The Mechanism Of Bubble-induced Endothelial Microparticles Formation And Its Role In Vascular Inflammtory Injuries During Decompression Sickness

Decompression sickness（DCS）is the critical medical problem for people who participate in diving,hyperbaric operation and spaceflight.The biogenesis for DCS is the inert gas bubble formation during and after a rapid reduction in ambient pressure.Conventional wisdom holds that the mechanical effects induced by intravascular bubbles,such as occlusion and shear stress are the root causes for DCS.With the further researches,more and more evidence found that extensive biochemical responses and vascular inflammatory damage triggered by intravascular bubbles,such as platelets aggregation,leukocytes activation,have played a key role in the development of DCS.Microparticles（MPs）are submicron vesicles（0.1¹.0μm in diameter）resulting from activated or apoptotic cells,harboring cell surface proteins,cytoplasmic constituents,mRNA and miRNA,and expressing specific surface markers of the parent cell.MPs,once they pinch off from the parent cell,can transfer the contents to the targeted cells and can lead to distinct phenotypic characteristics or functional regulations.Endothleial-derived microparticles（EMPs）are not only the biomarker for digonosis of endothelial injury,but also can play an important role in the development of vascular diseases.Madden et al.observed that the level of circulating EMPs in divers markedly increased after surfacing,indicating the endothelium was severely injured.Thom et al.found that the level of MPs in circulation was positively related with the amount of bubbles detected by Doppler ultrasound.The subtypes of circulating MPs significantly increased and promoted leucocytes adhesion through activating platelets and neutrophil,which can induce intravascular inflammatory responses and even result in central nervous dysfunction,which can be restored by MPs abatement strategy polyethylene glycol.All in all,these studies indicated that MPs may contribute to intravascular inflammatory injuries in DCS.Our previous study also found that circulating endothelial microparticles（EMPs）significantly increased in rats suffering a stimulated diving,with leukocytes sequestered in the brain,skeleton muscle and omentum majus,indicating an extensive vascular inflammatory injury.When pretreated with simvastatin,an endothelium protective agent,it can markedly decrease DCS morbidity,alleviate lung injury and reduce the level of circulating EMPs.We speculated that bubbles can activate or damage the endothelium by directly contacting with the endothelium or by change shear stress,meanwhile,EMPs formation and inflammatory responses occurred,which may play a pivotal role in DCS vascular inflammation.In order to observe the effects of bubble-induced EMPs in vascular inflammation injuries during DCS,an in vitro model was applied to simulate the effects of bubbles in DCS.The number of EMPs was analyzed after bubble stimulus to identify the degree of endothelial injury.Besides,bubble-induced EMPs were obtained and co-incubated with the normal endothelial cells or injected to the rats,which help us to identify the pro-inflammatory effects of bubble-induced EMPs in DCS（Part I）.Next,we explored the mechanisms in bubble-induced EMPs formation,which may provide us a potential therapy for DCS by precisely inhibiting EMPs generation（Part II）.Finally,the pharmacological agent aimed at ameliorating lung function and promoting gas exchange was applied to find an innovative method for DCS prevention.Part Ⅰ Effects of bubble-induced EMPs on endothelial cellsMethods:Rat pulmonary microvascular endothelial cells（PMVECs）were isolated and stimulated by bubbles for 30 min.Six hours after bubble stimulus,the cultural medium was collected,centrifuged at 10000 g for 30 min and then 100000 g for 60 min to pelleted EMPs.The number of EMPs was detected by flow cytometry.EMPs were co-incubated with FSN-100 to observe its abatement strategy.Bubble-induced EMPs were incubated with normal PMVECs in vitro and cell activity and function were observed.In vivo study,bubble-induced EMPs were intravenously injected to the rats to evaluate endothelial dysfunction.Results:In our study,bubble stimulus resulted in a significant increase of EMPs release.Bubble-induced EMPs significantly decreased cell viability and increased cell apoptosis.Moreover,abnormal increase of cell permeability and over-secretion of pro-inflammatory cytokines（ICAM-1,VCAM-1）appeared after EMPs co-incubation.Intracellular ROS production increased while NO production decreased.Finally,intravenous injection of bubble-induced EMPs caused increases of soluble thrombomodulin and pro-inflammatory cytokines in the circulation,indicating an extensive endothelial injury.These negative effects were remarkably attenuated when EMPs pretreated with surfactant FSN-100.Conclusions:Altogether,our results demonstrated that bubble-induced EMPs can mediate endothelial dysfunction in vitro and vivo,which can be attenuated by EMPs abatement strategy.Part Ⅱ Mechanisms involved in bubble-induced endothelial microparticles formationMethods:Human umbilical vein endothelial cells（HUVECs）were contacted by bubbles and EMPs level in supernatant were quantified by flow cytometry.Cytoplasmic calcium(Ca²⁺)was measured by the Ca²⁺binding dyes Fluo-3 AM.Flippase activity was assessed by translocation rate of fluorescent phosphatidylserine（PS）analogue NBD-PS.Protein levels of phospho-myosin light chain（MLC,a Rho kinase substrate）and phospho-extracellular signal-regulated kinase 1 or 2（ERK1/2）were determined by western blotting.The score of actin colocalization was assessed by phalloidin-FITC using an immunofluorescent microscopy.Results:Cytoplasmic Ca²⁺significantly elevated（P<0.05）after bubble stimulus and the number of bubble-induced EMPs increased in accordance with intracellular Ca²⁺（r=0.687,P<0.05）.Protein levels of phospho-MLC and phospho-ERK1/2 significantly increased（P<0.05,P<0.05）,and the score of actin colocalization markedly reduced（P<0.05）in bubble-stimulated HUVECs.Additionally,MLC phosphorylation was significantly inhibited and actin colocalization markedly increased by Rho kinase inhibitor（Y27632）pretreatment and more importantly,bubble-induced EMPs markedly decreased.Flippase activity decreased and PS exposure increased（P<0.05）.All the above changes except the increase in phospho-ERK1/2 can be reversed by Ca²⁺channel blocker LaCl₃.Conclusions:These results demonstrate that bubble stimulates EMPs formation by cytoplasmic Ca²⁺elevation and subsequently activating Rho kinase pathway and cytoskeleton reorganization.Simultaneously,cytoplasmic Ca²⁺elevation inhibits the flippase activity and subsequently increases phosphatidylserine exposure,which also contributes to EMPs formation.Part Ⅲ Beneficial effects of pulmonary surfactant on decompression sickness in ratsMethods:.Surfactant（10 mg for each）was administered by aerosolization 12 h before a stimulated dive in a DCS rat model.The rats were suffered with a simulated diving（7 ATA,90 min,2 ATA/min to surface）and after decompression,signs of DCS were recorded and intravascular bubble formation was detected by Doppler ultrasound bronchoalveolar lavage fluid（BALF）,lung tissue and blood samples were obtained to evaluate surfactant content,inflammatory responses,endothelial injury,and oxidative stress.Results:In surfactant treated rats,DCS morbidity and mortality markedly decreased（75%vs.46.4%,P<0.01;28.6%vs.14.3%,P<0.01;respectively）.The latency period to DCS delayed and survival time prolonged（P<0.05）.Besides,bubble loading was significantly inhibited（P<0.01）.BALF phospholipid and surfactant protein increased（P<0.05）and pro-inflammatory cytokines（interlukin-1β,interlukin-6）were ameliorated（P<0.05）.Endothelial injury（lung wet/dry ratio,BALF total protein,E-selectin,ICAM-1and EMPs）and oxidative stress were significantly restored by surfactant pretreatment（P<0.05）.Conclusions:These results indicate pulmonary surfactant has definite beneficial effects on DCS and might be a candidate drug for DCS prevention and treatment.It may prevent DCS by inhibiting inflammation,ameliorating the lung function and promoting inert gas exchange.