Lymphatic Endothelial C‐C Motif Chemokine Ligand 2 Enhances Lymphangiogenesis and Lymphatic Clearance of Macrophages Following Myocardial Infarction

Figure 1. The expression of Ccl2 is upregulated in lymphatic endothelial cells after acute myocardial infarction. A, A volcano plot illustrated the differential gene expression from RNA‐sequencing analysis in cardiac lymphatic endothelial cells at 3 d after myocardial infarction or sham operation. A total of 4381 downregulated genes (green) and 4888 upregulated genes (red) were identified (n=3, the n value represents biological replicates). B, Kyoto Encyclopedia of Genes and Genomes pathway analysis identified pathways enriched with differentially expressed genes between the sham control group and MI group. The chemokine signaling pathway exhibited the highest level of enrichment, as highlighted by the red box. (n=3, the n value represents biological replicates). C, The heat map highlighted the key differentially expressed genes between the sham control group and MI groups. Green represents down‐regulated genes, red represents upregulated genes. Ccl2, as one of the most significantly differentially expressed gene, is highlighted by the red box. (n=3, the n value represents biological replicates). D, Relative mRNA expression levels of Ccl2 in cardiac LECs of mice at 3 d after sham or left…

Figure 2. LEC‐Ccl2 deficiency worsens cardiac dysfunction after myocardial infarction. A, Representative images of cardiac echocardiography in WT and Ccl2 LECKO mice at 4 wk after sham or MI operation. B, Quantification of LVEF% in WT and Ccl2 LECKO mice at 4 wk after sham or MI operations (n=12, the n value represents the number of individual animals). C, Quantification of LVFS% in WT and Ccl2 LECKO mice at 4 wk after sham or MI operations (n=12, the n value represents the number of individual animals). D, The ratio of HW/BW (mg/g) in WT and Ccl2 LECKO mice at 4 wk after sham or MI operations (n=12, the n value represents the number of individual animals). E, Measurement of the LV mass in WT and Ccl2 LECKO mice at 4 wk after sham or MI operations (n=12, the n the number of individual animals). Data were shown as mean ± SEM. Two‐way ANOVA: (B, C, D, E). HW/BW indicates heart weight to bodyweight; LEC, lymphatic endothelial cell; LV, left ventricular; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; MI, myocardial infarction; and n.s., not significant.

Figure 3. LEC‐Ccl2 deficiency aggravates post‐MI adverse cardiac remodeling. A, Representative images of hematoxylin and eosin staining of serial sections from hearts in WT and Ccl2 LECKO mice at 28 d after sham or myocardial infarction operations. B, Representative images of Masson’s Trichrome staining of serial sections from hearts in WT and Ccl2 LECKO mice at 28 d after sham or myocardial infarction operations. C, with quantification of the percentage of infarct size in left ventricular myocardium in the MI group. (n=6, the n value represents the number of individual animals, and 3 serial sections/each mouse for quantification analysis). D, Representative images of wheat germ agglutinin staining of hearts in WT and Ccl2 LECKO mice at 28 d after myocardial infarction, with quantification of the cardiomyocyte cross‐section area in hearts (n=6, the n value represents the number of individual animals used in the experiment, and 5 randomly selected microscopic fields/ animal for analysis). E, Representative images of CD31 staining of hearts in WT and Ccl2 LECKO mice at 28 d after myocardial infarction, with quantification of capillary density in post‐MI myocardium (n=6, the n value…

Figure 4. The loss of LEC‐Ccl2 retards macrophage clearance in injured hearts. A, Representative images of immunostaining of Ly6G (neutrophil marker) from hearts in WT and Ccl2 LECKO mice at 1 d after myocardial infarction, with quantification of the number of neutrophils in myocardium of the indicated groups (n=6, the n value represents the number of individual animals, and 5 randomly selected microscopic fields/ animal for analysis). B, Representative images of immunostaining of F4/80 (macrophage marker) within hearts in WT and Ccl2 LECKO mice at 7 d after myocardial infarction, with quantification of the number of macrophages in myocardium of the indicated groups (n=6, the n value represents the number of individual animals, and 5 randomly selected microscopic fields/animal for analysis). C, Schematic diagram of the adoptive cell transfer approach. EGFP transgenic mice were used as splenic EGFP+ monocyte/macrophage donors, and recipient WT or Ccl2 LECKO adult mice received donor EGFP+ monocyte/macrophage via intramyocardial delivery at the time of left anterior descending coronary artery ligation. The trafficking of EGFP+ donor monocyte/macrophage was monitored in post‐MI hearts and…

Figure 5. CCL2 regulates lymphatic endothelial cell migration, proliferation, and angiogenic activity. A, RT‐qPCR analysis of CCL2 mRNA levels in HLECs in the indicated group (n=6, the n value represents biological replicates, and each biological replicate consists of 3 technical replicates). B, ELISA analysis of the CCL2 levels in HLECs supernatants in the indicated group (n=6, the n value represents biological replicates, and each biological replicate consists of 3 technical replicates). C, Scratch wound healing assay showed the cell migration ability in the indicated group, with quantification of the cell migration area (%) (n=6, the n value represents biological replicates). D, Boyden chamber assay showed the cell migration ability in the indicated group, with quantification of the number of migrated cells (n=6, the n value represents biological replicates). E, MTT assay showed cell proliferation ability of the indicated group (n=6, the n value represents biological replicates, and each biological replicate consists of 3 technical replicates). F, Tube formation assay showed the tube formation ability of HLECs in the indicated group, with quantification of the number of meshes,…

Figure 6. CCL2 boosts VEGFC expression in lymphatic endothelial cells via CCR2/AKT/ETS1 signaling pathway. A, Western blotting analysis of AKT and ETS1 activation status in the indicated groups with their quantification of the ratio of p‐AKT/T‐AKT and p‐ETS1/t‐ETS1 (n=6, the n value represents biological replicates). B, RT‐qPCR analysis of the VEGFC levels in HLECs in the indicated group (n=3, the n value represents biological replicates, and each biological replicate consists of 3 technical replicates). C, ELISA analysis of VEGFC levels in HLECs supernatants of the indicated group (n=6, the n value represents biological replicates, and each biological replicate consists of 5 technical replicates). Data were shown as mean ± SEM. One‐way ANOVA: (A through C). CCL2 indicates C‐C motif chemokine ligand 2; Control, empty lentivirus; ETS1, E26 oncogene homolog 1; LY294002, AKT inhibitor; OE, CCL2‐overexpressing lentivirus; RS102895, CCR2 inhibitor; RT‐qPCR, real‐time quantitative polymerase chain reaction; Scramble, scramble shRNA lentivirus; shRNA, CCL2 shRNA lentivirus; and VEGFC, vascular endothelial growth factor C.

Figure 7. CCL2 regulates lymphatic endothelial cell migration, proliferation, and angiogenic activity via CCR2/AKT/ETS1/VEGFC signaling pathway. A, Scratch wound healing assay showed the ability of cell migration, with quantification of cell migration area (%) of the indicated group (n=6, the n value represents biological replicate). B, Boyden chamber assay showed the ability of cell migration, with quantification of the number of migrated cells in the indicated group (n=6, the n value represents biological replicates). C, MTT assay showed the ability of cells proliferation in the indicated group (n=6, the n value represents biological replicates, and each biological replicate consists of 3 technical replicates). D, Tube formation assay showed the tube formation ability of HLECs, with quantification of the number of meshes, capillary length, branch points, and vascular network area coverage in the indicated group (n=6, the n value represents biological replicates, and each biological replicate consists of 5 technical replicates). Data were shown as mean±SEM. Scale Bars: A, B, D, 100 μm. One‐way ANOVA: (A through D). CCL2 indicates C‐C motif chemokine ligand 2; Control, empty lentivirus;…

Figure 8. Working model of lymphatic endothelial CCL2 enhances macrophage clearance via lymphatic system following myocardial infarction and promotes myocardial repair. CCL2 indicates C‐C motif chemokine ligand 2; ETS1, E26 oncogene homolog 1; and VEGF, vascular endothelial growth factor.