| Implications and objective:Hepatopulmonary syndrome(HPS)is a complex syndrome,mainly manifested as: a triad of primary liver disease-intrapulmonary microvascular dilatation(IPVD)-hypoxemia,the incidence rate of 4-47%in patients with liver cirrhosis.HPS is often accompanied by pulmonary hypertension,which is one of the main causes of perioperative pulmonary complications in patients with various types of liver diseases,and is also the key reason for the failure of liver transplantation.At present,most scholars believe that IPVD is an early core change in the process of HPS,and pulmonary vascular remodeling(PVR)is one main pathological feature of HPS.We used the classical common bile duct ligation(CBDL)rats model,found an interesting phenomenon: the normal rat pulmonary microvascular wall is composed of a layer of endothelial cells arranged orderly,and pulmonary microvascular wall of HPS rat is thickened abnormalities.Under microscope the thickening vessel walls were taken,cultured and identified,we has detected these cells were composed of some CD34 and factor VIII related antigen positive cells and some alpha-smooth muscle actin(SMA)staining positive cells,which suggesting that " smooth muscle like cells in the thickening vessel wall " may come from in situ myogenic differentiated PMVECs or ectopic PASMCs;these smooth muscle like cells appear in the intimal is difficult to explain with simple P MVECs of myogenic differentiation.Cell migration is a complex process that can be regulated by multiple signaling pathways,which involved in angiogenesis,vascular remodeling,tumor metastasis,wound healing,and so on.Secondly,cell migration is closely related to the expression of aquaporin1(AQP1)in the cell membrane,and the abnormal expression and distribution of AQP1 is often accompanied by abnormal cell migration.P38-MAPK,a member of MAPK family,can be activated by a variety of extracellular stimuli,and is a key factor for cell migration.Therefore,the present study investigated whether there is an abnormal p38-MAPK/AQP1 pathway in the pathological process of HPS,and induces PASMCs migration in the process of PVR.In our broaden research,we found that the loss of cell polarity would lead to uncontrolled cell proliferation,cell migration,cell polarity defect may be a key upstream event in PVR of HPS.Methods:Total five parts:1.CBDL rat modelHPS rat model was established by chronic CBDL.The pathological sections and blood gas analysis were performed after 4 weeks.2.Primary PASMCs culture and detection of PASMCs migrationNormal SD rats(7w age,160-200g),the main pulmonary artery,left and right pulmonary artery were taken and cultured,PASMCs were purified and identified;randomly divided into 2 groups: sham group and CBDL group,detected by transwell and scratch test respectively in cultured 48 h and 72 h 24h(T1,T2,T3),to observe the cell migration under microscope.3.Detection of the involvement of AQP1 in the regulation of CBDL rat serum inducePASMCs cell migrationAQP1 m RNA and protein expression in PASMCs were detected by RT-PCR and western blot method in T1,T2,T3 time points,immunohistochemical localization of AQP1 expression in rat pulmonary vascular,and immunofluorescence to detect the expression of AQP1 in PASMCs in vitro.Cells transfected with si AQP1,AQP1 mRNA and protein expression in PASMCs were detected by RT-PCR and western blot assay,and transwell assay was used to detect the changes of PASMCs migration.4.Detection of CBDL rat serum induce AQP1 dependent cell migration of PASMCsvia p38-MAPKDown regulation of p38-MAPK activity in PASMCs,the expression of AQP1 in PASMCs was detected by western blot assay and the migration ability was detected by transwell assay.5.Detection of the loss of cell polarity is involved in the regulation of PVR in HPSImmunofluorescence assay detected the location of cell polarity protein in pulmonary vascular intimal;the activity of cdc42 was measured by pull-down assay;F-actin staining served to test cytoskeletal remodeling;transwell assay and Ki67 staining were used to detect cell proliferation and migration after the loss of cell polarity.Results:1.Effects of CBDL rat serum on the migration of PASMCs cells.1)Under the inverted microscope,the cells were long spindle shaped,triang ular,interwoven into a network,clear cytoplasm and abundant cytoplasm.2)Compared with sham group,the number of migratory cell was increased in CBDL group(P<0.05),and in time dependent manner.2.The involvement of AQP1 in the regulation of CBDL rat serum induce PASMCs migration.1)Compared with sham group,the expression of AQP1 mRNA and protein were increased in CBDL group(P<0.05),and in time dependent manner;compared with sham group,the green fluorescent staining of AQP1 in PASMCs was enhanced in CBDL group.2)In the sham group,compared with si Ctrl group,the number of migratory cell was decreased in si AQP1 group(P<0.05);in CBDL group,compared with si Ctrl,the number of migratory cell was reduced significantly in si AQP1 group(P<0.01),and in a time dependent manner.3.CBDL rat serum induce AQP1 dependent cell migration of PASMCs via p38-MAPK.1)Compared with sham group,the expression of p38-MAPK protein were increased in CBDL group(P<0.05);2)Compared with CBDL group,the expression of p38-MAPKand AQP1 protein were decreased in CBDL+SB203580(1,5uM)group(P<0.05),and in a dosedependent manner;and SB203580 can effectively inhibit cell migration mediated by AQP1(P<0.05).4.The loss of cell polarity is involved in the regulation of PVR in HPS.1)The results of immunofluorescence in lung tissue showed that podocalyxin,the apical cell polarity protein,mainly distributed in the apicalmembrane of vascular intima cells,while ?-catenin mainly distributed in the basal-lateral side of vascular intima cells in sham group;while in CBDL group,cell polarity protein podocalyxin and ?-catenin were mis-located and distributed around the cells.2)In vitro,the activity of cdc42 were increased in PMVECs treated with CBDL rat serum;compared with CBDL group,CBDL+casin group could effectively inhibit the activity of cdc42(P<0.05).3)Compared with sham group,cytoskeletal remodeling occurred in PMVECs of CBDL group,and in CBDL+casin group,disorganization of the skeleton returned to normal;compared with sham group,the number of proliferative and migratory cells were increased in CBDL group(P<0.05),while in the CBDL+casin group the number of proliferative and migratory cells was inhibited(P<0.05).Conclusions:1.CBDL rat serum promoted cell migration of PASMCs.2.The expression of AQP1 in PASMCs was increased in mRNA and protein level induced by CBDL rats serum,and the siAQP1 can significantly inhibit cell migration of PASMCs,suggesting that the involvement of AQP1 in CBDL rats serum-induce PASMC migration.3.The activity of p38-MAPK was enhanced in PASMCs treated with CBDL rats serum,and the inhibition of p38-MAPK activity can reduce AQP1 mediated cell migration,suggesting that p38-MAPK signaling pathway is involved in AQP1-dependent cell migration,and plays an important role in the development of HPS.4.Cell polarity proteins were mis-located inpulmonary vascular intima cells of CBDL rat;in vitro,the activity of cdc42 was increased and cytoskeletal remodeling occurred in CBDL group;the inhibition of cdc42 activity can reduce cell proliferation and migration;it suggesting that cdc42 is involved in regulation of cell polarity and cell proliferation,migration after the loss of cell polarity,and plays an important role in the development of HPS. |
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