Mechanisms Of Stim1/Orai1 In Regulating Pulmonary Endothelial Permeability In Ventilator-induced Lung Injury

The expression of Stiml/Orail in ventilator-induced lung inj u ry ratsBackground:With the continuous improvement of medical technology and the acceleration of population aging,more and more patients with respiratory dysfunction need to receive mechanical ventilation adjuvant therapy and general anesthesia.However,researchers realized that mechanical ventilation not only brings benefits to patients but also cause acute lung injury,which is termed as ventilator-induced lung injury(VILI).The underlying mechanisms of VILI are not clearly delineated.During mechanical ventilation,pulmonary gas blood barrier was disrupted,while the alveolar lumen fluid exudation increased and severe pulmonary edema were observed.Mechanical ventilation can induce Ca2+ overload,which leads to increase of endothelial permeability.Stiml,a sensor of intracellular calcium located on endoplasmic reticulum(ER),regulates store-operated Ca2+ entry(SOCE)influx.Orail,located on the plasma membrane,is a prototypic store-operated Ca2+release-activated Ca2+ channel protein.Stiml and Orail mediate SOC activation,which is important for control of the endothelial barrier.The classical PKCa isoform in ECs,which contains both calcium binding domains,has an important role in pulmonary edema.We inferred that Stiml and Orail mediated Ca2+ influx,which activated PKCa and induced lung edema during mechanical ventilation.In this study,we investigate the roles of Stiml and Orail in VILI through ventilated rats model,detect the underlying mechanisms of ventilator-induced lung inj ury.Objective:The pathophysiological changes of ventilator-induced lung injury(VILI)are complex,but endothelial hyperpermeability and lung edema are the common evolutionary process.timl/Orail mediates store-operated Ca2+(SOC)influx,which modulates endothelial permeability,however the underlying mechanisms of Stiml/Orail signaling way in VILI are poorly understood.Methods:Forty male healthy Wistar rats(Laboratory Animal Center,Shandong University,China)weighing 200?220g were randomly divided into 5 groups(n=8 in each group):control group(without ventilation),low tidal volume(VT=7ml/kg,),high tidal volume(VT=40ml/kg),DMSO group(received 0.4ml/kg i.p.DMSO,2h prior to ventilation,VT=40ml/kg),BTP-2 group(received 1mg/kg i.p.BTP-2,2h prior to ventilation,VT=40ml/kg).Rats in control group did not perform ventilation,while the other four groups were ventilated for 4h using an ALC-V8 animal ventilator.Ventilation parameters were set as follows:a respiratory rate of 40 times/min,I/E ratio of 1:3,a fraction of inspired oxygen of 21%,and no PEEP.After ventilation,rats were sacrificed and the lungs were collected for further analysis.Lung wet/dry weight ratio,histological lung injury and bronchoalveolar lavage fluid(BALF)protein were measured.The protein expression was determined by western blotting.Results:HE staining showed that:compared with control group,low tidal volume didn't result in severe lung pathology changes,however high tidal volume mechanical ventilation induced alveolar wall thickening,severe congestion,hemorrhage,and neutrophils infiltration,and BTP2 pretreatment can protect lung from injury.The expression of Stim1,Orai1 and PKC?,lung wet/dry weight ratio,and the BALF protein level significantly increased in high tidal volume group than those in control group and low tidal volume group.Importantly,BTP2 pretreatment could alleviate the above mentioned effects induced by high tidal volume ventilation.Conclusions:High tidal volume ventilation induced severe lung injury including lung edema and acute inflammation,Stim1/Orai1 signaling way and PKC? were activated during the process,and BTP2,an inhibitor of Orai1channel,can block the process partly,protect lung from injury.The effects of cyclic stretching on expression of Stiml/Orail and pulmonary endothelial peameabilityBackgroud:Maintaining the structural integrity of the pulmonary gas-blood barrier is essential for pulmonary gas exchange.Dysfunction of gas-blood barrier causes pulmonary edema,even ARDS.Pulmonary endothelial cells are important components of gas-blood barrier.When exposed to inflammation,hypoxia,cyclic stretching,endothelial cells were damaged and cell permeability increased.Water,protein and other substances entered the alveolar space through endothelial cells,which leads to pulmonary edema.To simulate the process of mechanical ventilation,pulmonary endothelial cells seeded in specific cell culture plates were exposed to cyclic stretching.Large amplitude cyclic stretching directly activated Ca2+ channels and induced store-operated Ca2+ entry(SOCE).Stiml and Orail are essential for the activation of store-operated channels(SOC).The PKC family is a highly conserved group of kinases.Activation of PKCa is dependent on the increase of intracellular Ca2+,translocated from the cytoplasm to cell membrane,which induce hyperpermeability of endotheilial cells.We discuss that cyclic stretching regulated cell permeability through Stiml/Orail signal pathway,induced Ca2+ Influx and activated PKC?.Objective;To investigate the effects of cyclic stretching on expression of Stiml/Orail and endothelial peameability in pulmonary endothelial cells.Methods:Human lung microvascular endotheilial cells(HULEC)were purchased from ACTT and seeded at a density of 5×105 cells/well on collagen I coated flexible bottom BioFlex plates in MCDB131 medium supplemented with 10%FBS,lOng/ml Epidermal Growth Factor,1?g/ml Hydrocortisone and 10 mM Glutamine.The Flexcell Strain Unit was used for the cyclic stretching experiments.A pattern of cyclic stretching in the basement membrane surface area at a frequency 0.5Hz with a stretch-to-relaxation relation of 1:1 was used.Cells were exposed to 8%CS and 18%CS for 2h or 4h to determine optimal experimental conditions.Then Cells were divided into four groups:control group,18%CS group,18%CS+DMSO,18%CS+BTP2.Non-stretched cells were used in control group.In 18%CS+BTP2 and 18%CS+DMSO group,the 20?M of BTP2or 3?l/ml of DMSO was used for 2h prior to cyclic stretching.Endothelial permeability was determined by the influx of Evans blue-labeled albumin across the endothelial monolayer and intracellular calcium concentration were evaluated by flow cytometry in HULEC.The protein expression was determined by western blotting.Results:The relative density of Stiml has no statistical difference among 8%CS for 2h,4h groups and control group,however the level of Stiml in 18%CS group significantly increased for 2h(P<0.05)and 4h(P<0.01)to compare with control group.So endothelial cells were stretched for 4hs with 18%amplitude as experimental group.Compared with control group,the protein levels of Stim1,Orai1 and PKCa in HULEC significantly increased after exposure to 18%CS for 4h,whereas BTP2 pretreatment significantly inhibited the increase of Stiml,Orail and PKCa(P<0.05).BTP2 pretreatment also suppressed increasing of endothelial permeability and the intracellular calcium induced by 18%CIS(P<0.05).Conclusion:When exposed to large magnitude CS,Stim1 and Orai1 expression are up-regulated,which may further activate calcium-sensitive PKCa,result in calcium overload,endothelial hyperpermeability.