Differential Expression Of Endothelin Receptors Of The Liver Tissue In The Progression Of Liver Disease

Part 1 Differential expression of endothelin receptors in the liver hemangioma, liver cirrhosis,hepatocellular carcinoma and paraneoplastic tissuesBackground and Objective: Endothelin receptors(ETR) are a class of receptors which cause portal hypertension in liver cirrhosis,constituting a crucial part of endothelin system. Endothelin(ET) has been found to regulate multiple signaling pathways involved in fibrosis by activating ETR, endothelin receptor A(ETAR) involves in the invasion and metastasis of hepatocellular carcinoma(HCC), but few research has been studied on the differential expression of ETR in procession of cirrhosis-HCC. The paper means to explore the function of endothelin receptors in liver cirrhosis-HCC by detecting expression differences of endothelin receptors in the liver tissue of different disease progression.Method: Collecting clinical specimens from patients undergoing surgery of liver hemangioma and liver cancer,and liver biopsy tissue samples from patients(with early cirrhosis) in clinical trials and dividing them into four groups:liver hemangioma group(n = 13), cirrhosis group(n = 15), paraneoplastic group(n = 15) and liver cancer group(n = 15). Western blot is used to detect expression differences of ETAR, ETBR, α-SMA in the liver tissue of four groups,and One-way ANOVA is used to compare the significance of the relative gray value of the four groups.Results:In different liver tissue specimens, ETAR expression was most up-regulated in HCC,more-regulated in liver hemangioma,which showed a significant difference compared with paraneoplastic tissues(p<0.05)by relative gray value analysis. ETBR was most up-regulated in liver cirrhosis which showed a significant difference compared with liver hemangioma,paraneoplastic tissues,and hepatocellular carcinoma.Little significance was shown among four groups of α-SMA expression(p value> 0.05). Conclusion:The trend of ETAR expression is HCC >liver hemangioma >liver cirrhosis >paraneoplastic tissues,and the result may be due to the increased number of activated HSC, thus indicating that ETAR was involved in the growth and progression of tumor, cell proliferation,angiogenesis, tumor invasion and migration. ETBR was most-expressed in liver cirrhosis, which suggested a contrary trend of ETAR, may indicate that a quite relaxation effect of HSCs, a involvement in regulating sinusoidal blood flow changes, mediating hepatic sinusoidal dilation, reducing portal pressure and portal blood flow.Part 2 Differential expression of endothelin receptor-A distributed in hepatic stellate cells and endothelial cellsBackground and Objective: It has been demonstrated that endothelin receptors are expressed in the liver cells, with ETAR mainly distributing in vascular smooth muscle cells and hepatic stellate cells,and ETBR mainly in sinusoidal endothelial cells(LSECs) and hepatic stellate cells(HSCs). Endothelin receptors have almost all features of HSCs activation and play an important role in regulating HSC contraction, extrahepatic vascular resistance and portal hypertension. We can conclude that ETAR was closely related with cell proliferation,angiogenesis, the occurrence of tumor through the conclusion of chapter1. In this chapter we analyze different expression of endothelin receptors in liver cells- HSCs and LSECs to further explore the role of ETAR in liver disease progression.Method: The clinical specimens were divided into four groups as described in chapter 1:liver hemangioma, liver cirrhosis, paraneoplastic tissue and liver cancer. Positioning ETR hepatic stellate cells and sinusoidal endothelial cells by dual immunofluorescence staining and recording the positive cells and its percentage by using the Mann-Whitney test and Kruskal-Wallis test to analyze differential expression of ETR in HSCs and LSECs in different liver tissues and the significant difference.Results:The results show ETAR positive cells in hemangioma,cirrhosis,HCC and paraneoplastic tissues, HCC and hemangioma was significantly up-regulated compared with cirrhosis and paraneoplastic tissues(p<0.05).The trend of ETAR expression was HCC >liver hemangioma >liver cirrhosis >paraneoplastic tissues.ETAR and α-SMA expression was positive in the four groups, and numbers of hemangioma was significantly increased compared with liver cirrhosis and paraneoplastic tissues(p <0.05).The trend of ETAR and CD31 positive expression in the four groups.was liver hemangioma > HCC>liver cirrhosis >paraneoplastic tissues, and P value was of no significant differenceby comparing between groups(p> 0.05).Conclusion Highest level of ETAR was expressed in HCC, showing ETAR mainly distributed in vascular smooth muscle cells was involved in angiogenesis and tumorigenesis. The ETAR and α-SMA positive cells were more in normal control tissues than others, indicating ETAR was mainly expressed in vascular smooth muscle cells; and similarly ETAR and CD31 positive cells were more in angiogenic normal control tissues than others. Therefore, we considered that ETAR was closely related to angiogenesis and tumorigenesis.Part 3 Correlation between the expression of Endothelin receptor-A and clinical parametersBackground and Objective Endothelin receptors play an important role in the formation of portal hypertension in liver cirrhosis, and are involved in the progression of liver cancer. We aim at exploring the function of ETAR in the progression of liver cirrhosis to HCC by analyzing the correlation of ETAR-positive cells, co-expressed ETAR and CD31,ETAR and α-SMA cells by immunofluorescence staining in Chapter 2 and clinical indicators of liver cirrhosis such as platelet count(PLT), portal vein diameter, the thickness of spleen, liver cancer index like alpha-fetoprotein(AFP), liver function such as alanine aminotransferase(ALT), total bilirubin(TBIL), glutamyl peptidase(GGT) and so on.Method Statistically analyzing the correlation between positive cells in liver tissues Chapter 2 and clinical indicators by using SPSS software. We analyzed the statistical meaning by using the method of one-way ANOVA to conduct the comparison between groups. It represented a significant statistical meaning when p values were less than 0.05.Results There has been a correlation between ETAR positive cells and PLT,AFP,and portal vein diameter according to statistics, p values were 0.035, 0.009 and 0.008 respectively, both were less than 0.05. ETAR was positively correlated with the index of PLT and AFP,but significantly negatively correlated with portal vein diameter(p <0.01). ETAR and α-SMA positive cells were correlated with the indicator of ALT with p value 0.047(p<0.05), Pearson coefficient-0.449, being negatively correlated. ETAR and CD31 positive cells were correlated with the indicator of portal vein diameter(p <0.05), with Pearson coefficient-0.339, being negatively correlated.Conclusion The results may demonstrate ETAR of total liver cells and endothelial cells was closely related to the with portal hypertension in cirrhosis,and ETAR of the stellate cells was closely related.to inflammation.