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

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 pos...

Neuropsychological Deficits Chronically Developed after Focal Ischemic Stroke and Beneficial Effects of Pharmacological Hypothermia in the Mouse

“…Whether a small stroke restricted to cortical tissues could result in delayed psychological alterations is obscure. The small stroke model of mice induced by ligations of distal branches of the right MCA selectively damages the right sensorimotor cortex and is well known for its resultant sensorimotor deficits, specifically the dysfunction of the whisker barrel pathway [7][8][9]. In this investigation, we report chronically developed neuropsychological deficits after the small cortical ischemic stroke.…”

“…This effect of neurotensin involves both NTR1 and neurotensin receptor 2 (NTR2), but the hypothermia effect is exclusively dependent on NTR1. As an analog of NT [8][9][10][11][12][13], HPI-363 is a specific agonist on NTR1 [30,65]. To identify whether the effect observed with HPI-363 was due to the cooling action or other effects of NTR1 activation, physical cooling was tested as a control.…”

PKM2 regulates angiogenesis of VR‐EPCs through modulating glycolysis, mitochondrial fission, and fusion

“…VEGF downstream signaling pathways such as MAPKs, FAK, and AKT are known to regulate proliferation, migration, and tube formation (Cai et al, 2015; Medfai et al, 2019). For instance, ischemic stroke in adult mice is proven to be relieved by angiogenesis effect of PKM2 through activating the FAK signaling pathway (Chen et al, 2018). Our results exhibited that DASA‐58 activates VEFG downstream MAPK, FAK, and AKT signaling pathways in VR‐EPCs, meanwhile, C3k inhibits activation of those pathways induced by VEGF.…”

“…Transwell assay was performed as described previously (Chen et al, 2018). Briefly, VR‐EPCs (3 × 10 4 cells/well) were seeded into the upper chamber of the 24‐well Millicell transwell system (3422, Millipore) with serum‐free M199 medium containing DASA‐58 (30 μM) or C3k (0.3 μM) or only DMSO, and the lower chamber was filled with serum‐free M199 medium alone.…”

“…In brief, pyruvate kinase liver isoenzyme is located in liver cells, pyruvate kinase RBC isoenzyme basically localizes in red blood cells, pyruvate kinase muscle isoenzyme 1 (PKM1) mainly exists in highly differentiated tissues such as muscle, heart, and brain, pyruvate kinase muscle isoenzyme 2 (PKM2) is expressed in all cells with high nucleic acid synthesis rates, such as embryonic cells, normal proliferating cells, and tumor cells (MacDonald & Chang, 1985). Previous findings have indicated that PKM2 can regulate angiogenesis in tumorigenesis and wound healing (Azoitei et al, 2016; Chen et al, 2018; Y. Zhang, Li, Liu, & Liu, 2016). Specifically speaking, PKM2 exists in two forms, dimers that mainly transport to the nucleus and interact with the transcription factors and which are responsible for survival and proliferation (inactive form of the enzyme), and tetramers that primarily exert kinase activity in the cytoplasm (active form of the enzyme; C. H. Su, Hung, Hung, & Tarn, 2017; Wu et al, 2018; Y. Zhang et al, 2016).…”