Biologicals and biotherapeuticsMEPO promotes neurogenesis and angiogenesis but suppresses gliogenesis in mice with acute ischemic stroke
Introduction
Erythropoietin (EPO) is a hematopoiesis factor, which is mainly secreted by the kidney and liver that regulates erythropoiesis. As a neuroprotective agent in cerebral ischemia, its protective effects have been proved by large number of preclinical and some clinical studies in the last two decades (Ehrenreich et al., 2002, Wu et al., 2009, Nguyen et al., 2014, Tsai et al., 2015). Although in the German Multicenter Erythropoietin Stroke Trial (NCT00604630) and the following experimental studies indicated that the combination of rtPA and EPO treatment maybe not suitable for patients with cerebral ischemic stroke (Ehrenreich et al., 2009, Jia et al., 2010, Zechariah et al., 2010). However, only less than 5% of stroke patients fit the criteria for tPA therapy due to its seriously side effects of intracranial hemorrhage and its narrow therapeutic time-window (less than 6 h) (National Institute of Neurological and Stroke rt, 1995, Soeteman et al., 2017). Therefore, EPO is still an alternative neuroprotectant for most patients with acute cerebral ischemia (Ehrenreich et al., 2002).
Less than 1% efficient EPO can across the blood-brain barrier into the brain parenchyma through systemic administration, consequently, high-doses EPO are required to achieve effective therapeutically concentrations (Brines et al., 2000). However, high-dose and multiple administration of EPO was associated with unexpected side effects in clinical research, such as increases risk of thrombosis and secondary infarction, which restricts its application in clinical treatment. In light of recently studies have shown that EPO's hematological and tissue-protective activities were distinct and separate through interaction with different receptors (Brines et al., 2004, Leist et al., 2004). Accordingly, alternate strategies to abolish its erythropoietic activity and reserve the tissue-protective through chemical and genetic modification will greatly improve its clinical applicability for ischemic stroke treatment. Recent years, several types of EPO derivatives that lack hematopoietic but retain tissue-protection activity have been generated, which include asialo-EPO (Erbayraktar et al., 2003), carbamylated EPO (Leist et al., 2004), neuro-EPO (Parra and Rodriguez, 2012) and ARA290, a peptide derived from erythropoietin (Swartjes et al., 2014). However, the very short plasma half-life, complex chemical modifications and high production costs restricted its application in brain injuries. We constructed the MEPO (S104I-EPO) containing a single amino acid mutation within the erythropoietic motif, which completely lacks erythropoietic activity but retains neuroprotective effects against ischemic brain injury in mice, with an efficacy similar to that of wild-type EPO (Gan et al., 2012). In this study, we used a well-established mouse model of transient focal cerebral ischemia to further evaluate the effects of MEPO on neurogenesis, angiogenesis, and gliogenesis after cerebral ischemia. The results demonstrated that MEPO treatment enhanced the proliferation of neuronal precursors and mature neurons, increased the newly generated cells in blood vessels, and suppressed newly proliferating cells of astrocytes and microglia in peri-infarct area, thus improved neurological function and survival rate after cerebral ischemic injury. Our investigation will provide a reliable basis for clinical trials of MEPO.
Section snippets
Experiment animals and middle cerebral artery occlusion (MCAO) model
Male C57BL/6 mice (25–30 g, 2-month-old) were purchased from Vital River Laboratory Animal Technology Co. Ltd and approved by the Institutional Animal Care and Use Committee of Capital Medical University. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO), with a nylon filament silicon tip (0.19 mm diameter) inserted to the right middle cerebral artery (MCA) to obstruct the blood flow. Forty-five min after occlusion, the filament was removed to allow for reperfusion.
MEPO provides neuroprotective effects against cerebral ischemic injury
We first verified whether MEPO treatment could exert similar neuroprotective effects as EPO in the early recovery periods of cerebral ischemic injury. The result shown that MEPO treatment remarkedly improved the survival rate compared to I/R+Veh group until 28 days after MCAO surgery (Fig. 1A).
Neurobehavioral tests were performed at 1, 3, 7, 14, 21, and 28 days after MCAO by two investigators who were blinded to the experimental groups. The Rota-rod test indicated MEPO treatment obviously
Discussion
Exogenously administered EPO has reported to provide multiple neuroprotective effects in the animal models of cerebral ischemia and in ischemic stroke patients for many years, through the anti-inflammatory, anti-oxidant and anti-apoptotic effects, as well as through the stimulation of neurogenesis and angiogenesis (Villa et al., 2003, Iwai et al., 2007, Patel et al., 2012). Unfortunately, the serious adverse effects of wide type EPO or recombinant human EPO severely limits its clinical
Acknowledgements
This work was supported by the National Natural Science Foundation of China (81471340, 81801149, 81641054), China, and National Institutes of Health/NINDS grants NS079345, United States.
Declarations of interest
None.
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2022, Chemical Engineering JournalCitation Excerpt :While 3-MA-NLCs-treated MCAO rats compared to MCAO treated rats had considerably improved overall neurobehavioral function (Day 1 (P < 0.01 Sham vs. MCAO; P < 0.05 MCAO vs. MCAO + 3-MA-NLCs); Day 7 (P < 0.01 Sham vs. MCAO; P < 0.05 MCAO vs. MCAO + 3-MA-NLCs); Day 14 (<0.01 Sham vs. MCAO; P < 0.05 MCAO vs. MCAO + 3-MA-NLCs); Day 21 (P < 0.01 Sham vs. MCAO; P < 0.05 MCAO vs. MCAO + 3-MA-NLCs); Day 28 (P < 0.05 Sham vs. MCAO; P < 0.05 MCAO vs. MCAO + 3-MA-NLCs); Day 35 (P < 0.01 Sham vs. MCAO; P < 0.01 MCAO vs. MCAO + 3-MA-NLCs)) (Fig. 6A). The adhesive removal test was used to augment the neurobehavioral research by giving additional selectivity to identify changes in the frontal cortex through its sensitivity to sensory inputs [61,62]. To summarize, rats in the MCAO group took longer to remove adhesive tape from their forepaws after MCAO followed by reperfusion than sham group rats.
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2022, Current Research in Pharmacology and Drug DiscoveryCitation Excerpt :Due to its role with oxygen sensors, the effects of EPO under cerebral I/R conditions have been investigated. Previous studies demonstrated that pretreatment with EPO in cerebral I/R injury attenuated BBB breakdown, decreased pro-inflammatory cytokines (Liu et al., 2013), ameliorated lipid peroxidation (Bahcekapili et al., 2007), increased neuronal proliferation and maturation (Zhang et al., 2019), and decreased neuronal apoptosis (Khaksari et al., 2017), resulting in the reduction of brain infarct size, and the improvement of cognitive function. Taken together, all findings suggested the neuroprotective effects on the brain in cerebral I/R condition.
Mutant erythropoietin enhances white matter repair via the JAK2/STAT3 and C/EBPβ pathway in middle-aged mice following cerebral ischemia and reperfusion
2021, Experimental NeurologyCitation Excerpt :Compared with other types of EPO derivatives (asialo-EPO, carbamylated EPO, neuro-EPO, EPO-TAT) that have been generated in recent years (Erbayraktar et al., 2003; Leist et al., 2004; Parra and Rodriguez, 2012; Zhang et al., 2010), MEPO has several advantages such as a longer plasma half-life, lower production costs, and not requiring chemical modification (Gan et al., 2012). Our subsequent research demonstrated that MEPO could stimulate neurogenesis and angiogenesis but inhibit excessive gliogenesis comparable to recombinant human EPO (rhEPO) in cerebral ischemic mice during the recovery period (Zhang et al., 2019). Moreover, our previous study found that rhEPO improves white matter integrity through facilitating microglia to shift to the M2 phenotype following cerebral ischemia (Wang et al., 2017).
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2019, Neurobiology of AgingCitation Excerpt :In light of this, the potential of EPO as a neuroprotective agent is hampered by its canonical hematopoietic effects and other side effects (e.g., hypertension, thrombosis, and tumor progression) (Brines and Cerami, 2005; Maiese et al., 2012; Miskowiak et al., 2012, 2014), as well as its relatively large size of 30.4 kDa. One of the ways to circumvent these limitations is to use nonhematopoietic EPO modifications (i.e., carbamylated EPO, asialoEPO (Erbayraktar et al., 2003; Leist et al., 2004)), EPO mutants (Dhanushkodi et al., 2013; Zhang et al., 2019), or small peptide mimetics (pHBSP/ARA90; Epotris, Epobis; JM4) (Brines et al., 2008; Dmytriyeva et al., 2016; Pankratova et al., 2010, 2012; Wang et al., 2016). Importantly, the aforementioned EPO formulations and peptide mimetics can readily cross the BBB and have shown neurotrophic and neuroprotective functions, confirmed in various in vitro and preclinical models, further supporting the observation that the neuroprotective potential of EPO is unrelated to its hematopoietic effect (Siren et al., 2009).
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These authors contributed equally to the paper.