Cultured Brain Endothelial Cells Research Articles

Protective effects of vascular endothelial growth factor in cultured brain endothelial cells against hypoglycemia

Hypoglycemia is a common and serious problem among patients with type 1 diabetes receiving treatment with insulin. Clinical studies have demonstrated that hypoglycemic edema is involved in the initiation of hypoglycemic brain damage. However, the mechanisms of this edema are poorly understood. Vascular endothelial growth factor (VEGF), a potent regulator of blood vessel function, has been observed an important candidate hormone induced by hypoglycemia to protect neurons by restoring plasma glucose. Whether VEGF has a protective effect against hypoglycemia-induced damage in brain endothelial cells is still unknown. To investigate the effects of hypoglycemia on cerebral microvascular endothelial cells and assess the protective effect of exogenous VEGF on endothelial cells during hypoglycemia, confluent monolayers of the brain endothelial cell line bEnd.3 were treated with normal (5.5 mM glucose), hypoglycemic (0, 0.5, 1 mM glucose) medium or hypoglycemic medium in the presence of VEGF. The results clearly showed that hypoglycemia significantly downregulated the expression of claudin-5 in bEnd.3 cells, without affecting ZO-1 and occludin expression and distribution. Besides, transendothelial permeability significantly increased under hypoglycemic conditions compared to that under control conditions. Moreover, the hypoglycemic medium in presence of VEGF decreased endothelial permeability via the inhibition of claudin-5 degradation and improved hypoglycemia-induced cell toxicity. Furthermore, Glucose transporter-1 (Glut-1) and apoptosis regulator Bcl-2 expression were significantly upregulated. Taken together, hypoglycemia can significantly increase paraendocellular permeability by downregulating claudin-5 expression. We further showed that VEGF protected brain endothelial cells against hypoglycemia by enhancing glucose passage, reducing endothelial cell death, and ameliorating paraendocellular permeability.

Abstract W P406: Regulation Of MicroRNAs By Angiotensin II In Neurovascular Niche

In adult brain, neuronal stem cells (NSC) are located in the neurovascular niche therefore, vascular cells play an important role in creating a healthy environment for adult neurogenesis. Several laboratories including ours have demonstrated that Angiotensin II (AngII)-induced hypertension affects numerous vascular functions by causing oxidative stress, vascular inflammation and endothelial dysfunction. Further, the available evidence suggests that blood-brain barrier (BBB) deregulation is an indirect consequence of cerebrovascular inflammation. Of these, AngII effect on BBB dysfunction has been reported, and is implicated in the pathogenesis of stroke, small vessel disease, vascular dementia and Alzheimer’s disease. In this regard, miRNAs emerged as key players in the development of vascular and neurodegenerative diseases. In here, we studied the effects of AngII on adult NSC and brain endothelial cell (BEC) miRNAs regulation and their interaction focusing on a specific miRNAs that were selected due to their role in other vascular systems. We used primary mouse NSC and BEC culture model to study the effects of AngII on miR-15, -34a, -155 and -let7 expression using qPCR. AngII 100nm induced expression of miR-15 in both neurospheres and BEC by 2 and 2.5-fold respectively after one hr. miR-34a expression was induced by 3.5-fold in neurospheres and by 3-fold in BEC in 24-hr. Mir-155 expression was elevated in neurospheres by 2-fold and in BEC by 3-fold after 45-min. miR-let7 was increased in neurospheres and BEC by 4-fold in 15 min, and remained induced in EC for one hr. These miRNAs expression changes were accompanied by increased protein expression of C/EBPβ, pCREB, and p38MAPK. However, Ets1 expression was downregulated in both neurospheres and BEC. These results indicate that AngII has major effects on NSC and BEC through deregulating miRNAs and protein levels involved in this phenomenon. Further studies are needed to explore the mechanisms involved.

Abstract 642: Transcend, a protein vector for brain delivery, allows trastuzumab to reach the brain at effective concentration after incorporation to form BT2111

Abstract Trastuzumab, a monoclonal antibody against HER2, is an effective therapy for the treatment of peripheral HER2+ breast cancer. However a significant number of patients eventually succumb to metastases of the brain because the endothelial tight junctions of the blood-brain barrier prevent distribution of trastuzumab to the metastatic tumors. Improving distribution of trastuzumab to HER2+ brain tumors represents a major hurdle in the treatment of metastatic HER2+ breast cancer. Previous studies have shown that doxorubicin conjugated to melanotransferrin (MTf) increased significantly the survival of mice bearing intracranial tumors, while doxorubicin alone was not effective. In this study we examine the potential of MTf-trastuzumab conjugates (BT2111) to penetrate the blood-brain barrier. BT2111 and trastuzumab were labeled with fluorescent markers, either Cy5.5 or rhodamine. The cellular uptakes of these labeled proteins were studied with fluorescence microscopy in cultures of primary human brain endothelial cells (HBEC) and in HER2+ (A172, SKBR3, and BT474), and HER2- (MDA-MB-468) breast cancer cell lines. The fluorescent conjugates were also injected IV into mice and the distribution of BT2111 and trastuzumab to endothelial cells and brain parenchyma was compared with laser scanning confocal microscopy. To aid in the co-localization of BT2111 and trastuzumab to the brain vasculature, mice were injected with tomato lectin-FITC or anti CD31 IgG-FITC prior to sacrifice. The results showed that primary human brain endothelial cells (HBEC) took up BT2111 suggesting that BT2111 can interact with the MTf receptor and transport BT2111 into the intracellular compartment. In contrast, no specific staining associated with trastuzumab was observed, either on the surface or in the interior of HBECs. Uptake of both BT2111 and trastuzumab was observed on the surface of A172, SKBR3, and BT474 cells but not on MDA-MB-468 cells confirming that BT2111 retains HER2 binding activity. When BT2111 and trastruzumab were injected into mice, laser scanning confocal microscopy showed increased distribution of BT2111 in the brain parenchyma when compared to trastuzumab. 10 to 15 times more trastuzumab was delivered in the brain parenchyma when conjugated to MTf. Using a mice model characterized by the formation of brain metastasis after intracardiac administration of MDA-MB 231BR we show that BT2111 is homogenously distributed in normal brain and can reach therapeutical concentration in the brain using 10% of the predicted therapeutic IV dose when trastuzumab was administered as BT2111. These results indicate that BT2111 has the potential to become a new anti-cancer agent for treatment of HER2+brain metastases. Citation Format: Reinhard Gabathuler, Timothy Z. Vitalis, Mohamed I. Nounou, Chris E. Adkins, Paul R. Lockman, Wilfred A. Jefferies. Transcend, a protein vector for brain delivery, allows trastuzumab to reach the brain at effective concentration after incorporation to form BT2111. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 642. doi:10.1158/1538-7445.AM2014-642

In Vitro Models of the Blood–Brain Barrier for the Study of Drug Delivery to the Brain

The most important obstacle to the drug delivery into the brain is the presence of the blood-brain barrier, which limits the traffic of substances between the blood and the nervous tissue. Therefore, adequate in vitro models need to be developed in order to characterize the penetration properties of drug candidates into the central nervous system. This review article summarizes the presently used and the most promising in vitro BBB models based on the culture of brain endothelial cells. Robust models can be obtained using primary porcine brain endothelial cells and rodent coculture models, which have low paracellular permeability and express functional efflux transporters, showing good correlation of drug penetration data with in vivo results. Models mimicking the in vivo anatomophysiological complexity of the BBB are also available, including triple coculture (culture of brain endothelial cells in the presence of pericytes and astrocytes), dynamic, and microfluidic models; however, these are not suitable for rapid, high throughput studies. Potent human cell lines would be needed for easily available and reproducible models which avoid interspecies differences.

Exposure of human astrocytes to leukotriene C4 promotes a CX3CL1/fractalkine-mediated transmigration of HIV-1-infected CD4+ T cells across an in vitro blood–brain barrier model

Eicosanoids, including cysteinylleukotrienes (cysLTs), are found in the central nervous system (CNS) of individuals infected with HIV-1. Few studies have addressed the contribution of cysLTs in HIV-1-associated CNS disorders. We demonstrate that conditioned medium from human astrocytes treated with leukotriene C4 (LTC4) increases the transmigration of HIV-1-infected CD4+ T cells across an in vitro blood–brain barrier (BBB) model using cultured brain endothelial cells. Additional studies indicate that the higher cell migration is linked with secretion by astrocytes of CX3CL1/fractalkine, a chemokine that has chemoattractant activity for CD4+ T cells. Moreover, we report that the enhanced cell migration across BBB leads to a more important CD4+ T cell-mediated HIV-1 transfer toward macrophages. Altogether data presented in the present study reveal the important role that LTC4, a metabolite of arachidonic acid, may play in the HIV-1-induced neuroinvasion, neuropathogenesis and disease progression.

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.

Acute effects of short‐chain alkylglycerols on blood‐brain barrier properties of cultured brain endothelial cells

The blood-brain barrier (BBB) restricts drug penetration to the brain preventing effective treatment of patients suffering from brain tumours. Intra-arterial injection of short-chain alkylglycerols (AGs) opens the BBB and increases delivery of molecules to rodent brain parenchyma in vivo. The mechanism underlying AG-mediated modification of BBB permeability is still unknown. Here, we have tested the effects of AGs on barrier properties of cultured brain microvascular endothelial cells. The effects of two AGs, 1-O-pentylglycerol and 2-O-hexyldiglycerol were examined using an in vitro BBB model consisting of primary cultures of rat brain endothelial cells, co-cultured with rat cerebral glial cells. Integrity of the paracellular, tight junction-based, permeation route was analysed by functional assays, immunostaining for junctional proteins, freeze-fracture electron microscopy, and analysis of claudin-claudin trans-interactions. AG treatment (5 min) reversibly reduced transendothelial electrical resistance and increased BBB permeability for fluorescein accompanied by changes in cell morphology and immunostaining for claudin-5 and β-catenin. These short-term changes were not accompanied by alterations of inter-endothelial tight junction strand complexity or the trans-interaction of claudin-5. AG-mediated increase in brain endothelial paracellular permeability was short, reversible and did not affect tight junction strand complexity. Redistribution of junctional proteins and alterations in the cell shape indicate the involvement of the cytoskeleton in the action of AGs. These data confirm the results from in vivo studies in rodents characterizing AGs as adjuvants that transiently open the BBB.

Selective accumulation of aluminum in cerebral arteries in Alzheimer's disease (AD)

Once biologically available aluminum bypasses gastrointestinal and blood–brain barriers, this environmentally-abundant neurotoxin has an exceedingly high affinity for the large pyramidal neurons of the human brain hippocampus. This same anatomical region of the brain is also targeted by the earliest evidence of Alzheimer's disease (AD) neuropathology. The mechanism for the selective targeting and transport of aluminum into the hippocampus of the human brain is not well understood. In an effort to improve our understanding of a pathological aluminum entry system into the brain, this study examined the aluminum content of 8 arteries that supply blood to the hippocampus, including the aorta and several cerebral arteries. In contrast to age-matched controls, in AD patients we found a gradient of increasing aluminum concentration from the aorta to the posterior cerebral artery that supplies blood to the hippocampus. Primary cultures of human brain endothelial cells were found to have an extremely high affinity for aluminum when compared to other types of brain cells. Together, these results suggest for the first time that endothelial cells that line the cerebral vasculature may have biochemical attributes conducive to binding and targeting aluminum to selective anatomical regions of the brain, such as the hippocampus, with potential downstream pro-inflammatory and pathogenic consequences.

A detailed method for preparation of a functional and flexible blood–brain barrier model using porcine brain endothelial cells

The blood–brain barrier (BBB) is formed by the endothelial cells of cerebral microvessels and forms the critical interface regulating molecular flux between blood and brain. It contributes to homoeostasis of the microenvironment of the central nervous system and protection from pathogens and toxins. Key features of the BBB phenotype are presence of complex intercellular tight junctions giving a high transendothelial electrical resistance (TEER), and strongly polarised (apical:basal) localisation of transporters and receptors. In vitro BBB models have been developed from primary culture of brain endothelial cells of several mammalian species, but most require exposure to astrocytic factors to maintain the BBB phenotype. Other limitations include complicated procedures for isolation, poor yield and batch-to-batch variability. Some immortalised brain endothelial cell models have proved useful for transport studies but most lack certain BBB features and have low TEER. We have developed an in vitro BBB model using primary cultured porcine brain endothelial cells (PBECs) which is relatively simple to prepare, robust, and reliably gives high TEER (mean∼800Ωcm2); it also shows good functional expression of key tight junction proteins, transporters, receptors and enzymes. The model can be used either in monoculture, for studies of molecular flux including permeability screening, or in co-culture with astrocytes when certain specialised features (e.g. receptor-mediated transcytosis) need to be maximally expressed. It is also suitable for a range of studies of cell:cell interaction in normal physiology and in pathology. The method for isolating and growing the PBECs is given in detail to facilitate adoption of the model.This article is part of a Special Issue entitled Companion Paper.

Brain edema in acute liver failure: Role of neurosteroids

Brain edema is a major neurological complication of acute liver failure (ALF) and swelling of astrocytes (cytotoxic brain edema) is the most prominent neuropathological abnormality in this condition. Elevated brain ammonia level has been strongly implicated as an important factor in the mechanism of astrocyte swelling/brain edema in ALF. Recent studies, however, have suggested the possibility of a vasogenic component in the mechanism in ALF. We therefore examined the effect of ammonia on blood–brain barrier (BBB) integrity in an in vitro co-culture model of the BBB (consisting of primary cultures of rat brain endothelial cells and astrocytes). We found a minor degree of endothelial permeability to dextran fluorescein (16.2%) when the co-culture BBB model was exposed to a pathophysiological concentration of ammonia (5mM). By contrast, lipopolysaccharide (LPS), a molecule well-known to disrupt the BBB, resulted in an 87% increase in permeability. Since increased neurosteroid biosynthesis has been reported to occur in brain in ALF, and since neurosteroids are known to protect against BBB breakdown, we examined whether neurosteroids exerted any protective effect on the slight permeability of the BBB after exposure to ammonia. We found that a nanomolar concentration (10nM) of the neurosteroids allopregnanolone (THP) and tetrahydrodeoxycorticosterone (THDOC) significantly reduced the ammonia-induced increase in BBB permeability (69.13 and 58.64%, respectively). On the other hand, we found a marked disruption of the BBB when the co-culture model was exposed to the hepatotoxin azoxymethane (218.4%), but not with other liver toxins commonly used as models of ALF (thioacetamide and galactosamine, showed a 29.3 and 30.67% increase in permeability, respectively). Additionally, THP and THDOC reduced the effect of TAA and galactosamine on BBB permeability, while no BBB protective effect was observed following treatment with azoxymethane. These findings suggest that ammonia does not cause a significant BBB disruption, and that the BBB is intact in the TAA or galactosamine-induced animal models of ALF, likely due to the protective effect of neurosteroids that are synthesized in brain in the setting of ALF. However, caution should be exercised when using azoxymethane as an experimental model of ALF as it caused a severe breakdown of the BBB, and neurosteriods failed to protect against this breakdown.

Establishment of rat brain endothelial cells susceptible to rat cytomegalovirus ALL-03 infection

Endothelial cells have been implicated as key cells in promoting the pathogenesis and spread of cytomegalovirus (CMV) infection. This study describes the isolation and culture of rat brain endothelial cells (RBEC) and further evaluates the infectious potential of a Malaysian rat CMV (RCMV ALL-03) in these cultured cells. Brain tissues were mechanically fragmented, exposed to enzymatic digestion, purified by gradient density centrifugation, and cultured in vitro. Morphological characteristics and expression of von Willebrand factor (factor VIII-related antigen) verified the cells were of endothelial origin. RBEC were found to be permissive to the virus by cytopathic effects with detectable plaques formed within 7d of infection. This was confirmed by electron microscopy examination which proved the existence of the viral particles in the infected cells. The susceptibility of the virus to these target cells under the experimental conditions described in this report provides a platform for developing a cell-culture-based experimental model for studies of RCMV pathogenesis and allows stimulation of further studies on host cell responses imposed by congenital viral infections.

Initial contact of glioblastoma cells with existing normal brain endothelial cells strengthen the barrier function via fibroblast growth factor 2 secretion: a new in vitro blood-brain barrier model.

Glioblastoma multiforme (GBM) cells invade along the existing normal capillaries in brain. Normal capillary endothelial cells function as the blood-brain barrier (BBB) that limits permeability of chemicals into the brain. To investigate whether GBM cells modulate the BBB function of normal endothelial cells, we developed a new in vitro BBB model with primary cultures of rat brain endothelial cells (RBECs), pericytes, and astrocytes. Cells were plated on a membrane with 8μm pores, either as a monolayer or as a BBB model with triple layer culture. The BBB model consisted of RBEC on the luminal side as a bottom, and pericytes and astrocytes on the abluminal side as a top of the chamber. Human GBM cell line, LN-18 cells, or lung cancer cell line, NCI-H1299 cells, placed on either the RBEC monolayer or the BBB model increased the transendothelial electrical resistance (TEER) values against the model, which peaked within 72h after the tumor cell application. The TEER value gradually returned to baseline with LN-18 cells, whereas the value quickly dropped to the baseline in 24h with NCI-H1299 cells. NCI-H1299 cells invaded into the RBEC layer through the membrane, but LN-18 cells did not. Fibroblast growth factor 2 (FGF-2) strengthens the endothelial cell BBB function by increased occludin and ZO-1 expression. In our model, LN-18 and NCI-H1299 cells secreted FGF-2, and a neutralization antibody to FGF-2 inhibited LN-18 cells enhanced BBB function. These results suggest that FGF-2 would be a novel therapeutic target for GBM in the perivascular invasive front.

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.

Thrombin, a mediator of cerebrovascular inflammation in AD and hypoxia

Considerable evidence implicates hypoxia and vascular inflammation in Alzheimer's disease (AD). Thrombin, a multifunctional inflammatory mediator, is demonstrable in the brains of AD patients both in the vessel walls and senile plaques. Hypoxia-inducible factor 1α (HIF-1α), a key regulator of the cellular response to hypoxia, is also upregulated in the vasculature of human AD brains. The objective of this study is to investigate inflammatory protein expression in the cerebrovasculature of transgenic AD mice and to explore the role of thrombin as a mediator of cerebrovascular inflammation and oxidative stress in AD and in hypoxia-induced changes in brain endothelial cells. Immunofluorescent analysis of the cerebrovasculature in AD mice demonstrates significant (p < 0.01–0.001) increases in thrombin, HIF-1α, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases (MMPs), and reactive oxygen species (ROS) compared to controls. Administration of the thrombin inhibitor dabigatran (100 mg/kg) to AD mice for 34 weeks significantly decreases expression of inflammatory proteins and ROS. Exposure of cultured brain endothelial cells to hypoxia for 6 h causes an upregulation of thrombin, HIF-1α, MCP-1, IL-6, and MMP2 and ROS. Treatment of endothelial cells with the dabigatran (1 nM) reduces ROS generation and inflammatory protein expression (p < 0.01–0.001). The data demonstrate that inhibition of thrombin in culture blocks the increase in inflammatory protein expression and ROS generation evoked by hypoxia. Also, administration of dabigatran to transgenic AD mice diminishes ROS levels in brain and reduces cerebrovascular expression of inflammatory proteins. Taken together, these results suggest that inhibiting thrombin generation could have therapeutic value in AD and other disorders where hypoxia, inflammation, and oxidative stress are involved.

Design of Poly(ethylene Glycol)-Functionalized Hydrophilic Carbon Clusters for Targeted Therapy of Cerebrovascular Dysfunction in Mild Traumatic Brain Injury

Traumatic brain injury (TBI) involves the elaboration of oxidative stress that causes cerebrovascular dysfunction, including impairment of autoregulation of cerebral blood flow. Currently, there is no clinically effective antioxidant treatment for these pathologies. Most currently available antioxidants act through mechanisms in which the antioxidant either transfers the radical or requires regeneration, both of which are impaired in the toxic post-TBI environment. We previously reported that single-walled carbon nanotubes (SWCNTs) and ultrashort SWCNTs possess antioxidant activity, and their characteristics suggest that radical annihilation is the major mechanism. We have now developed a biologically compatible class of carbon-based nanovectors, poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs) that can be further functionalized with antibodies, and hence show promise as targeted drug delivery platforms. Here we report that PEG-HCCs possess innate antioxidant activity and can be rapidly targeted via an antibody to the P-selectin antigen in a model of injured cultured brain endothelial cells. One immediate application of this therapy is to vascular dysfunction that accompanies TBI and worsens outcome in the face of systemic hypotension. These in vitro results support the need for further investigation in animal models.

Silver nanoparticle-induced cytotoxicity in rat brain endothelial cell culture

Silver nanoparticles (AgNPs) are among the most widely commercialised engineered nanomaterials, because of their antimicrobial properties. They are already commonly used in medical devices, household products and industry. Concerns have been raised about potential adverse health effects due to increasing dispersion of AgNPs in the environment. The present study examined the cytotoxic effects of spherical, citrate-coated AgNPs (10, 50 and 100nm) in rat brain endothelial (RBE4) cells and investigated whether the observed effects can be explained by the intrinsic toxicity of the particles or the silver ions released from the particles. The results indicated that exposure of RBE4 cells to AgNPs lead to significant reduction in dye uptake as measured with the Neutral red (NR) assay. The effect was found to be related to particle size, surface area, dose and exposure time. In contrast, silver ions increased NR uptake (ca. 10%) in RBE4 cells after 1h, while a reduction in NR uptake was observed after 24h exposure at high concentrations (20–30μM). Colony formation, as an indicator of proliferation ability, was completely inhibited by AgNPs at concentrations higher than 1μg/ml. Silver ions had less effect on the colony formation of RBE4 cells than AgNPs.

Age-related decrease in cerebrovascular-derived neuroprotective proteins: Effect of acetaminophen

As the population ages, the need for effective methods to maintain brain function in older adults is increasingly pressing. Vascular disease and neurodegenerative disorders commonly co-occur in older persons. Cerebrovascular products contribute to the neuronal milieu and have important consequences for neuronal viability. In this regard vascular derived neuroprotective proteins, Such as vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), and pituitary adenylate cyclase activating peptide (PACAP) are important for maintaining neuronal viability, especially in the face of injury and disease. The objective of this study is to measure and compare levels of VEGF, PEDF and PACAP released from isolated brain microvessels of Fischer 344 rats at 6, 12, 18, and 24months of age. Addition of acetaminophen to isolated brain microvessels is employed to determine whether this drug affects vascular expression of these neuroprotective proteins. Experiments on cultured brain endothelial cells are performed to explore the mechanisms/mediators that regulate the effect of acetaminophen on endothelial cells. The data indicate cerebrovascular expression of VEGF, PEDF and PACAP significantly decreases with age. The age-associated decrease in VEGF and PEDF is ameliorated by addition of acetaminophen to isolated brain microvessels. Also, release of VEGF, PEDF, and PACAP from cultured brain endothelial cells decreases with exposure to the oxidant stressor menadione. Acetaminophen treatment upregulates VEGF, PEDF and PACAP in brain endothelial cells exposed to oxidative stress. The effect of acetaminophen on cultured endothelial cells is in part inhibited by the selective thrombin inhibitor hirudin. The results of this study suggest that acetaminophen may be a useful agent for preserving cerebrovascular function. If a low dose of acetaminophen can counteract the decrease in vascular-derived neurotrophic factors evoked by age and oxidative stress, this drug might be useful for improving brain function in the elderly.

Pro-Inflammatory Cytokine Regulation of P-glycoprotein in the Developing Blood-Brain Barrier

Placental P-glycoprotein (P-gp) acts to protect the developing fetus from exogenous compounds. This protection declines with advancing gestation leaving the fetus and fetal brain vulnerable to these compounds and potential teratogens in maternal circulation. This vulnerability may be more pronounced in pregnancies complicated by infection, which is common during pregnancy. Pro-inflammatory cytokines (released during infection) have been shown to be potent inhibitors of P-gp, but nothing is known regarding their effects at the developing blood-brain barrier (BBB). We hypothesized that P-gp function and expression in endothelial cells of the developing BBB will be inhibited by pro-inflammatory cytokines. We have derived brain endothelial cell (BEC) cultures from various stages of development of the guinea pig: gestational day (GD) 50, 65 (term ∼68 days) and postnatal day (PND) 14. Once these cultures reached confluence, BECs were treated with various doses (100–104 pg/mL) of pro-inflammatory cytokines: interleukin-1β (IL-1β), interleukin-6 (IL-6) or tumor necrosis factor- α (TNF-α). P-gp function or abcb1 mRNA (encodes P-gp) expression was assessed following treatment. Incubation of GD50 BECs with IL-1β, IL-6 or TNF-α resulted in no change in P-gp function. GD65 BECs displayed a dose-dependent decrease in function with all cytokines tested; maximal effects at 42%, 65% and 34% with IL-1β, IL-6 and TNF-α treatment, respectively (P<0.01). Inhibition of P-gp function by IL-1β, IL-6 and TNF-α was even greater in PND14 BECs; maximal effects at 36% (P<0.01), 84% (P<0.05) and 55% (P<0.01), respectively. Cytokine-induced reductions in P-gp function were associated with decreased abcb1 mRNA expression. These data suggest that BBB P-gp function is increasingly responsive to the inhibitory effects of pro-inflammatory cytokines, with increasing developmental age. Thus, women who experience infection and take prescription medication during pregnancy may expose the developing fetal brain to greater amounts of exogenous compounds – many of which are considered potentially teratogenic.

Lipopolysaccharide impairs amyloid beta efflux from brain: altered vascular sequestration, cerebrospinal fluid reabsorption, peripheral clearance and transporter function at the blood–brain barrier

BackgroundDefects in the low density lipoprotein receptor-related protein-1 (LRP-1) and p-glycoprotein (Pgp) clearance of amyloid beta (Aβ) from brain are thought to contribute to Alzheimer’s disease (AD). We have recently shown that induction of systemic inflammation by lipopolysaccharide (LPS) results in impaired efflux of Aβ from the brain. The same treatment also impairs Pgp function. Here, our aim is to determine which physiological routes of Aβ clearance are affected following systemic inflammation, including those relying on LRP-1 and Pgp function at the blood–brain barrier.MethodsCD-1 mice aged between 6 and 8 weeks were treated with 3 intraperitoneal injections of 3 mg/kg LPS at 0, 6, and 24 hours and studied at 28 hours. 125I-Aβ1-42 or 125I-alpha-2-macroglobulin injected into the lateral ventricle of the brain (intracerebroventricular (ICV)) or into the jugular vein (intravenous (IV)) was used to quantify LRP-1-dependent partitioning between the brain vasculature and parenchyma and peripheral clearance, respectively. Disappearance of ICV-injected 14 C-inulin from brain was measured to quantify bulk flow of cerebrospinal fluid (CSF). Brain microvascular protein expression of LRP-1 and Pgp was measured by immunoblotting. Endothelial cell localization of LRP-1 was measured by immunofluorescence microscopy. Oxidative modifications to LRP-1 at the brain microvasculature were measured by immunoprecipitation of LRP-1 followed by immunoblotting for 4-hydroxynonenal and 3-nitrotyrosine.ResultsWe found that LPS: caused an LRP-1-dependent redistribution of ICV-injected Aβ from brain parenchyma to brain vasculature and decreased entry into blood; impaired peripheral clearance of IV-injected Aβ; inhibited reabsorption of CSF; did not significantly alter brain microvascular protein levels of LRP-1 or Pgp, or oxidative modifications to LRP-1; and downregulated LRP-1 protein levels and caused LRP-1 mislocalization in cultured brain endothelial cells.ConclusionsThese results suggest that LRP-1 undergoes complex functional regulation following systemic inflammation which may depend on cell type, subcellular location, and post-translational modifications. Our findings that systemic inflammation causes deficits in both Aβ transport and bulk flow like those observed in AD indicate that inflammation could induce and promote the disease.

Regulation of Cerebral Vascular Function by Sirtuin 1

Endothelial dysfunction, associated with reduced nitric oxide bioavailability and oxidative stress, is a common feature of vascular-related diseases. Sirtuin 1 (SIRT1) is a protein deacetylase that has been shown to target endothelial nitric oxide synthase in large arteries and is protective during oxidative stress. However, within resistance-sized vessels, the expression and functional effects of SIRT1 remain unknown. Immunoblotting and immunohistochemistry were used to determine SIRT1 expression and localization in cultured brain endothelial cells and intact rat middle cerebral artery. The influence of SIRT1 on vascular function was then studied in intact middle cerebral arteries using pressure myography. We report for the first time that SIRT1 is expressed in the resistance-sized vessels in the brain and is present in both the endothelium and smooth muscle. Pharmacological inhibition of SIRT1 demonstrated reduced endothelium-dependent dilation mediated by nitric oxide. However, endothelium-independent dilations were comparable in the presence and absence of SIRT1 block. Our results support a role for SIRT1 in endothelium-dependent relaxation in the cerebral vasculature and reveal a potential for SIRT1 as a therapeutic target in vascular-related diseases by restoring endothelial function.