Dynamic Changes Of Plasma VWF In AMI Patients And The Effect And Mechanism Of Cadmium On VWF Expression

BackgroundAcute myocardial infarction(AMI)has become one of the most frequent and critical types of cardiac vascular diseases.Globally,nearly 3 million people suffer from acute ST-segment elevated myocardial infarction(STEMI)and more than 4 million suffer from non-ST-segment elevated myocardial infarction(NSTEMI)each year.AMI is an ischemic hypoxic response to coronary lumen occlusion that includes myocardial cell death,endothelial cell injury and dysfunction,and abnormal tissue repair resulting from fibrosis.To date,however,no clinical marker has been available to accurately assess this process.Von Willebrand factor(vWF)is an adhesive secreted glycoprotein produced by vascular endothelial cells and blood megakaryocytes,among which endothelial cells are the primary source of circulating vWF.The mature endothelial vWFcan be released through spontaneous constitutive secretory pathways.However,the vast majority of vWF is first stored in the Weibel-Palade secretory corpuscles.Once the endothelial cells are damaged by the stimulation of the microenvironment,they are quickly released into the vascular lumen.Therefore,vWF is considered as a specific marker of endothelial injury or dysfunction.Current animal studies have shown that vascular endothelial cell injury during AMI can lead to elevated plasma levels of vWF,suggesting that vWF has potential auxiliary diagnostic value for AMI.However,the current research results of plasma changes of vWF in clinical AMI patients are still inconsistent,leading to controversial conclusions.ObjectiveBy collecting clinical study data,meta-analysis was performed to confirm the changes of plasma vWF in AMI patients,so as to evaluate the application value of vWF measurementin the clinical diagnosis of AMI,and to providea new strategy for disease progression monitoring and prognosis evaluation in patients with AMI.MethodsPlasma vWF levels in AMI patients and healthy subjects or patients with normal coronary angiography were collected for meta-analysis.Data were obtained from studies collected in PubMed,Web of Science,Cochrane Library Database and CNKI electronic literature retrieval service platform until August 2016.Qualified literatures were screened by reading titles,abstracts and full texts.Two authors evaluated the methodological quality ofeligibleexperiments,and extracted key data and general informationrespectively.Data were analyzed by RevMan 5.3 software.The standard mean difference(SMD)and 95%confidence intervals(95%CI)were calculated using random-effects model,and the results areshown in the forest map.Sensitivity analysis was used to evaluate whether the overall effect of meta-analysis was stable.Funnel plots were used to determine whether the included studies had significant publication bias.Results1.Meta-analysis results showed that:Plasma vWF levels rise rapidly within 6 hours after AMI(SMD=1.94,95%CI=1.39-2.48,P<0.001).It continued to rise for 24 hours(SMD=1.17,95%CI=0.45-1.89,P=0.001).SMDof day 2 was 1.28(95%CI=0.11-2.45,P=0.03),and SMD of day 3-4 was 0.81(95%CI=0.29-1.34,P=0.002).It was still significantly higher than the control group on day 7(SMD=1.02,95%CI=0.54-1.50,P<0.001).However,the differencein plasma vWF was no longer statistically significant at the 14th day(SMD=0.44,95%CI=-0.14-1.02,P=0.14).2.Subgroup-analysis results showed that:For patients admitted within 6 hours aftar AMI,plasma vWF levels were significantly higher at admission(SMD=1.19,95%CI=0.90-1.48,P<0.001)and on the first day after admission(SMD=1.21,95%CI=0.11-2.31,P=0.03)than control group,while no significant difference as found on the 3-4 days(SMD=0.93,95%CI=-0.10-1.97,P=0.08)and the 7 days after admission(SMD=0.94,95%CI=-0.22-2.10,P=0.11);For patient admitted later than 6 hours from AMI,plasma vWF kept high levels till one week after admission.In patients who underwent PCI,plasma vWF kept high levels for one week;In patients without PCI,the elevation of vWF was statistically significant only on the first day after admission.For patients with TIMI grade ? on day 7 after admission,the combined effect size SMD was 0.49(95%CI=0.12-0.85,P=0.009);For those without TIMI grade ?,the combined effect size SMD wasl.29(95%CI=0.73-1.84,P<0.001).Conclusion1.Plasma levels of vWF were significantly elevated in AMI patients,with high levels lasting for at least one week and then decreasing gradually to normal levels two weeks later,suggestingthat vWF may be a new indicator for the assessment of the onset and progression of AMI.2.The time between the onset of AMI and admission to the hospital,PCI operation and postoperative coronary artery reflow were the factors that affected plasma vWF levels.Therefore,vWF can be used as an indicator to reflect the degree of myocardial injury and the status of reflow.BackgroundEpidemiological evidence suggests that elevated plasma vWF levels are associated with many cardiovascular diseases.vWF participates in blood coagulation and thrombosis and is an independent risk factor for secondary myocardial infarction in AMI patients.In addition,vWF is involved in the regulation of inflammatory response and vascular smooth muscle cell proliferation,thus is an pathogenic factor of atherosclerotic disease.The plasma vWF level is mainly determined by the expression and secretion of vWF.The expression of vWF is affected by both genetic and environmental factors,and is strictly regulated at the level of gene transcription.There are several transcriptional regulation sites on human vWF gene promoter,which can be bound by specific transcription factors to regulate the expression of vWF under physiological and pathological conditions.Cadmium(Cd) is a harmful substance widely distributed in environment.The abundance of Cd in nature is not high.But with the rapid development of modem industry,Cd pollution and related health hazards are emerging in endlessly.At present,Cd is recognized as the seventh heavy metal pollutant that damages human life and health.EpidemioIogical studies have shown that elevated plasma Cd levels significantly increase the risk of atherosclerotic cardiovascular disease and death in the population.For general population,low dose Cd exposure through cigarette smoking and contaminated water and food are the primary pattern of Cd exposure.After ingestion through the respiratory and digestive systems,Cd is absorbed into the blood through microcirculationand is transported to tissues and organs all over the body.The biological half-life of Cd is aslong as 10-30 years,leading to itsaccumulation in the body,especially in the liver and kidney.More and more researchs show that peripheral blood vessels are also the target organs for Cd accumulation.Vascular endothelial cells are directly contact with Cd in blood and vessel wall,which is an important target of Cd action.Low dose Cddose not destroy the activity of endothelial cells,but can cause endothelial dysfunction.In this study,the mouse vacular endothelium and humanumbilical vein endothelial cells(HUVECs)were stimulated by low dose CdCl₂ to investigate the effect of CdCl₂ on endothelial vWF expression and ralated regulational mechanisms in vivo and in vitro.ObjectiveTo explore the effect of low dose Cd exposure on vWF expression in vascular endothelial cells and its molecular mechanism,and to confirm whether low dose Cd exposure is a risk environmental factor for elevated plasma vWF level.Methods(1)Treatment of animalsC57BL6/J wild-type mice aged 10-12 weeks were fed with sterile drinking water containing 50mg/L CdCl₂ for three consecutive days.The animals were anesthetized with chloral hydrate and subjected to cardiac perfijsion.Tissue samples were taken for examination.(2)Treatment of cellsFor concentration gradient experiment,CdCl₂ at the final concentrations of 0, 0.1,1,10 l?m were added into the culture medium of HUVECs;For time gradient experiment 0,1,2,6,12 and 24h were selected for detection,respectively.Finally,CdCl₂ at a final concentration of l?m and stimulation of 24 hwere selected for subsequent experiments.In the experiment to confirm the transcriptional regulation pathway,the small interfering RNA of transcription factors was transfected into cells24 h before CdCl₂ stimulation.(3)Western Blot(WB)The total protein of HUVCs was extracted following stimulation by CdCl₂,and the changes of vWF and related transcription factors at protein level were observed by SDS-PAGE gel electrophoresis,transmembrane,specific antibody incubation and ECL luminescent liquid exposure.(4)Quantitative Real-time PCR(qRT-qPCR)Total RNA of HUVCS was extracted following stimulation by CdCb,and the genomic DNA was removed before reverse transcription.The cDNA generated by reverse transcription was used as the template for RT-qPCR amplification,and the changes in gene expression of vWF and related transcription factors in cells were detected.(5)ImmunofluorescenceThe cells grown onthe slides were incubated in culture medium containing l?m CdCl₂ for 24 hours.After cell fixation and antibody incubation,the expression intensity of vWF and related transcription factors in each group was observed under fluorescence microscope.(6)Enzyme-linked immunosorbent assay(ELISA)The cell culture supernatants of control group(PBS)and experimental group(l?m CdCl₂)were collected,and the precipitation was removed by centrifugation.The secretion of vWF protein in the culture medium was detected by ELISA assay.(7)ImmunohistochemistryLung and kidney tissues of mice treated with CdCl₂ and control group were taken,and then the tissues were sliced and incubated with vWF antibody,HRP-conjugated secondary antibody and substrate incubation.The expression and distribution of vWF in the above tissues were observed.(8)Chromatin Immunoprecipitation(ChIP)The HUVECs stimulated by CdCl₂ were collected for protein-DNA cross-linking,and the chromatin was lysed by ultrasound under the optimal conditions to obtain DNA fragments.The target DNA fragment was precipitated and purified by adding ERG specific antibody and agarose beads.The fragment was amplified with vWF promoter-specific primers,and the binding ability of ERG and vWF promoter was detected.Results(1)After exposure to low dose CdCl₂,vWF gene expression was significantly increased in lung and kidney tissues of mice.Immunohistochemical staining showed that the expression of vWF protein was specifically increased in the vasculature.(2)ELISA results showed that vWF secretion was significantly increased after 24 h stimulation with luM CdCl₂.The results of qRT-qPCR showed that the mRNA level of vWF increased significantly at 6h and continued to increase to 24 h.WB results also showed a time-dependent increase in vWF protein levels.Immunofluorescence showed that the expression intensity of vWF in CdCl₂-stimulated cells was significantly higher than that in the control group.(3)WB and immunofluorescence results showed that 1 l?m CdCl₂ stimulation for24 h had no effect on the activity of NF-kB signaling pathway in vascular endothelial cells.(4)After CdCl₂ stimulation,the expression of transcription factor GATA3 had no significant change.Knockdown of GATA3 can not block low dose Cd-inducedvWF expression.(5)After CdCl₂ stimulation,the expression of transcription factor ERG gene and protein in HUVECs was significantly increased.(6)After knockdown of transcription factor ERG in HUVECs by cell transfection,the induction effect of low dose Cd on vWF disappeared,confirming that ERG plays a regulatory role in Cd-induced vWF expression.(7)ChIP assay showed that transcription factor ERG interacted with-56 ETS site on vWF gene promoter in HUVECs,and its binding ability was significantly enhanced by CdCl₂ treatment.Conclusion1.Exposure to low dose(luM)Cd can induce the expression and secretion of vWF in endothelial cells,which is a risk factor for the elevation of vWF in plasma.2.Low dose Cd regulates vWF gene transcriptional through transcription factor ERG rather than NF-kB or GATA3,which is a new mechanism of Cd's effect on endothelial cells,and may be involved in the occurrence and development of CD-related cardiovascular diseases.