Permeability Of Brain Microvascular Endothelial Cells Research Articles

URB597 and Andrographolide Improve Brain Microvascular Endothelial Cell Permeability and Apoptosis by Reducing Oxidative Stress and Inflammation Associated with Activation of Nrf2 Signaling in Oxygen-Glucose Deprivation.

Ischemic stroke, a cerebrovascular disease worldwide, triggers a cascade of pathophysiological events, including blood-brain barrier (BBB) breakdown. Brain microvascular endothelial cells (BMECs) play a vital role in maintaining BBB function. The injury of BMECs may worsen neurovascular dysfunction and patients' prognosis. Therefore, uncover the principal molecular mechanisms involved in BBB disruption in stroke becomes pressing. The endocannabinoid system (ECS) has been implicated in increasingly physiological functions, both in neurometabolism and cerebrovascular regulation. Modulating its activities by the fatty acid amide hydrolase (FAAH) shows anti-inflammatory characteristics. Andrographolide (AG), one Chinese herbal ingredient, has also attracted attention for its role in immunomodulatory and as a therapeutic target in BBB disorders. Recently, the FAAH inhibitor URB597 and AG have important regulatory effects on neuronal and vascular cells in ischemia. However, the effects of URB597 and AG on BMEC permeability and apoptosis in oxygen-glucose deprivation (OGD) and the underlying mechanisms remain unclear. To address these issues, cultured BMECs (bEnd.3 cells) were exposed to OGD. The cell viability, permeability, tube formation, and apoptosis were assessed following treatment with URB597, AG, and cotreatment. Mitochondrial membrane potential (MMP), reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), proinflammatory factors, tight junction (TJ) proteins, and oxidative stress-mediated Nrf2 signaling were also investigated. Results revealed that OGD broke the endothelial barrier, cell viability, MMP, and tube formation, which was reversed by URB597 and AG. OGD-induced enhancement of ROS, MDA, and apoptosis was reduced after drug interventions. URB597 and AG exhibited antioxidant/anti-inflammatory and mitochondrial protective effects by activating Nrf2 signaling. These findings indicated that URB597 and AG protect BMECs against OGD-induced endothelial permeability impairment and apoptosis by reducing mitochondrial oxidative stress and inflammation associated with activation of Nrf2 signaling. URB597 and AG showing the vascular protection may have therapeutic potential for the BBB damage in ischemic cerebrovascular diseases.

Foxo1-induced miR-92b down-regulation promotes blood-brain barrier damage after ischaemic stroke by targeting NOX4.

The blood‐brain barrier (BBB) damage is a momentous pathological process of ischaemic stroke. NADPH oxidases 4 (NOX4) boosts BBB damage after ischaemic stroke and its expression can be influenced by microRNAs. This study aimed to probe into whether miR‐92b influenced the BBB damage after ischaemic stroke by regulating NOX4 expression. Here, miR‐92b expression was lessened in the ischaemic brains of rats and oxygen‐glucose deprivation (OGD)‐induced brain microvascular endothelial cells (BMECs). In middle cerebral artery occlusion (MCAo) rats, miR‐92b overexpression relieved the ameliorated neurological function and protected the BBB integrity. In vitro model, miR‐92b overexpression raised the viability and lessened the permeability of OGD‐induced BMECs. miR‐92b targeted NOX4 and regulated the viability and permeability of OGD‐induced BMECs by negatively modulating NOX4 expression. The transcription factor Foxo1 bound to the miR‐92b promoter and restrained its expression. Foxo1 expression was induced by OGD‐induction and its knockdown abolished the effects of OGD on miR‐92b and NOX4 expressions, cell viability and permeability of BMECs. In general, our findings expounded that Foxo1‐induced lessening miR‐92b boosted BBB damage after ischaemic stroke by raising NOX4 expression.

Exosomal shuttled miR-424-5p from ischemic preconditioned microglia mediates cerebral endothelial cell injury through negatively regulation of FGF2/STAT3 pathway

Exosomes secreted by microglia have been found to play a role in neurovascular unit injury under the ischemic/hypoxic state. However, the modulatory effect of exosomes shuttled miRNAs produced by microglia in endothelial cells remains undefined. Here, an oxygen-glucose deprivation (OGD) model was constructed both in microglia and brain microvascular endothelial cells (BMEC). The exosomes secreted by microglia were isolated, and the exosomal miRNA profile was detected. Next, gain- and loss- functions of miR-424-5p, one of the most differentially expressed miRNAs in microglia derived exosomes, were conducted in BMEC. The results demonstrated that exosomes from OGD-activated microglia aggravated OGD induced BMEC viability and integrity damage as well as the loss of vascular formation. While the damaging effects were markedly attenuated by inhibiting miR-424-5p. In addition, miR-424-5p overexpression significantly aggravated OGD induced BMEC damage and permeability. Mechanistically, bioinformatics analysis indicated that miR-424-5p targeted the FGF2 mediated STAT3 signaling pathway, which was verified via dual luciferase activity assay and RIP experiment. Furthermore, in vivo experiments in the middle cerebral artery occlusion (MCAO) model mice were conducted. The results revealed that inhibition of miR-424-5p markedly reduced neurological dysfunctions and endothelial cell injury induced by MCAO. The above results confirmed that exosomes from OGD activated microglia induced significant cell damage and permeability of BMEC, in which the upregulated miR-424-5p in the exosomes functioned by regulating FGF2/STAT3 pathway.

LncRNA Snhg3 contributes to dysfunction of cerebral microvascular cells in intracerebral hemorrhage rats by activating the TWEAK/Fn14/STAT3 pathway

LncRNA small nucleolar RNA host gene 3 (Snhg3) has been involved in cell proliferation and migration in malignant cells. However, its role in regulating functions of non-malignant cells has been hardly reported. Here, we found Snhg3 expression was sharply induced in primary brain microvascular endothelial cells (BMVECs) treated with oxygen-and-glucose-deprivation (OGD) plus hemin, an in vitro model of intracerebral hemorrhage (ICH). Downregulation of Snhg3 by siRNA transfection improved cell proliferation and migration abilities and reduced cell apoptosis and monolayer permeability in BMVECs under treatment with OGD plus hemin. Snhg3 overexpression suppressed cell proliferation and migration and increased cell apoptosis and monolayer permeability under normal condition. In ICH rats, downregulation of Snhg3 by siRNA injection improved behavioral and histological manifestations, including number of right turns, limb placement score, integrity of blood-brain barrier (BBB), brain water content and cell apoptosis in vivo. In the mechanism exploration, we found that, TWEAK and Snhg3 displayed a positive correlation with each other. Snhg3 overexpression increased expression of TWEAK protein and its receptor Fn14, that were also induced by OGD plus hemin, activating the downstream neuroinflammatory pathway STAT3 and enhancing the secretion of MMP-2/9. Finally, the TWEAK-siRNA, the Fn14 inhibitor ATA and the STAT3 blocker AG490 were respectively used to treat BMVECs under treatment with OGD plus hemin. Our results showed either TWEAK downregulation, Fn14 inhibition, or STAT3 blockade, could rescue Snhg3-induced impairment of BMVEC functions. In conclusion, the lncRNA Snhg3 contributes to dysfunction of cerebral microvascular cells in ICH rats by activating the TWEAK/Fn14/STAT3 pathway.

Selective Disruption of the Blood-Brain Barrier by Zika Virus.

The blood–brain barrier (BBB) selectively regulates the cellular exchange of macromolecules between the circulation and the central nervous system (CNS). Here, we hypothesize that Zika virus (ZIKV) infects the brain via a disrupted BBB and altered expression of tight junction (TJ) proteins, which are structural components of the BBB. To assess this hypothesis, in vitro and in vivo studies were performed using three different strains of ZIKV: Honduras (ZIKV-H), Puerto Rico (ZIKV-PR), and Uganda (ZIKV-U). Primary human brain microvascular endothelial cells (BMECs) were productively infected by all studied ZIKV strains at MOI 0.01, and were analyzed by plaque assay, immunofluorescence for NS1 protein, and qRT-PCR at 2 and 6 days post-infection (dpi). Compared to mock-infected controls, expression level of ZO-1 was significantly upregulated in ZIKV-H-infected BMECs, while occludin and claudin-5 levels were significantly downregulated in BMECs infected by all three studied viral strains. Interestingly, BMEC permeability was not disturbed by ZIKV infection, even in the presence of a very high viral load (MOI 10). All studied ZIKV strains productively infected wild-type C57BL/J mice after intravenous infection with 107 PFU. Viral load was detected in the plasma, spleen, and brain from 1 to 8 dpi. Peak brain infection was observed at 2 dpi; therefore, TJ protein expression was assessed at this time point. Claudin-5 was significantly downregulated in ZIKV-U-infected animals and the BBB integrity was significantly disturbed in ZIKV-H-infected animals. Our results suggest that ZIKV penetrates the brain parenchyma early after infection with concurrent alterations of TJ protein expression and disruption of the BBB permeability in a strain-dependent manner.

Mitochondrial Mechanisms of Nelfinavir Toxicity in Human Brain Microvascular Endothelial cells

Antiretroviral therapies with combinations of mechanistically different classes of drugs resulted in reduced HIV‐associated neurocognitive disorders, however, milder forms of cognitive disorders continue to be disabling in AIDS patients. Toxicity resulting from long‐term use of antiretroviral drugs have been implicated in the cognitive disorders but the exact underlying mechanisms are poorly understood. We hypothesized that nelfinavir, a protease inhibitor, causes the disruption of blood brain barrier (BBB) by promoting mitochondrial oxidative stress and impairing mitochondrial respiration. Our studies examined the effects of nelfinavir in human brain microvascular endothelial cells (BMECs). Cell viability following 48 h Nelfinavir treatment of hBMECs was determined using CCK‐8 assay. Measurements of superoxide levels were made by electron spin resonance spectrometery using spin probes for total (1‐Hydroxy‐3‐methoxycarbonyl‐2,2,5,5‐tetramethyl‐pyrrolidine) and mitochondrial superoxide (mito‐Tempo‐H). Oxygen consumption rates (OCR) were measured by Seahorse Analyzer. Transcellular endocytosis measurements were performed by using 40 kDa‐FITC‐dextran transfer in BMECs grown in transwells. Nelfinavir treatment caused an increase in cell proliferation of BMECs at low doses (0.5 μM‐1.5 μM) but decreased viability at high doses (5 μM‐10 μM). Nelfinavir at therapeutic doses promoted the generation of total and mitochondrial superoxide in BMECs. In addition, nelfinavir dose‐dependently diminished the basal OCR and reserve mitochondrial respiratory capacity in BMECs. Moreover, nelfinavir dose‐dependently promoted increased transcellular permeability in BMECs. Thus, nelfinavir induced mitochondrial oxidative stress promotes diminished mitochondrial respiratory capacity and decreased cell proliferation leading to the disruption of BBB.Support or Funding InformationSupport: PVK: Louisiana BoRSF‐RCS (LEQSF(2014‐17)‐RD‐A‐11) and AHA Scientist Development Grant (14SDG20490359); IR: AHA Postdoctoral Fellowship Grant (15POST23040005); and DWB: NIH grants (HL‐077731and HL093554).

The influence of brain microvascular endothelial cells permeability induced by extract of LPS pretreated PMNs

Objective To investigate the mechanism of the permeability change in the blood brain barrier(BBB)at early stage of infectious brain edema which was pretreated with LPS.Methods PMNs were isolated and purified.The concentration of Glu was measured in the supernatant without cells.The rat BMECs were primarily isolated and cultured.The permeability of BMECs waB evaluated by measuring the passing rate of 125 I-BSA via γ ray counter.The expression of NMDAR1 on BMECs was evaluated.Results The concentration of Glu in the LPS preconditioning PMNs subgroup at 5 rain time point was highest.In each group conditioned for 240 min,the permeability index of Bake average of the LPS preconditioning PMNs extract group was highest(P＜0.01).The expression of NMDARI increased in the LPS preconditioning PMNs extract group was more than that in other groups.Conclusions The research showed for the first time that the concentration of Glu increased after LPS preconditioning and the Glu excreted by PMNs can promote the permeability of BMECs and change the function of BBB,which possibly may be related the mechanism of NMDAR1 expression increased in BMECs. Key words: Lipepolysaccharides/PD; Neatrophils; Capillary permeability; Endothelial cells

Extracellular proteases of Acanthamoeba castellanii (encephalitis isolate belonging to T1 genotype) contribute to increased permeability in an in vitro model of the human blood–brain barrier

Objectives Granulomatous amoebic encephalitis (GAE) is a serious human infection with fatal consequences, however, the pathogenic mechanisms associated with this disease remain unclear. Several lines of evidence suggest that haematogenous spread is a prerequisite for Acanthamoeba encephalitis but it is not clear how circulating amoebae cross the blood–brain barrier to gain entry into the central nervous system. Objectives of this study were to determine the effects of Acanthamoeba on the permeability of an in vitro blood–brain barrier model and factors contributing to these changes. Methods Using human brain microvascular endothelial cells, an in vitro blood–brain barrier model was constructed in 24-well Transwell plates. Acanthamoeba (GAE isolate belonging to T1 genotype) or its conditioned media were used to determine permeability changes. Zymography assays were performed to characterise Acanthamoeba proteases. In addition, the ability of Acanthamoeba to bind brain microvascular endothelial cells was determined using adhesion assays. Results We observed that Acanthamoeba produced an increase of more than 45% in the blood–brain barrier permeability. Acanthamoeba-conditioned media exhibited similar effects indicating Acanthamoeba-mediated blood–brain barrier permeability is contact-independent. Prior treatment of conditioned media with phenylmethyl sulfonyl fluoride (PMSF, serine protease inhibitor), abolished permeability changes indicating the role of serine proteases. Of interest, methyl α- d-mannopyranoside inhibited Acanthamoeba binding to human brain microvascular endothelial cells but had no effect on Acanthamoeba-mediated blood–brain barrier permeability. Zymography assays revealed that Acanthamoeba produced two major proteases, one of which was inhibited by PMSF (serine protease inhibitor) and the second with 1,10-phenanthroline (metalloprotease inhibitor). Conclusions We have for the first time shown that Acanthamoeba produces human brain microvascular endothelial cells permeability, which can be blocked by PMSF. A metalloprotease of approx. molecular weight of 150 kDa is produced by A. castellanii (GAE isolate belonging to T1 genotype) and its role in the disease is suggested.

Secretion of interleukin-1beta by astrocytes mediates endothelin-1 and tumour necrosis factor-alpha effects on human brain microvascular endothelial cell permeability.

Evidence suggests that endothelin-1 (ET-1) plays an essential role in brain inflammation. However, whether ET-1 contributes directly to blood-brain barrier (BBB) breakdown remains to be elucidated. Using an in vitro BBB model consisting of co-cultures of human primary astrocytes and brain microvascular endothelial cells (BMVECs), we first investigated the expression of ET-1 by BMVECs upon stimulation with tumour necrosis factor (TNF)-alpha, which plays an essential role in the induction and synthesis of ET-1 during systemic inflammatory responses. Increased ET-1 mRNA was detected in the human BMVECs 24 h after TNF-alpha treatment. This was correlated with an increase in ET-1 levels in the culture medium, as determined by sandwich immunoassay. Both TNF-alpha and ET-1 increased the permeability of human BMVECs to a paracellular tracer, sucrose, but only in the presence of astrocytes. The increase in BMVEC permeability by TNF-alpha was partially prevented by antibody neutralization of ET-1 and completely by monoclonal antibody against IL-1beta. Concomitantly, TNF-alpha induced IL-1beta mRNA expression by astrocytes in co-culture and this effect was partially prevented by ET-1 antibody neutralization. In parallel experiments, treatment of human primary astrocytes in single cultures with ET-1 for 24 h induced IL-1beta mRNA synthesis and IL-1beta protein secretion in the cell culture supernatant. Taken together, these results provide evidence for paracrine actions involving ET-1, TNF-alpha and IL-1beta between human astrocytes and BMVECs, which may play a central role in BBB breakdown during CNS inflammation.