Aspirin-induced Repression Of GLUT1 Expression Inhibits Glycolysis In A Vascular Endothelial Cell Line

BackgroundThe process of forming new blood vessels from the development of existing blood vessels is so called angiogenesis.Physiologically,angiogenesis is involved in the repair of damaged tissues.In adults,angiogenesis is more common in pathological processes,especially in tumor growth and metastasis,which is considered to be closely related to tumor neovascularization.Vascular endothelial cells(ECs)are monolayer endothelial cells in vascular lumen,which control the function of blood vessels and directly participate in the pathological process of angiogenesis.The anaerobic oxidation of glucose,that is,the decomposition of glucose or glycogen into lactic acid and the production of a small amount of ATP under anaerobic or hypoxic conditions,is called glycolysis.In recent years,a large number of researches have shown that tumor-induced angiogenesis is related to the changes of ECs’ glucose metabolism,including increased glucose flux,increased lactate synthesis and the participation of key glycolytic enzymes in ECs.Glycolysis is also considered to be involved in resistance to current anti-VEGF therapies.Although a new therapeutic concept of targeting endothelial cell glycolysis to interfere angiogenesis has long been proposed,there is no clinically available glycolysis inhibitors.As a classical non-steroidal anti-inflammatory drug,aspirin has showed many new evidences in cancer prevention and treatment,and aspirin has attracted extensive attention for its inhibition of angiogenesis.However,the underlying mechanism remains to be elucidated,especially the effect of aspirin on glycolysis of vascular endothelial cells has not been reported yet.These results revealed GLUT1 has potential to be a target of aspirin and provided a new perspective to understand the pharmacological efficacy of aspirin on vascular ECs.ObjectiveIn this study,we cultured SEND cells(a mice vascular endothelial cell line)and administered different concentrations of aspirin in vitro to investigate the effect of aspirin on glucose metabolism of vascular endothelial cells and its possible mechanism.In vitro experiments were carried out to detect the effects of glycolysis-related gene expression,glucose transporter GLUT-1,glucose uptake,glycolysis product(ATP and lactate)in vascular endothelial cells after exposuring to different concentrations of aspirin.The possible mechanisms were preliminarily explored.These findings revealed GLUT1 has potential to be a target of aspirin and provided a new perspective to understand the pharmacological efficacy of aspirin on vascular ECs,and suggested the need for further investigation on the phenomenon.Methods 1.To investigate the effect of aspirin on SEND glycolysis gene expression in vascular endothelial cells1.1 The expression levels of glycolysis related genes were detected.SENDs were cultured in vitro and divided into four groups: normal control group,0.25 mM aspirin group,1 mM aspirin group and 4 mM aspirin group.The expression of GLUT1,PFKFB2,PFKFB3,LDHA,PKM2 and HK2 were detected by real-time fluorescence quantitative PCR(qRT-PCR).1.2 To observe the expression of GLUT1,PFKFB2,HK2 in SEND cells after exposuring to aspirin at different concentrations and time points.SEND cells were stimulated with aspirin at different time and concentration(0.25 mM,1 mM,4 mM).Then,4 mM aspirin was selected to stimulate SEND cells at different time points(3h,6h,12 h,24h,48 h,72h).Western Blot was used to detect the expression of GLUT1 after exposuring to aspirin.2.To explore the effect of aspirin on function of vascular endothelial cells2.1 To observe the expression levels of VE-Cadherin,ICAM-1 and VCAM-1 after exposuring to different concentrations of aspirin.SEND cells were stimulated with different concentrations of aspirin(0.25 mM,1 mM,4 mM,respectively)for 24 hours.Western Blot was used to detect the expression levels of VE-Cadherin,ICAM-1 and VCAM-1.2.2 The effects of different concentrations of aspirin(0.25 mM,1 mM,4 mM)on glucose uptake in SEND cells were examined.SEND cells were cultured in vitro and divided into four groups: normal control group,0.25 mM aspirin group,1 mM aspirin group and 4 mM aspirin group.The uptake of fluorescent glucose analogue 2-NBDG was detected by flow cytometry.2.3 The effects of high concentration aspirin(4mM)stimulation on glycolytic metabolites synthesis in SEND cells were detected.SEND cells were cultured in vitro and divided into normal control group and 4 mM aspirin group.The contents of lactate and ATP in cells were measured by colorimetry after a 24-hour stimulation.3.To explore the effect of aspirin on signaling pathway molecule expression in SENDsThe effects of different concentrations of aspirin(0.25 mM,1 mM,4 mM)on the expression of glycolysis-related signaling molecules in SEND cells were detected.SEND cells were cultured in vitro and divided into four groups: normal control group,0.25 mM aspirin group,1 mM aspirin group and 4 mM aspirin group.Western blot was used to detect the effects of aspirin on the expression of NF-κB,HIF-1a,Erk and Akt in SENDs after a 24-hour stimulation.Results1.The mRNA expression level of GLUT1 in SENDs was significantly down-regulated after exposuring to 4 mM aspirin for 12 hours(P < 0.001).Western Blot was used to detect the expression of glycolysis-related proteins including GLUT1,HK2 and PFKFB2.The inhibitory effect of single dose of aspirin administration on the expression of GLUT1 was most obviously down-regulated after a 24-hour stimulation.It suggests that aspirin may interfere glucose metabolism in SENDs by inhibiting the expression of GLUT1.2.Exposuring to aspirin increased the expression of VE-Cadherin in SENDs,while the expression levels of ICAM-1 and VCAM-1 did not show significant differeces.At the same time,the decrease of glucose uptake in SENDs was observed(P < 0.01).The intracellular lactate and ATP of SEND cells were significantly decreased after exposuring to 4 mM aspirin(P < 0.05).3.SENDs were treated with different concentrations of aspirin(0.25 mM,1 mM,4 mM),and the expression of HIF1α,NF-κB,Akt and Erk were detected.It was found that 4 mM aspirin significantly decreased the phosphorylation of NF-κB p65 in SEND cells.This result suggests that the effect of aspirin on SENDs may be related to its regulation of NF-κB.Conclusion1.Aspirin-induced down-regulation of GLUT1 expression results in an impaired glucose uptake capacity in SENDs.2.Aspirin has potential to inhibit glycolysis of SEND cells.Exposure to 4 mM aspirin can reduce the synthesis of ATP and lactate in SEND cells.3.Aspirin inhibits the phosphorylation of NF-κB p65 and up-regulates the expression of VE-Cadherin in SENDs.