Brain Vascular Endothelial Cells Research Articles

The protective effects of butorphanol tartrate against homocysteine-induced blood-brain barrier dysfunction

ABSTRACT A high concentration of homocysteine (Hcy) has been recently reported to be closely associated with the development of stroke, which is related to the Hcy-induced blood-brain barrier (BBB) dysfunction. Butorphanol tartrate is a promising analgesic agent that targets the opiate receptor and shows promising protective effects on ischemia/reperfusion injury. The present research proposes to investigate the protective effect of butorphanol tartrate on Hcy-induced BBB disruption to explore the potential application of butorphanol tartrate in treating Hcy-induced stroke. Hcy was utilized to establish both an in vivo animal model and in vitro human brain vascular endothelial cells (HBVECs) injury model. We found that the increased diffusion of sodium fluorescein and Evan’s blue, declined expression of Claudin-5, and increased production of interleukin- 6 (IL-6) and tumor necrosis factor-α (TNF-α) were observed in Hcy-treated mice, which were all significantly reversed by butorphanol tartrate. In Hcy-stimulated HBVECs, increased endothelial permeability and reduced expression levels of Claudin-5 and Krüppel-like factor 5 (KLF5) were observed, all of which were dramatically rescued by 2 and 5 µM butorphanol tartrate. Lastly, the protective function of butorphanol tartrate in Hcy-stimulated HBVECs was dramatically abolished by the knockdown of KLF5. Collectively, butorphanol tartrate showed protective effects on Hcy-induced BBB disruption by upregulating the KLF5/Claudin-5 axis.

Abstract WP210: Eicosapentaenoic Acid (EPA) Decreases Cytokine Release And Inflammatory Proteins In Brain Vascular Endothelium During Inflammation

Background: Brain vascular endothelial cell (EC) dysfunction is characterized by release of pro-inflammatory mediators that contribute to atherosclerosis and ischemic stroke. Treatment with icosapent ethyl (IPE), the ethyl ester of the omega-3 fatty acid, eicosapentaenoic acid (EPA), reduced first and total ischemic strokes each by 36%, without increasing hemorrhagic stroke, in statin-treated patients with elevated cardiovascular risk (REDUCE-IT). We tested the effects of EPA on cytokine release and expression of inflammatory proteins from brain microvascular ECs during inflammation. Methods: Human brain microvascular endothelial cells (HBECs) were pretreated with the cytokine IL-6 at 12 ng/ml for 2 h before treatment with EPA (40 μM) for 24 h. Proteomic analysis was performed using LC/MS to capture relative expression levels. Only significant changes in protein expression between treatment groups >1-fold were analyzed. Gene set enrichment analysis was also performed on all significantly regulated proteins to identify groups of proteins (pathways) that were affected. Levels of soluble intercellular adhesion molecule-1 (sICAM-1) and tumor necrosis factor-α (TNF-α) were measured by immunochemistry (ELISA). Results: IL-6 exposure produced increased levels of sICAM-1 and TNF-α by 102% and 147% (p<0.001), respectively, in brain ECs compared with vehicle. EPA treatment reduced release of sICAM-1 by 43% (p<0.001) and TNF-α by 52% (p<0.001) compared to IL-6 alone. EPA also downregulated 43 proteins involved in the “neutrophil degranulation” pathway in brain ECs (p-adjusted = 2.63 х 10 -12 ), including heat shock protein 90-alpha and beta by 1.1-fold each relative to IL-6 alone. Conclusions: In brain vascular ECs, EPA significantly reduced expression of pro-inflammatory mediators and proteins associated with neutrophil degranulation under inflammatory conditions. The ability of EPA to reverse brain EC dysfunction during inflammatory may contribute to reductions in stroke risk.

Zebularine protects against blood-brain-barrier (BBB) disruption through increasing the expression of zona occludens-1 (ZO-1) and vascular endothelial (VE)-cadherin

ABSTRACT Blood-brain-barrier (BBB) disruption is an important pathological characteristic of ischemic stroke (IS) and mainly results from dysfunction of brain vascular endothelial cells and tight junctions. Zebularine is a novel inhibitor of DNA methyltransferase (DNMT). Here, we assessed its effects on BBB disruption in IS. Firstly, we reported that Zebularine maintained BBB integrity in middle cerebral artery occlusion (MCAO) mice by increasing the expressions of zona occludens-1 (ZO-1) and vascular endothelial (VE)-cadherin. Importantly, we found that Zebularine reduced the production of pro-inflammatory cytokines, attenuated brain edema, and improved neurological deficits. In in vitro experiments, the bEnd.3 brain endothelial cells were exposed to oxygen and glucose deprivation/reoxygenation (OGD/R), and the protective effects of Zebularine were assessed. Our findings demonstrated that Zebularine prevented OGD/R-induced cytotoxicity by reducing the release of lactate dehydrogenase (LDH). Additionally, Zebularine protected bEnd.3 cells against OGD/R-induced hyper-permeability and reduction of trans-endothelial electrical resistance (TEER). Notably, we found that treatment with Zebularine activated the Adenosine 5ʹ-monophosphate (AMP)-activated protein kinase (AMPK) pathway by increasing the phosphorylation of adenosine monophosphate-activated protein kinase α (AMPKα). Blockage of AMPKα using its specific inhibitor compound C abolished the beneficial effects of Zebularine in mitigating endothelial hyper-permeability by reducing the expressions of ZO-1 and VE-cadherin. These findings suggest that the protective effects of Zebularine against OGD/R-induced endothelial hyper-permeability are mediated by the activation of AMPKα. In conclusion, our study sheds light on the potential application of Zebularine in the treatment of IS.

NMDA mediates disruption of blood-brain barrier permeability via Rho/ROCK signaling pathway

Glutamate can activate the N-methyl-D-aspartatic acid (NMDA) receptor (NMDAR), damage brain microvascular endothelial cells, and disturb the intercellular tight junctions (TJs). These result in changes in the permeability of the blood brain barrier (BBB). In neurons, the activation of Rho/ROCK signaling pathway is related to the activation of NMDAR，however, whether human brain vascular endothelial cells NMDAR mediates the Rho/ROCK pathway is not fully understood. The present study evaluates the effects of excessive NMDAR activation induced by NMDA (a glutamate analog) on the Rho/ROCK signaling pathway and the permeability of BBB by using a primary human brain microvascular endothelial cell (HBMEC) model. NMDAR subunit GluN1 was expressed in HBMECs and promoted by NMDA detected by Western blot and qRT-PCR. Furthermore, NMDA exposure decreased HBMEC viability, promoted HBMEC apoptosis, increased intracellular reactive oxygen species (ROS) levels, and destroyed the endothelial cytoskeleton. Additionally, NMDA exposure suppressed transendothelial electrical resistance (TEER) values and the expression of TJ proteins occludin and claudin5; it also promoted ROCK activated substrate myosin phosphatase target subunit-1 (MYPT)-1 phosphorylation and the transmittance of sodium fluorescein. In contrast, these effects were attenuated by ROCK inhibitor hydroxyfasudil (HF) and NMDAR antagonist MK801, respectively. Therefore, these results indicate that excessive endothelial NMDAR activation induced by NMDA may induce TJs and cytoskeleton damage, while HF attenuated NMDA-induced cytotoxicity in HBMECs by inhibiting the Rho/ROCK signaling pathway.

Ramelteon Mitigates Free Fatty Acid (FFA)-Induced Attachment of Monocytes to Brain Vascular Endothelial Cells.

Acute ischemic stroke is a challenging disease that threatens the life of older people. Dysfunction of brain endothelial cells is reported to be involved in the pathogenesis of acute ischemic stroke. Ramelteon is a novel agonist of melatonin receptor developed for the treatment of insomnia. Recently, the promising protective effect of Ramelteon on brain injury has been widely reported. The present study aims to investigate the protective effect of Ramelteon against free fatty acid (FFA)-induced damages in brain vascular endothelial cells and the underlying mechanism. Firstly, we discovered that Ramelteon administration remarkably reversed the decreased cell viability, increased LDH release, activated oxidative stress, and excessive released inflammatory factors caused by FFAs. Secondly, Ramelteon extensively suppressed the attachment of U937 monocytes to bEnd.3 brain endothelial cells induced by FFAs. In addition, the elevated expression of E-selectin and the reduced expression of KLF2 induced by FFAs were pronouncedly alleviated by Ramelteon. Lastly, silencing of KLF2 abolished the protective effects of Ramelteon against FFA-induced expression of E-selectin and the attachment of U937 monocytes to bEnd.3 brain endothelial cells. In conclusion, Ramelteon mitigated FFA-induced attachment of monocytes to brain vascular endothelial cells by increasing the expression of KLF2 and reducing the expression of E-selectin.

Peptide5 Attenuates rtPA Related Brain Microvascular Endothelial Cells Reperfusion Injury via the Wnt/β-Catenin Signalling Pathway.

Brain vascular endothelial cell dysfunction after rtPA treatment is a significant factor associated with poor prognosis, suggesting that alleviation of rtPA-related endothelial cell injury may represent a potential beneficial strategy along with rtPA thrombolysis. Thrombolysis with recombinant tissue plasminogen activator (rtPA) is beneficial for acute ischemic stroke but may increase the risk of Hemorrhagic Transformation (HT), which is considered ischemia-reperfusion injury. The underlying reason may contribute to brain endothelial injury and dysfunction related to rtPA against ischemic stroke. As previous studies have demonstrated that transiently blocked Cx43 using peptide5 (Cx43 mimetic peptide) during retinal ischemia reduced vascular leakage, it is necessary to know whether this might help decrease side effect of rtPA within the therapeutic time window. This study aims to investigate the effects of peptide5 on rtPA-related cell injury during hypoxia/reoxygenation (H/R) within the therapeutic time window. In this study, we established a cell hypoxia/reoxygenation H/R model in cultured primary Rat Brain Microvascular Endothelial Cells (RBMECs) and evaluated endothelial cell death and permeability after rtPA treatment with or without transient peptide5. In addition, we also investigated the potential signaling pathway to explore the underlying mechanisms preliminarily. The results showed that peptide5 inhibited rtPA-related endothelial cell death and permeability. It also slightly increased tight junction (ZO-1, occluding, claudin-5) and β-catenin mRNA expression, demonstrating that peptide5 might attenuate endothelial cell injury by regulating the Wnt/ β-catenin pathway. The following bioinformatic exploration from the GEO dataset GSE37239 was also consistent with our findings. This study showed that the application of peptide5 maintained cell viability and permeability associated with rtPA treatment, revealing a possible pathway that could be exploited to limit rtPA-related endothelial cell injury during ischemic stroke. Furthermore, the altered Wnt/β- catenin signaling pathway demonstrated that signaling pathways associated with Cx43 might have potential applications in the future. This study may provide a new way to attenuate HT and assist the application of rtPA in ischemic stroke.

Role of Neuroinflammation and Blood-Brain Barrier Permutability on Migraine

Currently, migraine is treated mainly by targeting calcitonin gene-related peptides, although the efficacy of this method is limited and new treatment strategies are desired. Neuroinflammation has been implicated in the pathogenesis of migraine. In patients with migraine, peripheral levels of pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α, are known to be increased. Additionally, animal models of headache have demonstrated that immunological responses associated with cytokines are involved in the pathogenesis of migraine. Furthermore, these inflammatory mediators might alter the function of tight junctions in brain vascular endothelial cells in animal models, but not in human patients. Based on clinical findings showing elevated IL-1β, and experimental findings involving IL-1β and both the peripheral trigeminal ganglion and central trigeminal vascular pathways, regulation of the Il-1β/IL-1 receptor type 1 axis might lead to new treatments for migraine. However, the integrity of the blood-brain barrier is not expected to be affected during attacks in patients with migraine.

Alzheimer's Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells

SummaryAmyloid β-protein (Aβ) may contribute to worsening of Alzheimer’s disease (AD) through vascular dysfunction, but the molecular mechanism involved is unknown. Using ex vivo blood vessels and primary endothelial cells from human brain microvessels, we show that patient-derived Aβ assemblies, termed amylospheroids (ASPD), exist on the microvascular surface in patients’ brains and inhibit vasorelaxation through binding to the α3 subunit of sodium, potassium-ATPase (NAKα3) in caveolae on endothelial cells. Interestingly, NAKα3 is also the toxic target of ASPD in neurons. ASPD-NAKα3 interaction elicits neurodegeneration through calcium overload in neurons, while the same interaction suppresses vasorelaxation by increasing the inactive form of endothelial nitric oxide synthase (eNOS) in endothelial cells via mitochondrial ROS and protein kinase C, independently of the physiological relaxation system. Thus, ASPD may contribute to both neuronal and vascular pathologies through binding to NAKα3. Therefore, blocking the ASPD-NAKα3 interaction may be a useful target for AD therapy.

Proteomic Profiling of Exosomes From Hemorrhagic Moyamoya Disease and Dysfunction of Mitochondria in Endothelial Cells.

Moyamoya disease (MMD) is a rare steno-occlusive and slowly progressing cerebrovascular disorder. The detailed mechanism of the underlying pathogenesis is still blurry. Tandem Mass Tag-labeled quantitative proteomics was performed on serum-derived exosomes (SDEs) extracted from adult patients diagnosed with pure ischemic MMD or hemorrhagic MMD and healthy controls. Then mouse brain vascular endothelial cell (EC), human umbilical vein EC, neuroblastoma cell, and human hepatocyte cell were treated with exosomes, and changes of the protein expression in mouse brain vascular EC cells were identified. Proteomics analysis results showed that 859 shared proteins were detected in SDEs from ischemic and hemorrhagic MMD patients with 231 differently expressed compared with healthy controls. Bioinformatic analysis revealed dysregulated cell growth and maintenance and indicated disturbed actin dynamics in MMD, with CFL1 (Cofilin-1) and ACTR2/3 (actin-related protein 2/3; also known as ARP2/3) downregulated in ischemic and hemorrhagic patients’ SDEs. We also found immunity dysfunction in hemorrhagic MMD. Following treatment with MMD SDEs, mouse brain vascular EC cells showed significantly higher levels of proliferation and more ethynyl-2-deoxyuridine-positive cells compared with the healthy control group, while there were no obvious changes in the human umbilical vein EC and human hepatocyte cell. Interestingly, we also found that SDEs from ischemic MMD promoted neuroblastoma cell proliferation. Proteomic analysis of mouse brain vascular EC cells suggested that SDEs from hemorrhagic MMD patients induced dysfunction of the mitochondria in cerebrovascular ECs. This study highlighted potential molecular mechanisms underlying the pathogenesis of MMD patients, thereby providing new therapeutic strategies for MMD.

The Protective Effects of Benzbromarone Against Propofol-Induced Inflammation and Injury in Human Brain Microvascular Endothelial Cells (HBMVECs).

It has been widely reported that severe neurotoxicity can be induced by the application of propofol, which is closely related to the disruption of the blood-brain barrier (BBB) induced by inflammation and injury in the human brain microvascular endothelial cells (HBMVECs). Benzbromarone is a classic anti-gout agent that has been recently reported to exert anti-inflammatory and anti-oxidative stress effects. In the present study, we aim to investigate the protective property of Benzbromarone against propofol-induced injury on HBMVECs and the underlying mechanism. CCK8 assay was used to detect the cell viability of treated HBMVECs. Oxidative stress in HBMVECs was evaluated by measuring the levels of MDA and mitochondrial ROS. ELISA and qRT-PCR assay were used to determine the production of IL-1β, IL-8, MCP-1, ICAM-1, and VCAM-1 by treated HBMVECs. Calcein-AM staining was utilized to evaluate the attachment of U937 monocytes to HBMVECs. The expression level of Egr-1 was determined by qRT-PCR and Western blot assay. Firstly, the decreased cell viability of HBMVECs induced by propofol was significantly elevated by treatment with Benzbromarone. The increased levels of MDA and mitochondrial ROS induced by propofol were dramatically suppressed by Benzbromarone. Secondly, the excessive production of inflammatory factors (IL-1β, IL-8, and MCP-1) and adhesion molecules (ICAM-1 and VCAM-1) triggered by propofol was pronouncedly inhibited by Benzbromarone. Benzbromarone ameliorated propofol-induced attachment of U937 monocytes to HBMVECs. Lastly, Benzbromarone downregulated propofol-induced expression of the transcriptional factor Egr-1 in HBMVECs. Benzbromarone protected against propofol-induced inflammation and injury through suppressing Egr-1 in human brain vascular endothelial cells.

MiR-124 affects the apoptosis of brain vascular endothelial cells and ROS production through regulating PI3K/AKT signaling pathway.

The article "MiR-124 affects the apoptosis of brain vascular endothelial cells and ROS production through regulating PI3K/AKT signaling pathway, by S.-W. Wang, L.-X. Deng, H.-Y. Chen, Z.-Q. Su, S.-L. Ye, W.-Y. Xu, published in Eur Rev Med Pharmacol Sci 2018; 22 (2): 498-505-DOI: 10.26355/eurrev_201801_14201-PMID: 29424909" has been withdrawn from the authors due to inaccuracies (there are some errors and incorrect data). The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/14201.

Celecoxib Alleviates Radiation-Induced Brain Injury in Rats by Maintaining the Integrity of Blood-Brain Barrier.

The underlying mechanisms of radiation-induced brain injury are poorly understood, although COX-2 inhibitors have been shown to reduce brain injury after irradiation. In the present study, the effect of celecoxib (a selective COX-2 inhibitor) pretreatment on radiation-induced injury to rat brain was studied by means of histopathological staining, evaluation of integrity of blood-brain barrier and detection of the expressions of inflammation-associated genes. The protective effect of celecoxib on human brain microvascular endothelial cells (HBMECs) against irradiation was examined and the potential mechanisms were explored. Colony formation assay and apoptosis assay were undertaken to evaluate the effect of celecoxib on the radiosensitivity of the HBMECs. ELISA was used to measure 6-keto-prostaglandin F1α (6-keto-PGF1α) and thromboxane B2 (TXB2) secretion. Western blot was employed to examine apoptosis-related proteins expressions. It was found that celecoxib protected rat from radiation-induced brain injury by maintaining the integrity of the blood-brain barrier and reducing inflammation in rat brain tissues. In addition, celecoxib showed a significant protective effect on HBMECs against irradiation, which involves inhibited apoptosis and decreased TXB2/6-keto-PGF1α ratio in brain vascular endothelial cells. In conclusion, celecoxib could alleviate radiation-induced brain injury in rats, which may be partially due to the protective effect on brain vascular endothelial cells from radiation-induced apoptosis.

Integrated Multi-Omic Analyses of the Genomic Modifications by Gut Microbiome-Derived Metabolites of Epicatechin, 5-(4'-Hydroxyphenyl)-γ-Valerolactone, in TNFalpha-Stimulated Primary Human Brain Microvascular Endothelial Cells.

Cerebral blood vessels are lined with endothelial cells and form the blood-brain barrier. Their dysfunction constitutes a crucial event in the physiopathology of neurodegenerative disorders and cognitive impairment. Epicatechin can improve cognitive functions and lower the risk for Alzheimer’s disease or stroke. However, molecular mechanisms of epicatechin on brain vascular endothelium are still unexplored. The objective of this study was to investigate the biological effects of gut microbiome-derived metabolites of epicatechin, 5-(4′-Hydroxyphenyl)-γ-valerolactone-3′-sulfate and 5-(4′-Hydroxyphenyl)-γ-valerolactone-3′-O-glucuronide, in TNF-α-stimulated human brain microvascular endothelial cells at low (nM) concentrations by evaluating their multi-omic modification (expression of mRNA, microRNA, long non-coding RNAs, and proteins). We observed that metabolites are biologically active and can simultaneously modulate the expression of protein-coding and non-coding genes as well as proteins. Integrative bioinformatics analysis of obtained data revealed complex networks of genomics modifications by acting at different levels of regulation. Metabolites modulate cellular pathways including cell adhesion, cytoskeleton organization, focal adhesion, signaling pathways, pathways regulating endothelial permeability, and interaction with immune cells. This study demonstrates multimodal mechanisms of action by which epicatechin metabolites could preserve brain vascular endothelial cell integrity, presenting mechanisms of action underlying epicatechin neuroprotective properties.

Anagliptin Protected against Hypoxia/Reperfusion-Induced Brain Vascular Endothelial Permeability by Increasing ZO-1.

Background and purpose: Cerebral ischemia-reperfusion injury is commonly induced during the treatment of ischemic stroke and is reported to be related to the blood–brain barrier destruction and brain vascular endothelial cell dysfunction. Anagliptin is a novel antidiabetic agent recently reported to protect neurons from oxidative stress. In the present study, we aim to investigate the protective property of anagliptin against oxygen–glucose deprivation and reperfusion (OGD/R)-induced injury on endothelial cells and clarify the potential underlying mechanism. Methods: OGD/R modeling was established on bEnd.3 brain endothelial cells. Cell viability was detected using the MTT assay, and the mitochondrial reactive oxygen species (ROS) level was measured using the mitoses red staining assay. The endothelial monolayer permeability was determined using an FITC-dextran permeation assay. The expression levels of NOX-4 and ZO-1 were evaluated using qRT-PCR and Western blot assays. The expressions of MLC-2, p-MLC-2, and myosin light chain kinase (MLCK) were determined using Western blot. Results: First, the decreased cell viability, upregulated NOX-4, and elevated mitochondrial ROS level in the endothelial cells induced by OGD/R were reversed by treatment with anagliptin. Second, the enlarged endothelial permeability and the decreased expression level of ZO-1 in the endothelial cells induced by OGD/R were alleviated by anagliptin. Third, the downregulation of ZO-1 and enlarged brain endothelial monolayer permeability induced by OGD/R were ameliorated by an MLCK inhibitor, ML-7. Lastly, the elevated expressions of MLCK and p-MLC-2 induced by OGD/R were suppressed by anagliptin. Conclusion: Anagliptin protected against hypoxia/reperfusion-induced brain vascular endothelial permeability by increasing the expression ZO-1, mediated by inhibition of the MLCK/MLC-2 signaling pathway.

The protective effects of Pimavanserin against cerebral ischemia-induced brain injury

ABSTRACT The integrity of the blood-brain barrier (BBB) is mainly maintained by the brain vascular endothelial cells and the tight junctions amongst them. Pimavanserin is a novel agent approved for the treatment of Parkinson’s disease and exerts neuroprotective properties. The present study aims to explore the possibility that Pimavanserin might be an effective agent used for the treatment of cerebral ischemia stroke. Middle cerebral artery occlusion (MCAO) was established in mice, and oxygen-glucose deprivation/reoxygenation (OGD/R) was established in brain bEND.3 endothelial cells. Mice were randomly divided into four groups: (1) Sham operation group; (2). Pimavanserin (1 mg/kg); (3). MCAO; (4). Pimavanserin+ MCAO. We found that compared to the Sham group, the elevated neurological deficit score and brain water content increased production of inflammatory factors, increased BBB permeability, and downregulated Claudin 5 expression were observed in the MCAO group and were all dramatically reversed by the administration of Pimavanserin. Brain bEND.3 endothelial cells were treated with Pimavanserin before the exposure to OGD/R. The significantly increased lactate dehydrogenase (LDH) release, declined cell viability, increased endothelial permeability, downregulated Claudin 5 and Krüppel-like factors 6 (KLF6) were observed in the OGD/R group and were all reversed by the introduction of Pimavanserin. Lastly, the effects of Pimavanserin on the expression level of Claudin 5 and endothelial permeability in OGD/R-challenged endothelial cells were both abolished by the knockdown of KLF6. Taken together, our data revealed that Pimavanserin protected against cerebral ischemia injury by regulating the BBB integrity in a KLF6-dependent manner.

Hyperbaric Oxygen Exposure Reduces ICAM-1 And HIF1? Expression in Brain Endothelial Cells from Experimental Cerebral Malaria Mice

This study aimed to reveal the role of ICAM-1 and HIF-1? in brain endothelial cell of cerebral malariamice model after exposure to 2.4 absolute atmospheres (ATA) hyperbaric oxygen (HBO). Thirty-nineC57BL/6 mice were divided into three groups: control negative (normal mice without any exposure),control positive (Plasmodium berghei ANKA [PbA] infection without HBO exposure), and treatment (PbAinfection and exposed to HBO for 10 sessions after the parasite grew). Parasitemia and clinical symptomswere observed every day. Brain tissues were isolated on day 13 post-infection for histopathological andimmunohistochemical examination (observed at 400x magnification in 10 visual fields). HBO decreasedHIF-1? and ICAM-1 expression in endothelial cells. There was a moderate correlation between HIF-1? andICAM-1 expression. Ten HBO sessions prevented cerebral malaria, as denoted by the decreased expressionof ICAM-1 and HIF-1? in brain vascular endothelial cells from the experimental mice.

Polyinosinic-Polycytidylic Acid Induces CXCL1 Expression in Cultured hCMEC/D3 Human Cerebral Microvascular Endothelial Cells

Objective: Brain microvascular endothelial cells are integral components of the blood-brain barrier and play a role in protecting the brain from invading microbes. CXC motif chemokine ligand 1 (CXCL1) induces the chemotaxis of neutrophils, and neutrophils are important in host defense in the brain. However, dysregulated neutrophil infiltration leads to brain diseases. Toll-like receptor 3 (TLR3) is a pattern recognition receptor that recognizes viral double-stranded RNA (dsRNA). The aim of this study was to investigate the effect of an TLR3 agonist on the expression of CXCL1 in brain vascular endothelial cells. Methods: hCMEC/D3 human cerebral microvascular endothelial cells were cultured and treated with polyinosinic-polycytidylic acid (poly IC), a potent synthetic dsRNA agonist for TLR3. The production of CXCL1 mRNA and protein was assessed by real-time RT-PCR and ELISA. The expression of CXCL1 was compared with that of CXCL8. The effect of pretreatment of cells with a NF-κB inhibitor (SN50), a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), an interferon (IFN) regulatory factor 3 inhibitor (MRT67307), and an anti-type I IFN-neutralizing antibody mixture was examined. Phosphorylation of p38 was examined using Western blotting. Results: Treating cultured hCMEC/D3 human cells with poly IC induced the expression of CXCL1 as well as another chemokine CXCL8. Pretreatment of cells with SN50, SB203580, and SP600125 decreased the induction of CXCL1 by poly IC. However, it was not affected by MRT67307 or by an anti-type I IFN-neutralizing antibody mixture. Pretreatment of cells with SN50 decreased the poly IC-induced phosphorylation of p38. Conclusions: Poly IC induces the expression of CXCL1 in hCMEC/D3 cells. NF-κB, p38 MAPK, and JNK are involved in this reaction. There is a cross-talk between NF-κB and p38, and NF-κB partially regulates phosphorylation of p38. CXCL1 produced by brain microvascular endothelial cells may contribute to the brain’s defense against viral infection and various neurological diseases associated with neutrophil accumulation.

Reduction of TNFR2 Expression in Brain Endothelial Cells in Cerebral Malaria Mice Models after Hyperbaric Oxygen Exposure

Tumour Necrosis Factor Receptor 2/ TNFR2 is the main receptor of TNF-α expressed by brain endothelial cells and contributes to Nuclear Factor Kappa Beta/ NFκB activation which induces sequestration of leucocyte cells. The role of hyperbaric oxygen/HBO as a regulator of the inflammatory process can be used as a supportive therapeutic approach in cerebral malaria infection. The research aimed to examine the impact of HBO administration on expression of TNFR-2 in brain endothelial cells of mice infected with P. berghei ANKA. P.berghei ANKA / PbA infected C57BL/6 divided into 3 groups with 13 mice per group: G1 (negative control, normal mice); G2 (positive control; mice infected with PbA without HBO); G3 (treatment group; mice infected with PbA with HBO). Observation of TNFR2 expression using immunohistochemical techniques on the last day of treatment. Study results revealed that there has been a significant decrease in TNFR2 expression in brain vascular endothelial cells in the group that received HBO (p <0.05). Exposure to HBO 2.4 ATA for 10 sessions can prevent cerebral malaria by decreasing TNFR2 expression in vascular endothelial cells of the brain of the mice model.

Abstract P2-20-04: Controlling tight junctions in the blood brain barrier (BBB) with artemisinin as a potential adjuvant in the treatment of metastatic disease

Abstract Introduction: Brain metastatic disease (BMD) is attributed to significant mortality and morbidity in breast cancer patients. Strict policing of molecular traffic across the blood-brain barrier (BBB) precludes brain parenchymal penetration of most small molecule drugs, chemotherapy included. Consequently, the treatment of BMD remains difficult, particularly if the BBB remains intact during disease progression. Artemisinin has been efficacious in treating multi-drug resistant cerebral malaria likely indicative of an ability to traverse the BBB. Thus, Artemisinin may be able to modulate the restrictive nature of the BBB and provide a potential adjunctive therapy to established drug therapy in the treatment of BMD. Emerging evidence suggests that Artemisinin itself may harbour potent anti-cancer properties. Aim: To investigate in vitro the effect on barrier function in particular paracellular permeability of brain vascular endothelial cells compared to non-brain vascular endothelial cells when treated with Artemisinin. Materials and Methods: Inter-individual BBB differences were modelled using the human brain vascular endothelial cell lines hCMEC-D3, TY09 and TY10. In addition to the brain cell lines, a HUVEC cell line was treated with incremental concentrations of artemisinin. Adhesion, migration and electrical resistance across cellular monolayers were quantified using Electric Cell-Substrate Impedance Sensing (ECIS). Trans- Endo/Epithelial Resistance (TEER) and Paracellular Permeability (PCP) were used to assess barrier function. Immunofluorescence was used to qualitatively assess tight junction (TJ) integrity of all cell lines. RNAseq was used to determine changes in message after treatment with Artemisinin in all 4 cell lines. Results: A significant reduction in electrical resistance across all cell lines was observed when treated with 1mM Artemisinin and may be suggestive of a cytotoxic concentration. In comparison when treated with 0.01mM Artemisinin the electrical resistance decreased which could suggest restrictive barrier properties were enhanced, in addition evidencing Artemisinin may exhibit biphasic activity. The reduction in electrical resistance across the brain endothelial cell lines was significant (p<0.05) but deemed not significant across HUVEC, perhaps mirroring Artemisinin’s pharmacological action at differing vascular interfaces in vivo. Higher Artemisinin concentrations significantly reduced TEER across the brain cell lines and increased paracellular permeability, the same trends were observed in HUVEC. IF demonstrated that Artemisinin was able to effect changes in TJ protein location which supports the hypothesis that Artemisinin reduces barrier function across the brain endothelial lines, suggested by an increased permeability in the presence of Artemisinin. Electrical resistance across all cell lines significantly reduced when treated with 1mM Artemisinin. When compared to negative control, there was a significant decrease in electrical resistance across all cell lines treated with 1mM Artemisinin. RNAseq comparisons revealed that in TY10 cells, 1167 genes were upregulated and 94 downregulated in comparison to HUVEC where 185 and 0 genes were up or downregulated respectively and that in all 3 brain endothelial cell lines that the TJ protein CLDN5 exhibited a significant reduction in expression at gene level (p=0.03). Conclusion: The results demonstrate that Artemisinin may have the capacity to transiently increase or decrease the permeability of the BBB. Therefore, Artemisinin may have a role in facilitating drug delivery to the cerebral parenchyma providing potential therapeutic benefit for the treatment of BMD or other related neurological conditions difficult to treat due to the BBB. Citation Format: Emily Appadurai, Fiona Ruge, Andrew Sanders, Wen G Jiang, Tracey A. Martin. Controlling tight junctions in the blood brain barrier (BBB) with artemisinin as a potential adjuvant in the treatment of metastatic disease [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-20-04.

Phoenixin-14 protects human brain vascular endothelial cells against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced inflammation and permeability

Stroke is one of the world's most deadly pathologies, and the rate of stroke recurrence is high. However, due to the complex nature of ischemia and reperfusion injury, there is presently no reliable treatment. The main factors driving brain damage from ischemic stroke are neuronal cell death resulting from oxidative stress, inflammation, and failure of the blood brain barrier. While under normal conditions, the blood brain barrier acts as a selectively permeable membrane allowing solutes and other substances to pass into the tissues of the central nervous system, ischemia and reperfusion alter the expression of tight junction proteins such as occludin, which leads to unmitigated perfusion and loss of homeostasis. Phoenixin-14 is a 14-amino acid neuropeptide that has been shown to play a role in regulating reproduction, blood sugar metabolism, pain, anxiety, and more recently, certain aspects of ischemic cardiac injury. In the present study, we found that phoenixin-14 confers protective effects against oxygen-glucose deprivation/reoxygenation (OGD/R) injury in bEnd.3 brain endothelial cells. Phoenixin-14 attenuated oxidative stress via downregulation of ROS and NOX1 and inhibited HMGB1 expression. Additionally, phoenixin-14 increased the expression of eNOS and NO, which play a protective role. Phoenixin-14 reduced endothelial monolayer permeability by increasing the expression of occludin. Finally, we found that the effects of phoenixin-14 on the expression of eNOS and occludin are dependent on the KLF2 transcriptional pathway, as evidenced by the results of our KLF2 knockdown experiment. Thus, phoenixin-14 may serve as a novel therapeutic agent for ischemic stroke.