The Effect Of Matrix Metalloproteinase-9 (MMP-9) On Blood Pressure And Ventricular Remodeling In Agtr1a Knockout Mouse

Background and purposeMatrix metalloproteinase 9(MMP-9),a member of the Matrix Metalloproteinase family(MMPs),is not only involved in physiological processes such as embryo implantation,fetal heart development,angiogenesis,wound healing,but also in pathological processes such as inflammation and tumor metastasis.MMP-9 is widely distributed in the cardiovascular system.Previous studies had found that MMP-9 was significantly increased and positively correlated with the degree of ventricular remodeling in a variety of cardiovascular diseases,moreover,MMP-9 gene knockout or MMP-9protein expression down-regulation could attenuate cardiovascular remodeling or postpone the occurrence and development of cardiovascular diseases.It suggesting that MMP-9 may promote the ventricular remodeling occurrence and development,however,its specific mechanism has not been fully clarified.The effect of MMP-9 on elevated blood pressure is rarely studied in the past.Our previous studies found that exogenous MMP-9injection could not only construct ventricular remodeling model successfully in rats,but also increase the blood pressure of the model rats.Further studies revealed that the model rats were accompanied with ACE,AngI and AngII levels rise,and Olmesartan,an AngII type 1 receptor(AT1)blocker,could improve left ventricular remodeling and lower blood pressure induced by MMP-9.It suggest that the effect of MMP-9 on promoting ventricular remodeling and elevating blood pressure may be partially with Renin-Angiotensin-Aldosterone system(RAAS)activation,however,it is not clear whether MMP-9 is independent of RAAS(i.e.AngII-Agtr1 pathwayactivation),which leads to blood pressure elevation and ventricular remodeling.AngII had been proved to elevate blood pressure and induce ventricular remodeling mainly by binding with AT1 R.There are three subtypes of AT1 R in rodents: angiotensin II type 1A receptor(Agtr1a),angiotensin II type 1B receptor(Agtr1b)and angiotensin II type 1C receptor(Agtr1c).Agtr1 a is equivalent to the effect of human AT1 A and is a pathway protein for pathological effects of.AngII such as elevated blood pressure and ventricular remodeling in rodents.Agtr1 a gene knockout will block the elevated blood pressure and ventricular remodeling effect of RAAS.This study intends to use Agtr1 a knockout mouse model,through the injection of exogenous MMP-9 and by adeno-associated virus(AAV9)as the carrier to endogenous MMP-9 overexpression method,investigate whether the MMP-9 can be independent of Ang?-Agtr1 pathways lead to elevated blood pressure and ventricular remodeling.It would provide a new understanding of the pathogenesis of hypertension and ventricular remodeling,and provide theoretical support for the prevention and treatment targets of hypertension and ventricular remodeling.Method:Experimental animals: Agtr1a(-/-)male mouse and Agtr1a(+/+)female mouse were bred to obtain Agtr1a(+/-),then the same generation of Agtr1a(+/-)and Agtr1a(-/-)were co-bred by female and male mouse.Agtr1a(+/+),Agtr1a(-/-)of 8 weeks old in F5 generation were used to inject exogenous recombinant mouse MMP-9 via tail veins.The 8 weeks old Agtr1a(+/+)and Agtr1a(-/-)of F6 generation were used for endogenous overexpression of MMP-9 in Adeno-Associated Virus 9(AAV9).Endogenous overexpression of MMP-9 was achieved by constructing cTNT to initiate cardiomyocyte-specific overexpression of AAV9.With half male and half female in each group.1.Experimental grouping and processing1 Exogenous MMP-9 infusion group: According to body weight and gender,Agtr1a(+ / +)and Agtr1a(-/-)mouse were randomly divided into Agtr1a(+ / +)control group(WT control group),Agtr1a(+ / +)low-dose group(WT 5 ? g / kg group),Agtr1a(+ / +)high-dose group(WT 10 ? g / kg group),and Agtr1a(-/-)control group(KO control group),Agtr1a(-/-)low-dose group(KO 5 ?g / kg group),Agtr1a(-/-)high-dose group(KO 10 ?g / kg group),n=6,WT control group and KO control group were injected with 200 ?l normal saline,WT 5 ? g / kg and KO5 ? g / kg were injected recombinant mouse MMP9 protein 5 ? g / kg + Normal saline(a total of 200?l)via tail veins.WT 10 g/kg and KO10 g/kg were injected recombinant mouse MMP9 protein 10 ? g / kg and normal saline(a total of 200 ? l)via tail veins,BIW(Each Monday and Thursday).(2)Endogenous MMP-9 overexpression group: According to body weight and gender,Agtr1a(+ / +)and Agtr1a(-/-)mouse were randomly divided into Agtr1a(+ / +)AAV9 empty control group(WT AAV9 control),Agtr1a(+ / +)AAV9-MMP-9 overexpression group(WT AAV9-MMP-9 group),Agtr1a(-/-)AAV9 empty control group(KO AAV9control),n=6,WT AAV9 control group and KO AAV9 control group were injected with 100? l AAV9 MMP-9empty virus via tail veins,WT AAV9-MMP-9 group and KO AAV9-MMP-9group were injected with 100 ?l AAV9-MMP-9 virus via tail veins.2.Observation items: Observation and detection items of exogenous MMP-9 infusion group and endogenous MMP-9 overexpression group are as follows: monitoring body weight,initial blood pressure,blood pressure at the end of the eighth week;It were measure by echocardiography that left ventricular diastolic anterior wall(LVAW d),left ventricular systolic anterior wall(LVAW s),left ventricular diastolic posterior wall(LVPW d),Left ventricular systolic posterior wall(LVPW d),left ventricular diastolic diameter(LVIDd),left ventricular systolic diameter(LVIDs),left ventricular weight(LVMass),ejection fraction(EF),Fractional shortening rate(FS),the ratio peak E/ peak A of mitral valve diastaolic Doppler blood flow(E/A),left ventricular diastolic volume(LV vold),left ventricular systolic volume(LV vols);Left ventricular mass index(LWVI)=left ventricular weight/body weight;Cardiomyocyte morphology,structure and myocardial collagen were determined by HE,Masson and Sirius red staining of myocardial pathological sections;MMP-9 localization and quantification in myocardium were observed by immunofluorescence staining;HE and Masson were used to observe the vascular morphology and collagen volume fraction of abdominal aorta and mesenteric artery;plasma MMP-9,TIMP-1,ACE,Ang II,ACE2,Ang(1-7),ET-1,eNOS content were determined by ELISA,myocardial MMP-9,TIMP-1 protein content were determined by Western blot,and myocardial MMP-9 and TIMP-1 mRNA were measured by Real Time-PCR.Results:1.Effect of exogenous MMP-9 infusion and endogenous overexpression MMP-9 on blood pressure in Agtr1 a knockout mouse(1)Baseline dataBoth in the exogenous MMP-9 infusion groups and endogenous overexpression MMP-9 groups,the survival rate of mouse was 100%,and the initial average body weight was not significantly different between the groups;Agtr1a(-/-)groups had significantly lower initial blood pressure(systolic blood pressure,diastolic blood pressure,mean arterial pressure)than Agtr1a(+/+)groups(p< 0.001),.while there was no significant difference in the initial blood pressure and heart rate between Agtr1a(+/+)and Agtr1a(-/-)groups respectively(p > 0.05).(2)The blood pressure at the end of the eighth week(1)After exogenous MMP-9 infusion,the systolic,diastolic and mean arterial blood pressures of Agtr1a(+/+)and Agtr1a(-/-)groups were significantly increased in a dose-dependent manner(p< 0.05),while the heart rate was not significantly different between groups.(p > 0.05).(2)After endogenous overexpression MMP-9,the systolic,diastolic and mean arterial blood pressures of Agtr1a(+/+)and Agtr1a(-/-)groups were significantly increased(p< 0.05).there was no significant difference in heart rate between groups(p > 0.05).2.Effect of exogenous MMP-9 infusion and endogenous overexpression MMP-9 on vessels in Agtr1 a knockout mouse(1)Lumen-wall ratio of mesenteric artery:(1)After exogenous MMP-9 infusion,the luminal wall ratio of mesenteric artery was significantly reduced in the Agtr1a(+/+)group(p < 0.05),while that of the Agtr1a(-/-)group showed a decreasing trend.(2)After endogenous overexpressionMMP-9,the luminal wall ratio of mesenteric artery was significantly reduced in the Agtr1a(+/+)group and Agtr1a(-/-)group(p < 0.05).(2)Pathological changes of abdominal aort(1)After exogenous MMP-9 infusion,the collagen volume fraction(CVF)of abdominal aorta was significantly reduced in Agtr1a(+/+)and Agtr1a(-/-)groups(p < 0.05),and the lumen-wall ratio of abdominal aortic cavity wall was not significantly changed between groups(p>0.05).(2)After endogenous overexpression MMP-9,the collagen volume fraction(CVF)of abdominal aorta was significantly reduced in Agtr1a(+/+)and Agtr1a(-/-)groups(p< 0.05),and the lumen-wall ratio of abdominal aortic cavity wall was not significantly changed between groups(p>0.05).3.Effect of exogenous MMP-9 infusion and endogenous overexpression MMP-9 on ventricular remodeling in Agtr1 a knockout mouse(1)Echocardiography(1)After exogenous MMP-9 infusion,ventricular wall hypertrophy was observed in Agtr1a(+/+)and Agtr1a(-/-)groups,but stroke volume,cardiac systolic function and diastolic function were not significantly different between groups(p > 0.05).(2)After endogenous overexpression MMP-9,ventricular wall hypertrophy was observed in Agtr1a(+/+)and Agtr1a(-/-)groups,stroke volume was significantly increased in Agtr1a(+/+)group(p < 0.05),but cardiac systolic and diastolic functions were not significantly different between groups(p > 0.05).(2)LVWI(1)After exogenous MMP-9 infusion,LVWI in Agtr1a(+/+)and Agtr1a(-/-)groupswere significantly increased(p < 0.05).(2)After endogenous overexpressionMMP-9,LVWI in Agtr1a(+/+)and Agtr1a(-/-)groupswere significantly increased(p < 0.05).(3)HE staining of myocardium(1)After exogenous MMP-9 infusion,the cardiomyocytes in Agtr1a(+/+)group and Agtr1a(-/-)group were enlarged,the arrangement of cardiomyocytes was disordered,some myocardial fibers were broken,the cytosol was dissolved and necrotic,and the boundary between cardiomyocytes was blurred.(2)After endogenous overexpressionMMP-9,the cardiomyocytes in Agtr1a(+/+)group and Agtr1a(-/-)group were enlarged,the arrangement ofcardiomyocytes was disordered,some myocardial fibers were broken,the cytosol was dissolved and necrotic,and the boundary between cardiomyocytes was blurred.(4)Collagen volume fraction of Myocardial(1)After exogenous MMP-9 infusion,the CVF of myocardium was significantly reduced in Agtr1a(+/+)group and Agtr1a(-/-)group.(2)After endogenous overexpressionMMP-9,myocardial CVF was significantly reduced in Agtr1a(+/+)group and Agtr1a(-/-)group.(5)Sirius red staining of myocardiumAfter exogenous MMP-9 infusion or endogenous overexpression MMP-9,there was no significant difference in type I collagen,type III collagen and type I/III collagen stained in myocardium4.Effect of exogenous MMP-9 infusion and endogenous overexpression MMP-9 on plasma levels of MMP-9,TIMP-1,Ang II,ACE2,Ang(1-7),Endothelin-1(ET-1),Endothelial nitric oxide synthase(eNOS)in Agtr1 a knockout mouse(1)After exogenous MMP-9 infusion,the plasma levels of MMP-9,MMP-9/TIMP-1 and Ang II were significantly increased,while,ACE2 and Ang(1-7)were significantly decreased in Agtr1a(+/+)and Agtr1a(-/-)groups;In addition the levels of ET-1 and eNOS were significantly decreased in the Agtr1a(-/-)group(p < 0.05).(2)After endogenous overexpressionMMP-9,the plasma levels of MMP-9and MMP-9/TIMP-1 were significantly increased,Ang(1-7)was significantly decreased in Agtr1a(+/+)and Agtr1a(-/-)groups;Ang II was significantly increased in Agtr1a(+/+)group,while ET-1 was significantly decreased in Agtr1a(-/-)group.(p< 0.05).5.Effect of exogenous MMP-9 infusion and endogenous overexpression MMP-9 on ventricular myocardiumMMP-9 expression in Agtr1 a knockout mouse(1)Myocardial MMP-9 distribution(Immunofluorescence)After exogenous MMP-9 infusion and endogenous overexpressionMMP-9,Myocardial MMP-9 prote distribution in Agtr1a(+ / +)and Agtr1a(-/-)groups was significantly increased,especially in disorderedand ruptured myocardial fiber.(2)Myocardial MMP-9 and TIMP-1(Western blot)(1)After exogenous MMP-9 infusion and endogenous overexpressionMMP-9,the levels of myocardial MMP-9 and MMP-9 /TIMP-1 were significantly increased in Agtr1a(+ / +)group and Agtr1a(-/-)group.(3)MMP-9 mRNA and TIMP1 mRNA in myocardium(Real time PCR):(1)After exogenous MMP-9 infusion,MMP-9 mRNA and MMP-9/TIMP1 mRNA in the myocardium of Agtr1a(+/+)group showed an up regulation trend;but MMP-9 mRNA and MMP-9/TIMP1 mRNA in the myocardium of Agtr1a(-/-)group were increased significantly.(2)After endogenous overexpression MMP-9,MMP-9 mRNA and MMP-9/ TIMP1 mRNA were increased significantly in Agtr1a(+ / +)and Agtr1a(-/-)groups.ConclusionMMP-9 can increase the blood pressure and induce ventricular remodeling in Agtr1 a knockout mouse,suggesting that MMP-9 may have the effect of elevating blood pressure and inducing ventricular remodeling independent of RAAS(namely Ang II-Agtr1 pathway activated).