Deletion of pyruvate dehydrogenase kinases reduces susceptibility to deep vein thrombosis in mice

Abstract

Neutrophils contribute to deep vein thrombosis (DVT) by releasing prothrombotic neutrophil extracellular traps (NETs). NET formation (known as NETosis) is an energy-intensive process that requires an increased rate of aerobic glycolysis. The metabolic enzymes pyruvate dehydrogenase kinases (PDKs) inhibit the pyruvate dehydrogenase complex to divert the pyruvate flux from oxidative phosphorylation toward aerobic glycolysis. Herein, we identified that the combined deletion of PDK2 and PDK4 (PDK2/4-/-) renders mice less susceptible to DVT (measured by thrombus incidence, weight, and length) in the inferior vena cava-stenosis model at day 2 after surgery. Compared with wild-type (WT) mice, the venous thrombus obtained from PDK2/4-/- mice exhibited reduced citrullinated histone content, a known marker of NETs. In line with in vivo observations, phorbol 12-myristate 13-acetate (PMA)-stimulated PDK2/4-/- neutrophils displayed reduced NETosis and secretion of cathepsin G and elastase compared with PMA-stimulated WT neutrophils. The formation of platelet aggregates mediated by PMA-stimulated PDK2/4-/- neutrophils were significantly reduced compared with PMA-stimulated WT neutrophils. Finally, PDK2/4-/- neutrophils exhibited reduced levels of intracellular Ca2+ concentration, extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation, and glycolytic proton efflux rate (a measure of aerobic glycolysis), known to facilitate NETosis. Together, these findings elucidate, to our knowledge, for the first time, the fundamental role of PDK2/4 in regulating NETosis and acute DVT.

Graphical abstract

Figure 1.

The combined deletion of PDK2/4 inhibits acute DVT. (A) Thrombus incidence in male PDK2/4^–/– mice and WT mice 48 hours after IVC stenosis; n = 8 mice per group. Statistical analysis was performed using Fisher exact test. (B) The representative image of IVC thrombus harvested from PDK2/4^–/– mice and WT mice 48 hours after IVC stenosis. The bar graphs show quantified mean thrombus weight and thrombus length. Values are mean ± SEM, n = 8 mice per group. Statistical analysis was performed using Mann-Whitney U test. (C) The level of Cit-H3 was measured in the thrombus isolated from PDK2/4^–/– mice and WT mice 48 hours after IVC stenosis. A representative western blot for Cit-H3 is shown. Ponceau S (Ponc. S) staining was used as a loading control. The bar graphs show densitometry analysis of immunoblots. Values are mean ± SEM, with n = 4 mice per group. Statistical analysis was performed using Mann-Whitney U test.

Figure 2.

PDK2/4^–/– neutrophils exhibit reduced release of NETs. NETs assay was performed by stimulating bone marrow–derived WT and PDK2/4^–/– neutrophils with PMA (10 nM) for 4 hours. Representative microphotographs of NETs stained with Sytox green (stains extracellular DNA, green) and counterstained with Hoechst (stains nuclei, blue) are shown. The bar graph shows the quantification of the percentage of cells releasing NETs. Values are mean ± SEM, n = 5 mice per group. Two different fields at 20× magnification were used to estimate the number of neutrophils releasing NETs, which was expressed as a percentage of total neutrophils (coverslip edges were avoided). Statistical analysis was performed using 2-way analysis of variance (ANOVA) followed by Tukey multiple comparisons test.

Figure 3.

Combined deletion of PDK2/4 results in reduced neutrophil-mediated platelet aggregation. The extent of (A) cathepsin G and (B) elastase release was measured using enzyme-linked immunosorbent assay in WT or PDK2/4^–/– neutrophils that were stimulated with PMA (50 nM) for 30 minutes. Values are mean ± SEM, n = 5 mice per group. Statistical analysis was performed using 2-way ANOVA followed by Tukey multiple comparisons test. (C) WT platelets were coincubated and stirred with WT or PDK2/4^–/– neutrophils in an aggregometer before the addition of PMA (10 nM). Results are expressed as the percent change in light transmission with respect to the blank (buffer without platelets and neutrophils) set at 100%. Left: the representative aggregation curve is shown. Right: the line graph shows the quantified data for platelet aggregation. Values are mean ± SEM, n = 5 mice per group. Statistical analysis was performed using 2-way ANOVA followed by Šidák multiple comparisons test. Neu, neutrophils; Plt, platelets.

Figure 4.

Double deletion of PDK2/4 inhibits intracellular Ca²⁺ concentration levels in PMA-stimulated neutrophils. (A) The intracellular Ca²⁺ concentration was measured in Fura-2AM–loaded WT or PDK2/4^−/− neutrophils that were stimulated with PMA (2 nM) in the presence of 1.3 mM CaCl₂. The representative curve for intracellular calcium Ca²⁺ concentration after stimulation with PMA is shown. The bar graphs show the peak Ca²⁺ level and the area under the curve (AUC). Values are mean ± SEM, n = 6 mice per group. Statistical analysis was performed using Mann-Whitney U test. (B) The Erk1/2 phosphorylation was measured in resting and PMA (50 nM)–stimulated WT and PDK2/4^−/− neutrophils. A representative western blot for phosphorylated Erk1/2 is shown. Total Erk1/2 was used as a loading control. The bar graphs show densitometry analysis of immunoblots. Values are mean ± SEM, n = 5 mice per group. Statistical analysis was performed using 2-way ANOVA followed by Tukey multiple comparisons test.

Figure 5.

Deletion of PDK2/4 inhibits aerobic glycolysis in PMA-stimulated neutrophils. The proton efflux rate (PER) and glycoPER were measured in PMA (100 nM)–stimulated WT or PDK2/4^−/− neutrophils using glycolytic rate assay. The line graph represents the PER, and compensatory glycoPER (after the sequential addition of mitochondrial inhibitors, rotenone and antimycin A [AA]). The middle and right panels show the quantification of PER and glycoPER. The bar graph shows the quantified data. Values are mean ± SEM, with n = 4 to 5 mice per group. Statistical analysis was performed using 2-way ANOVA followed by Tukey multiple comparisons test.

Figure 6.

A schematic depicting the role of the PDK/PDH axis in regulating neutrophil activation and NET formation. The deletion of PDK2/4 inhibits aerobic glycolysis, secretion, and intracellular Ca²⁺ levels in PMA-stimulated neutrophils, leading to reduced release of NETs.