A Study On The Role Of PTEN/Cdc42 Signaling Pathway In Serum-mediated Loss Of PMVECs Polarity In Hepatopulmonary Syndrome

Background and Aims:Hepatopulmonary syndrome?HPS?is a serious complication that occurs in patients with end-stage chronic liver disease.The incidence in patients with advanced cirrhosis is about 5%-32%.The main clinical features can be attributed to the triads of basic liver disease,intrapulmonary vasodilation?IPVD?and arterial oxygenation dysfunction excluding the existence of primary cardiopulmonary diseases.The possible pathogenesis mainly includes abnormal expansion of pulmonary microvasculature and pulmonary vascular remodeling?PVR?,but it has not yet been fully elucidated.The pathophysiological abnormalities of the lungs are inseparable from the changes of cell function.In the early studies of our team,it was found that abnormal proliferation and migration existed in the pulmonary microvascular endothelial cells?PMVECs?of HPS model animals,which seems to be related to the myoid differentiation of endothelial cells.Previous studies have also found that PMVECs change from a regular monolayer epithelial state to a disordered multilayer state in the rat model of Chronic bile duct ligation?CBDL?.However,this phenomenon cannot be fully explained by the concept of myoid differentiation alone.In many studies,it has been found that the establishment and maintenance of"Cell polarity"is essential for cell development,differentiation and maintenance of function,and is also involved in cell proliferation,migration and adhesion.Then,in HPS,does"polarity loss"exist in the pulmonary microvascular endothelium,which leads to the disordered arrangement and growth of the cells,and eventually causes changes in the pulmonary microvascular structure to cause pulmonary oxygenation disorders?In addition,some public reports on tumors showed that Phosphatase and tensin homolog?PTEN?as a tumor suppressor gene with phosphorylase function,is not only involved in cell growth,apoptosis,adhesion,migration,infiltration and other processes,but also have an important regulatory role in cell polarity.Celldivision cycle42?Cdc42?,as a small molecule GTPase in the Rho protein family,plays a key role in the intracellular polarization signaling pathway.We suspect that the PTEN/Cdc42 pathway may be involved in the process of loss of PMVECs polarity during PVR.This study is to confirm that HPS serum is involved in the pathological process of PVR due to the loss of polarity of PMVECs through PTEN/Cdc42 signaling pathway.Methods:1.Construction of HPS rat model and preparation of serum,culture and identification of rat primary PMVECs.Chronic bile duct ligation?CBDL?was used for the construction of HPS rat model.They were divided into sham operation group?Sham group?,CBDL 1 week group?CBDL-1w?,2weeks group?CBDL-2w?,3 weeks group?CBDL-3w?,and 4 weeks group?CBDL-4w?.Arterial blood was collected for blood gas analysis,lung tissue sections were stained with HE and serum of each group was prepared.Primary PMVECs were cultured?using tissue block method?,immunohistochemistry?IHC?staining was performed to identify specific antigens of endothelial cells.2.Detection of polarity-related proteins in vascular endothelial cells of rat lung tissue and cultured PMVECs,and changes in TEER after rats serum stimulated PMVECs.Immunofluorescence?IF?was used to detect the distribution of polaroid-related proteins Podocalyxin?PCX?,?-catenin and gp200 in lung tissue and PMVECs after serum stimulation in Sham group and CBDL-4w group.The TEER of PMVECs was measured at 0h,3h,6h,9h,12h,30h and 36h after treatment.3.Detection of changes in PTEN and Cdc42 activities in lung tissue of HPS rats,and the effect of PTEN-specific inhibitor Bpv?pic?on Cdc42 activity in PMVECs.The expression of p-PTEN?active?and total PTEN protein levels were detected by Western blot in the lung tissues of Sham group and CBDL1-4w rats.The Cdc42 activity was detected by Pull-down assay.PMVECs were pretreated with PTEN specific inhibitor Bpv?pic?in vitro,and the effect of Cdc42 activity on the activity of Cdc42 was detected after 24 hrs of serum stimulation.4.Detection of the effect of Cdc42 inhibitor CASIN on the distribution of polarity-related proteins,cytoskeletal proteins,cell migration and proliferation of PMVECs.PMVECs were divided into Sham group and CBDL group,and were stimulated with 5%Sham group serum or CBDL-4w group serum.Each group was given 5?mol/L Cdc42 specific inhibitor?CASIN+?or serum-free medium?CASIN-?.After 24hrs treatment,the distribution of polarity-related proteins PCX and gp200 in PMVECs was detected by immunofluorescence.Changes in the cytoskeletal protein F-actin were detected using phalloidin labeled with fluorescein isothiocyanate?FITC?.Transwell experiments were used to examine the effects of cell migration.The effect of cell proliferation was examined by Ki-67 fluorescence staining.5.Detection of annexin A2?AX2?involved in the role of the PTEN/Cdc42 pathway in the loss of PMVECs cell polarity.The annexin A2 gene in PMVECs was silenced by small interfering RNA?siRNA?transfection technique.The transfection efficiency was verified after 24hrs.The change of Cdc42 activity was detected by Pull-down assay 48hrs later.Results:1.Construction of HPS rat model,staining of rat lung tissue sections,identification of cultured PMVECs:?1?Compared with the Sham group,the results of blood gas analysis in the CBDL group showed a decrease in PaO₂?P<0.05?and an increase in P?A-a?DO₂?P<0.05?.?2?Microscopic observation showed that the arterial wall of the lung tissue of CBDL rats was thickened and the microvascular expansion was obvious;?3?Immunohistochemical staining showed that the cell CD31 antigen was positive;2.Changes in the distribution of polarity-related proteins and membrane resistivity in pulmonary microvascular endothelial cells of HPS rats:?1?IF results showed that in the lung tissue of rats and in PMVECs cultured in vitro,the cell polarity-related proteins PCX and gp200 in Sham group were mainly expressed in the apical surface of endothelial cells,and?-catenin was mainly distributed in the basal-lateral side of the cell.In the CBDL group,these polarity-related proteins were clearly ectopic in the endovascular cells and rescattered around the cells;?2?At 0h after treatment,there was no significant difference in TEER between PMVECs in Sham group and CBDL group?P>0.05?.After 3hrs,6hrs and 9hrs serum treatment,the TEER of serum-stimulated PMVECs in the CBDL group was significantly lower than that in the Sham group?P<0.05?.3.Changes in PTEN and Cdc42 activities in lung tissue of HPS rats:?1?Compared with the Sham group,the expression of p-PTEN and active Cdc42 were increased in the CBDL group?P<0.05?.?2?After treatment with PTEN inhibitor,Cdc42 activity in PMVECs was inhibited?P<0.05?.4.The effect of Cdc42 inhibitor CASIN on the distribution of polarity-related proteins,cytoskeletal proteins,cell migration and proliferation of PMVECs:?1?After 24 hrs of CASIN treatment,compared with CASIN?-?,after adding CASIN,the polarity-related protein PCX and gp200 in PMVECs of CBDL group were redistributed to the apical surface of cell membrane;?2?The CBDL rats serum-stimulated PMVECs showed an unstable phenotype with a large leading edge lamellipodia with filopodia.FITC-labeled phalloidin showed loose and disordered intracellular actin F-actin.After CASIN treatment,the filamentous and flaky pseudopods of PMVECs returned to normal state,and the expression of actin in the cells was enhanced.?3?Compared with the Sham group,the migrational and proliferational ability of PMVECs in the CBDL group was enhanced?P<0.05?.Compared with the CASIN?-?group,the migrational and proliferational ability of PMVECs was decreased after serum stimulation in the CASIN treatment group?P<0.05?;5.PTEN and AX2 co-localize during PMVECs polarity loss,and AX2 participates in the PTEN/Cdc42 pathway during PMVECs cell polarity loss:?1?.Co-immunoprecipitation experiments showed that PTEN and AX2 co-localized during the loss of PMVECs polarity;?2?.After transfection of silencing annexin A2 gene with siRNA for 36hrs,Cdc42 activity in PMVECs was inhibited compared with blank transfected control group?P<0.05?.Conclusion:The loss of PMVECs polarity is involved in the process of pulmonary vascular remodeling associated with HPS.This loss of polarity is mediated by the enhanced PTEN/AX2/Cdc42signaling pathway,and down-regulation of its activity can reduce the abnormal migration and proliferation of PMVECs stimulated by CDBL rats serum and restore cell polarity.