Immunomodulators and microRNAs as neurorestorative therapy for ischemic stroke.

Most of all strokes are ischemic due to occlusion of a vessel, and comprise two main types, thrombotic and embolic. Inflammation and immune response play an important role in the outcome of ischemic stroke. Pharmaceutical and cell-based therapies with immunomodulatory properties could be of benefit in treating ischemic stroke. Possible changes in microRNAs brought about by immunomodulatory treatments may be important. The pharmaceutical studies described in this review have identified several differentially regulated miRNAs associated with disregulation of mRNA targets or the upregulation of several neuroprotective genes, thereby highlighting the potential neuroprotective roles of specific miRNAs such as miR-762, -1892, -200a, -145. MiR-124, -711, -145 are the strongly associated miRNAs predicted to mediate anti-inflammatory pathways and microglia/macrophage M2-like activation phenotype. The cell-based therapy studies reviewed have mainly utilized mesenchymal stem cells or human umbilical cord blood cells and shown to improve functional and neurological outcomes in stroke animals. MiR-145 and miR-133b were implicated in nerve cell remodeling and functional recovery after stroke. Human umbilical cord blood cells decreased proinflammatory factors and promoted M2 macrophage polarization in stroke diabetic animals.

Introduction

Stroke is a potentially fatal or highly debilitating condition with multiple consequences for patients and their families, as well as healthcare providers and society. It is the second most common cause of death worldwide and the leading cause of disability in adults. Approximately 80% of all strokes are ischemic due to occlusion of a vessel, and comprise two main types, thrombotic and embolic. Thrombotic stroke accounts for almost 50% of all strokes. Thrombolysis and/or mechanical thromboectomy may be considered in patients with severe neurological symptoms and early presentation within 3–6 hours of symptom onset. Many stroke patients are not treated by intravenous thrombolysis because of the narrow window of time for treatment. In patients with minor stroke, while the immediate brain damage and associated symptoms are limited, there is a significant risk of a major stroke within 90 days (Emberson, 2014) even when taking aspirin antiplatelet therapy (Wang et al., 2013). Early intervention in stroke is desirable to limit the serious consequence of a major stroke and also to lower the risk of a minor stroke progressing to a seriously disabling stroke. Also extending the window for therapeutic intervention would enable a greater number of stroke patients to be treated.

Evidence indicates that inflammation and immune response play an important role in the outcome of ischemic stroke (Famakin, 2014). Inflammation after stroke involves leukocyte infiltration in brain parenchyma that contributes to cerebral damage. Peripherally derived mononuclear phagocytes, T lymphocytes, natural killer (NK) cells, and polymorphonuclear leukocytes, which produce and secrete cytokines, can all contribute to central nervous system (CNS) inflammation and gliosis (Brea et al., 2009). Blood-derived leukocytes and resident microglia are the more activated inflammatory cells, accumulating in the brain tissue after cerebral ischemia, leading to inflammatory injury (Akopov et al., 1996). Microglia, the major source of cytokines and other immune molecules of the CNS, are the first non-neuronal cells that respond to CNS injury, becoming phagocytic when fully activated by neuronal death. As cerebral inflammation is one of the earliest events in stroke, early intervention to modify the immune response may have a beneficial effect. We have searched the PubMed database for studies of pharmaceutical and cell-based therapies with immunomodulatory properties that have been used for treating ischemic stroke. We have also examined for possible changes in microRNAs brought about by immunomodulatory treatments on account of a possible role of microRNAs in ischemic stroke (Martinez and Peplow, 2015).

Immunomodulatory Therapies for Ischemic Stroke

Pharmaceutical therapies

The pharmaceutical therapies were with niacin, cytosine-phosphate-guanine (CpG), thymosin beta 4 (Tβ4), lipopolysaccharide (LPS), interleukin-4 (IL-4), MK801 (an NMDA antagonist), and microRNA-146a (miR-146a). These have all been shown to have immunomodulatory properties (niacin: Ganji et al., 2009; CpG: Tan et al., 2009; Tβ4: Badamchian et al., 2003; LPS: Jacobs et al., 1981; IL-4: Splawski et al., 1989; MK801: Esposito et al., 2011; miR-146a: Li et al., 2010). The seven animal studies utilizing these pharmaceutical agents are summarized in .

Niacin treatment of middle cerebral artery occlusion (MCAO) normal rats reduced infarct volume and improved neurological outcomes. It decreased apoptosis, attenuated tumor necrosis factor-α (TNF-α) expression, and increased vascular endothelial growth factor (VEGF), phosphoinositide 3-kinase (PI3K)/Akt activity in the ischemic brain (Shehadah, 2010). Conditioning with CpG prior to stroke in mice differentially regulated several miRNAs that were associated with neuroprotective genes (Vartanian et al., 2015). Tβ4 upregulated miR-200a level in the rat ischemic brain and may induce Akt activation and protect brain cells from brain ischemia-mediated apoptosis (Santra et al., 2016). Under LPS stimulation of mouse microglia, miR-155 was the most significantly upregulated miRNA and regulates the signal transducer and activator of transcription 3 signaling pathway enabling the late phase response to M1-skewing by LPS stimulation (Freilich et al., 2013; ). In IL-4 stimulated microglia, miR-145 was the most increased miRNA (Freilich et al., 2013). MiR-145 potentially regulates peripheral monocyte/macrophage differentiation and facilitates the M2 phenotype in microglia/macrophages by IL-4 stimulation (Liu et al., 2016; ). Treatment with MK801 reduced the infarct volume in rat ischemic brain and caused alterations in miRNA profile in brain and blood compared to control rats. MiR-132 was upregulated in MK801-treated rats (Lim et al., 2010). Elevation of miR-146a in mouse oligodendrocyte precursor cells (OPCs) promoted their differentiation, while in neural progenitor cells (NPCs), miR-146a enhanced differentiation of these cells into neuronal and oligodendrocyte lineage cells (Liu et al, 2017).

M1- and M2-like activation phenotypes induced by stimulating murine microglia with LPS and IL-4, respectively, and altered miRNA expressions (Freilich et al., 2013).

IL-4: Interleukin-4; LPS: lipopolysaccharide.

Cell-based therapies

The cell-based therapies were with mesenchymal stem cells (MSCs), human umbilical cord blood cells (HUCBCs), and endothelial progenitor cells (EPCs). They have all been associated with immunomodulatory properties (MSCs: Gao et al., 2016; Zhao et al., 2016; HUCBCs: Yu et al., 2009; EPCs: Nuzzolo et al., 2014; Bartaula-Brevik et al., 2016). The studies utilizing these cells are summarized in . The MSCs were isolated from bone marrow stroma, while HUCBCs and EPCs were obtained commercially. Human cord blood is a source of MSCs (Roura et al, 2012) and EPCs (Lin et al., 2011; Yoder, 2012).

MSC therapy

Four animal studies and one clinical trial were found. Administration of MSCs had positive benefit after stroke in normal rats (Chen et al., 2001a), and transfecting MSCs with miR-133b increased functional outcomes with exosomes-enriched extracellular particles being released from MSCs and transferred to adjacent astrocytes and neurons (Xin et al., 2013). One study showed that MSC treatment after stroke in type 1 diabetes mellitus (T1DM) rats did not improve functional outcomes (Chen et al., 2011). Interestingly treatment with MSCs from T1DM rats improved functional outcome and promoted neurorestorative effects in stroke T1DM rats (Cui et al., 2016). The neurorestorative effects were decreased by administration of MSCs from T1DM rats transfected with miR-145 (Cui et al., 2016). In a phase 1/2a clinical trial, administration of SB623 cells was shown to be safe and improved clinical outcome at 12 months in patients with stable chronic stroke (Steinberg et al., 2016).

HUCBC therapy

Five animal studies were found. HUCBC treatment after stroke of normal rats improved functional recovery, with the HUCBCs surviving and migrating after entering the brain (Chen et al., 2001b). Some HUCBCs were reactive for the astrocyte marker glial fibrillary acidic protein and the neuronal markers NeuN and microtubule-associated protein 2 (Chen et al., 2001b). In stroke rats with type 2 diabetes mellitus (T2DM), HUCBC treatment increased white matter and vascular remodeling, decreased proinflammatory factors Toll-like receptor 4 (TLR4), matrix metallopeptidase 9 (MMP-9), and RAGE expression, and promoted M2 macrophage polarization in the ischemic brain (Yan et al., 2015). Interestingly, HUCBC treatment in T2DM mice after stroke increased miR-126 expression in blood serum and ischemic brain tissue, and miR-126 may contribute to HUCBC-induced neurorestorative effects (Chen et al., 2016). Increased functional recovery occurred in T1DM rats treated with HUCBCs post-stroke, with increased white matter and vascular remodeling in the ischemic brain and increased angiopoietin 1 (Ang1) and decreased RAGE expression in the ischemic boundary zone (IBZ) (Yan et al., 2014). Administration of CD34+ or CD34– cells to spontaneously hypertensive rats after stroke improved functional and neurological outcomes (Boltze et al., 2008).

EPC therapy

One animal study was found. Administration of EPCs transfected with miR-145 promoted cell proliferation and migration and recanalization of arterial thrombosis in normal mice post-stroke (Chen et al., 2015).

Neurorestorative Effects in Stroke of Pharmaceutical and Cell-Based Therapies with Immunomodulatory Properties

Astrocytes and microglia are immune cells of the brain and elicit an inflammatory response by the production of inflammatory mediators (Ransohoff and Brown, 2012). Proinflammatory cytokines are increased during brain ischemia and lowering the levels of these cytokines has been shown to ameliorate ischemic brain injury (Lin et al., 2016; Shu et al., 2016). Cytokines regulate the expression of brain endothelial miRNAs that either promote or inhibit inflammatory pathways to orchestrate neuroinflammation in the brain (Lopez-Ramirez et al., 2016). Proinflammatory cytokines alter the levels of several important miRNA clusters. Members of the miRNA family have been shown to mediate many biological effects including induced cell proliferation and decreased apoptosis (Lopez-Ramirez et al., 2016). MiRNAs play an important role in the regulation of adult neurogenesis, and form an important class of epigenetic regulators that contribute to chronic inflammation in microglia of the brain causing the progression of neurological diseases such as ischemic stroke. Cytokines are likely to be involved in changes in brain capillaries with age and diabetes, as shown for renal capillaries (Bianchi et al., 2016), and increased risk of stroke.

The pharmaceutical studies described in this review have identified several differentially regulated miRNAs associated with disregulation of mRNA targets or the upregulation of several neuroprotective genes, and thereby implicating these miRNAs in neuroprotection. These have included miR-762, -1892, -200a, -145 (). IL-4 induced M2 polarization in microglia/macrophages (). MiR-124, -711, -145 are the strongly associated miRNAs predicted to mediate anti-inflammatory pathways and M2-like activation phenotype. Interestingly, niacin and Tβ4 treatment protected brain cells from stroke-mediated apoptosis, while niacin and the NMDA antagonist MK801 reduced the infarct volume in stroke rats. Niacin treatment attenuated the proinflammatory cytokine TNF-α and increased VEGF, PI3K/Akt activity in the ischemic brain. Transfection of neuroprogenitor cells with miR-146a enhanced their differentiation into neuronal and oligodendrocyte lineage cells. The cell-based therapy studies reviewed have mainly utilized MSCs or HUCBCs and shown to improve functional and neurological outcomes in stroke animals. MiR-145 and miR-133b were implicated in nerve cell remodeling and functional recovery after stroke. HUCBCs decreased proinflammatory factors and promoted M2 macrophage polarization in stroke diabetic animals ().

Future Perspectives

The in vivo stroke studies were mostly performed with young adult male animals. Future studies need to be made in female animals, and also in aged animals. The possible reasons for MSC therapy not improving functional outcomes in stroke type 1 diabetic rats require scientific clarification. Also the role of miRNAs in pharmaceutical and cell-based therapies in improving functional and neurological outcomes in ischemic stroke needs to be further investigated. Preconditioning of MSCs ex vivo by hypoxia, inflammatory stimuli, or other factors/conditions prior to their use in therapy as an adaptive strategy to prepare such cells to survive in the harsh environment at the site of tissue injury/inflammation should be examined (Saparov et al., 2016). This would also apply to HUCBCs. At the present time, the lack of a complete understanding of the mechanism of action mediating the observed therapeutic benefits of adult stem cell therapy in stroke is a critical limitation (Bang et al., 2016) and restricts their use in clinical studies. Also the role of specific microRNAs in inducing angiogenesis, neurogenesis and oligodendrogenesis could be examined by injection of a vector carrying the microRNA into the brain of normal and stroke animals. MicroRNAs with anti-inflammatory properties include miR-146a, -122, -let-7c (Alexander et al., 2015; Roy et al., 2015; Yu et al., 2016) and these should be trialed.

MiRNAs are major molecular regulators and appear to have pivotal roles in cell-based and possibly pharmacological restorative therapies for stroke. Targeting specific miRNAs may provide major restorative therapies for stroke. This will still hold many challenges due to possible delivery and potential off-target effects.

: Open peer review report 1.

Open peer review report 1 on “Immunomodulators and microRNAs as neurorestorative therapy for ischemic stroke”

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Table 1

Studies of pharmacological therapies with immunomodulatory properties to treat stroke

Table 2

Studies of cell-based therapies with immunomodulatory properties to treat stroke