SPNS2 Regulate HUVEC Proliferation,Migration And Tube Formation In Mitochondrial Pathway

Endothelial cells,as an important key link in angiogenesis,target to maintain their functional homeostasis,can effectively promote the establishment of collateral circulation in the area of myocardial infarction and improve the cardiac function after myocardial infarction(MI).Sphingosine 1-phosphate(S1P),an important metabolite of sphingomyelin,acts as a powerful regulator of endothelial function and endothelial injury response,is considered an ideal target for the treatment of vascular endothelial dysfunction with its central signaling axis,especially the S1PS1 P receptor(Sphingosine 1-phosphate receptoers,S1PRs)receptor pathway.But little is known about the regulation of vascular endothelial function by intracellular S1 P relative to the study of S1 P extracellular function.Spinster homolog 2(Spinster homolog 2,SPNS2),as the main transporter of S1 P in endothelial cells,whether and how its mediated changes in intracellular S1 P content and related signaling pathways are involved in the regulation of endothelial cell function and angiogenesis remains to be further investigated.This study confirms that SPNS2 regulates human umbilical vein endothelial cell(Human umbilical vein endothelial cells,HUVEC)proliferation,migration,and lumen-like structure generation by regulating extracellular S1P-S1PR1/3,especially intracellular sphingosine kinase 2(Sphingosine kinase 2,SPHK2)signaling,and that this intracellular pathway relies on mitochondrial SPHK2 mediated-mediated changes in mitochondrial function.Part Ⅰ Collateral circulation formation and SPNS2 expression in Myocardial infarction RatsAim: To observe the formation of collateral circulation and the expression of SPNS2 in myocardial infarction SD rats.Methods: 60 male SD rats aged 8 weeks were randomly divided into normal group(n=15),sham operation group(n=20),two dead and MI group(25),five dead,all of which were fed on a general diet.The MI group performed coronary artery left anterior descending(LAD)ligation to construct a myocardial infarction(MI)model.The sham operation group did thoracotomy without ligate.Post-operative electrocardiogram(ECG)monitoring showed obvious elevation of ST-segment,confirming MI successful ligation.MI SD rats in each group were subjected to ECG monitoring after 6 weeks of operation,and euthanized.Separate the heart,TTC detect myocardial infarction;hematoxylin-eosin staining(HE)to observe myocardial pathological changes,Masson staining to confirm the degree of myocardial fibrosis;western immunoblotting(WB)to detect the expression of platelet-endothelial cell adhesion molecules(CD31)and SPNS2,and immunofluorescence to observe the co-localization of CD31 and SPNS2.Results: Compared with the normal group and the sham operation group,the ECG results showed that the ST segment of the MI group was significantly elevated and pathological Q waves occurred;TTC staining showed a significant increase in the infarct size in the MI group;HE staining showed that myocardial necrosis increased in the MI group,Muscle fibers are wavy,sarcoplasmic agglomerates,and red-stained horizontal bands are formed.Masson staining shows a significant increase in collagen fibers and a higher degree of myocardial fibrosis in the MI group.WB results show that the expressions of CD31 and SPNS2 in the infarct area are significantly increased.Immunofluorescence showed that the CD31 and SPNS2 of MI region were co-localized,and the fluorescence intensity of vascular endothelial SPNS2 in MI region was significantly higher than that in control group and sham operation group.Conclusion: The MI model of rat was constructed successfully,and the generation of lateral branch circulation as well as the expression of endothelial cell SPNS2 in MI region were shown significantly increased.Part Ⅱ Downregulation role of SPNS2 sh RNA in human umbilical vein endothelial cells(HUVEC)proliferation,migration and lumen-like structure formation cannot rescued by extracellular S1P-S1PRsAim: To observe the effect of SPNS2 knockdown on the proliferation,migration,and lumen-like structure formation of human umbilical vein endothelial cells(HUVEC)and to explore the role and potential mechanism of SPNS2 on angiogenesis.Methods: Culture HUVEC and construct SPNS2 sh RNA lentiviral particles.Pre-experimentally confirm the concentration of lentiviral(MOI=50)transfected HUVEC.HUVEC were transfected with lentiviral empty vector and SPNS2 sh RNA lentivirus respectively(16h).Subsequently,48 h were treated with purinomycin(2.5 ug/ml)to construct stable transfer cell lines with lentiviral empty vector and SPNS2 sh RNA lentivirus,and then followed by PBS and S1 P treatment,respectively.The groups are as follows: HUVEC+phosphate buffer salt solution(Phosphate buffer saline,PBS),HUVEC+lentivirus-empty vector,HUVEC+ SPNS2 sh RNA lentivirus,HUVEC+SPNS2 sh RNA lentivirus+S1P.Immunofluorescence assay the construction of a transformation system;Quantitative Real-time PCR(RT-PCR)and WB detect SPNS2 expression;Ki67 detect HUVEC proliferation;Trans-well observed HUVEC migration;tubule formation experiments detect lumen-like structure formation,RTPCR and WB detect the expression of S1PR1 and S1PR3.Results: The levels of SPNS2 m RNA and protein,and the proliferation and migration ability of HUVEC were significantly decreased in SPNS2 sh RNA lentivirus group compared with normal and lentivirusempty vector group.Moreover,SPNS2 sh RNA significantly inhibited the formation of lumen-like structure.The further results showed that SPNS2 sh RNA down-regulated the expression of S1PR1 and S1PR3,while the SPNS2 sh RNA mediated impaired function of HUVEC was improved after recovery S1PR1 and S1PR3 expression by the addition of S1 P,but there was still significant statistical difference compared with the normal and the lentivirus empty vector group.Conclusion: SPNS2 sh RNA significantly inhibits HUVEC proliferation,migration,and formation of lumen-like structure,and further downregulates S1PR1 and S1PR3 expression.However,the impaired function of HUVEC is not entirely rescued even extracellular S1 P restored S1PR1 and S1PR3 expression.Part Ⅲ SPNS2 sh RNA down-regulates intracellular SPHK2 to regulate HUVEC proliferation,migration and lumen-like structure formationAim: To investigate whether SPNS2 knockdown affects HUVEC function and lumen-like structure formation and its possible mechanisms by regulating the levels and activities of Sphingosine Kinase 1(SPHK1)and Sphingosine Kinase 2(SPHK2)in HUVEC.Methods: HUVEC,SPNS2 sh RNA lentivirus stabilized strains and lentiviral empty-vector stabilized strains were cultured.The experimental groups were as follows: HUVEC+PBS,HUVEC+ lentivirus-empty vector,HUVEC+SPNS2 sh RNA lentivirus,HUVEC+SPNS2 sh RNA lentivirus +human-derived recombinant SPHK2 protein,HUVEC+SPNS2 sh RNA lentivirus+S1P.RT-PCR detect SPHK1 and SPHK2 expression;WB and immunofluorescence assay both protein expression and phosphorylation levels;Ki67 confirm HUVEC proliferation;Trans-well assay HUVEC migration;Tubule formation assay the ability of lumen-like structure formation.Results: SPNS2 sh RNA significantly decreased the m RNA and protein levels of SPHK2 rather than P-SPHK2/SPHK2 compared with normal group and lentivirus-empty vector group.But the m RNA and protein levels of SPHK1 and P-SPHK1/SPHK1 did not change significantly.Meanwhile,the SPNS2 sh RNA treatment group significantly reduced HUVEC viability,proliferation,migration,and lumen-like structure formation.However,when human recombinant SPHK2 protein recovered SPHK2 expression,the above trend was significantly reversed.And the expression of SPHK2 was not significantly different from that of SPNS2 sh RNA lentivirus group after the addition of S1 P to activated receptor on the basis of SPNS2 sh RNA lentivirus.Conclusion: SPNS2 sh RNA inhibits HUVEC proliferation,migration,and lumen-like structure formation by down-regulating SPHK2 m RNA and protein,and the down-regulation effect does not rely on extracellular S1 P signaling pathways.Part Ⅳ SPNS2 sh RNA regulates mitochondrial function via mitochondrial SPHK2Aim: To investigate whether SPNS2 sh RNA regulate HUVEC function by SPHK2 affecting respiratory dysfunction in mitochondria.Methods: HUVEC,SPNS2 sh RNA lentivirus stabilized strains and lentiviral empty-vector stabilized strains were cultured.,The experimental groups were as follows: HUVEC+PBS,HUVEC+ lentivirus-empty vector,HUVEC+SPNS2 sh RNA lentivirus,HUVEC+SPNS2 sh RNA lentivirus +human-derived recombinant SPHK2 protein.Mitochondrial fluorescence probes to detect mitochondrial integrity;ROS Assay Kit to detect mitochondrial ROS levels,and Tetramethylrhodamine ethyl ester perchlorate(TMRE)kit to detect mitochondrial membrane potential;Mitochondrial proteins were extracted using a mitochondrial protein extraction kit;and WB to assay PHB2 protein expression levels in mitochondria.Results: SPNS2 sh RNA significantly impaired mitochondrial integrity;decreased mitochondrial membrane potential levels,increased mitochondrial ROS production and down-regulated the expression of PHB2 in mitochondria compared with control group and empty vector group.The above phenomenon was effectively reversed after human recombinant SPHK2 protein was added on SPNS2 sh RNA basis.Conclusion: SPNS2 sh RNA reduces mitochondrial PHB2,affects mitochondrial respiratory chain function by downregulating mitochondrial SPHK2 expression.