Rat Brain Microvascular Endothelial Cells Research Articles

RhoA-Mediated Potential Regulation of Blood–Tumor Barrier Permeability by Bradykinin

The purpose of this work is to investigate the potential for the small G protein RhoA to play a role in bradykinin (BK)-induced actin cytoskeleton rearrangement, tight junction (TJ) protein disassembly, and an increase in blood-tumor barrier (BTB) permeability in rat brain microvascular endothelial cells (RBMECs). Our study used primary RBMECs as an in vitro BTB model and a RhoA inhibitor (C(3) exoenzyme) to establish whether RhoA played a role in the process of TJ disassembly, stress fiber formation, and increasing BTB permeability by BK. Data from the HRP flux and TEER assays revealed that BTB permeability was increased by BK induction. C(3) exoenzyme could partially inhibit endothelial leakage and restored normal TEER values in RBMECs. An obvious shift in occludin distribution from insoluble to soluble fractions was observed as assessed by Western blot, which was prevented by C(3) exoenzyme. In addition, C(3) exoenzyme inhibited BK-induced relocation of occludin from cellular borders into the cytoplasm, as well as stress fiber formation in RBMECs. A time-dependent increase in RhoA activity by BK administration was observed, which was inhibited by C(3) exoenzyme. RhoA activation is important for BK-induced increase in BTB permeability and appears to involve the ability for RhoA to mediate occludin disassembly and stress fiber formation.

Comparison of Two Blood-Brain Barrier In Vitro Systems: Cytotoxicity and Transfer Assessments of Malathion/Oxon and Lead Acetate

Toxicity and integrity disruption in response to transport through the blood-brain barrier (BBB) of the organophosphates malathion and malaoxon and heavy metal lead acetate were assessed in two in vitro barrier systems. One system was constructed using bovine brain microvascular endothelial cells (BMEC), while the other system was constructed with rat brain microvascular endothelial cells (RBE4); both were cocultured with rat astrocytes. We hypothesized that these models would respond differently to neurotoxic compounds. Concentrations of malathion, malaoxon, and lead acetate between 0.01 microM and 1 mM were assessed for their capacity to cause cytotoxicity to the astrocytes and endothelial cells utilized to construct the BBB systems, with the least cytotoxic concentrations chosen for transfer assessments of neurotoxicants through the barrier systems. Concentrations of malathion at 10 microM, malaoxon at 1 microM, and lead acetate at 1 and 10 microM were selected. Lead concentrations were measured in media of the abluminal and luminal sides of both systems using graphite furnace atomic absorption at the beginning of the treatment (T0) and 14 h later (T14). Passage of organophosphate compounds was determined utilizing inhibition of acetylcholinesterase enzyme in a neuroblastoma cell line (SH-SY5Y) localized below the barrier system. Transendothelial electrical resistance was assessed as a measurement of integrity of the barrier systems, with baseline values higher with the RBE4-astrocyte system than with the BMEC-astrocyte system. Metabolic capability, as measured by esterase activity, was higher in BMECs, which were more likely to retain lead than RBE4 cells. Results suggest that differences in endothelial cell source can affect the outcome of studies on toxicant transfer through in vitro BBB systems.

Potentiating effect of endothelial cells on astrocytic plasminogen activator inhibitor type-1 gene expression in an in vitro model of the blood–brain barrier

There is accumulating evidence of the importance of cellular communication between the cells that compose the blood-brain barrier (BBB). Astrocytes are known to affect the expression of tissue-type plasminogen activator (t-PA) and its inhibitor plasminogen activator inhibitor type-1 (PAI-1) in endothelial cells. We investigated the influence of endothelial cells on astrocytic gene expression of PAI-1, protease nexin-1 (PN-1) and t-PA using an in vitro model of the BBB. Primary rat astrocyte-enriched cultures were cocultured with primary adult rat brain microvascular endothelial cells on opposite sides of a transwell membrane. After coculturing for 9–11 days, the cultures were treated with lipopolysaccharide (LPS) for 8 h or 24 h. The levels of PAI-1, PN-1 and t-PA mRNA in untreated and treated monocultures and cocultures were analyzed by Real-Time RT-PCR. Cocultivation of astrocytes and endothelial cells increased astrocytic PAI-1 mRNA expression, and this response was further amplified by LPS treatment. The levels of PN-1 and t-PA mRNA expression in astrocytes were unaffected by cocultivation and/or LPS treatment. Analysis of endothelial PAI-1 and t-PA gene expression revealed increased PAI-1 mRNA levels in cocultured cells, whereas t-PA mRNA levels remained unchanged. These results demonstrate that the cocultivation of astrocytes and endothelial cells induces a pronounced increase in astrocytic PAI-1 gene expression, and that this effect is amplified by LPS treatment. These findings imply an important role for intercellular crosstalk in modulating PAI-1 gene expression within the BBB, under both physiologic and pathophysiologic conditions.

Lipopolysaccharide-activated microglia induce dysfunction of the blood-brain barrier in rat microvascular endothelial cells co-cultured with microglia.

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells, astrocytes, pericytes, microglia, and neurons. BBB disruption under pathological conditions such as neurodegenerative disease and inflammation is observed in parallel with microglial activation. To test whether activation of microglia is linked to BBB dysfunction, we evaluated the effect of lipopolysaccharide (LPS) on BBB functions in an in vitro co-culture system with rat brain microvascular endothelial cells (RBEC) and microglia. When LPS was added for 6 h to the abluminal side of RBEC/microglia co-culture at a concentration showing no effects on the RBEC monolayer, transendothelial electrical resistance was decreased and permeability to sodium-fluorescein was increased in RBEC. Immunofluorescence staining for tight junction proteins demonstrated that zonula occludens-1-, claudin-5-, and occludin-like immunoreactivities at the intercellular borders of RBEC were fragmented in the presence of LPS-activated microglia. These functional changes induced by LPS-activated microglia were blocked by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, diphenyleneiodonium chloride. The present findings suggest that LPS activates microglia to induce dysfunction of the BBB by producing reactive oxygen species through NADPH oxidase.

The impact of paracrine signaling in brain microvascular endothelial cells on the survival of neurons

Neurons depend for survival on local neurotrophic factors which act in an autocrine/paracrine manner. However, the effect of paracrine signaling of brain microvascular endothelial cells (BMECs) under pathological conditions on neuron survival is not fully understood. In this study we cultured rat BMECs and cortical neurons. BMECs were cultured in oxygen-glucose-deprived (OGD) conditions to mimic cerebral ischemia in vitro. The conditioned media of normal BMECs or OGD-injured BMECs were used to culture normal or injured neurons. Neuron activity, free Ca 2+ concentration, NMDA receptor status, mitochondrial membrane potential and cytochrome C release level were determined. The results showed: mitochondrial activity of injured neurons was significantly increased and lactate dehydrogenase (LDH) leakage was decreased ( P < 0.05) by grown in conditioned medium of normal BMECs. Inversely, mitochondrial activity of normal or injured neurons was decreased and LDH leakage was significantly increased ( P < 0.05) by grown in conditioned medium of injured BMECs. The changes in free Ca 2+ concentration, NMDA receptor status, mitochondrial membrane potential and cytochrome C release level were consistent with the changes in neuronal activity. These findings suggest that the conditioned medium of normal BMECs has a neuroprotective effect. However, this protective effect was lost after BMECs injury; in fact, the conditioned medium became neurotoxic. Therefore, it appears that early recovery of BMECs might be helpful for neuron survival.

RNAi Inhibits Coriaria Lactone-Induced MDR1b Overexpression in Rat Brain Microvascular Endothelial Cells

P-glyprotein (multidrug resistance gene 1, MDR1) was the first isolated and most extensively investigated refractory epilepsy-related drug-resistant protein. Coriaria lactone (CL) can induce P-glyprotein expression of brain capillary and astrocytes in vivo. We established the primary rat brain microvascular endothelial cell (BMECs) models overexpressing P-glycoprotein induced by CL successfully and infected them with adenovirus vector, which carry small interfering RNA (siRNA) designed to target MDR1b. MDR1b mRNA levels and P-glyprotein expression in experimental group decreased significantly 72 h after infection, and the fluorescence intensity of rhodamine within the cells of experimental group increased significantly. So, CL can induce P-glyprotein overexpression in BMECs in vitro, and siRNA can effectively inhibit expression of P-glyprotein and decrease its "pumping out" function. This may offer a new method and rationale for studying the mechanism of refractory epilepsy and relevant gene therapy.

Vasoactive intestinal peptide in rats with focal cerebral ischemia enhances angiogenesis

We studied the effect of vasoactive intestinal peptide (VIP) on angiogenesis in the ischemic boundary area after focal cerebral ischemia. Adult male Sprague–Dawley rats underwent middle cerebral artery occlusion for 2 h. A single dose of VIP was given via i.c.v. injection at the beginning of reperfusion. Immunohistochemistry and Western blotting were performed to assay angiogenesis and brain levels of vascular endothelial growth factor (VEGF) protein, respectively. In addition, the expression of VEGF and its receptors (flt-1 and flk-1), as well as endothelial proliferation, was measured using rat brain microvascular endothelial cells. Immunohistochemical analyses revealed significant (P<0.05) increases in the numbers of bromodeoxyuridine (BrdU) positive endothelial cells and microvessels at the boundary of the ischemic lesion in rats treated with VIP compared with rats treated with saline. Western blotting analysis showed that treatment with VIP significantly (P<0.05) raised VEGF levels in the ischemic hemisphere. In addition, treatment with VIP increased flt-1 and flk-1 immunoreactivity in endothelial cells. In vitro, incubation with VIP significantly (P<0.01) increased the proliferation of endothelial cells and induced the expression of VEGF, flt-1 and flk-1 in endothelial cells. The stimulatory effect of VIP on the proliferation of endothelial cells was significantly (P<0.01) inhibited by SU5416, a selective inhibitor of VEGF receptor tyrosine kinase. Our data suggest that treatment with VIP enhances angiogenesis in the ischemic brain, and this effect may be mediated by increases in levels of VEGF and its receptors.

Effects of preconditioning on tight junction and cell adhesion of cerebral endothelial cells

Brain ischemia can usually disrupt the blood-brain barrier (BBB), increasing the permeability of BBB. Ischemic preconditioning could not only reduce neurons damage, but also protect the functions of BBB during brain ischemia. In this study, we used BBB model in vitro to examine the effects of preconditioning on cell viability, BBB permeability, tight junction and cell adhesion of rat brain microvascular endothelial cells (BMECs). The rat BMECs were exposed to 2.5 h of oxygen glucose deprivation (OGD) following 3 h of reoxygenation to simulate ischemia/reperfusion in vivo, using 0.5 h OGD as preconditioning. Results showed that OGD/reoxygenation induced cell death, increased BBB permeability, changed the distribution of endothelial cells tight junction protein ZO-1 and cellular framework protein F-actin, and increased the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). OGD preconditioning could partially protect cell viability and BBB permeability, maintain the membrane location of ZO-1 and F-actin, and decrease the expression of ICAM-1 and VCAM-1. Our study suggests that OGD preconditioning could stabilize the tight junction protein and alleviate cell adhesion to protect BBB function during ischemic stress.

Effects of Qingkailing effective components on nuclear factor-kappa B in an ischemia-reperfusion injury model of rat brain microvascular endothelial cells in vitro

To establish an ischemia-reperfusion injury model of rat cerebral microvascular endothelial cells (MVECs) in vitro, and to explore the relationship between nuclear factor-kappa B (NF-kappaB) and the protective effects of Qingkailing effective components (hyocholic acid, taurocholic acid, baicalin, jasminoidin, Pinctada martensii) on MVECs. Brain MVECs of male rats were digested with trypsin and subcultured, then the content of MVECs was adjusted to 1x10 (5)/mL and the MVECs were divided into normal control group, untreated group, hyocholic acid group, taurocholic acid group, baicalin group, jasminoidin group, Pinctada martensii group and nimodipine group, with six holes in each group. Except for the normal control group, the MVECs in the other groups were exposed in oxygen and glucose deprivation (OGD) circumstance in vitro to simulate ischemia-reperfusion injury. Immunocytochemical staining and image analysis system were used to observe the expression of NF-kappaB protein. Under a light microscope, the nuclei of MVECs in the normal control group were blank. Staining intensity of NF-kappaB protein in the nucleus in the untreated group was much deeper than that in the endochylema, with NF-kappaB shifted to nucleus after activation; a small quantity of NF-kappaB protein were expressed in the border of nucleus next to endochylema in groups of Qingkailing effective components, and the NF-kappaB protein expression was weaker than that in the untreated group. With the image analysis, we found that transmittance of nucleus and endochylema in the untreated group was significantly lower than that in the normal control group (P<0.01). Transmittance of nucleus and endochylema in the treated groups was higher than that in the untreated group (P<0.05, P<0.01). Qingkailing effective components have significant effect in inhibiting NF-kappaB protein transferring from endochylema to nucleus in vitro.

The molecular mechanism of dexamethasone-mediated effect on the blood–brain tumor barrier permeability in a rat brain tumor model

This study was performed to determine whether dexamethasone (DEX) had an effect on calcium-activated potassium channels (KCa channels) and Occludin protein in blood–brain tumor barrier (BTB). Using a rat brain glioma model, we found that the expression of KCa channels protein and Occludin protein was significantly increased in brain tumor tissue after DEX treatment for 3 days. Compared with non-DEX-treated animals, Evans Blue levels were greatly attenuated in DEX-treated animals. These effects were significantly reversed by the glucocorticoid receptor antagonist RU38486. In addition, DEX treatment enhanced the density of IKCa in the rat brain microvascular endothelial cells (RBMECs) in vitro BTB. All of these results strongly suggest that DEX could be involved in the regulation of both transcellular and paracellular pathway.

Bradykinin selectively modulates the blood–tumor barrier via calcium‐induced calcium release

To investigate the underlying mechanism for the selective modulation of permeability of the blood-tumor barrier by small dose of BK, we established cell lines of rat brain microvascular endothelial cells (BMECs) and astrocytes by primary culture from neonatal rats. BMECs, astrocytes, or C6 glioma cells were treated with BK, and changes of intracellular NO and intracellular calcium level were measured with a fluorescent spectrophotometer. Similarly to the observations in astrocytes, although the initial application of BK easily triggered a ryanodine-mediated calcium-induced calcium release (CICR), we also detected a long-lasting intracellular nitric oxide (NO) elevation in C6 glioma cells upon BK treatment. However, BMECs are not the direct target of BK. Further study showed that ryanodine-mediated CICR contributes greatly to the secondary NO elevation induced by BK treatment. With an in vitro blood-tumor barrier (BTB) model, we demonstrated that NO generated in C6 glioma cells might act as an intercellular messenger and play an important role in the selective modulation of permeability of BMECs by BK. In conclusion, BK triggered CICR in C6 glioma cells, and the associated NO generation might be the underlying mechanism for the selective modulation of BTB permeability by BK.

Inhibition of Tissue Factor Expression in Brain Microvascular Endothelial Cells by Nanoparticles Loading NF-κB Decoy Oligonucleotides

To investigate a nuclear factor-kappa B decoy oligonucleotides strategy on the inhibition of tissue factor (TF) expression in cultured rat brain microvascular endothelial cells (BMECs) by polylactic acid (PLA) nanoparticles delivery system and to evaluate this new vector for in vitro gene therapy. Nanoparticles were formulated using poly D,L-polylactic acid with surface modifying by polysorbates 80. 3-[4,5-Dimethylthiazol-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assays showed that PLA nanoparticles were not toxic to the cultured BMECs.The decoy oligonuceotides (ODNs) loaded within nanoparticles was 6 μg/mg, encapsulation efficacy was (60.5±1.5)%. It was observed by flow cytometry that the cellular uptake of nanoparticles depended on the time of incubation and the concentration of nanoparticles in the medium. And confocal microscopy demonstrated that nanoparticles localized mostly in the BMECs cytoplasm. The released decoy oligonuceotides (ODNs) uptaked by BMECs retained their biologic activity and led to reduced level of tissue factor expression as compared to control cultures. These findings offer a potential therapeutic strategy in the control of TF expression in BMECs in vitro and suggest that PLA nanoparticles may be appropriate as delivery vehicles for decoy strategy in the gene therapy of cerebral thrombosis.

Hemolysate-induced expression of intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 expression in cultured brain microvascular endothelial cells via through ROS-dependent NF-kappaB pathways.

In order to determine the possible effects of hemolysate on brain microvascular endothelial cells (BMECs), we examined the effects of hemolysate on the expression of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1), generation of reactive oxygen species (ROS), and NF-kappaB activation in rat BMECs. Hemolysate induced the expression of ICAM-1 and MCP-1 in endothelial cells. In addition, hemolysate stimulated nuclear translocation of the p65 subunit of NF-kappaB, and NF-kappaB DNA-binding activity in BMECs. Furthermore, hemolysate increased ROS generation, and hemolysate-induced ICAM-1and MCP-1 expression and NF-kappaB activation were abrogated in the presence of the direct scavenger of ROS. Taken together, our results indicate that hemolysate can induce inflammatory responses that increase expression of ICAM-1 and MCP-1, through ROS-dependent NF-kappaB activation in BMECs.

P.1.c.035 The role of nitric oxide in PC12 cells death. Protective effect of 12-lipoxygenase and cyclooxygenases inhibitors

Inhibitors of PARP-1(Poly(ADP-ribose) polymerase-1) act by competing with NAD+, the enzyme physiological substrate, which play a protective role in many pathological conditions characterized by PARP-1 overactivation. It has been shown that PARP-1 also promotes tumor growth and progression through its DNA repair activity. Since angiogenesis is an essential requirement for these activities, we sought to determine whether PARP inhibition might affect rat brain microvascular endothelial cells (GP8.3) migration, stimulated by C6-glioma conditioned medium (CM). Through wound-healing experiments and MTT analysis, we demonstrated that PARP-1 inhibitor PJ-34 [N-(6-Oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide] abolishes the migratory response of GP8.3 cells and reduces their viability. PARP-1 also acts in a DNA independent way within the Extracellular-Regulated-Kinase (ERK) signaling cascade, which regulates cell proliferation and differentiation. By western analysis and confocal laser scanning microscopy (LSM), we analyzed the effects of PJ-34 on PARP-1 expression, phospho-ERK and phospho-Elk-1 activation. The effect of MEK (mitogen-activated-protein-kinase-kinase) inhibitor PD98059 (2-(2-Amino-3-methoxyphenyl)-4 H-1-benzopyran-4-one) on PARP-1 expression in unstimulated and in CM-stimulated GP8.3 cells was analyzed by RT-PCR. PARP-1 expression and phospho-ERK activation were significantly reduced by treatment of GP8.3 cells with PJ-34 or PD98059. By LSM, we further demonstrated that PARP-1 and phospho-ERK are coexpressed and share the same subcellular localization in GP8.3 cells, in the cytoplasm as well as in nucleoplasm. Based on these data, we propose that PARP-1 and phospho-ERK interact in the cytosol and then translocate to the nucleus, where they trigger a proliferative response. We also propose that PARP-1 inhibition blocks CM-induced endothelial migration by interfering with ERK signal-transduction pathway.

Tissue plasminogen activator enhances the hypoxia/reoxygenation-induced impairment of the blood-brain barrier in a primary culture of rat brain endothelial cells.

Hemorrhagic transformation is a major complication associated with tissue plasminogen activator (tPA) therapy for ischemic stroke. We studied the effect of tPA on the blood-brain barrier (BBB) function with our in vitro monolayer model generated using rat brain microvascular endothelial cells subjected either to normoxia or to hypoxia/reoxygenation (H/R) with or without the administration of tPA. The barrier function was evaluated by the transendothelial electrical resistance (TEER), the permeability of sodium fluorescein and Evans' blue-albumin (EBA), and the uptake of lucifer yellow (LY). The permeability of sodium fluorescein and EBA was used as an index of paracellular and transcellular transport, respectively. The administration of tPA increased the permeability of EBA and the uptake of LY under normoxia. It enhanced the increase in the permeability of both sodium fluorescein and EBA, the decrease in the TEER, and the disruption in the expression of ZO-1 under H/R conditions. Administration of tPA could cause an increase in the transcellular transport under normoxia, and both the transcellular and paracellular transport of the BBB under H/R conditions in vitro. Even in humans, tPA may lead to an opening of the BBB under non-ischemic conditions and have an additional effect on the ischemia-induced BBB disruption.

NADPH oxidase mediates radiation-induced oxidative stress in rat brain microvascular endothelial cells

The need to both understand and minimize the side effects of brain irradiation is heightened by the ever-increasing number of patients with brain metastases that require treatment with whole brain irradiation (WBI); some 200,000 cancer patients/year receive partial or WBI. At the present time, there are no successful treatments for radiation-induced brain injury, nor are there any known effective preventive strategies. Data support a role for chronic oxidative stress in radiation-induced late effects. However, the pathogenic mechanism(s) involved remains unknown. One candidate source of reactive oxygen species (ROS) is nicotinamide adenosine dinucleotide phosphate (NADPH) oxidase, which converts molecular oxygen (O 2) to the superoxide anion (O 2 – ) on activation. We hypothesize that brain irradiation leads to activation of NADPH oxidase. We report that irradiating rat brain microvascular endothelial cells in vitro leads to increased (i) intracellular ROS generation, (ii) activation of the transcription factor NFκB, (iii) expression of ICAM-1 and PAI-1, and (iv) expression of Nox4, p22 phox, and p47 phox. Pharmacologic and genetic inhibition of NADPH oxidase blocked the radiation-mediated upregulation of intracellular ROS, activation of NFκB, and upregulation of ICAM-1 and PAI-1. These results suggest that activation of NADPH oxidase may play a role in radiation-induced oxidative stress.

Increasing Ang1/Tie2 expression by simvastatin treatment induces vascular stabilization and neuroblast migration after stroke

In this study, we tested the hypothesis that the Angiopoietin 1 (Ang1)/Tie2 pathway mediates simvastatin-induced vascular integrity and migration of neuroblasts after stroke. Rats were subjected to 2 hrs of middle cerebral artery occlusion (MCAo) and treated, starting 1 day after stroke with or without simvastatin (1 mg/kg, daily) for 7 days. Simvastatin treatment significantly decreased blood–brain barrier (BBB) leakage and concomitantly, increased Ang1, Tie2 and Occludin expression in the ischaemic border (IBZ) compared to the MCAo control group. Simvastatin also significantly increased doublecortin (DCX, a marker of migrating neuroblasts) expression in the IBZ compared to control MCAo rats. DCX was highly expressed around vessels. To further investigate the signalling pathway of simvastatin-induced vascular stabilization and angiogenesis, rat brain microvascular endothelial cell (RBMEC) culture was employed. The data show that simvastatin treatment of RBMEC increased Ang1 and Tie2 gene and protein expression and promoted phosphorylated-Tie2 activity. Simvastatin significantly increased endothelial capillary tube formation, an index of angiogenesis, compared to non-treated control. Inhibition of Ang1 or knockdown of Tie2 gene expression in endothelial cells significantly attenuated simvastatin-induced capillary tube formation. In addition, simvastatin significantly increased subventricular zone (SVZ) explant cell migration compared to non-treatment control. Inhibition of Ang1 significantly attenuated simvastatin-induced SVZ cell migration. Simvastatin treatment of stroke increases Ang1/Tie2 expression and thereby reduces BBB leakage and promotes vascular stabilization. Ang1/Tie2 expression induced by simvastatin treatment promotes neuroblast micro-vascular coupling after stroke.

Dexamethasone increases expression and activity of multidrug resistance transporters at the rat blood-brain barrier

Brain edema is an important factor leading to morbidity and mortality associated with primary brain tumors. Dexamethasone, a synthetic glucocorticoid, is routinely prescribed with antineoplastic agents to alleviate pain associated with chemotherapy and reduce intracranial pressure. We investigated whether dexamethasone treatment increased the expression and activity of multidrug resistance (MDR) transporters at the blood-brain barrier. Treatment of primary rat brain microvascular endothelial cells with submicromolar concentrations of dexamethasone induced significantly higher levels of drug efflux transporters such as breast cancer resistance protein (abcg2), P-glycoprotein (P-gp; abcb1a/abcb1b), and MDR protein 2 (Mrp2; abcc2) as indicted by protein and mRNA levels as well as by functional activity. The effect of dexamethasone on transporter function was significant within 6 h of treatment, was dose dependent, and was reversible. Dexamethasone-induced upregulation of Bcrp and P-gp expression and function was partially abrogated by the glucocorticoid receptor (GR) antagonist RU486. In contrast, RU486 had no effect on the dexamethasone-induced upregulation of Mrp2, suggesting a GR-independent regulation of Mrp2, and a GR-dependent regulation of P-gp and Bcrp. In addition to the dexamethasone-induced upregulation of MDR transporters, we measured a dose-dependent and reversible increase in the expression of the nuclear transcription factor pregnane xenobiotic receptor (PXR). Administering dexamethasone to rats caused increased expression of PXR in brain microvessels within 24 h. These results suggest that adjuvant therapy with corticosteroids such as dexamethasone in the treatment of brain tumors may increase the expression of MDR transporters at the blood-brain barrier through pathways involving GR and PXR.

Induction of Nuclear Receptors and Drug Resistance in the Brain Microvascular Endothelial Cells Treated with Antiepileptic Drugs

Our work contributes to the understanding of the mechanisms of drug resistance in epilepsis. This study aimed to investigate i) the levels of expression of P-glycoprotein (P-gp), and multidrug resistance-associated proteins (MRP)1 and 2, ii) the activation of the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), and iii) the relationship between increased P-gp and MRPs expression and PXR and CAR activation, in immortalized rat brain microvascular endothelial cell lines, GPNT and RBE4, following treatment with the antiepileptic drugs (AEDs), topiramate, phenobarbital, carbamazepine, tiagabine, levetiracetam, and phenytoin, using Western blotting and immunocytochemistry methods. Carbamazepine, phenobarbital and phenytoin induced the highest levels of P-gp and MPRs expression that was associated with increased activation of PXR and CAR receptors as compared to levetiracetam, tiagabine and topiramate. We conclude that P-gp and MRPs are differently overexpressed in GPNT and RBE4 by various AEDs and both PXR and CAR are involved in the drug-resistant epilepsy induced by carbamazepine, phenobarbital and phenytoin.

HDL COUNTERACTS INFLAMMATORY PROPERTIES OF ELECTRONEGATIVE LDL (LDL(-))

We investigated the changes and the molecular mechanisms of cerebral vascular damage and tested the therapeutic effects of Niaspan in type-1 streptozotocin induced diabetic (T1DM) rats after stroke. T1DM-rats were subjected to transient middle cerebral artery occlusion (MCAo) and treated without or with Niaspan. Non-streptozotocin rats (WT) were also subjected to MCAo. Functional outcome, blood–brain-barrier (BBB) leakage, brain hemorrhage, immunostaining, and rat brain microvascular endothelial cell (RBEC) culture were performed. Compared to WT-MCAo-rats, T1DM-MCAo-rats did not show an increase lesion volume, but exhibited significantly increased brain hemorrhage, BBB leakage and vascular damage as well as decreased functional outcome after stroke. Niaspan treatment of stroke in T1DM-MCAo-rats significantly attenuated BBB damage, promoted vascular remodeling and improved functional outcome after stroke. T1DM-MCAo-rats exhibited significantly increased Angiopoietin 2 (Ang2) expression, but decreased Ang1 expression in the ischemic brain compared to WT-MCAo-rats. Niaspan treatment attenuated Ang2, but increased Ang1 expression in the ischemic brain in T1DM-MCAo-rats. In vitro data show that the capillary-like tube formation in the WT-RBECs marginally increased compared to T1DM-RBEC. Niaspan and Ang1 treatment significantly increased tube formation compared to non-treatment control. Inhibition of Ang1 attenuated Niacin-induced tube formation in T1DM-RBECs. Niaspan treatment of stroke in T1DM-rats promotes vascular remodeling and improves functional outcome. The Ang1/Ang2 pathway may contribute to Niaspan induced brain plasticity. Niaspan warrants further investigation as a therapeutic agent for the treatment of stroke in diabetics.