Fig 2: TRAM deficiency attenuates inflammatory polarization of BMMs in vitro.BM cells from WT C57 BL/6 mice and Tram-/- mice were cultured with M-CSF (10 ng/mL) in the presence of PBS or super-low-dose LPS (100 pg/mL) for 5 days. Surface expressions of CCR2 and SIRP-a on CD11b+Ly6C++ BMMs (A) and CD11b+Ly6C+ BMMs (B) were examined by flow cytometry. (C) The phosphorylation of STAT1, STAT5, and SRC in BMMs was examined by Western blotting, and relative levels were normalized to ß-actin. Data are representative of 3 independent experiments, and error bars represent means ± SEM. **P < 0.01, and ***P < 0.001; 1-way ANOVA (n = 3 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 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]).

Fig 5: Effects of endothelial loss of Gas on retinal angiogenesis and tumor angiogenesis. (a) Whole mount retinae of control and EC-Gas-KO mice at P7 stained with Isolectin B4. Quantification of vasculature parameters are presented as bar diagrams (n = 5 mice). (b) Immunohistochemistry of B16-F10 tumors dissected from control or EC-Gas-KO mice. Sections were analyzed for a marker of apoptosis (cleaved caspase3; green) or of hypoxia (Hif1a; red) and were stained with DAPI (blue; n = 6 mice for each genotype). (c–e) Immunohistochemistry of LLC1 tumors dissected from control or EC-Gas-KO mice. Sections were analyzed for markers of endothelial cells (PECAM1 or VE-Cad; green), lymphatic endothelial cell (Prox1; red), perivascular cells (a-SMA; red), proliferating cells (Ki67; red), hypoxia (Hif1 a; red), as well as M1 and M2 macrophages (CD68 and CD206), and were stained with DAPI (blue). The bar diagrams show the statistical evaluation (n = 6 mice for each genotype). (f) Flow cytometric analysis of immune cells in tumors. LLC1 tumors dissected from control or EC-Gas-KO mice were dissociated, and populations of neutrophils, B cells, CD8+ T cells, CD4+ T cells, and macrophages were analyzed by determining the expression of Ly6G, CD8, CD4, and F4/80, respectively. F4/80 expressing macrophage were also evaluated for their expression of the M2 marker CD206 or of the CCL2 receptor CCR2 (n = 10 mice per group). Bar length: 500 µm, 100 µm (a); 50 µm (b–e). Data represent mean values ± SEM; *, P = 0.05; **, P = 0.01; ***, P = 0.001 (two-tailed Student’s t test).