Brain Endothelial Permeability Research Articles

Ibrutinib disrupts blood-tumor barrier integrity and prolongs survival in rodent glioma model

In malignant glioma, cytotoxic drugs are often inhibited from accessing the tumor site due to the blood-tumor barrier (BTB). Ibrutinib, FDA-approved lymphoma agent, inhibits Bruton tyrosine kinase (BTK) and has previously been shown to independently impair aortic endothelial adhesion and increase rodent glioma model survival in combination with cytotoxic therapy. Yet additional research is required to understand ibrutinib’s effect on BTB function. In this study, we detail baseline BTK expression in glioma cells and its surrounding vasculature, then measure endothelial junctional expression/function changes with varied ibrutinib doses in vitro. Rat glioma cells and rodent glioma models were treated with ibrutinib alone (1–10 µM and 25 mg/kg) and in combination with doxil (10–100 µM and 3 mg/kg) to assess additive effects on viability, drug concentrations, tumor volume, endothelial junctional expression and survival. We found that ibrutinib, in a dose-dependent manner, decreased brain endothelial cell–cell adhesion over 24 h, without affecting endothelial cell viability (p < 0.005). Expression of tight junction gene and protein expression was decreased maximally 4 h after administration, along with inhibition of efflux transporter, ABCB1, activity. We demonstrated an additive effect of ibrutinib with doxil on rat glioma cells, as seen by a significant reduction in cell viability (p < 0.001) and increased CNS doxil concentration in the brain (56 ng/mL doxil alone vs. 74.6 ng/mL combination, p < 0.05). Finally, Ibrutinib, combined with doxil, prolonged median survival in rodent glioma models (27 vs. 16 days, p < 0.0001) with brain imaging showing a − 53% versus − 75% volume change with doxil alone versus combination therapy (p < 0.05). These findings indicate ibrutinib’s ability to increase brain endothelial permeability via junctional disruption and efflux inhibition, to increase BTB drug entry and prolong rodent glioma model survival. Our results motivate the need to identify other BTB modifiers, all with the intent of improving survival and reducing systemic toxicities.

Lifibrate attenuates blood-brain barrier damage following ischemic stroke via the MLCK/p-MLC/ZO-1 axis.

Dysfunction of tight junction proteins-associated damage to the blood-brain barrier (BBB) plays an important role in the pathogenesis of ischemic stroke. Lifibrate, an inhibitor of cholinephosphotransferase (CPT), has been used as an agent for serum lipid lowering. However, the protective effects of Lifibrate in ischemic stroke and the underlying mechanism have not been clearly elucidated. Here, we employed an in vivo mice model of MCAO and an OGD/R model in vitro. In the mice models, neurological deficit scores and infarct volume were assessed. Evans Blue solution was used to detect the BBB permeability. The TEER was examined to determine brain endothelial monolayer permeability. Here, we found that Lifibrate improved neurological dysfunction in stroke. Additionally, increased BBB permeability during stroke was significantly ameliorated by Lifibrate. Correspondingly, the reduced expression of the tight junction protein ZO-1 was restored by Lifibrate at both the mRNA and protein levels. Using an in vitro model, we found that Lifibrate ameliorated OGD/R-induced injury in human bEnd.3 brain microvascular endothelial cells by increasing cell viability but reducing the release of LDH. Importantly, Lifibrate suppressed the increase in endothelial monolayer permeability and the reduction in TEER induced by OGD/R via the rescue of ZO-1 expression. Mechanistically, Lifibrate blocked activation of the MLCK/ p-MLC signaling pathway in OGD/R-stimulated bEnd.3 cells. In contrast, overexpression of MLCK abolished the protective effects of Lifibrate in endothelial monolayer permeability, TEER, as well as the expression of ZO-1. Our results provide a basis for further investigation into the neuroprotective mechanism of Lifibrate during stroke.

Kv7 channel activation reduces brain endothelial cell permeability and prevents kainic acid-induced blood-brain barrier damage.

Ion channels in the blood-brain barrier (BBB) play a main role in controlling the interstitial fluid composition and cerebral blood flow, and their dysfunction contributes to the disruption of the BBB occurring in many neurological diseases such as epilepsy. In this study, using morphological and functional approaches, we evaluated the expression and role in the BBB of Kv7 channels, a family of voltage-gated potassium channels including five members (Kv7.1-5) that play a major role in the regulation of cell excitability and transmembrane flux of potassium ions. Immunofluorescence experiments showed that Kv7.1, Kv7.4, and Kv7.5 were expressed in rat brain microvessels (BMVs), as well as brain primary- and clonal (BEND-3) endothelial cells (ECs). Kv7.5 localized at the cell-to-cell junction sites, whereas Kv7.4 was also found in pericytes. The Kv7 activator retigabine increased transendothelial electrical resistance (TEER) in both primary ECs and BEND-3 cells; moreover, retigabine reduced paracellular dextran flux in BEND-3 cells. These effects were prevented by the selective Kv7 blocker XE-991. Exposure to retigabine also hyperpolarized cell membrane and increased tight junctions (TJs) integrity in BEND-3 cells. BMVs from rats treated with kainic acid (KA) showed a disruption of TJs and a selective reduction of Kv7.5 expression. In BEND-3 cells, retigabine prevented the increase of cell permeability and the reduction of TJs integrity induced by KA. Overall, these findings demonstrate that Kv7 channels are expressed in the BBB, where they modulate barrier properties both in physiological and pathological conditions.NEW & NOTEWORTHY This study describes for the first time the expression and the functional role of Kv7 potassium channels in the blood-brain barrier. We show that the opening of Kv7 channels reduces endothelial cell permeability both in physiological and pathological conditions via the hyperpolarization of cell membrane and the sealing of tight junctions. Therefore, activation of endothelial Kv7 channels might be a useful strategy to treat epilepsy and other neurological disorders characterized by blood-brain barrier dysfunction.

Abstract TP307: oxLDL Preconditioning Intensifies Ischemic-Like Injury Mediated Alterations in Human Male Brain Endothelial Cell Tight Junction Protein Levels and Proinflammatory Mediators

Introduction: Acute ischemic stroke (AIS) triggers endothelial activation and induces cerebrovascular inflammation which can result in vascular function and integrity loss leading to worsened stroke outcome. A clinically correlated risk factor for stroke shown to mediate vascular dysfunction and injury is elevated levels of oxidized low density lipoprotein (oxLDL). We hypothesized that oxLDL would exacerbate acute ischemic injury mediated alteration of endothelial proinflammatory mediator and integral barrier protein levels. Methods: Primary adult male human brain microvascular endothelial cells (HBMEC) were preconditioned with human oxLDL (50 or 100ug/dL; 6, 12, 24, or 36h) or vehicle, using a serum dose reported in AIS patients. HBMECs were then exposed to normoxia (21% O 2 ) or ischemic-like injury, hypoxia plus glucose deprivation (HGD; 1% O 2 ), for 3 or 6h. HBMEC levels or expression of barrier markers, adhesion molecules, and inflammatory mediators were examined using qRT-PCR, in cell western, and FIJI mediated immunocytochemical analysis. Secondary analysis was performed on RNA sequencing of adult male aortic endothelial cells (HAoECs) exposed to oxLDL (50ug/mL; 3, 6, 12, 24h) obtained from GEO dataset GSE13139. Results: HGD injury concomitantly decreased HBMEC claudin-5, occludin, and ZO-1 as well as increased IL-1b, ICAM-1, VCAM-1, iNOS, and TNF-a. OxLDL exacerbated an HGD mediated decrease in HBMEC barrier levels and increase in proinflammatory levels in a dose and time dependent manner. HAoEC barrier, adhesion, and proinflammatory levels were not altered by oxLDL. Conclusion: OxLDL may preferentially alter brain endothelial barrier and proinflammatory levels, as opposed to aortic endothelial cells in dose and time dependent manner. Mitigating the effect of oxLDL on brain endothelial permeability and inflammation, possibly via receptor inhibition may be a novel, viable therapeutic target in the treatment of AIS.

Abstract 12294: Poldip2 Mediates Brain Vascular Permeability by Regulating ZO-1 Phosphorylation and Localization at the Interendothelial Border

Introduction: Poldip2 is a novel regulator of cerebral vascular permeability involved in aggravating blood-brain barrier disruption following ischemic stroke; however, the underlying mechanisms are incompletely understood. Tight junctions are crucial for the formation of endothelial barriers and are composed of transmembrane proteins and adaptor proteins such as ZO-1. Cerebral ischemia and inflammation lead to ZO-1 redistribution resulting in vascular permeability. Hypothesis: We hypothesized that Poldip2 may mediate ZO-1 phosphorylation and promote brain endothelial monolayer permeability following cerebral ischemia. Methods: Cerebral ischemia was induced in endothelial-specific Poldip2 knockout and control mice. Vascular permeability was assessed by Evans blue dye extravasation. For in vitro analysis, primary rat brain microvascular endothelial cells were used to investigate the effect of Poldip2 depletion by siRNA on TNF-α-induced permeability and trans-endothelial electrical resistance (TEER). Immunocytochemistry was carried out to investigate the effects of Poldip2 depletion or overexpression on ZO-1 localization. Results: Endothelial-specific Poldip2 deletion abolished Evans blue dye extravasation after cerebral ischemia induction (p&lt;0.05). Poldip2 knockdown in vitro suppressed TNF-α-induced endothelial cell (EC) monolayer permeability (FITC-dextran and biotin-avidin assays, p&lt;0.05 and p&lt;0.001) and prevented decreases in TEER (p&lt;0.01). Poldip2 depletion prevented TNF-α-induced ZO-1 disruption at interendothelial junctions (p&lt;0.05). Poldip2 overexpression increased endothelial permeability (p&lt;0.05), decreased TEER (p&lt;0.05), reduced ZO-1 localization at cell-cell junctions (p&lt;0.0001) and enhanced reactive oxygen species (ROS) production (p&lt;0.05). Treatment with the antioxidant N-acetyl cysteine (NAC) abrogated Poldip2-induced ZO-1 disruption (p&lt;0.0001). Immunoprecipitation studies demonstrated that Poldip2 overexpression induced tyrosine phosphorylation of ZO-1 (p&lt;0.0001), an effect prevented by NAC treatment (p&lt;0.01). Conclusions: Poldip2 induces ROS-mediated tyrosine phosphorylation of the TJ protein ZO-1 and promotes EC permeability following cerebral ischemia

20 kDa isoform of connexin-43 augments spatial reorganization of the brain endothelial junctional complex and lesion leakage in cerebral cavernous malformation type-3

Cerebral cavernous malformation type-3 (CCM3) is a type of brain vascular malformation caused by mutations in programmed cell death protein-10 (PDCD10). It is characterized by early life occurrence of hemorrhagic stroke and profound blood-brain barrier defects. The pathogenic mechanisms responsible for microvascular hyperpermeability and lesion progression in CCM3 are still largely unknown. The current study examined brain endothelial barrier structural defects formed in the absence of CCM3 in vivo and in vitro that may lead to CCM3 lesion leakage. We found significant upregulation of a 20 kDa isoform of connexin 43 (GJA1–20 k) in brain endothelial cells (BEC) in both non-leaky and leaky lesions, as well as in an in vitro CCM3 knockdown model (CCM3KD-BEC). Morphological, biochemical, FRET, and FRAP analyses of CCM3KD-BEC found GJA1–20 k regulates full-length GJA1 biogenesis, prompting uncontrolled gap junction growth. Furthermore, by binding to a tight junction scaffolding protein, ZO-1, GJA1–20 k interferes with Cx43/ZO-1 interactions and gap junction/tight junction crosstalk, promoting ZO-1 dissociation from tight junction complexes and diminishing claudin-5/ZO-1 interaction. As a consequence, the tight junction complex is destabilized, allowing “replacement” of tight junctions with gap junctions leading to increased brain endothelial barrier permeability. Modifying cellular levels of GJA1–20 k rescued brain endothelial barrier integrity re-establishing the spatial organization of gap and tight junctional complexes. This study highlights generation of potential defects at the CCM3-affected brain endothelial barrier which may underlie prolonged vascular leakiness.

TRLS-03. IBRUTINIB DISRUPTS BLOOD-TUMOR BARRIER INTEGRITY TO ENHANCE CNS DRUG DELIVERY AND RODENT GLIOMA MODEL SURVIVAL

Abstract BACKGROUND In the setting of malignant glioma, cytotoxic drugs are often inhibited from accessing the tumor site due to the blood-brain-barrier (BBB). Identifying agents that transiently increase BBB permeability would advance glioma treatment. Ibrutinib, an FDA approved lymphoma treatment, has previously impaired aortic endothelial adhesion and in combination with cytotoxic therapy, increased rodent glioma model survival. In this study, we propose ibrutinib inhibits brain endothelial cell junctional expression to disrupt BBB integrity and increase chemotherapy delivery to malignant glioma cells. METHODS Using rat glioma cells, S635, we evaluated treatment effects with doxil (3mg/kg), ibrutinib (25mg/kg) and combination therapy. We measured effects with serial brain MRIs, doxil plasma and brain regional concentrations, along with 3kD dextran permeability. To evaluate the effects of ibrutinib on in vitro brain endothelial cells, we measured changes in cell-cell electrical impedance and rhodamine efflux, as a surrogate of Abcb1 transporter function, over time (0 to 8h) and at varied drug concentrations (1, 5, and 10µM ibrutinib). RESULTS Ibrutinib in combination with doxil, prolonged median survival in rodent glioma models (18 vs. 24days, p&amp;lt;0.05). MRI findings demonstrated a -14 or -53% vs -75% tumor volume change with doxil or ibrutinib alone vs. combination therapy, respectively (p&amp;lt;0.05). Comparing combination therapy vs. doxil alone, ibrutinib increased CNS concentrations of doxil in the tumor injected brain regions with statistical significance (56ng/mL vs. 74.6ng/mL, p&amp;lt;0.05). In in vitro studies, ibrutinib decreased brain endothelial cell-cell impedance maximally at 2 hours, in a dose dependently without affecting cell viability. Additionally, we observed ibrutinib dose dependently inhibit Abcb1 activity in both endothelial and glioma cells. CONCLUSION Our results suggest ibrutinib increases brain endothelial permeability by causing junctional disruption and inhibition of Abcb1, resulting in increased CNS drug entry and prolonged survival in glioma rat models.

Correlates of Zonulin and Claudin-5, markers of intestinal and brain endothelial permeability, in Parkinson's Disease: A pilot study

IntroductionIdiopathic Parkinson's Disease is a chronic, progressive, neurodegenerative disease that affects the substantia nigra pars compacta and dopaminergic neurons in the brain stem. Since zonulin and claudin-5 are involved in intestinal and brain endothelial permeability and it is hypothesized that the brain-gut axis is relevant in IPD, the aim of our study is to evaluate whether the relationship between Zonulin and Claudin-5 levels and Parkinson's Disease patients. MethodsA total of 139 patients, including 34 mild, 33 moderate, 39 severe stage patients and 33 healthy controls were included. The patients’ demographic data, age of onset, disease duration, disease type and drugs were noted. UPDRS and H&Y scores were evaluated. Prodromal stage symptoms and non-motor symptoms were noted. Zonulin and Claudin-5 levels in the serum were studied. ResultsThe mean Zonulin value was significantly higher in the IPD group compared to the control one (16.0 ± 10.5 vs. 11.1 ± 4.3; p = 0.0012). Likewise, the mean Claudin-5 value in the IPD group was again significantly higher than in the control group (8.4 ± 5.5 vs. 6.2 ± 3.4; p = 0.0003). The combined ROC curve, though, showed only modest albeit significant discriminant ability. Moreover, neither zonulin nor claudin-5 related to age, phenotype or disease duration, and in terms of non-motor symptoms there was only a significant association between zonulin and urine problems and between claudin-5 and sleep symptoms. ConclusionOur results suggest an association of these markers of intestinal and brain endothelial permeability and IPD, although these remain modest and preliminary and warrant further consideration in larger studies including prodromal cases.

TMIC-23. EXPLORING IBRUTINIB'S EFFECTS ON BLOOD-BRAIN BARRIER INTEGRITY AND GLIOMA PROGRESSION

Abstract BACKGROUND The blood-brain barrier (BBB), among malignant gliomas limits certain agents from impairing tumor growth. Thus, identifying agents that can transiently increase BBB permeability may prove useful in enhancing glioma treatment response. Previous ibrutinib studies showed it independently disrupted gut epithelial integrity and hampered glioma growth through BMX (bone marrow x-linked tyrosine kinase) inhibition. We propose BMX inhibition via ibrutinib disrupts BBB integrity while impairing glioma progression. METHODS To evaluate ibrutinib’s effect on brain endothelium, we evaluated electrical cell-cell impedance, junctional/cytoskeletal expression, downstream protein expression and functional ABC transporter activity; monitoring changes over time (0 to 8h) and at varied drug concentrations (1-10μM ibrutinib). Using rat glioma cells (S635) in vitro and in models, we examined cytotoxicity, apoptosis, model survival, and drug concentrations with ibrutinib alone or combined with poorly permeable Abcb1 substrate doxorubicin. RESULTS Ibrutinib dose-dependently decreased brain endothelial cell-cell impedance by 60% at 2h, without affecting cell viability. We observed decreased ZO-1 junctions, actin cytoskeletal rearrangement, and downstream pErk and pMek decreased expression optimally 2h (10μM ibrutinib). Dose-dependently ibrutinib inhibited Abcb1 efflux activity, further favoring a more permeable endothelium. Synergy with doxorubicin was seen in rat glioma cells treated with ibrutinib via increased apoptosis and cytotoxicity. However, while combined treatment resulted in decreased systemic doxorubicin concentrations, no differences in brain:plasma or tumor:plasma concentrations were evident. Combination therapy also did not increase rodent glioma model survival nor decrease tumor size. CONCLUSION Our results suggest that while ibrutinib induces brain endothelial permeability by junctional/cytoskeletal disruption and inhibition of Abcb1 efflux, no sustained effect can be seen in rodent glioma models in combination with a cytotoxic agent. Additional studies exploring similar agents that can enhance BBB permeability while slowing glioma growth are warranted, so as to improve upon current bleak malignant glioma treatment options.

Increased Type I interferon signaling and brain endothelial barrier dysfunction in an experimental model of Alzheimer’s disease

Blood–brain barrier (BBB) dysfunction is emerging as a key pathogenic factor in the progression of Alzheimer’s disease (AD), where increased microvascular endothelial permeability has been proposed to play an important role. However, the molecular mechanisms leading to increased brain microvascular permeability in AD are not fully understood. We studied brain endothelial permeability in female APPswe/PS1∆E9 (APP/PS1) mice which constitute a transgenic mouse model of amyloid-beta (Aβ) amyloidosis and found that permeability increases with aging in the areas showing the greatest amyloid plaque deposition. We performed an unbiased bulk RNA-sequencing analysis of brain endothelial cells (BECs) in female APP/PS1 transgenic mice. We observed that upregulation of interferon signaling gene expression pathways in BECs was among the most prominent transcriptomic signatures in the brain endothelium. Immunofluorescence analysis of isolated BECs from female APP/PS1 mice demonstrated higher levels of the Type I interferon-stimulated gene IFIT2. Immunoblotting of APP/PS1 BECs showed downregulation of the adherens junction protein VE-cadherin. Stimulation of human brain endothelial cells with interferon-β decreased the levels of the adherens junction protein VE-cadherin as well as tight junction proteins Occludin and Claudin-5 and increased barrier leakiness. Depletion of the Type I interferon receptor in human brain endothelial cells prevented interferon-β-induced VE-cadherin downregulation and restored endothelial barrier integrity. Our study suggests that Type I interferon signaling contributes to brain endothelial dysfunction in AD.

MiR-27a-3p regulates expression of intercellular junctions at the brain endothelium and controls the endothelial barrier permeability.

BackgroundThe brain endothelial barrier permeability is governed by tight and adherens junction protein complexes that restrict paracellular permeability at the blood-brain barrier (BBB). Dysfunction of the inter-endothelial junctions has been implicated in neurological disorders such as multiple sclerosis, stroke and Alzheimer’s disease. The molecular mechanisms underlying junctional dysfunction during BBB impairment remain elusive. MicroRNAs (miRNAs) have emerged as versatile regulators of the BBB function under physiological and pathological conditions, and altered levels of BBB-associated microRNAs were demonstrated in a number of brain pathologies including neurodegeneration and neuroinflammatory diseases. Among the altered micro-RNAs, miR-27a-3p was found to be downregulated in a number of neurological diseases characterized by loss of inter-endothelial junctions and disruption of the barrier integrity. However, the relationship between miR-27a-3p and tight and adherens junctions at the brain endothelium remains unexplored. Whether miR-27a-3p is involved in regulation of the junctions at the brain endothelium remains to be determined.MethodsUsing a gain-and-loss of function approach, we modulated levels of miR-27a-3p in an in-vitro model of the brain endothelium, key component of the BBB, and examined the resultant effect on the barrier paracellular permeability and on the expression of essential tight and adherens junctions. The mechanisms governing the regulation of junctional proteins by miR-27a-3p were also explored.ResultsOur results showed that miR-27a-3p inhibitor increases the barrier permeability and causes reduction of claudin-5 and occludin, two proteins highly enriched at the tight junction, while miR-27a-3p mimic reduced the paracellular leakage and increased claudin-5 and occludin protein levels. Interestingly, we found that miR-27-3p induces expression of claudin-5 and occludin by downregulating Glycogen Synthase Kinase 3 beta (GSK3ß) and activating Wnt/ß-catenin signaling, a key pathway required for the BBB maintenance.ConclusionFor the first time, we showed that miR-27a-3p is a positive regulator of key tight junction proteins, claudin-5 and occludin, at the brain endothelium through targeting GSK3ß gene and activating Wnt/ß-catenin signaling. Thus, miR-27a-3p may constitute a novel therapeutic target that could be exploited to prevent BBB dysfunction and preserves its integrity in neurological disorders characterized by impairment of the barrier’s function.

Increased activation product of complement 4 protein in plasma of individuals with schizophrenia

Structural variation in the complement 4 gene (C4) confers genetic risk for schizophrenia. The variation includes numbers of the increased C4A copy number, which predicts increased C4A mRNA expression. C4-anaphylatoxin (C4-ana) is a C4 protein fragment released upon C4 protein activation that has the potential to change the blood–brain barrier (BBB). We hypothesized that elevated plasma levels of C4-ana occur in individuals with schizophrenia (iSCZ). Blood was collected from 15 iSCZ with illness duration < 5 years and from 14 healthy controls (HC). Plasma C4-ana was measured by radioimmunoassay. Other complement activation products C3-ana, C5-ana, and terminal complement complex (TCC) were also measured. Digital-droplet PCR was used to determine C4 gene structural variation state. Recombinant C4-ana was added to primary brain endothelial cells (BEC) and permeability was measured in vitro. C4-ana concentration was elevated in plasma from iSCZ compared to HC (mean = 654 ± 16 ng/mL, 557 ± 94 respectively, p = 0.01). The patients also carried more copies of the C4AL gene and demonstrated a positive correlation between plasma C4-ana concentrations and C4A gene copy number. Furthermore, C4-ana increased the permeability of a monolayer of BEC in vitro. Our findings are consistent with a specific role for C4A protein in schizophrenia and raise the possibility that its activation product, C4-ana, increases BBB permeability. Exploratory analyses suggest the novel hypothesis that the relationship between C4-ana levels and C4A gene copy number could also be altered in iSCZ, suggesting an interaction with unknown genetic and/or environmental risk factors.

Evans blue dye as an indicator of albumin permeability across a brain endothelial cell monolayer in vitro.

An increase in the brain endothelial (BEnd) cell permeability of blood albumin is often seen as an early sign of blood-brain barrier (BBB) disruption and can precede increases in the BEnd permeability of small molecules and other plasma proteins in the course of brain disease. Therefore, Evans blue dye (EBD), an albumin-binding fluorescent tracer that is simple to detect and quantify, has been widely utilized for studying BEnd permeability during BBB disruption. Here, we investigated whether EBD is a suitable indicator of albumin permeability across mouse BEnd cell monolayers, alone or cocultured with mouse cortical astrocytes, in an in-vitro permeability assay; given the strong affinity of EBD for albumin, we further asked whether EBD can affect albumin permeability and vice versa. Albumin and EBD readily crossed membrane cell culture inserts with pore diameters of no less than 1 µm in the absence of a cellular barrier, and their permeability was substantially reduced when the membranes were overlaid with a monolayer of BEnd cells. In line with albumin binding, the BEnd permeability of EBD was substantially reduced by the presence of albumin. While EBD at an EBD-to-albumin ratio similar to those typically used in in vivo BBB experiments had little effect on the BEnd permeability of albumin, a much higher concentration of EBD augmented the BEnd permeability of albumin. In conclusion, we investigated the use of EBD as an indicator of albumin permeability in vitro, explored some of its drawbacks and further demonstrated that EBD at the concentration used in vivo does not affect albumin permeability.

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

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

Impact of Wnt/β-catenin signaling on ethanol-induced changes in brain endothelial cell permeability.

Chronic exposure to ethanol is associated with enhanced leakiness in the brain microvessel endothelial cells that form the blood-brain barrier (BBB). As previous studies suggested Wnt/β-catenin signaling could improve the BBB phenotype of brain endothelial cells, we examined the extent to which Wnt signaling is altered following ethanol exposure, using both a cell culture model of the BBB and mice exposed to ethanol, and the ability of Wnt activation to reverse the permeability effects of ethanol. The human brain endothelial cells, hCMEC/D3, were exposed to ethanol (17-200mM) for various periods of time (0-96hr) and Wnt signaling, as well as expression of downstream genes influencing BBB integrity in the cell monolayers were monitored. Determination of Wnt signaling in both brain homogenates and brain microvessels from mice exposed to ethanol was also performed. The effects of ethanol on the permeability of the hCMEC/D3 monolayers were examined using both small molecular weight (sodium fluorescein) and large molecular weight (IRdye 800CW PEG) fluorescent markers. Exposure of hCMEC/D3 to ethanol (50mM) caused a down-regulation of Wnt/β-catenin signaling, a reduction of tight junction protein expression and up-regulation of plasmalemma vesicle associated protein (PLVAP). A similar reduction in Wnt/β-catenin activity in both cortical brain homogenates and isolated cortical cerebral microvessels were observed in mice. Other areas such as cerebellum and striatum displayed as much as 3-6 fold increases in Dkk-1, an endogenous Wnt inhibitor. Ethanol exposure caused significant changes in both sodium fluorescein and IRdye 800CW PEG permeability (2-fold compared to control). The ethanol-induced increases in permeability were attenuated by treatment with known Wnt activators (i.e. LiCl or Wnt3a). Additional screens of CNS active agents with possible Wnt activity indicated fluoxetine could also prevent the permeability effects of ethanol. These studies suggest that ethanol-induced changes in brain microvessel permeability can be reversed through activation of Wnt signaling.

Paracellular permeability changes induced by multi-walled carbon nanotubes in brain endothelial cells and associated roles of hemichannels

Multi-walled carbon nanotubes (MWCNTs) have promising applications in neurology depending on their unique physicochemical properties. However, there is limited understanding of their impacts on brain microvascular endothelial cells, the cells lining the vessels and maintaining the low and selective permeability of the blood-brain barrier. In this study, we examined the influence of pristine MWCNT (p-MWCNT) and carboxylated MWCNT (c-MWCNT) on permeability and tight junction tightness of murine brain microvascular endothelial cells, and investigated the potential mechanisms in the sight of hemichannel activity. Treatment with p-MWCNT for 24 h at subtoxic concentration (20 μg/mL) decreased the protein expression of occludin, disrupted zonula occludens-1 continuity, and elevated monolayer permeability as quantified by transendothelial electrical resistance and paracellular flux of 4000 Da fluorescein isothiocyanate-dextran conjugates. Moreover, p-MWCNT exposure also increased hemichannel activity with upregulated protein expression and altered subcellular localization of connexin (Cx)43 and pannexin (Panx)1. p-MWCNT-induced elevation in endothelial permeability could be prevented by hemichannel inhibitor carbenoxolone and peptide blocker of Cx43 and Panx1, indicating the crucial role of activated Cx43 and Panx1 hemichannels. Furthermore, Cx43 and Panx1 hemichannel-mediated ATP release might be involved in p-MWCNT-induced rise in endothelial permeability. In contrast, the above effects caused by p-MWCNT were not observed in cells treated with c-MWCNT, the functionalized form with more stable dispersion and a lower tendency to aggregate. Our study contributes further understanding of the impact of MWCNTs on brain endothelial tightness and permeability, which may have important implications for the safety application of MWCNTs in nanomedicine.

Human brain microvascular endothelial cell pairs model tissue-level blood-brain barrier function.

The blood-brain barrier plays a critical role in delivering oxygen and nutrients to the brain while preventing the transport of neurotoxins. Predicting the ability of potential therapeutics and neurotoxicants to modulate brain barrier function remains a challenge due to limited spatial resolution and geometric constraints offered by existing in vitro models. Using soft lithography to control the shape of microvascular tissues, we predicted blood-brain barrier permeability states based on structural changes in human brain endothelial cells. We quantified morphological differences in nuclear, junction, and cytoskeletal proteins that influence, or indicate, barrier permeability. We established a correlation between brain endothelial cell pair structure and permeability by treating cell pairs and tissues with known cytoskeleton-modulating agents, including a Rho activator, a Rho inhibitor, and a cyclic adenosine monophosphate analog. Using this approach, we found that high-permeability cell pairs showed nuclear elongation, loss of junction proteins, and increased actin stress fiber formation, which were indicative of increased contractility. We measured traction forces generated by high- and low-permeability pairs, finding that higher stress at the intercellular junction contributes to barrier leakiness. We further tested the applicability of this platform to predict modulations in brain endothelial permeability by exposing cell pairs to engineered nanomaterials, including gold, silver-silica, and cerium oxide nanoparticles, thereby uncovering new insights into the mechanism of nanoparticle-mediated barrier disruption. Overall, we confirm the utility of this platform to assess the multiscale impact of pharmacological agents or environmental toxicants on blood-brain barrier integrity.

Oligodendrocytes upregulate blood-brain barrier function through mechanisms other than the PDGF-BB/PDGFRα pathway in the barrier-tightening effect of oligodendrocyte progenitor cells

White matter lesions are associated with impairment of the blood-brain barrier (BBB), an essential component of the cerebrovasculature. The BBB allows the brain to maintain its highly specialized microenvironment by restricting entry of blood-borne substances including molecules that induce myelin damage. Accumulating evidence suggests that interactions between brain endothelial cells and neighboring cells, including oligodendrocyte progenitor cells (OPCs), are required for the induction and maintenance of BBB function. Here, we compared the ability of OPCs and oligodendrocytes to modulate BBB integrity using co-cultures of rat brain endothelial cells with OPCs or oligodendrocytes. We found that OPCs lowered the brain endothelial permeability to sodium fluorescein, and this enhancement of BBB function was prevented by treatment with AG1296 (a PDGFRα inhibitor). Oligodendrocytes also enhanced BBB integrity. Pharmacological inhibition of PDGFRα did not affect the oligodendrocyte-induced BBB facilitation. These data indicate that oligodendrocytes enhance BBB integrity through pathways other than PDGF-BB/PDGFRα signaling triggered by the brain endothelial cell-derived PDGF-BB. Therefore, our findings suggest that oligodendrocytes constitutively support BBB integrity through soluble factors. Crosstalk between brain endothelial cells and oligodendrocytes could play a facilitatory role in maintaining BBB integrity in the white matter.

Interplay of Plasmodium falciparum and thrombin in brain endothelial barrier disruption

Recent concepts suggest that both Plasmodium falciparum factors and coagulation contribute to endothelial activation and dysfunction in pediatric cerebral malaria (CM) pathology. However, there is still limited understanding of how these complex inflammatory stimuli are integrated by brain endothelial cells. In this study, we examined how mature-stage P. falciparum infected erythrocytes (IE) interact with tumor necrosis factor α (TNFα) and thrombin in the activation and permeability of primary human brain microvascular endothelial cell (HBMEC) monolayers. Whereas trophozoite-stage P. falciparum-IE have limited effect on the viability of HBMEC or the secretion of pro-inflammatory cytokines or chemokines, except at super physiological parasite-host cell ratios, schizont-stage P. falciparum-IE induced low levels of cell death. Additionally, schizont-stage parasites were more barrier disruptive than trophozoite-stage P. falciparum-IE and prolonged thrombin-induced barrier disruption in both resting and TNFα-activated HBMEC monolayers. These results provide evidence that parasite products and thrombin may interact to increase brain endothelial permeability.

Effects of lipocalin-2 on brain endothelial adhesion and permeability.

Lipocalin-2 (LCN2) is a stress protein, and can be hyper-produced by many kinds of cells after exposure to injury or disease conditions. In this study, we asked whether LCN2 may play a protective role in cerebral endothelium. After focal cerebral ischemia in rats, plasma levels of LCN2 were significantly elevated at 6, 12, and 24 hrs, and persisted until 3 days post-stroke. To assess the vascular mechanisms of LCN2, we used brain endothelial cell cultures to investigate its effects on neutrophil adhesion and endothelial barrier integrity. LCN2 did not affect neutrophil adhesion to endothelial cells either under normal conditions or after TNFα stimulation. TNFα significantly increased endothelial permeability, and LCN2 rescued endothelial permeability. Concomitantly, LCN2 restored the membrane distribution of the tight junction protein ZO-1 and the adherens junction protein VE-cadherin. Our findings suggest that elevated LCN2 in the blood after ischemic stroke might affect endothelial function, in part by reducing damage to endothelial junctional proteins and maintain blood-brain barrier integrity.