The Role And Mechanism Of Autophagy In Vascular Endothelial Injuries Induced By Bubbles

With the rapid development of economy and the continuous advance of ocean strategy,commercial diving,military diving and recreational diving have been advanced by leaps and bounds.Some physiological inert gases will be dissolved in body when divers are underwater due to high pressure.However,these gases could escape after undergoing an amiss decompression and lead the formation of bubbles,which are the main cause of decompression sickness（DCS）.DCS is characterized by rapid onset,complex symptoms,difficult treatment and serious consequences.Prevention and treatment of DCS are crucial to ensure diving security.Elucidating the pathogenesis of DCS is the basis for finding effective prevention and treatment methods.Vascular endothelial cell（VEC）,which plays a vital role in maintaining homeostasis of internal environment,is widely involved in the regulation of vasoconstriction funtion,blood coagulation and immuno-inflammatory responses,etc.VEC injuries are usually shown by increased stiffness of peripheral blood vessels and changes of endothelial markers in DCS patients.Previous studies found that Escin and Simvastatin,endothelial protective agents,could alleviate VEC injuries and secondary inflammatory response and decrease significantly the incidence of DCS in rats,suggesting that VEC injuries and secondary inflammatory reaction are important pathogenic factors of DCS.Further studies have found that bubble is the trigger factor for VEC injuries in DCS,and its secondary responses further aggravate cell injuries.VEC injuries are triggered by bubbles,however the specific mechanism is not clear yet.Autophagy is the process in almost all mammalian cells,in which aging or damaged organelles and biological macromolecules are encapsulated in a specific bilayer membrane structure and are transported to lysosome for degradation and recycling.Autophagy is widely involved in various physiological and pathological activities,protecting or promoting VEC injuries.VEC autophagy can be induced by various of factors,such as shear stress,Ca²⁺,ROS.Bubbles in vessels could induced shear stress changed.A number of studies including our group have shown that Ca²⁺ increased in VEC by bubbles.Antioxidants also improved vascular endothelial function.Therefore,we speculated that autophagy might be involved in VEC injuries induced by bubbles.In order to verify this hypothesis,this study took human umbilical vein endothelial cells（HUVEC）as the research object.By establishing a cell-multibubble contact model,we systematically studied the effect and law of VEC autophagy induced by bubbles,clarified the role and mechanism of autophagy which in VEC injuries,and provided theoretical basis for exploring effective prevention and treatment methods of DCS.Part Ⅰ:The Role of Autophagy in Vascular Endothelial Injuries Induced by bubbles1.The law of VEC autophagy induced by bubblesMethods:With the cell layer-multibubble contact device established,exposing HUVEC to bubbles for different durations,detecting autophagy marker proteins,and analyzing possible relationship between them.Results:After 30 min of bubble contact,LC3-Ⅱ/Ⅰ ratio and Beclin-1 began to rise at 2h,peaked at 8 h,and still were higher than normal at 24 h in HUVEC.P62 began to decrease at 4 h,most obviously at 8 h,and was still below the normal at 24 h.8 h after bubble contact was selected for detecting autophagy in VEC.The number of autophagosomes increased observed under the electron microscopy in HUVEC at 8 h following bubble contact for 30min.LC3-Ⅱ/Ⅰ ratio and Beclin-1 increased as well as P62 decreased in HUVEC after 1,5,10,20,30 min of bubble contacts,.It was found that LC3-Ⅱ/Ⅰ ratio and Beclin-1 positively,while P62 negatively,correlated with bubble contact durations.LC3-Ⅱ/Ⅰ ratio was most obviously correlated with bubble contact durations,which was selected as a representative of autophagy.Conclusions:VEC autophagy was induced by bubbles,and the level of which closely correlated to bubble contact durations.LC3-Ⅱ/Ⅰ ratio was selected as a representative of autophagy.2.The role of autophagy in VEC injuries induced by bubblesMethods:According to the above methods,HUVEC was exposed to bubbles for different durations,cell damage indexes were detected,and the correlations between which and LC3-Ⅱ/Ⅰ ratio were analyzed.Then we repeated the above steps with the pretreatment of autophagy inhibitor 3-methylpurine（3-MA）.Results:Cell viability decreased,apoptosis rate,the expressions of cytochrome C and cleaved caspase-3 increased after 10,20 or 30 min of bubble contact,but no significant changes of these indexes were detected after 1 or 5 min of bubble treatment.The analysis showed that cell injury indexes closely correlated with LC3-Ⅱ/Ⅰ ratio.Compared with 10,20 or 30 min of bubble treatment alone,3-MA increased cell viability,decreased apoptosis rate as well as cytochrome C and cleaved caspase-3.Compared with 1 or 5 min of bubble contact alone,3-MA decreased cell viability,increased apoptosis rate as well as cytochrome C and cleaved caspase-3.These results suggested that mild autophagy activated by short time （1,5min） bubble contact protected VEC from damaging,while severe autophagy by long-period（10,20 or 30 min）bubble stimulation promoted cell injuries.To confirm the result above,we further observed the effects of two successive bubble contact on VEC injuries,and in which explored the role of autophagy.According to the relationship between VEC injures and bubble contact durations,bubble contacting for 1 min and 30 min represented mild and severe stimuli.The peak time of autophagy induced by bubbles in VEC,namely 8 h,was used as the interval between two successive bubble contacts.Since the above cell damage indicators significantly correlated with bubble contact durations,cell viability and apoptosis rate were selected to evaluate the degree of cell injures.The results showed that 1 min+30 min（bubble contact for 1 min,8 h interval,30 min again）increased cell viability and decreased apoptosis rate,which was inhibited by 3-MA.30min+30 min（bubble contact for 30 min,8 h interval,30 min again） further reduced cell viability and increased apoptosis rate,which was reversed by 3-MA.Conclusions:Autophagy could play a dural role in VEC injuries induced by bubbles.Mild autophagy,induced by short time（1,5 min） bubble contact,could protect VEC from damaging.While severe autophagy,induced by long period（10,20,30 min） bubble treatment,and further promoted VEC injuries.Part Ⅱ:The mechanism of endothelial autophagy induced by bubblesMethods:With the cell-multibubble contact device established,exposing HUVEC to bubbles for different durations,Ca²⁺,reactive oxygen species（ROS） and critical autophagy related signaling molecules were detected.The role of these molecules and the relationships between them were detected in bubble-induced VEC autophagy,using calcium chelator 1,2-aminophenoxy-ethane-N,N,N’N’-tetraacetic acid（BAPTA）,antioxidant N-acetyl-L-cysteine（NAC） and specific inhibitors or agonists.Results:Compared with the normal,Ca²⁺ increased after 1,5,10,20,30 min of bubble contact in HUVEC,the level of which positively correlated with bubble contact durations and LC3-Ⅱ/Ⅰ ratio,and which was significantly reduced by BAPTA.ROS increased significantly after 10,20 or 30 min of bubble contact,but no significant change of which was detected in 1 or 5 min groups.HUVEC was pretreated with BAPTA and NAC,respectively,and then the levels of Ca²⁺ and ROS were detected by bubble contact for 30min.The results showed that compared with 30 min of bubble contact alone,BAPTA significantly decreased ROS level,and NAC did not significantly affect Ca²⁺ but reduced LC3-Ⅱ/Ⅰ ratio.It was further found that calmodulin-dependent protein kinase-β（CaMKK-β）,adenylate-activated protein kinase（AMPK） and extracellular signal-regulated protein kinase（ERK） were activated as well as protein kinase B（AKT） and mammalian rapamycin target protein（mTOR） were inhibited by bubble contact for 30 min in HUVEC.Following bubble contact for 30 min,mTOR was significantly up-regulated and LC3-Ⅱ/Ⅰ ratio was decreased by CaMKK-βinhibitor STO-609,AMPK inhibitor Compound C and AKT agonist SC79,except ERK inhibitor PD98059.CaMKK-βand AMPK were activated after 1 or 5 min of bubble contacts,besides that,AKT and mTOR were inhibited by bubble contacts for 10,20,30 min.Finally,BAPTA significantly inhibited CaMKK-βas well as up-regulated AKT and mTOR;NAC also significantly inhibited AMPK as well as up-regulated AKT and mTOR,but had no significant effect on CaMKK-βin HUVEC following bubble contact for 30 min.Conclusions:Short time（1,5 min） bubble contact moderately increased Ca²⁺,and induced mild autophagy via CaMKK-β/AMPK pathway in VEC.Long period（10,20,30min） bubble contact further increased Ca²⁺,besides via CaMKK-β/AMPK pathway,further activated AMPK and inhibited AKT via increasing ROS,and then inhibited mTOR to induce severe autophagy.