Murine Brain Endothelial Cells Research Articles

Cathepsin L acutely alters microvessel integrity within the neurovascular unit during focal cerebral ischemia.

During focal cerebral ischemia, the degradation of microvessel basal lamina matrix occurs acutely and is associated with edema formation and microhemorrhage. These events have been attributed to matrix metalloproteinases (MMPs). However, both known protease generation and ligand specificities suggest other participants. Using cerebral tissues from a non-human primate focal ischemia model and primary murine brain endothelial cells, astrocytes, and microglia in culture, the effects of active cathepsin L have been defined. Within 2 hours of ischemia onset cathepsin L, but not cathepsin B, activity appears in the ischemic core, around microvessels, within regions of neuron injury and cathepsin L expression. In in vitro studies, cathepsin L activity is generated during experimental ischemia in microglia, but not astrocytes or endothelial cells. In the acidic ischemic core, cathepsin L release is significantly increased with time. A novel ex vivo assay showed that cathepsin L released from microglia during ischemia degrades microvessel matrix, and interacts with MMP activity. Hence, the loss of microvessel matrix during ischemia is explained by microglial cathepsin L release in the acidic core during injury evolution. The roles of cathepsin L and its interactions with specific MMP activities during ischemia are relevant to strategies to reduce microvessel injury and hemorrhage.

Acute Stimulation of GLUT1 Cell Surface Localization in Blood‐Brain Barrier Endothelial Cells

The human brain relies on constant uptake of glucose from the blood stream for its energy needs. However, the blood‐brain barrier (BBB) prevents simple diffusion of glucose and other substrates from circulation into the brain interstitial fluid. Thus, glucose must be transported across the BBB endothelial cells (ECs) by the glucose transporter, GLUT1. Misregulation of BBB GLUT1 expression or function results in severe cognitive and developmental disorders. Until recently, BBB GLUT1 was believed to be predominantly at the cell surface, and that increased GLUT1‐mediated glucose transport was simply achieved by incorporation of newly synthesized GLUT1 into the plasma membrane. However, recent data from our lab suggest that AMP kinase (AMPK) controls GLUT1 localization between intracellular and cell surface membranes. To understand how AMPK regulate GLUT1 trafficking in BBB ECs, we are using a reversible cell surface biotinylation technique to study GLUT1 kinetics in the presence or absence of AMPK activation. From our preliminary data in basal, murine brain endothelial (bEnd.3) cells we obtain a first order rate constant of exocytosis (kex) and endocytosis (ken) of 0.091min‐1 and 0.436min‐1 respectively for GLUT1 trafficking. Next, we will determine if AMPK stimulates GLUT1 kex or inhibits GLUT1 ken. This will aid in identifying downstream signaling partners in AMPK‐regulated GLUT1 trafficking. Finally, we will test whether signals that couple neuronal activation to increased BBB glucose transport, act via the AMPK pathway. Findings from this study will provide fundamental insights into cerebral glucose transporter misregulation in diseases and may reveal mechanisms for pharmacologic manipulation of the BBB permeability.

Biochanin A inhibits endothelial cell functions and proangiogenic pathways: implications in glioma therapy.

Malignant gliomas, such as glioblastoma multiforme, are highly vascularized tumors of the central nervous system. A rich network of angiogenic vessels supporting glioma growth is an important therapeutic target in glioma therapy. In the past few years, small molecules have gained interest as multitargeting therapies for cancer. Biochanin A is a small, natural dietary isoflavone known for its anticancer potential. Previously, we have found that biochanin A inhibits invasion in human glioblastoma cells. In this study, we elucidated the antiangiogenic mechanisms of biochanin A using rat brain tumor (C6) and murine brain endothelial (bEnd.3) cells and an ex-vivo chick chorioallantoic membrane model. Biochanin A inhibited endothelial cell functions such as cell viability, migration, and invasion, as analyzed using MTT, scratch wound, and gelatin zymography assays. Activation of proangiogenic proteins (ERK/AKT/mTOR) was inhibited. Biochanin A also inhibited chemical hypoxia-inducible factor-1α and vascular endothelial growth factor in C6 cells. Results of chick chorioallantoic membrane assay showed that biochanin A inhibited blood vessel formation ex vivo. As these results suggest that biochanin A directly targets different facets of angiogenesis in vitro and ex vivo, this study provides a rationale for future preclinical evaluation of its efficacy against angiogenic gliomas.

Dabigatran abrogates brain endothelial cell permeability in response to thrombin.

Atrial fibrillation (AF) increases the risk and severity of thromboembolic stroke. Generally, antithrombotic agents increase the hemorrhagic risk of thromboembolic stroke. However, significant reductions in thromboembolism and intracerebral hemorrhage have been shown with the antithrombin dabigatran compared with warfarin. As thrombin has been implicated in microvessel injury during cerebral ischemia, we hypothesized that dabigatran decreases the risk of intracerebral hemorrhage by direct inhibition of the thrombin-mediated increase in cerebral endothelial cell permeability. Primary murine brain endothelial cells (mBECs) were exposed to murine thrombin before measuring permeability to 4-kDa fluorescein isothiocyanate-dextran. Thrombin increased mBEC permeability in a concentration-dependent manner, without significant endothelial cell death. Pretreatment of mBECs with dabigatran completely abrogated the effect of thrombin on permeability. Neither the expressions of the endothelial cell β1-integrins nor the tight junction protein claudin-5 were affected by thrombin exposure. Oxygen-glucose deprivation (OGD) also increased permeability; this effect was abrogated by treatment with dabigatran, as was the additive effect of thrombin and OGD on permeability. Taken together, these results indicate that dabigatran could contribute to a lower risk of intracerebral hemorrhage during embolism-associated ischemia from AF by protection of the microvessel permeability barrier from local thrombin challenge.

Stability of murine bradykinin type 2 receptor despite treatment with NO, bradykinin, icatibant, or C1-INH.

Little is known about factors which trigger and/or contribute to hereditary angioedema or ACE-inhibitor-mediated angioedema including variations in bradykinin type 2 receptor (B2R) expression and activity. Protein and mRNA expression of B2R and the increase of intracellular calcium (iCa) in response to bradykinin were monitored in porcine and murine endothelial cells in response to NO donors or bradykinin. B2R protein expression was evaluated in skin, heart, and lung of (i) mice with endothelial-specific overexpression of eNOS (eNOS(tg) ), (ii) in eNOS(-/-) mice and (iii) in C57BL/6 mice treated with the NO donor pentaerythritol tetranitrate (PETN), the NOS inhibitor l-nitroarginine (L-NA), plasma pool C1-INH, and the B2R antagonist icatibant. Aortic reactivity to bradykinin was investigated including eNOS(-/-) mice. B2R protein and mRNA expression remained unchanged in cells subjected to L-NA, NO donors, and bradykinin in a time- and concentration-dependent manner. Likewise, increases of iCa in murine brain endothelial cells remained unchanged. B2R protein levels were similar in eNOS(tg) and eNOS(-/-) as compared to transgene-negative littermates. Likewise, treatment of C57BL/6 mice with PETN, L-NA, C1-INH or icatibant did not change B2R protein expression. In aortic rings of C57BL/6 mice, bradykinin induced B2R-dependent constrictions which were attenuated by endothelial NO and abolished by diclofenac indicating the functional importance of B2R-induced activation of endothelial NO synthase and cyclooxygenase. These data suggest that alterations of B2R protein expression induced by NO, bradykinin, C1-INH, or icatibant unlikely contribute to bradykinin-induced angioedema. This finding does not rule out a role for NO in bradykinin-induced extravasation and/or angioedema.

Aβ1-42 reduces P-glycoprotein in the blood-brain barrier through RAGE-NF-κB signaling.

The reduced clearance of amyloid-β peptide (Aβ) from the brain partly accounts for the neurotoxic accumulation of Aβ in Alzheimer's disease (AD). Recently, it has been suggested that P-glycoprotein (P-gp), which is an efflux transporter expressed on the luminal membrane of the brain capillary endothelium, is capable of transporting Aβ out of the brain. Although evidence has shown that restoring P-gp reduces brain Aβ in a mouse model of AD, the molecular mechanisms underlying the decrease in P-gp expression in AD is largely unknown. We found that Aβ1–42 reduced P-gp expression in the murine brain endothelial cell line bEnd.3, which was consistent with our in vivo data that P-gp expression was significantly reduced, especially near amyloid plaques in the brains of five familial AD mutations (5XFAD) mice that are used as an animal model for AD. A neutralizing antibody against the receptor for advanced glycation end products (RAGE) and an inhibitor of nuclear factor-kappa B (NF-κB) signaling prevented the decrease in Aβ1–42-induced P-gp expression, suggesting that Aβ reduced P-gp expression through NF-κB signaling by interacting with RAGE. In addition, we observed that the P-gp reduction by Aβ was rescued in bEnd.3 cells receiving inductive signals or factors from astrocytes making contacts with endothelial cells (ECs). These results support that alterations of astrocyte–EC contacts were closely associated with P-gp expression. This suggestion was further supported by the observation of a loss of astrocyte polarity in the brains of 5XFAD mice. Taken together, we found that P-gp downregulation by Aβ was mediated through RAGE–NF-κB signaling pathway in ECs and that the contact between astrocytes and ECs was an important factor in the regulation of P-gp expression.

Abstract 161: Effects of Radiation Dose on Gene Expression in Cultured Murine Brain Endothelial Cells

Brain arteriovenous malformations (AVMs) are direct connections between arteries and veins that allow high pressure arterial blood to flow into fragile cerebral veins. AVMs are the leading cause of hemorrhagic stroke in children and young adults. Treatment of large and deep AVMs is challenging; new treatment methods are required. It is proposed that stereotactic radiosurgery could be used to selectively alter the AVM endothelial cell phenotype, allowing targeted molecular therapies which stimulate thrombosis. We have previously shown that irradiation of endothelial cell cultures with 25Gy results in up-regulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). In this study we hypothesise that a lower dose of radiation is similarly effective at causing up-regulation in ICAM-1 and VCAM-1 expression. Murine brain endothelial cell cultures were irradiated with 15Gy (n = 4) or 25Gy (n = 4) using a linear accelerator; non-irradiated cells were used as controls (n = 4). The relative gene expression at 96, 120, 144 and 168h after radiation was measured by quantitative real-time PCR. Genes encoding for ICAM-1 and VCAM-1 were found to be significantly up-regulated post-radiation with either 15Gy or 25Gy (p <0.001). Maximum gene expression of ICAM-1 and VCAM-1 was observed at 144h (7-fold) and at 120h (23-fold), respectively. There was no significant difference between the two doses. These results support the hypothesis that a 15Gy dose is as effective as a 25Gy dose. Therefore a lower dose of radiation might be equally effective at causing changes in endothelial cell phenotype. This is important from a clinical perspective because lower doses of radiation will be safer for the treatment of large AVMs.

Chondroitin Sulfate is the Primary Receptor for a Peptide-Modified AAV That Targets Brain Vascular Endothelium In Vivo

Recently, we described a peptide-modified AAV2 vector (AAV-GMN) containing a capsid-displayed peptide that directs in vivo brain vascular targeting and transduction when delivered intravenously. In this study, we sought to identify the receptor that mediates transduction by AAV-GMN. We found that AAV-GMN, but not AAV2, readily transduces the murine brain endothelial cell line bEnd.3, a result that mirrors previously observed in vivo transduction profiles of brain vasculature. Studies in vitro revealed that the glycosaminoglycan, chondroitin sulfate C, acts as the primary receptor for AAV-GMN. Unlike AAV2, chondroitin sulfate expression is required for cell transduction by AAV-GMN, and soluble chondroitin sulfate C can robustly inhibit AAV-GMN transduction of brain endothelial cells. Interestingly, AAV-GMN retains heparin-binding properties, though in contrast to AAV2, it poorly transduces cells that express heparan sulfate but not chondroitin sulfate, indicating that the peptide insertion negatively impacts heparan-mediated transduction. Lastly, when delivered directly, this modified virus can transduce multiple brain regions, indicating that the potential of AAV-GMN as a therapeutic gene delivery vector for central nervous system disorders is not restricted to brain vascular endothelium.

Cytoadherence of Plasmodium berghei-Infected Red Blood Cells to Murine Brain and Lung Microvascular Endothelial CellsIn Vitro

Sequestration of infected red blood cells (iRBC) within the cerebral and pulmonary microvasculature is a hallmark of human cerebral malaria (hCM). The interaction between iRBC and the endothelium in hCM has been studied extensively and is linked to the severity of malaria. Experimental CM (eCM) caused by Plasmodium berghei ANKA reproduces most features of hCM, although the sequestration of RBC infected by P. berghei ANKA (PbA-iRBC) has not been completely delineated. The role of PbA-iRBC sequestration in the severity of eCM is not well characterized. Using static and flow cytoadherence assays, we provide the first direct in vitro evidence for the binding of PbA-iRBC to murine brain and lung microvascular endothelial cells (MVEC). We found that basal PbA-iRBC cytoadherence to MVECs was significantly higher than that of normal red blood cells (NRBC) and of RBC infected with P. berghei K173 (PbK173-iRBC), a strain that causes noncerebral malaria (NCM). MVEC prestimulation with tumor necrosis factor (TNF) failed to promote any further significant increase in mixed-stage iRBC adherence. Interestingly, enrichment of the blood for mature parasites significantly increased PbA-iRBC binding to the MVECs prestimulated with TNF, while blockade of VCAM-1 reduced this adhesion. Our study provides evidence for the firm, flow-resistant binding to endothelial cells of iRBC from strain ANKA-infected mice, which develop CM, and for less binding of iRBC from strain K173-infected mice, which develop NCM. An understanding of P. berghei cytoadherence may help elucidate the importance of sequestration in the development of CM and aid the development of antibinding therapies to help reduce the burden of this syndrome.

Wild-type Measles Virus Infection Upregulates Poliovirus Receptor-Related 4 and Causes Apoptosis in Brain Endothelial Cells by Induction of Tumor Necrosis Factor-Related Apoptosis-lnducing Ligand

Small numbers of brain endothelial cells (BECs) are infected in children with neurologic complications of measles virus (MV) infection. This may provide a mechanism for virus entry into the central nervous system, but the mechanisms are unclear. Both in vitro culture systems and animal models are required to elucidate events in the endothelium. We compared the ability of wild-type (WT), vaccine, and rodent-adapted MV strains to infect, replicate, and induce apoptosis in human and murine brain endothelial cells (HBECs and MBECs, respectively). Mice also were infected intracerebrally. All MV stains productively infected HBECs and induced the MV receptor PVRL4. Efficient WT MV production also occurred in MBECs. Extensive monolayer destruction associated with activated caspase 3 staining was observed in HBECs and MBECs, most markedly with WT MV. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not Fas ligand, was induced by MV infection. Treatment of MBECs with supernatants from MV-infected MBEC cultures with an anti-TRAIL antibody blocked caspase 3 expression and monolayer destruction. TRAIL was also expressed in the endothelium and other cell types in infected murine brains. This is the first demonstration that infection of low numbers of BECs with WT MV allows efficient virus production, induction of TRAIL, and subsequent widespread apoptosis.

Temporal and spatial distribution of Nrf2 in rat brain following stroke: quantification of nuclear to cytoplasmic Nrf2 content using a novel immunohistochemical technique

Activation of the redox-sensitive transcription factor NF-E2 related factor 2 (Nrf2) affords protection against cerebral ischaemia-reperfusion injury via the upregulation of antioxidant defence genes. We have quantified for the first time Nrf2 content in brains from rats subjected to stroke and from cultured bEnd.3 brain endothelial cells using a novel immunohistochemical technique. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion for 70 min followed by reperfusion for 4, 24 or 72 h. Coronal brain sections were incubated with anti-Nrf2 primary and biotinylated-horseradish peroxidase-conjugated secondary antibody, after which sections were reacted with 3,3-diaminobenzidine (DAB) in the presence of hydrogen peroxide. The initial rates of DAB polymer formation were directly proportional to the Nrf2 protein concentration. Image processing was used to determine the temporal and spatial distribution of Nrf2 in nuclear and cytoplasmic compartments in stroke-affected and contralateral hemispheres. Nuclear to cytoplasmic Nrf2 ratios were increased in the stroke region after 24 h reperfusion and declined after 72 h reperfusion. Pretreatment with the Nrf2 inducer sulforaphane reduced total cellular Nrf2 levels in peri-infarct and core regions of the stroke hemisphere after 24 h reperfusion. Treatment of cultured murine brain endothelial cells with sulforaphane (2.5 μm) increased nuclear accumulation of Nrf2 over 1-4 h. We report the first quantitative measurements of spatial and temporal nuclear Nrf2 expression in rat brains following stroke, and show that sulforaphane pretreatment affects Nrf2 distribution in the brain of naïve rats and animals subjected to cerebral ischaemia. Our findings provide novel insights for targeting endogenous redox-sensitive antioxidant pathways to ameliorate the damaging consequences of stroke.

Prostaglandin E2‐induced intercellular adhesion molecule‐1 expression is mediated by cAMP/Epac signalling modules in bEnd.3 brain endothelial cells

Prostaglandin E₂ (PGE₂) has been implicated in the regulation of adhesion molecules, leukocyte adhesion and infiltration into inflamed site. However, the underlying mechanism therein involved remains ill-defined. In this study, we explored its cellular mechanism of action in the regulation of the intercellular adhesion molecule-1 (ICAM-1) expression in the brain endothelial cells. bEnd.3 cells, the murine cerebrovascular endothelial cell line and primary mouse brain endothelial cells were treated with PGE₂ with or without agonists/antagonists of PGE₂ receptors and associated signalling molecules. ICAM-1 expression, Akt phosphorylation and activity of NF-κB were determined by reverse transcription polymerase chain reaction (RT-PCR), immunoblot analysis, luciferase assay and immunocytochemistry. PGE₂ significantly up-regulated the expression of ICAM-1, which was blocked by EP4 antagonist (ONO-AE2-227) and knock-down of EP4. PGE₂ effects were mimicked by forskolin, dibutyryl cAMP (dbcAMP) and an exchange protein directly activated by cAMP (Epac) activator (8-Cpt-cAMP) but not a protein kinase A activator (N⁶-Bnz-cAMP). PGE₂-induced ICAM-1 expression was reduced by knock-down of Epac1. A PI3K specific inhibitor (LY294002), Akt inhibitor VIII (Akti) and NF-κB inhibitors (Bay-11-7082 and MG-132) attenuated the induction of ICAM-1 by PGE₂. PGE₂, dbcAMP and 8-Cpt-cAMP induced the phosphorylation of Akt, IκB kinase and IκBα and the translocation of p65 to the nucleus and increased NF-κB dependent reporter gene activity, which was diminished by Akti. Our findings suggest that PGE₂ induces ICAM-1 expression via EP4 receptor and Epac/Akt/NF-κB signalling pathway in bEnd.3 brain endothelial cells, supporting its pathophysiological role in brain inflammation.

Cooling Treatment Transiently Increases the Permeability of Brain Capillary Endothelial Cells Through Translocation of Claudin-5

The blood-brain-barrier (BBB) is formed by different cell types, of which brain microvascular endothelial cells are major structural constituents. The goal of this study was to examine the effects of cooling on the permeability of the BBB with reference to tight junction formation of brain microendothelial cells. The sensorimotor cortex above the dura mater in adult male Wistar rats was focally cooled to a temperature of 5°C for 1h, then immunostaining for immunoglobulin G (IgG) was performed to evaluate the permeability of the BBB. Permeability produced by cooling was also evaluated in cultured murine brain endothelial cells (bEnd3) based on measurement of trans-epithelial electric resistance (TEER). Immunocytochemistry and Western blotting of proteins associated with tight junctions in bEnd3 were performed to determine protein distribution before and after cooling. After focal cooling of the rat brain cortex, diffuse immunostaining for IgG was observed primarily around the small vasculature and in the extracellular spaces of parenchyma of the cortex. In cultured bEnd3, TEER significantly decreased during cooling (15°C) and recovered to normal levels after rewarming to 37°C. Immunocytochemistry and Western blotting showed that claudin-5, a critical regulatory protein for tight junctions, was translocated from the membrane to the cytoplasm after cooling in cultured bEnd3 cells. These results suggest that focal brain cooling may open the BBB transiently through an effect on tight junctions of brain microendothelial cells, and that therapeutically this approach may allow control of BBB function and drug delivery through the BBB.

Solubilization of flurbiprofen into aptamer-modified PEG–PLA micelles for targeted delivery to brain-derived endothelial cells in vitro

Novel aptamer-functionalized polyethylene glycol–polylactic acid (PEG–PLA) (APP) micelles were developed with the objective to target the transferrin receptor on brain endothelial cells. Flurbiprofen, a potential drug for therapeutic management of Alzheimer’s disease (AD), was loaded into the APP micelles using the co-solvent evaporation method. Results indicated that 9.03% (w/w) of flurbiprofen was entrapped in APP with good retention capacity in vitro. Targeting potential of APPs was investigated using the transferring receptor-expressing murine brain endothelial bEND5 cell line. APPs significantly enhanced surface association of micelles to bEND5 cells as quantified by fluorescence spectroscopy. Most importantly, APPs significantly enhanced intracellular flurbiprofen delivery when compared to unmodified micelles. These results suggest that APP micelles may offer an effective strategy to deliver therapeutically effective flurbiprofen concentrations into the brain for AD patients.

DABIGATRAN ABROGATES BRAIN ENDOTHELIAL CELL PERMEABILITY IN RESPONSE TO THROMBIN

Atrial fibrillation (AF) increases the risk and severity of thromboembolic stroke. Generally, antithrombotic agents increase the hemorrhagic risk of thromboembolic stroke. However, significant reductions in thromboembolism and intracerebral hemorrhage have been shown with the antithrombin dabigatran compared with warfarin. As thrombin has been implicated in microvessel injury during cerebral ischemia, we hypothesized that dabigatran decreases the risk of intracerebral hemorrhage by direct inhibition of the thrombin-mediated increase in cerebral endothelial cell permeability. Primary murine brain endothelial cells (mBECs) were exposed to murine thrombin before measuring permeability to 4-kDa fluorescein isothiocyanate-dextran. Thrombin increased mBEC permeability in a concentration-dependent manner, without significant endothelial cell death. Pretreatment of mBECs with dabigatran completely abrogated the effect of thrombin on permeability. Neither the expressions of the endothelial cell β1-integrins nor the tight junction protein claudin-5 were affected by thrombin exposure. Oxygen-glucose deprivation (OGD) also increased permeability; this effect was abrogated by treatment with dabigatran, as was the additive effect of thrombin and OGD on permeability. Taken together, these results indicate that dabigatran could contribute to a lower risk of intracerebral hemorrhage during embolism-associated ischemia from AF by protection of the microvessel permeability barrier from local thrombin challenge.

Abstract 184: Irradiation Induces Gene Expression Changes In Cultured Murine Brain Endothelial Cells

Brain arteriovenous malformations (AVMs) are abnormal connections between arteries and veins and are the leading cause of hemorrhagic stroke in children and young adults. Treatment of large and deep AVMs remains challenging, therefore new and safe treatment methods are required. Our hypothesis is that stereotactic radiosurgery can be used to selectively alter AVM endothelial cell phenotype, allowing targeted molecular therapies that spare normal vessels. This study examined the post-radiation time course of gene expression changes of potential molecular targets. A murine brain endothelial cell culture (bEnd.3) was treated with 25 Gy using a linear accelerator; non-irradiated cells were used as controls. Quantitative real-time PCR was used to measure the relative gene expression at 1, 6, 12, 24, 48, 72, 96, 120, 144, and 168 h after radiation. Genes encoding for intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were found to be significantly up-regulated post irradiation (p<0.0001), with the maximum level of gene expression for ICAM-1 being evident at 144 h (9-fold) and for VCAM-1 at 120 h (23-fold). Additionally, leukocyte-endothelial cell adhesion molecule 2 (E-selectin) and leukocyte-endothelial cell adhesion molecule 3 (P-selectin) showed significantly increased gene expression at 24 h with 1.5-fold (p<0.001) and 2-fold (p<0.0001) increases respectively. These data support the hypothesis that stereotactic radiosurgery can significantly alter endothelial cell phenotype in AVMs, potentially enabling the use of ligand-based molecular targeting therapies such as the stimulation of thrombosis for AVM treatment.

Cerebral ischemia reduces expression of Hs1-associated protein X-1 (Hax-1) in mouse brain

Hax-1, a multi-functional protein, recently was found to be involved in apoptosis and nerve system development. The purpose of this study was to detect the effect of cerebral ischemia on Hax-1 expression. We have detected the expression of Hax-1 in normal brain tissue and in ischemic brain tissue. Hax-1 was expressed in all brain regions detected with a level similar to the level of β-actin. There were no differences in the expression of Hax-1 in different brain regions detected. The confocal images confirmed that neurons expressed Hax-1. The results of ischemic stroke in vivo indicated that Hax-1 level was significantly reduced at 24h after ischemia in the ischemic hemisphere, which was only 37%±4.8 of healthy hemisphere (p<0.05), and there was a strong reverse correlation between the level of Hax-1 and infarct size indicated by the regress analysis (R2=0.84). The expression of Hax-1 was also reduced in the cells subjected to oxygen/glucose deprivation (OGD) (p<0.01). The expression of Hax-1 was 87%±4.6, 78%±4.9 and 54%±8.2 of control in the murine brain endothelial cell (bEND5 cell) at 1h, 2h and 16h OGD, respectively. The Hax-1 level was 82%±7.3 and 61%±8.1 of control in neuronal cell line (neuro-2a cells) at 5h and 12h OGD, respectively. The percentage of neuro-2a cell death was 40%±11 induced by a 5h of OGD compared to only 10%±4.2 cell death in the control group (p<0.01). Our present study provides preliminary evidence of the effect of cerebral ischemia on Hax-1 expression. The expression of Hax-1 in normal brain tissue and reduction of Hax-1 in ischemic brain tissue indicate its possible involvement in pathophysiological functions in the brain.

Agmatine promotes the migration of murine brain endothelial cells via multiple signaling pathways

AimsThe combination of adhesion and migration of endothelial cells (ECs) is an integral process for evolution, organization, repair and vessel formation in living organisms. Agmatine, a polycationic amine existing in brain, has been investigated to exert neuroprotective effects. Up to date, there are no studies reporting that agmatine modulates murine brain endothelial (bEnd.3) cells migration. In the present study, we intend to investigate the role of agmatine in bEnd.3 cells migration and the molecular mechanism mediating this action. Main methodsThe effect of agmatine on the bEnd.3 cells migration was examined by migration assay, and the mechanism involved for this effect was investigated by western blot analysis and NO contents measurements. Key findingsAgmatine treatment (50, 100 and 200μM) significantly accelerated bEnd.3 cells migration in a concentration-dependent manner. Western blotting revealed that agmatine treatment significantly induced vascular endothelial growth factor (VEGF), VEGF receptor 2 (Flk-1/KDR or VEGFR2), phosphatidylinositol 3-kinase (PI3K), Akt/protein kinase B (also known as PKB, PI3K downstream effector protein), endothelial nitric oxide synthase (eNOS) nitric oxide (NO; product by eNOS) and intercellular adhesion molecule 1 (ICAM-1) expressions during bEnd.3 cells migration. The expression of ICAM-1 and migration of bEnd.3 cells, induced by agmatine, were significantly attenuated by treatment of wortmannin, a specific PI3K inhibitor. SignificanceTaken together, we provide the first evidence that activation of VEGF/VEGFR2 and the consequential PI3K/Akt/eNOS/NO/ICAM-1 signaling pathways are serial events, through which the treatment of agmatine could lead to bEnd.3 cells migration.

Membrane configuration optimization for a murine in vitro blood–brain barrier model

A powerful experimental tool used to study the dynamic functions of the blood–brain barrier (BBB) is an in vitro cellular based system utilizing cell culture inserts in multi-well plates. Currently, usage of divergent model configurations without explanation of selected variable set points renders data comparisons difficult and limits widespread understanding. This work presents for the first time in literature a comprehensive screening study to optimize membrane configuration, with aims to unveil influential membrane effects on the ability of cerebral endothelial cells to form a tight monolayer. First, primary murine brain endothelial cells and astrocytes were co-cultured in contact and non-contact orientations on membranes of pore diameter sizes ranging from 0.4μm to 8.0μm, and the non-contact orientation and smallest pore diameter size were shown to support a significantly tighter monolayer formation. Then, membranes made from polyethylene terephthalate (PET) and polycarbonate (PC) purchased from three different commercial sources were compared, and PET membranes purchased from two manufacturers facilitated a significantly tighter monolayer formation. Models were characterized by transendothelial electrical resistance (TEER), sodium fluorescein permeability, and immunocytochemical labeling of tight junction proteins. Finally, a murine brain endothelial cell line, bEnd.3, was grown on the different membranes, and similar results were obtained with respect to optimal membrane configuration selection. The results and methodology presented here on high throughput 24-well plate inserts can be translated to other BBB systems to advance model understanding.

Blood–brain barrier disruption and enhanced vascular permeability in the multiple sclerosis model EAE

Multiple sclerosis (MS) is a demyelinating disease characterized by the breakdown of the blood–brain barrier (BBB), and accumulation of inflammatory infiltrates in the central nervous system. Tight junctions are specialized cell–cell adhesion structures and critical components of the BBB that have previously been shown to be abnormally distributed in MS tissue. To evaluate whether experimental autoimmune encephalomyelitis (EAE) provides a suitable model for this aspect of MS disease, we examined the expression and distribution of ZO-1 over the course of disease in EAE. We observed a dramatic relocalization of ZO-1 which precedes overt clinical disease and correlates with the sites of inflammatory cell accumulation. Treatment of in vitro cultures of murine brain endothelial cells with components of EAE induction provided similar findings, with relocalization of ZO-1 and increased permeability of endothelial monolayers. BBB disruption in the EAE model appears to parallel disease progression in MS, with direct effects on the cerebrovascular endothelium, making it an ideal tool for future evaluation of tight junction breakdown and repair in MS-like pathology.