The Effect And Mechanism Of Simulated Microgravity On Pulmonary Microvascular Endothelial Cells

Objective Endothelial cell is one of the components of vascular wall and plays an important role in regulating vascular function.In animals,the microvascular is abundant,and the area of the endothelium covered by the inner wall of microvascular is about 50 times as large as the total of that in large vascular.Therefore,abnormal microvascular ECs will have a huge impact on the function of vascular system.The aim of this study is to study the effect of simulated microgravity on pulmonary microvascular endothelial cell(PMEC)using human PMEC as a model.The expression profiles of miRNA in human PMECs under simulated microgravity were analyzed.The effect and mechanism of miR-503-5p in human PMECs under microgravity were studied.Finally,animal experiments were carried out to confirm the effect of simulated microgravity on dog PMECs.The effects of simulated microgravity on blood pressure and electrocardiogram in dogs were also evaluated.Eventually,the effects of simulated microgravity on microvascular endothelial cells were comprehensively and scientifically studied at molecular,cellular and overall animal levels,and the cardiovascular effects of simulated microgravity environment were also systematically explored,which can provide experimental basis for ensuring the safety of space flight.Methods Human PMEC was used as a cell model.Microgravity effect was simulated by clinorotation for 72 hours.The apoptosis of cells was detected by Annexin-V Fluoresceine Isothiocyanate(FITC)and Propidium Iodide(PI)double staining method.The effect of simulated microgravity on the mitochondrial membrane potential was detected by JC-1 method.The Caspase-Glo 3/7 reagent kit was used to detect the activity of apoptotic execution factors Caspase 3/7.The effects of simulated microgravity on the ultrastructure of human PMECs were detected by transmission electron microscopy.The Agilent human miRNA(8*60K)V19.0 chip was used to analyze the expression profile of miRNAs in human PMECs under simulated microgravity.The differential expression of miRNAs was verified by quantitative real-time polymerase chain reaction(qRT-PCR).The online softwares of TargetScan 7.1,TarBase v7.0,and miRDB 2016 were used to predict the target genes of the differentially expressed miRNAs.The online software GeneCoDis3 was used to analyze the biological processes and signal pathways of the predicted target genes.miR-503-5p mimic and inhibitor(LNA-antimiR-503-5p)were transfected by HiPerFect Transfection Reagent to overexpress or silence miR-503-5p in human PMECs.The mRNA and protein expression of miR-503-5p target gene Bcl-2 were analyzed by qRT-PCR and Western blot,respectively.Beagle dog was used as an animal model.Microgravity effect was simulated by hindlimb unloading for 7 days.On day 8,2 hindlimb unloading dogs and 2 normal gravity dogs were necropsied.The lung tissue was used for transmission electron microscopy.On the same day,the remaining animals get into the recovery period without hindlimb unloading.During the experiment,the animal body weight and food consumption were measured.The animal hematology and serum chemistry parameters were analyzed.The effect of hindlimb unloading on the PMECs was observed by transmission electron microscope and the changes of blood pressure and electrocardiogram were detected by DSI telemetry system.Results When compared to the normal gravity control group,significantly increased apoptosis rate(P<0.01),decreased mitochondrial membrane potential(P<0.05),and increased activity of Caspase 3/7(P<0.01)were observed in human PMECs after 72 hours of clinorotation.Under transmission electron microscopy,typical cell apoptosis changes were observed in human PMECs after 72 hours clinorotation,including chromatin margination and apoptotic bodies formation.By using strict screening criteria(the change fold is more than 2,P value is less than 0.05,and the Flag value / Call value is shown as P),a total of 8 differentially expressed miRNAs were screened by chip analysis,of which miR-503-5p,miR-424-5p,miR-4485,miR-630,miR-5703,and miR-937-5p were up-regulated(fold changes were 6.92,2.07,2.28,3.08,2.41 and 2.44,respectively).The expression of miR-1973 and miR-4430 was down-regulated(fold changes were 0.39 and 0.48 respectively).The results qRT-PCR were consistent with the results of the chip.miR-503-5p is the most significantly up-regulated miRNA.A total of 9186 target genes of the differentially expressed miRNAs were predicted and mainly involved in the biological processes including DNA dependent transcription regulation,signal transduction,multi cell organ development,transmembrane transport,cell apoptosis process,positive regulation of transcription from RNA polymerase II transcriptional promoter,transshipment,cell adhesion.They also involved in the signal pathway of cancer,MAPK signaling pathway,local adhesion,cytokine receptor interaction,endocytosis,actin cytoskeleton regulation,neuroactive ligand receptor interaction,chemokine signal transduction pathway,Wnt signaling pathway,calcium signaling pathway and so on.Under normal gravity,the apoptosis rate of miR-503-5p mimic transfected cells was significantly higher than that of miR-503-5p mimic negative control group(NC group)(P<0.01)and culture only group(Control group)(P<0.01).After 72 hours clinorotation,the apoptosis rate of miR-503-5p mimic group was significantly higher than that of miR-503-5p mimic NC group(P<0.01)and only clinorotation group(P<0.01).After 72 hours clinorotation,the apoptosis rate of LNA-miR-503-5p group was significantly lower than that of LNA-miR-503-5p-NC(P<0.01)and only rotation culture group(P<0.01).After 72 hours clinorotation(high expression of miR-503-5p),the expression of Bcl-2 mRNA and protein in human PMECs was significantly lower than that of normal gravity control group(P<0.01).After 72 hours clinorotation,the levels of Bcl-2 mRNA and protein expression in the LNA-miR-503-5p group were significantly higher than that of LNA-miR-503-5p-NC group(P< 0.05 and P<0.01,respectively)and clinorotation only group(P<0.01).The results of Western blot also showed that after 72 hours clinorotation,the expression of IGF-1R and pAKT protein in human PMECs was lower than that of normal gravity control group(P<0.01).Conversely,after 72 hours clinorotation,the level of IGF-1R and pAKT protein in the LNA-miR-503-5p group was higher than that of LNA-miR-503-5p-NC group(P<0.01 and P<0.05,respectively)and clinorotation only group(P<0.01).Compared to pre-hindlimb unloading,a slight body weight loss was observed in the dogs from day 3 of clinorotation.The most obvious change was observed on day 7.The body weight was gradually recovered at the end of the hindlimb unloading.The decrease of food consumption was earlier than that of body weight change.The most food consumption decrease was observed on day 3 of clinorotation,and gradually recovered on day 7.The food consumption was recovered to the control group after the hindlimb unloading.Hindlimb unloading had no significant effect on hematology and serum chemistry parameters.After 7 days hindlimb unloading,the early changes of apoptosis including endothelial swelling,chromatin margination and mitochondria swollen were observed in the dog PMECs.In the early stage of hindlimb unloading(on the first day),the blood pressure(systolic pressure,diastolic pressure,mean arterial blood pressure)and heart rate were increased and the heart rate was accelerated.Although no statistical difference was found on the day 3,the blood pressure of the hindlimb unloading group was still lower than that of the normal gravity group.A transient decrease in the systolic pressure,diastolic pressure and mean arterial blood pressure was observed at 0.5 hours after the removal of the hindlimb unloading.Although no statistical difference was observed when compared with the normal gravity group,the decrease was obvious.A slight increase in heart rate was also noted.The phenomenon is recovered at about 1-2 hours after removal of the hindlimb unloading.Conclusion Under simulated microgravity conditions,apoptosis of human PMECs was observed in vitro.Differential expression of multiple miRNAs is involved in the apoptosis of PMECs under simulated microgravity.in which miR-503-5p is most significantly up-regulated miRNA and plays a key role in the process.Bcl-2,the confirmed target gene of miR-503-5p and IGF-1R/AKT pathway may involve in the effect of miR-503-5p on the apoptosis of PMECs under microgravity.Microgravity simulated by 7 days of hindlimb unloading caused damage to the pulmonary microvascular endothelial cells in Beagle dogs,which mainly manifested early changes of apoptosis including endothelial swelling,chromatin margination and mitochondria swollen.In addition,after 3 days of hindlimb unloading,the blood pressure and heart rate of Beagle dogs can be significantly increased,and then the blood pressure decreased slightly.After the hindlimb unloading removal,the blood pressure was decreased shortly and recovered quickly.