Ferroptosis of brain microvascular endothelial cells contributes to hypoxia-induced blood-brain barrier injury.

Hypoxia is pivotal to the pathogeneses of myriad disorders, especially hypoxic cerebropathy. Much is known about the damage to the blood-brain barrier (BBB) in response to hypoxia. Studies have shown that endothelial cell death is closely linked to functional impairment of BBB. Mounting evidences have demonstrated that ferroptosis, a new pathway regulating cell death, is implicated in brain injury. However, whether ferroptosis is involved in hypoxia-induced BBB disruption remains ambiguous. Here, we utilized in vivo zebrafish and in vitro bEnd.3 cells to explore the correlation between endothelial ferroptosis and hypoxia-induced BBB damage. We found that hypoxic treatment for 45 min can induce BBB disruption by triggering down-regulation of claudin-5 (CLDN5) both in zebrafish cerebrovascluar endothelial cells and bEnd.3 cells. Besides, in vitro and in vivo studies revealed the cysteine/glutamate antiporter xCT (also known as solute carrier family 7 member 11; SLC7A11) decrease, glutathione peroxidase 4 (GPX4) and glutathione (GSH) reduction, 4-Hydroxynonenal (4-HNE) increasement, malondialdehyde (MDA) upregulation and reactive oxygen species (ROS) accumulation in hypoxia group. Further mechanism studies indicated that hypoxia-induced BBB damage might associate with microvascular endothelial cellular ferroptosis, since hypoxic exposure significantly activated the expression of ferroptosis-related genes (Ptgs2, Por, Lpcat3, Alox5, Alox12, Nfe2l2, and Ncoa4) and inhibited the expression of Slc7a11. Additionally, the application of 20 µM ferrostatin-1 (Fer-1), a ferroptosis inhibitor, could partially alleviate BBB disruption under hypoxia, suggesting that inhibition of ferroptosis might be a potential strategy for some neurological diseases with BBB defect.

Heat-shock protein A12A attenuates oxygen-glucose deprivation/reoxygenation-induced human brain microvascular endothelial cell dysfunction via PGC-1α/SIRT3 pathway.

Ischemic stroke is a life-threatening brain disease with the leading cause of disability and mortality worldwide. Heat-shock protein A12A (HSPA12A) is recognized as a neuroprotective target for treating ischemic stroke; however, its regulatory mechanism has been not fully elucidated yet. Human brain microvascular endothelial cells (hBMECs) were induced by oxygen-glucose deprivation/reoxygenation (OGD/R) to mimic ischemic stroke. Gain- and loss-of-function experiments were conducted to explore the regulation of HSAPA12 and PGC-1α. Cell viability, apoptosis, and permeability were assessed by CCK-8, TUNEL, and transendothelial electrical resistance (TEER) assays, respectively. The expression of HSPA12A and corresponding proteins was measured by western blot. Cell immunofluorescence was adopted to evaluate ZO-1 expression. THP-1 cells were applied to adhere hBMECs in vitro to simulate leukocyte adhesion in the brain. HSPA12A was downregulated in OGD/R-treated hBMECs. HSPA12A overexpression significantly suppressed OGD/R-induced cell viability loss and apoptosis in hBMECs. Meanwhile, HSPA12A overexpression attenuated blood-brain barrier (BBB) integrity in OGD/R-induced hBMECs, evidenced by the restored TEER value and the upregulated ZO-1, occludin, and claudin-5. HSPA12A also restricted OGD/R-induced attachment of THP-1 cells to hBMECs, accompanied with downregulating ICAM-1 and VCAM-1. Additionally, OGD/R-caused downregulation of PGC-1α/SIRT3 in hBMECs was partly restored by HSPA12A overexpression. Furthermore, the above effects of HSPA12A on OGD/R-induced hBMECs injury were partly reversed by PGC-1α knockdown. HSPA12A plays a protective role against OGD/R-induced hBMECs injury by upregulating PGC-1α, providing a potential neuroprotective role of HSPA12A in ischemic stroke.

Tanshinone IIA ameliorates Aß transendothelial transportation through SIRT1-mediated endoplasmic reticulum stress.

BACKGROUND: The disruption of blood-brain barrier (BBB), predominantly made up by brain microvascular endothelial cells (BMECs), is one of the characteristics of Alzheimer's disease (AD). Thus, improving BMEC function may be beneficial for AD treatment. Tanshinone IIA (Tan IIA) has been proved to ameliorate the cognitive dysfunction of AD. Herein, we explored how Tan IIA affected the function of BMECs in AD. METHODS: Aß1-42-treated brain-derived endothelium cells.3 (bEnd.3 cells) was employed for in vitro experiments. And we performed molecular docking and qPCR to determine the targeting molecule of Tan IIA on Sirtuins family. The APPswe/PSdE9 (APP/PS1) mice were applied to perform the in vivo experiments. Following the behavioral tests, protein expression was determined through western blot and immunofluorescence. The activities of oxidative stress-related enzymes were analyzed by biochemically kits. Nissl staining and thioflavin T staining were conducted to reflect the neurodegeneration and Aß deposition respectively. RESULTS: Molecular docking and qPCR results showed that Tan IIA mainly acted on Sirtuin1 (SIRT1) in Sirtuins family. The inhibitor of SIRT1 (EX527) was employed to further substantiate that Tan IIA could attenuate SIRT1-mediated endoplasmic reticulum stress (ER stress) in BMECs. Behavioral tests suggested that Tan IIA could improve the cognitive deficits in APP/PS1 mice. Tan IIA administration increased SIRT1 expression and alleviated ER stress in APP/PS1 mice. In addition, LRP1 expression was increased and RAGE expression was decreased after Tan IIA administration in both animals and cells. CONCLUSION: Tan IIA could promote Aß transportation by alleviating SIRT1-mediated ER stress in BMECs, which ameliorated cognitive deficits in APP/PS1 mice.

BMECs Ameliorate High Glucose-Induced Morphological Aberrations and Synaptic Dysfunction via VEGF-Mediated Modulation of Glucose Uptake in Cortical Neurons.

It has been demonstrated that diabetes cause neurite degeneration in the brain and cognitive impairment and neurovascular interactions are crucial for maintaining brain function. However, the role of vascular endothelial cells in neurite outgrowth and synaptic formation in diabetic brain is still unclear. Therefore, present study investigated effects of brain microvascular endothelial cells (BMECs) on high glucose (HG)-induced neuritic dystrophy using a coculture model of BMECs with neurons. Multiple immunofluorescence labelling and western blot analysis were used to detect neurite outgrowth and synapsis formation, and living cell imaging was used to detect uptake function of neuronal glucose transporters. We found cocultured with BMECs significantly reduced HG-induced inhibition of neurites outgrowth (including length and branch formation) and delayed presynaptic and postsynaptic development, as well as reduction of neuronal glucose uptake capacity, which was prevented by pre-treatment with SU1498, a vascular endothelial growth factor (VEGF) receptor antagonist. To analyse the possible mechanism, we collected BMECs cultured condition medium (B-CM) to treat the neurons under HG culture condition. The results showed that B-CM showed the same effects as BMEC on HG-treated neurons. Furthermore, we observed VEGF administration could ameliorate HG-induced neuronal morphology aberrations. Putting together, present results suggest that cerebral microvascular endothelial cells protect against hyperglycaemia-induced neuritic dystrophy and restorate neuronal glucose uptake capacity by activation of VEGF receptors and endothelial VEGF release. This result help us to understand important roles of neurovascular coupling in pathogenesis of diabetic brain, providing a new strategy to study therapy or prevention for diabetic dementia. Hyperglycaemia induced inhibition of neuronal glucose uptake and impaired to neuritic outgrowth and synaptogenesis. Cocultured with BMECs/B-CM and VEGF treatment protected HG-induced inhibition of glucose uptake and neuritic outgrowth and synaptogenesis, which was antagonized by blockade of VEGF receptors. Reduction of glucose uptake may further deteriorate impairment of neurites outgrowth and synaptogenesis.

Exosomal Mir-3613-3p derived from oxygen-glucose deprivation-treated brain microvascular endothelial cell promotes microglial M1 polarization.

BACKGROUND: Brain microvascular endothelial cell (BMEC) injury can affect neuronal survival by modulating immune responses through the microenvironment. Exosomes are important vehicles of transport between cells. However, the regulation of the subtypes of microglia by BMECs through the exosome transport of microRNAs (miRNAs) has not been established. METHODS: In this study, exosomes from normal and oxygen-glucose deprivation (OGD)-cultured BMECs were collected, and differentially expressed miRNAs were analyzed. BMEC proliferation, migration, and tube formation were analyzed using MTS, transwell, and tube formation assays. M1 and M2 microglia and apoptosis were analyzed using flow cytometry. miRNA expression was analyzed using real-time polymerase chain reaction (RT-qPCR), and IL-1ß, iNOS, IL-6, IL-10, and RC3H1 protein concentrations were analyzed using western blotting. RESULTS: We found that miR-3613-3p was enriched in BMEC exosome by miRNA GeneChip assay and RT-qPCR analysis. miR-3613-3p knockdown enhanced cell survival, migration, and angiogenesis in the OGD-treated BMECs. In addition, BMECs secrete miR-3613-3p to transfer into microglia via exosomes, and miR-3613-3p binds to the RC3H1 3' untranslated region (UTR) to reduce RC3H1 protein levels in microglia. Exosomal miR-3613-3p promotes microglial M1 polarization by inhibiting RC3H1 protein levels. BMEC exosomal miR-3613-3p reduces neuronal survival by regulating microglial M1 polarization. CONCLUSIONS: miR-3613-3p knockdown enhances BMEC functions under OGD conditions. Interfering with miR-3613-3p expression in BMSCs reduced the enrichment of miR-3613-3p in exosomes and enhanced M2 polarization of microglia, which contributed to reduced neuronal apoptosis.

Role of Orai3-Mediated Store-Operated Calcium Entry in Radiation-Induced Brain Microvascular Endothelial Cell Injury.

Radiation-induced brain injury is a serious complication with complex pathogenesis that may accompany radiotherapy of head and neck tumors. Although studies have shown that calcium (Ca2+) signaling may be involved in the occurrence and development of radiation-induced brain injury, the underlying molecular mechanisms are not well understood. In this study, we used real-time quantitative polymerase chain reaction and Western blotting assays to verify our previous finding using next-generation sequencing that the mRNA and protein expression levels of Orai3 in rat brain microvascular endothelial cells (rBMECs) increased after X-ray irradiation. We next explored the role of Orai3 and Orai3-mediated store-operated Ca2+ entry (SOCE) in radiation-induced brain injury. Primary cultured rBMECs derived from wild-type and Orai3 knockout (Orai3(-/-)) Sprague-Dawley rats were used for in vitro experiments. Orai3-mediated SOCE was significantly increased in rBMECs after X-ray irradiation. However, X-ray irradiation-induced SOCE increase was markedly reduced in Orai3 knockout rBMECs, and the percentage of BTP2 (a nonselective inhibitor of Orai channels)-inhibited SOCE was significantly decreased in Orai3 knockout rBMECs. Functional studies indicated that X-ray irradiation decreased rBMEC proliferation, migration, and tube formation (a model for assessing angiogenesis) but increased rBMEC apoptosis, all of which were ameliorated by BTP2. In addition, occurrences of all four functional deficits were suppressed in X-ray irradiation-exposed rBMECs derived from Orai3(-/-) rats. Cerebrovascular damage caused by whole-brain X-ray irradiation was much less in Orai3(-/-) rats than in wild-type rats. These findings provide evidence that Orai3-mediated SOCE plays an important role in radiation-induced rBMEC damage and brain injury and suggest that Orai3 may warrant development as a potential therapeutic target for reducing or preventing radiation-induced brain injury.

Ferroptosis in brain microvascular endothelial cells mediates blood-brain barrier disruption after traumatic brain injury.

Ferroptosis is a newly recognized form of regulated cell death. Recently, growing evidence has shown that ferroptosis is involved in the pathogenesis of traumatic brain injury (TBI). However, less attention has been paid to its role in brain microvascular endothelial cells (BMVECs) and blood-brain barrier (BBB) damage, the central pathological process in secondary brain injury of TBI. Here, we established a mechanical stretch injury bEnd.3 model and a Controlled Cortical Impact (CCI) mouse model to explore the ferroptosis-related markers in brain endothelial cells after TBI in vitro and in vivo. From the results of RNA-seq analysis, RT-qPCR and immunostaining, glutathione peroxidase 4 (GPX4) downregulation, Cyclooxygenase-2 (COX-2) upregulation, and iron accumulation were observed in brain endothelial cells after TBI both in vitro and in vivo. Furthermore, we utilized Ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, to investigate the protective effects of ferroptosis inhibition on BBB disruption and neurological deficits. From the results of immunostaining, transmission electron microscopy (TEM), and western blotting, we demonstrated that Fer-1 significantly reduced BMVECs death, BBB permeability, and tight junction loss at 3 days after TBI. The neurological tests including grid walking, rotarod test, and wire-hanging test showed that Fer-1 administration exerted neuroprotective effects in the early stage of TBI. Our findings provided evidences for inhibition of BMVECs ferroptosis as a promising therapeutic target against TBI-induced BBB disruption.

Central Nervous System-Infecting Pathogens Escherichia coli and Cryptococcus neoformans Exploit the Host Pdlim2 for Intracellular Traversal and Exocytosis in the Blood-Brain Barrier.

Microbial penetration of the blood-brain barrier, a prerequisite for the development of central nervous system (CNS) infection, involves microbial invasion, intracellular traversal, and exocytosis. Microbial invasion of the blood-brain barrier has been investigated, but the molecular basis for microbial traversal and exit from the blood-brain barrier remains unknown. We performed transcriptome analysis of human brain microvascular endothelial cells (HBMEC) infected with Escherichia coli and Cryptococcus neoformans, representative bacterial and fungal pathogens common in CNS infections. Among the targets upregulated in response to E. coli and C. neoformans infection, PDLIM2 was knocked down by small hairpin RNA (shRNA) in HBMEC for further investigation. We demonstrated that Pdlim2 specifically regulated microbial traversal and exit from HBMEC by assessing microbial invasion, transcytosis, intracellular multiplication, and egression. Additionally, the defective exocytosis of internalized E. coli cells from the PDLIM2 shRNA knockdown cells was restored by treatment with a calcium ionophore (ionomycin). Moreover, we performed proximity-dependent biotin labeling with the biotin ligase BioID2 and identified 210 potential Pdlim2 interactors. Among the nine Pdlim2 interactors enriched in response to both E. coli and C. neoformans infection, we selected MPRIP and showed that HBMEC with knockdown of MPRIP mimicked the phenotype of PDLIM2 knockdown cells. These results suggest that the CNS-infecting microbes hijack Pdlim2 and Mprip for intracellular traversal and exocytosis in the blood-brain barrier.

Influence of basal media composition on barrier fidelity within human pluripotent stem cell-derived blood-brain barrier models.

It is increasingly recognized that brain microvascular endothelial cells (BMECs), the principal component of the blood-brain barrier (BBB), are highly sensitive to soluble cues from both the bloodstream and the brain. This concept extends in vitro, where the extracellular milieu can also influence BBB properties in cultured cells. However, the extent to which baseline culture conditions can affect BBB properties in vitro remains unclear, which has implications for model variability and reproducibility, as well as downstream assessments of molecular transport and disease phenotypes. Here, we explore this concept by examining BBB properties within human-induced pluripotent stem cell (iPSC)-derived BMEC-like cells cultured under serum-free conditions in DMEM/F12 and Neurobasal media, which have fully defined compositions. We demonstrate notable differences in both passive and active BBB properties as a function of basal media composition. Further, RNA sequencing and phosphoproteome analyses revealed alterations to various signaling pathways in response to basal media differences. Overall, our results demonstrate that baseline culture conditions can have a profound influence on the performance of in vitro BBB models, and these effects should be considered when designing experiments that utilize such models for basic research and preclinical assays.

Dimethyl fumarate reduces TNF and Plasmodium falciparum induced brain endothelium activation in vitro.

BACKGROUND: Cerebral malaria (CM) is associated with morbidity and mortality despite the use of potent anti-malarial agents. Brain endothelial cell activation and dysfunction from oxidative and inflammatory host responses and products released by Plasmodium falciparum-infected erythrocytes (IE), are likely the major contributors to the encephalopathy, seizures, and brain swelling that are associated with CM. The development of adjunctive therapy to reduce the pathological consequences of host response pathways could improve outcomes. A potentially protective role of the nuclear factor E2-related factor 2 (NRF2) pathway, which serves as a therapeutic target in brain microvascular diseases and central nervous system (CNS) inflammatory diseases such as multiple sclerosis was tested to protect endothelial cells in an in vitro culture system subjected to tumour necrosis factor (TNF) or infected red blood cell exposure. NRF2 is a transcription factor that mediates anti-oxidant and anti-inflammatory responses. METHODS: To accurately reflect clinically relevant parasite biology a unique panel of parasite isolates derived from patients with stringently defined CM was developed. The effect of TNF and these parasite lines on primary human brain microvascular endothelial cell (HBMVEC) activation in an in vitro co-culture model was tested. HBMVEC activation was measured by cellular release of IL6 and nuclear translocation of NFκB. The transcriptional and functional effects of dimethyl fumarate (DMF), an FDA approved drug which induces the NRF2 pathway, on host and parasite induced HBMVEC activation was characterized. In addition, the effect of DMF on parasite binding to TNF stimulated HBMVEC in a semi-static binding assay was examined. RESULTS: Transcriptional profiling demonstrates that DMF upregulates the NRF2-Mediated Oxidative Stress Response, ErbB4 Signaling Pathway, Peroxisome Proliferator-activated Receptor (PPAR) Signaling and downregulates iNOS Signaling and the Neuroinflammation Signaling Pathway on TNF activated HBMVEC. The parasite lines derived from eight paediatric CM patients demonstrated increased binding to TNF activated HBMVEC and varied in their binding and activation of HBMVEC. Overall DMF reduced both TNF and CM derived parasite activation of HBMVEC. CONCLUSIONS: These findings provide evidence that targeting the NRF2 pathway in TNF and parasite activated HBMVEC mediates multiple protective pathways and may represent a novel adjunctive therapy to improve infection outcomes in CM.

FGF21 Protects against Aggravated Blood-Brain Barrier Disruption after Ischemic Focal Stroke in Diabetic db/db Male Mice via Cerebrovascular PPARγ Activation.

Recombinant fibroblast growth factor 21 (rFGF21) has been shown to be potently beneficial for improving long-term neurological outcomes in type 2 diabetes mellitus (T2DM) stroke mice. Here, we tested the hypothesis that rFGF21 protects against poststroke blood-brain barrier (BBB) damage in T2DM mice via peroxisome proliferator-activated receptor gamma (PPARγ) activation in cerebral microvascular endothelium. We used the distal middle cerebral occlusion (dMCAO) model in T2DM mice as well as cultured human brain microvascular endothelial cells (HBMECs) subjected to hyperglycemic and inflammatory injury in the current study. We detected a significant reduction in PPARγ DNA-binding activity in the brain tissue and mRNA levels of BBB junctional proteins and PPARγ-targeting gene CD36 and FABP4 in cerebral microvasculature at 24 h after stroke. Ischemic stroke induced a massive BBB leakage two days after stroke in T2DM mice compared to in their lean controls. Importantly, all abnormal changes were significantly prevented by rFGF21 administration initiated at 6 h after stroke. Our in vitro experimental results also demonstrated that rFGF21 protects against hyperglycemia plus interleukin (IL)-1ß-induced transendothelial permeability through upregulation of junction protein expression in an FGFR1 activation and PPARγ activity elevation-dependent manner. Our data suggested that rFGF21 has strong protective effects on acute BBB leakage after diabetic stroke, which is partially mediated by increasing PPARγ DNA-binding activity and mRNA expression of BBB junctional complex proteins. Together with our previous investigations, rFGF21 might be a promising candidate for treating diabetic stroke.

[Effect of baicalin on inflammatory response and TLR4/NF-κB signaling pathway of human brain microvascular endothelial cell after hypoxia-reoxygenation injury].

In this study, the oxygen-glucose deprivation(OGD) model in the human brain microvascular endothelial cell(HBMEC) was used to simulate the ischemic neuronal damage and observe the inflammatory response, explore the possible mechanisms for treating cerebral ischemia/reperfusion and improving memory impairment from the view point of inhibiting inflammatory response, which is of great reference significance for related Chinese medicine treatment of ischemic diseases. HBMECs were given with drugs at the same time of OGD injury, and reoxygenated for 2 h after 4 h treatment. Cell supernatant was then collected, and the inflammatory factors in cell supernatant were detected. Immunofluorescence assay was used to detect HBMECs morphology and expression of p-nuclear factor kappa-light-chain-enhancer of activated B(p-NF-κB); Western blot was used to detect expression changes of Toll like receptor 4(TLR4), myeloid differentiation primary response 88(MYD88) and p-NF-κB. The results showed that, after OGD modeling, the levels of interleukin 6(IL-6), IL-1α, IL-1ß and tumor necrosis factor-α(TNF-α) were significantly increased; baicalin protected HBMEC, inhibited intranuclear transcription of p-NF-κB, significantly decreased HBMEC release of inflammatory factors caused by OGD injury, and inhibited the expression of TLR4, MYD88, and p-NF-κB. The studies suggested that baicalin had obvious protective effect on HBMECs damaged by OGD, and could inhibit inflammatory response. Its protection mechanism may be related to inhibiting TLR4 signaling pathways.

UHPLC-QTOF/MS-based metabolomics investigation for the protective mechanism of Danshen in Alzheimer's disease cell model induced by Aß1-42.

INTRODUCTION: Alzheimer's disease (AD) is a chronic neurodegenerative disorder with neither definitive pathogenesis nor effective therapy so far. Danshen, the dried root and rhizome of Salvia miltiorrhiza Bunge, is used extensively in Alzheimer's disease treatment to ameliorate the symptoms, but the underlying mechanism remains to be clarified. OBJECTIVES: To investigate potential biomarkers for AD and elucidate the protective mechanism of Danshen on AD cell model. METHODS: An ultra high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-QTOF/MS)-based approach combined with partial least squares discriminant analysis (PLS-DA) has been developed to discriminate the metabolic modifications between human brain microvascular endothelial cell (hBMEC) and AD cell model induced by amyloid-ß protein (Aß1-42). To further elucidate the pathophysiology of AD, related metabolic pathways have been studied. RESULTS: Thirty-three distinct potential biomarkers were screened out and considered as potential biomarkers corresponding to AD, which were mostly improved and partially restored back to normalcy in Danshen pre-protection group. It was found that AD was closely related to disturbed arginine and proline metabolism, glutathione metabolism, alanine aspartate and glutamate metabolism, histidine metabolism, pantothenate and CoA biosynthesis, phenylalanine tyrosine and tryptophan biosynthesis, citrate cycle and glycerophospholipid metabolism, and the protective mechanism of Danshen in AD cell model may be related to partially regulating the perturbed pathways. CONCLUSIONS: These outcomes provide valuable evidences for therapeutic mechanism investigation of Danshen in AD treatment, and such an approach could be transferred to unravel the mechanism of other traditional Chinese medicine (TCM) and diseases.

Role of pregnancy and female sex steroids on aneurysm formation, growth, and rupture: a systematic review of the literature.

OBJECTIVE: Women have been shown to have a higher risk of cerebral aneurysm formation, growth, and rupture than men. The authors present a review of the recently published neurosurgical literature that studies the role of pregnancy and female sex steroids, to provide a conceptual framework with which to understand the various risk factors associated with cerebral aneurysms in women at different stages in their lives. METHODS: The PubMed database was searched for "("intracranial" OR "cerebral") AND "aneurysm" AND ("pregnancy" OR "estrogen" OR "progesterone")" between January 1980 and February 2019. A total of 392 articles were initially identified, and after applying inclusion and exclusion criteria, 20 papers were selected for review and analysis. These papers were then divided into two categories: 1) epidemiological studies about the formation, growth, rupture, and management of cerebral aneurysms in pregnancy; and 2) investigations on female sex steroids and cerebral aneurysms (animal studies and epidemiological studies). RESULTS: The 20 articles presented in this study include 7 epidemiological articles on pregnancy and cerebral aneurysms, 3 articles reporting case series of cerebral aneurysms treated by endovascular therapies in pregnancy, 3 epidemiological articles reporting the relationship between female sex steroids and cerebral aneurysms through retrospective case-control studies, and 7 experimental studies using animal and/or cell models to understand the relationship between female sex steroids and cerebral aneurysms. The studies in this review report similar risk of aneurysm rupture in pregnant women compared to the general population. Most ruptured aneurysms in pregnancy occur during the 3rd trimester, and most pregnant women who present with cerebral aneurysm have caesarean section deliveries. Endovascular treatment of cerebral aneurysms in pregnancy is shown to provide a new and safe form of therapy for these cases. Epidemiological studies of postmenopausal women show that estrogen hormone therapy and later age at menopause are associated with a lower risk of cerebral aneurysm than in matched controls. Experimental studies in animal models corroborate this epidemiological finding; estrogen deficiency causes endothelial dysfunction and inflammation, which may predispose to the formation and rupture of cerebral aneurysms, while exogenous estrogen treatment in this population may lower this risk. CONCLUSIONS: The aim of this work is to equip the neurosurgical and obstetrical/gynecological readership with the tools to better understand, critique, and apply findings from research on sex differences in cerebral aneurysms.

Fatty Acid-Binding Protein 5 at the Blood-Brain Barrier Regulates Endogenous Brain Docosahexaenoic Acid Levels and Cognitive Function.

Fatty acid-binding protein 5 (FABP5) at the blood-brain barrier contributes to the brain uptake of docosahexaenoic acid (DHA), a blood-derived polyunsaturated fatty acid essential for maintenance of cognitive function. Given the importance of DHA in cognition, the aim of this study was to investigate whether deletion of FABP5 results in cognitive dysfunction and whether this is associated with reduced brain endothelial cell uptake of exogenous DHA and subsequent attenuation in the brain levels of endogenous DHA. Cognitive function was assessed in male and female FABP5+/+ and FABP5-/- mice using a battery of memory paradigms. FABP5-/- mice exhibited impaired working memory and short-term memory, and these cognitive deficits were associated with a 14.7 ± 5.7% reduction in endogenous brain DHA levels. The role of FABP5 in the blood-brain barrier transport of DHA was assessed by measuring 14C-DHA uptake into brain endothelial cells and capillaries isolated from FABP5+/+ and FABP5-/- mice. In line with a crucial role of FABP5 in the brain uptake of DHA, 14C-DHA uptake into brain endothelial cells and brain capillaries of FABP5-/- mice was reduced by 48.4 ± 14.5% and 14.0 ± 4.2%, respectively, relative to those of FABP5+/+ mice. These results strongly support the hypothesis that FABP5 is essential for maintaining brain endothelial cell uptake of DHA, and that cognitive deficits observed in FABP5-/- mice are associated with reduced CNS access of DHA. SIGNIFICANCE STATEMENT: Genetic deletion of fatty acid-binding protein 5 (FABP5) in mice reduces uptake of exogenous docosahexaenoic acid (DHA) into brain endothelial cells and brain capillaries and reduces brain parenchymal levels of endogenous DHA. Therefore, FABP5 in the brain endothelial cell is a crucial contributor to the brain levels of DHA. Critically, lowered brain DHA levels in FABP5-/- mice occurred in tandem with cognitive deficits in a battery of memory paradigms. This study provides evidence of a critical role for FABP5 in the maintenance of cognitive function via regulating the brain uptake of DHA, and suggests that upregulation of FABP5 in neurodegenerative diseases, where brain DHA levels are possibly diminished (e.g., Alzheimer's disease), may provide a novel therapeutic approach for restoring cognitive function.

Let-7i attenuates human brain microvascular endothelial cell damage in oxygen glucose deprivation model by decreasing toll-like receptor 4 expression.

The let-7 family of microRNAs (miRNAs) plays an important role on endothelial cell function. However, there have been few studies on their role under ischemic conditions. In this study, we demonstrate that let-7i, belonging to the let-7 family, rescues human brain microvascular endothelial cells (HBMECs) in an oxygen-glucose deprivation (OGD) model. Our data show that the expression of let-7 family miRNAs was downregulated after OGD. Overexpression of let-7i significantly alleviated cell death and improved survival of OGD-treated HBMECs. Let-7i also protected permeability in an in vitro blood brain barrier (BBB) model. Further, let-7i downregulated the expression of toll-like receptor 4 (TLR4), an inflammation trigger. Moreover, overexpression of let-7i decreased matrix metallopeptidase 9 (MMP9) and inducible nitric oxide synthase (iNOS) expression under OGD. Upon silencing TLR4 expression in HBMECs, the anti-inflammatory effect of let-7i was abolished. Our research suggests that let-7i promotes OGD-induced inflammation via downregulating TLR4 expression.

Atg7 Regulates Brain Angiogenesis via NF-κB-Dependent IL-6 Production.

The formation of brain vasculature is an essential step during central nervous system development. The molecular mechanism underlying brain angiogenesis remains incompletely understood. The role of Atg7, an autophagy-related protein, in brain angiogenesis was investigated in this study. We found that the microvessel density in mice brains with endothelial-specific knockout of Atg7 (Atg7 EKO) was significantly decreased compared to wild-type control. Consistently, in vitro angiogenesis assays showed that Atg7 knockdown impaired angiogenesis in brain microvascular endothelial cells. Further results indicated that knockdown of Atg7 reduced interleukin-6 (IL-6) expression in brain microvascular endothelial cells, which is mediated by NF-κB-dependent transcriptional control. Interestingly, exogenous IL-6 restored the impaired angiogenesis and reduced cell motility caused by Atg7 knockdown. These results demonstrated that Atg7 has proangiogenic activity in brain angiogenesis which is mediated by IL-6 production in a NF-κB-dependent manner.

Effects of Fullerenols on Mouse Brain Microvascular Endothelial Cells.

Fullerenols, water-soluble C60-fullerene derivatives, have been shown to exert neuroprotective effects in vitro and in vivo, most likely due to their capability to scavenge free radicals. However, little is known about the effects of fullerenols on the blood-brain barrier (BBB), especially on cerebral endothelial cells under inflammatory conditions. Here, we investigated whether the treatment of primary mouse brain microvascular endothelial cells with fullerenols impacts basal and inflammatory blood-brain barrier (BBB) properties in vitro. While fullerenols (1, 10, and 100 µg/mL) did not change transendothelial electrical resistance under basal and inflammatory conditions, 100 µg/mL of fullerenol significantly reduced erk1/2 activation and resulted in an activation of NFκB in an inflammatory milieu. Our findings suggest that fullerenols might counteract oxidative stress via the erk1/2 and NFκB pathways, and thus are able to protect microvascular endothelial cells under inflammatory conditions.

EGFR transactivation contributes to neuroinflammation in Streptococcus suis meningitis.

BACKGROUND: Streptococcus suis serotype 2 (SS2) is an important zoonotic bacterial pathogen in both humans and animals, which can cause high morbidity and mortality. Meningitis is one of the major clinical manifestations of SS2 infection. However, the specific process of SS2 meningitis and its molecular mechanisms remain unclear. Epidermal growth factor receptor (EGFR) has been reported to initiate transduction of intracellular signals and regulate host inflammatory responses. Whether and how EGFR contributes to the development of S. suis meningitis are currently unknown. METHODS: The tyrosine phosphorylation of cellular proteins, the transactivation of EGFR, as well as its dimerization, and the associated signal transduction pathways were investigated by immunoprecipitation and western blotting. Real-time quantitative PCR was used to investigate the transcriptional level of the ErbB family members, EGFR-related ligands, cytokines, and chemokines. The secretion of cytokines and chemokines in the serum and brain were detected by Q-Plex™ Chemiluminescent ELISA. RESULTS: We found an important role of EGFR in SS2 strain SC19-induced meningitis. SC19 increasingly adhered to human brain microvascular endothelial cells (hBMEC) and caused inflammatory lesions in the brain tissues, with significant induction and secretion of proinflammatory cytokines and chemokines in the serum and brains. SC19 infection of hBMEC induced tyrosine phosphorylation of cellular EGFR in a ligand-dependent manner involving the EGF-like ligand HB-EGF, amphiregulin (AREG), and epiregulin (EREG) and led to heterodimerization of EGFR/ErbB3. The EGFR transactivation did not participate in SS2 strain SC19 adhesion of hBMEC, as well as in bacterial colonization in vivo. However, its transactivation contributed to the bacterial-induced neuroinflammation, via triggering the MAPK-ERK1/2 and NF-κB signaling pathways in hBMEC that promote the production of proinflammatory cytokines and chemokines. CONCLUSIONS: We investigated for the first time the tyrosine phosphorylation of cellular proteins in response to SS2 strain SC19 infection of hBMEC and demonstrated the contribution of EGFR to SS2-induced neuroinflammation. These observations propose a novel mechanism involving EGFR in SS2-mediated inflammatory responses in the brain, and therefore, EGFR might be an important host target for further investigation and prevention of neuroinflammation caused by SS2 strains.

Fingolimod (FTY720-P) Does Not Stabilize the Blood-Brain Barrier under Inflammatory Conditions in an in Vitro Model.

Breakdown of the blood-brain barrier (BBB) is an early hallmark of multiple sclerosis (MS), a progressive inflammatory disease of the central nervous system. Cell adhesion in the BBB is modulated by sphingosine-1-phosphate (S1P), a signaling protein, via S1P receptors (S1P1). Fingolimod phosphate (FTY720-P) a functional S1P1 antagonist has been shown to improve the relapse rate in relapsing-remitting MS by preventing the egress of lymphocytes from lymph nodes. However, its role in modulating BBB permeability-in particular, on the tight junction proteins occludin, claudin 5 and ZO-1-has not been well elucidated to date. In the present study, FTY720-P did not change the transendothelial electrical resistance in a rat brain microvascular endothelial cell (RBMEC) culture exposed to inflammatory conditions and thus did not decrease endothelial barrier permeability. In contrast, occludin was reduced in RBMEC culture after adding FTY720-P. Additionally, FTY720-P did not alter the amount of endothelial matrix metalloproteinase (MMP)-9 and MMP-2 in RBMEC cultures. Taken together, our observations support the assumption that S1P1 plays a dual role in vascular permeability, depending on its ligand. Thus, S1P1 provides a mechanistic basis for FTY720-P-associated disruption of endothelial barriers-such as the blood-retinal barrier-which might result in macular edema.