The Effect Of Monocyte-derived Macrophages On Myocardial Fibrosis In The Early Stage Of Pressure Overload In Mice

Background: Myocardial fibrosis is common in hypertension,myocardial infarction,rheumatic heart diseases and other cardiovascular diseases.Currently,due to unclear regulatory mechanism of the formation and development of myocardial fibrosis,there are still lack of effective interventions for it.Studies found that macrophages involved in the process of myocardial fibrosis by secreting inflammatory cytokines,growth factors,chemokines,matrix metalloproteinases and other factors.According to their originations,macrophages in myocardial tissue can be divided into monocyte-derived macrophages and yolk-sac-derived macrophages.Recent studies have confirmed that these two kinds of macrophages had significantly different role in cardiomyocytes proliferation and myocardial angiogenesis,but the role in myocardial fibrosis have not reported.Therefore,it is necessary to elucidate the role of different origination of macrophages in myocardial fibrosis.Objective: We established the TAC model to study the effects of macrophages from different originations on myocardial fibrosis in the early stage of pressure overload in mice.Methods:Part 11.1.40 male C57BL/6J mice(8～10weeks,20～25g)were divided into 4 groups(n=10):(1)sham operation group(SHAM),(2)3 days after TAC surgery(TAC3D),(3)7days after TAC surgery(TAC7D),(4)14 days after TAC surgery(TAC14D).1.2.The mice heart function was evaluated by the ejection fraction(EF)and left ventricular fractional shortening(FS)measured by Trans Thoracic Echocardiography.1.3.The changes of myocardial structure were evaluated by the ratio of heart weight to body weight and the diameter of cardiomyocytes.1.4.The degree of myocardial fibrosis was evaluated by Masson staining.1.5.The hypoxic degree of myocardial microenvironment was evaluated byhypoxyprobe.1.6.Graphs were generated with Graph Pad Prism 6(Graph Pad Software Inc.,La Jolla,CA,USA),and the data in the figures are expressed as the mean ± standard deviation.The differences among groups was tested with one-way analysis of variance(ANOVA)and between two groups with t-test.p<0.05 indicate a statistically significant difference.Part 22.1.40 male C57BL/6J mice(8～10weeks,20～25g)were divided into 4 groups(n=10):(1)Vehicle + sham operation group(Vehicle + SHAM),(2)Vehicle+ 14 days after TAC surgery(Vehicle + TAC14D),(3)CCR2 Inhibitor+ sham operation group(CCR2 Inhibitor+ SHAM),(4)CCR2 Inhibitor+14 days after TAC surgery(CCR2 Inhibitor + TAC14D).2.2.Macrophages were labeled with anti-F4/80 antibody and endothelial cells were labeled with anti-CD31 antibody,then we observed the distribution of macrophages.2.3.The proportion of macrophage subtypes from different originations were detected by the flow cytometry.2.4.We evaluated the degree of myocardial fibrosis by Masson staining.2.5.The hypoxic degree of myocardial microenvironment was evaluated by hypoxyprobe.2.6.Graphs were generated with Graph Pad Prism 6(Graph Pad Software Inc.,La Jolla,CA,USA),and the data in the figures are expressed as the mean ± standard deviation.The differences among many groups was tested with one-way analysis of variance(ANOVA)and two groups with t-test.p<0.05 indicate a statistically significant difference.Results:Part 1 The pathology changes of the myocardial fibrosis in the early phase of pressure overload1.1.The death rate of the mice with TAC surgery was 6.7%(2/30)after 2 weeks,and the SHAM group had no death.1.2.There was no significant difference in EF and FS between SHAM group and TAC14D(p>0.05).1.3.Myocardial structure occured changes induced by pressure overload in the early stage.The ratio of heart weight to body weight revealed significant cardiac growth in TAC hearts by day 3,and this increased further at 14 days(p<0.05).Myocyte width measured byWGA-stained cross-sections was significant changed(p<0.05).1.4.Masson staining showed the main characteristic was perivascular fibrosis in the early stage of pressure overload.Compared with the sham operation group,every groups with TAC surgery showed perivascular fibrosis(p<0.05).1.5.We used hypoxyprobe revealed the degree of hypoxia of myocardial microenvironment.Compared with the SHAM group,every groups with TAC surgery showed the degree of hypoxia of myocardial microenvironment significant serious(p<0.05).Part 2 The Effect of Macrophages from Different Sources on Myocardial Fibrosis in the Early Stage of Pressure Overload in the Heart2.1.Macrophages were labeled with anti-F4/80 antibodies and vascular endothelial cells were labeled with anti-CD31 antibodies,immunofluorescence staining results showed that the macrophages mainly appeared in the periphery of vessels,compared with SHAM group(p<0.05).2.2.The proportion of CCR2+ macrophages of the SHAM group and TAC 14 D group were detected with flow cytometry.Compared with SHAM groups,the proportion of CCR2+macrophages were significantly increased in TAC 14 D groups(p<0.05).2.3.With the intervention of CCR2 inhibitor,we observed that it could relieve the aggregation of macrophages in the periphery of vessels;it could reduce the proportion of CCR2+ macrophages(p<0.05),it could relieve perivascular fibrosis(p<0.05),it could relieve the degree of hypoxia of myocardial microenvironment(p<0.05).Conclusion:1.Perivascular fibrosis was a significant pathological change in the early stage of ventricular pressure overload.2.In the early stage of pressure overload,the macrophages mainly appeared in the periphery of vessels.At the same time,the proportion of monocyte-derived macrophages significantly increased.And if we reduced the proportion of them,the perivascular fibrosis could be alleviated.The monocyte-derived macrophages maybe a new target cell on treating cardiac fibrosis.