Fig 2: TRAM-deficient BMMs propagate its resolving nature to neighboring monocytes through CD200R.(A) BMMs prepared from WT C57 BL/6 mice and Tram-/- mice, which were both CD45.2+, were transfected with CD200R siRNA or control siRNA with Lipofectamine. The CD45.2+ donor cells were then cocultured with recipient BMMs prepared from B6 SJL mice, which were CD45.1+, for 2 days. Surface expressions of CCR2, SIRP-a, and CD200R on CD45.1+ recipient BMMs were examined by flow cytometry. (B) Donor BMMs were prepared from WT C57BL/6 mice and Tram-/- mice, and recipient BMMs were prepared from B6 SJL mice. The donor cells were either directly cocultured with recipient cells or cultured in the upper chamber of a Transwell insert with recipient cells in the lower chamber. After 2 days, surface expressions of CCR2, SIRP-a, and CD200R on CD45.1+ recipient BMMs were examined by flow cytometry. Data are representative of 3 independent experiments, and error bars represent means ± SEM. **P < 0.01, and ***P < 0.001; 1-way ANOVA (n = 5 for each group).

Fig 3: TRAM deficiency reduces monocyte inflammatory state in atherosclerotic mice.Apoe-/- Tram+/+ mice and Apoe-/- Tram-/- mice were fed with HFD for 8 weeks. Surface expressions of CCR2 and SIRP-a on CD11b+Ly6C++ monocytes in the peripheral blood (A), BM (B), spleen (C), and aorta (D) were examined by flow cytometry. Data are representative of 2 independent experiments, and error bars represent means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001; Student’s 2-tailed t test (n = 5 to 10 for each group).

Fig 4: Endothelial CCL2 functions as a regulator of adrenomedullin expression by tumor cells in vivo. (a and b) 1.25 × 105 (a; n = 5 mice per group) or 2.5 × 105 (b; n = 6 mice per group) B16-F10 tumor cells were injected subcutaneously in control animals (a and b), EC-Ccl2-KO (a), EC-Gas-KO (b), or EC-Gas/Ccl2-dKO (b) mice, and tumor growth was determined. (c) Western blot analysis of mammary gland lysates dissected from control, EC-Gas-KO or EC-Gas/Ccl2-dKO mice showing protein levels of ADM and CCL2. Determination of tubulin protein levels served as a loading control. (d) Immunohistochemistry of B16-F10 tumors dissected from control, EC-Gas-KO, or EC-Gas/Ccl2-dKO mice. Sections were analyzed for markers of endothelial cells (PECAM1; green), perivascular cells (a-SMA; red) or proliferating cells (Ki67; red) and were stained with DAPI (blue). The bar diagrams show the statistical evaluation (n = 6). (e) B16-F10 cells transduced with scrambled control shRNA (control B16) or shRNA directed against CCR2 (CCR2-KD-B16) were injected subcutaneously in control or EC-Gas-KO mice, and tumor growth was determined (n = 5 mice for each condition). (f) Schematic representation showing the angiocrine role of CCL2 in the regulation of adrenomedullin expression by tumor cells and the effects of tumor cell–derived adrenomedullin on the proliferation of tumor and endothelial cells as well as endothelial CCL2 expression. Dashed lines indicate potential additional mechanisms of tumor growth regulation through CCL2. Bar length: 50 µm (d). Shown are mean values ± SEM; *, P = 0.05; **, P = 0.01; ***, P = 0.001 (two-way ANOVA and Bonferroni’s post hoc test [a, b, and e], and one-way ANOVA and Tukey’s post hoc test two-tailed Student’s t test [c and d]).

Fig 5: Endothelial CCL2 regulates adrenomedullin expression and release in tumor cells in vitro. (a–d) HUVECs were transfected with scrambled control siRNA or siRNA directed against GNAS and/or CCL2 and were cultured with GFP-MeWo for the indicated time periods. Endothelial cell proliferation was determined by staining for Ki67 (a), and the number of GFP-expressing tumor cells was determined by immunofluorescence (b) or determined as the protein level of GFP by Western blot analysis (c). In addition, expression of ADM was analyzed by immunoblotting (d). The bar diagrams show the statistical evaluation (a and b; n = 3 independent experiments) or the relative densitometric values based on Fiji software (c and d; n = 3 independent experiments). (e) HUVECs were transfected with control siRNA or siRNA directed against GNAS and cells were co-cultured for 24 h with MeWo cells transfected with control siRNA or siRNA directed against CCR2. Thereafter, the expression level of ADM was determined by Western blot analysis. The bar diagrams represent the relative densitometric values of the band recognized by the anti-ADM antibody based on Fiji software (n = 3 independent experiments). Bar length: 100 µm (a and b). Data represent mean values ± SEM; *, P = 0.05; **, P = 0.01; ***, P = 0.001 (two-way ANOVA and Bonferroni’s post hoc test [c and e], and one-way ANOVA and Tukey’s post hoc test [a, b, and d]).