Brain Endothelial Monolayers Research Articles

Reversible Ca2+ signaling and enhanced paracellular transport in endothelial monolayer induced by acoustic bubbles and targeted microbeads

Ultrasound and microbubble mediated blood brain barrier opening is a non-invasive and effective technique for drug delivery to targeted brain region. However, the exact mechanisms are not fully resolved. The influences of Ca2+ signaling on sonoporation and endothelial tight junctional regulation affect the efficiency and biosafety of the technique. Therefore, an improved understanding of how ultrasound evokes Ca2+ signaling in the brain endothelial monolayer, and its correlation to endothelial permeability change is necessary. Here, we examined the effects of SonoVue microbubbles or integrin-targeted microbeads on ultrasound induced bioeffects in brain microvascular endothelial monolayer using an acoustically-coupled microscopy system, where focused ultrasound exposure and real-time recording of Ca2+ signaling and membrane perforation were performed. Microbubbles induced robust Ca2+ responses, often accompanied by cell poration, while ultrasound with microbeads elicited reversible Ca2+ response without membrane poration. At the conditions evoking reversible Ca2+ signaling, intracellular Ca2+ release and reactive oxygen species played key roles for microbubbles induced Ca2+ signaling while activation of mechanosensitive ion channels was essential for the case of microbeads. Trans-well diffusion analysis revealed significantly higher trans-endothelial transport of 70 kDa FITC-dextran for both integrin-targeted microbeads and microbubbles compared to the control group. Further immunofluorescence staining showed disruption of cell junctions with microbubble stimulation and reversible remodeling of many cell junctions by ultrasound with integrin-targeted microbeads. This investigation provides new insights for ultrasound induced Ca2+ signaling and its influence on endothelial permeability, which may help develop new strategies for safe and efficient drug/gene delivery in the vascular system.

APPLICATION OF IN VITRO MODELS: INCORPORATING BLOOD BRAIN BARRIER MODELS WITH GLIOMA, AND THE USE OF MICROflUIDICS

Abstract AIMS The blood brain barrier (BBB) presents one of the main obstacles for the development of novel glioblastoma treatments. The various transporters and enzymes within the BBB reduce the disposition of novel therapies into the brain. There is a need for an in vitro BBB-glioma model with proven efficacy of predicting CNS delivery at earlier stages in the drug discovery pipeline. METHOD A BBB-glioma lab-on-a-chip model could enable rapid identification of novel therapies that can cross the BBB at earlier stages of the drug development process, before going to clinical trials. The application of microfluidic technology allows for the detection of soluble tissue-derived factors and the assessment of the levels of soluble markers released from malignant tissue. Adding ‘flow’ to the model, is more reflective of the dynamic flow present at the BBB. RESULTS Validation studies of the current BBB model and converting this model over to a lab-on-a-chip model have shown the presence of an endothelial cell barrier and the tight junction proteins Zo-1 and Claudin-1, through western blotting, immunocytochemistry, and permeability experiments using varying molecular weights of FitC-dextran. These validation studies have been completed using brain endothelial monolayers on polycarbonate transwell inserts, with the immortalised cell line HCMEC/d3, and the short-term culture cells HBMEC. Also, validation studies using a co-culture of endothelial cells, astrocytes and pericytes have begun, using the same techniques to show a barrier has formed, on both the current BBB model and the lab-on-a-chip model. CONCLUSION The aim of this study is to combine the use of an all-human BBB-glioma model with lab-on-a-chip technology to produce a high throughput model to test novel therapies at an earlier stage. The lab-on-a-chip model will be validated for the purpose of measuring permeability and drug response and to investigate the expression of novel targets verses expression in conventional models.

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.

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<0.05). Poldip2 knockdown in vitro suppressed TNF-α-induced endothelial cell (EC) monolayer permeability (FITC-dextran and biotin-avidin assays, p<0.05 and p<0.001) and prevented decreases in TEER (p<0.01). Poldip2 depletion prevented TNF-α-induced ZO-1 disruption at interendothelial junctions (p<0.05). Poldip2 overexpression increased endothelial permeability (p<0.05), decreased TEER (p<0.05), reduced ZO-1 localization at cell-cell junctions (p<0.0001) and enhanced reactive oxygen species (ROS) production (p<0.05). Treatment with the antioxidant N-acetyl cysteine (NAC) abrogated Poldip2-induced ZO-1 disruption (p<0.0001). Immunoprecipitation studies demonstrated that Poldip2 overexpression induced tyrosine phosphorylation of ZO-1 (p<0.0001), an effect prevented by NAC treatment (p<0.01). Conclusions: Poldip2 induces ROS-mediated tyrosine phosphorylation of the TJ protein ZO-1 and promotes EC permeability following cerebral ischemia

β1 integrin is essential for blood–brain barrier integrity under stable and vascular remodelling conditions; effects differ with age

BackgroundMaintaining a tight blood–brain barrier (BBB) is an important prerequisite for the preservation of neurological health, though current evidence suggests it declines with age. While extracellular matrix-integrin interactions play critical roles in regulating the balance between vascular stability and remodeling, it remains to be established whether manipulation of integrin function weakens or strengthens vascular integrity. Indeed, recent reports have generated conflicting outcomes in this regard.MethodsHere, in young (8–10 weeks) and aged (20 months) mice, we examined the impact of intraperitoneal injection of a function-blocking β1 integrin antibody, both under normoxic conditions, when the BBB is stable, and during chronic mild hypoxic (CMH; 8% O2) conditions, when a vigorous vascular remodeling response is ongoing. Brain tissue was examined by immunofluorescence (IF) for markers of vascular remodeling and BBB disruption, and microglial activation and proliferation. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-hoc test.ResultsIn both young and aged mice, β1 integrin block greatly amplified hypoxia-induced vascular disruption, though it was much less under normoxic conditions. Interestingly, under both normoxic and hypoxic conditions, β1 integrin antibody-induced BBB disruption was greater in young mice. Enhanced BBB breakdown was associated with increased levels of the leaky BBB marker MECA-32 and with greater loss of endothelial tight junction proteins and the adherens protein VE-cadherin. Surprisingly, β1 integrin blockade did not reduce hypoxia-induced endothelial proliferation, nor did it prevent the hypoxia-associated increase in vascularity. Commensurate with the increased vascular disruption, β1 integrin blockade enhanced microglial activation both in young and aged brain, though the impact was much greater in young brain. In vitro studies revealed that β1 integrin blockade also reduced the integrity of a brain endothelial monolayer and triggered disruptions in tight junction proteins.ConclusionsThese data demonstrate that β1 integrin plays an essential role in maintaining BBB integrity, both under stable normoxic conditions and during hypoxia-induced vascular remodeling. As β1 integrin blockade had a greater disruptive effect in young brain, effectively shifting the BBB phenotype of young brain towards that of the aged, we speculate that enhancing β1 integrin function at the aged BBB may hold therapeutic potential by reverting the deteriorating BBB phenotype back towards that of the young.

Enhanced brain delivery of hypoxia-sensitive liposomes by hydroxyurea for rescue therapy of hyperacute ischemic stroke.

Ischemic stroke is characterized by high morbidity, disability, and mortality. Unfortunately, the only FDA-approved pharmacological thrombolytic, alteplase, has a narrow therapeutic window of only 4.5 h. Other drugs like neuroprotective agents have not been clinically used because of their low efficacy. To improve the efficacy of neuroprotective agents and the effectiveness of rescue therapies for hyperacute ischemic stroke, we investigated and verified the variation trends of the blood-brain barrier (BBB) permeability and regional cerebral blood flow over 24 h in rats that had ischemic strokes. Hypoperfusion and the biphasic increase of BBB permeability are still the main limiting factors for lesion-specific drug distribution and drugbrain penetration. Herein, the nitric oxide donor hydroxyurea (HYD) was reported to downregulate the expression of tight junction proteins and upregulate intracellular nitric oxide content in the brain microvascular endothelial cells subjected to oxygen-glucose deprivation, which was shown to facilitate the transport of liposomes across brain endothelialmonolayer in an in vitro model. HYD also increased the BBB permeability and promoted microcirculation in the hyperacute phase of stroke. The neutrophil-like cell-membrane-fusogenic hypoxia-sensitive liposomes exhibited excellent performance in targeting the inflamed brain microvascular endothelial cells, enhancing cell association, and promoting rapid hypoxic-responsive release in the hypoxic microenvironment. Overall, the combined HYD and hypoxia-sensitive liposome dosing regimen effectively decreased the cerebral infarction volume and relieved neurological dysfunction in rats that had ischemic strokes; these therapies were involved in the anti-oxidative stress effect and the neurotrophic effect mediated by macrophage migration inhibitory factor.

Human T-bet+ B cell development is associated with BTK activity and suppressed by evobrutinib.

Recent clinical trials have shown promising results for the next-generation Bruton’s tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of multiple sclerosis (MS). BTK has a central role in signaling pathways that govern the development of B cells. Whether and how BTK activity shapes B cells as key drivers of MS is currently unclear. Compared with levels of BTK protein, we found higher levels of phospho-BTK in ex vivo blood memory B cells from patients with relapsing-remitting MS and secondary progressive MS compared with controls. In these MS groups, BTK activity was induced to a lesser extent after anti-IgM stimulation. BTK positively correlated with CXCR3 expression, both of which were increased in blood B cells from clinical responders to natalizumab (anti–VLA-4 antibody) treatment. Under in vitro T follicular helper–like conditions, BTK phosphorylation was enhanced by T-bet–inducing stimuli, IFN-γ and CpG-ODN, while the expression of T-bet and T-bet–associated molecules CXCR3, CD21, and CD11c was affected by evobrutinib. Furthermore, evobrutinib interfered with in vitro class switching, as well as memory recall responses, and disturbed CXCL10-mediated migration of CXCR3+ switched B cells through human brain endothelial monolayers. These findings demonstrate a functional link between BTK activity and disease-relevant B cells and offer valuable insights into how next-generation BTK inhibitors could modulate the clinical course of patients with MS.

Toxoplasma effector-induced ICAM-1 expression by infected dendritic cells potentiates transmigration across polarised endothelium.

The obligate intracellular parasite Toxoplasma gondii makes use of infected leukocytes for systemic dissemination. Yet, how infection impacts the processes of leukocyte diapedesis has remained unresolved. Here, we addressed the effects of T. gondii infection on the trans-endothelial migration (TEM) of dendritic cells (DCs) across polarised brain endothelial monolayers. We report that upregulated expression of leukocyte ICAM-1 is a feature of the enhanced TEM of parasitised DCs. The secreted parasite effector GRA15 induced an elevated expression of ICAM-1 in infected DCs that was associated with enhanced cell adhesion and TEM. Consequently, gene silencing of Icam-1 in primary DCs or deletion of parasite GRA15 reduced TEM. Further, the parasite effector TgWIP, which impacts the regulation of host actin dynamics, facilitated TEM across polarised endothelium. The data highlight that the concerted action of the secreted effectors GRA15 and TgWIP modulate the leukocyte-endothelial interactions of TEM in a parasite genotype-related fashion to promote dissemination. In addition to the canonical roles of endothelial ICAM-1, this study identifies a previously unappreciated role for leukocyte ICAM-1 in infection-related TEM.

Regulation and Release of Vasoactive Endoglin by Brain Endothelium in Response to Hypoxia/Reoxygenation in Stroke.

In large vessel occlusion stroke, recanalization to restore cerebral perfusion is essential but not necessarily sufficient for a favorable outcome. Paradoxically, in some patients, reperfusion carries the risk of increased tissue damage and cerebral hemorrhage. Experimental and clinical data suggest that endothelial cells, representing the interface for detrimental platelet and leukocyte responses, likely play a crucial role in the phenomenon referred to as ischemia/reperfusion (I/R)-injury, but the mechanisms are unknown. We aimed to determine the role of endoglin in cerebral I/R-injury; endoglin is a membrane-bound protein abundantly expressed by endothelial cells that has previously been shown to be involved in the maintenance of vascular homeostasis. We investigated the expression of membranous endoglin (using Western blotting and RT-PCR) and the generation of soluble endoglin (using an enzyme-linked immunosorbent assay of cell culture supernatants) after hypoxia and subsequent reoxygenation in human non-immortalized brain endothelial cells. To validate these in vitro data, we additionally examined endoglin expression in an intraluminal monofilament model of permanent and transient middle cerebral artery occlusion in mice. Subsequently, the effects of recombinant human soluble endoglin were assessed by label-free impedance-based measurement of endothelial monolayer integrity (using the xCELLigence DP system) and immunocytochemistry. Endoglin expression is highly inducible by hypoxia in human brain endothelial monolayers in vitro, and subsequent reoxygenation induced its shedding. These findings were corroborated in mice during MCAO; an upregulation of endoglin was displayed in the infarcted hemispheres under occlusion, whereas endoglin expression was significantly diminished after transient MCAO, which is indicative of shedding. Of note is the finding that soluble endoglin induced an inflammatory phenotype in endothelial monolayers. The treatment of HBMEC with endoglin resulted in a decrease in transendothelial resistance and the downregulation of VE-cadherin. Our data establish a novel mechanism in which hypoxia triggers the initial endothelial upregulation of endoglin and subsequent reoxygenation triggers its release as a vasoactive mediator that, when rinsed into adjacent vascular beds after recanalization, can contribute to cerebral reperfusion injury.

Hypoxia increases expression of selected blood\u2013brain barrier transporters GLUT-1, P-gp, SLC7A5 and TFRC, while maintaining barrier integrity, in brain capillary endothelial monolayers

BackgroundBrain capillary endothelial cells (BCECs) experience hypoxic conditions during early brain development. The newly formed capillaries are tight and functional before astrocytes and pericytes join the capillaries and establish the neurovascular unit. Brain endothelial cell phenotype markers P-gp (ABCB1), LAT-1(SLC7A5), GLUT-1(SLC2A1), and TFR(TFRC) have all been described to be hypoxia sensitive. Therefore, we hypothesized that monolayers of BCECs, cultured under hypoxic conditions, would show an increase in LAT-1, GLUT-1 and TFR expression and display tight endothelial barriers.Methods and resultsPrimary bovine BCECs were cultured under normoxic and hypoxic conditions. Chronic hypoxia induced HIF-1α stabilization and translocation to the nucleus, as judged by immunocytochemistry and confocal laser scanning imaging. Endothelial cell morphology, claudin-5 and ZO-1 localization and barrier integrity were unaffected by hypoxia, indicating that the tight junctions in the BBB model were not compromised. SLC7A5, SLC2A1, and TFRC-mRNA levels were increased in hypoxic cultures, while ABCB1 remained unchanged as shown by real-time qPCR. P-gp, TfR and GLUT-1 were found to be significantly increased at protein levels. An increase in uptake of [3H]-glucose was demonstrated, while a non-significant increase in the efflux ratio of the P-gp substrate [3H]-digoxin was observed in hypoxic cells. No changes were observed in functional LAT-1 as judged by uptake studies of [3H]-leucine. Stabilization of HIF-1α under normoxic conditions with desferrioxamine (DFO) mimicked the effects of hypoxia on endothelial cells. Furthermore, low concentrations of DFO caused an increase in transendothelial electrical resistance (TEER), suggesting that a slight activation of the HIF-1α system may actually increase brain endothelial monolayer tightness. Moreover, exposure of confluent monolayers to hypoxia resulted in markedly increase in TEER after 24 and 48 h, which corresponded to a higher transcript level of CLDN5.ConclusionsOur findings collectively suggest that hypoxic conditions increase some BBB transporters' expression via HIF-1α stabilization, without compromising monolayer integrity. This may in part explain why brain capillaries show early maturation, in terms of barrier tightness and protein expression, during embryogenesis, and provides a novel methodological tool for optimal brain endothelial culture.

Abstract 11953: Hemorrhagic Insults And Secondary Takotsubo Syndrome: A New In-vitro Model

Introduction: Hemorrhagic insults result in very devastating forms of cerebral damage. The effects on the blood-brain barrier are so far examined only ex vivo and show reduced integrity. Hypothesis: There is a connection between hemorrhagic bleeding and secondary takotsubo syndrome (TTS), leading to a poor outcome in comorbidity. Our hypothesis is that through the elevated catecholamine levels and proinflammatory cytokine leves demonstrated to be present in the serum of TTS patients, BBB integrity will be impaired by TTS blood exposure more pronounced and/or early than by control group blood exposure (potentially fueling cardiac arrhythmia/ TTS at the brain-heat axis). Methods: Our study aims to establish an in-vitro cell culture model of blood exposure from heathy and TTS patients and measure the time-dependent effect on integriy of brain capillary cell monolayers as a basis to further examine the connection between hemorrhagic insults and TTS. We perfomed blood and serum exposure from healthy and TTS subjects to murine brain microvascular endothelial cells (cerebEND) derived from the cerebellum as an established model of the blood brain barrier and evaluated effects on barrier components by PCR and western blot. Results: Decreased monolayer integrity was readily detectable, reduced levels of Claudin-5 and Occludin were confirmed by RT-qPCR and Westernblot. Both tight-junction proteins show a time-dependent reduction. Conclusions: the development of the model seems successful, as ex-vivo studies previously showed matching results. Exposure of monolayer to TTS patient blood clearly showed a faster and more pronounced disintegration of brain endothelial monolayer.

Insights Into the Mechanisms of Brain Endothelial Erythrophagocytosis.

The endothelial cells which form the inner cellular lining of the vasculature can act as non-professional phagocytes to ingest and remove emboli and aged/injured red blood cells (RBCs) from circulation. We previously demonstrated an erythrophagocytic phenotype of the brain endothelium for oxidatively stressed RBCs with subsequent migration of iron-rich RBCs and RBC degradation products across the brain endothelium in vivo and in vitro, in the absence of brain endothelium disruption. However, the mechanisms contributing to brain endothelial erythrophagocytosis are not well defined, and herein we elucidate the cellular mechanisms underlying brain endothelial erythrophagocytosis. Murine brain microvascular endothelial cells (bEnd.3 cells) were incubated with tert-butyl hydroperoxide (tBHP, oxidative stressor to induce RBC aging in vitro)- or PBS (control)-treated mouse RBCs. tBHP increased the reactive oxygen species (ROS) formation and phosphatidylserine exposure in RBCs, which were associated with robust brain endothelial erythrophagocytosis. TNFα treatment potentiated the brain endothelial erythrophagocytosis of tBHP-RBCs in vitro. Brain endothelial erythrophagocytosis was significantly reduced by RBC phosphatidylserine cloaking with annexin-V and with RBC-ROS and phosphatidylserine reduction with vitamin C. Brain endothelial erythrophagocytosis did not alter the bEnd.3 viability, and tBHP-RBCs were localized with early and late endosomes. Brain endothelial erythrophagocytosis increased the bEnd.3 total iron pool, abluminal iron levels without causing brain endothelial monolayer disruption, and ferroportin levels. In vivo, intravenous tBHP-RBC injection in aged (17–18 months old) male C57BL/6 mice significantly increased the Prussian blue-positive iron-rich lesion load compared with PBS-RBC-injected mice. In conclusion, RBC phosphatidylserine exposure and ROS are key mediators of brain endothelial erythrophagocytosis, a process which is associated with increased abluminal iron in vitro. tBHP-RBCs result in Prussian blue-positive iron-rich lesions in vivo. Brain endothelial erythrophagocytosis may provide a new route for RBC/RBC degradation product entry into the brain to produce iron-rich cerebral microhemorrhage-like lesions.

Integrin-dependent migratory switches regulate the translocation of Toxoplasma-infected dendritic cells across brain endothelial monolayers

Multiple cellular processes, such as immune responses and cancer cell metastasis, crucially depend on interconvertible migration modes. However, knowledge is scarce on how infectious agents impact the processes of cell adhesion and migration at restrictive biological barriers. In extracellular matrix, dendritic cells (DCs) infected by the obligate intracellular protozoan Toxoplasma gondii undergo mesenchymal-to-amoeboid transition (MAT) for rapid integrin-independent migration. Here, in a cellular model of the blood–brain barrier, we report that parasitised DCs adhere to polarised endothelium and shift to integrin-dependent motility, accompanied by elevated transendothelial migration (TEM). Upon contact with endothelium, parasitised DCs dramatically reduced velocities and adhered under both static and shear stress conditions, thereby obliterating the infection-induced amoeboid motility displayed in collagen matrix. The motility of adherent parasitised DCs on endothelial monolayers was restored by blockade of β1 and β2 integrins or ICAM-1, which conversely reduced motility on collagen-coated surfaces. Moreover, parasitised DCs exhibited enhanced translocation across highly polarised primary murine brain endothelial cell monolayers. Blockade of β1, β2 integrins, ICAM-1 and PECAM-1 reduced TEM frequencies. Finally, gene silencing of the pan-integrin-cytoskeleton linker talin (Tln1) or of β1 integrin (Itgb1) in primary DCs resulted in increased motility on endothelium and decreased TEM. Adding to the paradigms of leukocyte diapedesis, the findings provide novel insights in how an intracellular pathogen impacts the migratory plasticity of leukocytes in response to the cellular environment, to promote infection-related dissemination.

Comparison of Leading Biosensor Technologies to Detect Changes in Human Endothelial Barrier Properties in Response to Pro-Inflammatory TNFα and IL1β in Real-Time.

Electric Cell-Substrate Impedance Sensing (ECIS), xCELLigence and cellZscope are commercially available instruments that measure the impedance of cellular monolayers. Despite widespread use of these systems individually, direct comparisons between these platforms have not been published. To compare these instruments, the responses of human brain endothelial monolayers to TNFα and IL1β were measured on all three platforms simultaneously. All instruments detected transient changes in impedance in response to the cytokines, although the response magnitude varied, with ECIS being the most sensitive. ECIS and cellZscope were also able to attribute responses to particular endothelial barrier components by modelling the multifrequency impedance data acquired by these instruments; in contrast the limited frequency xCELLigence data cannot be modelled. Consistent with its superior impedance sensing, ECIS exhibited a greater capacity than cellZscope to distinguish between subtle changes in modelled endothelial monolayer properties. The reduced resolving ability of the cellZscope platform may be due to its electrode configuration, which is necessary to allow access to the basolateral compartment, an important advantage of this instrument. Collectively, this work demonstrates that instruments must be carefully selected to ensure they are appropriate for the experimental questions being asked when assessing endothelial barrier properties.

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

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

EXTH-19. IBRUTINIB MODULATES BLOOD-BRAIN BARRIER INTEGRITY TO ENHANCE CHEMOTHERAPY PERMEABILITY

Abstract BACKGROUND The blood-brain barrier (BBB) poses a major treatment challenge for malignant brain tumors. Ibrutinib is an FDA approved Bruton’s tyrosine kinase (BTK) and bone marrow x-linked tyrosine kinase (BMX) inhibitor indicated for the treatment of B-cell lymphomas. BMX is highly expressed in endothelial cells and is suggested to regulate cytoskeletal organization and barrier function in epithelial cells. We hypothesize that inhibition of BMX by ibrutinib in brain endothelial cells will disrupt the integrity of the BBB to increase drug permeability. METHODS Protein expression and phosphorylation of BMX was confirmed in immortalized mouse brain endothelial cells (bEnd.3). Endothelial barrier integrity was evaluated with increasing doses of ibrutinib (1–30μM) for 24hrs via the ACEA xCELLigence system (impedance) and the ECIS TEER24 system (resistance). Paracellular permeability of doxorubicin was measured by fluorescent spectrophotometry. Junctional protein expression of VE-Cadherin and F-actin cytoskeletal organization were detected by fluorescent staining. Ibrutinib’s effect on efflux pumps, ABCB1 and ABCG2, was performed by flow cytometry using specific substrates, valspodar and FTC, respectively. RESULTS Ibrutinib decreased endothelial cell impedance in a dose-dependent manner over 24hrs, with transient decrease by 46% at 30min with an optimal dose of 1μM, and partial recovery by 4hrs. TEER was decreased by 60% at 3hrs. Ibrutinib enhanced doxorubicin permeability by 30%; attributed to the transient disorganized nature of actin cytoskeletal filaments 2hrs post treatment. No effects on VE-cadherin expression or localization were observed. Ibrutinib successfully inhibited activity of ABCB1 and ABCG2 in a dose-dependent manner. CONCLUSION Our results suggest ibrutinib causes transient disruption of the brain endothelial monolayer due to cytoskeletal disorganization and ABC transporter inhibition to enhance permeability of doxorubicin. Further studies using ibrutinib and cytotoxic agents are warranted in glioma bearing animals. We propose that using ibrutinib as an adjunctive therapy may enhance drug delivery to patients with malignant brain tumors.

Transient Photoinactivation of Cell Membrane Protein Activity without Genetic Modification by Molecular Hyperthermia.

Precise manipulation of protein activity in living systems has broad applications in biomedical sciences. However, it is challenging to use light to manipulate protein activity in living systems without genetic modification. Here, we report a technique to optically switch off protein activity in living cells with high spatiotemporal resolution, referred to as molecular hyperthermia (MH). MH is based on the nanoscale-confined heating of plasmonic gold nanoparticles by short laser pulses to unfold and photoinactivate targeted proteins of interest. First, we show that protease-activated receptor 2 (PAR2), a G-protein-coupled receptor and an important pathway that leads to pain sensitization, can be photoinactivated in situ by MH without compromising cell proliferation. PAR2 activity can be switched off in laser-targeted cells without affecting surrounding cells. Furthermore, we demonstrate the molecular specificity of MH by inactivating PAR2 while leaving other receptors intact. Second, we demonstrate that the photoinactivation of a tight junction protein in brain endothelial monolayers leads to a reversible blood-brain barrier opening in vitro. Lastly, the protein inactivation by MH is below the nanobubble generation threshold and thus is predominantly due to the nanoscale heating. MH is distinct from traditional hyperthermia (that induces global tissue heating) in both its time and length scales: nanoseconds versus seconds, nanometers versus millimeters. Our results demonstrate that MH enables selective and remote manipulation of protein activity and cellular behavior without genetic modification.

Enterovirus A71 capsid protein VP1 increases blood\u2013brain barrier permeability and virus receptor vimentin on the brain endothelial cells

Enterovirus A71 (EV-A71) is the major cause of severe hand-foot-and-mouth diseases (HFMD), especially encephalitis and other nervous system diseases. EV-A71 capsid protein VP1 mediates virus attachment and is the important virulence factor in the EV-A71pathogenesis. In this study, we explored the roles of VP1 in the permeability of blood–brain barrier (BBB). Sera albumin, Evans blue, and dextran leaked into brain parenchyma of the 1-week-old C57BL/6J mice intracranially injected with VP1 recombinant protein. VP1 also increased the permeability of the brain endothelial cells monolayer, an in vitro BBB model. Tight junction protein claudin-5 was reduced in the brain tissues or brain endothelial cells treated with VP1. In contrast, VP1 increased the expression of virus receptor vimentin, which could be blocked with VP1 neutralization antibody. Vimentin expression in the VP1-treated brain endothelial cells was regulated by TGF-β/Smad-3 and NF-κB signal pathways. Moreover, vimentin over-expression was accompanied with compromised BBB. From these studies, we conclude that EV-A71 virus capsid protein VP1 disrupted BBB and increased virus receptor vimentin, which both may contribute to the virus entrance into brain and EV-A71 CNS infection.

Brain Endothelial Cells Release Apical and Basolateral Microparticles in Response to Inflammatory Cytokine Stimulation: Relevance to Neuroinflammatory Stress?

Microparticles (MP) are regarded both as biomarkers and mediators of many forms of pathology, including neurovascular inflammation. Here, we characterized vectorial release of apical and basolateral MPs (AMPs and BMPs) from control and TNF-α/IFN-γ treated human brain endothelial monolayers, studied molecular composition of AMPs and BMPs and characterized molecular pathways regulating AMP and BMP release. The effects of AMPs and BMPs on blood-brain barrier properties and human brain microvascular smooth muscle tonic contractility in vitro were also evaluated. We report that human brain microvascular endothelial cells release MPs both apically and basolaterally with both AMP and BMP release significantly increased following inflammatory cytokine challenge (3.5-fold and 3.9-fold vs. control, respectively). AMPs and BMPs both carry proteins derived from parent cells including those in BBB junctions (Claudin−1, −3, −5, occludin, VE-cadherin). AMPs and BMPs represent distinct populations whose release appears to be regulated by distinctly separate molecular pathways, which depend on signaling from Rho-associated, coiled-coil containing protein kinase (ROCK), calpain as well as cholesterol depletion. AMPs and BMPs modulate functions of neighboring cells including BBB endothelial solute permeability and brain vascular smooth muscle contractility. While control AMPs enhanced brain endothelial barrier, cytokine-induced AMPs impaired BBB. Cytokine-induced but not control BMPs significantly impaired human brain smooth muscle contractility as early as day 1. Taken together these results indicate that AMPs and BMPs may contribute to neurovascular inflammatory disease progression both within the circulation (AMP) and in the brain parenchyma (BMP).

Human Placental Stem Cell Therapy in Stroke: Endothelial / Smooth Muscle Mechanisms Underlying Protection?

Following thrombolytic therapy for stroke, ischemia/reperfusion (I/R) mediated inflammation often disrupts the blood brain barrier (BBB). This can enhance expression of endothelial adhesion markers and perturb normal blood flow regulation. Proposed benefits of stem cell therapy (SCT) in stroke include secretion of protective and trophic factors which limit acute brain I/R injury. We evaluated human placenta mesenchymal stem cells (hPMSC) as potential ameliorative SCT in an acute ischemic stroke model. We hypothesized that potential benefits of hPMSCs, in acute stroke injury, reflect site‐specific suppression of post‐ischemic immune cell adhesion, transvascular leukocyte emigration and preservation of the BBB. This can limit the progression of post –ischemic stroke injury. We found that intraperitoneal (IP) administration of hPMSCs at the time of reperfusion, using the transient middle cerebral artery occlusion (MCAO)/reperfusion model, produced highly significant protection of the ipsilateral hemisphere. We then examined hPMSC support of BBB integrity via mobilization of junctional proteins in human brain endothelial cells (hCMEC‐D3) in conjunction with a biotin/FITC‐avidin permeability assay coupled with western blotting and immunolocalization of junctional proteins. We also assessed the ability of hPMSC to suppress inflammation‐driven hCMEC‐D3 binding of macrophages (RAW264.7) and transmigration of PMNs. In addition, we used collagen gels to study the influence of hPMSC on human brain vascular smooth muscle cells (hBVSMC) tonic contractility.We observed that hPMSC administration significantly (p<0.001) enhanced the barrier integrity of brain endothelial monolayers, at 24h, in an oxygen/glucose deprived/reperfused (OGDR) condition. We found that hCMEC‐D3, which were independently co‐cultured with hPMSCs, expressed significantly higher levels of VE‐cadherin and α‐catenin under OGDR conditions (p<0.001). Additionally, the spatial localization of VE‐cadherin and α‐catenin at hCMEC‐D3 junctions was increased, in the OGDR state, following hPMSC treatment. We also observed that hPMSC treatment significantly (p<0.0001) reduced binding of RAW264.7 to TNF‐α/ LPS activated hCMEC‐D3 monolayers. The expression of VCAM‐1 was increased in TNF‐α/ LPS activated hCMEC‐D3 while this was significantly (p<0.01, P<0.05) decreased by hPMSC treatment. hPMSC also inhibited (p<0.05) PMN transmigration across hCMEC‐D3 monolayers. Lastly, we found that hBVSMC contraction was significantly increased by hPMSC treatment (p=0.0001). This effect was reversed by angiotensin II Type 1 receptor blockade (Losartan, 50μM), angiotensin converting enzyme (Captopril, 50μM), thromboxane A2 synthesis (Ozagrel, 10μM), and endothelin converting enzyme inhibition (SM19712, 100μM).We conclude that pleiotropic factors associated with hPMSC administration can have a favorable impact on BBB integrity, immune cell interaction, and vasoregulation potentially alleviating the detrimental effects of ischemic stroke.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.