There is a critical need for cerebro-protective interventions to improve the suboptimal outcomes of patients with ischemic stroke who have been treated with reperfusion strategies. We found that nuclear pyruvate kinase muscle 2 (PKM2), a modulator of systemic inflammation, was upregulated in neutrophils after the onset of ischemic stroke in both humans and mice. Therefore, we determined the role of PKM2 in stroke pathogenesis by using murine models with preexisting comorbidities. We generated novel myeloid cell–specific PKM2−/− mice on wild-type (PKM2fl/flLysMCre+) and hyperlipidemic background (PKM2fl/flLysMCre+Apoe−/−). Controls were littermate PKM2fl/flLysMCre– or PKM2fl/flLysMCre–Apoe−/− mice. Genetic deletion of PKM2 in myeloid cells limited inflammatory response in peripheral neutrophils and reduced neutrophil extracellular traps after cerebral ischemia and reperfusion, suggesting that PKM2 promotes neutrophil hyperactivation in the setting of stroke. In the filament and autologous clot and recombinant tissue plasminogen activator stroke models, irrespective of sex, deletion of PKM2 in myeloid cells in either wild-type or hyperlipidemic mice reduced infarcts and enhanced long-term sensorimotor recovery. Laser speckle imaging revealed improved regional cerebral blood flow in myeloid cell–specific PKM2-deficient mice that was concomitant with reduced post-ischemic cerebral thrombo-inflammation (intracerebral fibrinogen, platelet [CD41+] deposition, neutrophil infiltration, and inflammatory cytokines). Mechanistically, PKM2 regulates post-ischemic inflammation in peripheral neutrophils by promoting STAT3 phosphorylation. To enhance the translational significance, we inhibited PKM2 nuclear translocation using a small molecule and found significantly reduced neutrophil hyperactivation and improved short-term and long-term functional outcomes after stroke. Collectively, these findings identify PKM2 as a novel therapeutic target to improve brain salvage and recovery after reperfusion.

Background: The glycolytic enzyme PKM2 (pyruvate kinase muscle 2) is upregulated in monocytes/macrophages of patients with atherosclerotic coronary artery disease. However, the role of cell type-specific PKM2 in the setting of atherosclerosis remains to be defined. We determined whether myeloid cell-specific PKM2 regulates efferocytosis and atherosclerosis. Methods: We generated myeloid cell-specific PKM2 ^−/− mice on Ldlr (low-density lipoprotein receptor)-deficient background (PKM2 ^mye-KO Ldlr ^−/− ). Controls were littermate PKM2 ^WT Ldlr ^−/− mice. Susceptibility to atherosclerosis was evaluated in whole aortae and cross sections of the aortic sinus in male and female mice fed a high-fat Western diet for 14 weeks, starting at 8 weeks. Results: PKM2 was upregulated in macrophages of Ldlr ^−/− mice fed a high-fat Western diet compared with chow diet. Myeloid cell-specific deletion of PKM2 led to a significant reduction in lesions in the whole aorta and aortic sinus despite high cholesterol and triglyceride levels. Furthermore, we found decreased macrophage content in the lesions of myeloid cell-specific PKM2 ^−/− mice associated with decreased MCP-1 (monocyte chemoattractant protein 1) levels in plasma, reduced transmigration of macrophages in response to MCP-1, and impaired glycolytic rate. Macrophages isolated from myeloid-specific PKM2 ^−/− mice fed the Western diet exhibited reduced expression of proinflammatory genes, including MCP-1, IL (interleukin)-1β, and IL-12. Myeloid cell-specific PKM2 ^−/− mice exhibited reduced apoptosis concomitant with enhanced macrophage efferocytosis and upregulation of LRP (LDLR-related protein)-1 in macrophages in vitro and atherosclerotic lesions in vivo. Silencing LRP-1 in PKM2-deficient macrophages restored inflammatory gene expression and reduced efferocytosis. As a therapeutic intervention, inhibiting PKM2 nuclear translocation using a small molecule reduced glycolytic rate, enhanced efferocytosis, and reduced atherosclerosis in Ldlr ^−/− mice. Conclusions: Genetic deletion of PKM2 in myeloid cells or limiting its nuclear translocation reduces atherosclerosis by suppressing inflammation and enhancing efferocytosis.

Fibronectin–splice variant containing extra domain A (Fn-EDA) is associated with smooth muscle cells (SMCs) following vascular injury. The role of SMC-derived Fn-EDA in SMC phenotypic switching or its implication in neointimal hyperplasia remains unclear. Herein, using human coronary artery sections with a bare metal stent, we demonstrate the expression of Fn-EDA in the vicinity of SMC-rich neointima and peri-strut areas. In mice, Fn-EDA colocalizes with SMCs in the neointima of injured carotid arteries and promotes neointima formation in the comorbid condition of hyperlipidemia by potentiating SMC proliferation and migration. No sex-based differences were observed. Mechanistic studies suggested that Fn-EDA mediates integrin- and TLR4-dependent proliferation and migration through activation of FAK/Src and Akt1/mTOR signaling, respectively. Specific deletion of Fn-EDA in SMCs, but not in endothelial cells, reduced intimal hyperplasia and suppressed the SMC synthetic phenotype concomitant with decreased Akt1/mTOR signaling. Targeting Fn-EDA in human aortic SMCs suppressed the synthetic phenotype and downregulated Akt1/mTOR signaling. These results reveal that SMC-derived Fn-EDA potentiates phenotypic switching in human and mouse aortic SMCs and neointimal hyperplasia in the mouse. We suggest that targeting Fn-EDA could be explored as a potential therapeutic strategy to reduce neointimal hyperplasia.

Triacylglycerol (TG) accumulation caused by adipose triglyceride lipase (ATGL) deficiency or very low-density lipoprotein (VLDL) loading of wild-type (Wt) macrophages results in mitochondrial-mediated apoptosis. This phenotype is correlated to depletion of Ca²⁺ from the endoplasmic reticulum (ER), an event known to induce the unfolded protein response (UPR). Here, we show that ER stress in TG-rich macrophages activates the UPR, resulting in increased abundance of the chaperone GRP78/BiP, the induction of pancreatic ER kinase-like ER kinase, phosphorylation and activation of eukaryotic translation initiation factor 2A, the translocation of activating transcription factor (ATF)4 and ATF6 to the nucleus and the induction of the cell death executor CCAAT/enhancer-binding protein homologous protein. C16:0 ceramide concentrations were increased in Atgl–/– and VLDL-loaded Wt macrophages. Overexpression of ceramide synthases was sufficient to induce mitochondrial apoptosis in Wt macrophages. In accordance, inhibition of ceramide synthases in Atgl–/– macrophages by fumonisin B1 (FB1) resulted in specific inhibition of C16:0 ceramide, whereas intracellular TG concentrations remained high. Although the UPR was still activated in Atgl–/– macrophages, FB1 treatment rescued Atgl–/– macrophages from mitochondrial dysfunction and programmed cell death. We conclude that C16:0 ceramide elicits apoptosis in Atgl–/– macrophages by activation of the mitochondrial apoptosis pathway.

Resting platelets rely on oxidative phosphorylation (OXPHOS) and aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen) to generate adenosine triphosphate, whereas activated platelets exhibit a high level of aerobic glycolysis, suggesting the existence of metabolic flexibility in platelets. Mitochondrial pyruvate dehydrogenase kinases (PDK 1-4) play a pivotal role in metabolic flexibility by inhibiting pyruvate dehydrogenase complex. We determined whether metabolic reprogramming, diverting metabolism from aerobic glycolysis back to OXPHOS, would inhibit platelet function. PDKs activity in human and mouse platelets was inhibited with dichloroacetic acid (DCA), a potent inhibitor of all 4 forms of PDK. Human and mouse platelets pretreated with DCA exhibited decreased platelet aggregation to suboptimal doses of collagen, convulxin, thrombin, and adenosine diphosphate concomitant with decreased glucose uptake. Bioenergetics profile revealed that platelets pretreated with DCA exhibited decreased aerobic glycolysis in response to convulxin only. Furthermore, DCA inhibited ATP secretion, thromboxane A2 generation, and tyrosine phosphorylation of Syk and PLCγ2 in response to collagen or convulxin in human and mouse platelets (P < .05 vs vehicle treated). In the flow chamber assay, human and mouse blood pretreated with DCA formed smaller thrombi when perfused over collagen for 10 minutes at an arterial shear rate of 1500 s⁻¹ (P < .05 vs control). Wild-type mice pretreated with DCA were less susceptible to thrombosis in the FeCl₃-induced carotid and laser injury–induced mesenteric artery thrombosis models (P < .05 vs vehicle control), without altering hemostasis. Targeting metabolic plasticity with DCA may be explored as a novel strategy to inhibit platelet function. Key Points DCA, an inhibitor of PDKs, impedes glucose uptake and aerobic glycolysis in activated platelets. DCA inhibits agonist-induced human and mouse platelet aggregation and arterial thrombosis without altering hemostasis in mice. Visual Abstract

Objective—: Cellular fibronectin containing extra domain A (EDA ⁺ -FN) is abundant in the arteries of patients with atherosclerosis. Several in vitro studies suggest that EDA ⁺ -FN interacts with Toll-like receptor 4 (TLR4). We tested the hypothesis that EDA ⁺ -FN exacerbates atherosclerosis through TLR4 in a clinically relevant model of atherosclerosis, the apolipoprotein E–deficient ( Apoe ^−/− ) mouse. Approach and Results—: The extent of atherosclerosis was evaluated in whole aortae and cross sections of the aortic sinus in male and female EDA ^−/− Apoe ^−/− mice (which lack EDA ⁺ -FN), EDA ^fl/fl Apoe ^−/− mice (which constitutively express EDA ⁺ -FN), and control Apoe ^−/− mice fed a high-fat Western diet for 14 weeks. Irrespective of sex, EDA ^fl/fl Apoe ^−/− mice exhibited a 2-fold increase in atherosclerotic lesions (aorta and aortic sinus) and macrophage content within plaques, whereas EDA ^−/− Apoe ^−/− mice exhibited reduced atherosclerotic lesions ( P <0.05 versus Apoe ^−/− , n=10–12 mice/group), although cholesterol and triglyceride levels and circulating leukocytes were similar. Genetic ablation of TLR4 partially reversed atherosclerosis exacerbation in EDA ^fl/fl Apoe ^−/− mice ( P <0.05) but had no effect on atherosclerotic lesions in EDA ^−/− Apoe ^−/− mice. Purified cellular FN, which contains EDA, potentiated dose-dependent NFκB-mediated inflammation (increased phospho-NFκB p65/NFκB p65, tumor necrosis factor-α, and interleukin-1β) in bone marrow–derived macrophages from EDA ^−/− Apoe ^−/− mice but not from EDA ^−/− TLR4 ^−/− Apoe ^−/− mice. Finally, using immunohistochemistry, we provide evidence for the first time that EDA ⁺ -FN colocalizes with macrophage TLR4 in murine aortic lesions and human coronary artery atherosclerotic plaques. Conclusions—: Our findings reveal that TLR4 signaling contributes to EDA ⁺ -FN–mediated exacerbation of atherosclerosis. We suggest that EDA ⁺ -FN could be a therapeutic target in atherosclerosis.