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THE ROLE OF NEUTROPHILS IN PRESSUREOVERLOAD-INDUCED CARDIAC DYSFUNCTIONObjective: To investigate the activation and role of neutrophils in non-ischemic myocardial injury.Methods: A mouse transverse aortic arch constriction(TAC)model was used to establish pressure overload-induced cardiac hypertrophy and heart failure,and injection of Ly6 G antibody was used to deplete neutrophils.(1)Divided into sham operation group and TAC group,the number of Lin-Sca1+c Kit+(LSK)and granulocyte-monocyte progenitor cells(GMP)in bone marrow,and the number of neutrophils,monocytes and macrophages recruited in the heart were detected by flow cytometry 3days after TAC.Heart weight was measured.(2)Divided into isotype control + sham group,isotype control + TAC group,Ly6 G Ab + TAC group.The mice were subjected to TAC surgery,and the neutrophils were depleted by injecting Ly6 G antibodies at the corresponding time points of TAC surgery(-2,1,4,7,10,13 days).Cardiac function parameters(PWTd,LVDd,LVDs,FS)were detected by ultrasound,q PCR was used to detect the gene expression levels of ANP,BNP,?/? MHC,immunofluorescence staining was used to detect myocardial myocyte cross-sectional area,and heart weight and tibia length were measured.(3)Divided into isotype control + TAC group,Ly6 G Ab.+ TAC group.The mice were subjected to TAC surgery,and the neutrophils were depleted by injecting Ly6 G antibody at the corresponding time point of TAC surgery(-2,-1,1 day).Flow cytometry was used to detect the number of Ly6 Chi monocytes and macrophages recruited in the heart.(4)divided into isotype control + sham operation group,isotype control + TAC group,Ly6 G antibody + TAC group.The mice were subjected to TAC surgery,and the neutrophils were eliminated by injecting Ly6 G antibody at the corresponding time point of TAC surgery(-2,-1,1 day).The expression levels of Il1?,Il6,Cxcl1,Cxcl2,Cxcl5 and Ccl2 genes were detected by q PCR.Results:(1)Three days after TAC,the number of LSK cells and GMP cells in the bone marrow of the TAC group was significantly increased compared with the sham(p<0.01).(2)Three days after TAC,compared with sham group,the number of neutrophils(p<0.01)and monocytes(p<0.001)recruited in the hearts of TAC mice increased significantly,and macrophages showed Increased trend(p=0.0519).(3)There was no significant difference in heart weight between the sham and TAC group 3days after TAC.(4)At 14 days after TAC,the heart weight/tibia ratio(HW/TL)of the isotype control + TAC group was significantly higher than that of the isotype control + sham group(p<0.0001),and the HW/TL of the Ly6 G antibody + TAC group was significantly lower(p < 0.001)compared to the isotype control + TAC group of mice.(5)14 days after TAC,the left ventricular posterior wall thickness(PWTd),left ventricular end diastolic diameter(LVDd)and left ventricular end systolic diameter(LVDs)of the isotype control + TAC group were higher than the baseline level,and significantly higher than the isotype control + sham group(p <0.01),the left ventricular short axis shortening rate(FS)was significantly lower than the baseline level and lower than the isotype control + sham group(p<0.0001).The PWTd and LVDs of the Ly6 G antibody + TAC group were significantly lower than those of the isotype control + TAC group(p<0.05),and FS was significantly increased(p<0.05),which was not significantly different from the isotype control + sham group.(6)14 days after TAC,compared with the isotype control + sham group,the expression levels of myocardial injury factors ANP,BNP,?/?MHC in the heart of the isotype control + TAC group were significantly increased(p<0.01).The expression level of myocardial injury factor in the Ly6 G antibody + TAC group was significantly lower than that of the isotype control + TAC group(p < 0.05),and no significant difference compared to the isotype control + sham operation group.(7)14 days after TAC,the cardiac myocyte cross-sectional area of the isotype control + TAC group was significantly increasedcompared with the isotype control + sham group(p < 0.0001).The cross-sectional area of cardiac myocytes in the Ly6 G Ab+ TAC group was significantly lower than that in the isotype control + TAC group(p <0.0001).(8)Two days after TAC,the number of Ly6 Chi monocytes and macrophages recruited in the heart of Ly6 G antibody + TAC group was significantly lower than that of the isotype control + TAC group(p<0.01).(9)Two days after TAC,compared with the isotype control + sham group,the expression levels of inflammatory factors Il1?,Il6,and chemokines Cxcl1,Cxcl2,Cxcl5,and Ccl2 were significantly increased in the heart of the isotype control + TAC group.Expression level of these gene in the heart of Ly6 G antibody + TAC group were significantly lower than those of isotype control + TAC group,and the expression of Il1? and Ccl2 genes showed decrease trend,no difference compared with the isotype control +sham group.Conclusions: Neutrophils are activated under pressure overload and recruited to the heart.Depletion of neutrophils attenuates the inflammatory response and cardiac dysfunction induced by pressure overload.PART ? REGULATION AND MECHANISM OF MYELOID-DERIVED WNT5 A ON NEUTROPHILFUNCTIONObjective: To investigate the regulation and potential mechanism of myeloid-derived Wnt5 a on neutrophil function.Methods:(1)Establish a mouse TAC and myocardial infarction(MI)model,using flow sorter to sort neutrophils,Ly6 Chi monocytes and Ly6 Clo monocytes from mouse peripheral blood,and sort fibroblasts,neutrophils,monocytes and macrophages from heart at 3 days post-TAC and 3 days post-MI,and their Wnt5 a gene expression levels were detected by q PCR.(2)From the peripheral blood,neutrophils,Ly6 Chi monocytes and Ly6 Clo monocytes were sorted by flow sorter,and the gene expression level of Frizzled(Fzd1-10)receptor was detected by q PCR.(3)Neutrophils were isolated from mouse bone marrow,stimulated with different concentrations of Wnt5a(0,100,500 ?g/ml),and the migration of neutrophils was detected by modified Boyden chamber method.(4)Neutrophils were isolated from mouse bone marrow,and Wnt5 a stimulation(400 ng/ml)was given at different time points(0,10,30,60 min),and protein phosphorylation of PI3 K,JNK1/2,Erk were detected by western blotting.Results:(1)The gene expression level of Wnt5 a is easily detected in cardiac fibroblasts,cardiac neutrophils at 3 days after TAC,and cardiacneutrophils at 3 days after MI;only significantly lower Wnt5 a gene expression was detected in cardiac macrophages post-TAC/MI;Wnt5a expression was not detected in cardiac Ly6 Chi monocytes after TAC/MI;and Wnt5 a gene expression was not detected in neutrophils,Ly6 Chi monocytes cells,Ly6 Clo monocytes from blood.(2)High expression of the Fzd5 receptor gene was detected only in peripheral blood neutrophils,which was significantly higher than that in Ly6 Chi monocytes and Ly6 Clo monocytes.(3)Exogenous addition of Wnt5 a significantly stimulated neutrophil migration in a dose-dependent manner compared with the untreated group(p<0.05).(4)Exogenous addition of Wnt5 a significantly increased the phosphorylated protein expression levels of PI3 K,JNK and ERK compared to the untreated group,and peaked at 30 min of stimulation.Conclusions: The myeloid cell source of Wnt5 a is activated neutrophils.Wnt5 a may act as an autocrine regulator of neutrophils,regulating the migration of neutrophils by activating the PI3K/JNK/ERK signaling pathway.PART ? EFFECT OF MYELOID-SPECIFIC WNT5ADEFICIENCY ON CARDIAC HYPERTROPHY AND HEART FAILURE INDUCED BY PRESSURE OVERLOADObjective: To explore the role of myeloid-specific Wnt5 a deficiency in cardiac hypertrophy and heart failure induced by pressure overload model.Methods:(1)Construction of Wnt5 a myeloid cell-specific knockout(Myelo-KO)mice.Myelo-KO mouse bone marrow-derived neutrophils and neutrophils in the heart 3 days after TAC were extracted,and genomic DNA was purified.The gene recombination of Wnt5 a was detected by PCR.(2)Divided into control + TAC group,Wnt5 a Myelo-KO + TAC group.The hearts were isolated at 0,3,7,and 28 days after TAC and the number of neutrophils,Ly6 Chi monocytes,and macrophages in the heart was measured by flow cytometry.(3)The mice were divided into control + TAC group,Wnt5 a Myelo-KO+TAC group.The inflammatory cytokines Il1?,Il6,and chemokine Cxcl1,Cxcl2,Cxcl5,Ccl2 genes were detected by q PCR 3 days after TAC.(4)The mice were divided into control + TAC group,Wnt5 a Myelo-KO + TAC group.The heart weight/tibia length ratio(HW/TL)and lung weight/tibia length ratio(LW/TL)were measured at different time points(0,2,and 8 weeks)after TAC.(5)The mice were divided into control + TAC group,Wnt5 a Myelo-KO + TAC group.Thecardiac function parameters LVPWTd,LVDd,LVDs,and FS were measured by ultrasound at different time points(0,1,4,and 8 weeks)after TAC.(6)Sham operation and TAC-8 weeks treatment were performed in control and Wnt5 a Myelo-KO mice,and cardiac myocyte cross-sectional area was detected by immunofluorescence staining.(7)Sham operation and TAC-8 weeks treatment were performed in control and Wnt5 a Myelo-KO mice,and cardiac interstitial fibrosis area was detected by immunohistochemical staining.Results:(1)It was confirmed that gene recombination of Wnt5 site occurred in bone marrow-derived neutrophils of Myelo-KO mice,and exon2 of Wnt5 a was removed.Myelo-KO mice recruited neutrophils to the heart after TAC escaped recombinase-mediated gene recombination.(2)Compared with the control group,the number of Ly6 Chi monocytes recruited into the heart was significantly decreased in Myelo-KO mice 3and 7 days after TAC(p<0.0001),the amount of macrophages recruited to the heart was significantly reduced 7 days after TAC(p < 0.001).(3)Three days after TAC,the expression levels of inflammatory cytokines and chemokines in Myelo-KO mice were significantly lower than those in the control group(p<0.001).(4)Compared with the control group,HW/TL of Myelo-KO mice decreased significantly at 2 and 8 weeks after TAC(p<0.0001);LW/TL decreased significantly at 8 weeks after TAC(p<0.0001)).(5)Myelo-KO mice had significantly lower LVPWTd,LVDd,and LVDs at different time points(1,4,and 8 weeks)than those in the control group(p<0.01);FS was significantly higher than the control group(p< 0.0001).(6)The cross-sectional area of Myelo-KO mice at 8 weeks after TAC was significantly lower than that of the control group(p<0.001).(7)The area of cardiac interstitial fibrosis in Myelo-KO mice at 8 weeks after TAC was significantly lower than that in the control group(p<0.001).Conclusions: Myeloid-specific Wnt5 a knockout significantly reduced cardiac inflammatory response induced by pressure overload,significantly ameliorating cardiac hypertrophy and heart failure.PART ? EFFECT OF MYELOID-SPECIFIC WNT5 A OVEREXPRESSION ON CARDIAC HYPERTROPHY AND HEART FAILURE INDUCED BY PRESSURE OVERLOADObjective: To investigate the effect of myeloid-specific overexpression of Wnt5 a on cardiac hypertrophy and heart failure induced by pressure overload model.Methods:(1)Construction of Wnt5 a myeloid cell-specific overexpressing(Myelo-TG)mice.The bone marrow-derived neutrophils of control and Myelo-TG mice were extracted.The expression of Wnt5 a gene was detected by q PCR and the expression of Wnt5 a protein was detected by western blotting.(2)Sham and TAC-2 week treatment were performed in control and Wnt5 a Myelo-TG mice,and the number of Mac2 cells in the heart was detected by immunohistochemical staining.(3)Sham and TAC-7day treatment were performed in control and Wnt5 a Myelo-TG mice,and the number of neutrophils,monocytes,and macrophages recruited into the heart was measured by flow cytometry.(4)TAC was performed in control and Wnt5 a Myelo-TG mice.The expression levels of inflammatory cytokines Il1?,Il6,and chemokines Cxcl1,Cxcl2,Cxcl5,and Ccl2 genes were detected by q PCR at 3 days after surgery.(5)The mice were divided into control + TAC group,Wnt5 a Myelo-TG + TAC group.HW/TL,LW/TL were detected at different time points(0,2,8 weeks)after TAC.(6)The mice were divided into control + TAC group,Wnt5 a Myelo-TG + TAC group.The cardiac function parameters LVPWTd,LVDd,LVDs,and FS were measured by ultrasound at different time points(0,1,4,and 8 weeks)after TAC.(7)Sham and TAC-8 weeks treatment were performed in control and Wnt5 a Myelo-TG mice,and cardiac myocyte cross-sectional area was detected by immunofluorescence staining.(8)Sham and TAC-8 weeks treatment were performed in control and Wnt5 a Myelo-TG mice,and cardiac interstitial fibrosis area was detected by immunohistochemical staining.Results:(1)Overexpression of Wnt5 a gene and protein levels in bone marrow-derived neutrophils of Myelo-TG mice was confirmed.(2)Compared with the control group,the number of Mac2+ positives in Myelo-TG mice recruited to the heart 2 weeks after TAC was significantly increased(p<0.0001).(3)Compared with the control group,Myelo-TG mice recruited neutrophils to the heart 7 days after TAC,and the number of monocytes and macrophages increased significantly(p<0.05).(4)Compared with the control group,the expression levels of inflammatory cytokiens Il1?,Il6 and chemokine Cxcl2,Ccl2 genes in Myelo-TG mice were significantly increased(P<0.05).The chemokines Cxcl1 and Cxcl5 showed an increase trend.(5)Compared with the control group,HW/TL of Myelo-TG mice increased significantly at 2 and 8 weeks after TAC(p<0.01);LW/TL increased significantly at 8 weeks after TAC(p<0.001)).(6)Myelo-TG mice had significantly higher LVPWTd,LVDd,and LVDs at different time points(1,4,and 8 weeks)than those in the control group(p<0.001);FS was significantly lower than that of control group(p<0.0001).(7)The cross-sectional area of ? ? Myelo-TG mice at 8 weeks after TAC was significantly increased compared with the control group(p<0.001).(7)Myelo-TG mice had a significant increase in cardiac interstitial fibrosis area at 8 weeks after TAC compared with the control group(p<0.001).Conclusions: Myeloid-specific Wnt5 a overexpression significantly increased cardiac inflammatory response induced by pressure overload,significantly promotes cardiac hypertrophy and heart failure.PART ? EFFECT OF NEUTROPHIL DEPLETION ON CARDIAC EFFECT OF MYELOID-SPECIFIC WNT5A OVEREXPRESSION IN PRESSURE OVERLOAD INDUCTIONObjective: To investigate the effect of neutrophil depletion on cardiac effects of myeloid-specific overexpression of Wnt5 a in a pressure overload model.Methods:(1)A TAC model was established in control and Myelo-TG mice for 2 weeks,and each genotype mouse was homogenized at different time points(-2,1,4,7,10,13 days)of TAC surgery.Control or Ly6 G antibody injection eliminates neutrophils.(1)The HW/TL of each group of mice was measured.(2)The cardiac function parameters LVPWTd,LVDd,LVDs,FS of each group of mice after TAC 2 weeks were detected by ultrasound.(3)q PCR was used to detect the expression levels of myocardial injury factor ANP,?/?MHC gene in the hearts of mice in each group after 2 weeks of TAC.(4)The cross-sectional area of cardiac myocytes in each group of mice was detected by immunofluorescence staining.Results:(1)In WT mice and Myelo-TG mice,HW/TL at 2 weeks after TAC was significantly lower in mice injected with Ly6 G antibody than in the control group(p<0.05),and the degree of reduction inMyelo-TG mice was significantly higher.(2)In Myelo-TG mice,the injection of Ly6 G antibody reduced the elevation of LVPWTd,LVDd,LVDs to normal level(p<0.001),and the decrease of FS to normal level(p<0.0001),which was small compared with the control group.There was no significant difference in the rats.(3)In Myelo-TG mice,injection of Ly6 G antibody significantly decreased the expression levels of elevated myocardial injury factor ANP,?/?MHC gene(p<0.01).(4)In WT mice and Myelo-TG mice,the cross-sectional area of cardiac myocytes was significantly decreased in mice injected with Ly6 G antibody 2 weeks after TAC(p<0.0001),eliminating the difference between control group and Myelo-TG group.Conclusions: Neutrophil ablation reverses the exacerbated cardiac effect of overexpression of myeloid-specific Wnt5 a under pressure overload. |
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