The Study On Protective Effect Of Apelin On Pulmonary Microvascular Endothelial Cells

Partâ… :The effect of angiotensinâ…¡on expression of Apelin-APJ mRNA in pulmonary microvascular endothelial cellsObjective Exploring the effect of angiotensinâ…¡(Angâ…¡) on expression of Apelin-APJ mRNA in pulmonary microvascular endothelial cells(PMVECs).Methods Pulmonary microvascular endothelial cells from neonatal rat of 24h after birth were cultured.Expressions of Apelin-APJ mRNA in endothelial cells were determined by reverse transcription-polymerase chain reaction(RT-PCR).PMVECs were used between passages 2 and 4.5×10⁵ cells grown in 6-well plates at subconfluence were stimulated with various concentrations of Angâ…¡(0,10^-9,10^-8,10^-7,10^-6M) for analyzing apelin and APJ mRNA expressions by RT-PCR at 24hr and with Angâ…¡of 10^-7 M for apelin and APJ mRNA expression at different time points of 0hr,1hr,6hr, 12hr,24hr,48hr.At the same time analyzing their expressions in blank groups at three time points of 0hr,24hr and 48hr without stimulation of Angâ…¡.every experience was repeated for 4 times.Resultsâ‘ PMVECs expressed apelin and APJ mRNA determined by RT-PCR.â‘¡compared with control group(OM Angâ…¡),10^-9 M Angâ…¡induced upregulation of apelin mRNA expressions in PMVECs at 24hr(P＜0.01), higher concentrations of Angâ…¡(10^-8,10^-7,10^-6M) induced concentrationdependent downregulation of apelin mRNA expressions(P＜0.05 or P＜0.01); different concentrations of Angâ…¡(10^-9,10^-8,10^-7,10^-6M) concentrationdependently downregulated expressions of APJ mRNA.â‘¢10^-7 M Angâ…¡upregulated apelin mRNA expressions in short time(within 6hr,P＜0.05) with a peak at 1hr(P＜0.01),and markedly downregulated them 6 hrs later (P＜0.01);10^-7M AnglI also markedly time-dependently downregulated APJ mRNA expressions after 12 hrs(P＜0.01);apelin and APJ mRNA expressions did not significantly change in PMVECs without treatment with Angâ…¡at 3 time points of 0hr,24hr and 48hr.Conclusion Angâ…¡can markedly effect on apelin and APJ mRNA expressions in PMVECs,and apelin-APJ system may involve in the mechanism of injury of Angâ…¡on endothelial cells.Partâ…¡:Effects of Apelin on proliferation and apoptosis in pulmonary vascular endothelial cellsobjective To investigate the effects of apelin on proliferation and apoptosis in pulmonary vascular endothelial cells.Methods Pulmonary microvascular endothelial cells(PMVECs) between passage 2 and 4 cultured from neonatal rat of 24 hours after birth were starved serum-free for 24hours,and then were used to following studies:â‘ 2×10³ cells per well grown in 96-well plate were divided randomly into 3 groups:Control group(C group) was added with equal volume of PBS;Lower concentration group(10^-8 M group) with 10^-8 M of apelin; Higher concentration group(10^-6 M group) with 10^-6 M of apelin.MTT assay was used to assess cell proliferation rates after cells were continuously incubated for 48 hours.â‘¡5×10⁵ cells grown in cultured-bottles of 25 ml were divided into 5 groups:Control group were added with equal volume of PBS;Lower concentration group(10^-8 M group) with 10^-8 M of apelin; Higher concentration group(10^-6 M group) with 10^-6 M of apelin; Angiotensinâ…¡(Angâ…¡) group with 10^-7 M of Angâ…¡;Angâ…¡+Apelin group with 10^-7 M of Angâ…¡and 10^-8M of apelin.The apoptosis rates of PMVECs in each group were detected by flow cytometry after the cells were incubated for 24 hours.Resultsâ‘ The proliferation rates of the PMVECs significantly increased in both lower and higher concentration of apelin groups compared to control group(P＜0.01),and there were not significantly different between lower and higher concentration of apelin groups.â‘¡The apoptosis rates of the PMVECs significantly decreased in both lower and higher concentration groups but dramatically increased in Angâ…¡group,compared to control group(P＜0.05 or P＜0.01);The apoptosis rates were lower in Angâ…¡+Apelin group than in Angâ…¡group(P＜0.01).Conclusion Apelin can promote proliferation of PMVECs and inhibit apoptosis.Partâ…¢:Protective effects of Apelin on pulmonary microvascular endothelial cells and the mechanism of it' s promoting NO releaseObjective To investigate the effects of Apelin on release of ET-1 induced by Angiontensinâ…¡and it' s mechanism of stimulating NO release in pulmonary microvascular endothelial cells(PMVECs).Methods PMVECs between passage 2 and 4 cultured from neonatal rat of 24 hours after birth were starved serum-free for 24 hours,and then used to following studies.â‘ The effects of Apelin on release of NO in PMVECs at different times: 1×10⁵ cells per well grown in 24-well plate were divided randomly into 2 groups:Apelin group was added with 10^-8 M Apelin;control goup with equal volume of serum-free DMEM.Then concentration of NO in cultural medium were detected in both groups at five different time points of 0min,2min,5min,15min and 30min.â‘¡The effects of Apelin on ET-1 release in PMVECs induced by Angiotensinâ…¡:PMVECs grown in 24-well plats were randomly divided into 4 groups: Angâ…¡group was added with 10^-7M Apelin;Angâ…¡+10^-8M Apelin group incubated with 10^-8M Apelin at 5 min prior to stimulation of 10^-7M Angâ…¡; Angâ…¡+10^-6 M Apelin group with 10^-6M Apelin 5min prior to 10^-7M Angâ…¡; control group with equal volume of serum-free DMEM.ET-1 concentrations in cultural medium were measured in all groups at different time points of 30min,6hr and 24hr.â‘¢The effects of Apelin on phosphorylations of endothelial nitric-oxide synthase and Akt:5×10⁵ cells per well grown in 6-well plates were divided into 3 groups:Apelin group was added with 10^-8M of Apelin; Apelin+Akt inhibitor group was pretreated with 5uM of Akt inhibitor 30min prior to incubated with Apelin;control group with equal volume of serum-free DMEM.5min later western blot was used to assay protein expressions of phosphorylations of endothelial nitric-oxide synthase and Akt.Resultsâ‘ The effects of Apelin on release of NO in PMVECs at different times: compared with control group,concentrations of NO in cultural medium increased at the three time points of 2min,5min,and 15min(P＜0.01 or P＜0.05),with the peak at the point of 5min(P＜0.01);but concentration of NO did not changed statistically at the point of 30min compared to control group.â‘¡The effects of Apelin on ET-1 release in PMVECs induced by Angiotensinâ…¡:1) Compared with control group,concentrations of ET-1 in cultural medium of Angâ…¡group at three points of 30min,6hr and 24hr increased significantly(P＜0.05).2) Compared with Angâ…¡group,concentratins of ET-1 in cultural medium of Angâ…¡+10^-8 Apelin group at the three points decreased significantly(P＜0.05),and close to control group.3) Compared with Angâ…¡group,concentratins of ET-1 in cultural medium of Angâ…¡+10^-6 Apelin group trended to decrease but without statistical significance.â‘¢The effects of Apelin on phosphorylations of endothelial nitric-oxide synthase and Akt:1) Compared with control group,Apelin induced increase of protein expressions of phosphorylation of both Akt and eNOS significantly in Apelin group(P＜0.01).2) Compared with Apelin group,protein expressions of phosphorylation of eNOS significantly decreased(P＜0.01).Conclusion Apelin can induced NO release and attenuated ET-1 release induced by Angâ…¡in PMEVCs.Promoting phosphorylation of Akt/eNOS was one of mechanisms to induce NO release and may be involved in protective effect on PMVECs for Apelin.