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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.

Introduction:Current treatment regimen for deep vein thrombosis (DVT) includes anticoagulants or surgical intervention. While effective, anticoagulants exhibit a bleeding risk that limits their use. Activated platelets release pro-thrombotic chemokines such as platelet factor 4 (PF4) from their α-granules and are known to potentiate DVT via promoting neutrophil extracellular traps (NETs). Recently, we have shown that dimeric pyruvate kinase M2 (PKM2) regulates platelet function and arterial thrombosis without affecting hemostasis. However, the role of platelet-specific PKM2 in modulating NETosis and DVT remains unexplored.Methods:Susceptibility to DVT was evaluated in the flow-restricted inferior vena cava (IVC) stenosis model in PKM2fl/flPF4Cre+/-mice. The littermate PKM2fl/flPF4Cre-/-(abbreviated as PKM2fl/fl) was used as control. Next, we evaluated whether wild-type mice infused with a specific inhibitor of dimeric-PKM2, ML265, reduced DVT susceptibility in the IVC stenosis model.Results:The platelet-specific PKM2-/-mice were less susceptible to DVT in the IVC stenosis model. The PF4 secretion from α-granules was significantly inhibited in agonist-stimulated platelets from PKM2fl/flPF4Cre+ mice compared to the platelets from PKM2fl/flmice. SNAP-23 phosphorylation regulates SNARE protein complex-mediated mediated secretion from α granules. Therefore, we determined the mechanistic role of PKM2 in modulating SNAP-23-mediated PF4 secretion in platelets. In agonist-stimulated PKM2-/- platelets, SNAP23 phosphorylation and PF4 secretion were reduced compared to agonist-stimulated WT platelets. In addition, neutrophils treated with stimulated platelet releasates from PKM2fl/flPF4Cre+ mice exhibited significantly reduced NET formation in contrast to PKM2fl/flmice. Finally, we demonstrate that ML265-treated mice showed reduced susceptibility to DVT in the IVC stenosis model.Conclusions:Our results suggest an essential role of PKM2 in regulating SNAP-23 mediated α-granule release, NETosis, and subsequent development of DVT.

Rationale:The glycolytic enzyme pyruvate kinase muscle 2 (PKM2) 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.Objective:We determined whether myeloid cell-specific PKM2 regulates efferocytosis and atherosclerosis.Methods and Results:We generated novel myeloid cell-specific PKM2-/-mice on Ldlr-deficient background (PKM2mye-KOLdlr-/-). Controls were littermate PKM2WTLdlr-/-mice. To rule out sex-based differences, male and female mice were placed on a high-fat "Western" diet for 14 weeks, starting at eight weeks. PKM2 was upregulated in macrophages of Ldlr-/-mice fed the Western diet compared with a control 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 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 pro-inflammatory genes, including MCP-1, IL-1β, and IL-12. Myeloid cell-specific PKM2-/-mice exhibited reduced apoptosis concomitant with enhanced macrophage efferocytosis and upregulation of LRP1 in macrophages in vitroand atherosclerotic lesions in vivo. Silencing LRP1 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.Conclusion:Genetic deletion or limiting PKM2 nuclear translocation in myeloid cells reduces atherosclerosis by suppressing inflammation and enhancing efferocytosis.