| Background and Purposes:Hepatopulmonary syndrome?HPS?is a kind of a severe pulmonary complication caused by intrapulmonary micro-vessel dilation?IPVD?,gas exchange disorders and abnormal arterial oxygenation in the setting of chronic liver disease and/or portal hypertension.It significantly increases the perioperative mortality of liver disease patients,and is mainly responsible for postoperative liver dysfunction and pulmonary infection after liver transplantation,which has aroused widespread concern among hepatology and critical care experts both at home and abroad.According to reports,the median survival time of cirrhosis patients without HPS after liver transplantation was 87 months,and the 5-year survival rate was 63%,while the the median survival time of cirrhosis patients with HPS was only 24 months,and the 5-year survival rate was 23%.China is a country with a high incidence of liver diseases,and the occurrence of HPS will impose a heavier medical burden on patients,hospitals and the society.Angiogenesis is a process involving many cell types and signaling pathways,which play an important role in the development of cancer,rheumatoid arthritis,and diabetic retinopathy.Pathological angiogenesis is similar to normal blood vessels which deliver oxygen and nutrients to cells through the blood.Except for certain special physiological conditions,such as pregnancy and wound healing,pathological angiogenesis often causes disease.Previous studies demonstrate that pathological angiogenesis is one of the most important pathological features of severe HPS,making it urgent to further study its molecular mechanisms.In addition,recent studies have shown that exosomes play an important role in the pathophysiological processes of many diseases.However,the role of exosomes in the development and progression of HPS remains unclear.Therefore,common bile duct ligation?CBDL?rat model was used to study the role of serum exosomes on angiogenesis during HPS.The results of the study are of great significance for further understanding the pathogenesis of HPS and provide a theoretical basis for the treatment of HPS.In addition,collective cell migration plays an important role in the process of angiogenesis.In order to better understand the regulatory mechanism of collective migration,drosophila border cell migration was observed in our study due to the feasibility of in vivo genetic modifications and ex vivo live imaging during the entire migratory period.Methods:1.CBDL rat model was constructed,serum exosomes were extracted and identified,and the uptake of serum exosomes by PMVECs was observed.The HPS model was induced by chronic bile duct ligation?CBDL?,which is validated by the blood gas test,liver function test as well as liver and lung tissue pathological section analysis after 5 weeks.Serum exosomes from sham-operated rats and HPS rats were isolated by differential centrifugation and identified by western blot,TEM and NTA,followed by the measurement of exosomes concentration by BCA.After that,PMVECs were incubated with PKH67-labeled exosomes,and the uptake of exosomes was visualized by confocal microscopy.2.The effect of HPS rat serum exosomes?HEs?and sham-operated rat serum exosomes?SEs?on the proliferation,migration and tube formation of PMVECs were observed.HEs and SEs were co-cultured with PMVECs,along with the analysis of the proliferation ability of PMVECs detected by flow cytometry,the migration capabilities of PMVECs detected by transwell assay,the angiogenesis ability of PMVECs detected by tube formation assay,and the directional collective cell migration ability detected by wound healing assay.3.miRNA microarray chip was used to screen the expression miRNAs profile within HEs and SEs,qRT-PCR were performed to validate the microarray data.4.The roles of miR-194 on the proliferation,migration and tube formation of PMVECs were observed.Mir-194 mimics and inhibitors were transfected into PMVECs,along with the analysis of the proliferation ability of PMVECs detected by flow cytometry,the migration capabilities of PMVECs detected by transwell assay,the angiogenesis ability of PMVECs detected by tube formation assay,and the directional collective cell migration ability detected by wound healing assay.5.Bioinformatics analysis and validation of potential miR-194 target genesThe target genes of miR-194 were predicted by using the Targetscan database,and the Amigo database was used to collect the genes negatively regulating angiogenesis.The target genes of miR-194 and the direct binding relationships were further validated by dual-luciferase reporter assay.6.Analyze the secreting cells of exosomes increased in serum during HPSScreening was performed for cells which might lead to an increase in serum exosomes and miR-194 in exosomes,and the cells which were screened treated with bile acids to observe changes in secreted exosomes and miR-1947.Verify the role of P53 pathway-mediated exosome secretion in HPS angiogenesis in vivo and in vitroThe effects of P53 inhibitors on TASP6 expression,exosome concentration and miR-194 in hepatocytes after bile acid treatment were investigated by using P53 inhibitors?pifithrin-??and P53 RNAi.CBDL model rats were injected with pifithrin-?,GW4869 or anta-miR-194 intravenously to observe their effects on pulmonary angiogenesis and survival.The correlation between HPS patients and serum extracellular miR-194 levels and P?A-a?O2 in rats was further analyzed.8.By using live imaging and optogenetics in drosophila egg chamber,we tried to observe the specific role of Myo-II pulse in collective cell migration in multicellular environments.Results:1.Our results validated the availability of CBDL for establishing experimental HPS model.The concentration of exosomes derived from HPS rat serum was markedly higher than that of exosomes derived from Sham-operated rat serum.Serum exosomes labeled with fluorescent PKH67 were internalized by unstained PMVECs over time.2.Exosomes derived from HPS rat serum promote PMVECs proliferation,migration and tube formation3.The microarray data and verified that miR-194 was the most significantly upregulated miRNA in exosomes derived from HPS rat serum.4.miR-194 enhance PMVECs proliferation,migration and tube formation5.MiR-194 targeting THBS1,STAT1 and LIF may be involved in promotes pulmonary angiogenesis6.Both the levels of exosome and exosomal miR-194 were significantly increased in the hepatocyte culture medium within 24?h of the bile acid treatment7.Bile acid overload induced p53 nuclear translocation promotes the production of exosomes from hepatocyte.Both pifithrin-?and P53 RNAi markedly inhibited the TASP6expression,exosome and exosomal miR-194 secretion.The groups treated with pifithrin-?,GW4869 or anta-miR-194 showed lower levels of serum exosomal miR-194 and microvessel counts,and higher survival rates when compared with CBDL group.8.We identify two distinct myosin flows,tightly coupled to E-cadherin,that control front protrusion extension versus rear detachment of border cells as they move between nurse cells.Bidirectional myosin and E-cadherin flows in leader cells correlate with protruding and retrograde F-actin flows,and they in turn control polarized F-actin dynamics for protrusion growth.In contrast,in follower border cells unidirectional pulsed myosin and E-cadherin flows promote dynamic separation from nurse cells for transient detachment.Conclusion:1.In conclusion,we demonstrated that HPS rat serum exosomal miR-194 mediates the cross-talk between hepatocytes and PMVECs and contributes to PMVEC proliferation,migration and tube formation.The exosome/miR-194 axis plays a critical pathologic role in pulmonary angiogenesis.The findings suggest that exosomal miR-194 may represent a new therapeutic target for inhibiting the progression of HPS.2.Distinct myosin flows as a pivotal biomechanical mechanism that governs front protrusions and rear detachment in collective cell migration within multicellular environments. |
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