Barrier-Protective Effects of Activated Protein C in Human Alveolar Epithelial Cells.docVIP

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Barrier-Protective Effects of Activated Protein C in Human Alveolar Epithelial Cells.doc

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Barrier-Protective Effects of Activated Protein C in Human Alveolar Epithelial Cells

Barrier-ProtectiveEffectsofActivatedProteinCin HumanAlveolarEpithelialCells FerrandaPuig1,GemmaFuster1,Me′lanieAdda2,Llu?′sBlanch1,2,RamonFarre1,3,4,DanielNavajas1,3,5 , AntonioArtigas1,2 * 1CIBERdeEnfermedadesRespiratorias,Bunyola,Spain,2CriticalCareCenter,HospitaldeSabadell,InstitutUniversitariParcTaul?′,UniversitatAuto`nomadeBarcelona, Sabadell,Spain,3UnitatdeBiof?′sicaiBioenginyeria,FacultatdeMedicina,UniversitatdeBarcelona,Barcelona,Spain,4Institutd’InvestigacionsBiome`diques AugustPii Sunyer(IDIBAPS),Barcelona,Spain,5InstitutdeBioenginyeriadeCatalunya(IBEC),Barcelona,Spain Abstract Acutelunginjury(ALI)isaclinicalmanifestationofrespiratoryfailure,causedbylunginflammationandthedisruptionof the alveolar-capillary barrier. Preservation of the physical integrity of the alveolar epithelial monolayer is of critical importancetopreventalveolaredema.Barrierintegritydependslargelyonthebalancebetweenphysicalforcesoncell-cell andcell-matrixcontacts,andthisbalancemightbeaffectedbyalterationsinthecoagulationcascadeinpatientswithALI. WeaimedtostudytheeffectsofactivatedproteinC(APC)onmechanicaltensionandbarrierintegrityinhumanalveolar epithelial cells (A549) exposed to thrombin. Cells were pretreated for 3h with APC (50 mg/ml) or vehicle (control). Subsequently,thrombin(50nM)ormediumwasaddedtothecellculture.APCsignificantlyreducedthrombin-inducedcell monolayerpermeability,cellstiffening,andcellcontraction,measuredbyelectricalimpedance,opticalmagnetictwisting cytometry, and traction microscopy, respectively, suggesting a barrier-protective response. The dynamics of the barrier integrity was also assessed by western blotting and immunofluorescence analysis of the tight junction ZO-1. Thrombin resultedinmoreelongatedZO-1aggregatesatcell-cellinterfaceareasandinducedanincreaseinZO-1membraneprotein content.APCattenuatedthelengthoftheseZO-1aggregatesandreducedtheZO-1membraneproteinlevelsinducedby thrombin. In conclusion, pretreatment with APC reduced the disruption of barr