Intravenous antagomiR-494 lessens brain-infiltrating neutrophils by increasing HDAC2-mediated repression of multiple MMPs in experimental stroke

Abstract Background: Neutrophil infiltration and phenotypic transformation are believed to contribute to neuronal damage and clinical outcome in ischemic stroke. Emerging evidence suggests that HDAC2 is an epigenetic regulator of inflammatory cells. Here, we investigated whether miR-494 affects HDAC2-mediated neutrophil infiltration and phenotypic shift. Methods: The miR-494 levels in neutrophils from AIS patients were detected by real-time PCR. C57BL/6J mice were subjected to transient middle cerebral artery occlusion, and the N1/N2 neutrophil shift was examined. Cortical neurons were subjected to oxygen-glucose deprivation and stimulated with supernatant from differently treated neutrophils or were cocultured with neutrophils; neuronal injury was detected, and ChIP-Seq was performed to clarify which genes are the binding targets of HDAC2. Finally, a transwell assay was conducted to examine neutrophil migration. Results: Compared to the control subjects, AIS patients had increased neutrophil expression of miR-494, and in AIS patients, elevated miR-494 expression in neutrophils was a predictor of worse neurological outcomes. MiR-494 correlates with the upregulation of adhesion molecules in neutrophils of AIS patients. Systemically administered antagomiR-494 partly shifts neutrophils into the N2 phenotype in MCAO mice. AntagomiR-494-treated neutrophils exert a neuroprotective role in vitro. ChIP-seq revealed that HDAC2 targets multiple MMP genes in neutrophils of AIS patients. Further in vitro and in vivo experiments showed that antagomiR-494 repressed expression of MMP genes, including MMP7, MMP10, MMP13, and MMP16, to reduce the number of brain-infiltrating neutrophils by regulating HDAC2. Conclusion: MiR-494 may serve as an alternative predictive biomarker of the outcome of AIS patients, and antagomiR-494 treatment decreased the expression of multiple MMPs and the infiltration of neutrophils partly by targeting HDAC2.

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Cerebroprotective effect of xantohumol sensu experimental stroke model: study design and preliminary results

Saratov Medical Journal ◽

10.15275/sarmj.2021.0103 ◽

2021 ◽

Vol 2 (1) ◽

Author(s):

Aigul Saitgareyeva ◽

Leyla Akhmadeyeva

Keyword(s):

Ischemic Stroke ◽

Cortical Neurons ◽

Glucose Deprivation ◽

Experimental Stroke ◽

Control Group ◽

Stroke Model ◽

Sprague Dawley ◽

Preliminary Results

The objective of our study was to evaluate the cerebroprotective effect of xanthohumol (ХN) on experimental models of acute ischemic stroke in vivo and in vitro. Materials and methods. We used middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation (OGD) as in vivo and in vitro models. Our study subjects were Sprague-Dawley rats, which were randomly assigned to three groups: the control group and two MCAO groups with and without XN. The primary culture of cortical neurons was obtained from newborn rats. We employed the Bederson test and the corner test to evaluate neurological disorders. Results. The preliminary results indicated a possible cerebroprotective effect of XN in an ischemic stroke model. Conclusion. Preventive administration of XN before cerebral ischemia in an experiment can effectively reduce the volume of cerebral infarction and improve neurologic deficit 24 hours after MCAO.

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Modulator of apoptosis-1 is a potential therapeutic target in acute ischemic injury

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x18794839 ◽

2018 ◽

Vol 39 (12) ◽

pp. 2406-2418 ◽

Cited By ~ 3

Author(s):

Su Jing Chan ◽

Hui Zhao ◽

Kazuhide Hayakawa ◽

Chou Chai ◽

Chong Teik Tan ◽

...

Keyword(s):

Cortical Neurons ◽

Infarct Volume ◽

Neuronal Loss ◽

Ischemic Injury ◽

Glucose Deprivation ◽

Artery Occlusion ◽

In Vivo Models ◽

The Brain

Modulator of apoptosis 1 (MOAP-1) is a Bax-associating protein highly enriched in the brain. In this study, we examined the role of MOAP-1 in promoting ischemic injuries following a stroke by investigating the consequences of MOAP-1 overexpression or deficiency in in vitro and in vivo models of ischemic stroke. MOAP-1 overexpressing SH-SY5Y cells showed significantly lower cell viability following oxygen and glucose deprivation (OGD) treatment when compared to control cells. Consistently, MOAP-1−/− primary cortical neurons were observed to be more resistant against OGD treatment than the MOAP-1+/+ primary neurons. In the mouse transient middle cerebral artery occlusion (tMCAO) model, ischemia triggered MOAP-1/Bax association, suggested activation of the MOAP-1-dependent apoptotic cascade. MOAP-1−/− mice were found to exhibit reduced neuronal loss and smaller infarct volume 24 h after tMCAO when compared to MOAP-1+/+ mice. Correspondingly, MOAP-1−/− mice also showed better integrity of neurological functions as demonstrated by their performance in the rotarod test. Therefore, both in vitro and in vivo data presented strongly support the conclusion that MOAP-1 is an important apoptotic modulator in ischemic injury. These results may suggest that a reduction of MOAP-1 function in the brain could be a potential therapeutic approach in the treatment of acute stroke.

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Proof-of-concept clinical trial of etokimab shows a key role for IL-33 in atopic dermatitis pathogenesis

Science Translational Medicine ◽

10.1126/scitranslmed.aax2945 ◽

2019 ◽

Vol 11 (515) ◽

pp. eaax2945 ◽

Cited By ~ 29

Author(s):

Yi-Ling Chen ◽

Danuta Gutowska-Owsiak ◽

Clare S. Hardman ◽

Melanie Westmoreland ◽

Teena MacKenzie ◽

...

Keyword(s):

Atopic Dermatitis ◽

Therapeutic Potential ◽

Neutrophil Migration ◽

Neutrophil Infiltration ◽

Severity Index ◽

Fundamental Biology ◽

Targeted Inhibition

Targeted inhibition of cytokine pathways provides opportunities to understand fundamental biology in vivo in humans. The IL-33 pathway has been implicated in the pathogenesis of atopy through genetic and functional associations. We investigated the role of IL-33 inhibition in a first-in-class phase 2a study of etokimab (ANB020), an IgG1 anti–IL-33 monoclonal antibody, in patients with atopic dermatitis (AD). Twelve adult patients with moderate to severe AD received a single systemic administration of etokimab. Rapid and sustained clinical benefit was observed, with 83% achieving Eczema Area and Severity Index 50 (EASI50), and 33% EASI75, with reduction in peripheral eosinophils at day 29 after administration. We noted significant reduction in skin neutrophil infiltration after etokimab compared with placebo upon skin challenge with house dust mite, reactivity to which has been implicated in the pathogenesis of AD. We showed that etokimab also inhibited neutrophil migration to skin interstitial fluid in vitro. Besides direct effects on neutrophil migration, etokimab revealed additional unexpected CXCR1-dependent effects on IL-8–induced neutrophil migration. These human in vivo findings confirm an IL-33 upstream role in modulating skin inflammatory cascades and define the therapeutic potential for IL-33 inhibition in human diseases, including AD.

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Ras-like Gem GTPase induced by Npas4 promotes activity-dependent neuronal tolerance for ischemic stroke

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2018850118 ◽

2021 ◽

Vol 118 (32) ◽

pp. e2018850118

Author(s):

Hiroo Takahashi ◽

Ryo Asahina ◽

Masayuki Fujioka ◽

Takeshi K. Matsui ◽

Shigeki Kato ◽

...

Keyword(s):

Ischemic Stroke ◽

Cortical Neurons ◽

Glucose Deprivation ◽

Artery Occlusion ◽

Small Gtpase ◽

Neuroprotective Effects ◽

Necessary And Sufficient ◽

Activity Dependent

Ischemic stroke, which results in loss of neurological function, initiates a complex cascade of pathological events in the brain, largely driven by excitotoxic Ca2+ influx in neurons. This leads to cortical spreading depolarization, which induces expression of genes involved in both neuronal death and survival; yet, the functions of these genes remain poorly understood. Here, we profiled gene expression changes that are common to ischemia (modeled by middle cerebral artery occlusion [MCAO]) and to experience-dependent activation (modeled by exposure to an enriched environment [EE]), which also induces Ca2+ transients that trigger transcriptional programs. We found that the activity-dependent transcription factor Npas4 was up-regulated under MCAO and EE conditions and that transient activation of cortical neurons in the healthy brain by the EE decreased cell death after stroke. Furthermore, both MCAO in vivo and oxygen-glucose deprivation in vitro revealed that Npas4 is necessary and sufficient for neuroprotection. We also found that this protection involves the inhibition of L-type voltage-gated Ca2+ channels (VGCCs). Next, our systematic search for Npas4-downstream genes identified Gem, which encodes a Ras-related small GTPase that mediates neuroprotective effects of Npas4. Gem suppresses the membrane localization of L-type VGCCs to inhibit excess Ca2+ influx, thereby protecting neurons from excitotoxic death after in vitro and in vivo ischemia. Collectively, our findings indicate that Gem expression via Npas4 is necessary and sufficient to promote neuroprotection in the injured brain. Importantly, Gem is also induced in human cerebral organoids cultured under an ischemic condition, revealing Gem as a new target for drug discovery.

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Effects of human neutrophil elastase (HNE) on neutrophil function in vitro and in inflamed microvessels

Blood ◽

10.1182/blood.v82.7.2188.2188 ◽

1993 ◽

Vol 82 (7) ◽

pp. 2188-2195 ◽

Cited By ~ 45

Author(s):

RC Woodman ◽

PH Reinhardt ◽

S Kanwar ◽

FL Johnston ◽

P Kubes

Keyword(s):

Neutrophil Elastase ◽

Human Neutrophil ◽

Neutrophil Migration ◽

Specific Inhibitor ◽

Neutrophil Infiltration ◽

Cathepsin G ◽

Neutrophil Adhesion ◽

Human Neutrophil Elastase

Abstract The primary objective of this study was to test the hypothesis that human neutrophil elastase (HNE) affects neutrophil infiltration (adhesion and emigration) into inflamed vessels. To determine whether HNE contributes to neutrophil adhesion in vivo, intravital microscopy was used to study neutrophil-endothelial cell interactions in single inflamed postcapillary venules. Superfusion of platelet-activating factor (PAF) (100 nmol/L) onto the mesentery caused an increase in neutrophil-neutrophil interactions, neutrophil adhesion to postcapillary venules, and cellular emigration out of the vasculature. Both L658 758 (an elastase-specific inhibitor), and Eglin C (an elastase and cathepsin G inhibitor) significantly attenuated all of these parameters in vivo. To further characterize the mechanism(s) involved, various in vitro parameters were assessed. HNE, but not trypsin, caused a dose-dependent (0.01 to 1.0 microgram/mL) increase in the expression of the beta subunit (CD18) of the CD11/CD18 adhesive glycoprotein complex on neutrophils. An HNE-dependent increase in CD11b expression was also observed; however, HNE did not affect the expression of other neutrophil adhesion molecules (L-selectin), superoxide production, or degranulation. PAF-enhanced CD18 expression on neutrophils and neutrophil migration were both abolished by L658 758 but PAF-induced neutrophil adhesion to endothelial monolayers was not affected by the antiproteinase. The in vitro data suggest that the antiproteinases do not directly prevent neutrophil adhesion in vivo but may be important in other CD18-dependent events such as neutrophil- neutrophil interaction or neutrophil infiltration (chemotaxis). These results translate into an important, rate-limiting role for elastase in the process of leukocyte infiltration and accumulation in inflamed microvessels.

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Metoprolol exerts a non-class effect against ischaemia–reperfusion injury by abrogating exacerbated inflammation

European Heart Journal ◽

10.1093/eurheartj/ehaa733 ◽

2020 ◽

Vol 41 (46) ◽

pp. 4425-4440 ◽

Cited By ~ 3

Author(s):

Agustín Clemente-Moragón ◽

Mónica Gómez ◽

Rocío Villena-Gutiérrez ◽

Doménica V Lalama ◽

Jaime García-Prieto ◽

...

Keyword(s):

Conformational Changes ◽

In Silico ◽

Adrenergic Receptor ◽

Neutrophil Migration ◽

Neutrophil Infiltration ◽

Ischaemia Reperfusion Injury ◽

Ischaemia Reperfusion ◽

Β Blockers

Abstract Aims Clinical guidelines recommend early intravenous β-blockers during ongoing myocardial infarction; however, it is unknown whether all β-blockers exert a similar cardioprotective effect. We experimentally compared three clinically approved intravenous β-blockers. Methods and results Mice undergoing 45 min/24 h ischaemia–reperfusion (I/R) received vehicle, metoprolol, atenolol, or propranolol at min 35. The effect on neutrophil infiltration was tested in three models of exacerbated inflammation. Neutrophil migration was evaluated in vitro and in vivo by intravital microscopy. The effect of β-blockers on the conformation of the β1 adrenergic receptor was studied in silico. Of the tested β-blockers, only metoprolol ameliorated I/R injury [infarct size (IS) = 18.0% ± 0.03% for metoprolol vs. 35.9% ± 0.03% for vehicle; P < 0.01]. Atenolol and propranolol had no effect on IS. In the three exacerbated inflammation models, neutrophil infiltration was significantly attenuated only in the presence of metoprolol (60%, 50%, and 70% reductions vs. vehicle in myocardial I/R injury, thioglycolate-induced peritonitis, and lipopolysaccharide-induced acute lung injury, respectively). Migration studies confirmed the particular ability of metoprolol to disrupt neutrophil dynamics. In silico analysis indicated different intracellular β1 adrenergic receptor conformational changes when bound to metoprolol than to the other two β-blockers. Conclusions Metoprolol exerts a disruptive action on neutrophil dynamics during exacerbated inflammation, resulting in an infarct-limiting effect not observed with atenolol or propranolol. The differential effect of β-blockers may be related to distinct conformational changes in the β1 adrenergic receptor upon metoprolol binding. If these data are confirmed in a clinical trial, metoprolol should become the intravenous β-blocker of choice for patients with ongoing infarction.

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Effects of human neutrophil elastase (HNE) on neutrophil function in vitro and in inflamed microvessels

Blood ◽

10.1182/blood.v82.7.2188.bloodjournal8272188 ◽

1993 ◽

Vol 82 (7) ◽

pp. 2188-2195

Author(s):

RC Woodman ◽

PH Reinhardt ◽

S Kanwar ◽

FL Johnston ◽

P Kubes

Keyword(s):

Neutrophil Elastase ◽

Human Neutrophil ◽

Neutrophil Migration ◽

Specific Inhibitor ◽

Neutrophil Infiltration ◽

Cathepsin G ◽

Neutrophil Adhesion ◽

Human Neutrophil Elastase

The primary objective of this study was to test the hypothesis that human neutrophil elastase (HNE) affects neutrophil infiltration (adhesion and emigration) into inflamed vessels. To determine whether HNE contributes to neutrophil adhesion in vivo, intravital microscopy was used to study neutrophil-endothelial cell interactions in single inflamed postcapillary venules. Superfusion of platelet-activating factor (PAF) (100 nmol/L) onto the mesentery caused an increase in neutrophil-neutrophil interactions, neutrophil adhesion to postcapillary venules, and cellular emigration out of the vasculature. Both L658 758 (an elastase-specific inhibitor), and Eglin C (an elastase and cathepsin G inhibitor) significantly attenuated all of these parameters in vivo. To further characterize the mechanism(s) involved, various in vitro parameters were assessed. HNE, but not trypsin, caused a dose-dependent (0.01 to 1.0 microgram/mL) increase in the expression of the beta subunit (CD18) of the CD11/CD18 adhesive glycoprotein complex on neutrophils. An HNE-dependent increase in CD11b expression was also observed; however, HNE did not affect the expression of other neutrophil adhesion molecules (L-selectin), superoxide production, or degranulation. PAF-enhanced CD18 expression on neutrophils and neutrophil migration were both abolished by L658 758 but PAF-induced neutrophil adhesion to endothelial monolayers was not affected by the antiproteinase. The in vitro data suggest that the antiproteinases do not directly prevent neutrophil adhesion in vivo but may be important in other CD18-dependent events such as neutrophil- neutrophil interaction or neutrophil infiltration (chemotaxis). These results translate into an important, rate-limiting role for elastase in the process of leukocyte infiltration and accumulation in inflamed microvessels.

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The AAA + ATPase Thorase is neuroprotective against ischemic injury

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x18769770 ◽

2018 ◽

Vol 39 (9) ◽

pp. 1836-1848 ◽

Cited By ~ 2

Author(s):

Jianmin Zhang ◽

Jia Yang ◽

Huaishan Wang ◽

Omar Sherbini ◽

Matthew J Keuss ◽

...

Keyword(s):

Glucose Deprivation ◽

Artery Occlusion ◽

Glutamate Excitotoxicity ◽

Experimental Stroke ◽

Dependent Manner ◽

Aaa Atpase ◽

Neurologic Disorders ◽

Cerebral Artery Occlusion

Neuronal preconditioning in vitro or in vivo with a stressful but non-lethal stimulus leads to new protein expression that mediates a profound neuroprotection against glutamate excitotoxicity and experimental stroke. The proteins that mediate neuroprotection are relatively unknown and under discovery. Here we find that the expression of the AAA + ATPase Thorase is induced by preconditioning stimulation both in vitro and in vivo. Thorase provides neuroprotection in an ATP-dependent manner against oxygen–glucose deprivation (OGD) neurotoxicity or glutamate N-Methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity in vitro. Knock-down of Thorase prevents the establishment of preconditioning induced neuroprotection against OGD or NMDA neurotoxicity. Transgenic overexpression of Thorase provides neuroprotection in vivo against middle cerebral artery occlusion (MCAO)-induced stroke in mice, while genetic deletion of Thorase results in increased injury in vivo following stroke. These results define Thorase as a neuroprotective protein and understanding Thorase signaling could offer a new therapeutic strategy for the treatment of neurologic disorders.

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Transporters MRP1 and MRP2 Regulate Opposing Inflammatory Signals To Control Transepithelial Neutrophil Migration duringStreptococcus pneumoniaeLung Infection

mSphere ◽

10.1128/msphere.00303-18 ◽

2018 ◽

Vol 3 (4) ◽

Cited By ~ 1

Author(s):

Andrew Zukauskas ◽

Randall J. Mrsny ◽

Paula Cortés Barrantes ◽

Jerrold R. Turner ◽

John M. Leong ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Neutrophil Migration ◽

Neutrophil Infiltration ◽

Mortality Rates ◽

Positive Bacterium ◽

Immunomodulatory Agents ◽

Content Type ◽

Mouse Lungs

ABSTRACTStreptococcus pneumoniaeremains a source of morbidity and mortality in both developed and underdeveloped nations of the world. Disease can manifest as pneumonia, bacteremia, and meningitis, depending on the localization of infection. Interestingly, there is a correlation in experimental murine infections between the development of bacteremia and influx of neutrophils into the pulmonary lumen. Reduction of this neutrophil influx has been shown to improve survivability during infection. In this study, we usein vitrobiotinylation and neutrophil transmigration andin vivomurine infection to identify a system in which two epithelium-localized ATP-binding cassette transporters, MRP1 and MRP2, have inverse activities dictating neutrophil transmigration into the lumen of infected mouse lungs. MRP1 effluxes an anti-inflammatory molecule that maintains homeostasis in uninfected contexts, thus reducing neutrophil infiltration. During inflammatory events, however, MRP1 decreases and MRP2 both increases and effluxes the proinflammatory eicosanoid hepoxilin A3. If we then decrease MRP2 activity during experimental murine infection withS. pneumoniae, we reduce both neutrophil infiltration and bacteremia, showing that MRP2 coordinates this activity in the lung. We conclude that MRP1 assists in depression of polymorphonuclear cell (PMN) migration by effluxing a molecule that inhibits the proinflammatory effects of MRP2 activity.IMPORTANCEStreptococcus pneumoniaeis a Gram-positive bacterium that normally inhabits the human nasopharynx asymptomatically. However, it is also a major cause of pneumonia, bacteremia, and meningitis. The transition from pneumonia to bacteremia is critical, as patients that develop septicemia have ~20% mortality rates. Previous studies have shown that while neutrophils, a major bacterium-induced leukocyte, aid inS. pneumoniaeelimination, they also contribute to pathology and may mediate the lung-to-blood passage of the bacteria. Herein, we show that epithelium-derived MRP1 and MRP2 efflux immunomodulatory agents that assist in controlling passage of neutrophils during infection and that limiting neutrophil infiltration produced less bacteremia and better survival during murine infection. The importance of our work is twofold: ours is the first to identify an MRP1/MRP2 axis of neutrophil control in the lung. The second is to provide possible therapeutic targets to reduce excess inflammation, thus reducing the chances of developing bacteremia during pneumococcal pneumonia.

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GATA3 (GATA-Binding Protein 3)/KMT2A (Lysine-Methyltransferase-2A) Complex by Increasing H3K4-3me (Trimethylated Lysine-4 of Histone-3) Upregulates NCX3 (Na + -Ca 2+ Exchanger 3) Transcription and Contributes to Ischemic Preconditioning Neuroprotection

Stroke ◽

10.1161/strokeaha.121.034637 ◽

2021 ◽

Vol 52 (11) ◽

pp. 3680-3691

Author(s):

Natascia Guida ◽

Luigi Mascolo ◽

Angelo Serani ◽

Ornella Cuomo ◽

Serenella Anzilotti ◽

...

Keyword(s):

Ischemic Preconditioning ◽

Cortical Neurons ◽

Promoter Region ◽

Glucose Deprivation ◽

Artery Occlusion ◽

Histone 3 ◽

Lysine Methyltransferase ◽

Cerebral Artery Occlusion

Background and Purpose: NCX3 (Na + -Ca 2+ exchanger 3) plays a relevant role in stroke; indeed its pharmacological blockade or its genetic ablation exacerbates brain ischemic damage, whereas its upregulation takes part in the neuroprotection elicited by ischemic preconditioning. To identify an effective strategy to induce an overexpression of NCX3, we examined transcription factors and epigenetic mechanisms potentially involved in NCX3 gene regulation. Methods: Brain ischemia and ischemic preconditioning were induced in vitro by exposure of cortical neurons to oxygen and glucose deprivation plus reoxygenation (OGD/Reoxy) and in vivo by transient middle cerebral artery occlusion. Western blot and quantitative real-time polymerase chain reaction were used to evaluate transcripts and proteins of GATA3 (GATA-binding protein 3), KMT2A (lysine-methyltransferase-2A), and NCX3. GATA3 and KMT2A binding on NCX3 gene was evaluated by chromatin immunoprecipitation and Rechromatin immunoprecipitation experiments. Results: Among the putative transcription factors sharing a consensus sequence on the ncx3 brain promoter region, GATA3 was the only able to up-regulate ncx3. Interestingly, GATA3 physically interacted with KMT2A, and their overexpression or knocking-down increased or downregulated NCX3 mRNA and protein, respectively. Notably, site-direct mutagenesis of GATA site on ncx3 brain promoter region counteracted GATA3 and KMT2A binding on NCX3 gene. More importantly, we found that in the perischemic cortical regions of preconditioned rats GATA3 recruited KMT2A and the complex H3K4-3me (trimethylated lysine-4 of histone-3) on ncx3 brain promoter region, thus reducing transient middle cerebral artery occlusion–induced damage. Consistently, in vivo silencing of either GATA3 or KMT2A prevented NCX3 upregulation and consequently the neuroprotective effect of preconditioning stimulus. The involvement of GATA3/KMT2A complex in neuroprotection elicited by ischemic preconditioning was further confirmed by in vitro experiments in which the knocking-down of GATA3 and KMT2A reverted the neuroprotection induced by NCX3 overexpression in cortical neurons exposed to anoxic preconditioning followed by oxygen and glucose deprivation plus reoxygenation. Conclusions: Collectively, our results revealed that GATA3/KMT2A complex epigenetically activates NCX3 gene transcription during ischemic preconditioning.

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