Article Figures & Data
Figures
- FIGURE 1.
GM-CSF augments 2-deoxyglucose uptake and glycolytic flux in human macrophages via PFKFB3. (A and B) Human monocytes were differentiated to macrophages. Cells were exposed to human GM-CSF (10 ng/mL) for the specified time duration, and phosphorylation of STAT5 in Y694 (A) and levels of glycolytic enzymes (B) were determined. Addition of GM-CSF induced significant expression of hexokinase-1, hexokinase-2, and mainly PFKFB3. (C) Cells exposed to native GM-CSF demonstrated increased PFKFB3 expression compared with incubation with heat-inactivated GM-CSF (10 min at 80°C). (D) GM-CSF–mediated increase in PFKFB3 expression (right) was decreased with addition of silencer to PFKFB3 but not by selective PFKFB3 inhibiter 3PO, demonstrating specificity of silencer. Cells were treated with mixture of siPFKFB3 for silencing PFKFB3 18 h before challenge with GM-CSF. Results show mean ± SD. *P < 0.05 vs. same condition in control (absence of GM-CSF or 0 h). **P < 0.01 vs. same condition in control (absence of GM-CSF or 0 h). ##P < 0.01 for scRNA vs. siPFKFB3 with or without 3PO. a.u. = arbitrary units; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; HK = hexokinase; scRNA = scrambled RNA.
- FIGURE 2.
PFKFB3 mediates increase in 2-deoxyglucose uptake in human macrophages treated with GM-CSF. (A) Intracellular levels of Fru-2,6-P2 were determined at the indicated times with and without GM-CSF and in absence or presence of PFKFB3 inhibitor 3PO (5 μM). GM-CSF augments Fru-2,6-P2 levels, which are attenuated in presence of 3PO, demonstrating critical role of expression of PFKFB3 after GM-CSF treatment. (B) Lactate accumulation in culture medium was determined at 18 h with and without native or heat-inactivated GM-CSF and in absence or presence of 3PO (5 μM). Lactate levels (by-product of glycolysis) increase with GM-CSF exposure and are dampened by 3PO. (C) TNF-α levels were determined in culture medium after challenge with native or heat-inactivated GM-CSF. (D) Intracellular levels of Fru-2,6-P2 were determined at the indicated times with GM-CSF and in presence of neutralizing anti-TNF-α antibody. GM-CSF augments Fru-2,6-P2 levels, which are attenuated in presence of neutralizing anti-TNF-α antibody. (E) 2-deoxyglucose uptake was determined at the indicated times with and without GM-CSF and in absence or presence of neutralizing anti-TNF-α antibody (20 ng/mL) or PFKFB3 inhibitor 3PO (5 μM). 2-deoxyglucose significantly increases with addition of GM-CSF. This effect was diminished with neutralizing anti-TNF-α antibody and with 3PO. (F) To analyze contribution of PFKFB3 to enhancement of glycolytic flux elicited by GM-CSF, macrophages were transfected for 18 h with specific siRNA to silence PFKFB3 or with corresponding inactive RNA control (scRNA) and in absence or presence of 3PO, followed by activation for 18 h with GM-CSF. 2-deoxyglucose uptake was significantly decreased after silencing PFKFB3 compared with corresponding control (scRNA) or inhibition with 3PO. Data are mean ± SD. *P < 0.05 vs. same condition in control (absence of GM-CSF or 0 h). **P < 0.01 vs. same condition in control (absence of GM-CSF or 0 h). #P < 0.05 for GM-CSF vs. heated GM-CSF, with 3PO plus GM-CSF. ##P < 0.01 for GM-CSF vs. heated GM-CSF, with 3PO plus GM-CSF. IgG vs. anti-TNF-α antibody or scRNA vs. siPFKFB3 with or without 3PO. scRNA = scrambled RNA; siRNA = silencer RNA.
- FIGURE 3.
In vivo augmentation of 18F-FDG uptake after GM-CSF administration in mice. (A) 18F-FDG uptake in atherosclerotic mice (n = 30). Animals received siPFKFB3 (n = 10) or scRNA (n = 8) at days 3, 7, 10, and 12 after high-fat/high-cholesterol administration. GM-CSF (37.5 μg/kg; n = 15) or saline (n = 15) was intravenously administered on day 12, and 18F-FDG and PET analysis was performed on day 14. (B) Aortas were stained with oil red and images evaluated with Image J. (C) Average target and background SUVs from A were compared (n = 15 animals for each group). Target SUVs were higher with than without GMCSF (3.38 ± 0.46 vs. 2.70 ± 0.26; P = 0.046). Background SUVs were unchanged with vs. without GMCSF (0.54 ± 0.10 vs. 0.55 ± 0.10; P = 0.53). (D) In parallel experiment, aortas from untreated (n = 4) or GM-CSF–treated animals (n = 4) as described in A were isolated and RNA extracted for analysis of the indicated genes representative of endothelial cells (F8), smooth muscle cells (Sm22a, Myh11, Col1a1), and macrophages (Lxra). Data are mean ± SD. *P < 0.05 vs. same condition in absence of GM-CSF. ##P < 0.01 vs. same condition with scRNA. a.u. = arbitrary units; scRNA = scrambled RNA.
- FIGURE 4.
In vivo augmentation of 18F-FDG uptake after GM-CSF administration in rabbits. (A) 18F-FDG uptake in rabbits (n = 9) before and after challenge with cytokine GM-CSF or saline shows significant increase in 18F-FDG uptake in atherosclerotic rabbits (P < 0.001). 18F-FDG uptake is expressed as TBR compared with baseline imaging. (B) Representative image of 18F-FDG signal enhancement demonstrating increased 18F-FDG uptake in descending aorta before and after GM-CSF.
- FIGURE 5.
Relationship between arterial 18F-FDG uptake (by PET/CT) and histologic macrophage accumulation. Rabbits that had received GM-CSF (4 atherosclerotic and 2 control) were imaged using 18F-FDG PET/CT. Arterial 18F-FDG uptake was recorded as TBR. Subsequently, the animals were sacrificed and macrophage concentration within aortic wall was determined histologically (%RAM11 staining). There was significant relationship between macrophage staining and TBR (R = 0.76, P = 0.002).
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