Partial Inhibition of Glycolysis Reduces Atherogenesis Independent of Intraplaque Neovascularization in Mice

Abstract

Objective: Intraplaque neovascularization is an important feature of unstable human atherosclerotic plaques. However, its impact on plaque formation and stability is poorly studied. Because proliferating endothelial cells generate up to 85% of their ATP from glycolysis, we investigated whether pharmacological inhibition of glycolytic flux by the small-molecule 3PO (3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one) could have beneficial effects on plaque formation and composition. Approach and Results: ApoE^-/- (apolipoprotein E deficient) mice treated with 3PO (50 µg/g, ip; 4×/wk, 4 weeks) showed a metabolic switch toward ketone body formation. Treatment of ApoE^-/-Fbn1^C1039G+/- mice with 3PO (50 µg/g, ip) either after 4 (preventive, twice/wk, 10 weeks) or 16 weeks of Western diet (curative, 4×/wk, 4 weeks) inhibited intraplaque neovascularization by 50% and 38%, respectively. Plaque formation was significantly reduced in all 3PO-treated animals. This effect was independent of intraplaque neovascularization. In vitro experiments showed that 3PO favors an anti-inflammatory M2 macrophage subtype and suppresses an M1 proinflammatory phenotype. Moreover, 3PO induced autophagy, which in turn impaired NF-κB (nuclear factor-kappa B) signaling and inhibited TNF-α (tumor necrosis factor-alpha)-mediated VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) upregulation. Consistently, a preventive 3PO regimen reduced endothelial VCAM-1 expression in vivo. Furthermore, 3PO improved cardiac function in ApoE^-/-Fbn1^C1039G+/- mice after 10 weeks of treatment.

Conclusions: Partial inhibition of glycolysis restrained intraplaque angiogenesis without affecting plaque composition. However, less plaques were formed, which was accompanied by downregulation of endothelial adhesion molecules-an event that depends on autophagy induction. Inhibition of coronary plaque formation by 3PO resulted in an overall improved cardiac function.

Keywords: animals; atherosclerosis; autophagy; glycolysis; mice.

Figure 1.

3PO (3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one) inhibits intraplaque neovascularization in the carotid artery of ApoE^−/−Fbn1C1039G± mice. A, Differences in the occurrence (number of mice) and the number of intraplaque microvessels of control (ctrl) and 3PO-treated mice that underwent a curative or preventive 3PO regimen. *P<0.05, **P<0.01 vs ctrl (χ² [top] or Mann-Whitney U test [bottom]; ctrl, n=8–10; 3PO, n=9–13). B, Anti–Ter-119 staining of atherosclerotic plaques in the carotid artery of ctrl and 3PO-treated mice (curative regimen). Microvessels are marked by arrows. Scale bar=50 µm. M indicates media; and P, plaque.

Figure 2.

3PO (3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one) inhibits atherosclerotic plaque formation in ApoE^−/−Fbn1C1039G± mice. A, Plaque formation index of the right carotid artery as a measure of plaque occurrence in control (ctrl) and 3PO-treated mice that underwent a curative or preventive 3PO regimen. Plaque formation index is shown for all animals (left) of the ctrl group (n=12) and 3PO-treated group (n=9), for mice with intraplaque microvessels (middle; 10/12 ctrl mice and 3/9 treated mice) or for mice without intraplaque microvessels (right; 2/12 ctrl and 6/9 treated mice). *P<0.05, **P<0.01 vs ctrl (Mann-Whitney U test). B, En face Oil Red O staining of the carotid artery and aortic arch of ctrl (n=6–7) and 3PO-treated vessels (n=6–7). Scale bar=1.5 mm. *P<0.05, ***P<0.001 vs ctrl (independent samples t test). IP indicates intraplaque.

Figure 3.

3PO (3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one) stimulates autophagosome formation in endothelial cells independent of mTOR. A, Human aortic endothelial cells (HAOECs) were stimulated with 3PO (5–20 µmol/L) for 16 h followed by Western blot analysis for the autophagosomal marker protein LC3. Protein levels of LC3-I and LC3-II were quantified relative to the reference protein β-actin. *P<0.05 vs untreated control (ctrl) cells (1-way ANOVA, followed by Dunnett test, n=3). B, Detection and quantification of autophagic vacuoles (arrows) in 3PO-treated HAOECs (20 µmol/L 3PO, 16 h) using transmission electron microscopy. Scale bar=500 nm. ***P<0.001 (Mann-Whitney U test; n=3). C, Evaluation of the phosphorylation status of mTOR and its downstream target p70S6K in 3PO-treated HAOECs via Western blotting. Everolimus (EV; 10 µmol/L) was used as a positive control. LC3 indicates microtubule-associated protein light chain 3; and mTOR, mammalian target of rapamycin.

Figure 4.

3PO (3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one) impairs TNF-α (tumor necrosis factor-alpha)–mediated upregulation of VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) in endothelial cells via autophagy. A, Quantitative PCR and Western blot analysis of VCAM-1, ICAM-1, and ATG7 (autophagy-related 7) expression, either in wild-type (siCTRL-treated) human aortic endothelial cells (HAOECs; left) or siATG7-treated HAOECs (right) and exposed to hTNF-α (20 ng/mL) in the presence or absence of 3PO (10–20 µmol/L) for 24 h. β-actin was used as a reference gene. *P<0.05, **P<0.01 (1-way ANOVA followed by Dunnett test, n=3). B, Quantification of VCAM-1–positive endothelial cells in the carotid artery of ApoE^−/−Fbn1^C1039G+/− and the aortic arch of ApoE^−/− (apolipoprotein E deficient) mice treated with 3PO or solvent (control [ctrl]) for 10 wk (preventive regimen). *P<0.05, ***P<0.001 vs ctrl (unpaired Student t test, n=7–12).

Figure 5.

3PO (3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one) promotes a macrophage M2 phenotype. Bone marrow–derived macrophages from ApoE^−/− (apolipoprotein E deficient) mice were treated in vitro with 3PO (10–20 μM) or a mixture of IFN-γ (interferon-gamma; 20 ng/mL) and LPS (lipopolysaccharide; 100 ng/mL) for 24 h. Subsequently, mRNA expression of M1 exclusive genes CD38 (cluster of differentiation 38) and Gpr18 (G-protein−coupled receptor 18; A) or the M2-specific genes Erg2 (early growth response 2) and Arg1 (arginase 1; B) was analyzed by real-time polymerase chain reaction. *P<0.05 (Kruskal-Wallis followed by Mann-Whitney U test, n=4).