Inflammatory stimuli and hypoxia on atherosclerotic plaque thrombogenicity: Linking macrophage tissue factor and glycolysis

Abstract

Background: The thrombogenic potential of cells within atherosclerotic plaques is critical in the formation of a coronary thrombus. We hypothesized that a combination of inflammatory and hypoxic stimuli enhances tissue factor (TF) expression and glycolysis in cells in atherosclerotic plaques and contributes to coronary thrombus formation.

Aims: To identify TF- and hexokinase (HK)-II-expressing cells in coronary atherosclerotic plaques and thrombi and determine the effects of combined inflammatory and hypoxic stimuli and glycolysis on TF expression in peripheral blood mononuclear cell-derived macrophages.

Methods: We immunohistochemically assessed TF and HK-II expression in stable (n = 20) and unstable (n = 24) human coronary plaques and aspirated acute coronary thrombi (n = 15). The macrophages were stimulated with tumor necrosis factor-α, interferon-γ, or interleukin-10 under normoxic (21% O2) or hypoxic (1% O2) conditions, and TF expression was assessed.

Results: TF and HK-II expression were increased in unstable plaques compared with stable plaques. The number of CD68- and HK-II-immunopositive cells positively correlated with the number of TF-immunopositive cells. TF- and HK-II-expressing macrophages, which expressed M1- or M2-like markers, were involved in platelet-fibrin thrombus formation in ruptured plaques. The combination of inflammatory and hypoxic conditions additively augmented TF expression and procoagulant activity in the cultured macrophages. Inhibition of glycolysis with 2-deoxyglucose reduced the augmented TF expression and procoagulant activity.

Conclusion: Combined inflammatory and hypoxic conditions in atherosclerotic plaques may markedly enhance procoagulant activity in macrophages and contribute to coronary thrombus formation following plaque disruption. Macrophage TF expression may be associated with glycolysis.

Fig 1. Expression of TF and HK-II in stable and vulnerable coronary atherosclerotic plaques.

(A) Representative histological and immunohistochemical images of a fibrous cap atheroma (stable lesions). In the high-magnification images (square), the stable plaque shows a mild infiltrate of CD68-immunopositive cells around the necrotic core and modest expression of TF and HK-II. (B) Representative histological and immunohistochemical images of a thin-cap fibroatheroma (vulnerable lesions). In the high-magnification images (square), the vulnerable plaque shows the presence of CD68-immunopositive cells in the fibrous cap and necrotic core. TF- and HK-II-immunopositive cells are predominantly localized in macrophage-rich areas. (C) Representative immunohistochemical high-magnification images of a thin-cap fibroatheroma (vulnerable lesions). CD68-immunopositive cells are immunopositive for TF and HK-II (arrow) but not SMA.

Fig 2. Immunohistochemical expression of HK-II, TF, and vascular cells in stable and vulnerable lesions of human coronary arteries.

(A) HK-II, TF, CD68, CD31, and CD3 expression in stable and vulnerable human coronary lesions. Mann–Whitney U test. (B) Relationships among CD68, HK-II, CD3, CD31, and TF expression in the coronary artery. Spearman rank correlation analysis.

Fig 3. Representative histological and immunohistochemical images of aspirated thrombus with ruptured plaque in patients with ACS.

The ruptured plaque and thrombus interface demonstrated the involvement of CD68-, HK-II-, and TF-triple immunopositive cells in fibrin and platelet aggregation (arrows). Fibrin formation is localized in the ruptured plaque and a part of the thrombus (asterisks). Platelet aggregation is observed on ruptured plaque (arrowheads). The dotted lines indicate the boundary between thrombus (T) and ruptured plaque (P).

Fig 4. TF expression and procoagulant activity in PBMC-derived macrophages under inflammatory stimuli and hypoxia.

PBMC-derived macrophages were stimulated by TNFα (10 mg/mL) and INF-γ (20 ng/mL), hypoxia (1% O2), or IL-10 (10 ng/mL) for 4 h (TF mRNA expression) or 24 h (TF protein expression and procoagulant activity). Data are expressed as fold changes in comparison with the nonstimulated control. One-way analysis of variance was performed with Sidak’s multiple-comparison tests (A, TF mRNA and TF protein; B, TF mRNA). Kruskal–Wallis test was performed with Dunn’s multiple-comparison test (A, procoagulant activity; B, TF protein, procoagulant activity).

Fig 5. HK-II expression and lactate production in PBMC-derived macrophages under inflammatory stimuli and hypoxia.

PBMC-derived macrophages were stimulated by TNFα (10 mg/mL) and INF-γ (20 ng/mL), hypoxia (1% O2), or IL-10 (10 ng/mL) for 4 h (A, HK-II mRNA expression) or 24 h (B, C, lactate production). Data are expressed as fold changes in comparison with the nonstimulated control. Kruskal–Wallis test was performed with Dunn’s multiple-comparison test (HK-II mRNA), and a one-way analysis of variance was performed with Sidak’s multiple-comparison test (lactate levels).

Fig 6. Lactate production, TF expression, and procoagulant activity in PBMC-derived macrophages under inflammatory stimuli, hypoxia, and inhibition of glycolysis.

PBMC-derived macrophages were stimulated by TNFα (10 mg/mL) and INF-γ (20 ng/mL) and hypoxia (1% O₂) for 4 h (TF mRNA expression) or 24 h (lactate production, TF protein expression, and procoagulant activity). Data are expressed as fold changes in comparison with the nonstimulated control. (A) Lactate production and TF expression in PBMC-derived macrophages under inflammatory stimuli, hypoxia, and inhibition of glycolysis. A one-way analysis of variance was performed with Sidak’s multiple-comparison test (lactate levels, TF mRNA upper, and TF proteins), and the Kruskal–Wallis test was performed with Dunn’s multiple-comparison test (TF mRNA lower). (B) Procoagulant activity in PBMC-derived macrophages under inflammatory stimuli, hypoxia, and inhibition of glycolysis. One-way analysis of variance was performed with Sidak’s multiple-comparison test or Student’s t-test.