Effects Of HIF-2α/SPHK1 On Endothelial Cell Migration And Lumen Formation After Ischemiaand Hypoxia

Backgrounds and aims:During the long-term lack of blood supply and hypoxia due to myocardial infarction（MI）,it is particularly important to restore collateral circulation in infarct area in order to maintain the homeostasis and repair of body function.Endothelial cells play a key role in angiogenesis.Promoting endothelial cell migration and tube formation and accelerating the recovery of collateral circulation are important for healingandremodelingofmyocardialinfarction.Sphingosine-1-Phosphate（S1P）is a regulator of endothelial cell function synthesized by sphingosine kinase（SPHK）and regulated by S1P receptor（S1PRs）in vitro.S1P has powerful and specific angiogenesis,especially in inducing angiogenesis after tissue injury.Local tissue autocrine S1P is more helpful to improve tissue vascular repair and regeneration than S1P,systemic circulatory system.Previous studies have shown that ischemia and hypoxia can regulate SPHK1 expression,but the mechanism of SPHK1 and angiogenesis and collateral circulation in myocardial infarction need to be further explored.This paper explores the changes of endothelial cell SPHK1 expression level and its influence on cell migration and tube formation under ischemia and hypoxia,and studies the mechanism of increased SPHK1 expression level caused by ischemia and hypoxia.Methods:Human umbilical vein endothelial cells（HUVECs）were first grown in a high-sugar medium CO₂10%fetal bovine serum（5%CO₂）and then HUVECs in human umbilical vein endothelial cells.HypoxiainDMEM（3%O₂）atdifferenttime（0h,4h,8h,12h,16h,20h,24h）.Cell morphology was observed,and cell morphology was observed at different time points of hypoxia after collecting total cell protein.CCK-8 cell survival rate of seven groups under different hypoxia time was measured.cell mobility and tube formation in Control and Hypoxia groups were calculated using cell scratch experiments and tube formation experimental data.（2）SPHK1inhibitor PF-543（100n M）was added to the HUVECs of hypoxia to inhibit the effect of SPHK1,and HIF-2αinhibitor（50n M）was added to the HUVECs of hypoxia.Through Western Blot to explore whether HIF-2αand SPHK1 affect each other and the relationship between them under hypoxia.（3）The cell viability of Control、Hypoxia、Hypoxia PT2385 and Hypoxia PF-543 groups was detected by CCK-8experiments.（4）The cell mobility and tube formation in the Control、Hypoxia、Hypoxia PT2385 and Hypoxia PF-543 groups were detected by cell scratch test and matrix hose formation test.（5）To establish a SD model of myocardial infarction in rats SD intraperitoneal injection of PF-543（SPHK1 inhibitor,1.2 mg/kg）after myocardial infarction.CD31proteins detected by tissue immunofluorescence assay.Results:（1）With the prolongation of hypoxia time,cell viability gradually decreased and decreased to about 50%at 16 h.while compared with Control,the protein expression levels of SPHK1 and p-SPHK1increased gradually with the increase of hypoxia time,until the peak reached 12 hours,and then the expression level decreased gradually.（2）Western Blot results show that,with HIF-2αinhibitor PT-2385,HUVECs SPHK1 and p-SPHK1 protein expression levels decreased significantly.The immunofluorescence results showed that the expression level of SPHK1 decreased significantly after HIF-2αinhibition.There was no change in protein expression levels of SPHK2 and p-SPHK2 in Hypoxia and group.There was no change in histone expression level Hypoxia PF-543 group.（3）The cell viability in the Hypoxia group decreased,while the cell viability in the Hypoxia PF-543 group and Hypoxia PT2385 group decreased significantly compared with that in the Hypoxia group.（4）The HUVECs mobility and tube formation in Hypoxia group were significantly higher than those in Control group.Conclusion:（1）Ischemia and hypoxia regulate HUVECs SPHK1levels and promote migration and tube formation.（2）HIF-2αregulate the expression level HUVECs SPHK1 ischemia and hypoxia.（3）HIF-2α/SPHK1 involvement in regulating ischemia-hypoxia-induced migration and tube formation...