| BackgroundHypertension,which plays a vital role in the development of cardiovascular diseases,has been identified as the leading risk factor for death.In China,systolic blood pressure>140 mmHg and/or diastolic blood pressure?90 mmHg are defined as hypertension.At present,the rates of awareness and control for hypertensive patients are relatively low in China.It is urgent to improve the diagnosis and treatment of hypertension.The pathogenesis of hypertension is complex,including hypersympathetic nerve activity,renin-angiotensin system activation,renal sodium retention,vascular remodeling,impaired endothelial cell function,insulin resistance,etc.Various complicated neurohumoral factors control blood pressure by affecting blood volume,cardiac output,and peripheral resistance ultimately.Among them,vascular resistance depends on the balance between contraction and relaxation of vascular smooth muscle cells(VSMCs).VSMCs could directly drive the contraction of the vascular wall by controlling the diameter of vessels in response to biomechanical stress,vasoactive physiological and pathophysiologic stimuli.Abnormalities of the vascular smooth muscle contractile state are sufficient to cause disorders of blood pressure.The increase in intracellular Ca2+is a key step in increasing the contractile activity of VSMCs.Depolarization of the VSMC membrane activates voltage-gated Ca2+channels,resulting in Ca2+influx;Vasocontraction agonists bind to G-protein-coupled receptors(GPCRs),thereby catalyzing the conversion of phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-trisphosphate(IP3).The latter binds to the IP3 receptor on the sarcoplasmic reticulum,leading to Ca2+channels open and Ca2+ release.The increase in cytosolic Ca2+binds to calmodulin and activates myosin light chain kinase(MLCK),thereby resulting in phosphorylation of MLCK and subsequent smooth muscle contraction.MLCK deficiency leads to impaired smooth muscle contraction and reduced blood pressure.GPCRs is abundant in the cardiovascular system and plays a crucial role in physiological processes such as vasoconstriction.The GPCRs signaling can be inhibited by RGS(regulator of G-protein signaling)protein(s)binding activated(GTP bound)G? subunits and accelerating GTP hydrolysis.RGS proteins is known to be involved in the physiological and pathological processes of the nervous system,cardiovascular system as well as the occurrence and development of tumors.Many of the GPCRs which responsible for vasoconstriction in the cardiovascular system bind to Gaq subunits,a member of the G protein family Gq/11.There are no less than 30 RGS proteins in mammals,and more than half of them exhibit GTPase-activating protein(GAP)activity against Gaq subunits and may affect vascular tone.Three members of the RGS protein family,RGS2,RGS4 and RGS5,are highly expressed in hearts and blood vessels.The genes encoding these proteins are clustered on a locus of both human and mouse chromosome 1,which is associated with variations in blood pressure.RGS2 deficiency increased activation of G protein induced by angiotensin receptors and led to hypertension.RGS4 and RGS5 have been implicated as key inhibitory proteins of cardiac hypertrophy and fibrosis.RGS5 was recently identified to prevent smooth muscle cell proliferation and attenuate neointima formation.RGS proteins are important regulators of the GPCRs signaling pathway,the study on the upstream regulatory mechanism of RGS proteins is very important.However,the mechanisms involved in regulating RGS expression have not been well studied.Gene expression is regulated at both transcriptional and posttranscriptional levels.The posttranscriptional mechanisms include the export,edition,turnover,storage and translation of precursor mRNA.HuR(human antigen R)is a member of the embryonic lethal visual abnormality family.It is widely expressed in eukaryotic cells and involved in many physiological and pathological processes.HuR increased mRNA stability by binding to adenine(A)and uracil(U)rich elements(AREs)in target mRNAs with high affinity and selectivity.HuR has a large number of target RNAs that can participate in regulating the cell survival,apoptosis and other physiological processes by stabilizing the mRNA of various growth factors and apoptosis factors.In addition,HuR is involved in the pathological process of many diseases.One of the most widely studied is the relationship between HuR and tumors.HuR is highly expressed in the oral cavity,stomach,colorectum,lung,breast,and skin cancer and closely related to tumor development and prognosis.HuR is also involved in chronic inflammation and pathophysiological processes of the cardiovascular,nervous and muscular systems.HuR is reported to be associated with myocardial infarction and myocardial fibrosis.However,the mechanism of HuR in cardiovascular disease remains unclear.To investigate the potential role of HuR in VSMCs,we constructed smooth muscle-specific HuR knockout(HuRSMKO)mice.We found that HuRSMKO mice displayed elevated blood pressure and increased vasoconstrictor response to vasoconstrictor substances in adulthood.RGS proteins play an important role in GPCRs-mediated vasoconstriction and we found that RGS mRNAs have AREs in 3'UTR that bind to HuR.Therefore,the following hypothesis was proposed in this study:Smooth muscle specific knockout of HuR increases GPCRs-mediated contraction of mesenteric artery smooth muscle and elevated blood pressure through down-regulation of RGS proteins.In this study,?-SMA-Cre/loxP system was applied to construct HuRSMKO mice.We explore the role and mechanism of HuR in hypertension and vascular smooth muscle contraction to provide new ideas and methods for clinical diagnosis and treatment of hypertension.Objectives1.To explore the role of smooth muscle HuR in hypertension2.To explore the role and mechanism of smooth muscle HuR in vasoconstriction.Methods1.Experimental subjects:1.1.Animal model construction:?-SMA-Cre/loxP system was used to construct smooth muscle-specific HuR knockout mice.1.2.SHRs(spontaneously hypertensive rats)and Wistar rats:Ten-week-old male SHRs and Wistar rats,weighs about 270 to 300 g,were purchased from Beijing Vital River Laboratory Animal Technology,Co.2.Cell model:2.1.Mouse smooth muscle cell line(Movas):Movas were bought from American Type Culture Collection.2.2.Mouse primary aortic smooth muscle cell:Mouse primary SMCs were extracted from aortas of 4-6 weeks old mice by substrate-attached explant methods.3.Clinical specimen collection:Blood vessel specimens were collected from blood vessel tissues of hypertensive and non-hypertensive patients requiring surgical resection of partial blood vessels.The use of human tissues was approved by the Medical Institutional Ethics Committee of Qilu Hospital,Shandong University,China.4.Identification of knockout efficiency and tissue specificity of HuRSMKO mice:Tissue genomic DNA extraction kit was used to extract DNA of mouse tail.HuR and Cre sequences were amplified by PCR,and 2%agarose gel electrophoresis was used to identify mouse genotypes.The expression of HuR in aorta and mesenteric arteries of mice was detected by western blot,immunofluorescence and immunohistochemical staining.Western blot was performed on brain,heart,liver,skeletal muscle and adipose tissues to detect the HuR expression.5.Investigate the phenotypic changes of HuRSMKO mice:Monitor the body weight and heart rate of mice;Tail-cuff manometer was used to measure systolic blood pressure,diastolic blood pressure and mean arterial blood pressure of control(CTR)and HuRSMKO mice of different age and sex.6.Explore the effect of smooth muscle HuR knockout on cardiac hypertrophy:Ejection fraction(%EF),percentage fractional shortening(%FS),interventricular septal thickness at diastole(IVS;d),interventricular septal thickness at systole(IVS;s),diastolic left ventricular volume(LV Vol;d),systolic left ventricular volume(LV Vol;s),ratio of E and A velocity in mitral valve(MV E/A)and left ventricular mass(LV mass)of CTR and HuRSMKO were measured by ultrasound.The ratio of heart weight to tibia length was measure,and the ratio of heart weight to body weight was also measured.The heart size of CTR and HuRSMKO mice was compared by H&E staining in paraffin section of heart tissue.H&E staining and immunohistochemical staining for ?-SMA in paraffin sections of mouse vascular tissue were used to observe and compare the thickness of vascular media.7.Explore the expression of HuR in clinical samples and SHRs:Immunohistochemical staining of paraffin sections of aorta in non-hypertensive and hypertensive patients was used to detect HuR.Blood pressure of rats were measured;Western blot analysis of HuR expression in the aorta of Wistar rats and SHRs.8.Investigate the effect of exogenous HuR on blood pressure in HuRSMKO mice and SHRs:CTR and HuRSMKO mice were injected with adenovirus expressing GFP or HuR,and blood pressure was monitored continuously for one week.Immunohistochemical staining and western blot were used to detect the expression of HuR in aorta and mesenteric arteries of CTR and HuRSMKO mice one week after injection of adenovirus.Wistar rats and SHRs were injected with adenovirus expressing GFP or HuR,and blood pressure was monitored continuously for one week.HuR expression was detected in Aorta and mesenteric arteries of Wistar and SHRs by immunohistochemical staining and western blot.9.Explore the effect of HuR on peripheral vasoconstriction:The secondry mesenteric artery segments of CTR and HuRSMKO mice were seperated and stimulated with norepinephrine(NE),endothelin(ET-1),U46619 and angiotensin ?(Ang ?)to detect the response of mesenteric artery to vasoconstrictor drugs.10.Investigate the regulatory effect of HuR on RGS:RNA immunoprecipitation was used to determine whether HuR could directly bind to RGS mRNA;Half-life experiment was used to observe whether HuR overexpression affected the stability of RGS mRNA.RNA and protein were extracted from the aorta of CTR and HuRSMKO mice,and the expressions of RGS2,RGS4 and RGS5 were detected by real-time quantitative PCR(RT-PCR)and western blot.The expressions of RGS2,RGS4 and RGS5 in mouse aortic smooth muscle were detected by immunohistochemistry.11.Explore the mechanism of HuR regulating change of intracellular calcium ion:Phenylproterenol(PE)was used to stimulate primary smooth muscle cells of CTR and HuRSMKO mice,and Ca2+indicator Fluo-4 AM was used to detect the change of intracellular Ca2+.RGS2,RGS4 or RGS5 were overexpressed in primary smooth muscle cells of CTR and HuRSMKO mice.And changes of Ca2+were also detected after Fluo-4 AM load and PE stimulation.12.Investigate the effects of exogenous RGS2 and RGS5 on blood pressure in HuRSMKO mice:CTR and HuRSMKO mice were injected with adenovirus-expressing RGS2 or RGS5,and blood pressure was monitored continuously for a week.Immunohistochemical staining and western blot were used to detect RGS2 or RGS5 levels in aorta and mesenteric arteries of CTR and HuRSMKO mice after the injection of adenovirus.13.Study the influence of HuR overexpression on protein levels of RGS2,RGS4 and RGS5Adeno-virus expressing GFP or HuR was injected into the tail vein of CTR and HuRSMKO mice,and RGS2,RGS4 and RGS5 protein levels in the mesenteric arteries of the mice were detected.14.Statistical analysisAll data were expressed as the mean ± SEM.Data were analyzed by using GraphPad Prism 6.0 and SPSS 23.0(IBM software).All data have passed normality and equal variance test.Student t test was used to compare 2 groups.Repeated measures ANOVA was applied for the statistical analysis of blood pressure.P<0.05 was considered statistically significant.Results1.Generation of HuRSMKO miceHuRflox/flox mice were crossbred with ?-SMA-Cre transgenic mice to obtain HuRflox/flox/Cre+mice(referred to as HuRSMKO).Littermate HuRflox/flox/Cre-mice were used as controls(referred to as CTR).Immunofluorescence was used to detect HuR and?-SMA expression in mesenteric arteries isolated from mice.HuR was expressed in the smooth muscle of mesenteric arteries from CTR mice but not HuRSMKO mice.Western blot and immunohistochemical staining results confirmed that HuR protein expression n the mesenteric artery and aorta of HuRSMKO mice was significantly reduced.However,the expression of HuR was not changed in the brain,liver,heart,skeletal muscle and fat from HuRSMKO mice.The above results show that the knockout effect of smooth muscle HuR in HuRSMKO mice is significant,and it has obvious smooth muscle specificity.2.HuRSMKO mice display elevated blood pressure and cardiac hypertrophyThere were no significant differences in body weight and heart rate between CTR and HuRSMKO mice at 2 and 4 months of age.We also found no significant difference in blood pressure between 2-month-old CTR and HuRSMKO mice.However,from 3 months,the systolic arterial pressure was higher for HuRSMKO than CTR mice.Echocardiography revealed increased left ventricular mass and interventricular septal thickness in HuRSMKO mice.The ratio of heart weight to mouse tibial length and the ratio of heart weight to body weight were greater for HuRSMKO than CTR mice.H&E staining showed hearts were enlarged for HuRSMKO than CTR mice in 3-month-old.However,there were no significant differences for the cardiac morphology and function between 2-month-old CTR and HuRSMKO mice.HuR knockout also did not affect vascular medial thickness.Overall,these results suggest that HuR deficiency in SMCs resulted in elevated blood pressure and cardiac hypertrophy.3.HuR overexpression lowers high blood pressure in HuRSMKO mice and SHRsHuR expression were decreased in aortic tissues from hypertensive patients as compared with normotensive controls.Also,HuR levels were lower in SHRs with stable hereditary hypertension than Wistar rats without hypertension.After overexpression of HuR,blood pressures of HuRSMKO mice and SHRs were decreased.4.Deletion of HuR enhanced contractile responses of vascular smooth muscle Upon stimulation with NE,Ang II,U46619,or ET-1,mesenteric arteries from HuRSMKO mice showed greater maximal force.We further examined the dose-response effect of NE on force development.At 10,30.and 100 ?M NE,the corresponding force was significantly greater for HuR-deficient than control arteries.However,there was no significant difference in the contractile responsiveness to 124mM KC1 between CTR and HuRSMKO mice.These results confirm that the loss of HuR enhances the response of mesenteric arteries to vasoconstrictor,and the regulation of HuR on vasoconstrictor may be through the GPCRs pathway rather than membrane potential depolarization.5.RGS2,RGS4 and RGS5 are the HuR target genesRNA immunoprecipitation verified that HuR binds to RGS2,RGS4.and RGS5 mRNAs.RNA stability assay showed that overexpression of HuR increased the half-lives of RGS2,RGS4 and RGS5 mRNAs compared with control GFP.Consistently,the mRNA levels of RGS2.RGS4,and RGS5 were significantly reduced with HuR deletion.Western blot and immunohistochemical staining showed that the protein levels of RGS2,RGS4 and RGS,in the aorta of HuRSMKO mice were significantly decreased compared with those of CTR mice.In summary,HuR can bind to RGS2,RGS4 and RGS5 mRNAs and increase their stability,thereby increasing the expression of RGS2,RGS4 and RGS5 proteins.The expressions of RGS4 and RGS5 were decreased in aortic smooth muscle of patients with hypertension,but the expression of RGS2 was increased.The levels of RGS2 and RGS4 in SHRS aortic smooth muscle were lower,while the expression of RGS5 was increased.These results indicate that the pathogenesis of hypertension is very complex,and RGS protein expression is regulated by other proteins besides HuR.6.HuR regulates GPCRs-mediated intracellular calcium increase through RGSPrimary VSMCs from CTR and HuRSMKO mice were treated with 1 mM PE,and cytosolic Ca2+content was measured by green fluorescent calcium indicator Fluo-4 AM.The addition of PE evoked a transient increase in cytosolic calcium from both control and HuR-deficient VSMCs.However,the fluorescent intensity was stronger for HuR-deficient than control cells.The increase in Ca2+after PE stimulation was significantly reduced in HuR-deficient VSMCs with overexpression of RGS2,RGS4,and RGS5,which suggests that HuR regulates GPCR-mediated Ca2+release through RGS.7.Administration of RGS2 or RGS5 lowers high blood pressure in HuRSMKO miceOverexpression of RGS2 or RGS5 significantly reduced systolic blood pressure of HuRSMKO mice.Immunohistochemical staining and western blot analysis showed that the RGS2 or RGS5 protein levels in endothelial-denuded aorta and mesenteric arteries of HuRSMKO mice could be corrected back to the wild-type levels by the corresponding adenovirus.The administration of HuR increased protein levels of RGS2,RGS4 and RGS5 in mesenteric arteries of HuRSMKO mice.Conclusion1.Lack of HuR caused hypertension and cardiac hypertrophy.2.HuR knockout increased contraction of vascular smooth muscle.3.Mechanically,HuR could directly bind to RGS2,RGS4 and RGS5 mRNAs,and improve their stability,thereby promoting the expression of RGS2,RGS4 and RGS5 proteins,and negatively regulating GPCRs-mediated intracellular calcium increase.4.Exogenous HuR,RGS2 and RGS5 could lower high blood pressure in HuRSMKO mice.Background Atherosclerosis is the most common pathology of coronary artery,peripheral artery and cerebrovascular diseases.Atherosclerosis is initiated by endothelial dysfunction and vascular inflammation caused by cardiovascular risk factors such as hyperlipidemia and hypertension.Lipoproteins in the blood enter the arterial wall from the damaged endothelial cells Inflammatory factors can stimulate macrophages and vascular smooth muscle cells(VSMCs)to engulf oxidized low-density lipoprotein(oxLDL)and form foam cells.Disordered lipid metaboIism? inflammation and endothelial injury all seem to play major roles in atherosclerosis.VSMCs are involved in the process of atherosclerosis as the main cells of vascular wall.Since VSMCs are highly differentiated and static physiologically,compared to skeletal muscle and cardiomyocytes,they retain dedifferentiation potential and plasticity.In pathological condition,aberrant proliferation and migration followed by phenotypic switching of VSMCs are involved in the formation of foam cells.VSMCs produce the extracellular matrix,which forms the fibrous cap to prevent plaque rupture in the early stages of atherosclerosis.The death and senescence of VSMCs not only participate in the formation of atherosclerotic plaques,but also promote the instability of late plaque.However,the specific regulatory mechanism of VSMCs in atherosclerosis is not clear.Autophagy is a highly conserved energy and material cycling process in eukaryotes.It plays an important role in cell survival and organelle quality control by degrading organelles,proteins and macromolecules and recovering the decomposition products.Autophagy is a multi-step process that requires a variety of autophagy-related proteins that take part in nucleation,expansion and finally fusion with lysosomes of autophagosomes.Currently,more than 30 different autophagy-related genes(ATGs)have been identified in yeast genes,most of which are highly conserved in slime molds,plants,mites,flies and mammals.Many studies have demonstrated the important role of autophagy in cardiovascular diseases.For example,autophagy has a protective effect on mild ischemia of myocardial cells,and autophagy is also involved in the process of cardiac hypertrophy and heart failure.Accumulating evidence suggested that autophagy is involved in the occurrence and development of atherosclerosis and related diseases.ATG5 in macrophages has an anti-atherosclerosis effect.Smooth muscle specific deletion of ATG7 can promote atherosclerosis.And the loss of ATG5 and ATG7 in endothelial cells can also lead to the formation of atherosclerotic plaques.However,the role of autophagy in atherosclerosis and its regulatory mechanism are complex and need to be further studied.Adenosine (?)-monophosphate(AMP)-activated protein kinase(AMPK)is a key protein that regulates cell metabolism and energy homeostasis.AMPK is a heterotrimer composed of a catalytic subunit and two regulatory subunits.Each AMPK subunit has multiple subtypes,and different subtypes affect the localization and function of AMPK.AMPK is involved in the regulation of autophagy in yeast and mammalian cells.AMPK directly phosphorylates and activates the ATG1 homologue ULK1 to induce autophagy.Besides,AMPK can also indirectly activate autophagy by inhibiting the activity of mammalian Target of Rapamycin(mTOR).AMPK also phosphorylates core autophagy-related proteins such as ATG9,Beclinl,VPS34 and RACK1 to regulate autophagy.Therefore,AMPK is critical in autophagy and autophagy-related diseases.At present^ the regulation mechanism of AMPK subunit expression is not clear,and further study is needed.The regulation of gene expression is known to occur at the transcriptional and post-transcriptional levels.Human antigen R(HuR)? as an RNA-binding protein,can bind to target mRNAs to exert post-transcriptional regulation.Our study found that the expression of HuR was decreased in aherosclerotic plaques.We used smooth muscle specific HuR knockout mice(HuRSMKO)to study the role and mechanism of smooth muscle HuR in atherosclerosis.As compared with control mice,HuRSMK0 showed increased plaque burden in the atherosclerotic model.Because AMPK is involved in multiple autophagy processes,and we found that AMPKal and AMPKa2 mRNAs have AREs on the 3-UTR.Therefore,this study proposed the following hypothesis: mouse smooth muscle specific knockout of HuR induces autophagy deficiency and promotes atherosclerosis by down-regulating AMPKal and AMPKa2 proteins.Our study provides new idea for the diagnosis and treatment of atherosclerosis.Objectives1.To investigate the role and mechanism of smooth muscle HuR in atherosclerosis.2.To study the regulatory mechanism of smooth muscle HuR in autophagy.Methods1.Experimental subjects:1.1.Construction of smooth muscle-specific HuR knockout atherosclerosis model:HuRsmko mice were generated as described above.Male CTR and HuRSMKO mice at 8 weeks old were given a single tail-vein injection with rAAV/D377Y-mPCSK9 at1.5*10U vg for each mouse and fed a paigen diet for 12 weeks.1.2.Apolipoprotein E-deficient mice(ApoE(?):Male ApoE(?) mice at 8 weeks old were from Vital River(Beijing)and were divided into two groups for normal diet(ND)or high fat diet(HFD)feeding.2.Cell model:2.1.Mouse smooth muscle cell line(Movas): Movas were bought from American Type Culture Collection.2.2.Mouse primary aortic smooth muscle cell: Mouse primary SMCs were extracted from aortas of 4-6 weeks old mice by substrate-attached explant methods.3.Study the expression of HuR in atherosclerotic plaques:ApoE(?) mice were fed with ND or HFD for 12 weeks.HuR protein expression in atherosclerotic plaques were determined by western blot,RT-PCR and immunohistochemical staining.VSMCs were stimulated with oxidized low density lipoprotein(ox-LDL)and HuR expression was detected by western blot.4.Explore the influence of HuR on atherosclerosis:CTR and HuRSMK0 mice single injected with rAAV/D377Y-mPCSK9 and fed with a Paigen diet for 12 weeks.Serum was isolated for determining levels of total cholesterol(TC),triglycerides(TG)S high-density lipoprotein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C).Hearts and aortas were removed and fixed in 4% paraformaldehyde.Oil red O staining was used to observe the area of atherosclerotic plaques in aorta and aortic root.The contents of macrophages and smooth muscle cells were detected by immunohistochemical staining.Masson staining was used to detect collagen content.Apoptosis was detected by immunohistochemical staining with cleaved caspase-3 and terminal UTP nick end-labeling(TUNEL).Proteins extracted from aorta for MMP2 and cleaved-caspase detection by western blot.The plaque vulnerability index was calculated as follows:(macrophage staining % + lipid staining %)/(SMC staining % + collagen staining %).5.Investigate the influence of HuR on autophagy:The content of autophagosomes in VSMCs after overexpression of HuR or lacZ was observed by electron microscopy.After VSMCs overexpressed or knockdown of HuR,VSMCs were infected with ad-GFP-mRFP-LC3 II for 48 hr.LC3 II spots were observed by fluorescence microscopy.6.Explore the rulatory effect of HuR on AMPKal and AMPKa2:RNA immunoprecipitation was used to determine whether HuR could directly bind to AMPKal and AMPKa2 mRNAs.Half-life assay was conducted to determine whether overexpression or inhibition of HuR affected the mRNA stability of AMPKal and AMPKa2.The expressions of AMPKal and AMPKa2 were detected by western blot after VSMCs overexpression or inhibition of HuR.RT-PCR and western blot were used to detect the expression of AMPKal and AMPKa2 in the aorta of CTR and HuRSMK0 mice.The protein expressions of AMPKal and AMPKa2 in aorta were detected by immunohistochemistry.7.Explore the regulatory effect of HuR on autophagy-related proteins:The expressions of p-AMPKa? P62 and LC3 II were detected by western blot after VSMCs overexpression or inhibition of HuR.The aorta of CTR and HuRSMK0 mice were isolated,and the expressions of p-AMPKa,P62 and LC3 II were detected by western blot.The expressions of AMPKa and LC3 II were detected by ox-LDL stimulation of VSMCs after overexpression or inhibition of HuR.8.Investigate tbe effects of pharmacological AMPK activation on autophagy and atherosclerosis:After knocked down of HuR,VSMCs were stimulated with AMPK agonist A769662,and the expressions of p-AMPKa and LC3 II were detected by western blot.CTR and HuRSMKO mice were injected with rAAV/D377Y-mPCSK9,then a paigen diet fed and 30mg/kg A769662 injected intraperitoneally daily for 12 weeks,the changes of plaque area,blood lipid and number of apoptotic cells were observed.9.Statistical analysis:Data were expressed as mean ± SEM and were analyzed by using GraphPad Prism6.0.All data were tested for normal distribution and equal variances.Student t test was used to compare 2 groups.One-way ANOVA was used to 4 groups.Statistical significance was set at ? < 0.05.Results1.HuR levels were reduced in atherosclerotic plaque ApoE(?) mice were fed with a ND or HFD for 12 weeks.Western blot results showed that HuR protein level was decreased in aortas from the HFD group.As compared with the ND group,for the HFD group,HuR was down-regulated in atherosclerotic lesions by immunohistochemical staining.As well,HuR mRNA level was significantly reduced in aortas from the HFD group.HuR protein level was decreased in VSMCs induced by ox-LDL at 24,48,72 and 96 hr.HuRSMKO mice were used to further investigate the role and mechanism of HuR in atherosclerosis.Western blot and immunofluorescence results confirmed that the expression of HuR protein was decreased in aorta from HuRSMKO mice.2.HuR deletion in smooth muscle exacerbated atherosclerosis CTR and HuRSMK0 mice were injected with rAAV/D377Y-mPCSK9 then fed a paigen diet for 12 weeks.The proportion of atherosclerotic surface lesions was greater in HuRSMK0 than control mice.Furthermore,deletion of HuR increased macrophage accumulation and matrix metalloproteinase 2(MMP2)level,decreased collagen content.However,VSMC content and total monocyte/macrophages did not differ from controls.From the above results,we calculated the plaque vulnerability index,which was elevated after HuR deficiency.Taken together,lack of HuR in VSMCs promoted the development of atherosclerosis.3.Loss of HuR promoted apoptosis in atherosclerosisTo determine whether HuR knockout affected apoptosis,aortic root sections underwent TUNEL staining.Loss of HuR markedly increased the TUNEL-positive SMCs in HuRsmko versus control mice.Meanwhile,the expression of the apoptosisrelated protein cleaved caspase-3 was up-regulated in aortic roots of HuRSMKO mice.Besides,HuR deletion also increased the level of cleaved caspase-3 in SMCs.Also,the serum levels of TC,TG,LDL-C were higher in HuRSMKO than control mice.4.HuR deletion resulted in defective autophagy To detect whether HuR could regulate autophagy,we used transmission electron microscopy after VSMCs were infected with ad-HuR or ad-LacZ for 48 hr.HuR overexpression increased the number of autophagosomes.Also,autophagic flux was monitored in VSMCs by infection with ad-GFP-mRFP-LC3 II.As compared with controls,VSMCs with HuR overexpression by ad-HuR infection showed increased number of GFP+/RFP+ and GFP7RFP+ LC3 II puncta.However,VSMCs knocked down by HuR siRNA transfection showed decreased number of GFP+/RFP+ and GFP"/RFP+LC3II puncta as compared with controls.Thus,HuR overexpression induced autophagic flux,and loss of HuR suppressed autophagic flux and resulted in defective autophagy.5.AMPKal and AMPKa2 were the target genes of HuR The mRNA levels of AMPKal and AMPKa2 were decreased in aortas from HuRSMKOmice.RNA immunoprecipitation showed that HuR could bind to the mRNAs of AMPKal and AMPKa2.Meanwhile,the stability of AMPKal and AMPKa2 mRNAs was increased by HuR overexpression,but decreased by HuR deficiency.AMPKal and AMPKa2 protein levels in VSMCs were reduced after HuR knockdown by CMLD-2 treatment or HuR siRNA transfection.In contrast,the levels of AMPKal and AMPKa2 were increased in VSMCs with HuR overexpression by ad-HuR infection or HuR recombinant protein stimulation.The protein levels of AMPKal and AMPKa2 were significantly reduced in HuRSMKO versus control mice by western blot and immunohistochemical staining.Therefore,AMPKal and AMPKa2 are the target genes of HuR.6.HuR positively regulates autophagy HuR inhibition with CMLD-2 or HuR siRNA decreased levels of p-AMPKa and LC3 II and increased p62 level in VSMCs.In contrast,levels of p-AMPKa and LC3 II were elevated and that of p62 was decreased with HuR overexpression by ad-HuR infection or HuR recombinant-protein stimulation.Furthermore,as compared with control mice,HuRSMKO mice showed lower levels of p-AMPK and LC3 II and higher level of p62.The effect of HuR on AMPKal,AMPKa2 and LC3 II expression was further confirmed by oxLDL-stimulated SMCs after HuR knockdown or expression.Thus,HuR positively regulates autophagy.7.Pharmacological AMPK activation suppressed atherosclerosis in CTR and HuRSMKO miceVSMCs were transfected with CTR or HuR siRNA then treated with the AMPK activator A769662.The levels of p-AMPK and LC3 II were increased with A769662 in control and HuR-deficient VSMCs.In animal experiments,CTR and HuRSMK0 mice were given an intraperitoneal injection of A769662 daily and fed a paigen diet for 12 weeks after single intravenous injection of rAAV/D377Y-mPCSK9.The plaque area was significantly decreased after A769662 treatment in CTR and HuRsmk0 mice.Also,A769662 reduced the number of apoptotic cells.Furthermore,only TC and LDL-C blood levels were slightly reduced with A769662 treatment.Thus,smooth-muscle HuR protects against the development of atherosclerosis via AMPK-mediated autophagy.Conclusion1.HuR level was reduced in atherosclerotic plaque.2.Smooth muscle knockout of HuR exacerbated atherosclerosis.3.HuR deficiency in VSMCs caused autophagy deficiency.4.AMPKal and AMPKa2 are the target genes of HuR.5.Activation of AMPK inhibited atherosclerosis in CTR and HuRSMKO mice. |
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