Effects Of Dihydroartemisinin And Cadmium On Endothelial Cells

Background:Angiogenesis is the physiological process of new capillary development from pre-existing vessels.Angiogenesis is a multi-step process involving endothelial cell activation,proliferation,migration,differentiation,maturation and tube formation.The endothelial cells play a key role during angiogenesis.Uncontrolled angiogenesis is associated with angiogenic diseases such as caner,rheumatism,and diabetes.Inhibition of abnormal angiogenesis has become an important treatment for angiogenesis related dieases.Artemisinin is a sesquiterpene lactone endoperoxide extracted from the green artemisia plant,and is widely used as an antimalarial-agent.Artemisinin and its derivatives show strong anti-angiogenic effects in cell culture experiments and animal models.Dihydroartemisinin(DHA)is a semi-synthetic derivative of artemisinin with high water-solubility and few side effects in antimalarial treatment.The anti-angiogenic effects of DHA have been found in solid tumor models,but the underlying molecular mechanisms has not been clear,restricting its application in clinical treatment.Vascular endothelial growth factor(VEGF)is the major angiogenesis stimulator.Upon VEGF binding,VEGFR2 on endothelial cells activates downstream signaling pathways and promotes angiogenesis.In addition,extracellular signal-regulated kinase(ERK)signaling pathway induces endothelial cell proliferation.This study hypothesized that DHA may inhibit the activation of VEGFR and ERK signaling pathways in endothelial cells to exert an anti-angiogenic effect.Objective:(1)To confirm the effect of DHA on the proliferation and migration of HUVECs in vitro;(2)To explore the molecular mechanisms of the anti-angiogenic of DHA in vitro;(3)To verifiy the anti-angiogenic effects of DHA and the underlying mechanisms in a mouse retinal neovascularization model.Methods:(1)Plasmid constructionThe pGL3-basic polyclonal vector was used to construct a plasmid containing the VEGFR2 promoter fragment and a NF-?B mutant plasmid,which the VEGFR2 promoter was selected from the-115 to +296 fragment and the-90 NF-?B mutation of VEGFR2 promoter was mutated from GGAGAGCCCC to AAAGAGCCTT.(2)Transfection and stimulation of cellsFor the dose experiments,final concentrations of 0,1,5,25 and 100 ?M DHA were stimulated HUVECs separately;For the time experiments,0,0.5,1,3,6,12 and 24 hours after DHA stimulation for further testing;To verify whether DHA affects the NF-?B signaling pathway,a final concentration of 100?M PDTC(NF-?B signaling pathway inhibitor)was added 1 hour earlier to inhibit NF-?B pathway;In order to verify whether DHA affects ERK signaling,a final concentration of 10 ?M,PD98059(ERK signaling pathway inhibitor)was added 1 hour earlier to inhibit ERK pathway;For plasmid transfection experiments,0.75 ?g plasmid per well was added to each well of a 6-well plate and transfected with lipo3000 transfection reagent for further testing.(3)MTT experimentMTT proliferation assay was used to detect the proliferation of HUVECs;For NF-?B signaling pathway,cells were divided into control group(DMSO),DHA group(25 ?M),PDTC group(100 ?M),and PDTC+DHA group(100 ?M+25 ?M);For ERK signaling pathway control group(DMSO),DHA group(20 pM),PD98059 group(10?M)and PD98059 + DHA group(10 ?M + 20 pM).The effect of each group on the proliferation of HUVECs was examined by MTT.(4)Cell migration experimentHUVECs were seeded in the upper chamber and were divided into control group(DMSO),DHA group(25 ?M),PDTC group(100 ?M),and PDTC+DHA group(100?M+25 ?M).Cell migration was analyzed by crystal violet staining after 12 hours of stimulation.(5)Real-time quantitative PCR(qRT-PCR)After extraction of total RNA from HUVECs stimulated by DHA group(25 ?M)or PDTC+DHA group,the expression of VEGFR1 and VEGFR2 genes in HUVECs was detected by reverse transcription and RT-PCR;After extraction of total RNA from HUVECs stimulated by DHA group(20 ?M),the expressions of ERK1,ERK2,c-fos and c-myc in HUVECs were detected by reverse transcription and RT-PCR.(6)Western BlotAfter extraction of total protein and nuclear protein from HUVECs stimulated by DHA(25 ?M)or PDTC + DHA group(100 ?M + 25 ?M).The target protein of VEGFR1,VEGFR2,I?B-?,NF-?B p65 and phosphorylated ERK1/2(p-ERK1/2),ERK1/2,c-fos,c-myc were detected by Western blot.(7)ImmunofluorescenceAfter cells were seeded and cells were stimulated by control group(DMSO),DHA group,EGF group(100 ?M)and EGF+DHA group(100 ?M+25 ?M).Cells were fixed and incubated with primary antibody and secondary antibody.Microscopic images and other steps were taken to visually observe the expression and distribution of NF-?B p65 protein in HUVECs stimulated by DHA and/or EGF.(8)Electric cell-substrate impedance sensing(ECIs)HUVECs were seeded into ECIs.For NF-?B signaling,cells were divided into control group(DMSO),DHA group(25 ?M),PDTC group(100 ?pM),and PDTC+DHA group(100 p.M+25 ?M).The changes of endothelial cell permeability or cell migration ability were compared by detecting the real-time resistance or electrode stimulation of each group;For ERK signaling pathway,the cells were divided into control group(DMSO)and DHA group(20 ?M),PD98059 group(10 ?M)and PD98059+DHA group(10 pM+20 ?M),and the changes of endothelial proliferation ability were detected by the real-time resistance of each group.(9)Chromatin Immunoprecipitation(ChIP)To obtain the optimum length DNA fragment,ultrasound was used to lyse HUVECs by DHA treatment.After adding NF-?B p65 antibody to obtain target DNA and purification,VEGFR2 promoter was used for PCR amplification to detect the binding of NF-?B p65 and VEGFR2 promoter.(10)Retinal neovascularization animal modelA model of mouse retinal neovascularization has been established.Intravitreal injections were performed daily from P12 to P14 by delivering 0.5 ml of 0.1 mM DHA diluted in PBS to the left eye and PBS alone to the right eye,using a 32-gauge needle at 200 mm posterior to the limbus,A second group of mice received 0.5 ml of 0.1 mM DHA and 0.5 mM of PDTA to the left eye,and 0.5 mM of PDTA to the right eye.The mice were sacrificed at P17 and the mechanism of anti-angiogenic effect were further examined through retinal vascular density testing experiments in vivo.(11)Statistical analysisAll experimental data are presented as mean ± SD and analyzed using SPSS 19.0 software.Paired t-test was used to compare between groups,all experiments were repeated at least 3 times,P<0.05 was statistically significant.Results:(1)MTT assay and Transwell chamber migration experiments showed that DHA inhibits the proliferation and migration of human umbilical vein endothelial cells(HUVECs)in a dose-dependent manner,and the effect of DHA at the final concentration of 20-25 ?M or high concentration are obvious.(2)Western blot and qRT-PCR experiments showed that DHA specifically down-regulated the expression of VEGFR2 protein and mRNA in endothelial cells,but had no significant effect on VEGFR1;In addition,DHA inhibited the activation of ERK signaling pathway by down-regulated ERK1/2,phospho-ERK1/2,c-fos and c-myc protein and mRNA expression.(3)Western blot analysis showed that the expression of IKB-a protein in cytoplasm was significantly increased,while the expression of NF-?B p65 protein in nucleus was significantly reduced.The results of immunofluorescence were found that DHA can inhibit the nuclear translocation of NF-?B p65 to inhibit the activation of NF-?B(4)Dual luciferase reporter assays revealed that the activity of VEGFR2 promoter was significantly reduced after treatment with DHA.Co-immunoprecipitation experiments finally confirmed that DHA could specifically reduce the binding of NF-?B p65 with VEGFR2 promoter,reduce the activity of the VEGFR2 promoter,and reduce its expressiont.(5)Western blot and qRT-PCR showed that DHA down regulated the expression of VEGFR2 by inhibited NF-?B signaling.(6)MTT assay and ECIs real-time proliferation experiments showed that DHA regulates endothelial cell proliferation by inhibiting NF-?B and ERK signaling pathways.(7)The density of blood vessels in the retina treated with DHA(0.1 mM)was decreased by 36.56%compared with control(PBS)group;The density of retinal vessels in PDTC(0.5 mM)was decreased of 48.23%compared with the control group(PBS);While there was no statistically significant reduction in PDTC+DHA group(0.5 mM +0.1 mM)compared with PDTC group(0.5 mM).It confirmed that DHA exerts an anti-angiogenic effect by regulating the NF-?B signaling pathway.Conclusion:Dihydroartemisinin targets VEGFR2 via the NF-?B pathway in endothelial cells to inhibit angiogenesis;Dihydroartemisinin inhibit the proliferation of endothelial cells via the ERK pathway.Background:With the development of industry,heavy metal pollution has become one of the most serious environmental problems in China.Cadmium is an environmental toxin,and listed as the sixth toxic substance of human health by the American Toxicity Management Committee(ATSDR),with a half-life of 10-30 years.It accumulates in the body,especially in the liver and kidney.Cadmium may be uptake through contaminated water and food,cigarette smoking.Accumulation of Cd damages multiple organs including the lung,liver,testis,and kidney depending on the dose,route and duration of exposure.Cd enters organs and tissues through the circulation system.The vascular endothelium is an organ that directly interact with Cd.Following absorption,Cd travels in the bloodstream as free ions or bounded with albumin or metallothioneins,and distributes into tissues all over the body.Vascular endothelial cells are an important target for cadmium.High concentrations of Cd induces cell death of vascular endothelial cells.Although low concentrations of Cd does not afect on the viability of endothelial cells,but it may cause damage to vessel barrier.Previously most studies focused on long-term cadmium accumulation,and there have been few studies on the damage of vascular endothelial cells caused by short term,low dose cadmium exposure.Vascular permeability is mainly dependent on the integrity of the endothelium.The adheren junction plays a major role in maintaining the permeability of vascular endothelial cells.The vascular endothelial cell cadherin(VE-cadherin)forms a linker complex with ?-catenin and is an adhesion-linked scaffold protein.Separation of VE-cadherin and ?-catenin from cell adhesion junctions results in an increase in endothelial cell permeability.The p38 MAPK signaling pathway regulates endothelial cell permeability.This study hypothesized that heavy metal cadmium affects vascular endothelial cell permeability through the p38 MAPK signaling pathway.Objective:(1)To confirm the effects of cadmium on endothelial cell permeability;(2)To confirm to role of p38 MAPK siginaling in mediating the effects of CdCl2 on endothelial cell permeability.Methods:(1)Stimulation of cellsFor the dose experiments,stimulation of human umbilical vein endothelial cells(HUVECs)with CdCl2 at final concentrations of 0,1,4,10,50,and 100.M,respectively;For the time experiments,0,1,2,6,12,24 and 48 hours were as the checkpoints after Cd added;To confirm the impact of the p38 MAPK signaling pathway on HUVECs,an inhibitor of the p38 MAPK pathway SB203850 was added 1 hour earlier than CdCl2 at concentration of 10?M.(2)Trypan blue stainingTrypan blue staining was used to detect the effect of different concentrations of CdC2 on cell viability at 24 or 48 hours.(3)MTT experimentMTT proliferation assay was used to detect the proliferation ability of HUVECs exposed to cadmium at a concentration of 4 ?M for different time points.(4)FITC-dextran transwellThe cells were divided into control group(ddH20),CdCl2 group(4 ?M,SB203850 group(10 ?M)and SB203850+CdCl2 group(10 ?M +4 ?M).After stimulating HUVECs,they were seeded into the upper chamber and cell permeability were analyzed by FITC-dextran filtration and fluorescence microplate reader.(5)Western BlotExtract total protein from HUVECs which stimulated by different concentrations of CdCl2,detected the expression of phosphor-p38(p-p38)or p38 protein by gel electrophoresis,transmembrane,primary antibody,secondary antibody incubation and ECL exposure.(6)ImmunofluorescenceCells were divided into control group(ddH2O),CdCl2(4 ?M)and SB203850 ?CdCl2 groups(10 ?M +4 ?M).After each group HUVECs were stimulated for 24 hours,the cells were fixed and incubated with antibody.And observe the expression and distribution of VE-cadherin and ?-catenin protein in each group by microscopy.(7)Electric cell-substrate impedance sensing(ECIs)Cells were divided into control group(ddH20),CdCl2 group(4 ?M),SB203850 group(10 ?M)and SB203850+ CdCl2 group(10 ?M +4 ?M).Each group were seeded into ECIs plates.The changes of endothelial cell permeability or cell migration ability were compared by measuring the transcellular resistance of cells after stimulated.(8)Enzyme-linked immunosorbent assay(ELISA)The cell culture medium was stimulated with control group(ddH20),CdCl2(4 ?M)and SB203850 + CdCl2 group(10?M +4 ?M).The secretion of TNF-a protein in each group was detected by enzyme-linked immunosorbent assay.(9)Real-time fluorescence quantitative PCR(qRT-PCR)Total RNA of HUVECs stimulated with ddH20,CdCl2(4 ?M)and SB203850 +CdCl2 groups(10 ?M +4 ?M)were extracted,and TNF-a gene expression of was detected by reverse transcription and RT-PCR.(10)Statistical analysisAll experimental data are presented as mean ± SD and analyzed using SPSS 19.0 software.Paired t-test was used to compare between groups,all experiments were repeated at least 3 times,P<0.05 was statistically significant.Results:(1)MTT and trypan blue experiments show that when the concentration of cadmium ions is lower than 4 ?M,CdC12 has no obvious effect on the proliferation ability and viability to HUVECs;(2)The results of Trasswell FITC-dextran experiment showed that the final concentration of 4 ?M CdCl2 could increase the permeability of HUVECs;(3)Western Blotting experiments showed that the expression of phosphorylated p38 and p38 were significantly increased,and cadmium could activate p38 MAPK signaling pathway;(4)SB203850 could effectively block the increasing permeability caused by CdCl2 through ECIs permeability test and Trasswell FITC-dextran experiment;(5)Visualized by immunofluorescence experiments,the final concentration of ?M cadmium can redistribute VE-cadherin and ?-catenin on the cell membrane,reducing the distribute of VE-cadherin at the junction of cells.While inhibit p38 MAPK signaling pathway can block Cd-induced relocalization of VE-cadherin and p-catenin proteins in the cell membrane and recover cell permeability;(6)ELISA results showed that after cadmium stimulation,the secretion of TNF-a in HUVECs was significantly increased.While the expression level of TNF-a gene in HUVECs were detected by qRT-PCR.But inhibition of p38 MAPK signaling pathway could block cadmium-induced secretion and expression of TNF-a in HUVECs.Conclusion:Cadmium induces vascular permeability via activation of the p38 MAPK pathway.Targeted inhibition of p38 MAPK signaling pathway may provide a new therapeutic strategy for cadmium-related diseases.Background:Leukocyte differentiation antigen CD 109 is a glycosylphosphatidylinositolanchored protein cell surface antigen.In recent years,it has been found that CD 109 is highly expressed in a variety of human malignant tumors and is also highly expressed in circulating endothelial cells of breast cancer and glioblastoma patients.Studies have shown that decreased expression of CD 109 in endothelial cells increases angiogenesis and metastasis of hepatocellular carcinoma.Angiogenesis plays an important role in the development of tumors.However,the expression of CD 109 in vascular endothelial cells of squamous carcinoma has not been examined.In this study,we examined the expression of CD 109 in vascular endothelial cells from squamous cell carcinomas microarrays.Objective:To determine the expression of CD 109 in vascular beds of squamous carcinoma.Methods:(1)ImmunohistochemistryPurchasing normal thymic tissue microarrays,squamous cell carcinoma tissue microarrays from the esophagus,penis,skin,gallbladder,and the microarrays were stained with CD 109 to observe the expression in these tissues.(2)Real-time quantitative PCR(qRT-PCR)Fresh penis squamous cell carcinoma specimens were collected and total RNA was extracted.The expression of CD 109 gene in penis squamous cell carcinoma was detected by reverse transcription and RT-PCR.(3)Western BlotFresh penis squamous cell carcinoma specimens were collected,and extact total protein,perform gel electrophoresis,transfer membrane,primary antibody and secondary antibody incubation,ECL luminescence exposure,to confirm the expression of CD 109 in penis squamous cell carcinoma.(4)ImmunofluorescenceThe fresh penis squamous cell carcinoma tissues were collected,frozen and embedded in sections.The sections were stained with CD 109 antigen,labeled with fluorescent secondary antibodies,and photographed with a microscope.The expression and distribution of CD 109 in each tissues were visually observed.(5)Statistical analysisSame as the first partResults:(1)CD 109 was highly expressed in epithelial cells not in vascular endothelial cells of normal thymic tissue.(2)Immunohistochemical analysis revealed that CD 109 expressed weakly in normal skin and was highly expressed in cancer cells of skin SCC,esophagus SCC,gallbladder SCC and penis SCC,and negatively correlated with tumor grade.There was no expression in the vascular endothelial cells of SCCs.Conclusion:CD 109 is highly specifically expressed in cancer cells of squamous cell carcinoma and is negatively correlated with tumor grade.