黄芩苷作用于脑微血管内皮细胞和星形胶质细胞对

背景：实验室前期研究表明黄芩苷可以提高体外培养的神经干细胞分化为神经元的比例。但考虑到体内实验中血脑屏障的存在,黄芩苷能否通过血脑屏障主要细胞成分而发挥诱导神经干细胞定向分化为神经元的能力还不清楚。 目的：分别将大鼠脑微血管内皮细胞、星形胶质细胞与神经干细胞共培养,观察在模拟体内复杂微环境条件下,黄芩苷能否定向诱导神经干细胞向神经元分化并促进分化神经元的成熟。 设计、时间及地点：细胞水平的体外对照观察,于2007-09/2008-03在天津中医药大学中医药研究院中药药理学重点实验室和南开大学医学院完成。 材料：取孕14 d SD大鼠,用以分离培养神经干细胞。 方法：利用Transwell装置,分别将脑微血管内皮细胞、星形胶质细胞和神经干细胞共培养。用含有10 μmol/L黄芩苷的培养基作用7 d,并设置空白对照组。以β-tubulinⅢ标记未成熟神经元,MAP-2标记成熟神经元,胶质纤维酸性蛋白标记星形胶质细胞。 主要观察指标：应用细胞免疫荧光化学染色检测神经干细胞分化后β-tubulinⅢ、MAP-2和胶质纤维酸性蛋白阳性细胞比例,以实时荧光定量反转录-聚合酶链反应技术检测黄芩苷对脑微血管内皮细胞和星形胶质细胞血管内皮细胞生长因子、神经生长因子和血小板衍生生长因子 mRNA表达的影响。 结果：与脑微血管内皮细胞共培养条件下,与空白对照组比较,黄芩苷可显著增加β-tubulinⅢ阳性细胞比例（P < 0.05）。与星形胶质细胞共培养条件下,与空白对照组比较,黄芩苷对β-tubulinⅢ、MAP-2和胶质纤维酸性蛋白阳性细胞比例均无明显影响（P > 0.05）。与脑微血管内皮细胞、星形胶质细胞共培养条件下,与空白对照组比较,黄芩苷可显著增加MAP-2阳性细胞比例（P < 0.01）。黄芩苷作用于脑微血管内皮细胞48 h,可以显著上调血小板衍生生长因子基因表达（P < 0.01）;作用72 h可显著上调星形胶质细胞血管内皮细胞生长因子、神经生长因子和血小板衍生生长因子基因表达（P < 0.01）。 结论：黄芩苷作用于脑微血管内皮细胞可诱导神经干细胞向神经元分化,黄芩苷同时作用于脑微血管内皮细胞和星形胶质细胞可诱导神经干细胞向神经元定向分化并促进其成熟,可能与黄芩苷调控脑微血管内皮细胞和星形胶质细胞生长因子分泌,改善微环境有关。     

Immunocompetence of human microvascular brain endothelial cells: cytokine regulation of IL-1β, MCP-1, IL-10, sICAM-1 and sVCAM-1

Endothelia from the brains of four patients undergoing neurosurgery, including one multiple sclerosis (MS) patient, were studied in vitro to determine cytokine and chemokine production; the release of soluble adhesion molecules was also investigated. The same procedure was repeated on human umbilical vein endothelial cells (HUVECs) in order to detect possible district-specific differences. After isolation, the endothelium was cultured and stimulated with γ-interferon (IFN), tumour necrosis factor alpha (TNF-α) and LPS. The results showed that brain endothelium, in our experimental conditions, does not produce interleukin (IL)-10 and produces lower amounts of IL-1β and soluble intercellular adhesion molecule-1 (sICAM-1) than HUVECs do; no differences were detected in soluble vascular cell adhesion molecule-1 (sVCAM-1) production. MCP-1 mRNA was detected both without and after stimulation with TNF-α and γ-IFN in HUVECs and MS human brain endothelial cells (HBECs), while in non-MS-HBECs it was found only after γ-IFN stimulation. Received: 24 June 1997 Received in revised form: 20 February 1998 Accepted: 5 March 1988     

Calcium signaling in and between brain astrocytes and endothelial cells

Two in vitro co-culture models of the blood-brain barrier (BBB) were developed using primary rat cortical astrocytes and ECV304 endothelial cells or primary rat brain capillary endothelial cells. We showed that intercellular calcium (Ca2+) waves can mediate bidirectional astrocyte-endothelial Ca2+ signal communication in both co-culture models. It appears that two signaling pathways are involved. A first mechanism is related to intercellular diffusion of the Ca2+ mobilizing messenger inositoltrisphosphate (IP3) through gap junctions as studied with flash photolysis of caged-IP3. A second pathway involves extracellular diffusion of a purinergic messenger as studied with the purinergic inhibitors apyrase and suramin. Using gap junction-deficient HeLa cells and connexin-GFP transfected HeLa cells, the existence of the two Ca2+ signaling pathways mentioned was confirmed and their subcellular characteristics and differences were further studied. We also suggest that astrocytic intercellular Ca2+ waves may be accompanied by and contribute to some of the ionic shifts observed in the brain upon traumatic brain injury. Further work will be needed to confirm our data in brain slices or even in the brain in vivo, and to establish the role and function of intercellular Ca2+ signals at the BBB.     

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.     

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.     

Expression of the beta-chemokines RANTES and MIP-1 beta by human brain microvessel endothelial cells in primary culture

The mechanisms that regulate inflammatory cell recruitment across the blood-brain barrier (BBB) during CNS inflammation have not been fully characterized. Likely players in this process include the chemokines, small secondary messengers of inflammation capable of subset-specific leukocyte activation and chemoattraction. Primary cultures of human brain microvessel endothelial cells (HBMEC) were examined for their in vitro expression of the beta chemokines RANTES and MIP-1beta. Untreated HBMEC expressed low levels of RANTES and MIP-1beta RNA that were significantly upregulated following cytokine treatment. Parallel studies performed on human umbilical vein endothelial cells (HUVEC) showed induction of RANTES but not MIP-1beta RNA. Following stimulation with LPS, TNF-alpha, IFN-gamma, and IL-1beta alone or in combination, HBMEC released significant amounts of RANTES and MIP-1beta into the culture supernatants. RANTES secretion by HUVEC could be induced only with TNF-alpha/IFN-gamma. Both RANTES and MIP-1beta were detected by immunocytochemistry on the apical and basal surfaces of HBMEC, as well as bound to basal lamina-like material under the basal cell surface. Cytokine stimulation induced significant increase of RANTES and MIP-1beta molecules associated with the EC surface and subendothelial matrix. The expression of RANTES and MIP-1beta by HBMEC suggests that these chemokines may play an important role in mediating inflammatory responses and leukocyte trafficking across the BBB.     

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.     

Organic anion transporting polypeptide 2 transports valproic acid in rat brain microvascular endothelial cells

Objectives: Abnormal drug transporter expression or function in the brain may lead to decreased concentrations of antiepileptic drugs (AEDs) in the central nervous system in patients with drug-resistant epilepsy. We previously showed the influx transporter organic anion transport polypeptide 2 (Oatp2) was expressed in rat brain microvascular endothelial cells (BMECs). Seizures decrease expression of Oatp2, but it remains unclear whether Oatp2 transports AEDs. In this study, we utilized rat BMECs as an in vitro model of the blood–brain barrier (BBB) to study Oatp2-mediated transport of valproic acid (VPA), the most common clinically used AEDs.

Methods: In vivo injection of pregnenolone-16-carbonitrile was used to induce high expression of Oatp2 in isolated BMECs. Small interfering RNA treatment was used to silence Oatp2, and uptake of VPA was assessed.

Results: Increased expression of Oatp2 in BMECs increased the uptake of VPA, while inhibition of Oatp2 reduced VPA uptake.

Discussion: This study indicates Oatp2 transports VPA across the BBB, and suggests altered Oatp2 expression may contribute to resistance to VPA in patients with drug-resistant epilepsy.     

HIV-1 viral proteins gp120 and Tat induce oxidative stress in brain endothelial cells

The blood-brain barrier (BBB) has an important role in the development of AIDS dementia. The HIV-1 envelope glycoprotein (gp120) and transregulatory protein (Tat) of HIV-1 are neurotoxic and cytotoxic and have been implicated in the development of HIV dementia. They are known to cause oxidative stress and are associated with disruption of the BBB. Here, we used an immortalized endothelial cell line from rat brain capillaries, RBE4, to determine whether gp120 and Tat can induce oxidative stress in an in vitro model of the BBB. RBE4 cells were exposed to gp120 or Tat and the levels of reduced glutathione (GSH), oxidized glutathione (GSSG), catalase (CAT) activity, glutathione peroxidase (GPx) activity, and glutathione reductase (GR) activity, and malondialdehyde (MDA) used as measures of oxidative stress. Both gp120 and Tat significantly decreased the levels of intracellular GSH, GPx, and GR and increased the levels of MDA in RBE4 cells, showing that the cells were oxidatively challenged. The ratio of GSH/GSSG, a widely accepted indicator of oxidative stress, was also significantly decreased. These studies show that both of these viral proteins can induce oxidative stress in immortalized BBB endothelial cells.     

Development of membrane interactions between brain endothelial cells and astrocytes in vitro

To ascertain whether there is a mutual influence on the structure of their cell membranes, brain endothelial cells and their closest neighbor, astrocytes, were grown alone or together in vitro and freeze-fractured. When cultured separately, the brain endothelial cells had a low frequency of short, fragmented tight junctions. Many gap junctions, which are absent from mature brain capillaries in vivo, intercalated among the tight junctional strands, or were separate from them. The separately cultured astrocytes had low concentrations of randomly distributed assemblies (1-30/micron2) in their membranes. When the two cell types were co-cultured, the endothelial tight junctions were greatly enhanced in frequency, length, width and complexity, and the gap junctional area enclosed by the tight junctional strands were markedly reduced. Thus, the in vitro endothelial junctional complex resembled their in vivo counterpart, the tight junctions of brain capillaries, when co-cultured with astrocytes. Reciprocally, brain endothelial cells induced the astrocytic membrane assemblies to increase in concentrations by approximately 5 fold, and sometimes to form aggregates with very high concentrations (400/micron 2) which approached the concentration of the perivascular astrocytic membranes in vivo. Substituting astrocytes with fibroblasts or smooth muscle cells in co-cultures did not enhance the tight junctions in the brain endothelium. On the other hand, substituting brain endothelium with endothelium from pulmonary artery or aorta in co-cultures did not increase the concentration or induce aggregation of the assemblies in the astrocytes. Thus, the two close neighbors in vivo, brain endothelium and astrocytes, interact specifically in vitro to induce development of membrane specializations which resemble those at the site of the blood-brain barrier.     

Astrocytes induce manganese superoxide dismutase in brain capillary endothelial cells

Astrocytes induce blood-brain barrier (BBB) properties in brain endothelial cells (EC)*O(2)*, generated in blood and EC, opens the BBB. Hence, high activity of superoxide dismutase (SOD) is a prerequisite for normal BBB function. Therefore, the influence of rat astrocytes on the expression of manganese (Mn)SOD in rat EC was investigated in two coculture models of the BBB, allowing either exchange of soluble factors or additionally cellular contacts. Activity, protein content and mRNA expression of endothelial MnSOD were significantly increased in both coculture models in comparison to monoculture by soluble astrocytic factors, such as cytokines. High activity of endothelial MnSOD may be considered as a further essential property of the BBB, which is induced and maintained by astrocytes.     

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.     

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.     

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     

Long-term culture of microvascular endothelial cells derived from Mongolian gerbil brain

A method for long-term culture of microvascular endothelial cells from Mongolian gerbil brain and their biologic properties in vitro are described. Microvessels were isolated from Mongolian gerbil brain by a combination of enzymatic treatment, filtration, and centrifugation and were seeded onto a gelatin-coated dish. A morphologically homogeneous cell plaque showing a cobblestone appearance was removed 2 to 3 weeks after the seeding, and the cells were subcultured. The cultured cells grew as monolayers of flat polygonal cells and were carried for more than 20 passages without morphologic change. These cells synthesized prostacyclin and retained an endothelial specific marker, factor VIII-related antigen. When the cells were cultured in a collagen gel, they rapidly formed capillarylike tubular structures without endothelial cell growth factor or special substrata. Long-term culture of purified microvascular endothelial cells derived from Mongolian gerbil brain will facilitate the study of the function of microvascular endothelial cells in human brain under normal and pathologic conditions.