Oxygen-glucose Deprivation Conditions Research Articles

MicroRNA-210-3p Targets RGMA to Enhance the Angiogenic Functions of Endothelial Progenitor Cells Under Hypoxic Conditions.

Endothelial progenitor cells (EPCs) are multipotential stem cells considered to have immense clinical value for revascularization. However, the clinical application of EPCs has been hampered by their clinical potency in ischemic anoxic environments. This study aimed to explore the effect of microRNA-210 (miR-210) on EPCs under oxygen-glucose deprivation (OGD) conditions. We generated a model of EPCs cultured under OGD conditions to simulate ischemia and explore the expression of miR-210 in vitro. With longer exposure to hypoxia, we found that miR-210-3p expression was highly upregulated in OGD groups compared to that in controls from 4 to 24 h, but not miR-210-5p. We then transfected a miR-210-3p mimic and inhibitor into EPCs, and after 24 h, we exposed them to OGD conditions for 4 h to simulate ischemia. We detected miR-210 by real-time polymerase chain reaction (RT-PCR) and tested the proliferation, migration, and tube formation of normal EPCs and OGD-treated EPCs by CCK-8, transwell chamber, and Matrigel assays, respectively. The direct targets of miR-210-3p were predicted using miRWalk. Compared to that in normal EPCs, higher miR-210-3p expression was found in OGD-treated EPCs (p < 0.05). Moreover, upregulation of miR-210-3p was found to promote proliferation, migration, and tube formation in EPCs under normal and OGD conditions (p < 0.05), whereas down-regulation inhibited these abilities in OGD-treated EPCs (p < 0.05). Repulsive guidance molecule A (RGMA), a negative regulator of angiogenesis, was predicted to be a target of miR-210-3p. Accordingly, upregulation of miR-210-3p was found to inhibit its expression at the protein level in OGD-treated EPCs, whereas downregulation of miR-210-3p inhibited its expression (p < 0.05). A dual-luciferase reporter system confirmed that RGMA is a direct target of miR-210-3p. MicroRNA-210-3p overexpression enhances the angiogenic properties of OGD-treated EPCs by inhibiting RGMA.

A Novel Role of VEGFC in Cerebral Ischemia With Lung Injury.

Cerebral ischemia (CI) is a severe brain injury resulting in a variety of motor impairments combined with secondary injury in remote organs, especially the lung. This condition occurs due to insufficient blood supply to the brain during infancy. However, it has a molecular linkage that needs to be thoroughly covered. Here, we report on the role of vascular endothelial growth factor C (VEGFC) in lung injury induced by CI. The middle cerebral artery occlusion (MCAO) was depended to establish the animal model of CI. Rats were used and brain ischemia was confirmed through TTC staining. Serum was used for protein chip analysis to study the proteomic interaction. Immunohistochemistry analyses were used to quantify and locate the VEGFC in the lung and brain. The role of VEGFC was detected by siVEGFC technology in SY5Y, HUCEV, and A549 cell lines, under normal and oxygen glucose deprivation (OGD) conditions in vitro. As a result, the TTC staining demonstrated that the model of brain ischemia was successfully established, and MPO experiments reported that lung damage was induced in MCAO rats. VEGFC levels were up-regulated in serum. On the other hand, immunohistochemistry showed that VEGFC increased significantly in the cytoplasm of neurons, the endothelium of small trachea and the lung cells of CI animals. On a functional level, siVEGFC effectively inhibited the proliferation of SY5Y cells and decreased the viability of HUVEC cells in normal cell lines. But under OGD conditions, siVEGFC decreased the growth of HUVEC and increased the viability of A549 cells, while no effect was noticed on SYSY cells. Therefore, we confirmed the different role of VEGFC played in neurons and lung cells in cerebral ischemia-reperfusion injury. These findings may contribute to the understanding the molecular linkage of brain ischemia and lung injury, which therefore provides a new idea for the therapeutic approach to cerebral ischemia-reperfusion.

Combining Human Umbilical Cord Blood Cells With Erythropoietin Enhances Angiogenesis/Neurogenesis and Behavioral Recovery After Stroke.

Disruption of blood flow in the brain induces stroke, the leading cause of death and disability worldwide. However, so far the therapeutic options are limited. Thus, the therapeutic efficacy of cell-based approaches has been investigated to develop a potential strategy to overcome stroke-induced disability. Human umbilical cord blood cells (hUCBCs) and erythropoietin (EPO) both have angiogenic and neurogenic properties in the injured brain, and their combined administration may exert synergistic effects during neurological recovery following stroke. We investigated the therapeutic potential of hUCBC and EPO combination treatment by comparing its efficacy to those of hUCBC and EPO alone. Adult male Sprague-Dawley rats underwent transient middle cerebral artery occlusion (MCAO). Experimental groups were as follows: saline (injected once with saline 7 d after MCAO); hUCBC (1.2 × 107 total nucleated cells, injected once via the tail vein 7 d after MCAO); EPO (500 IU/kg, injected intraperitoneally for five consecutive days from 7 d after MCAO); and combination of hUCBC and EPO (hUCBC+EPO). Behavioral measures (Modified Neurological Severity Score [mNSS] and cylinder test) were recorded to assess neurological outcomes. Four weeks after MCAO, brains were harvested to analyze the status of neurogenesis and angiogenesis. In vitro assays were also conducted using neural stem and endothelial cells in the oxygen-glucose deprivation condition. Performance on the mNSS and cylinder test showed the most improvement in the hUCBC+EPO group, while hUCBC- and EPO-alone treatments showed superior outcomes relative to the saline group. Neurogenesis and angiogenesis in the cortical region was the most enhanced in the hUCBC+EPO group, while the findings in the hUCBC and EPO treatment alone groups were better than those in the saline group. Astrogliosis in the brain tissue was reduced by hUCBC and EPO treatment. The reduction was largest in the hUCBC+EPO group. These results were consistent with in vitro assessments that showed the strongest neurogenic and angiogenic effect with hUCBC+EPO treatment. This study demonstrates that combination therapy is more effective than single therapy with either hUCBC or EPO for neurological recovery from subacute stroke. The common pathway underlying hUCBC and EPO treatment requires further study.

Anti‐edematous effect of neurolysin in ischemic stroke

During ischemic stroke brain injury occurs due to oxygen and nutrient deficiency, which initiates secondary injury cascades, including intracellular sodium and calcium accumulation, release of neurotoxic dopamine and glutamate, ion gradient dissipation, accumulation of reactive oxygen species, mitochondrial dysfunction, and excitotoxicity. All of these pathophysiologic processes contribute to cellular and vasogenic edema, which can be a life‐threatening effect of stroke and the primary reason for patient mortality after stroke.Neuropeptides similar to neurotransmitters play a significant role in different signaling pathways. They act on the cellular surface after releasing into the extracellular spaces and their functions are terminated by endogenous neuropeptidases. Neurolysin (Nln), a zinc metallopeptidase, has been reported to be up‐regulated during stroke and plays a crucial role in the improvement of stroke outcomes. Functional importance of this enzyme in post‐stroke brain repair is due to its capability in processing a diverse group of neuropeptides, such as substance P, bradykinin and neurotensin, which all have distinct functions in the development of inflammation, excitotoxicity, and cell death after stroke. The objective of our present study was to investigate the role of Nln through the deactivation of the above neuropeptide substrates and subsequent reduction of brain edema after a model of ischemic stroke.For an ex‐vivo model of ischemia, an acute brain slice model has been used. Briefly, 5–7 month old CD1 male mouse brain was extracted and 400 μm slices were exposed to 1 hour of oxygen glucose deprivation (OGD) condition followed by reperfusion (R) for 2 hours in the presence and absence of Nln substrates, i.e., substance P, bradykinin and neurotensin. To study the role of Nln, Nln substrates were co‐incubated with Nln during OGD/R. We observed that the 21% water content in slices increased when exposed to OGD/R compared to normoxia which further increased in the presence of Nln substrates during reperfusion, suggesting the role of Nln substrate in edema formation. Interestingly, the presence of rNln attenuated the enhanced edema formation by Nln substrates. These results indicated the role of Nln in reducing brain edema during ischemic stroke. Furthermore, to confirm that the effect of Nln is through deactivation of neuropeptides, we confirmed the hydrolysis of neuropeptides in the presence of Nln by using the LC‐MS/MS method.These results imply that cellular edema formation after stroke can be worsened by common Nln substrates (substance P, bradykinin, and neurotensin), which all have well‐documented neurovascular effects that exacerbate brain edema formation, and treatment with rNln can significantly attenuate cerebral swelling. This study is instrumental in explaining the possible role of Nln substrates and their deleterious effect in edema formation during a stroke and further studies are needed to study the positive role of Nln activation in stroke recovery and reduction of brain edema.Support or Funding InformationThis research was supported by NIH R01 NS106879 to VK and TA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Myo‐inositol in Ischemic Stroke Outcome in a Preclinical Tobacco Smoke Exposed Mouse Model

Tobacco smoking is a preventable risk factor for ischemic stroke in both males and females and contributes up to 14% of all stroke deaths. It has been reported that an increase in brain edema after ischemic stroke worsens long‐term clinical outcome, such as seen in tobacco smokers. Myo‐inositol (MI) is a major organic “non‐perturbing” osmolyte that promotes cell membrane stability and also a precursor for phosphoinositide signaling pathway. Our previous work reported that MI attenuated brain infarct and edema ratio in an ischemic stroke model coupled to preclinical diabetes and suggests that MI and MI transporters provide neuroprotection during/following stroke both in non‐diabetic and diabetic conditions through protection from brain edema. Since our lab has shown that tobacco smoke exposure worsens stroke edema similarly to diabetic conditions, the aim of the present study was to assess the neuroprotective effects of MI associated with ischemic mice exposed to tobacco smoke. In this study, we also investigated the role of MI transporters during stroke and post‐stroke outcomes especially related to the role of astrocytes cells, which are the most abundant glial cells in the brain and play an important role in post‐stroke reorganization, and indirectly protecting neuronal metabolic support. Primary mouse astrocytes were incubated with tobacco smoke extract (TSE) overnight prior for in vitro studies. Toxicity of TSE was measured using MTT assay after incubating astrocytes with TSE 6hr and 24hr. Therapeutic efficacy of MI was evaluated in a transient‐middle cerebral artery occlusion (tMCAO) mouse model after smoking 3R4F research cigarettes for 10 days. Uptake studies showed an increase of MI uptake increased with longer incubation of TSE in normoxic conditions. Additionally, it was also observed that uptake of MI increased during Oxygen Glucose Deprivation(OGD) conditions with prior exposure of TSE. These results are in support with our previous reports, where we found an increased expression of MI transports, Sodium Myo‐inositol Transporter 1 (SMIT1) and Sodium Dependent Glucose Transporter 6 (SGLT6) during OGD exposure. Preliminary animal studies using middle cerebral artery occlusion mouse model for in vivo stroke studies, demonstrated that post‐stroke administration of MI attenuated infarction size and edema ratios in tobacco smoke exposed animals compared to non‐tobacco smoked ischemic animals and also improved motor function in behavioral tests. Taken together with previous work, this study provides an insight into how MI may play a neuroprotective role in ischemic stroke injury and could significantly improve stroke outcome, especially in tobacco smoke populations. Further studies are required to understand the neuroprotective mechanisms of MI in ischemic stroke injury in reducing brain edema.Support or Funding InformationR01NS076012 and R01DA029121This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Human brain microvascular endothelial cells produce CXCL9 after IFN gamma stimulation through JAK2/STAT1 Activation

BackgroundEmergent large vessel occlusion (ELVO) is the deadliest form of stroke and is caused by a blockage within a major a cerebral artery, usually the middle cerebral artery. This condition triggers edema, water movement into the brain, thus pressuring the brain producing massive damage resulting in disability and death. Leukemia inhibitory factor (LIF), a neuroprotective and anti‐inflammatory cytokine, decreases neurodegeneration and increases survival in a rat model of ELVO. Previously we have reported IFNγ is the primary inflammatory mediator in this stroke model and treatment with LIF decreases its expression. This study examined the presence of IFNγ on human endothelial cells by measuring the expression of the IFNγ‐induced CXCL9 and determine LIF's ability to block IFNγ signaling.MethodsFor cells under normoxia conditions, cells were plated and allowed to mature for 7 days, then stimulated with 1 ng/ml IFNγ. At time points 6, 24, 48 and 72 hours after stimulation the supernatant was taken and used for ELISA to determine the amount of CXCL9 released. For OGD conditions, cells were allowed to grow for a week and given glucose free media and placed in oxygen free chamber for 6 hours. The cells were then perfused with media with glucose and 1 ng/ml IFNγ. The supernatant was collected the same time points previously mentioned and CXCL9 was determined as stated previously. To determine the effects inhibitors had on CXCL9 release after being treated with inhibitors to JAK2 and STAT1 under normoxic conditions, cells were treated with IFNy the same as above along coupled with 50 μM and 100 M of fludarabine and AG490.ResultsExposing cerebral microvascular endothelial cells to 1 ng/ml IFNγ significantly induced expression of CXCL9 under both normoxia and oxygen‐glucose deprivation (OGD). Inhibitors to JAK2 and STAT1 significantly blocked the induction of CXCL9. Under normoxic conditions, the addition of LIF (200 ng/ml) with IFNγ significantly augmented the increase in CXCL9 in endothelial cells exposed to OGD.ConclusionCerebral microvascular endothelial cells are a major source of CXCL9, which is upregulated by IFNγ and LIF in a JAK2/STAT1‐dependent manner. The LIF signal transduction pathway in these cells has previously been shown to be dependent upon kinases such as ERK and Akt. CXCL9 is known to break down the blood‐brain barrier, which leads to edema. One potential explanation for this induction of CXCL9 is to open the blood‐brain barrier and allow for anti‐inflammatory leukocytes to enter the ischemic brain and promote repair after stroke.Support or Funding InformationThis project was funded by NINDS Award # 5R0INS091146‐04This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The protective effect of glutaredoxin 1/DJ-1/HSP70 signaling in renal tubular epithelial cells injury induced by ischemia

AimsGluaredoxin1 (GRX1) is an important protein of the cellular antioxidant defense system, but its role in renal epithelial cell injury caused by ischemia remains unclear. In this study, we aimed to gain insight into the role of GRX1 in HK-2 cells with oxygen glucose deprivation (OGD) injury, which served as an in vitro cell model of renal epithelial cell ischemic injury. We investigated the underlying regulation of GRX1, DJ-1, and HSP70 as well as the role of the GRX1/DJ-1/HSP70 signaling pathway in this model. Materials and methodsThe protein and mRNA expressions were measured by Western blot and qRT-PCR assays, respectively. GRX1 was overexpressed by transfection of pcDNA.3.1-GRX1 and DJ-1 was inhibited by transfection with DJ-1 siRNA. Cell apoptosis, caspase-3 activity, lactate dehydrogenase (LDH) leakage, or superoxide dismutase (SOD) content was tested by the related detection kit. Reactive oxygen species (ROS) level was detected via carboxy-H2DCF-DA. Key findingsWe found that GRX1 was distinctly down-regulated in HK-2 cells after incubation under the OGD condition. GRX1 overexpression markedly constrained cell apoptosis, caspase-3 activity, LDH leakage, and the ROS level, while SOD content was elevated. GRX1 up-regulation increased DJ-1 and HSP70 protein expression, while DJ-1 inhibition significantly offset the effect of GRX1 overexpression on HSP70, indicating that GRX1 could regulate HSP70 via control of DJ-1. Moreover, we observed that HSP70 inhibition removed the constraints imposed by GRX1 overexpression on ROS level, LDH leakage, and caspase-3 activity. SignificanceOverall, this study showed that GRX1 minimizes cell injury and apoptosis in HK-2 cells under OGD conditions via regulation of DJ-1 and HSP70 expression.

LncRNA MALAT1 up-regulates VEGF-A and ANGPT2 to promote angiogenesis in brain microvascular endothelial cells against oxygen-glucose deprivation via targetting miR-145.

Stroke is one of the leading causes of death and long-term disability around the world. Angiogenesis is supposed to protect brain microvascular endothelial cells (BMECs) from oxidative and ischemic stress. Previous studies indicated that interaction between metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and miR-145 was involved in myocardial ischemia reperfusion, suggesting MALAT1 and miR-145 were also mediated with the progress of angiogenesis and cell migration in oxygen–glucose deprivation (OGD)-induced BMECs. The present study aimed to investigate the functional roles of MALAT1 in regulating miR-145 and its downstream pro-angiogenesis factors, vascular endothelial growth factor (VEGF)-A and Angiopoietin-2 (ANGPT2) during the progress of angiogenesis in OGD-induced BMECs. An in vitro OGD model was employed in mouse BMECs to mimic brain hypoxic and ischemic conditions; MTT was used to determine cell viability. qRT-PCR was used to determine the expression of long non-coding RNA (lncRNA)-MALAT1 and miR-145 under OGD conditions; in vitro tube formation assay was used to investigate angiogenic effect of MALAT1 and miR-145. The relationship between lncRNA-MALAT1/miR-145 and miR-145/VEGF-A/ANGPT2 was evaluated by qRT-PCR and Western blot, and direct binding was assessed using dual luciferase assay. Results showed that the levels of lncRNA-MALAT1 and miR-145 were up-regulated in OGD-induced BMECs. miR-145 functioned as an anti-angiogenic and pro-apoptotic factor in OGD treated BMECs via down-regulating VEGF-A and ANGPT2 directly. While lncRNA-MALAT1 enhanced the expressions of VEGF-A and ANGPT2 by targetting miR-145 to promote angiogenesis and proliferation of BMECs under OGD conditions. Our present study revealed the inhibitory functions of miR-145 on angiogenesis through direct targetting on VEGF-A and ANGPT2 for the first time and proved the protective role of lncRNA-MALAT1 for BMECs under OGD conditions through the direct regulation of miR-145.

Leukemia Inhibitory Factor Receptor Is Involved in Apoptosis in Rat Astrocytes Exposed to Oxygen-Glucose Deprivation.

Leukemia inhibitory factor (LIF) and leukemia inhibitory factor receptor (Lifr) protect CNS cells, specifically neurons and myelin-sheath oligodendrocytes, in conditions of oxygen-glucose deprivation (OGD). In the case of astrocyte apoptosis resulting from reperfusion injury following hypoxia, the function of the Lifr remains to be fully elucidated. This study established models of in vivo ischemia/reperfusion (I/R) using an in vitro model of OGD to investigate the direct impact of silencing the Lifr on astrocyte apoptosis. Astrocytes harvested from newborn Wistar rats were exposed to OGD. Cell viability and apoptosis levels were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and annexin V/propidium iodide (PI) staining assays, respectively. Apoptosis was further investigated by the TdT-mediated dUTP nick-end labelling (TUNEL) assay. A standard western blotting protocol was applied to determine levels of the protein markers Bcl2, Bax, p-Akt/Akt, p-Stat3/Stat3, and p-Erk/Erk. The cell viability assay (MTT) showed that astrocyte viability decreased in response to OGD. Furthermore, blocking RNA to silence the Lifr further reduces astrocyte viability and increases levels of apoptosis as detected by annexin V/PI double staining. Likewise, western blotting after Lifr silencing demonstrated increased levels of the apoptosis-related proteins Bax and p-Erk/Erk and correspondingly lower levels of Bcl2, p-Akt/Akt, and p-Stat/Stat3. The data gathered in these analyses indicate that the Lifr plays a pivotal role in the astrocyte apoptosis induced by hypoxic/low-glucose environments. Further investigation of the relationship between apoptosis and the Lifr may provide a potential therapeutic target for the treatment of neurological injuries.

Taxifolin protects neurons against ischemic injury in vitro via the activation of antioxidant systems and signal transduction pathways of GABAergic neurons

Cerebral blood flow disturbances lead to the massive death of brain cells. The death of >80% of cells is observed in hippocampal cell cultures after 40 min of oxygen and glucose deprivation (ischemia-like conditions, OGD). However, there are some populations of GABAergic neurons which are characterized by increased vulnerability to oxygen-glucose deprivation conditions. Using fluorescent microscopy, immunocytochemical assay, vitality tests and PCR-analysis, we have shown that population of GABAergic neurons are characterized by a different (faster) Ca2+ dynamics in response to OGD and increased basal ROS production under OGD conditions. A plant flavonoid taxifolin inhibited an excessive ROS production and an irreversible cytosolic Ca2+ concentration increase in GABAergic neurons, preventing the death of these neurons and further excitation of a neuronal network; neuroprotective effect of taxifolin increased after incubation of 24 h and correlated with increased expression of antiapoptocic and antioxidant genes Stat3 Nrf-2 Bcl-2, Bcl-xL, Ikk2, and genes coding for AMPA and kainate receptor subunits; in addition, taxifolin decreased expression of prooxidant enzyme NOS and proinflammatory cytokine IL-1β.

Mi R -15a/15b Cluster Modulates Survival of Mesenchymal Stem Cells to Improve Its Therapeutic Efficacy of Myocardial Infarction.

BackgroundThe poor viability of transplanted mesenchymal stem cells (MSCs) hampers their therapeutic efficacy for ischemic heart disease. MicroRNAs are involved in regulation of MSC survival and function. The present study was designed to investigate the molecular effects of miR‐15a/15b on MSC survival, focusing on the role of vascular endothelial growth factor receptor 2.Methods and ResultsWe first harvested donor luc(Luciferase)‐MSCs (5×105) isolated from the luciferase transgenic mice with FVB background. Luc‐MSCs were transfected with miR‐15a/15b mimics or inhibitors and cultured under oxygen glucose deprivation condition for 12 hours to mimics the harsh microenvironment in infarcted heart; they were subjected to MTT (3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide?Thiazolyl Blue Tetrazolium Bromide) assay, bioluminescence imaging, quantitative reverse transcription–polymerase chain reaction, transferase‐mediated deoxyuridine triphosphate–digoxigenin nick‐end labeling assay, and flow cytometry. Furthermore, the levels of vascular endothelial growth factor receptor 2, protein kinase B, p(Phosphorylate)‐protein kinase B, Bcl‐2, Bax, and caspase‐3 proteins were available by Western blotting assay. In vivo, acute myocardial infarction was induced in 24 mice by coronary ligation, with subsequent receipt of Luc‐MSCs, Luc‐MSCs+miR‐15a/15b inhibitors, or PBS treatment. The therapeutic procedure and treatment effects were tracked and assessed using bioluminescence imaging and echocardiographic measurement. Next, ex vivo imaging and immunohistochemistry were conducted to verify the distribution of MSCs. We demonstrated that miR‐15a/15b targeted vascular endothelial growth factor receptor 2 to modulate MSC survival, possibly via phosphatidylinositol 3‐kinase/protein kinase B signaling pathway, which was proved by bioluminescence imaging, immunohistochemistry analysis, and echocardiographic measurement.ConclusionsLuc‐MSCs could be followed dynamically in vitro and in vivo by bioluminescence imaging, and the role of miR‐15a/b could be inferred from the loss of signals from luc‐MSCs. This finding may have practical clinical implications in miR‐15a/15b–modified MSC transplantation in treating myocardial infarction.

NMDA receptor-driven calcium influx promotes ischemic human cardiomyocyte apoptosis through a p38 MAPK-mediated mechanism.

N-methyl-D-aspartate receptor (NMDAR) activity plays a key role in cerebral ischemia. Although NMDAR is also expressed in cardiomyocytes, little research has been performed on NMDAR activity in myocardial ischemia. Here, using an in vitro oxygen-glucose deprivation (OGD) cardiomyocyte model, we evaluated the effects of NMDAR activity upon calcium influx, viability, apoptosis, and investigated the roles of several key mitogen-activated protein kinases (MAPKs). Primary human neonatal cardiomyocytes were cultured under OGD conditions to mimic in vivo ischemic conditions. Enhancing NMDAR activity via NMDA significantly promoted calcium influx, decreased cell viability, increased apoptosis, and enhanced p38 MAPK phosphorylation in OGD cardiomyocytes (all P < 0.05). These effects were rescued by several calcium-channel blockers (ie, MK-801, La3+ , Gap26 peptide, 18β-glycyrrhetinic acid) but most potently rescued via the NMDAR-specific antagonist MK-801 or removal of extracellular free calcium (all P < 0.05). Knocking-down p38 MAPK activity by small-molecule inhibition or genetic methods significantly increased cell viability and reduced apoptosis (all P < 0.05). Enhancing p38 MAPK activity abolished MK-801's apoptosis-reducing effects in a p38 MAPK-dependent manner. In conclusion, NMDAR-driven calcium influx promotes apoptosis in ischemic human cardiomyocytes, an effect which can be attributed to enhanced p38 MAPK activity.

Repetitive Transcranial Magnetic Stimulation Improves Neurological Function after Cerebral Ischemia in Rats by Increasing CREB-Regulated TrkB &lt;i&gt;via&lt;/i&gt; Activation of cAMP/PKA and Ca &lt;sup&gt;2+&lt;/sup&gt;/CaMKIV Signaling Pathways

Cerebral ischemia is the most prevalent form of clinical stroke. Repetitive transcranial magnetic stimulation (rTMS) can modulate excitability of the cerebral cortex, and this effect is maintained after the stimulation is terminated. However, the underlying mechanisms of rTMS in cerebral ischemia remain unclear. Herein, we identified the effect of rTMS on cerebral ischemia. The in vitro model was established using primary cultured neurons under the condition of oxygen-glucose deprivation (OGD), followed by 1 Hz or 10 Hz rTMS treatment. The levels of CREB, PKA and CaMKIV were depleted in neurons to explore the underlying regulation mechanisms of TrkB by rTMS via CREB. Rat models with cerebral ischemia were established by middle cerebral artery occlusion (MCAO) and stimulated with 1 Hz or 10 Hz rTMS to investigate the effect of rTMS on neurobehavior, CREB expression, cAMP/PKA and Ca2+/CaMKIV pathways. rTMS was observed to promote the nerve recovery ability in rats with cerebral ischemia, which was accompanied by high expression of TrkB. Besides, in OGD-treated neurons, rTMS activated CREB by upregulating cAMP/PKA and Ca2+/CaMKIV pathways. Moreover, rTMS induced the activation of CREB to upregulate TrkB. In MACO rats, rTMS promoted CREB expression and the content of CREB, cAMP and PKA phosphorylation, increased the CaMKIV phosphorylation, and promoted the interaction between CREB and TrkB. Taken together, rTMS promoted CREB expression and up-regulated TrkB to improve the neurological function in rats with cerebral ischemia by activating cAMP/PKA and Ca2+/CaMKIV pathways, which could be of great significance for cerebral ischemia therapy. Funding Statement: The authors declare: None. Declaration of Interests: The authors declare: None. Ethics Approval Statement: The animal experiment was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Animal Ethics Committee of North China University of Science and Technology Affiliated Hospital.

Ethanol exposed maturing rat cerebellar granule cells show impaired energy metabolism and increased cell death after oxygen-glucose deprivation.

Alcohol, a widely abused drug, has deleterious effects on the immature nervous system. This study investigates the effect of chronic in vitro ethanol exposure on the metabolism of immature rat cerebellar granular cells (CGCs) and on their response to oxygen-glucose deprivation (OGD). Primary CGC cultures were exposed to ethanol (100 mM in culture medium) or to control ethanol-free medium starting day one in vitro (DIV1). At DIV8, the expression of ATP synthase gene ATP5g3 was quantified using real-time PCR, then cultures were exposed to 3 hours of OGD or normoxic conditions. Subsequently, cellular metabolism was assessed by a resazurin assay and by ATP level measurement. ATP5g3 expression was reduced by 12-fold (P = 0.03) and resazurin metabolism and ATP level were decreased to 74.4 ± 4.6% and 55.5 ± 6.9%, respectively after chronic ethanol treatment compared to control values (P < 0.01). Additionally, after OGD exposure of ethanol-treated cultures, resazurin metabolism and ATP level were decreased to 12.7 ± 1.0% and 9.0 ± 2.0% from control values (P < 0.01). These results suggest that chronic ethanol exposure reduces the cellular ATP level, possibly through a gene expression down-regulation mechanism, and increases the vulnerability to oxygen-glucose deprivation. Thus, interventions which improve metabolic function and sustain ATP-levels could attenuate ethanol-induced neuronal dysfunction and should be addressed in future studies.

LncRNA NEAT1 facilitates survival and angiogenesis in oxygen-glucose deprivation (OGD)-induced brain microvascular endothelial cells (BMECs) via targeting miR-377 and upregulating SIRT1, VEGFA, and BCL-XL

ObjectiveThe present study was designed to investigate the mechanism by which lncRNA NEAT1 regulates survival and angiogenesis in oxygen-glucose deprivation (OGD)-induced brain microvascular endothelial cells (BMECs). MethodsOGD-treated BMECs were used to mimic cerebral ischaemia in vitro. The expression of lncRNA NEAT1 and miR-377 and proteins including VEGFA, SIRT1, and BCL-XL were measured by real-time quantitative PCR (qRT-PCR) and western blot, respectively. Cell viability and caspase 3 activity of BMECs under different conditions were determined using MTT and caspase activity assays, respectively. Matrigel-based angiogenesis assays were employed to evaluate the effect of lncRNA NEAT1 on angiogenesis. A dual-luciferase reporter assay was used to validate direct binding of miR-377 to putative targets. ResultsOGD exposure reduced the cell viability of BMECs. Upregulation of lncRNA NEAT1 and downregulation of miR-377 were also observed under OGD conditions. Knockdown of lncRNA NEAT1 inhibited angiogenesis and aggravated apoptosis in OGD-induced BMECs. Meanwhile, the expression level of miR-377 was upregulated while its downstream targets (VEGFA, SIRT1, and BCL-XL) were downregulated after lncRNA NEAT1 knockdown. Furthermore, miR-377 inhibited the angiogenesis and survival of OGD-induced BMECs. The expression of VEGFA, SIRT1, and BCL-XL were all attenuated by miR-377 overexpression. The dual-luciferase reporter assay proved miR-377 targeted the 3′ UTR sequences of lncRNA NEAT1, VEGFA, SIRT1, and BCL-XL. ConclusionlncRNA NEAT1 facilitated the survival and angiogenesis of OGD-induced BMECs via targeting miR-377 and promoting the expression of VEGFA, SIRT1, and BCL-XL, suggesting that lncRNA NEAT1 could be a promising target for cerebral ischaemia treatment.

AMPK promotes survival and adipogenesis of ischemia-challenged ADSCs in an autophagy-dependent manner

Some studies have shown that transplanted fat tissues usually cannot survive for long if adipose-derived stem cells (ADSCs) are removed from the tissues in advance. It is more meaningful to explore the mechanism mediating survival and differentiation of ADSCs in the transplanted microenvironment. AMP-activated protein kinase (AMPK) has been shown to be one of the energy receptors that regulate many aspects of cellular metabolism. AMPK activation has been implicated in models of adult ischemic injury, but the mechanism and the regulating effects of AMPK on survival and adipogenesis of transplanted ADSCs are still little known. In this study, we simulated the transplanted microenvironment using oxygen-glucose deprivation (OGD) to test the survival and adipogenesis of ADSCs. We found that OGD treatment triggered significant apoptosis and promoted autophagy. Simultaneously, OGD hindered the differentiation of ADSCs into mature adipocytes. After inhibiting AMPK, the OGD-induced apoptosis rate increased but autophagy was inhibited. The adipogenesis level also decreased. To show that the effects of AMPK on apoptosis and adipogenesis were autophagy-dependent, we pre-inhibited or pre-promoted autophagy with siATG7 or rapamycin while blocking AMPK. We found that inhibiting or improving autophagy exacerbated or alleviated the role of AMPK prohibition in apoptosis and adipogenesis. Furthermore, we showed that AMPK inhibition significantly lowered ULK1 activity but promoted mTOR activity, so that to inhibit autophagy. Our study shows that AMPK plays a protective role in maintaining survival and adipogenesis of OGD-challenged ADSCs partly by positively regulating autophagy. AMPK positively regulates autophagy by inhibiting mTOR but promoting ULK1 activity in OGD condition.

Hepatology Basic Science

Hepatology Basic Science

ETS-domain containing protein (Elk1) suppression protects cortical neurons against oxygen-glucose deprivation injury

ETS-domain containing protein (Elk1), which is a transcription factor, is reported to be closely related to the apoptosis of primary neurons and could be activated by hypoxia in human microvascular endothelial cells. In this study, we aimed to investigate the role of Elk1 in cortical neurons under oxygen-glucose deprivation (OGD) conditions. The OGD model of cortical neurons was established the anoxia/hypoglycemia-induced injury and the in vivo model was established by middle cerebral artery occlusion (MCAO). Elk1 mRNA and protein expression was significantly up-regulated in neurons exposed to OGD for 24 h, and mRNA expression was also markedly increased in cerebral cortex of rats with MCAO after 10 days. The knockdown of Elk1 in neurons without OGD obviously constrained Fra-1 and promoted Nrf2 expression. Also, Elk1 inhibition suppressed neuronal apoptosis, caspase-3 activity, LDH leakage, and MDA and SOD contents, while it increased cell viability in the neurons with OGD. The overexpression of Fra-1 showed a reverse effect on caspase-3 activity, cell viability and SOD contents in neurons under OGD conditions compared with Elk1 knockdown. Thus, Elk1 inhibition has a protective effect on neurons against OGD-induced injury.

Expression changes of the notch signaling pathway of PC12 cells after oxygen glucose deprivation

Ischemic stroke is caused by obstructed blood supply to the brain. It is a common as well as a serious health problem worldwide, which is often linked to disability and mortality. Here we studied, under the conditions of oxygen glucose deprivation (OGD), the expression of Notch signaling pathway proteins in PC12 cells. PC12 cells were stimulated and converted into neuron-like cells by nerve growth factor. Exposure to OGD was used as an in vitro model of cerebral hypoxia-ischemia. Our findings demonstrate that, after 3 h of OGD exposure, the expression of Notch1, Hes1 and Hes5 significantly increased, on both mRNA and protein levels. This effect gradually reduced with continuous OGD treatment, but the expression levels of these three genes remained higher, compared to untreated controls, even after 24 h of OGD exposure. Our results suggest that OGD exposure up-regulates the expression of Notch1, Hes1 and Hes5, which are important participants in Notch signaling pathway. Since their regulatory roles appear to change dynamically with the extension of OGD, the activation of the Notch pathway may play an important role in cerebral ischemic injury.

Catalpol Promotes the Survival and VEGF Secretion of Bone Marrow-Derived Stem Cells and Their Role in Myocardial Repair After Myocardial Infarction in Rats.

Bone mesenchymal stem cells (BMSCs) transplantation has been recognized as an effective method for the treatment of myocardial infarction (MI). However, its efficacy is always restricted by the low survival of transplanted BMSCs in the ischemic myocardium. The aim of this study was to investigate the effect of catalpol pre-treatment on the survival and vascular endothelial growth factor (VEGF) secretion of BMSCs under oxygen glucose deprivation (OGD) condition and their role in myocardial repair in a rat model of MI. According to our results, pre-treatment with catalpol enhanced VEGF secretion and survival of OGD-treated BMSCs. Moreover, the apoptosis of BMSCs induced by OGD was restrained by catalpol as evidenced by increased level of B-cell lymphoma-2 (Bcl-2) and decreased levels of BCL2-associated X (Bax) and cleaved caspase-3. In vivo study suggested that the survival of transplanted BMSCs was improved by catalpol pre-treatment. The myocardial fibrosis and apoptosis was further inhibited in catalpol pre-treated BMSCs group. Cardiac function detected by echocardiography was obviously improved by catalpol pre-treated BMSCs transplantation. Finally, angiogenesis and VEGF expression in the ischemic myocardium were significantly promoted in catalpol pre-treated BMSCs group. In conclusion, catalpol pre-treatment may facilitate the survival and VEGF secretion of BMSCs and improve their therapeutic effect on MI.