| Objectives:In modern society,accumulating evidences documented that the chronic psychological stress is closely linked to the incidence of many diseases.Exposure to chronic psychosocial stress is a major risk factor for cardiovascular disease.Previous studies have demonstrated that peptidyl peptidase-?(DPP-IV)participate in many disease initiation and progression(including inflammatory diseases,metabolic syndrome and cardiovascular diseases)through glucagon-like peptide-1(GLP-1)-dependent and-independent pathways.Recently,we have observed that chronic stress can increase plasma DPP-IV levels and cause a series of pathophysiological changes.The purposes of this study were as follows:1)To investigate the effect of chronic stress on high-fat(HF)diet-induced atherogenesis in ApoE deficiency ApoE-/-mice and its mechanism:2)To evaluate the changes in plasma DPP-IV and GLP-1 levels in chronic stress-induced atherosclerosis initiation and progression in ApoE-/-mice fed with HF diet;3)To investigate DPP-IV inhibition-mediated anti-atherosclerosis effect and its mechanism.Methods:Part ? Animal experimentsExperiment 1:Four week-old male ApoE-/-mice(n=30)fed with a standard diet for two weeks.First,for the evaluation of the effects of chronic stress on atherosclerosis,ApoE-/-mice fed with a HF diet(Western-type diet containing 21.0%fat from lard and 0.15%cholesterol)were randomly assigned to either the HF-C group(the non-stress control group)or the HF-S group(the stress group/daily 4-h immobilization stress)for 12 weeks(n= 15 for each group).At the end day of the stress protocol,we collected blood,bone marrow,subcutaneous and visceral fat,and the aortas for the following experiments:1)HE staining,immunostaining,P-Galactosidase staining,and oil red 0 staining were applied to investigate chronic stress-mediated harmful effects on atherosclerotic plaque formation,vascular senescence,inflammatory cell infiltration,lipid accumulation,extracellular matrix(ECM)degradation and vascular aging;2)flow cytometry was used to evaluate of the effects of chronic stress on hematopoietic stem cells mobilization;3)The levels of plasma adiponectin(APN),GLP-1 and leptin were analyzed by ELISA;the levels of low-density lipoprotein(LDL),high-density lipoprotein(HDL),creatinine,nonesterified fatty acid(NEFA),and blood urine nitrogen(BUN)were also analyzed by SRL company(Tokyo,Japan);4)Plasma and tissue levels of DPP-? were detected by aminoluciferin substrate luminescence;and the tissue NADPH activity was detected by lucigenin-based enhanced chemiluminescence assay;5)Real-time PCR was applied to evaluate the levels of the targeted mRNAs including monocyte chemoattractant protein-1(MCP-1),C-X-C chemokine receptor type 4(CXCR 4),toll-like receptor-2(TLR-2),TLR-4,NADPH oxidase components(gp91phox,p47phox,and p67phox),matrix metalloproteinase-2(MMP-2),MMP-9,cathepsin K(CatK),and CatS;Western blotting assay was applied to evaluate the levels of the targeted proteins such as phospho-AMP activated kinase(p-AMPK),P21,P16INK4A,peroxisome proliferator activated receptor-a(PPAR-a),angiotensin ? ?type receptor(ATR1),and gp91phox;6)Gelatin zymography was used to evaluate MMP-2,and MMP-9 activities.Experiment 2:In separate DPP-4 inhibition experiments,ApoE-/-mice(n= 28)fed with the HF diet were randomly assigned to one of two groups and given(by oral gavage)vehicle(distilled water;stress group)or DPP-IV inhibitor anagliptin(30 mg/kg,a generous gift from Sanwa Kagakuz Pharmaceutical,Mie,Japan;S-Ana group)twice daily for 12 weeks under continued daily-4 immobilization stress.At the end day of stress protocol,the blood,the bone marrow,the subcutaneous and visceral fat,and the aortas were collected for the biological and morphological analyses as above.Experiment 3:For the APN blocking experiments,the mice(n=10)fed with the HF-diet were randomly assigned to one of two groups and administered rabbit IgG(Control,450 ?g/kg/d,ab27472,Cambridge,UK)or rabbit neutralizing APN antibody(S-NAPN,450 ?g/kg/d,ab3455)by subcutaneous injection every week under immobilization stress conditions for 8 weeks.At the end day of stress protocol,the blood,the bone marrow,the subcutaneous and visceral fat,and the aortas were collected for the biological and morphological analyses as above.Part ? Cell experimentMouse 3T3-L1 cells(American Type Culture Collection,Rockville,MD)were cultured in Dulbecco's modified Eagle's medium(DMEM).Differentiated 3T3-L1 adipocytes were generated.Following cultured in serun-free DMEM for overnight,the cells were treated with GLP-1R agonist exenatide(0,5 nM,and 25 nM)and anagliptin(0,10,and 20 ?M)for 24h,and then were subjected to the real-time PCR assay.Results:1.Effect of chronic stress on body weight(BW),fat volume,plasma lipid profile and DPP4 levels.(1)Compared to the HF diet-alone mice,chronic stress reduced the BW of the mice significantly throughout follow-up period.The mice that underwent the 12-week stress protocol lost significant amounts of abdominal subcutaneous fat and inguinal fat.Furthermore,chronic stress significantly increased the levels of serum DPP-?(649±13ng/mL vs.997±40 ng/mL,P<0.01),and significantly decreased the levels of APN and GLP-las compared with that of the controls.(2)There was no significant difference in plasma creatinine,BUN,LDL-C,and HDL-C levels between the two experimental groups.2.Stress aggravated the diet-induced vascular senescence and atherosclerotic plaque growth and component alteration.(1)The intima volume was significantly higher in the aortic roots of HF-S mice as compared to that of the HF-C mice(284±14×103/?m2 vs.557±21×103/?m2,P<0.05).Likewise,the stressed aortas had a significantly increased ratio of the neointima area to the media in these lesions(1.3±0.1 vs2.3±0.2,P<0.05).(2)Chronic stress promoted atherosclerotic plaque inflammation.The result of immunohisto-chemical staining showed that as compared to the HF-C group,the plaque macrophage infiltration,osteopontin expression,and neovessel formation were increased in the HF-S group(22.±1.7%vs.34.5±2.4%;23.6±1.2%vs.30.4±1.0%:5.6±0.9%vs.9.3±1.3%;P<0.05,respectively).(3)Chronic stress increased oxidative stress production in the atherosclerotic plaque.The result of NADPH oxidase-dependent oxidative stress production assay revealed that the NADPH activity was significantly increased in the HF-S group as compared to the HF-C group(130.2=1=7.8 RLU/mg vs.210.0±15.1 RLU/mg,P<0.05).The levels of targeted genes(gp91phox,P47phox,P67phox)and protein(gp91phox)were also increased significantly.(4)Chronic stress accelerated plaque ECM degradation and instability.As compared with the HF-C group,the content of collagen and smooth muscle cells were reduced significantly in the HF-S group(32.1±1.3%vs.18.5±2.3%;27.3±1.0%vs.24.4±0.6%;P<0.05,respectively).The results of the EVG staining showed that the destruction of the elastic lamina in the aortic root of the stress group was more serious than that in the control group.It is consistently that the levels of the targeted proteolytic enzyme mRNAs(MMP-2,MMP-9,CatK and CatS)were significantly higher in the HF-S group compared to the HF-C group.Moreover,the gelatin zymography assay revealed that the gelatinolytic activities of MMP-2 and MMP-9 were enhanced in the stressed mice.(5)Chronic stress accelerated vascular aging.The result of ?-Gal staining showed the stressed aortas had enhanced senescence-associated expressions of ?-galactosidase activity(13.5±2.1×103/?m2 vs 33.5±3.5×103/?m2,P<0.05).Likewise,Western blotting assays showed that the levels of the senescence-related proteins(p 16INK4A and p21)were also upregulated in the stressed mice.3.DPP4 inhibition alleviated stress-related vascular aging and atherosclerosis.(1)Anagliptin treatment ameliorated stress-related atherogenesis in ApoE-/-mice fed with the HF diet.Anagliptin treatment decreased serum DPP_? levels(976±4ng/mL vs.477±22ng/mL,P<0.01)and significantly reduced neointimal area(506.0×103/?m2±22.0 vs.147.0±12.0×103/?m2,P<0.01).(2)The administration of anagliptin inhibited plaque macrophage infiltration,lipid accumulation,neovessel promotion,elastin destruction,vascular aging and increased the content of collagen.Consistently,DPP-? inhibition improved the ch a ges in the levels of targeted genes(TLR-2 and TLR-4,CXCR4,MMP-2,MMP-9,CatS,and Cat K)and proteins(osteopontin,PPAR-?,gene p16INK4A,and gp91phox)as compared to the control mice.(3)APN blocking with its neutralizing antibody abrogated the DPP4 inhibitor-related beneficial changes.Quantitative data.of the atherosclerotic lesions revealed that the neointimal area and the ratio of intima/media were bigger than that in the S-Ana-C group(198±11×103 vs 392±23×103;0.9±0.1 vs 1.7±0.1,P<0.05,respectively).4 GLP-1R agonist stimulated APN expression in cultured 3T3-L1 cells.In vitro,we found that GLP-1R agonist exenatide stimulated APN expression in mouse 3T3-L1 cells in a dose-dependent manner,whereas DPP-IV inhibition with anagliptin had no effect on itConclusioins:1.Chronic stress promotes atherogenesis accompanied with unbalance between plasma DPP-?GLP-1 levels in ApoE-/-mice,indicating that the changes in plasma DPP-? and GLP-1 may be new biological marker of the atherosclerosis-based cardiovascular diseases.2.Anagliptin treatment prevented HF-induced atherosclerosis and vascular senescence in mice under chronic stress;these effects were diminished by the APN blocking with its neutralizing antibody,indicating that DPP-? inhibition appear to produce a vascular benefit through the activation of GLP-1/GLP-R-APN signaling in ApoE-/-mice under our experimental condition.3.DPP-? will be a novel therapeutic target for the treatment of stress-related cardiovascular disease. |
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