Effect Of TRPV4 Channel On Vascular Permeability Of PMVECs In Rats With Chronic Hypoxic Pulmonary Hypertension

Objective: To investigate the changes of TRPV4 channel and the permeability on pulmonary microvascular endothelial cells?PMVECs?in chronic hypoxic pulmonary hypertension rats,To clarify the relationship between changes in TRPV4 channel function and changes in endothelial cell permeability.Methods:?1?Clean grade male SD rats were randomly divided into 2 groups: normal control group?CON?and chronic hypoxia group?CH?: hemodynamics measured the variation of rats right ventricular mass index?RVMI?and right ventricular systolic pressure?RVSP?,and using paraffin embedded section method and HE staining method to determine whether the hypoxic was successful;?2?The primary cultivation of PMVECs in CON and CH rats by Tissue Block Attachment,and identificated PMVECs by immunofluorescence and FITC-BSI Binding Assays;?3?Transmission electron microscopy detected changes in gap junctions between PMVECs in CH group compared with CON group;?4?For permeability measurements,PMVECs were seeded in transwell at a density of 1.5 x 105 cells per well,Cells were monitored until a stable monolayer formed with 100% confluence.Using fluorescein isothiocyanate-dextran to detect PMVECs permeability in CON and CH groups.?5?Real-time quantitative RT-PCR was used to detect the differences of m RNA expression levels of TRPV4,TJ-related proteins?Occludin,ZO-1,etc.?in the Pulmonary Microvascular Endometrium of rats in CON and CH groups,and to identify the changes in TJ-related protein expression in CH group.?6?Western blot was used to detect the expression of TRPV4 and TJ-related proteins in the pulmonary microvessels in CON and CH groups,and clarify the changes of TJ-related protein expression in CH group.?7?Fluo-3 fluorescence detecte [Ca²⁺]i and measure the change of [Ca²⁺]i in PMVECs,and detect the effect of specific agonists and inhibitors of TRPV4 channel on PMVECs [Ca²⁺]i,then determine whether the changes in intracellular calcium in CH group are caused by the change of TRPV4 channel;?8?In addition,in order to clarify the correlation between the changes in permeability of PMVECs and TRPV4 channals,cells were treated with 10.0 n M GSK101 in the presence/absence of the selective antagonist HC-067?0.5,1 ?M?,and then perform calcium fluorescence detection and permeability test.Results:?1?Compared with the normal group,RVSP?CON: 27±2.61,CH: 49±3.67,P<0.01?and corresponding RVMI?CON: 27.8 ± 1.9,CH: 47.3 ± 2.3?both were increased significantly,the smooth muscle of the pulmonary arterial wall was thickened and the lumen was narrow,suggesting that the CH-PH model was successfully prepared;?2?Endothelial cell emigration was performed 24 hours after adherent culture of pulmonary marginal tissue blocks,after 48 hours the tissue blocks were covered and the tissues were removed after 62 hours,after 1 week endothelial cells in the flask bottom were fused into a single layer;Immunofluorescence technique showing positive expression of v WF and CD31,and FITC-BSI-binding activity containing test cytoplasm yellow-green fluorescence,suggesting successful cultivation of high-purity PMVECs.?3?The m RNA and protein expression levels of TRPV4,TJ-related proteins?Occludin,ZO-1,etc.?in PMVECs of CH-PH rat model were significantly changed.?4?Intravenous calcium and calcium influx were significantly increased in PMVECs in CH rats,in addition,we used the TRPV4-specific agonist GSK101 and inhibitor HC-067 to measure [Ca²⁺]i changes and showed that PMVECs [Ca²⁺]i significantly increased or decreased,suggesting that TRPV4 function is enhanced in PMVECs in CH-PH model group.?5?The result of transmission electron microscopy showed that the gap junction between PMVECs in CH group was significantly larger than that in CON group,suggesting that the permeability of pulmonary microvascular intima was increased in CH group.?6?TER method confirmed PMVECs confluence monolayer,the permeability was measured by FITC-dextran method showed Pd value??l/cm2/h?significantly increased?CON: 12.5±1.87,CH:26.2±3.6,P< 0.01?,Pretreated with 10 n M GSK101 in the presence/absence of the selective antagonist HC-067.Conclusion:?1?Chronic hypoxia increase the permeability of PMVECs in PH rats,and down regulate the expression of major TJ proteins?like Occludin,ZO-1,etc.?;?2?Chronic hypoxia induce up regulate the expression of TRPV4 m RNA and protein and functional level in PMVECs of PH rats;?3?TRPV4 activation significantly increased the permeability of PMVECs monolayer.This study provides new research ideas for the research and prevention of PH pathogenesis.