| Background Atherosclerosis(AS)is a chronic inflammatory and autoimmune disease that can cause severe cardiovascular and cerebrovascular events such as acute myocardial infarction(AMI)and acute cerebral infarction.The morbidity and mortality of AS are increasing worldwide.Vascular endothelial cells(VECs)injury caused by inflammation,oxidative stress and increased lipid load initiates and runs through the whole course of AS.AS the most effective antigen presenting cells in the immune system.,Dendritic cells(DCs)exists in the arterial wall in immature form under physiological conditions and increases significantly in atherosclerotic plaques.DCs play an important role in AS by promoting macrophage activation,inducing T cell activation and proliferation.The maturity of DCs is a key step in the role of DCs in the AS process.Therefore,intervention of AS process by inhibiting the maturation of DCs has become a hot research topic.At present,it is believed that there is some signal transduction from endothelial cell injury to DCs migration and maturation.Exosomes(Exos)are small vesicles delivered by many cells of the organism and have recently been recognized as important mediators of intercellular communication by transmitting and exchanging donor cell-specific proteins,m RNA,Non-coding RNA(nc RNA)including long non-coding RNA(lnc RNA),and so on.Furthermore,exosomes have been proved to be deeply involved in development and complications of AS.Recent studies have shown that overexpression of one prominent lnc RNA,known as metastasis-associated lung adenocarcinoma transcript 1(MALAT1)induces tolerogenic DCs and immune tolerance in heart transplantation and autoimmune disease.MALAT1 can activate nuclear factor erythroid 2-related factor(Nrf2)signaling pathway in vascular endothelial cells,and Nrf2 is also involved in regulating DCs activation,maturation and immune tolerance.However,whether MALAT1 can participate in the important process from vascular endothelial injury to DCs maturation through exosomes in AS,and what role Nrf2 plays in it is still unclear.To explore the mechanism of DCs maturation in AS will be conducive to provide experimental basis and theoretical support for disease intervention.Objectives In the present study,we investigated the changes of exosomes MALAT1 in endothelial cells treated with oxidized low-density lipoprotein(ox-LDL),and subsequent regulation of DCs maturation through MALAT1,and explored the possible mechanism of Nrf2 signaling pathway in it.Methods 1.To observe MALAT1 expression in exosomes from AS-sera and ox-LDL-HUVECs Exosomes were isolated from sera from normal individuals(Normal group,n=20)and AS patients(AS group,n=25),also from HUVECs treated with phosphate buffered saline(PBS)(PBS group)or ox-LDL(ox-LDL group).The protein expression of exosomal surface markers in isolated exosomes in each group were examined by western blot.Exosomal MALAT1 expression in 4 groups was detected by quantitative real time polymerase chain reaction(q RT-PCR)analysis.2.To investigate whether exogenous overexpression of MALAT1 from ox-LDLHUVECs-Exos can inhibit the maturation of DCs We transfected MALAT1 overexpression vector Lv-MALAT1 into HUVECs.MALAT1 expression was upregulated in exosomes derived from Lv-MALAT1-transfected HUVECs.The HUVECs-Exos were labeled with the lipophilic fluorescent dye Di O(green)and co-incubated with DCs transfected with m Cherry plasmid(red).The intake of exosomes by immature DCs(i DCs)was analyzed under a laser confocal microscope.The i DCs were co-cultured with PBS(control of exosomes),exosomes derived from control HUVECs(Exos),exosomes derived from ox-LDL-treated HUVECs(ox-LDLExos),exosomes derived from ox-LDL-treated HUVECs that have been transfected with Lv-Ctrl(ox-LDL-ExosLv-ctrl),exosomes derived from ox-LDL-treated HUVECs that have been transfected with Lv-MALAT1(ox-LDL-ExosLv-MALAT1).Relative MALAT1 expression in DCs was detected by q RT-PCR analysis.The i DCs were co-cultured with lipopolysaccharide(LPS)(2 ?g/m L),PBS,or the exosomes mentioned,and endocytosis of DCs was measured by flow cytometry(FCM)analysis as the cellular uptake of fluorescein thiocarbamoyl dextrans(FITC-dextran).The expression of DCs cell surface markers CD80,CD86,and human leukocyte antigen DR(HLA-DR)were measured by FCM analysis.3.To observe whether Nrf2 signaling pathway activation exists in the above process The i DCs were co-cultured with LPS(2 ?g/m L),PBS or the indicated Exos(HUVECsExos,ox-LDL-HUVECs-Exos,ox-LDL-HUVECs-ExosLv-ctrl,ox-LDL-HUVECsExosLv-MALAT1).The protein levels of Nrf2,heme oxygenase1(HO-1),and NAD(P)H: quinine oxidoreductase 1(NQO1)in total cell lysates,nuclear Nrf2 in the nuclear fraction lysates were evaluated by Western blot.Reactive oxygen species(ROS)content was detected by the ROS kit.4.To verify that MALAT1 affects DCs maturation through Nrf2 signaling pathway The interaction between MALAT1 and Nrf2 protein was evaluated by RNA pull down assay.The interaction between MALAT1 and Nrf2 protein was further validated by RNA Immunoprecipitation(RIP).Effect of MALAT1 overexpression and effect of MALAT1 knockdown on the protein expression of Nrf2,HO-1,and NQO1 in total cell lysates as well as nuclear Nrf2 in the nuclear fraction lysates was evaluated by Western blot.5.To observe the effect of MALAT1 in exogenous exosome on atherosclerosis in mice Mice were randomly divided into five groups(n = 10/each group): Control,AS,AS+PBS,AS+VECs-Exos,and AS+ox-LDL-VECs-Exos.The Apo E-/-mice were fed with a highfat diet containing 21% fat and 0.15% cholesterol for 12 weeks to establish a mouse model of AS.The mice in the control group received an ordinary diet instead.At the beginning of the twelfth week,mice in the AS+PBS,AS+VECs-Exos,and AS+ox-LDL-VECs-Exos group received an intravenous injection of either PBS(control),exosomes from mouse VECs(VECs-Exo;1.2 ?g/g),or exosomes from ox-LDL-treated mouse VECs(ox-LDLVECs-Exos;1.2 ?g/g),respectively,twice for a week.At the end of the twelfth week,when Exos had been injected for one week,these animals were sacrificed and their serum samples were prepared for detection of Malondialdehyde(MDA),ROS,interleukin-10(IL-10),interleukin-12(IL-12),interleukin-6(IL-6),and transforming growth factor-?(TGF-?).The aortas were cut into sections for histological examination.Results 1.MALAT1 expression is decreased in exosomes from AS-sera and ox-LDL-HUVECs.2.LPS treatment significantly decreased cell endocytosis activity and increased expression of DCs markers CD80,CD86,and HLA-DR.HUVECs-Exos attenuated the LPS-induced DCs maturation.Furthermore,Exogenous overexpression of MALAT1 from ox-LDL-HUVECs-Exos inhibits DCs maturation.3.LPS downregulated protein levels of Nrf2 as well as Nrf2 signaling downstream genes and decreased Nrf2 nuclear translocation.HUVECs-Exos treatment attenuated the LPS-mediated inhibition of Nrf2 signaling.Ox-LDL-HUVECs-Exos showed weaker attenuation when compared with HUVECs-Exos group.Compared with oxLDL-HUVECs-ExosLv-ctrl,ox-LDL-HUVECs-ExosLv-MALAT1 significantly upregulated protein expression of Nrf2,HO-1,and NQO1,and increased Nrf2 nuclear translocation.In addition,compared with ox-LDL-HUVECs-ExosLv-ctrl group,ox-LDL-HUVECs-ExosLv-MALAT1 significantly decreased ROS content.4.The interaction between MALAT1 and Nrf2 protein was evaluated by RNA pull down assay,and was further validated by RIP assay.Data revealed that MALAT1 overexpression significantly upregulated protein levels of Nrf2,HO-1,and NQO1 and increased Nrf2 nuclear translocation.In contrast,MALAT1 knockdown exerted the opposite effects.5.Data from animal experiments showed that mouse VECs-Exos treatment alleviated AS progression,as evidenced by less atheromatous plaques and inflammatory cells infiltration decreased serum levels of oxidative stress indexes and pro-inflammatory cytokines(IL-12 and IL-6),as well as increased anti-inflammatory cytokines(IL-10 and TGF-?).Compared with the AS+VECs-Exos group,the mice in the AS+ox-LDLVECs-Exos group showed more atheromatous plaques and inflammatory cells infiltration,as well as increased serum levels of oxidative stress indexes and proinflammatory cytokines,as well as increased anti-inflammatory cytokines.MALAT1 expression was significantly decreased in the AS group compared with the control group.Furthermore,serum MALAT1 expression was significantly higher in the AS+VECs-Exos group than that in the AS+PBS group.Moreover,serum MALAT1 expression was decreased in the AS+ox-LDL-VECs-Exos group when compared with the AS+VECs-Exos group.Conclusions 1.In vivo and in vitro studies showed that the expression of MALAT1 in HUVECsExos was decreased after oxidative stress injury.2.Overexpression of exogenous MALAT1 in HUVECs-Exos treated with ox-LDL can inhibit the maturation of dendritic cells.3.ox-LDL-HUVECs-Exos exogenous MALAT1 overexpression activates Nrf2 signaling pathway and regulates the expression of pro-inflammatory factors to inhibit ROS accumulation,thereby inhibiting the maturation of dendritic cells.4.Exos-MALAT1 interacts with Nrf2 and activates Nrf2 signaling pathway in DCs,ultimately interfering with atherosclerosis pathological process. |
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