Pro-inflammatory macrophages coupled with glycolysis remodel adipose vasculature by producing platelet-derived growth factor-B in obesity

Figure 1. Impact of adipose macrophage deletion on gene expression and tissue weight in the WAT of HFD-fed mice. (a) Experimental protocol for the liposome injection. Liposome-encapsulated clodronate (Clod) or PBS (Veh) was administered twice a week for 2 or 6 weeks to mice fed HFD for 6 weeks, and maintained on HFD during the administration protocol. (b) Representative scatter plots of flow cytometric analyses for macrophages in the SVF of eWAT from mice given Veh or Clod for 2 (upper) and 6 (lower) weeks. (c) Percentage of living CD45+F4/80+ macrophages (ATMs) in the SVF of eWAT from mice given Veh or Clod for 2 (left) and 6 (right) weeks. (d–g) Relative expression levels of Pdgfb and Vegfa mRNA in eWAT from mice given Veh or Clod for 2 and 6 weeks. (h–k) Body and eWAT weights of mice after the 2- or 6-week administration protocol. Regarding the 2-week treatment, n = 7–9; for the 6-week treatment, n = 8–12. Data are shown as means ± S.E. *p < 0.05 and **p < 0.01, significantly different from Veh.

Figure 2. Impact of adipose macrophage deletion on pericyte coverage and vascular density in the eWAT of HFD-fed mice. (a,c) Representative images of pericytes and endothelial cells in eWAT from mice given Veh or Clod for 2 and 6 weeks. Pericytes and endothelial cells were stained with anti-CD13 (green) and anti-PECAM-1 (red) antibodies, respectively. (b,d) Percentage of the pericyte association with endothelial cells in eWAT from mice given Veh or Clod for 2 and 6 weeks. Percentages were quantified by the ratio of the associated area (yellow) to the endothelial area. (e,f) Representative images of endothelial cells stained with the anti-PECAM-1 antibody and quantified vessel area in the eWAT of mice given Veh or Clod for 6 weeks. Regarding the 2-week treatment, n = 7–9; for the 6-week treatment, n = 8–12. Data are shown as means ± S.E. *p < 0.05, significantly different from Veh.

Figure 3. Impact of high glucose conditions on Pdgfb expression in macrophages. (a) Pdgfb mRNA expression levels in peritoneal macrophages treated with 100 ng/mL LPS or 10 ng/mL IL-4 under 11 mM glucose for 24 hours. n = 4 (b) Representative scatter plot of living CD45+F4/80+ peritoneal macrophages and a histogram of 2-NBDG in these cells by flow cytometry. (c) Relative changes in the mean fluorescent intensity (MFI) of 2-NBDG in living CD45+F4/80+ peritoneal macrophages treated with 100 ng/mL LPS for 24 hours. n = 3. (d–f) Relative expression levels of Itgax, Mrc1, and Pdgfb mRNA in RAW264.7 cells treated with several glucose concentrations for 24 hours. Mean of data among six independent experiments. n = 14–15. (g) Relative expression levels of Pdgfb mRNA in RAW264.7 cells treated with 100 ng/mL LPS for 3 hours under several glucose concentrations. Mean of data among three independent experiments. n = 6. (h) Relative lactic acid contents in the cultured medium of RAW264.7 cells treated with 100 ng/mL LPS for 3 hours under several glucose concentrations. Mean of data among three independent experiments. n = 4. Data are shown as means ± S.E. *p < 0.05 and **p < 0.01, among two groups, as…

Figure 4. Importance of glycolysis for LPS-induced Pdgfb mRNA expression in macrophages. (a) Schematic pathway of glycolytic metabolism. The terminals of half lines represent the targets of inhibitors. (b) Relative expression levels of Pdgfb mRNA in peritoneal macrophages pretreated with 10 mM 2-DG for 0.5 hours and stimulated with 100 ng/mL LPS or 10 ng/mL IL-4 for 24 hours under 11 mM glucose. n = 4. (c–f) Relative expression levels of Pdgfb, Tnfa, and Il6 mRNA in RAW264.7 cells pretreated with 2-DG or HA for 0.5 hours and stimulated with LPS for 3 hours under 11 mM glucose. Mean of data among three or four independent experiments. n = 6 or 5. (g) Representative images blotted with anti-GAPDH and anti-α-Tubulin antibodies, and relative signal density of GAPDH normalized with that of α-Tubulin in siGapdh-transfected RAW264.7 cells. Mean of data among three independent experiments. n = 6. (h) Relative expression levels of Pdgfb mRNA in RAW264.7 cells stimulated with LPS for 3 hours under 11 mM glucose after siGapdh transfection. Mean of data among three independent experiments. n = 6. (i) Relative expression levels of Pdgfb mRNA in RAW264.7 cells pretreated with 10 mM 2-DG for 0.5 hours,…

Figure 5. Relevance of p65 and mTORC1 signaling pathways to LPS-induced Pdgfb mRNA expression coupled with glycolysis in macrophages. (a) Representative images blotted with anti-phospho-p65 and anti-p65 antibodies, and relative signal density of phospho-p65 normalized with that of p65 in RAW264.7 cells pretreated with 10 mM 2-DG for 0.5 hour and stimulated with 100 ng/mL LPS for 0.5 hours. Mean of data among four independent experiments. n = 4. Statistical analyses were performed under the same glucose concentration. (b) Representative images blotted with anti-phospho-p65 and anti-p65 antibodies, and relative signal density of phospho-p65 normalized with that of p65 in RAW264.7 cells pretreated with HA for 0.5 hours and stimulated with 100 ng/mL LPS for 3 hours. Mean of data among three independent experiments. n = 4. (c) Representative images blotted with anti-p65 and anti-α-Tubulin antibodies, and relative signal density of p65 normalized with that of α-Tubulin in siRela-transfected RAW264.7 cells. Mean of data among three independent experiments. n = 6. (d,e) Relative expression levels of Il6 and Pdgfb mRNA in RAW264.7 cells stimulated with 100 ng/mL LPS for 3 hours after siRela…

Figure 6. Relevance of MAPK signaling pathways to LPS-induced Pdgfb mRNA expression coupled with glycolysis in macrophages. (a-c) Representative images blotted with anti-phospho-ERK1/2, anti-ERK, anti-phospho-JNK, anti-JNK, anti-phospho-p38, and anti-p38 antibodies, and relative signal density of phosphorylated protein normalized with that of each total protein in RAW264.7 cells pretreated with 10 mM 2-DG for 0.5 hours and stimulated with 100 ng/mL LPS for 3 hours. Mean of data among four independent experiments. n = 4. Statistical analyses were performed among the same glucose concentration. (d) Representative images blotted with anti-phospho-ERK1/2 and anti-ERK1/2 antibodies and the relative signal density of phospho-ERK2 normalized with that of ERK2 in RAW264.7 cells pretreated with HA for 0.5 hours and stimulated with 100 ng/mL LPS for 3 hours. Mean of data among three independent experiments. n = 4. (e) Relative expression levels of Pdgfb mRNA in RAW264.7 cells pretreated with U0126 for 2 hours and stimulated with 100 ng/mL LPS for 3 hours. Mean of data among three independent experiments. n = 6. (f) Representative images blotted with anti-ERK1/2 and anti-α-Tubulin antibodies, and the…