| ObjecivePart one:To determine the effect of melatonin (MEL) on interleukin-1β(IL-1β)-induced impairment of permeability from human umbilical vein endothelial cells (HUVECs). Part two:To determine the effect of MEL on IL-1β-induced damage of endothelial adherens junctions and actin remodeling.Part three:To explore the possible receptors and cell signalling pathways by which melatonin exerts endothelial barrier protective effects.MethodsPart one:Primary HUVECs were isolated from fresh human umbilical veins and cultured after immunomagnetic separation by anti-CD31 antibody coated Dynabeads. For identification of endothelial cells, the cells were incubated with mouse anti-von Willebrand Facto (vWF) and FITC-conjugated goat anti-mouse secondary antibody. The signals were detected by fluorescence microscope. Paracellular permeability was studied in a Transwell system by measuring the flux of FITC-dextran across the endothelial monolayer.Part two:To detect actin and VE-cadherin,the cells were incubated with FITC-conjugated phalloidin and anti-VE-cadherin antibody, then observed by fluorescence microscope and confocal imaging system.Part three:Using Rac activation assays to test the involvement of small GTPase Rac in the melatonin-induced endothelial barrier protective effects as well as cell contact reorganization. Luzindole, MEL membrane receptor antagonist and NSC-23766, a specific inhibitor to Rac were used to explore the possible receptors and cell signalling pathways. ResultsPart one:(1) Primary HUVECs were isolated sucessfully from fresh human umbilical veins and cultured after purified by immunomagnetic separation. The endothelial origin was confirmed by the positive labeling of vWF with immunofluorescence staining. (2) Treatment with IL-1β(5,10 and 20ng/ml) increased the relative permeability of HUVECs from 1.5±0.1 to 4.8±0.3,5.2±0.4 and 9.8±0.8 (P<0.01); MEL (10 and 100μmol/L) attenuated IL-1β(10ng/ml) induced increase of permeability from 5.2±0.4 to 2.8±0.3 and 2.7±0.3 (P<0.01). This result is consistent with the time course analyses of cell permeability.Part two:(1) Fluorescence microscope:By VE-cadherin staining, treatement with IL-1βcaused a clear disruption of adherens junctions. Obvious signs of disruption of adherens junctions after the addition of melatonin in advance were not found. (2) Confocal microscope:A clear change of the actin cytoskeleton is observed from filamentous actin at the cell periphery in normal cells to stress fibers spanning the cell body in the IL-1β-induced cells. Pretreatment with melatonin induced significant reduction in central stress fibers compared with IL-1βtreated cells. (3) Western blotting: The protein level of VE-cadherin was reduced in IL-1β-treated cells, while that of the reduction was attenuated by melatonin.Part three:(1) Luzindole effectively inhibited the protective effect of melatonin on IL-1β-induced increase of endothelial permeability. (2) The inactivation responses of the Rac in HUVECs were investigated by stimulation with IL-1β. Melatonin pretreatment inhibited the IL-1β-induced decrease in the GTPbound form of Rac. Luzindole prevented the inhibitory effects of melatonin on IL-1β-induced Rac inactivation. (3) NSC-23766 abolished the effects of melatonin on IL-1β-induced adherens junctions, cytoskeletal remodeling and the expression of VE-cadherin.Conclusions:Our results provide evidence that L-1βtreatment disrupted HUVECs adherens junctions and induced stress fiber and paracellular gap formation. These changes increased the permeability of the endothelial monolayer. Melatonin inhibits IL-1β-induced endothelial permeability by tightening intercellular junctions and reducing IL-1β-induced stress fiber and paracellular gap formation via activation of Rac.
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