The Effects Of GLP-1 Receptor Agonists For The Hypertensive Cardiac Fibrosis Induced By AngⅡ

1.IntroductionCardiac fibrosis is associated with a wide variety of heart diseases,including hypertension,ischemic heart disease and valvular heart disease.The current epidemiological data in China and abroad suggested that the morbidity of hypertension was increasing every year.As there are 266 million people suffering from hypertension in the population>15 years old in China,and hypertensive heart disease has become the main cause of cardiac fibrosis.The cardiac fibrosis induced by hypertension presents as the disturbed balance between collagen generation and degration in heart tissue,and excessive deposition of collagen in extracellular matrix results in myocardial stiffening and hypertrophy,leading to the disability of properly cardiac relaxation and systole with the final heart failure.AngiotensinⅡ（AngⅡ）plays an important role in the process of cardiac fibrosis,through binding to the typeⅠangiotensin receptor（AT1R）to induce contraction of vascular smooth muscles,inflammatory reaction of the system and redundant expression of collagen from fibroblasts,which are key points for cardiac fibrosis.Therefore,the current treatment for cardiac fibrosis induced by AngⅡis due to the application of AngⅡconverting enzyme inhibitors（ACEIs）and AT1R blockers（ARBs）,which can reduce the production of AngⅡand suppress the activation of AT1R,respectively.The glucagon-like peptide-1（GLP-1）is an incretin hormone generated by intestinal endocrine L-cell as a key hormone for the glucose homestasis in response to diet.Endogenous GLP-1 with a short half-life between 1 to 2 minutes,is degraded by the enzyme named dipeptidyle peptidase-4（DDP-4）.Exenatide and liraglutide are the two kinds of long-acting GLP-1 receptor agonists for the treatment of type 2 diabetes.The clinical observations showed that GLP-1R agonist provided benefits for human lipid metabolism,arteriosclerosis and cardiac protection beyond the anti-diabetic effect of glucose control and insulin resistance improvement,indicating its dual aspects of cardiovascular protection in patients with type 2 diabetes.Experiments in vivo suggested that GLP-1R agonists could significantly reduce the infarction area,improve ischemia-reperfusion injury and cardiac function,and inhibit the cardiac fibrosis after myocardial infarction in animal models.In animal models of cardiac fibrosis induced by AngⅡ,GLP-1R agonist inhibited the elevation of blood pressure and cardiac hypertrophy besides of its suppression of cardiac fibrosis.Application of GLP-1R agonists also reduced the cardiac fibrosis in patients with chronic heart failure,myocardial infarction or hypertension complicated with type 2diabetes.However,there are no parallel comparison of the effect of liraglutide and exenatide on cardiac fibrosis induced by hypertension.In addition,few studied reported the effects of GLP-1R agonist on different cell types involved in the process of cardiac fibrosis,including myocardial cell,fibroblast and macrophage.It is still controversial whether GLP-1R agonist could directly influence cardiac fibrosis via its effect on fibroblasts.In our research,we attempted to explain the questions above by establishing a mouse model with infusion AngⅡand cell study involving culture of cardiomyoblast,fibroblast,macrophag in vitro under the treatment exenatide and liraglutide.2.Objectives（1）To compare the effects of liragutide and exenatide on the cardiac fibrosis in hypertensive mice induced by chronic infusion of AngⅡand investigate the potential molecular mechanism.（2）To investigate the influence of liraglutide and exenatide in cardiomyoblasts,macrophages and fibroblasts cultured in vitro under the stimulation of AngⅡ.（3）To investigate whether GLP-1R agonists can affect the interaction between different cell types involved in the process of cardiac fibrosis by use of cell co-culture.3.Methods3.1 Establishment of animal models and drug intervention Forty male C57BL/6J mice（eight-week old,weight 21-26g）were fed regular chow and maintained under standard conditions for one week.At the 2nd week,the osmotic pumps with relevant agents were implanted subcutaneously in each mouse for 28days.Based on different treatment,mice were divided into 4 groups randomly（n=10per group）:control group,AngⅡgroup,AngⅡ+liraglutide group and AngⅡ+exenatide group.The mice in control group received infusion of saline,and the mice in the left 3 groups all received infusion of AngⅡ（1.44mg/kg/day）.Mice in AngⅡ+liraglutide group also received liraglutide 0.4mg/kg/day i.p.,while the mice in AngⅡ+exenatide group received exenatide 0.1mg/kg/day i.p..Afer 28 days,all the mice were weighted,tested with blood glucose before euthanasia.Cardiac tissue of all animals was extracted for further investigation.3.2 Echocardiography for assessment of heart structure and functionAt 28th day,all the mice underwent transthoracic echocardiography,that is using the 2D and M-mode echocardiography to measure the left ventricular internal dimension in systole（LVIDs）,Left ventricular internal dimension in diastole（LVIDd）,interventricular septum in systole（IVSs）,interventricular septum in diastole（IVSd）,left ventricular posterior wall in systole（LVPWs）,left ventricular posterior wall in diastole（LVPWd）,left ventricular ejection fraction（LVEF）and left ventricular fraction shortening（LVFS）to evaluate the structure and function of the mouse heart.3.3 Systolic blood pressure measurementSystolic blood pressure of mice was measured every week before and after the implantation of osmotic pumps.Blood pressure was measured 3 consecutive times and presented as a mean.3.4 Histological pathological stainingAt the end of experiment,the cardiac tissue extracted from all mice were paraffin-embedded and sectioned as 5μm pieces for HE staining and Masson staining to observe the morphological characterics of myocardial cell and collagen deposition.3.5 Isolation and culture of cardiac fibroblast from miceHearts were taken out of mice aged 2-3days and cut into pieces for collagenase digestion.Cardiac fibroblasts were isolated using a differential adhesion method and cultured in DMEM after purified.Passage 2 or 3 was harvested for further investigation.3.6 Cell culture and treatment in vitroCardiac fibroblasts of mice,mouse macrophage（RAW264.7）and myocardioblasts of rats（H9C2）were used for in vitro study.Cells were randomly divided into the 4groups including the control group（no treatment）,AngⅡgroup(AngⅡ10^-6mol/L),AngⅡ+liraglutide group(AngⅡ10^-6mol/L+liraglutide 100nmol/L)and AngⅡ+exenatide group(AngⅡ10^-6mol/L+exenatide 100nmol/L).Cells were stimulated after 24 hours serum starvation and with 80%confluence seeded in 6-wells.3.7 Cell co-cultureTranswell chambers were used for co-culture of mouse macrophage and cardiac fibroblast to investigate the interaction between these two cell types.The treatments and grouping were same as step 3.6.3.8 Western blotTotal proteins were extracted from the cardiac tissue or cultured cells.Western blot was carried out to detect the expression of typeⅠcollagen,typeⅢcollagen,MMP-2and MMP-9 in myocardioblasts and cardiac fibroblasts as well as the expression of IL-6,IL-1βand TGF-β1 in macrophages.3.9 Statistical analysisThe experimental data was analyzed by software SPSS16.0.Continuous variables were presented as mean±SD,and categorical variables were presented as percentage.Based on the characteristics of the variables,one-way ANOVA or LSD post-hoc was utilized for comparisons among groups.P<0.05 was considered statically significant.4.Results4.1 Body weight and blood glucoseAt the end of experiment,the mean body weights of AngⅡ+liraglutide group（26.18±1.40g）and AngⅡ+exenatide group（26.51±1.38g）were slightly lower than that of the control group（27.65±0.97g）（p<0.05）.Fasting blood glucose was similar among the 4 groups（p>0.05）.4.2 Systolic blood pressureLiraglutide and exenatide have decreased the elevation of systolic blood pressure induced by AngⅡ.The final systolic blood pressure of mice treated with AngⅡ+liraglutide（139.0±2.9mm Hg）or AngⅡ+exenatide（141.7±5.4mm Hg）were obvious lower than those treated with AngⅡ（p<0.05）.4.3 Echocardiography measurementLiraglutide and exenatide protected mice against cardiac hypertrophy,ventricular dilatation and cardiac dysfunction induced by AngⅡinfusion.Compared to AngⅡgroup,the mice in liraglutide or exenatide group showed smaller LVIDd（4.64±0.30mm vs.3.59±0.38mm and 3.43±0.63mm）and LVIDs（3.00±0.22mm vs.2.01±0.26mm and 1.94±0.41mm）,thinner IVSd（0.89±0.06mm vs.0.59±0.08mm and 0.69±0.04mm）and LVPWd（0.79±0.04mm vs.0.82±0.05mm and 0.87±0.04mm）,and better LVEF（64.5±4.62%vs.76.3±3.06%and 75.6±5.23%）and FS（35.5±3.24%vs.44.4±2.17 and 44.3±2.75%）（p<0.05）.4.4 Histological HE staining and Masson stainingHE staining showed that mice in the AngⅡgroup had the thicker heart walls,dilated cardiac chambers,larger cardiomyocytes arranged disorderly,narrower intercellular spaces in comparesion to the control,liraglutide and exenatide group.Masson staining presented that the percentage of blue collagen was higher in the AngⅡgroup than that in the left 3 groups（p<0.05）.4.5 Effect of GLP-1R agonist on H9C2 myocardioblastsLiraglutide and exenatide stimulation significantly decreased the upregulation of MMP-2 and MMP-9 induced by AngⅡstimulation in myocardioblasts（p<0.05）.4.6 Effect of GLP-1R agonist on macrophageWestern blot showed that the expression of IL-6,IL-1βand TGF-β1 wasupregulated in RAW264.7 cells stimulated by AngⅡ（p<0.05 vs.control）,while the expression of TGF-β1 could be suppressed by liraglutide and exenatide treatments(（p<0.05 vs.AngⅡ+liraglutide group and AngⅡ+exenatide group,separately）.4.7 Effect of GLP-1R agonist on cardiac fibroblastsAngⅡinduced increased expression of typeⅠcollagen,typeⅢcollagen,MMP-2 and MMP-9 in fibroblast（p<0.05 vs.control）.However,stimulation of liraglutide or exenatide showed no effect on these upregulations（p<0.05 vs.AngⅡgroup）.4.8 Effect of GLP-1R agonist on the co-culture of macrophage and cardiac fibroblastMacrophages stimulated by AngⅡincreased the expression of collagen,MMP-2and MMP-9 in fibroblasts（p<0.05 vs.control）,while treatment with liraglutide or exenatide reversed their upregulation induced by AngⅡ-stimulated macrophages co-culture（p<0.05 vs.AngⅡgroup）.5.Conclusions5.1 Liraglutide and exenatide reduce the elevation of mouse systolic blood pressure induced by chronic AngⅡinfusion,with no effect on fasting blood glucose.5.2 Liraglutide and exenatide improve cardiac fibrosis,remodeling and heart dysfunction of hypertensive mice induced by AngⅡ.5.3 Liraglutide and exenatide inhibit the expression of MMP-2 and MMP-9 in myocardioblasts of rat,and TGF-β1 in mouse macrophage stimulated by AngⅡin vitro,while had no effects on the expression of collagen,MMP-2 and MMP-9 from mouse cardiac fibroblasts.5.4 Macrophages simulated by AngⅡ,after treated with liraglutide and exenatide,reduce the expression of collagen,MMP-2 and MMP-9 in myofibroblast,indicating the interaction of different cells in the process of cardiac fibrosis.