Figure 7

The inhibition of CCR2 positive monocyte infiltration changed the cardiac proportion of L⁻ and L⁺ macrophage subsets and modified the lymphatic network. (A) Number of monocytes positive for CCR2 after one week of TAC in heart of mice treated with either vehicle (TAC veh) or RS504393 (TAC RS) *P < 0.05 by t test. (B) Quantification of L⁺ macrophages present in heart of vehicle (TAC veh) and RS504393 (TAC RS) treated mice. **P < 0.01 by t test. (C) Flow cytometry quantification of Ki67 positive L⁺ macrophages in cardiac tissue. *P < 0.05 by t test. (D) Flow cytometry quantification of L⁻ macrophages present in heart of vehicle (TAC veh) and RS504393 (TAC RS) treated mice. (E) Quantification of lymphatic endothelial cells by flow cytometry identified as CD45⁻CD31⁺LYVE-1⁺ in cardiac tissue of TAC vehicle and TAC RS504393. *P < 0.05 by t test (Gating strategy detailed in supplementary Figure 3C). (F) Percentage of Ki67 positive lymphatic endothelial cells identified and evaluated by flow cytometry in cardiac tissue of TAC vehicle and TAC RS504393. (G) Immunofluorescence quantification of lymphatic vessel area in cardiac tissue of mice submitted to 1 week of TAC and treated with either vehicle (TAC veh) or RS504393 (TAC RS). *P < 0.05; by t test. (H) Flow cytometry determination of LYVE-1 expression (mean fluorescence intensity or MFI) on CD45^-CD31⁺LYVE-1⁺ cardiac endothelial lymphatic cell surface after 1 week of TAC of mice treated with either vehicle (TAC veh) or RS504393 (TAC RS). Data are presented in mean ± SEM. Significance *P < 0.05; by t test. (I) Relative expression of MMP12 in heart of mice submitted to 1 week of TAC and treated with either vehicle (TAC veh) or RS504393 (TAC RS). mRNA expression of gene was normalized to TAC RS, *P < 0.05; by t test. (J,K) Representative images of Sirius red staining of heart after 1 week of TAC from mice treated with either vehicle (TAC veh) or RS504393 (TAC RS) and quantification of fibrosis content (K) . **P < 0.01; by t test.