noninvasive indicators of portal hypertension for timely treatment of cirrhosis by early diagnosis,shows great clinical significanceLiver cirrhosis is characterized by the deposition of extracellular matrix proteins and increased intrahepatic angiogenesis induced by the activation of hepatic stellate cells(HSCs)and hepatic sinusoidal endothelial cells(HSECs).Abnormal intrahepatic angiogenesis can cause the reconstruction of pathological vascular structures,blood flow disorder,increase of intrahepatic vascular resistance(IVR),and can further aggravate the deposition of extracellular matrix proteins.Studies have shown that intrahepatic angiogenesis plays an important role in promoting cirrhosis.Therefore,intrahepatic angiogenesis is a potential therapeutic target for the treatment of cirrhosis.However,the specific pathophysiology of portal hypertension has not been fully elucidated,especially whether intrahepatic angiogenesis could account for increased IVR and the mechanisms behind itIt is well known that vascular endothelial growth factors(VEGFs)play an important role in the regulation of angiogenesis via the receptors VEGFRs and NRPs,among them VEGFR2 is the major member of VEGFRs and the major effector receptor of VEGF.NRP-1,which is the first member of NRPs,was originally described as a class Ⅲ semaphoring binding protein(SEMA3),which regulated axon guidance.Later studies revealed that NRP-1 interacts with VEGFs,collaborating with VEGFR2 to enhance cell angiogenesis,and was implicated in embryonic and tumor angiogenesis.Previous study indicated that NRP-1 promoted liver cirrhosis progression by promoting activation of HSC,serving as a co-receptor of platelet-derived growth factor(PDGF)and transforming growth factor-β(TGF-β)While,the role of NRP-1 in intrahepatic angiogenesis in HSEC remains unclear and needs further research.As a transmembrane glycoprotein,NRP-1 forms a complex with VEGF as well as VEGFR2.Currently,it is widely accepted that NRP-1 promotes angiogenesis of endothelial cell in the extracellular domain through VEGF-NRP-1 interaction,which further enhances the binding and affinity of VEGF to its receptor VEGFR2,thereby promoting VEGFR2 activity and downstream signaling.However,other studies have also shown that specific blocking of NRP-1/VEGF binding by NRP-1 fl-/-mutant does not affect angiogenesis of embryonic and adult mouse,suggesting that NRP-1 may act independently of binding VEGF.In addition,the proangiogenic effect of VEGFR2 modulated by NRP-1 involving multiple intracellular signal transduction pathways,such as p38,extracellular regulated protein kinases1/2(ERK1/2),phosphatidylinositol 3’-kinase(PI3K)/protein kinase B(Akt),and Src/focal adhesion kinase(FAK).In one word,the mechanism and intracellular signaling pathways of NRP-1 in HSEC-induced intrahepatic angiogenesis,especially its interaction with VEGFR2 remain largely unknownPortal hypertension could be caused by abnormal intrahepatic angiogenesis and the deposition of extracellular matrix proteins.Studies have indicated that the early diagnosis of portal hypertension is necessary for timely treatment.Currently,measurement of the hepatic venous pressure gradient(HVPG)is considered the gold standard for portal hypertension assessment in clinic.However,HVPG measurement is an invasive procedure that requires technical expertise,which is available in only a few centers.Therefore,the need for simple and convenient noninvasive alternatives has become urgent.Among the noninvasive alternative methods,serum markers are simple and easily evaluated in the clinic,von Willebrand Factor(vWF),which reflects intrahepatic angiogenesis,has been used as a non-invasive surrogate marker for HVPG prediction in patients with cirrhosis in our previous study.In this study,we intend to evaluate the diagnostic efficacy of serum liver fibrosis indexes,which reflect liver fibrosis,for prediction of portal hypertension.The whole study including three parts,In the first part,we first collected the clinical specimens and data of cirrhotic patients,and confirmed that intrahepatic angiogenesis is positively correlated with the Child-Pugh classes,and the fibrosis degree,which could further increase portal hypertension.In the second part,we further confirmed NRP-1 is increased in HSEC and positively correlated with intrahepatic angiogenesis level in human and mouse cirrhotic liver tissues.And we found that NRP-1promotes intrahepatic angiogenesis via VEGFR2-dependent PI3K/Akt signaling pathway in HSEC in vitro.Meanwhile,liver histoculture indicated that NRP-1 inhibitor suppressed angiogenesis and fibrosis associated factors.In the third part of the study,we analyzed the diagnostic efficacy of noninvasive serum liver fibrosis markers for prediction of portal hypertension.Collectively,our results revealed that intrahepatic angiogenesis plays an important role in cirrhosis and portal hypertension.NRP-1 aggravated cirrhosis by promoting intrahepatic angiogenesis via VEGFR2-dependent PI3K/Akt signaling pathway in HSEC,which might be a valuable therapeutic target against angiogenesis in cirrhosis.This study provides a theoretical basis for anti-angiogenesis therapy of cirrhosis and portal hypertension,and provides a non-invasive first-line prediction model for early diagnosis of portal hypertension,which has an extensive clinical signification.Part 1 Intrahepatic angiogenesis increases portal hypertension in patient with cirrhosisAimIntrahepatic angiogenesis plays an important role in promoting liver cirrhosis,however the role of intrahepatic angiogenesis in portal hypertension remains unclear.We aim to investigate the relationship between intrahepatic angiogenesis and portal hypertension in hepatitis B patients with cirrhosis in this study.Methods1.60 hepatitis B patients with cirrhosis and 40 healthy subjects were included in this study.Child-Pugh classification was examined according to patients’laboratory,clinical,and imaging examinations.2.Intrahepatic angiogenesis markers VEGFR2 and vWF were observed by immunohistochemistry(IHC)staining in all enrolled liver tissues.Correlations between levels of intrahepatic angiogenesis and Child-Pugh classes were examined.3.Fibrosis marker alpha-smooth muscle actin(a-SMA)was observed by IHC staining in all enrolled liver tissues.Sirius-Red staining was also used to determine liver fibrosis degree.Correlations between levels of intrahepatic angiogenesis and liver fibrosis degree were examined.4.HVPG score was used to evaluate patients’ HVPG.The relationship between levels of intrahepatic angiogenesis and portal vein pressure were examined.5.We also analysed the correlations between levels of intrahepatic angiogenesis and complications of portal hypertension,including EV,ascites and hypersplenismResults1.Intrahepatic angiogenesis levels increased in cirrhotic patients,liver tissue and were correlated positively with Child-Pugh classes.The VEGFR2 expressions in the cirrhotic liver tissue were increased and correlated with the density of microvessels shown by vWF staining(r=0.85,p<0.01).Correlation was observed between the levels of VEGFR2(r=0.590,p<0.01),vWF(r=0.524,p<0.01)in tissue and Child-Pugh classes,respectively.2.Intrahepatic angiogenesis levels were positively associated with the liver fibrosis degree in cirrhotic patients’ liver tissue.Significant correlations were observed between levels of VEGFR2 and a-SMA(r=0.710,p<0.01),VEGFR2 and Sirius-Red(r=0.841,p<0.01),vWF and a-SMA(r=0.768,p<0.01),vWF and Sirius-Red(r=0.825,p<0.01).3.Intrahepatic angiogenesis may increase portal vein pressure in hepatitis B patients with cirrhosis.Patients with HVPG>12 mmHg showed higher levels of VEGFR2 and vWF expression compared to those who with HVPG<12mmHg(2.60±1.28%vs.1.09±0.73%;5.85±2.45%vs.2.31±1.34%,p<0.01),respectively4.Intrahepatic angiogenesis may influence the occurrence of complications of portal hypertension.The complications of PH,including size of EV,presence of ascites and spleen volume were significantly affected by the levels of intrahepatic angiogenesis(p<0.01)ConclusionIntrahepatic angiogenesis increases portal hypertension in hepatitis B patients with cirrhosis.Further works are needed to investigate the key molecular in intrahepatic angiogenesis as valuable therapeutic target for the treatment of cirrhosisPart 2 NRP-1 promotes intrahepatic angiogenesis via VEGFR2-dependent PI3K/Akt signaling pathwayAimIt was implicated that NRP-1 collaborating with VEGFR2 promoted cell angiogenesis in embryonic and tumor angiogenesis.NRP-1 promoted liver cirrhosis progression through PDGF/TGF-β pathway in HSCs.We aimed to investigate the proangiogenic effect of NRP-1 on HSEC and its intrinsic mechanism,to provide therapeutic target for anti-angiogenesis treatment of cirrhosis.Methods1.Human liver samples were divided into 2 group(cirrhosis group and normal group).The liver cirrhosis model was induced by intraperitoneal inj ection of CCl4 in C57BL/6 mice,which were randomly divided into 3 groups(a control group,CC14-treated 6 weeks group,and CC14-treated 8 weeks group).Hematoxylin-eosin(HE)and Masson staining were used to reflect the cirrhosis degree.Expression of NRP-1,VEGFR2 and markers of angiogenesis and fibrosis in human and mouse liver tissues were confirmed by IHC staining,Western blot,and quantitative real-time polymerase chain reaction(qRT-PCR)analysis.Correlations between the expression of NRP-1 and VEGFR2,and between the expression of NRP-1 and levels of intrahepatic angiogenesis were examined.2.The HSEC and Human umbilical vein endothelial cell(HUVEC)were used for in vitro study.Lentivirus transfection and transfection efficiency verification:Lentivirus transfection with GFP tag to down-regulate or over-express NRP-1 was performed in HSEC,HUVEC according to the manufacturer’s instructions using 20 MOI of lentiviruses encoding target genes.The ratio of green fluorescent cells and fluorescent intensity were confirmed by confocal microscope.NRP-1 mRNA and protein expression were tested qRT-PCR and Western blot analysis.3.Cell functional experiments:the ability of angiogenesis of HSEC,HUVEC were tested by tube formation assay.The ability of migration of HSEC,HUVEC were measured by Transwell assay.Cells proliferation were measured by CCK8 assay.4.Signaling pathways:Western blot analysis was used to identify the downstream signaling pathways of HSEC and HUVEC.And we used the indicated inhibitors and agonists to confirm the effect.5*Liver histoculture:Fibrotic patients’ liver tissues was cultured on sponge,followed by daily administration of NRP-1 inhibitor for one week with different concentrations.The survival rates of all cultured fibrotic tissue were tested by CCK-8 assay.The intrahepatic angiogenesis and fibrosis associated factors were tested by qRT-PCR analysis to identify the NRP-1 function in treatment of liver cirrhosis.Results1.NRP-1 expression was significantly increased in HSEC during cirrhosis and correlated positively with VEGFR2 expression and intrahepatic angiogenesis level.The cirrhosis degree of liver samples was confirmed by HE and Masson staining.Expression of NRP-1,VEGFR2 and markers of angiogenesis and fibrosis were increased in human and mouse cirrhotic liver tissues confirmed by immunohistochemistry staining,Western blot,and qRT-PCR analysis(p<0.05).The expression of NRP-1 was upregulated in conjunction with VEGFR2(r = 0.829,p<0.05)and correlated positively with levels of intrahepatic angiogenesis(r = 0.783,p<0.05)during cirrhosis progression.2.NRP-1 was down-regulated with lentivirus in HSEC and HUVEC,a significant reduction of NRP-1 mRNA and protein expression was detected in NRP-1-RNAi group compared to control group,accompanied by reduced ability of tube formation,migration and proliferation in endothelial cells(ECs)(p<0.05);A significant increase of NRP-1 mRNA and protein expression was detected in Lenti-NRP-1 group compared to Lenti-control group,accompanied by increased ability of angiogenesis and migration and proliferation of ECs(p<0.05).3.NRP-1 promoted angiogenesis by upregulating VEGFR2 via FAK in HSEC.The down-regulation of NRP-1 decreased not only the phosphorylated VEGFR2 but also the VEGFR2 protein and mRNA expression in ECs(p<0.05).On the contrary,NRP-1 overexpression significantly increased the phosphorylated and total VEGFR2 protein,as well as the VEGFR2 mRNA expression in ECs compared to Lenti-control cells.Mechanistically,NRP-1 modulated the expression of VEGFR2 by regulating FAK and its kinase activity.4.NRP-1 promoted VEGFR2-dependent angiogenesis via PI3K/Akt pathway in HSEC.Western blot showed that along with the increased phosphorylation of VEGFR2,increased PI3K and Akt activation were evidenced in Lenti-NRP-1 HUVEC and HSEC group compared with the Lenti-control group.5.Blocking NRP-1 function suppressed intrahepatic angiogenesis and fibrosis associated factors in vitro liver histoculture.NRP-1 inhibitor treated fibrotic tissue showed an increased survival rate compared with vehicle-treated tissue,which was in a concentration-dependent manner(p<0.05).The difference between vehicle and NRP-1 inhibitor treated tissue was evidenced by quantification of angiogenesis markers CD31 and vWF mRNA,and fibrosis factors α-SMA,fibronectin(Fn)and collagen 1 type 1(Coll)mRNA levels(p<0.05)ConclusionNRP-1 was unregulated in HSECs,which aggravates liver cirrhosis by promoting angiogenesis via VEGFR2-dependent PI3K/Akt pathway.Mechanistically,NRP-1 modulated the expression and activation of VEGFR2 by regulating FAK and its kinase activity.Therefore,NRP-1 might be a valuable therapeutic target against angiogenesis in cirrhosis.Part 3 Diagnostic efficacy of serum liver fibrosis indexes in predicting portal hypertension in patients with cirrhosisAimIntrahepatic angiogenesis levels were correlated positively with fibrosis degree,collaboratively leading to increases in cirrhosis and portal hypertension.Intrahepatic angiogenesis factor vWF has been used as a non-invasive surrogate marker of HVPG in our previous study.In this study,we intend to evaluate the diagnostic efficacy of serum liver fibrosis indexes in predicting portal hypertension,to provide a non-invasive first-line prediction model for early diagnosis of portal hypertension.Methods1.A total of 238 cirrhotic patients,who underwent hepatic venous pressure gradient(HVPG)evaluation and relevant clinical data,were enrolled in this study.The baseline characters and relevant serum parameters were retrospectively analyzed.Factors associated with HVPG grade were calculated by univariate and multivariate analysis and were used to construct the HVPG prediction model.The diagnostic efficacy of the HVPG prediction model was evaluate.2.Seven serum liver fibrosis indexes(AAR,APRI,FI,FIB-4,Forns’ index,King’s score and Lok index)were calculated according to the publishd paper.Receiver operator characteristic(ROC)were plotted and each area under the ROC curve(AUC)was calculated to assess the diagnostic performance of each fibrosis indexes in predicting HVPG.3.In order to increase the performance of the single liver fibrosis indexes,combinations of two fibrosis indexes that showed the best performance were calculated.The diagnostic efficacy of the combined model in predicting HVPG was evaluate.4.All the enrolled patients were divided in two different subgroups according to Child-Pugh classes(Child-Pugh A and Child-Pugh B/C)and etiologies(Viral cause and Non-viral cause).A subgroup analysis was conducted to investigate the influence of different clinical parameters on the performance of fibrosis indexes for the prediction of HVPG.Results1.The results of univariate and multivariate analysis of serum markers showed that aspartae aminotransferase(AST)values,platelet(PLT)count and albumin(ALB)were associated with HVPG grade.However,the performance of the conducted model for predicting clinically significant portal hypertension(CSPH)and severe portal hypertension(SPH)were not quite satisfactory:0.780AUC(0.722-0.831 95%CI)vs.0.769 AUC(0.711-0.821 95%Cl).2.Among the seven liver fibrosis indexes,King’s score,APRI and the Lok index showed modest diagnostic accuracy in predicting CSPH and SPH,as indicated by AUC of 0.755 and 0.742,0.740 and 0.742,and 0.722 and 0.717,respectively.3.Combination of King’s score(cutoff 23.47)and Lok index(cutoff 1.30)predicted presence of CSPH,with the highest PPV(95.38%)and +LR(5.49).4.A subgroup analysis indicated that the serum fibrosis indexes may be more applicable to patients with cirrhosis of viral etiology.ConclusionSerum liver fibrosis indexes exhibited modest diagnostic accuracy for portal hypertension in cirrhotic patients.These indexes may not be able to replace HVPG measurements but may be used as first-line prediction of CSPH for early diagnosis of portal hypertension in cirrhotic patients.