The glycolytic enzyme PKM2 bridges metabolic and inflammatory dysfunction in coronary artery disease

Abstract

Abnormal glucose metabolism and enhanced oxidative stress accelerate cardiovascular disease, a chronic inflammatory condition causing high morbidity and mortality. Here, we report that in monocytes and macrophages of patients with atherosclerotic coronary artery disease (CAD), overutilization of glucose promotes excessive and prolonged production of the cytokines IL-6 and IL-1β, driving systemic and tissue inflammation. In patient-derived monocytes and macrophages, increased glucose uptake and glycolytic flux fuel the generation of mitochondrial reactive oxygen species, which in turn promote dimerization of the glycolytic enzyme pyruvate kinase M2 (PKM2) and enable its nuclear translocation. Nuclear PKM2 functions as a protein kinase that phosphorylates the transcription factor STAT3, thus boosting IL-6 and IL-1β production. Reducing glycolysis, scavenging superoxide and enforcing PKM2 tetramerization correct the proinflammatory phenotype of CAD macrophages. In essence, PKM2 serves a previously unidentified role as a molecular integrator of metabolic dysfunction, oxidative stress and tissue inflammation and represents a novel therapeutic target in cardiovascular disease.

Figure 1.

Hyperinflammatory monocytes in CAD patients. PBMCs isolated from healthy controls and CAD patients were stained with CD14, CD16, and HLA-DR, and frequencies of monocyte subsets were measured by flow cytometry. Representative dot plots (A) and frequencies of the three monocyte subpopulations (B) are shown. (C) Monocytes from seven controls and seven CAD patients were stimulated with LPS/IFN-γ, and RNA expression of genes was measured by quantitative RT-PCR. Heat map displays expression of genes with data presented as the log₂ value. (D) Classical and intermediate monocytes were sorted by flow cytometry, differentiated into macrophages, and stimulated with LPS/IFN-γ, and then cytokine RNA expression was measured by RT-PCR (n = 6). Values are mean ± SEM. *, P < 0.05; **, P < 0.01.

Figure 2.

CAD macrophages produce excessive IL-6 and IL-1β. (A) Monocytes were isolated from five healthy controls and five CAD patients, differentiated into macrophages using macrophage colony-stimulating factor (M-CSF) and polarized into M1 and M2 cells. Gene expression was measured using quantitative RT-PCR. Heat map displays expression of genes with data presented as the log₂ values. (B) Macrophages from healthy controls and CAD patients were stimulated with LPS/IFN-γ for 6 and 24 h. CD14 expression was analyzed by flow cytometry. Representative histograms are shown. (C) Frequencies of CD14^low macrophages in five healthy controls and five CAD patients at indicated time points after stimulation. (D) Production of IL-1β, IL-6, and TNF was measured by intracellular staining using flow cytometry in M0 (dashed lines) and M1 macrophages (solid lines). Percentage of cytokine producing macrophages (E) and mean fluorescent intensities (MFI) of intracellular cytokine stains (F) in the CD14^low population 6 h after LPS/IFN-γ stimulation (15 controls [blue] and 16 CAD patients [red]). (G) Cytokine production at the indicated time points after LPS/IFN-γ stimulation assessed by intracellular cytokine staining in healthy controls (blue; n = 8) and CAD patients (red; n = 7). (H) Frequencies of cytokine-producing macrophages were compared in healthy individuals not taking medications (n = 14) and those taking the indicated medications (n = 5 per treatment). Values are mean ± SEM. *, P < 0.05; **, P < 0.01.

Figure 3.

IL-6–producing macrophages in patients with CAD. (A) Frozen sections of carotid atheromas were immunostained with anti–IL-6 (green), anti-CD68 (red), and DAPI (blue), and analyzed by fluorescence microscopy. One representative of three independent experiments. Bar, 20 µm. (B) Plasma high-sensitivity CRP (hsCRP) is correlated with the frequency of ex vivo–generated IL-6–producing macrophages for each individual patient. (C) Patients are stratified according to the absence and presence of type 2 diabetes mellitus (DM), hypertension (HTN), and hyperlipidemia (HL). Each symbol represents the frequency of IL-6–producing macrophages from each individual patient. (D) The frequency of IL-6–producing macrophages in each individual patient is correlated with the number of comorbidities (DM, HTN, or HL). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.

ROS scavenging corrects the inflammatory phenotype of CAD macrophages. (A) Resting or activated monocytes were loaded with 5 µM CellROX Deep Red and intracellular ROS levels were analyzed by flow cytometry. Summarized mean fluorescent intensities (MFI) from n = 10 healthy controls and n = 10 CAD patients are shown. (B and C) Macrophages from controls and CAD patients were stimulated with LPS/IFN-γ for 2, 4, 6, and 24 h. Cells were then loaded with CellROX Deep Red and intracellular ROS levels were analyzed by flow cytometry. Representative dot plots (B) and MFI from macrophages of n = 7 healthy controls and n = 7 CAD patients (C) are shown. (D and E) CAD macrophages were stimulated for 6 h under M1-polarizing conditions in the absence (red) and presence (green) of the ROS scavenger Tempol (50 µM) and intracellular cytokines were measured by flow cytometry. (F) Total RNA was purified from ex vivo–generated macrophages, and expression of NOXs was measured by qRT-PCR (n = 3). (G) Macrophages were transfected with either siControl RNA or siNOX2 RNA, and total RNA was purified at the indicated times. NOX2 expression was measured by qRT-PCR (n = 3). (H) CAD macrophages were transfected with control or NOX2 siRNA and intracellular cytokines were measured by flow cytometry after 6 h of stimulation (n = 5). (I) CAD macrophages were stimulated in the absence and presence of gp91dstat (50 µM). Intracellular cytokines were measured by flow cytometry (n = 5). (J and K) CAD macrophages were treated with or without Mitotempo (20 µM). Intracellular cytokines were analyzed after 6 h of stimulation (n = 6). All data are mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5.

Glucose deprivation disrupts proinflammatory effector functions and ROS production. M0 macrophages were generated from CAD patients, stimulated with LPS/IFN-γ for 6 h and intracellular cytokines were measured by flow cytometry. Glycolytic activity was suppressed with 10 mM of 2-DG (A and B) or glucose-free medium (C and D). Representative histograms of intracellular cytokine stains and frequencies of cytokine-producing macrophages from five independent experiments are shown. (E) CAD macrophages were stimulated with LPS and IFN-γ for 6 h in the absence or presence of 10 mM of 2-DG. Macrophages were then stained with 7-AAD and viability of cells was measured by flow cytometry. Representative histograms from four independent experiments are shown. (F) CAD macrophages were stimulated with LPS and IFN-γ for 6 h in the absence or presence of 10 mM of 2-DG or 50 µM of Tempol. Expression of IL-1β and IL-6 was measured by qRT-PCR (n = 4). (G) Glucose uptake in monocytes was measured using the fluorescence-labeled glucose analogue, 2-NBDG. Summarized MFI from 12 independent experiments are shown. (H) Glucose uptake in macrophages was measured using the 2-NBDG. Summarized MFI from 10 independent experiments are shown. (I) Macrophages from CAD patients were stimulated for 4 h, and loaded with 5 µM of MitoSOX to quantify mtROS levels. Data represent ΔMFI compared with resting cells from four independent experiments. All data are mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

Mitochondrial respiration and glycolysis assays in monocytes and macrophages. (A–C) OCRs (A) and ECARs (B) were measured with the Seahorse Bioscience XF96 analyzer in monocytes 3 h after LPS/IFN-γ stimulation. Summarized graph from seven healthy controls and eight CAD patients is shown. Mitochondrial function was probed by the serial addition of oligomycin, FCCP, and antimycin A/rotenone as indicated. (C) ECAR-OCR ratios in controls and CAD patients. (D–F) OCR (D) and ECAR (E) were determined with the Seahorse Bioscience XF96 analyzer in macrophages 3 h after LPS/IFN-γ stimulation. Summarized graph from four healthy controls and six CAD patients is presented. Oligomycin, FCCP, and antimycin A/rotenone were added serially as in A and B. (F) ECAR-OCR ratios calculated for controls and patients. All data are mean ± SEM. *, P < 0.05; **, P < 0.01.

Figure 7.

Overexpression of the glycolytic enzyme PKM2 in CAD macrophages. (A) Ex vivo–generated macrophages from five controls and five CAD patients were stimulated with LPS/IFN-γ (M1) or IL-4/IL-13 (M2) for 48 h and genes related to glucose metabolism were quantified by RT-PCR. Heat map displays the fold change increase of gene expression presented as the log₂ value of relative mRNA expression (see color scale). (B) Total RNA was purified from macrophages, and expression of the glucose transporters GLUT1-4 was measured by qRT-PCR (n = 3). (C) Gene expression of the PKM1 and PKM2 isoforms in CAD macrophages was assessed by RT-PCR in three independent experiments. (D) Confocal images were acquired in ex vivo–generated macrophages stimulated with LPS/IFN-γ for 3 h and stained with anti-PKM2 (green). Nuclei were localized by DAPI (blue). Bars, 20 µm. (E) Bar graph represents averaged data from experiments quantifying the fluorescent signal within nuclei (n = 4 healthy controls and n = 7 CAD patients). All data are mean ± SEM. **, P < 0.01. (F) Frozen sections of carotid atheromas were stained with anti-PKM2 (green), anti-CD68 (red), and DAPI (blue) and analyzed by fluorescence microscopy. One representative of four independent experiments is shown. Bars, 100 µm; (inset) 20 µm.

Figure 8.

Dimeric PKM2 in inflammatory CAD monocytes and macrophages. (A) Monocytes from healthy controls and CAD patients were activated with LPS/IFN-γ. Cells were treated with 5 mM of di-succinimidyl suberate for 30 min before lysis, and protein extracts were analyzed by immunoblotting to identify oligomeric forms of PKM2. (B) Bar graph represents averaged data from n = 8 healthy controls and n = 9 CAD patients. (C) Macrophages from healthy controls and CAD patients were stimulated with LPS/IFN-γ, and protein extracts were analyzed by immunoblotting to identify oligomeric forms of PKM2. GAPDH was used as a loading control. (D) Bar graph represents averaged immunoblot data from seven independent experiments. (E) CAD macrophages were stained with anti-PKM2 (green) as indicated. Aliquots were treated with Tempol (50 µM) or 2-DG (10 mM). Distribution of PKM2 was analyzed by confocal microscopy. Nuclei were localized by DAPI (blue). Bar, 50 µm. (F) The bar graphs represent averaged data of the fluorescent signal within the nucleus (n = 5–6). (G) Nuclear protein extracts from CAD macrophages were analyzed by immunoblotting to identify PKM2. Lamin A/C was used as loading control. One representative of two independent experiments is shown. (H) Tetrameric assemblies of PKM2 were resolved by immunoblotting protein extracts of CAD macrophages prepared under nonreducing conditions. Aliquots were stimulated in the presence of Tempol or 2-DG. One representative of three independent experiments is shown. (I and J) PKM2 dimerization was inhibited in CAD macrophages by treating with ML265 (50 µM). Intracellular cytokines were measured by flow cytometry. Representative histograms (I) and summary of 8 independent experiments (J) are shown. Values are mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 9.

PKM2 regulates inflammatory effector functions via phosphorylation of STAT3. (A) CAD macrophages were stimulated for 6 h with or without the HIF-1α inhibitor CAS 934593–90-5 (10 µM). Intracellular cytokines were measured by flow cytometry in n = 5 experiments. (B) CAD macrophages were stimulated for 0, 3, and 24 h, and phosphorylation of STAT3 (Y705) was analyzed by flow cytometry. One representative of 3 independent experiments is shown. (C and D) CAD macrophages were stimulated for 6 h with or without the STAT3 inhibitor Stattic (5 µM) and intracellular cytokines were measured by flow cytometry. Representative histograms and results from 7 independent experiments are shown. (E–G) pSTAT3 was quantified by flow cytometry in CAD macrophages 3 h after stimulation in the absence or presence of Tempol (E; n = 5), 2-DG (F; n = 5), or glucose-free medium (G; n = 6). The fold change in MFI is plotted. (H and I) CAD macrophages were treated with ML265, stimulated and analyzed for pSTAT3 by flow cytometry. Representative histograms and results from 5 independent experiments. (J) Co-localization of pSTAT3 and PKM2 analyzed by confocal microscopy. Resting (M0) or M1-stimulated CAD macrophages in the absence (M1) and presence of Tempol, 2-DG, or ML265, were stained with anti-pSTAT3 and anti-PKM2. Close proximity (<40 nm) of pSTAT3 and PKM2 produces a red fluorescent signal. The blue signal indicates DAPI-stained nuclei. Bar, 50 µm. (K) Quantification of the fluorescent signal produced by pSTAT3/PKM2 co-localization (n = 4). Values represent mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.