Cells bEnd Research Articles

Design and characterization of a novel dimeric blood-brain barrier penetrating TNFα inhibitor.

Tumor necrosis factor-alpha (TNFα) inhibitors could prevent neurological disorders systemically, but their design generally relies on molecules unable to cross the blood-brain barrier (BBB). This research was aimed to design and characterize a novel TNFα inhibitor based on the angiopeptide-2 as a BBB shuttle molecule fused to the extracellular domain of human TNFα receptor 2 and a mutated vascular endothelial growth factor (VEGF) dimerization domain. This new chimeric protein (MTV) would be able to trigger receptor-mediated transcytosis across the BBB via low-density lipoprotein receptor-related protein-1 (LRP-1) and inhibit the cytotoxic effect of TNFα more efficiently because of its dimeric structure. Stably transformed CHO cells successfully expressed MTV, and its purification by Immobilized-Metal Affinity Chromatography (IMAC) rendered high purity degree. Mutated VEGF domain included in MTV did not show cell proliferation or angiogenic activities measured by scratch and aortic ring assays, which corroborate that the function of this domain is restricted to dimerization. The pairs MTV-TNFα (Kd 279 ± 40.9nM) and MTV-LRP1 (Kd 399 ± 50.5nM) showed high affinity by microscale thermophoresis, and a significant increase in cell survival was observed after blocking TNFα with MTV in a cell cytotoxicity assay. Also, the antibody staining in CHOK1 and bEnd3 cells demonstrated the adhesion of MTV to the LRP1 receptor located in the cell membrane. These results provide compelling evidence for the proper functioning of the three main domains of MTV individually, which encourage us to continue the research with this new molecule as a potential candidate for the systemic treatment of neurological disorders.

Hydrogen sulfide prevents ethanol-induced ZO-1 CpG promoter hypermethylation-dependent vascular permeability via miR-218/DNMT3a axis.

Ethanol (ET) causes cerebrovascular dysfunction by altering homocysteine (Hcy) metabolism and by causing oxidative stress. However, there are no strategies to prevent ET-induced epigenetic deregulation of tight junction protein (hyper-methylation) and endothelial cell permeability to date. Hydrogen sulfide (H2 S) has an antioxidative, antiapoptotic, and anti-inflammatory effect. Here, we investigated the protective role of H2 S in ET-induced endothelial permeability through epigenetic changes in mouse brain endothelial cells (bEnd3). The bEnd3 cells were exposed to 50 mM ET treatment in the presence or absence of 50 μM NaHS (H2 S donor). The result demonstrates that ET-induced cellular toxicity increased intracellular Hcy levels, which further intensified mitochondrial dysfunction and energy defects. Using miScript microRNA (miRNA) polymerase chain reactionarray-based screening, we identified a particular miRNA, miR-218, as a novel target of ET-induced DNA methyltransferase-3a (DNMT3a) activation. miR-218 influences CpG island methylation of the zonula occludens 1 (ZO-1) promoter in the endothelial cells. We discovered that ET suppressed miR-218 levels and induced endothelial permeability via DNMT3a-mediated ZO-1 hyper-methylation. Treatment with mito-TEMPO (mitochondria-targeted antioxidant), 5'-azacitidine (DNMT inhibitor), or miR-218 overexpression was shown to protect endothelial cells against ET-induced permeability. Also, bEnd3 cells pretreated with NaHS attenuated ET-induced vascular permeability and prevented CpG island methylation at the promoter. In conclusion, our data provide evidence that H2 S treatment protects vascular integrity from ET-induced stress by mitigating CpG (ZO-1 promoter) DNA hyper-methylation. This finding uncovers a new mechanistic understanding of NaHS/H2 S, that may have therapeutic potential in preventing or diminishing ET-induced brain vascular permeability and dysfunction induced by alcoholism.

Long noncoding RNA SNHG1 protects brain microvascular endothelial cells against oxygen-glucose deprivation/reoxygenation-induced injury by sponging miR-298 and upregulating SIK1 expression.

Growing evidence shows that long non-coding RNAs (lncRNAs) are widely involved in the progression of multiple diseases, including ischemic stroke. The aim of this study was to explore the function and underlying mechanism of lncRNAs small nucleolar RNA host gene 1 (SNHG1) in ischemic stroke. SNHG1 and salt-induced kinase 1 (SIK1) were upregulated in oxygen-glucose deprivation/reperfusion (OGD/R)-induced bEnd3 cells. SNHG1 downregulation promoted OGD/R-induced injury through decreasing cell proliferation and increasing apoptosis, which was reversed by upregulating SIK1 or downregulating miR-298. Moreover, SIK1 interference had similar functions with SNHG1 knockdown in OGD/R-treated bEnd3 cells. In addition, miR-298 was a direct target of SNHG1 and could specifically bind to SIK1. Furthermore, SNHG1 functioned as a molecular sponge of miR-298 to regulate SIK1 expression. SNHG1 knockdown enhanced OGD/R-induced injury in bEnd3 cells by regulating miR-298/SIK1 axis, which might provide promising therapeutic target for treatment of ischemic stroke.

Lychee seed polyphenol protects the blood-brain barrier through inhibiting Aβ(25-35)-induced NLRP3 inflammasome activation via the AMPK/mTOR/ULK1-mediated autophagy in bEnd.3 cells and APP/PS1 mice.

Blood-brain barrier (BBB) dysfunction has been implicated in Alzheimer's disease (AD) and is closely linked to the release of proinflammatory cytokines in brain capillary endothelial cells. We have previously reported that lychee seed polyphenols (LSP) exerted anti-neuroinflammatory effect. In this study, we aimed to explore the protective effect of LSP on BBB integrity. The monolayer permeability of bEnd.3 cells, and the mRNA level and protein expression of tight junction proteins (TJs), including Claudin 5, Occludin, and ZO-1, were examined. In addition, the inhibition of Aβ(25-35)-induced NLRP3 inflammasome activation, and the autophagy induced by LSP were investigated by detecting the expression of NLRP3, caspase-1, ASC, LC3, AMPK, mTOR, and ULK1. Furthermore, the cognitive function and the expression of TJs, NLRP3, caspase-1, IL-1β, and p62 were determined in APP/PS1 mice. The results showed that LSP significantly decreased the monolayer permeability and inhibited the NLRP3 inflammasome in Aβ(25-35)-induced bEnd3 cells. In addition, LSP induced autophagy via the AMPK/mTOR/ULK1 pathway in bEnd.3 cells, and improved the spatial learning and memory function, increased the TJs expression, and inhibited the expression of NLRP3, caspase-1, IL-1β, and p62 in APP/PS1 mice. Therefore, LSP protects BBB integrity in AD through inhibiting Aβ(25-35)-induced NLRP3 inflammasome activation via the AMPK/mTOR/ULK1-mediated autophagy.

Cabozantinib, a Multityrosine Kinase Inhibitor of MET and VEGF Receptors Which Suppresses Mouse Laser-Induced Choroidal Neovascularization.

Choroidal neovascularization (CNV) is a leading cause of blindness in the elderly in developed countries and is particularly associated with age-related macular degeneration (AMD). Cabozantinib (CBZ) hinders the activation of multiple receptor tyrosine kinases involved in tumor angiogenesis, such as hepatocyte growth factor receptor (MET) and vascular endothelial growth factor receptor 2 (VEGFR2). We aimed to investigate the role and mechanism of CBZ in a mouse laser-induced CNV model. In zebrafish embryos, CBZ perturbed intersegmental vessel (ISV) formation without obvious neurodevelopment impairment. In the mouse laser-induced CNV model, phosphorylated hepatocyte growth factor receptor (p-MET) and phosphorylated vascular endothelial growth factor receptor 2 (p-VEGFR2) were increased in the CNV region. CBZ intravitreal injection or oral gavage alleviated CNV leakage and the CNV lesion area without obvious intraocular toxicity, as well as disturbed the phosphorylation of MET and VEGFR2. Additionally, CBZ downregulated the expression of the hepatocyte growth factor (HGF) with no effect on the expression of the vascular endothelial growth factor (VEGF). CBZ downregulated HGF, p-MET, and p-VEGFR2 expressions in vitro, as well as inhibited the proliferation, migration, and tube formation of b-End3 cells. In summary, CBZ alleviates mouse CNV formation possibly via inhibiting the activation of MET and VEGFR2. The findings provide a novel potential therapy method for CNV patients.

HIF-1α is involved in blood–brain barrier dysfunction and paracellular migration of bacteria in pneumococcal meningitis

Bacterial meningitis is a deadly disease most commonly caused by Streptococcus pneumoniae, leading to severe neurological sequelae including cerebral edema, seizures, stroke, and mortality when untreated. Meningitis is initiated by the transfer of S. pneumoniae from blood to the brain across the blood–cerebrospinal fluid barrier or the blood–brain barrier (BBB). The underlying mechanisms are still poorly understood. Current treatment strategies include adjuvant dexamethasone for inflammation and cerebral edema, followed by antibiotics. The success of dexamethasone is however inconclusive, necessitating new therapies for controlling edema, the primary reason for neurological complications. Since we have previously shown a general activation of hypoxia inducible factor (HIF-1α) in bacterial infections, we hypothesized that HIF-1α, via induction of vascular endothelial growth factor (VEGF) is involved in transmigration of pathogens across the BBB. In human, murine meningitis brain samples, HIF-1α activation was observed by immunohistochemistry. S. pneumoniae infection in brain endothelial cells (EC) resulted in in vitro upregulation of HIF-1α/VEGF (Western blotting/qRT-PCR) associated with increased paracellular permeability (fluorometry, impedance measurements). This was supported by bacterial localization at cell–cell junctions in vitro and in vivo in brain ECs from mouse and humans (confocal, super-resolution, electron microscopy, live-cell imaging). Hematogenously infected mice showed increased permeability, S. pneumoniae deposition in the brain, along with upregulation of genes in the HIF-1α/VEGF pathway (RNA sequencing of brain microvessels). Inhibition of HIF-1α with echinomycin, siRNA in bEnd5 cells or using primary brain ECs from HIF-1α knock-out mice revealed reduced endothelial permeability and transmigration of S. pneumoniae. Therapeutic rescue using the HIF-1α inhibitor echinomycin resulted in increased survival and improvement of BBB function in S. pneumoniae-infected mice. We thus demonstrate paracellular migration of bacteria across BBB and a critical role for HIF-1α/VEGF therein and hence propose targeting this pathway to prevent BBB dysfunction and ensuing brain damage in infections.

4163 Aging and Smoking Exacerbates Post-Stroke Complement Driven Neuroinflammation

OBJECTIVES/GOALS: Following stroke, complement-dependent neuroinflammation exacerbates secondary injury and worsens acute and chronic outcomes. We have shown that an injury site-targeted complement inhibitor (B4Crry), that targets specifically to the ischemic brain, inhibits complement activation leading to improved outcomes. Stroke comorbidities have been shown to promote a pro-inflammatory environment in the brain and systemically, and to exacerbate inflammatory responses after injury. We investigated the impact of age and smoking on acute outcomes after stroke and assessed whether increased complement activation contributes to the worsening outcomes with these stroke comorbidities. METHODS/STUDY POPULATION: Mouse brain endothelial cells (bEnd3) were exposed to hypoxia followed by exposure to serum that was derived from either cigarette smoke (CS)-exposed mice or naïve mice, and IgM and C3d deposition assessed. Adult (12 weeks) and aged (1 year) mice were subjected to 1h transient middle cerebral artery occlusion. Animals were exposed to CS for 3-6 months (5hr/day, 5days/week) by burning 3R4F cigarettes using a smoking machine. Animals were treated with B4Crry or vehicle intravenously 2h post-MCAO. Survival analysis and neurological deficit scores were performed up to 7 days. Brains were extracted for histological and molecular analyses. RESULTS/ANTICIPATED RESULTS: Following hypoxia, bEnd3 cells exposed to serum from CS-exposed mice had higher C3d and IgM deposition compared to naïve serum. Older and CS-exposed mice had significantly worse neurological deficits and mortality compared to younger adults post-MCAO. B4Crry reduced mortality and motor deficits in young, old and old+CS mice with a higher effect size in comorbid animals. Age and/or CS exposure resulted in larger infarct volumes, and increased levels of C3d deposition and microglial activation compared to young adults, but aged/CS animals treated with B4Crry fared comparable to young adults. DISCUSSION/SIGNIFICANCE OF IMPACT: The pro-inflammatory effects of aging and smoking contribute to worse stroke outcomes, and these effects can be successfully mitigated by injury site-targeted complement inhibition.

Inertial-Force-Assisted, High-Throughput, Droplet-Free, Single-Cell Sampling Coupled with ICP-MS for Real-Time Cell Analysis.

Single-cell analysis facilitates perception into the most essential processes in life's mysteries. While it is highly challenging to quantify them at the single-cell level, where precise single-cell sampling is the prerequisite. Herein, a real-time single-cell quantitative platform was established for high-throughput droplet-free single-cell sampling into time-resolved (TRA) ICP-MS and real-time quantification of intracellular target elements. The concentrated cells (2 × 106 cells mL-1) were spontaneously and orderly aligned in a spiral microchannel with 104 periodic dimensional confined micropillars. The quantification is conducted simultaneously by internal standard inducing from another branch channel in the chip. The flow-rate-independent feature of single-cell focusing into an aligned stream within a wide range of fluidic velocities (100-800 μL min-1) facilitates high-throughput, oil-free, single-cell introduction into TRA-ICP-MS. The system was used for real-time exploration of intracellular antagonism of Cu2+ against Cd2+. an obvious antagonistic effect was observed for the MCF-7 cell by culturing for 3, 6, 9, and 12 h with 100 μg L-1 Cd2+ and 100 μg L-1 Cu2+, and a rivalry rate of 12.8% was achieved at 12 h. At identical experimental conditions, however, limited antagonistic effect was encountered for a bEnd3 cell within the same incubation time period, with a rivalry rate of 4.81%. On the contrary, an antagonistic effect was not observed for the HepG2 cell by culturing for 6 h, while an obvious antagonistic effect was found by further culturing to 12 h, with a rivalry rate of 10.43%. For all three cell lines, significant heterogeneity was observed among individual cells.

Activation of CRHR1 contributes to cerebral endothelial barrier impairment via cPLA2 phosphorylation in experimental ischemic stroke

The activation of corticotrophin-releasing hormone receptor (CRHR) 1 is implicated in neuronal injury in experimental stroke. However, little is known about the relationship between CRHR1 activation and brain endothelial barrier impairment after ischemia and reperfusion (I/R). Recently we have demonstrated that the activation of extracellular signal-regulated kinase (Erk) 1/2 as well as p38 is required for hydrogen peroxide (H2O2)-increased cytosolic phospholipase A2 (cPLA2) phosphorylation in bEnd3 cells. Using this in vitro ischemic-like model, we found that both blockade and interference of CRHR1 inhibited H2O2-enhancd p38, Erk1/2 and cPLA2 phosphorylation and in turn suppressed monolayer hyperpermeability and ZO-1 redistribution. Then using the transient middle cerebral artery occlusion (tMCAO) mouse model, we revealed that CRHR1 antagonist NBI27914 pretreatment attenuated cPLA2 phosphorylation, Evans blue dye (EBD) extravasation, tight junction disruption and mitochondrial cytochrome c release. CRHR1 interference also inhibited cortical vascular hyperpermeability. Furthermore, NBI27914 administration attenuated neurovascular injury. After 30 min MCAO with 7 days reperfusion CRHR1 interference alleviated hippocampal blood-brain barrier (BBB) leakage and improved spatial cognitive dysfunction. Thus, our study demonstrates that during ischemic stroke the activation of endothelial CRHR1 contributes to BBB impairment via cPLA2 phosphorylation.

Erg mediates downregulation of claudin-5 in the brain endothelium of a murine experimental model of cerebral malaria.

Cerebral malaria (CM) is a severe complication with brain vascular hyperpermeability. Claudin-5 is the major component of tight junctions. To investigate the expression of claudin-5 in CM, we established a murine experimental cerebral malaria (ECM) model and an invitro model by treating murine brain endothelial cells (bEnd3) with plasma from ECM mice. Expression of claudin-5 and the ETS transcription factor Erg was reduced in the brain endothelium of ECM mice. In bEnd3 cells exposed to ECM plasma, decreased expression of claudin-5 and Erg, and increased permeability were observed. Silencing of Erg significantly reduced Cldn5 expression. ChIP assays indicated that Erg binds to the -813 ETS motif of the murine Cldn5 gene promoter, and the binding is decreased by treatment with ECM plasma.

Role of acute-phase protein ORM in a mice model of ischemic stroke.

The only Food and Drug Administration-approved treatment for acute ischemic stroke is tissue plasminogen activator, and the discovery of novel therapeutic targets is critical. Here, we found orosomucoid (ORM), an acute-phase protein mainly produced by the liver, might act as a treatment candidate for an ischemic stroke. The results showed that ORM2 is the dominant subtype in mice normal brain tissue. After middle cerebral artery occlusion (MCAO), the level of ORM2 is significantly increased in the ischemic penumbra compared with the contralateral normal brain tissue, whereas ORM1 knockout did not affect the infarct size. Exogenous ORM could significantly decrease infarct size and neurological deficit score. Inspiringly, the best administration time point was at 4.5 and 6 hr after MCAO. ORM could markedly decrease the Evans blue extravasation, and improve blood-brain barrier-associated proteins expression in the ischemic penumbra of MACO mice and oxygen-glucose deprivation (OGD)-treated bEnd3 cells. Meanwhile, ORM could significantly alleviate inflammation by inhibiting the production of interleukin 1β (IL-1β), IL-6, and tumor necrosis factor α(TNF-α), reduce oxidative stress by improving the balance ofmalondialdehyde (MDA) and superoxide dismutase (SOD), inhibit apoptosis by decreasing caspase-3 activity in ischemic penumbra of MCAO mice and OGD-treated bEnd.3 cells. Because of its protective role at multiple levels, ORM might be a promising therapeutic target for ischemic stroke.

711 Propranolol exhibits activity against hemangiomas independent of beta blockade

Propranolol is a widely used beta-blocker that consists of a racemic mixture of R and S stereoisomers, but only the S-stereoisomer has significant activity against the beta-adrenergic receptor. Infants receiving propranolol for the treatment of hemangiomas require frequent monitoring to ensure that they do not suffer from side effects related to beta-blockade. The exact mechanism of activity of propranolol in hemangiomas of infancy is unknown. In this study, we treated hemangioma stem cells with both beta-blockade active (S) and inactive (R) propranolol and searched for genes that were coordinately regulated by this treatment. Angiopoietin-like 4 (Angptl4) was among the most downregulated. We confirmed that propranolol isomers downregulated Angptl4 in endothelial cells, with greater downregulation of Angptl4 using the beta-blockade inactive R-propranolol. Notably, Angptl4 is present in human hemangiomas of infancy. R-propranolol inhibited the growth of b.End3 hemangioma cells in vivo. Both isomers of propranolol converted the glycolytic phenotype of bend3 cells to a more respiratory phenotype. Finally, we established that EGR1 and APOA1, genes responsible for tumor suppression and lipid metabolism, were the most upregulated genes in cells treated with R-propranolol when compared with untreated cells. The implication of these findings is that hemangioma growth can be blocked without the side effects of beta-blockade. Given that humans have been exposed to racemic propranolol for decades and thus to R-propranolol, we propose the clinical development of R-propranolol for hemangiomas of infancy and other angiogenic diseases.

VEGF Antagonism Attenuates Cerebral Ischemia/Reperfusion-Induced Injury via Inhibiting Endoplasmic Reticulum Stress-Mediated Apoptosis.

Endoplasmic reticulum (ER) stress-mediated apoptosis pathway is considered to play a vital role in mediating stroke and other cerebrovascular diseases. Previous studies have showed that vascular endothelial growth factor (VEGF) antagonism reduced cerebral ischemic-reperfusion (CI/R) damage, but whether attenuation of ER stress-induced apoptosis is contributing to its mechanisms remains elusive. Our study aimed to investigate the protective effect of VEGF antagonism on CI/R-induced injury. First, oxygen-glucose deprivation and re-oxygenation (OGD/R) BEND3 cell model was constructed to estimate small interfering RNA (siRNA)-VEGF on damage of endothelial cells. Next, in animal model, CI/R mice were induced by middle cerebral artery occlusion (MCAO) for 2 h followed by 24 h reperfusion to investigate cerebral tissue damage. For treatment group, mice received 100 µg/kg anti-VEGF antibodies at 30 min before MCAO, followed by 24 h reperfusion. Our findings demonstrated that pre-administration of siRNA-VEGF before OGD/R changed the biological characteristics of BEND3 cells, reversed the levels of X-box binding protein-1 (XBP-1) and glucose-regulated protein 78 (GRP78), showing siRNA-VEGF attenuated, at least in part, the oxidative damage in OGD/R cell by down-regulating ER stress. In mice experiment, pre-administration of anti-VEGF antibody reduced the brain infarct volume and edema extent and improved neurological scores outcome of CI/R injury mice. Pathological and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining results also confirmed this protective effect. The expressions of VEGF, CATT/EBP homologous protein (CHOP), inositol requiring enzyme 1α (IRE-1α), and cleaved-caspase12 and c-jun N-terminal kinase (JNK) phosphorylation were also prominently decreased. These results suggested that inhibition of endogenous VEGF attenuates CI/R-induced injury via inhibiting ER stress-mediated apoptosis.

Abstract TP120: Inhibition of α5β1 Integrin With the Small Peptide ATN-161 Reduces Inflammation, Stabilizes the Blood-Brain Barrier, and is Neuroprotective After Experimental Ischemic Stroke

Endothelial cell integrin receptors, specifically the β 1 subtype, play a direct role in inflammation and blood-brain barrier (BBB) dysfunction, both implicated in poor ischemic stroke outcomes. We hypothesize that inhibition of a particular β1 integrin subtype, α5β1, (a pro-angiogenic fibronectin receptor acutely expressed after ischemic stroke) could be therapeutic in ischemic stroke via BBB stabilization. Methods: 3-month-old male, C57BL/J6 wildtype mice underwent transient middle cerebral artery occlusion for 1 hour. Mice were treated intraperitoneally with the small peptide α5β1 integrin inhibitor ATN-161 (1 mg/kg) upon reperfusion, post stroke day (PSD) 1 and PSD2. On PSD3, infarct volume (TTC and T2-weighted MRI) and vasogenic edema (MRI) were assessed. Neuroscore, an 11-point functional assessment, was performed on PSD1-14. Expression of MMP-9, claudin-5 and IL-1β levels were determined by qPCR. On PSD3, immunohistochemical analyses of α5 integrin, collagen IV, claudin-5, CD45, and DAPI expression were performed. In vitro oxygen-glucose deprivation (OGD) experiments on brain endothelial (bEND3) cells were also performed, evaluating ATN-161 administration (10 μM) effects on BEND3 layer permeability via FITC-dextran migration, and on immunocytochemistry of α5 integrin and claudin-5 expression. Results: Acute post-stroke administration of ATN-161 significantly decreased infarct volume and vasogenic edema, and improved functional recovery. Additionally, ATN-161 appeared to stabilize the BBB by increasing expression of the tight junction protein claudin-5, while decreasing expression of the extracellular matrix proteinase, MMP-9, inflammatory cytokine (IL-1β), and infiltrating leukocytes (CD45). Additionally, permeability was conserved with ATN-161 administration following OGD. Conclusion: Administration of the α5β1 inhibitor ATN-161 stabilizes the BBB barrier, and reduces inflammatory infiltration, while reducing infarct volume, vasogenic edema, and behavioral dysfunction following experimental stroke. Collectively, these results suggest inhibition of α5β1 via ATN-161 could represent a promising novel therapeutic approach for ischemic stroke.

Recent Expansions on Cellular Models to Uncover the Scientific Barriers Towards Drug Development for Alzheimer's Disease.

Globally, the central nervous system (CNS) disordersappear as the most critical pathological threat with no proper cure. Alzheimer's disease (AD) is one such condition frequently observed with the aged population and sometimes in youth too. Most of the research utilizes different animal models for in vivo study of AD pathophysiology and to investigate the potency of the newly developed therapy. These in vivo models undoubtably provide a powerful investigation tool to study human brain. Although, it sometime fails to mimic the exact environment and responses as the human brain owing to the distinctive genetic and anatomical features of human and rodent brain. In such condition, the in vitro cell model derived from patient specific cell or human cell lines can recapitulate the human brain environment. In addition, the frequent use of animals in research increases the cost of study and creates various ethical issues. Instead, the use of in vitro cellular models along with animal models can enhance the translational values of in vivo models and represent a better and effective mean to investigate the potency of therapeutics. This strategy also limits the excessive use of laboratory animal during the drug development process. Generally, the in vitro cell lines are cultured from AD rat brain endothelial cells, the rodent models, human astrocytes, human brain capillary endothelial cells, patient derived iPSCs (induced pluripotent stem cells) and also from the non-neuronal cells. During the literature review process, we observed that there are very few reviews available which describe the significance and characteristics of in vitro cell lines, for AD investigation. Thus, in the present review article, we have compiled the various in vitro cell lines used in AD investigation including HBMEC, BCECs, SHSY-5Y, hCMEC/D3, PC-2 cell line, bEND3 cells, HEK293, hNPCs, RBE4 cells, SK-N-MC, BMVECs, CALU-3, 7W CHO, iPSCs and cerebral organoids cell lines and different types of culture media such as SCM, EMEM, DMEM/F12, RPMI, EBM and 3D-cell culture.

Epidermal growth factor treatment has protective effects on the integrity of the blood-brain barrier against cerebral ischemia injury in bEnd3 cells.

Tight junctions (TJs) serve an important role in maintaining the integrity of the blood-brain barrier (BBB), while neurological disorders, including ischemic stroke, induce TJ disruption and increase BBB permeability; results include edema formation and hemorrhage transformation. Cerebral endothelium protection presents a promising approach in ischemic stroke therapy. In the current study, protective effects of the epidermal growth factor (EGF) on ischemia-induced disruption of BBB integrity were examined using an oxygen-glucose deprivation (OGD) model in bEnd3 cells. Expression levels of claudin-5 and TJ protein-1 (ZO-1) were determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis. Cell viability was evaluated by cell counting kit-8 assay and the endothelial permeability of Lucifer yellow (LY) was assessed using Transwell assays. The results revealed that post-ischemia administration of EGF (250 ng/ml) significantly attenuated the decrease in mRNA (P<0.05) and protein (P<0.01) expression levels of claudin-5 and ZO-1, and the increase in endothelial permeability of LY (P<0.05) induced by 4 h OGD exposure followed by 24 h reoxygenation. In addition, EGF did not significant affect cell viability. The current study suggested a potential of EGF to improve BBB integrity against ischemic injury by upregulating the expression of TJ proteins and reducing endothelial permeability.

Glutamate triggers intracellular Ca2+ oscillations and nitric oxide release by inducing NAADP- and InsP3 -dependent Ca2+ release in mouse brain endothelial cells.

The neurotransmitter glutamate increases cerebral blood flowby activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca2+ concentration ([Ca2+ ]i ) in the target cells, which activates the Ca2+ /Calmodulin-dependent nitric oxide (NO) synthaseto release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca2+ ]i and NO production. The current study assessed whether and how glutamate drives Ca2+ -dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca2+ ]i , which was maximally activated at 200 μM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca2+ oscillations were triggered by rhythmic endogenous Ca2+ mobilization and maintained over time by extracellular Ca2+ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca2+ release was mediated by InsP3 -sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca2+ entry mediated Ca2+ entry during ongoing Ca2+ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca2+ signals. Of note, glutamate induced Ca2+ -dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca2+ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.

S1PR2 antagonist alleviates oxidative stress-enhanced brain endothelial permeability by attenuating p38 and Erk1/2-dependent cPLA2 phosphorylation

Both sphingosine-1-phosphate receptor-2 (S1PR2) and cytosolic phospholipase A2 (cPLA2) are implicated in the disruption of cerebrovascular integrity in experimental stroke. However, the role of S1PR2 in induction of cPLA2 phosphorylation during cerebral ischemia-induced endothelial dysfunction remains unknown. This study investigated the effect of S1PR2 blockade on oxidative stress-induced cerebrovascular endothelial barrier impairment and explored the possible mechanisms. In bEnd3 cells, cPLA2 inhibitor CAY10502 as well as S1PR2 antagonist JTE013 profoundly suppressed hydrogen peroxide (H2O2)-induced changes of paracellular permeability and ZO-1 localization. Besides p38, extracellular signal-regulated kinase (Erk) 1/2 is required for H2O2-increased cPLA2 phosphorylation and endothelial permeability. Pharmacological and genetic inhibition of S1PR2 significantly suppressed their phosphorylation in response to H2O2. Especially lentivirus-mediated knockdown of S1PR2 inhibited H2O2-induced ZO-1 redistribution and paracellular hyperpermeability. Using the permanent middle cerebral artery occlusion (pMCAO) mouse model, we found JTE013 pretreatment markedly reduced Evans blue dye (EBD) extravasation and reversed the decrease in VE-cadherin, occludin, claudin-5 and CD31 expression in infarcted hemisphere. Lentivirus-mediated S1PR2 knockdown also attenuated EBD extravasation. Furthermore, JTE013 pretreatment attenuated neurological deficit, brain edema and infarction volume. Therefore, our findings suggest the protective effect of JTE013 on brain endothelial barrier integrity is likely mediated by suppressing p38 and Erk1/2-dependent cPLA2 phosphorylation under oxidative stress.

Nootropic nanocomplex with enhanced blood-brain barrier permeability for treatment of traumatic brain injury-associated neurodegeneration

Traumatic brain injury (TBI) is an intracranial injury which can induce immediate neuroinflammation and long-term neurological deficits. Methylene blue (MB) as a nootropic has a great potential to treat neurodegeneration after TBI because of its anti-inflmmatory and neuroprotective functions. However, its limited accumulation to the brain across the blood-brain barrier (BBB) remains a major hurdle to be overcome. In this paper, we present a polymer surfactant-encapsulated nanocomplex of MB as a delivery system with high BBB permeability for efficacious treatment of TBI-induced neurodegeneration. MB was formulated via electrostatically/hydrophobically directed assembly with fatty acid and Pluronic surfactant (F-127 or F-68) to construct nanocomplexes of two different colloidal sizes (<10 nm and ~108 nm in hydrodynamic diameter for NanoMB-127 and NanoMB-68, respectively). Compared to uncomplexed free MB, formulation into the ultrasmall nanocomplex (NanoMB-127) significantly enhanced the uptake of MB by blood-brain vascular endothelial bEnd3 cells in vitro, and indeed improved its BBB penetration upon systemic administration to normal mice in vivo. However, large-size NanoMB-68 showed negligible BBB crossing despite the efficient bEnd3 cell internalization in vitro, probably due to the unfavorable pharmacokinetic profile associated with its large particle size. By virtue of the efficient BBB penetration and cellular uptake, ultrasmall NanoMB-127 was shown to distinctively reduce the expression level of an inflammatory cytokine with no notable toxicity in vitro and also considerably prevent the neurodegeneration after TBI in mice at much lower doses than free MB. Overall, the Pluronic-supported nanocomplexation method allows efficient brain delivery of MB, offering a novel way of enhancing the efficacy of neurotherapeutics to treat brain diseases.

Paracellular tightness and the functional expression of efflux transporters P-gp and BCRP in bEnd3 cells

Objective The blood–brain barrier (BBB), regulating brain homeostasis and limiting the entry of most drugs, is characterized by intercellular tight junctions and the presence of transporters. In this study, the paracellular tightness and functional expression of efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) were evaluated in mouse brain immortalized cell line bEnd3 to prove it as a useful BBB-mimicking system for biological and pharmacological research.Methods The presence of P-gp, BCRP and tight junction proteins occludin, claudin-5 and ZO-1 were validated by RT-PCR and Western blot. The tightness of bEnd3 monolayers was evaluated by measuring the permeability of hydrophilic marker Lucifer yellow. The P-gp functionality was identified by intracellular uptake assay using Rhodamine 123 (R123) as P-gp substrate and verapamil as P-gp inhibitor. The BCRP functionality was identified by flow cytometric analysis of mitoxantrone accumulation and fluorescence microscopic analysis of Hoechst 33342 accumulation using Ko-143 as BCRP inhibitor.Results The bEnd3 cells demonstrated the expression of P-gp, BCRP and tight junction proteins occludin, claudin-5 and ZO-1 at mRNA and protein levels. The permeability coefficient of Lucifer yellow was 1.3 ± 0.13 × 10−3 cm/min, indicating the moderate paracellular tightness barrier formed by bEnd3 cells. The verapamil induced a higher cellular uptake of Rhodamine 123, and Ko-143 significantly elevated cellular accumulation of mitoxantrone and Hoechst 33342, suggesting the P-gp and BCRP functionality shown by bEnd3 cells.Conclusions The bEnd3 cell line represents a useful in vitro tool for studying BBB characteristics and drug transport mechanisms at the BBB.