, Volume 15, Issue 3, pp 770–784 | Cite as

Pyruvate Kinase M2 Increases Angiogenesis, Neurogenesis, and Functional Recovery Mediated by Upregulation of STAT3 and Focal Adhesion Kinase Activities After Ischemic Stroke in Adult Mice

  • Dongdong Chen
  • Ling Wei
  • Zhi-Ren Liu
  • Jenny J. Yang
  • Xiaohuan Gu
  • Zheng Z. Wei
  • Li-Ping Liu
  • Shan Ping Yu
Original Article


Ischemic stroke remains a serious threat to human life. Generation of neuronal and vascular cells is an endogenous regenerative mechanism in the adult brain, which may contribute to tissue repair after stroke. However, the regenerative activity is typically insufficient for significant therapeutic effects after brain injuries. Pyruvate kinase isoform M2 (PKM2) is a key regulator for energy metabolism. PKM2 also has nonmetabolic roles involving regulations of gene expression, cell proliferation, and migration in cancer cells as well as noncancerous cells. In a focal ischemic stroke mouse model, recombinant PKM2 (rPKM2) administration (160 ng/kg, intranasal delivery) at 1 h after stroke showed the significant effect of a reduced infarct volume of more the 60%. Delayed treatment of rPKM2, however, lost the acute neuroprotective effect. We then tested a novel hypothesis that delayed treatment of PKM2 might show proregenerative effects for long-term functional recovery and this chronic action could be mediated by its downstream STAT3 signaling. rPKM2 (160 ng/kg) was delivered to the brain using noninvasive intranasal administration 24 h after the stroke and repeated every other day. Western blot analysis revealed that, 7 days after the stroke, the levels of PKM2 and phosphorylated STAT3 and the expression of angiogenic factors VEGF, Ang-1, and Tie-2 in the peri-infarct region were significantly increased in the rPKM2 treatment group compared with those of the stroke vehicle group. To label proliferating cells, 5-bromo-2′-deoxyuridine (BrdU, 50 mg/kg, i.p.) was injected every day starting 3 days after stroke. At 14 days after stroke, immunohistochemistry showed that rPKM2 increased cell homing of doublecortin (DCX)-positive neuroblasts to the ischemic cortex. In neural progenitor cell (NPC) cultures, rPKM2 (0.4–4 nM) increased the expression of integrin β1 and the activation/phosphorylation of focal adhesion kinase (FAK). A mediator role of FAK in PKM2-promoted cell migration was verified in FAK-knockout fibroblast cultures. In the peri-infarct region of the brain, increased numbers of Glut-1/BrdU and NeuN/BrdU double-positive cells indicated enhanced angiogenesis and neurogenesis, respectively, compared to stroke vehicle mice. Using Laser Doppler imaging, we observed better recovery of the local blood flow in the peri-infarct region of rPKM2-treated mice 14 days after stroke. Meanwhile, rPKM2 improved the sensorimotor functional recovery measured by the adhesive removal test. Inhibiting the STAT3 phosphorylation/activation by the STAT3 inhibitor, BP-1-102 (3 mg/kg/day, o.g.), abolished all beneficial effects of rPKM2 in the stroke mice. Taken together, this investigation provides the first evidence demonstrating that early treatment of rPKM2 shows an acute neuroprotective effect against ischemic brain damage, whereas delayed rPKM2 treatment promotes regenerative activities in the poststroke brain leading to better functional recovery. The underlying mechanism involves activation of the STAT3 and FAK signals in the poststroke brain.

Key Words

Ischemic stroke neuroprotection pyruvate kinase isoform M2 STAT3 FAK neuroblasts proliferation angiogenesis neurogenesis LCBF sensorimotor function 



This work was partly supported by NIH grants NS085568 (LW/SPY), NS091585 (LW), and NS073378 (SPY) and VA Merit Award RX000666, RX001473 (SPY).

Authors’ Contributions

DC contributed to experimental design, performed many experiments, and participated in manuscript formation; LW contributed to concept development, experimental design, immunohistochemical staining and imaging, and data analysis, and participated in manuscript formation; ZL contributed to concept development, data analysis, and manuscript editing; JY and LPL contributed to concept development, data analysis, and experimental design; ZZW and XG contributed to animal surgery and data collection and analysis; SPY contributed to concept development, experimental design, data analysis, grant supports, and the writing/revising of the manuscript.

Compliance with Ethical Standards

All animal experiments and surgery procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Emory University.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

13311_2018_635_MOESM1_ESM.pdf (1.2 mb)
ESM 1 (PDF 1225 kb)
13311_2018_635_Fig8_ESM.png (5.3 mb)
Supplemental Figure 1

Knocking out FAK slowed down the migration of MEFs. A. Immunocytochemistry staining of cytoskeleton marker Acti-stain 555 phalloidin (red) and nuclei marker Hoechst 33,342 (blue) showed the change of cytoskeleton expression in FAK−/− MEFs. B. Transwell migration assay was performed in FAK+/+ MEFs and FAK−/− MEFs. At 2 h, 7 h and 15 h after plating, the cells on the bottom membrane of inserts were stained with Acti-stain 555 phalloidin (red) and Hoechst 33,342 (blue). C. Cell counting data showed that there were significantly less FAK−/− MEFs migrating to the bottom membrane of inserts 2 h and 7 h after plating, however, there were no differences between FAK+/+ MEFs and FAK−/− MEFs 15 h after plating. *p < 0.05 vs. FAK+/+ group. (GIF 238 kb)

13311_2018_635_MOESM2_ESM.tif (7.6 mb)
High resolution image (TIF 7762 kb)


  1. 1.
    Wei L, Wei ZZ, Jiang MQ, Mohamad O, Yu SP, Stem cell transplantation therapy for multifaceted therapeutic benefits after stroke. Prog Neurobiol, 2017. 157: p. 49–78.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Sahota P and S I Savitz, Investigational therapies for ischemic stroke: neuroprotection and neurorecovery. Neurotherapeutics, 2011. 8: p. 434–51.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Bluemlein K, Grüning NM, Feichtinger RG, Lehrach H, Kofler B, Ralser M, No evidence for a shift in pyruvate kinase PKM1 to PKM2 expression during tumorigenesis. Oncotarget, 2011. 2: p. 393–400.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Yang W and Z Lu, Pyruvate kinase M2 at a glance. J Cell Sci, 2015. 128: p. 1655–1660.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Desai S, Ding M, Wang B, et al., Tissue-specific isoform switch and DNA hypomethylation of the pyruvate kinase PKM gene in human cancers. Oncotarget, 2014. 5: p. 8202–8210.CrossRefPubMedGoogle Scholar
  6. 6.
    Gao X, Wang H, Yang JJ et al., Reciprocal regulation of protein kinase and pyruvate kinase activities of pyruvate kinase M2 by growth signals. J Biol Chem, 2013. 288: p. 15971–15979.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Liu V M and MG Vander Heiden, The role of pyruvate kinase M2 in cancer metabolism. Brain Pathol, 2015. 25: p. 781–783.CrossRefPubMedGoogle Scholar
  8. 8.
    Alves-Filho J C and E M Palsson-McDermott, Pyruvate kinase M2: a potential target for regulating inflammation. Front Immunol, 2016. 7: p. 145.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Yang W, Xia Y, Ji H, et al., Nuclear PKM2 regulates β-catenin transactivation upon EGFR activation. Nature, 2011. 480: p. 118–122.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Gupta V, Wellen KE, Mazurek S, Bamezai RN, Pyruvate kinase M2: regulatory circuits and potential for therapeutic intervention. Curr Pharm Des, 2014. 20: p. 2595–2606.CrossRefPubMedGoogle Scholar
  11. 11.
    Dong G, Mao Q, Xia W, et al., PKM2 and cancer: the function of PKM2 beyond glycolysis. Oncol Lett, 2016. 11: p. 1980–1986.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Gao X, Wang H, Yang JJ, Liu X, Liu ZR, Pyruvate kinase M2 regulates gene transcription by acting as a protein kinase. Mol Cell, 2012. 45: p. 598–609.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Vander Heiden M G, Locasale JW, Swanson KD, et al., Evidence for an alternative glycolytic pathway in rapidly proliferating cells. Science, 2010. 329: p. 1492–1499.CrossRefPubMedGoogle Scholar
  14. 14.
    Kim D, Fiske BP, Birsoy K, et al., SHMT2 drives glioma cell survival in ischaemia but imposes a dependence on glycine clearance. Nature, 2015. 520: p. 363–367.CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Zhao Y, Liu H, Riker AI, et al., Emerging metabolic targets in cancer therapy. Front Biosci (Landmark Ed), 2011. 16: p. 1844–1860.CrossRefGoogle Scholar
  16. 16.
    Lunt SY and M.G. Vander Heiden, Aerobic glycolysis: meeting the metabolic requirements of cell proliferation. Annu Rev Cell Dev Biol, 2011. 27: p. 441–464.CrossRefPubMedGoogle Scholar
  17. 17.
    Tech K, M Deshmukh, and TR Gershon, Adaptations of energy metabolism during cerebellar neurogenesis are co-opted in medulloblastoma. Cancer Lett, 2015. 356: p. 268–272.CrossRefPubMedGoogle Scholar
  18. 18.
    Cheon JH, Kim SY, Son JY, et al., Pyruvate kinase M2: a novel biomarker for the early detection of acute kidney injury. Toxicol Res, 2016. 32: p. 47–56.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Yang L, Lee MM, Leung MM, et al., Regulator of G protein signaling 20 enhances cancer cell aggregation, migration, invasion and adhesion. Cell Signal, 2016. 28: p. 1663–1672.CrossRefPubMedGoogle Scholar
  20. 20.
    Li L, Zhang Y, Qiao J, Yang JJ, Liu ZR, Pyruvate kinase M2 in blood circulation facilitates tumor growth by promoting angiogenesis. J Biol Chem, 2014. 289: p. 25812–25821.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Zhang Y, Li L, Liu Y, Liu ZR, PKM2 released by neutrophils at wound site facilitates early wound healing by promoting angiogenesis. Wound Repair Regen, 2016. 24: p. 328–336.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Chen D, Lee J, Gu X, Wei L, Yu SP, Intranasal delivery of apelin-13 is neuroprotective and promotes angiogenesis after ischemic stroke in mice. ASN Neuro, 2015. 7.Google Scholar
  23. 23.
    Wermeling DP, Intranasal delivery of antiepileptic medications for treatment of seizures. Neurotherapeutics, 2009. 6: p. 352–358.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Wei L, CM Rovainen, and TA Woolsey, Ministrokes in rat barrel cortex. Stroke, 1995. 26: p. 1459–1462.CrossRefPubMedGoogle Scholar
  25. 25.
    Espinera AR, Ogle ME, Gu X, Wei L, Citalopram enhances neurovascular regeneration and sensorimotor functional recovery after ischemic stroke in mice. Neurosci, 2013. 247: p. 1–11.CrossRefGoogle Scholar
  26. 26.
    Wang LL, Chen D, Lee J, et al., Mobilization of endogenous bone marrow derived endothelial progenitor cells and therapeutic potential of parathyroid hormone after ischemic stroke in mice. PLoS One, 2014. 9: p. e87284.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Li Y, Lu Z, Keogh CL, Yu SP, Wei L, Erythropoietin-induced neurovascular protection, angiogenesis, and cerebral blood flow restoration after focal ischemia in mice. J Cereb Blood Flow Metab, 2007. 27: p. 1043–1054.CrossRefPubMedGoogle Scholar
  28. 28.
    Bouet V, Boulouard M, Toutain J et al., The adhesive removal test: a sensitive method to assess sensorimotor deficits in mice. Nat Protoc, 2009. 4: p. 1560–1564.CrossRefPubMedGoogle Scholar
  29. 29.
    Gu H, Yu SP, Gutekunst CA, Gross RE, Wei L, Inhibition of the Rho signaling pathway improves neurite outgrowth and neuronal differentiation of mouse neural stem cells. Int J Physiol Pathophysiol Pharmacol, 2013. 5: p. 11–20.PubMedPubMedCentralGoogle Scholar
  30. 30.
    Wei JF, Wei L, Zhou X, et al., Formation of Kv2.1-FAK complex as a mechanism of FAK activation, cell polarization and enhanced motility. J Cell Physiol, 2008. 217: p. 544–557.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Avraamides CJ, B Garmy-Susini, and JA Varner, Integrins in angiogenesis and lymphangiogenesis. Nat Rev Cancer, 2008. 8: p. 604–617.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Kim S, M Harris, and JA Varner, Regulation of integrin alpha vbeta 3-mediated endothelial cell migration and angiogenesis by integrin alpha5beta1 and protein kinase A. J Biol Chem, 2000. 275: p. 33920–33928.CrossRefPubMedGoogle Scholar
  33. 33.
    Lakshmikanthan S, Sobczak M, Chun C, et al., Rap1 promotes VEGFR2 activation and angiogenesis by a mechanism involving integrin alphavbeta(3). Blood, 2011. 118: p. 2015–2026.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Somanath PR, NL Malinin, and TV Byzova, Cooperation between integrin alphavbeta3 and VEGFR2 in angiogenesis. Angiogenesis, 2009. 12: p. 177–185.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Schaeferhoff K, Michalakis S, Tanimoto N, et al., Induction of STAT3-related genes in fast degenerating cone photoreceptors of cpfl1 mice. Cell Mol Life Sci, 2010. 67: p. 3173–3186.CrossRefPubMedGoogle Scholar
  36. 36.
    Pereira L, Font-Nieves M, Van den Haute C, et al., IL-10 regulates adult neurogenesis by modulating ERK and STAT3 activity. Front Cell Neurosci, 2015. 9: p. 57.PubMedPubMedCentralGoogle Scholar
  37. 37.
    Couillard-Despres S, Winner B, Schaubeck S, et al., Doublecortin expression levels in adult brain reflect neurogenesis. Eur J Neurosci, 2005. 21: p. 1–14.CrossRefPubMedGoogle Scholar
  38. 38.
    Niu G, Wright KL, Huang M, et al., Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis. Oncogene, 2002. 21: p. 2000–2008.CrossRefPubMedGoogle Scholar
  39. 39.
    Lv L, Xu YP, Zhao D, et al., Mitogenic and oncogenic stimulation of K433 acetylation promotes PKM2 protein kinase activity and nuclear localization. Mol Cell, 2013. 52: p. 340–352.CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Yang W, Zheng Y, Xia Y, et al., ERK1/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect. Nat Cell Biol, 2012. 14: p. 1295–1304.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Jiang Y, Li X, Yang W, et al., PKM2 regulates chromosome segregation and mitosis progression of tumor cells. Mol Cell, 2014. 53: p. 75–87.CrossRefPubMedGoogle Scholar
  42. 42.
    Caravas J and D.E. Wildman, A genetic perspective on glucose consumption in the cerebral cortex during human development. Diabetes Obes Metab, 2014. 16: p. 21–5.CrossRefPubMedGoogle Scholar
  43. 43.
    Iqbal MA, Siddiqui FA, Gupta V, et al., Insulin enhances metabolic capacities of cancer cells by dual regulation of glycolytic enzyme pyruvate kinase M2. Mol Cancer, 2013. 12: p. 72.CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Sun Q, Chen X, Ma J, et al., Mammalian target of rapamycin up-regulation of pyruvate kinase isoenzyme type M2 is critical for aerobic glycolysis and tumor growth. Proc Natl Acad Sci U S A, 2011. 108: p. 4129–4134.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Larsen M, ML Tremblay, and KM Yamada, Phosphatases in cell-matrix adhesion and migration. Nat Rev Mol Cell Biol, 2003. 4: p. 700–711.CrossRefPubMedGoogle Scholar

Copyright information

© The American Society for Experimental NeuroTherapeutics, Inc. 2018
corrected publication June 2018

Authors and Affiliations

  • Dongdong Chen
    • 1
  • Ling Wei
    • 1
  • Zhi-Ren Liu
    • 2
  • Jenny J. Yang
    • 2
  • Xiaohuan Gu
    • 1
  • Zheng Z. Wei
    • 1
    • 3
  • Li-Ping Liu
    • 4
  • Shan Ping Yu
    • 1
    • 3
  1. 1.Department of AnesthesiologyEmory University School of MedicineAtlantaUSA
  2. 2.Department of BiologyGeorgia State UniversityAtlantaUSA
  3. 3.Center for Visual and Neurocognitive RehabilitationVeteran’s Affair Medical CenterAtlantaUSA
  4. 4.Department of  Neurology, Beijing Tiantan HospitalCapital Medical UniversityBeijingChina

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