Research On Mechanisms Of Polychlorinated Biphenyl Quinones Accelerating Atherogenesis Through Inducing CAV1 Phosphorylation And MiR-7-5p Expression

Polychlorinated biphenyls?PCBs?are a class of persistent organic pollutants,which were widely applied in industry due to the stable physicochemical properties.Although the production of PCBs had been banned in 1970s,they were difficult to be decomposed in the environment.PCBs can be accumulated in organisms and metabolized to polychlorinated biphenyl quinones?PCBQs?.PCBQs generate a lot of ROS,causing oxidative stress,leading to multiple toxic effects in organisms.Atherosclerosis?AS?is the primary cause of coronary heart disease and stroke.In recent years,numerous studies have found that PCBs have a strong link with the progression of cardiovascular diseases,and is one of the risk factors for inducing AS.In this article,the effects of PCB29-pQ,the typical metabolite of PCB quinones on AS and the molecular mechanisms.Part?:This section is aimed to investigate the effects of PCB29-pQ on the formation of atherosclerosis.ApoE^-/-mice were fed with high-fat diet to construct a model of atherosclerosis and intervened with PCB29-pQ.The mice were randomly assigned to the control or PCB29-pQ group and fed with a high-fat diet.The mice were injected PCB29-pQ or equal volumes of corn oil by intraperitoneal?ip?injection once a week for 10 weeks.The entire aortas were stained with Oil red O,and the result showed that PCB29-pQ remarkably augmented the plaque area in aortas.Hematoxylin and eosin?H&E?staining showed increased lipid core size in aortic wall of PCB29-pQ group.Fluorescent immunohistochemistry for CD68 indicated PCB29-pQ promoted macrophage infiltration located to aortic wall.In addition,more TUNEL-positive cells were found at aortic intima in PCB29-pQ group compared to the control.These results suggested PCB29-pQ accelerated atherogenesis.Part?:This section explored the molecular mechanisms of the involvement of Caveolin-1?CAV1?in the formation of atherosclerosis promoted by PCB29-pQ.Vascular endothelial cells play an important role in maintaining homeostasis of vessels,which are closely related to AS pathology.Thus in vitro experiments were performed using HUVECs.The expression of inflammatory cytokines and adhesion molecules including TNF-?,IL-1?,IL-6,VCAM-1 and ICAM-1 were upregulated in a manner that was time-dependent in HUVECs after being stimulated with PCB29-pQ.PCB29-pQ increased the phosphorylation level of p65 and the translocation of p65 into the nucleus.Meanwhile,the expression of p-CAV1 and its upstream p-SRC was up-regulated,and immunoprecipitation showed that the combination of eNOS and CAV1 was enhanced.The amount of monocyte THP-1 adhering to HUVECs significantly increased after PCB29-pQ exposure compared with the untreated cells.CAV1 phosphorylation was suppressed by PP2,leading to decreased upregulation of adhesion molecules and pro-inflammatory cytokines,suppressing activation of p65 and combination of eNOS and CAV1 induced by PCB29-pQ,which implied the important of CAV1phosphorylation.CAV1 siRNA reduced PCB29-pQ-induced upregulation of adhesion molecules and pro-inflammatory cytokines and p65 activation,while eNOS inhibitor L-NAME restored the expression of adhesion molecules and pro-inflammatory cytokines in HUVECs,indicating eNOS is a negative regulator in PCB29-pQ-induced vascular endothelial inflammation and its activity is suppressed through interaction with CAV1.Moreover,PCB29-pQ induced the binding of TLR4 to MyD88 and CAV1 and the dissociation of IRAK1 and CAV1,and both of them were inhibited by PP2,indicating that PCB29-pQ activated TLR4 signaling through CAV1 phosphorylation.CAV1 deficiency in ApoE^-/-mice alleviated plaque formation induced by PCB29-pQ,reduced the expression of p-p65 in artery and inflammatory cytokines in serum.Taken together,PCB29-pQ induced CAV1 phosphorylation to inhibit the activity of eNOS and activate TLR4 signaling pathway and p65,causing inflammation in endothelial cells,which further promoted atherogenesis.Part?:The first part proved that PCB29-pQ promoted the formation of atherosclerosis and the apoptosis of aortic intima.Endothelial injury and apoptosis are considered to be the critical events in the AS,and the role of microRNA in the development of AS has been paid much attention.This section investigated the damage of PCB29-pQ to endothelial cells and the critical microRNA involved.Tube formation assay indicated that PCB29-pQ damaged the angiogenic ability of endothelial cells.The apoptosis of HUVECs induced by PCB29-pQ was detected by flow cytometry and Mitochondrial membrane potential assay with JC-1.Differentially expressed miRNAs in HUVECs exposed with PCB29-pQ were identified by small RNA sequencing.miR-7-5p was found to be the most sensitive to PCB29-pQ,as it highly upregulated during PCB29-pQ exposure.Furthermore,miR-7-5p is closely related to the function of endothelial cells.Upregulation of miR-7-5p was found to mediate PCB29-pQ-induced endothelial cell injury and apoptosis by transfecting miR-7-5p mimic or inhibitor.Two target genes PPME1 and TGF-?2 were predicted using microRNA-related database and correlation analysis of microRNA-mRNA sequencing,further validated by qRT-PCR and Western blot.Dual-luciferase reporter assay confirmed that miR-7-5p directly interact with the 3'UTR of PPME1 and TGF-?2 to suppress their expression.miR-7-5p inhibitor weakened PCB29-pQ-induced apoptosis.However,co-transfection of siRNA with PPME1 and TGF-?2 abolished the effect of miR-7-5p inhibitor and exacerbated apoptosis.In vivo experiments showed that PCB29-pQ upregulated the expression of miR-7-5p in the aortic intima of ApoE^-/-mice detected by in-situ-hybridization and suppressed the expression of PPME1 and TGF-?2 detected by fluorescent immunohistochemistry,which was consistent with the in vitro experiment.In summary,PCB29-pQ induces endothelial cell apoptosis and further promotes the formation of atherosclerosis through upregulating miR-7-5p and suppressing the expression of PPME1 and TGF-?2.