1.Effects And Mechanism Of Exenatide On High Fat Diet And Chronic Stress Induced Vascular Aging And Atherosclerotic Plaque Growth In Apoe^-/- Mice 2.Clinical Significance Of Plasma Dipeptidy Peptidase-4 Activities In Patients With Coronary Arter

Objective:To investigate the beneficial effects and mechanism of GLP-1R activation withexenatide on diet-induced vascular aging and atherosclerosis in apolipoprotein E-deficient(ApoE-/-)mice under chronic stress.Materials and methods:Animals:The animal study protocol(protocol no.27304)was approved by the Institutional Animal Care and Use Committee of Nagoya University.All of the animal studies were conducted in accord with the animal care guidelines of the Nagoya University Graduate School of Medicine.Four-week-old male ApoE-/-mice(KOR/Stm Slc-Apoesshl)were obtained from Japan SLC(Hamamatsu,Japan).All mice were maintained in a 22? room with a 12-hr light/dark cycle and received drinking water ad libitum.For the stress experiments,5-week-old ApoE-/-mice were fed a Western-type diet containing 21.00%fat from lard and 0.15%cholesterol for 12 weeks.Experimental grouping and stress model:1.Mice(n=24)fed the Western-type diet were divided to the non-stress and the stress groups.Non-stress control mice were allowed contact with each other and left undisturbed,whereas the stressed mice were subjected to a 4-hr session in an immobilization stress tube(Cat.551-BSRR,Natsume Seisakusho,Tokyo)once daily(between 9:00 a.m.and 1:00 p.m.)for 7 days/week for 12 weeks.2.In separate GLP-1 analogue experiments,mice(n=22)fed the high-fat(HF)diet were divided to one of two groups and given(by subcutaneous injection)vehicle(saline,Stress)or the GLP-1R agonist exenatide(S-Exe,5 ?g/kg;AstraZeneca,London,UK)twice daily for 12 weeks under continued daily 4-hr immobilization stress.Experimental method:Before the start of the experiment,and every weekend after the experiment to measure the body weight,blood pressure,heart rate and other basic indicators of all mice.In the twelfth weekend of the experiment,Cardiac blood and arterial histology samples were collected from each group,using ELIS A measured Adiponectin(APN),leptin(leptin),and GLP-1 activity.Tlie levels of BUN,NEFA,LDL-C,HDL-C and TG were detected by automatic biochemical analyzer.DPP-4 activity was determined by chemiluminescence method;Using Oil red O staining,routine HE staining,beta galactosidase(Gal)staining,collagen staining and elastin staining were used to observe the changes of atherosclerotic plaque and vascular aging as well as the composition of atherosclerotic plaque.Using Gelatin zymography?RT-PCR to observe the oxidative and inflammatory,factors p22phox,gp91phox,MMP-9,MMP-2,TIMP-1,TIMP-2,CatS,CatK,CatL,TLR-4,TLR-2,SDF-1 alpha,CXCR4,eNOS and the expression of adiponectin gene expression.Western Blot method was used to detect the expression levels of PPAR-,AT1R and gp91phox protein.Result:1)Impact of chronic stress on body weight(BW)and blood biological parameters1.The mice that underwent the 12-week stress protocol lost marked amounts of inguinal fat and subcutaneous fat.2.The stressed mice had significantly increased levels of plasma 'DPP4(823±34 vs.305±28 ng/ml,p<0.01)and reduced levels of nonesterified fatty acid(NEFA)compared to the non-stress mice.3.Stress had no influence on the levels of plasma BUN,LDL-C,HDL-C,or creatinine.2)The chronic stress accelerated the diet-induced vascular aging and atherosclerotic lesion formation and component change1.The chronic stress significantly enhanced the area of the intima in the aortic roots compared to the non-stress controls(root:438±25 vs.213±13×103 ?m2,p<0.01).2.The stress also significantly increased the ratio of the neointima area to the media area in the lesions compared to the corresponding controls(root:1.9±0.2 vs.1.0 ±0.1,p<0.01).3.Oil red O staining was applied to assess the lipid content of atherosclerotic plaques.We observed that the Oil red O+ staining area was significantly higher in the aortic roots of the stressed mice compared fo those of the non-stress mice(209±20 vs.117±10×103 ?m2,p<0.01).4.The PRS staining showed a significant reduction of the collagen content in the stressed mice compared to the non-stress mice(collagen content:17.0±1.9 vs.38.9±2.3%,p<0.01).5.The EVG staining revealed that the degradation of the elastic lamina in the aortic root of the stress mice was significantly more serious than that in thenon-stress mice(no.of breaks:15.6±1,5 vs.4.5±0.7,p<0,01).6.Stress significantly decreased the contents of atherosclerotic lesion vascular smooth muscle cells(SMC content:13.3±0.8 vs.19.5±1.4%,p<0.05).7.The(3-Gal staining showed that the stressed aortas had enhanced senescence-associated expressions of ?-galactosidase activity(29.0±4.1 vs.10.1 ± 1.9,p<0.01).3)The impact of the chronic stress on inflammation,oxidative stress,and proteolysis1.The quantitative immunostaining revealed that the stressed plaque had significantly increased macrophage infiltration(25.5±2,6%vs.10.2±1.3%),osteopontin expression(25.6±2.1%vs.9.3±1.0%),and neovessel density(7.2±1.1 vs.;3.1 ± 0.5,p<0.01 for each).2.The expressions of the inflammation-and oxidative stress-related genes(i.e.,TLR-2,TLR-4,CXCR4,SDF-1?,p22phox,and gp91phox)in the aortas were analyzedby quantitative real-time PCR.We also observed that except for the eNOS mRNA levels,the mRNA expressions of MMP-9,MMP-2,TIMP-1,TIMP-2,CatL,CatS and CatK were also significantly elevated in the stressed mice compared to the non-stress mice.3.The quantitative gelatin zymography assay demonstrated that thegelatinolytic activities of MMP-9 and MMP-2 were enhanced in the stressed mice.4.The results of Western blot gel image and quantitative analysis showed thatthe expression of gp91,AT1R and PPAR-were significantly enhanced in the stress group.4)The impact of the chronic stress on the levels of plasma GLP-1,leptin and APN proteins and adipose APN gene expressionThe ELISA results demonstrated that the stressed mice had significantlyreduced levels of plasma GLP-1(9.2±0.8 vs.15.9± 1.1 pM,p<0.01),APN(5619 ±598 vs.7577 ± 382 ng/ml,p<0.05)and leptin(177±25 vs.402±45 pg/ml,p<0.01)compared to the non-stress mice.Consistently,the stress caused a reduction in APN gene expression in the subcutaneous fat.5)GLP-1R activation mitigated the HF diet-induced vascular aging and atherosclerotic plaque growth1.The quantitative Oil red O staining analyses results showed exenatide reduced the atherosclerotic lesions compared to the stress mice(145±11 vs.390±19×103/um2,p<0.01).2.By comparison,the collagen contents were significantly increased in thestressed mice treated with exenatide(28.1 ±3.1%vs.15.2± 1.2%,p<0.01;).As expected,the EVG staining revealed that exenatide significantly improved the numbers of broken-elastin(2.4±0.5 vs.9.0±1.6,p<0.01)in the aortic roots.We also observed that ?-Gal+ areas in the aortas were lower in S-Exe mice than in that of the control mice(9.8±1.3%vs.34.1±4.0%,p<0.01).3.Thus,GLP-1R activation appears to prevent vascular aging and atherosclerotic lesion formation and its vulnerability in mice fed a high-fat diet.However,exenatide had no effect on other lipid and kidney function parameters,BW or inguinal fat and subcutaneous fat.6)The effects of exenatide on inflammation and proteolytic enzyme expression and activity1.The immunostaining against ASMA,Mac3,and osteopontin revealed that exenatide had significant beneficial effects on the contents of vascular SMC(17.1±2.0%vs.12.7±1.3%,p<0.05),the accumulation of macrophages(26.2±1.8%vs.45.2±4.0%),and osteopontin expression(16.4±2.0%vs.38.4±1.4%)in the atherosclerotic plaques of' the stressed aortic roots compared to the stress-alone aortic roots.The immunohistochemistry against vWF also revealed that exenatide significantly reduced the neovessel density of the atherosclerotic plaques in the aortas compared to the control aortas(total neovessel numbers:2.3±0.3 vs.6.1 ± 0.9,P<0.01).2.The mRNA expressions of TLR-4,TLR-2,SDF-la,CXCR 4,p22phox and gp91Phox of the aorta were reduced significantly in the S-Exe mice compared to the stress-alone mice.Exenatide also significantly decreased the mRNA expressions of MMP-9,MM P-2,TIMP-1,TIMP-2,CatL,CatS and CatK compared to those of the control mice.In contrast,the levels of eNOS mRNA were increased in the S-Exe group compared to the control nice.3.The quantitative analysis revealed that exenatide reduced the gelatinolytic activities of MMP-9 and MMP-2.4.The Western blotting assay revealed that the.S-Exe mice had decreased levels of gp91phox protein.In addition,the Western blot results showed that exenatide treatment inhibited the protein expressions 'of AT1R and PPAR-a in the aortas of the stressed mice.7)DPP4 inhibition increased the levels of plasma leptin and APN and the adipose APN expression in the stressed miceThe results of the ELISA revealed that exenatide significantly increased the levels of plasma APN(8065±634 vs.5619±598 ng/ml,p<0.05),GLP-1R activation resulted in an increase in the subcutaneous adipose APN mRNA expression in the stressed mice.Conclusion:Chronic stress promoted DPP4-mediated GLP-1 truncation,which lowed adipose APN expression and production and promoted plaque inflammation,oxidative stress production,and proteolysis,leading to an aggravation of diet-induced vascular senescence and atherosclerosis inception and progression in ApoE-/-mice.This study is the first report,an incretin-based pharmacological intervention ameliorated stress-related vascular aging and atherosclerosi's.It is suggested that the GLP-1 analogue exenatide will be a new target for the treatment of stress related cardiovascular diseases.Objective:To explor the relationship among DPP4 activity,CAD and its risk factors,and to provide a experimental basis for new biomarker diagnosis of CAD.Materials and Methods:Study population and definitionResearch object:1.From May 2014 to December 2015,496 patients with chest pain through coronary angiography were divided into two groups:coronary heart disease(CHD)group(n = 362)and non coronary heart disease group(n = 134).The CAD patients were sub-grouped into the acute coronary syndrome(including the acute myocardial infarction[AMI]and the unstable angina pectoris[UAP]n = 299)and the stable angina pectoris(SAP n= 63)groups based on their symptoms and clinical examination marks.2.According to the CAD patients with and without DM,the CAD patients were divided into CAD/DM(+)group of 84 cases and CAD/DM(-)group of 278 cases.3.According to the control subjects with and without DM,Non CHD patients were divided into nonCAD/DM(+)group(22 cases)and nonCAD/DM(-)group according to whether or not suffering from diabetes.This study protocol was approved by the Ethics Committee of Yanbian University Hospital,and written informed consent was obtained from all patients.Exclusion condition:Patients were excluded if they had prior evidence of congenital heart disease,primary valvular disease,cardiomyopathy,secondary cardiac muscle disease caused by any known systemic condition,or end-stage kidney disease.The patients with DM who were taking a DPP4 inhibitor were also excluded.Experimental method:1.All selected patients were collected including age,gender,smoking history,medication history,and clinical history(hypertension,diabetes,previous MI,previous PCI,previous bypass surgery,and previous cerebrovascular disease).The measurement of body weight index(BMI)and blood pressure were as described.Venous blood samples were obtained prior to PCI and stored at-80?.2.Plasma DPP4 activity analysisWe measured each subject's DPP4 activity by using the DPP4 Glo Protease Assay(Promega,Madison,WI)with an aminoluciferin substrate.The plasma was isolated using VENOJect? vacuum blood collection tubes containing anticoagulants without a serine protease inhibitor(Terumo,Tokyo),and 'the plasma was then diluted in 0.1 mM Tris-HCl buffer(pH 8.0)by 30-fold.Equal amountsof diluted plasma(25?l)were subjected to a DPP4 Glo assay(Promega)in the presence or absence of the DPP4 inhibitor anagliptin(20 ?mol/L).2.Quantitative coronary angiogram(QCA)The quantitative coronary angiogram(QCA)was evaluated from angiography exhibiting the maximal degree of stenosis.We did the QCA analysis using a contour detection minimum cost algorithm(DSA Artis Zee Biplane;Siemens,Erlangen,Germany).Patients with CAD had severe stenosis defined as the presence of 50%diameter stenosis of at least one major artery.The reference segment diameter was obtained the averages from 5-mm long angiographically.normal segments proximal to the lesion,if a normal proximal segment could not be identified,a distal angiographically normal segment was analyzed as described.3.Laboratory examinationEach subject's plasma levels of hemoglobin Ale(HbAlc),high sensitive C-reactive protein(hs-CRP),low-density lipoprotein cholesterol(LDL-C),creatinine,and high-density lipoprotein cholesterol(HDL-C)were examined at the clinical laboratory of Yanbian University Hospital(Clinical Laboratory,Yanji,China).Result:Analysis of patients1.The baseline characteristics of the CAD subjects(n = 362)and nonCAD subjects(n = 134)were presented.There was a significant difference in the gender distribution of two experimental groups,but no significant between-group difference in age or BM1.2.The CAD patients showed a significantly higher prevalence of hypertension;these patients also showed a significantly higher numbers of current smokers and to have had cerebrovascular disease or a previous MI,or to have undergone PCI(P>0.05 for comparison).3.The frequencies of the CAD patients under intervention with anti-diabetic(insulin),anti-platelet,anti-lipid,and antihypertensive drugs were all higher than those of the nonCAD subjects.4.There were no significant differences in age or BMI between the SAP and UAP+AMI groups(P>0.05 for each comparison).There was no significant difference in the medication use and clinical histories between both experimental sub-groups(P>0.05 for each comparison).Atherosclerotic lesion characteristics1.L There was no significant difference in the target lesion locations of the left main artery(P>0.05 for each analysis).2.The UPA+AMI group had a higher ratio of target lesion location in the left anterior descending artery,left circumflex artery,and right coronary artery comparedto the SAP patients(P<0.05 for all comparisons).3.In the QCA analysis of target lesions,with the exception of reference vessel diameter(P>0.05 for each analysis)the patients with UAP or AMI had significantly greater diameter stenosis(87.3 ± 9.2 vs.74.3 ± 7.1 mm,P = 0.04)and lesion length(19.1 ± 4.0 vs.15.0 ± 4.4 mm,P<0.01)as well as Syntax score(17.8 ± 7.9 vs.11.9 ± 6.3,P<0.01)compared to the patients with SAP.Circulating Biomarkers1.The CAD group had significantly increased plasma DPP4 levels(11.8 ± 3.1 vs.6.9 ± 3.5 ng/L,P<0.01)compared to the nonCAD group.2.Levels of hs-CRP(15.2 ± 28.9 vs.3.3 ± 9.0 ng/mL,P = 0.04)and hemoglobin Alc(6.0 ± 1.5 vs.5.5± 1.1%,P<0.01)were significantly higher in the CAD patients compared to the nonCAD subjects(P<0.01 for each comparison).3.There was no significant difference in the levels of creatinine(1.0 ± 0.3 vs.0.9 ± 0.1 mg/dl),LDL-C(102.3 ± 31.2 vs.93.8 ± 28.9 mg/dl),or HDL-C(43.8 ±19.5 vs.47.4 ± 28.5 mg/dl)between the CAD and nonCAD groups(P>0.05 for each parameter analysis).4.The UAP or AMI patients had significantly increased levels of circulating DPP4(13.1 ± 4.2 vs.9.4 ± 3,8 ng/L,P<0.01).5.The UAP+AMI group patients had also significantly increased levels of plasma LDL-C(104.0 ± 31.4 vs.95.5 ± 29.4 mg/dl,P=0.04)and hs-CRP(17.8 ±31.0 vs.3.0 ± 7.2 ng/mL,P<0.01)and hemoglobin Ale(6.1 ± 1.5 vs.5.6 ±1.0%,P=0.01)compared to the patients with SAP,whereas there was no significant dif-ference in the HDL-C or creatinine levels between two experimental groups,(P>0.05 for all comparisons).Impacts of diabetes and CAD on plasma DPP4 activities1.Our findings indicated that DM significantly increased plasma DPP4 activity in the nonCAD con-trol subjects(8.0 ± 2.9 vs.6.4 ± 3.1 ng/L,P<0.01).2.There was no significant difference between CAD/DM(+)group and CAD/DM(-)group in DPP4 activity.(12.2±4.4 vs.10.9±4.9 ng/L,P>0.05).3.Without DM had increased plasma DPP4 activities as compared with nonCAD subjects(10.9 ± 4.9 vs.6.4 ± 3.1,ng/L,P<0.01).Association between DPP4 activity and CAD patients with and without DM1.The DPP4 activities positively correlated with the levels of both LDL7C(r ?0.23,P<0.01)and hs-CRP(r = 0.41,P<0.01),whereas they were not correlated with the HDL-C lev-els(r = 0.05,P>0.05).The DPP4 activities were also positively correlated with the stenosis(r = 0.24,P<0.05)and lesion length(r = 0.19,P<0.05)shown by the CAG in patients with CAD.2.The logistic regression analysis revealed that CAD significantly associ-atedwith age,gender,hs-CRP,hypertension,LDL-C,and DPP4.The data from the multiple logistic regression analysis show DPP4 level was an independent predictor of coronary heart disease(odds ratio 1.56;95%confidence interval,1.19(?)1.73;P<0.01).Conclusion:The results of the present study demonstrated that increased blood DPP4 levels are positively and independently associated with CAD,even without DM.increased DPP4 activity levels are associated with the presence of CAD and that the plasmaDPP4 level serves as a novel biomarker for CAD even without DM.There is a close relationship between DPP4 level and coronary heart disease,which may be a new biomarker for the diagnosis of coronary heart disease.