Exposure To Concentrated Ambient Fine Particulate Matter PM2.5 Induces Vascular Endothelial Dysfunction Via MiR-21

Objective:Vascular endothelial permeability transition does not cause significant lesions,but enhanced permeability may result in vascular diseases,eg.coronary heart disease,atherosclerosis and even cancer.Some reports domastrated that vascular endothelial permeability change is the initial link of vascular disease,vascular elasticity decrease because of the increase of permeability,and the blood lipid deposit under the vascular endothelial cell,eventually lead to thickening and hardening of the blood vessel,inducing a series of vascular lesions.This study research endothelial permeability changes in vivo and in vitro while PM2.5 exposed,so as to study the mechanism of vascular endothelial permeability increase,expounding the mechanism came down to inflammatory factor,transcription factors,and microRNA.All these research could provid a novel approach that the mechanism of PM2.5 induce endothelial permeability increase is involved miR-21,giving a new idea for the future response to the vascular diseases which caused by PM2.5.Methods:The effect of does and time of PM2.5 on cell viability of HUVECs was detecter by Cell counting kit-8.FITC-dextran transwell assay was used to detect the permeability of HUVECs monolayers under PM2.5 expose or not.Microarray expression profile of miRNAs and Real-time PCR was used to detect the microRNA transcription level.TIMP3 3'UTR has the target sequence of mi R-21,p-STAT3 can directly binding in miR-21 promoter by bioinformatics software,and the dual luciferase reporter assay system were used to analyze luciferase activity,also the Chromatin immunoprecipitation in the test of promoter binding site.Using the way of giving PM2.5 tracheal instillation to build a model,and endothelial permeability assays were performed through extraction of Evans Blue.Western blot assay,Hematoxylineosin staining,Immunoblot analysis,ELISA were used to value the changes of inflammatory factor,transcription factor and proteins related to endothelial permeability in vivo or vitro.Endothelial permeability associated protein expression in HUVECs were assayed by western blotting post-transfection with miR-21 inhibitor.Result:Following,the viability of HUVECs reducingdependent on time and concentration exposure to PM2.5;HUVECs monolayer permeability increases under PM2.5 expose with time-dependently;miRNA array showed PM2.5 exposure can cause 84 microRNA transcriptional differences,including the most obvious difference microRNA miR-21;Bioinformatics prediction and the Luciferase assay confirmed the miR-21 targeted TIMP3 3 'UTR.Western blot and the miR-21 inhibitors transfect assay found TIMP3 expression regulation by miR-21,and MMP9,inhibited by TIMP3,higher expression and direct effect on vascular endothelial permeability;PM2.5 exposure can cause inflammation factor IL-6 increase and the increasing of the IL-6 leading a rise in p-STAT3;Bioinformatics prediction,the Luciferase assay and chromatin precipitation experiments confirmed that p-STAT3 can combined with miR-21 promoter,and cause its transcription increases;The inflammation,transcription factors,miR-21 and the protein involved in permeability in the body of animal models exposed to PM2.5,shown vascular endothelial permeability increases relate to IL-6/p-STAT3/miR-21/TIMP3/MMP9.Conclusion:We found that the effect occurred mainly through induction of STAT3 phosphorylation,further transcriptional regulation of miR-21 and promotion of miR-21 expression.These changes post-transcriptionally repress TIMP3 and promote MMP9 expression.This work provides evidence that PM2.5 exerts direct inhibitory action on vascular endothelial barrier function and might give rise to a number of vascular diseases.