Expression of various multidrug resistance-associated protein (MRP) homologues in brain microvessel endothelial cells

Multidrug resistance-associated protein (MRP) actively transports a broad range of anionic compounds out of the cell. To date, six different homologues of MRP (i.e. MRP1-MRP6) have been identified. The current study examines the expression of the various MRP homologues in both primary cultured bovine brain microvessel endothelial cells (BBMEC) and the capillary-enriched fraction from bovine brain homogenates. RT-PCR analysis demonstrated the presence of MRP1, MRP4, MRP5 and MRP6 in both BBMEC and the capillary-enriched fractions of brain homogenates. While low levels of MRP3 were detected in the BBMEC, it was not observed in the capillary-enriched fraction. In addition, RT-PCR and Western blot studies indicated an absence of MRP2 expression in both blood-brain barrier preparations. The presence of several different MRP homologues in the brain microvessel endothelial cells may be important in controlling the permeability of the blood-brain barrier to organic anions.     

Expression of the multidrug transporter P-glycoprotein in brain capillary endothelial cells and brain parenchyma of amygdala-kindled rats

Summary: Purpose: Based on data from brain biopsy samples of patients with pharmacoresistant partial epilepsy, overexpression of the multidrug transporter P‐glycoprotein (PGP) in brain capillary endothelium has recently been proposed as a potential mechanism of resistance to antiepileptic drugs (AEDs). We examined whether PGP is overexpressed in brain regions of amygdala‐kindled rats, a widely used model of temporal lobe epilepsy (TLE), which is often resistant to AEDs. Methods: Rats were kindled by stimulation of the basolateral amygdala (BLA); electrode‐implanted but nonkindled rats and naive (not implanted) rats served as controls. PGP was determined by immunohistochemistry either 1 or 2 weeks after the last kindled seizure, by using a monoclonal anti‐PGP antibody. Six brain regions were examined ipsi‐ and contralateral to the BLA electrode: the BLA, the hippocampal formation, the piriform cortex, the substantia nigra, the frontal and parietal cortex, and the cerebellum. Results: In both kindled rats and controls, PGP staining was observed mainly in microvessel endothelial cells and, to a much lesser extent, in parenchymal cells. The distribution of PGP expression across brain regions was not homogeneous, but significant differences were found in both the endothelial and parenchymal expression of this protein. In kindled rats, ipsilateral PGP expression tended to be higher than contralateral expression in several brain regions, which was statistically significant in the piriform cortex and parietal cortex. However, compared with controls, no significant overexpression of PGP in capillary endothelial cells or brain parenchyma of kindled rats was seen in any ipsilateral brain region, including the BLA. For comparison with kindled rats, kainate‐treated rats were used as positive controls. As reported previously, kainate‐induced seizures significantly increased PGP expression in the hippocampus and other limbic brain regions. Conclusions: Amygdala‐kindling does not induce any lasting overexpression of PGP in several brain regions previously involved in the kindling process. In view of the many pathophysiologic and pharmacologic similarities between the kindling model and TLE, these data may indicate that PGP overexpression in pharmacoresistant patients with TLE is a result of uncontrolled seizures but not of the processes underlying epilepsy. It remains to be determined whether transient PGP overexpression is present in kindled rats shortly after a seizure, and whether pharmacoresistant subgroups of kindled rats exhibit an increased expression of PGP. Furthermore, other multidrug transporters, such as multidrug resistance–associated protein, might be involved in the resistance of kindled rats to AEDs.     

Glycosphingolipid composition of primary cultured human brain microvascular endothelial cells

Glycosphingolipid (GSL) antigens have been considered to be involved in the pathogenesis of autoimmune neurologic disorders including multiple sclerosis. To establish the GSL pattern specific for endothelial cells forming blood-brain barrier (BBB), we established a method to yield sufficient quantities of highly purified human brain microvascular endothelial cells (HBMECs) and compared their GSL composition to that of human umbilical cord vein endothelial cells (HUVECs), as the representative of endothelial cells not forming BBB. The major gangliosides were GM3 and sialyl paragloboside (LM1), and the major neutral GSLs were lactosylceramide (LacCer), globotriaosylceramide (Gb3), and globoside (Gb4). Trace amounts of GM1, GD1a, GD1b, GT1b, and sulfoglucuronosyl paragloboside (SGPG) could be detected by the high performance thin layer chromatography-overlay method. SGPG was detected only at a nonconfluent state in an amount almost 1/30 that of in nonconfluent HUVECs. Conversely, GM3 and LM1 increased significantly after confluency. The amount of Gb3 in HBMECs was almost as twice that in HUVECs. The significance of these differences in GSL content between HBMECs and HUVECs and between confluent and nonconfluent states is obscure. It might be related, however, to the defense mechanism at the BBB and to the susceptibility of the central nervous system in some disorders that target cell surface GSL, such as hemolytic uremic syndrome.     

Anti-angiogenic activity of the mutant Dutch A(beta) peptide on human brain microvascular endothelial cells

Cerebral amyloid angiopathy is a common pathological feature of patients with Alzheimer's disease (AD) and it is also the hallmark of individuals with a rare autosomal dominant disorder known as hereditary cerebral hemorrhage with amyloidosis-Dutch type. We have shown previously that wild type A(beta) peptides are anti-angiogenic both in vitro and in vivo and could contribute to the compromised cerebrovascular architecture observed in AD. In the present study, we investigated the potential anti-angiogenic activity of the Dutch A(beta)(1-40) (E22Q) peptide. We show that compared to wild type A(beta), freshly solubilized Dutch A(beta) peptide more potently inhibits the formation of capillary structures induced by plating human brain microvascular endothelial cells onto a reconstituted basement membrane. Aggregated/fibrillar preparations of wild type A(beta) and Dutch A(beta) do not appear to be anti-angiogenic in this assay. The stronger anti-angiogenic activity of the Dutch A(beta) compared to wild type A(beta) appears to be related to the increased formation of low molecular weight A(beta) oligomers in the culture medium surrounding human brain microvascular endothelial cells. Using oligonucleotide microarray analysis of human brain microvascular endothelial cells, followed by a genome-scale computational analysis with the Ingenuity Pathways Knowledge Base, networks of genes affected by an anti-angiogenic dose of Dutch A(beta) were identified. This analysis highlights that several biological networks involved in angiogenesis, tumorigenesis, atherosclerosis, cellular migration and proliferation are disrupted in human brain microvascular endothelial cells exposed to Dutch A(beta). Altogether, these data provide new molecular clues regarding the pathological activity of Dutch A(beta) peptide in the cerebrovasculature.     

大鼠脑微血管内皮细胞的体外培养

目的探讨大鼠脑微血管内皮细胞的培养方法.方法取Wistar大鼠乳鼠脑组织,采用筛网过滤、胶原酶消化、离心等技术获取脑微血管内皮细胞,并进行培养.通过形态学、免疫细胞化学方法进一步鉴定,采用MTT方法测定生长曲线.结果经形态学、免疫细胞化学方法鉴定所培养的细胞为脑微血管内皮细胞,观察到原代脑微血管内皮细胞有3种表型,细胞呈单层生长,并可传代培养.结论建立大鼠脑微血管内皮细胞的培养方法可为体外研究脑血管病提供有益帮助.     

Pertussis toxin induces angiogenesis in brain microvascular endothelial cells

Pertussis toxin (PTX) is an ancillary adjuvant used to elicit experimental allergic encephalomyelitis (EAE), the principal autoimmune model of multiple sclerosis. One mechanism whereby PTX potentiates EAE is to increase blood-brain barrier (BBB) permeability. To elucidate further the mechanism of action of PTX on the BBB, we investigated the genomic and proteomic responses of isolated mouse brain endothelial cells (MBEC) following intoxication. Among approximately 14,000 mouse genes tracked by cDNA microarray, 34 showed altered expression in response to PTX. More than one-third of these genes have roles in angiogenesis. Accordingly, we show that intoxication of MBEC induces tube formation in vitro and angiogenesis in vivo. The global effect of PTX on signaling protein levels and phosphorylation in MBEC was investigated by using Kinex antibody microarrays. In total, 113 of 372 pan-specific and 58 of 258 phospho-site-specific antibodies revealed changes >or=25% following intoxication. Increased STAT1 Tyr-701 and Ser-727 phosphorylation; reduced phosphorylation of the activating phospho-sites in Erk1, Erk2, and MAPKAPK2; and decreased phosphorylation of arrestin beta1 Ser-412 and Hsp27 Ser-82 were confirmed by Kinetworks multi-immunoblotting. The importance of signal transduction pathways on PTX-induced MBEC tube formation was evaluated pharmacologically. Inhibition of phospholipase C, MEK1, and p38 MAP kinase had little effect, whereas inhibition of cAMP-dependent protein kinase, protein kinase C, and phosphatidylinositol 3-kinase partially blocked tube formation. Taken together, these findings are consistent with the concept that PTX may lead to increased BBB permeability by altering endothelial plasticity and angiogenesis.     

PDE4 regulates tissue plasminogen activator expression of human brain microvascular endothelial cells

Introduction

Factors regulating brain tissue plasminogen activator (tPA) are pertinent for stroke. Recent observations have suggested a role for the phosphodiesterase-4 (PDE4) pathway in stroke pathogenesis, via an uncertain mechanism. We studied PDE4 regulation of tPA expression by human brain microvascular endothelial cells in a variety of conditions, including an in vitro model of ischemia.

Materials and Methods

We analyzed tPA antigen and mRNA of human brain microvascular endothelial cells (HBECs) during normoxia and oxygen-glucose deprivation (OGD) following inhibition of PDE4 and PDE4D, using HBEC monocultures and co-cultures with astrocytes and pericytes, and analyzed relevant signal transduction pathways.

Results

PDE4 inhibitor rolipram enhanced OGD effects on endothelial tPA release in endothelial monocultures and co-cultures with astrocytes; there was a 54 ± 10% (p < 0.001) reduction of tPA release in astrocyte-endothelial co-cultures under OGD. PDE4D siRNA reduced endothelial tPA mRNA to 40-55% of control (p < 0.05). Use of Epac inducer mimicked, while use of Epac siRNA inhibited, these effects.

Conclusions

Inhibition of PDE4 and PDE4D reduced expression of tPA by HBEC via Epac pathway.     

Human immunodeficiency virus type 1 Tat-mediated cytotoxicity of human brain microvascular endothelial cells

Human immunodeficiency virus (HIV)-1 infection is often complicated with neurologic disorders, but the pathogenesis of HIV-1 encephalopathy is incompletely understood. Tat (HIV-1 transactivator protein) is released from HIV-1-infected cells and has been detected in the sera and cerebrospinal fluid of HIV-1-infected patients. Tat, along with increased inflammatory cytokines such as interferon-gamma (IFN-gamma), have been implicated in the pathogenesis of HIV-1-associated blood-brain barrier dysfunction. The present study examined the effects of Tat and IFN-gamma on human brain microvascular endothelial cells (HBMECs), which constitute the blood-brain barrier. Tat produced cytotoxicity of HBMECs, but required IFN-gamma. IFN-gamma treatment of HBMECs up-regulates vascular endothelial growth factor receptor-2 (VEGFR2/KDR), which is known to be the receptor for Tat. Tat activated KDR in the presence of IFN-gamma, and Tat-mediated cytopathic changes involve its interaction with KDR and phosphatidylinositol 3-kinase (PI3K). Further understanding and characterization of Tat-HBMEC interactions should help us understand HIV-1 neuropathogenesis and develop strategies to prevent HIV-1 encephalopathy.     

P-glycoprotein and multidrug resistance-associated protein are involved in the regulation of extracellular levels of the major antiepileptic drug carbamazepine in the brain.

Despite considerable advances in the pharmacotherapy of epilepsy, about 30% of epileptic patients are refractory to antiepileptic drugs (AEDs). In most cases, a patient who is resistant to one major AED is also refractory to other AEDs, although these drugs act by different mechanisms. The mechanisms that lead to drug resistance in epilepsy are not known. Recently, over-expression of multidrug transporters, such as P-glycoprotein (PGP) and multidrug resistance-associated protein (MRP), has been reported in surgically resected epileptogenic human brain tissue and suggested to contribute to the drug resistance of epilepsy. However, it is not known to what extent multidrug transporters such as PGP or MRP are involved in transport of AEDs. In the present study, we used in vivo microdialysis in rats to study whether the concentration of carbamazepine in the extracellular fluid of the cerebral cortex can be enhanced by inhibition of PGP or MRP, using the PGP inhibitor verapamil and the MRP inhibitor probenecid. Local perfusion with verapamil or probenecid via the microdialysis probe increased the extracellular concentration of carbamazepine. The data indicate that both PGP and MRP participate in the regulation of extracellular brain concentrations of the major AED carbamazepine.     

脑微血管内皮细胞和脑肿瘤干细胞共培养的体外实验研究

目的 研究体外培养条件下,脑微血管内皮细胞(brain microvascular endothelial cells,BMECs)对脑肿瘤干细胞(braintumor stem cells,BTSCs)增殖、自我更新及分化功能的影响.方法 应用Transwell小室建立BMECs与BTSCs共培养模型,并建立对照组,共培养14 d后,测量肿瘤球(brain tumor sphere,BTS)直径大小,检测BTSCs增殖曲线,测定单细胞克隆形成率.在含血清的培养基中培养10 d后,行CD133、Nestin、GFAP、DAPI免疫荧光染色,计数两组的CD133、Nestin、GFAP染色的细胞数及同一视野的DAPI染色细胞核数,观察两组之间的差异.结果 共培养组BTS直径是对照组的5.05倍,增殖快,共培养组有61.4%能形成下一代BTSCs,而对照组为39.0%.在含血清的培养基中培养10 d后,共培养组的GFAP阳性率较对照组低,而CD133、Nestin阳性率较对照组高.结论 BMECs能够促进BTSCs增殖、自我更新能力,保持BTSCs未分化状态.     

Rapid morphogenesis of brain microvascular endothelial cells in culture system]

Endothelial cells of bovine brain microvascular vessels (BBECs), carotid artery (BCECs) and aorta (BAECs) were cultured on type I collagen and Matrigel. BBECs make tubular structures and BCECs and BAECs grow and make confluent monolayer on type I collagen gel. But BCECs and BAECs make tubular structures when second layer was overlaid. BBECs, BCECs and BAECs make tubular structures on Matrigel. These morphological changes were not affected by basic fibroblast growth factor. These results suggest that BBECs have more potent angiogenic ability than BCECs and BAECs.     

Secretion of brain-derived neurotrophic factor from brain microvascular endothelial cells

The cerebral microvasculature has recently been identified as a source of factors that can influence the generation and survival of neurons, including brain-derived neurotrophic factor (BDNF). However, relatively little is known about signals that regulate secretion of endothelial cell derived BDNF. To approach this issue the present study examined BDNF secretion from brain endothelial cells in response to reduced oxygen availability (hypoxia), using the mouse brain microvascular endothelial cell line, bEnd.3. We found that exposure of bEnd.3 cells to either sustained or intermittent hypoxia (IH) stimulates BDNF expression and release and that IH is the more potent stimulus. IH-induced BDNF release can be partially inhibited by either N-acetyl-L-cysteine, a scavenger of reactive oxygen species, or by the stable superoxide dismutase mimetic manganese(III)tetrakis1-methyl-4-pyridylporphyrin, indicating that oxyradical formation contributes to enhanced secretion of BDNF. In addition, we found that IH-induced BDNF release requires Ca2+ mobilization from internal stores through ryanodine- and inositol (1,4,5-triphosphate) IP3 receptors and is completely blocked by SKF 96365, a nonselective inhibitor of transient receptor potential (TRP) channels. These data demonstrate that bEnd.3 cells respond to oxidative stress by increasing BDNF secretion and, in addition, highlight TRP channels as potential therapeutic targets for enhancing BDNF availability from the cerebral microvasculature.     

Dynamic observation of oxygenation-induced contraction of and transient fiber-network formation-disassembly in cultured human brain microvascular endothelial cells.

Oxygenation-induced contraction of nonconfluent cultured human brain microvascular endothelial cells (HBECs, n = 30) was examined by video-enhanced contrast-differential interferential contrast microscopy. After administering a continuous gentle blow of pure oxygen gas to the surface of the medium just above the flattened HBEC, the plasma membrane exhibited tensioning and wrinkling, resulting in a strong contraction of the cell body by 14 +/- 7% (P < 0.001). When the cell stopped contracting, transient formation of a fiber network starting from certain spots (possibly adhesion plaques, though these were not visible in the majority of cases) and expanding to the whole cell was observed. The occurrence of fiber network formation was statistically significant (26 of 30 separate cells, P < 0.05). After cessation of oxygen delivery, the observed network of fibers broke up rapidly (in a period of 3.3 +/- 1.2 seconds) into small particles of <0.5 microm in diameter, which subsequently fused into the cellular structure. The HBEC completely recovered the control appearance. The sequential process was completed within 30 seconds and was reproduced in individual cells each time that oxygen gas was supplied. The authors conclude that the HBEC strongly contracts in response to a transient oxygenation stimulus, followed by rapid formation/disassembly of a network structure.     

Aluminum-induced alteration of surface anionic sites in cultured brain microvascular endothelial cells

Confluent monolayers of cultured microvascular endothelial cells from goat brain were used as an in vitro model system of the blood-brain barrier to study the effect of aluminum on the anionic sites located on the cell surface. Four experimental groups were used: (1) untreated (control 1) cells growing in normal medium; (2) cells growing in a medium containing 50 M maltol (control 2); (3) cells growing in a medium containing 50 M aluminum maltolate; and (4) cells growing in a medium containing 6 M cadmium chloride as a known cytotoxic substance. The cell cultures were exposed to the substances listed above for 4 days. The anionic sites were detected at the ultrastructural level with cationic colloidal gold and cationized ferritin in non-fixed and fixed cells (prefixation and postfixation procedures). In the applied experimental conditions, aluminum maltolate was found to affect the surface density of anionic sites, as manifested by their redistribution and segmental disappearance from the apical plasmalemma of the endothelial cells. These changes were more pronounced in the non-fixed than the fixed cells and are reversible. Most probably, aluminum, because of its unique properties as a cross-linker, facilitates the lateral migration of anionic sites induced by cationic probes. Whether these alterations contribute to in vivo aluminum-induced blood-brain barrier dysfunction requires further elucidation.Supported in part by funds from the New York State Office of Mental Retardation and Developmental Disabilities and the fund for the Center for Trace Metal Studies and Environmental Neurotoxicology     

Interactions between HIV-infected monocyte-derived macrophages and human brain microvascular endothelial cells result in increased expression of CC chemokines

The presence of perivascular monocytic infiltration is a major hallmark of HIV-1-associated dementia. Since CC chemokines are chemoattractant cytokines that are able to attract T cells and monocytes/macrophages to sites of inflammation, and since infiltrating monocytes/macrophages remain in close contact with the brain endothelium, we investigated whether interactions between HIV-1-infected macrophages and brain endothelium result in an altered chemokine production. We found an increased mRNA expression of monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, and RANTES by macrophages after HIV-1 infection. Interactions between HIV-infected macrophages and brain microvascular endothelial cells resulted in an additional upregulation of chemokine mRNA expression, during cell-cell contact as well as in a trans-well system. Since IL-1 beta can function as a modulator of chemokine expression we investigated if interleukin-1 beta could be involved in the regulation of chemokine induction. Coculturing of HIV-infected macrophages and endothelial cells resulted in immune-activation as indicated by increased mRNA expression of IL-1 beta. Subsequently, addition of a neutralizing antibody against IL-1 beta resulted in altered chemokine expression by macrophages, but not by endothelial cells. Thus, IL-1 beta appears to play a major role in the regulation of chemokines during cellular interactions in HIV-associated dementia, but other factors may also be involved.     

Antiheparin activity in human endothelial cells

Human endothelial cells possess antiheparin activity that neutralizes the anticoagulant action of heparin as measured by different tests of the clotting system. The antiheparin activity appears to be associated with an acid-soluble basic protein present in the particulate fraction of the endothelial cell cytoplasm. This finding might have some relevance in the maintenance of hemostasis. Furthermore, it might also have a pharmacological role in terms of resistance to exogenously infused heparin in patients with thromboembolic disorders.     

P-glycoprotein expression in brain capillary endothelial cells after focal ischaemia in the rat

Abstract

P-glycoprotein (P-gp) is expressed not only in tumour cells but also in some normal tissues including brain capillaries. We investigated whether or not P-gp was expressed in the capillary, endothelial cells of a rat focal ischaemic brain. The brains were immunohistochemically studied for Factor VIII, glial fibrillary acidic protein (GFAP), and P-gp. Endothelial γ-glutamyl transpeptidase (γ-GTP) activity; which is thought to be induced by glial cells, was also studied histochemically. The P-gp positive endothelial cells disappeared in the ischaemic lesion by post-ischaemic day 3. Factor Vlll-positive regenerating capillaries were first observed on day 3 without P-gp expression.. The P-gp positive endothelial cells began to reappear on day 5, and were detected in all the endothelial cells by day 8. The P-gp expression in endothelial cells showed a similar pattern as that of y-GTP, and seemed to correlate with GFAP-positive reactive astrocytes. The newly-formed brain capillaries thus appeared to have a potential to express P-gp in abnormal pathogenic conditions as cerebral infarction, and our present study also suggested that P-gp in the brain capillaries might therefore be expressed in conjunction with glial cells. [Neurol Res 1994; 16: 217–223]