| Background:Inflammation-induced lymphangiogenesis is a widely accepted concept.In healthy adults,lymphatic vessels system are stationary.However,lymphatic vessels dilate rapidly under inflammatory conditions.Atherosclerosis is a chronic inflammatory process.At the early stage of atherosclerosis,the density of adventitial lymphatics increases with the progression of the disease,but the exact mechanism remains unclear.As the research progresses,the activation of lymphatic function on the one hand not only interferes with the inflammatory process.On the other hand,the reverse transport of plaque cholesterol also depends on lymphatic vessel transport.It has been reported that sphingosine-1-phosphate?S1P?is not only closely related to the chronic inflammatory process of atherosclerosis,but also affects angiogenesis.Therefore,the purpose of this paper is to investigate whether S1P affects lymphangiogenesis and its mechanisms.Methods:?1?Human lymphatic endothelial cells?HLECs?were grown in DMEM containing 10%fetal bovine serum?5%CO2,37°C?and used between passage 2 and passage 8.Cells were starved for 10 h in DMEM containing 1%FBS prior to treatment with the reagents.The cells were then incubated with different concentrations of S1P?0,50,100,200 and 400 nM?for 24 h.Cell proliferation was detected by CCK-8 assay,cell migration was detected by cell scratch assay,and cell tubu formation was detected by Matrigel tubule formation.?2?The cells are incubated with different concentrations?or different times?of S1P.The secretion of IL-1?and TNF-?was detected by ELISA kit.In addition,Western blot was used to detect protein levels of IL-1?and TNF-?.Next,cells were treated with different concentrations of S1P?0,50,100,200,and 400 nM?,and protein expression levels of VEGF-C and VEGF-D were detected by Western blot and ELISA.?3?The targeted siRNA was transfected into HLECs using DharmaFECT 3Transfection Reagent.After 24 h of transfection,the cells were treated with S1P for the corresponding time,cell proliferation was detected by CCK-8method,cell migration was detected by cell scratch method,and cell tube formation was detected by matrigel tube formation.In addition,western blot was used to detect protein levels of IL-1?,TNF-?,and S1P receptors.At the same time,IL-1?and TNF-?were overexpressed,and the tube formation ability of the cells was examined.?4?Real-time PCR and Western blot were used to detect the expression level of S1P receptor in cells without treatment.?5?Cells were pretreated with W146?a selective antagonist of S1PR1?,CAY-10444?a specific antagonist of S1PR3?,and VPC23019?a specific antagonist of S1PR1/S1PR3?for 30 minutes prior to stimulation with S1P.The ELISA kit detects the secretion levels of IL-1?and TNF-?.In addition,Western blot was used to detect IL-1?and TNF-?protein levels.?6?HLECs were cultured,and cells were treated with S1PR1 antagonist?W146?or NF-?B inhibitor?BAY11-7082?for 30 minutes,respectively,and the cells were incubated with S1P for 12 h.Then,Western blot was used to detect I?B?,p-I?B?,NF-?B P65 and p-P65 protein levels,immunofluorescence detection of NF-?B nuclear translocation.?7?Cells were treated with W146or BAY11-7082,cell migration was detected by cell scratching,and cell tube formation was detected by matrigel tube formation.ApoE-/-mice high fat diet?HFD?was used to make an atherosclerosis model,mice were injected with 5 mg/kg/day fingolimod?FTY720?,mice were injected with 5mg/kg/day BAY11-7082.Three months later,the mice were sacrificed,and the aorta?ascending aorta?of the control and treatment groups were removed,and the number of lymphatic vessels in the ascending aorta was detected by Immunohistochemistry.Results:?1?S1P promotes lymphatic endothelial cell proliferation,migration,and tubule formation,and the best effect was observed at 200 nM.?2?S1P promoted an increase in the levels of TNF-?and IL-1?in HLECs in a dose manner,and the maximum effect was observed at 200 nM?12 h?.S1P did not affect the changes of VEGF-C and VEGF-D protein levels in HLECs.?3?S1P induces proliferation,migration and tube formation of lymphatic endothelial cells.Transfection of TNF-?siRNA or IL-1?siRNA revealed that S1P-induced HLEC proliferation,migration and tube formation were weakened.After overexpression of TNF-?and IL-1?,cell tube forming ability was found to be enhanced.?4?HLECs mainly express S1PR1 and S1PR3,but hardly express S1PR2.?5?Pretreatment of HLECs with W146?S1PR1 antagonist?and VPC23019?S1PR1/S1PR3 antagonist?significantly inhibited S1P-induced TNF-?and IL-1?release,but CAY-10444?S1PR3specific antagonist?had no effect on TNF-?and IL-1?release.?6?S1P significantly increased I?B?and NF-?B P65 phosphorylation levels.In addition,W146 and S1PR1 siRNA blocked phosphorylation of P65 in S1P treated cells.At the same time,S1PR1 siRNA inhibited the secretion of TNF-?and IL-1?in S1P-induced HLECs.Treatment of HLECs with W146and BAY11-7082 revealed that the secretion of TNF-?and IL-1?in HLECs was significantly lower than that of the control group,and the nuclear translocation of NF-?B was also significantly reduced.?7?Treatment of cells with W146 or BAY11-7082 revealed that S1P-induced HLEC migration and tube formation were inhibited.In animal experiments,the number of aortic adventitial lymphatic vessels in the mice injected with FTY720 was significantly higher than that in the HDF,HDF+W146antagonist and HDF+BAY11-7082 inhibitor groups.Conclusions:?1?S1P promotes inflammation-induced HLEC proliferation,migration,and tube formation.?2?S1PR1/NF-?B is involved in S1P-induced inflammatory effects of HLECs.?3?S1PR1/I?B?/NF-?B is involved in S1P proinflammatory-induced HLEC tube formation. |
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