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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
  • 265 Downloads

Abstract

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 

Notes

Acknowledgments

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)

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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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