West Nile Virus-Induced Cell Adhesion Molecules on Human Brain Microvascular Endothelial Cells Regulate Leukocyte Adhesion and Modulate Permeability of the In Vitro Blood-Brain Barrier Model

Characterizing the mechanisms by which West Nile virus (WNV) causes blood-brain barrier (BBB) disruption, leukocyte infiltration into the brain and neuroinflammation is important to understand the pathogenesis of WNV encephalitis. Here, we examined the role of endothelial cell adhesion molecules (CAMs) in mediating the adhesion and transendothelial migration of leukocytes across human brain microvascular endothelial cells (HBMVE). Infection with WNV (NY99 strain) significantly induced ICAM-1, VCAM-1, and E-selectin in human endothelial cells and infected mice brain, although the levels of their ligands on leukocytes (VLA-4, LFA-1and MAC-1) did not alter. The permeability of the in vitro BBB model increased dramatically following the transmigration of monocytes and lymphocytes across the models infected with WNV, which was reversed in the presence of a cocktail of blocking antibodies against ICAM-1, VCAM-1, and E-selectin. Further, WNV infection of HBMVE significantly increased leukocyte adhesion to the HBMVE monolayer and transmigration across the infected BBB model. The blockade of these CAMs reduced the adhesion and transmigration of leukocytes across the infected BBB model. Further, comparison of infection with highly neuroinvasive NY99 and non-lethal (Eg101) strain of WNV demonstrated similar level of virus replication and fold-increase of CAMs in HBMVE cells suggesting that the non-neuropathogenic response of Eg101 is not because of its inability to infect HBMVE cells. Collectively, these results suggest that increased expression of specific CAMs is a pathological event associated with WNV infection and may contribute to leukocyte infiltration and BBB disruption in vivo. Our data further implicate that strategies to block CAMs to reduce BBB disruption may limit neuroinflammation and virus-CNS entry via ‘Trojan horse’ route, and improve WNV disease outcome.     

Electroporation of Brain Endothelial Cells on Chip toward Permeabilizing the Blood-Brain Barrier

The blood-brain barrier, mainly composed of brain microvascular endothelial cells, poses an obstacle to drug delivery to the brain. Controlled permeabilization of the constituent brain endothelial cells can result in overcoming this barrier and increasing transcellular transport across it. Electroporation is a biophysical phenomenon that has shown potential in permeabilizing and overcoming this barrier. In this study we developed a microengineered in vitro model to characterize the permeabilization of adhered brain endothelial cells to large molecules in response to applied pulsed electric fields. We found the distribution of affected cells by reversible and irreversible electroporation, and quantified the uptaken amount of naturally impermeable molecules into the cells as a result of applied pulse magnitude and number of pulses. We achieved 81 ± 1.7% (N = 6) electroporated cells with 17 ± 8% (N = 5) cell death using an electric-field magnitude of ∼580 V/cm and 10 pulses. Our results provide the proper range for applied electric-field intensity and number of pulses for safe permeabilization without significantly compromising cell viability. Our results demonstrate that it is possible to permeabilize the endothelial cells of the BBB in a controlled manner, therefore lending to the feasibility of using pulsed electric fields to increase drug transport across the BBB through the transcellular pathway.     

Hypothalamic Extract Influences a Blood-Brain Barrier Model of Porcine Brain Capillary Endothelial Cells

The purpose of this study was to investigate the influence of hypothalamic extract, astrocyte co-culture, and astrocyte-conditioned medium on the barrier function of an in vitro model of the blood-brain barrier. Porcine brain capillary endothelial cells were grown on polycarbonate membranes suspended between two chambers of media, representing the capillary lumen and brain interstitium. Endothelial cells grown alone and cocultured with astrocytes were cultured in growth medium with or without 50 g/mL hypothalamic extract. An additional treatment consisted of endothelial cells cultured in growth medium that was first conditioned by astrocytes. Coculture consisted of a noncontact model with astrocytes attached to the bottom of the abluminal chamber. Barrier function of the endothelial cells was tested on days 1 through 9 post-seeding by measuring permeability to macromolecules (albumin) and small ions (electrical resistance). Resistance to the passage of macromolecules and small ions was greatest for endothelial cells grown without astro-cytes in growth medium supplemented with hypothalamic extract. This barrier was maximal during days 4 through 7 post-seeding and was significantly less permeable than the barrier formed by endothelial cells grown in un-supplemented growth medium, in coculture with astrocytes, or in astrocyte-conditioned medium. These results demonstrate that a noncontact coculture with astro-cytes did not enhance the integrity of this in vitro BBB model employing porcine brain capillary endothelial cells, but barrier function was increased when the model's medium was supplemented with hypothalamic extract.     

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. ¹). Human tissue samples are available only in a restricted number of laboratories or companies ^2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND ⁴ (from cerebral cortex) and cerebEND ⁵ (from cerebellar cortex), were isolated according to this procedure in the Förster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- ^4,6-9 and estrogen-treatment ¹⁰ as well as to pro-infammatory mediators, such as TNFalpha ^5,8. Moreover, we have studied the pathology of multiple sclerosis ¹¹ and hypoxia ^12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.     

大鼠脑微血管内皮细胞培养及其药物转运体Oatp2和P-gp的表达

贴块法培养脑微血管内皮细胞(BMECs),倒置显微镜动态观察细胞生长及形态,Ⅷ因子相关抗原、CD34免疫细胞化学联合鉴定细胞并确定纯度。免疫细胞化学和Western印迹法检测药物转运体有机阴离子转运多肽亚型2(Oatp2)及P-糖蛋白(P-gp)在培养内皮细胞上的表达。结果显示,获得的BMECs呈多角形或铺路石形,单层贴壁生长;培养细胞Ⅷ因子相关抗原免疫细胞化学、CD34免疫荧光染色均为阳性,细胞纯度90%;培养细胞有Oatp2及P-gp表达,且二者均主要表达于BMECs细胞膜。提示贴块法可获得原代培养BMECs,方法简便易行,细胞纯度较高。原代培养的BMECs上有药物转运体Oatp2及P-gp的表达,为血脑屏障上药物转运体的体外研究提供了可能途径。     

Edaravone Protects against Methylglyoxal-Induced Barrier Damage in Human Brain Endothelial Cells

Background

Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line) treated with methylglyoxal.

Methodology

Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging.

Principal Findings

Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM) provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound.

Conclusion

These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases.     

Synthesis of Apolipoprotein A-1 in Pig Brain Microvascular Endothelial Cells

In an approach toward the identification of hitherto unknown proteins involved in the function of the blood-brain barrier, we constructed a pig brain microvessel-derived cDNA library that is enriched in blood-brain barrier specific sequences by means of subtractive cloning. Sequence analysis of selected clones revealed that one of the cDNAs encoded porcine apolipoprotein (apo) A-1. The identity of apo A-1 mRNA was further confirmed by in vitro translation of RNA from brain microvascular endothelial cells and subsequent immunoprecipitation with an antibody against human apo A-1. We further investigated the expression of apo A-1 mRNA in several tissues and in endothelial cells of the pig. It is shown that cultured brain microvascular endothelial cells provide an in vitro model to study the expression and function of apo A-1 in the microvasculature of the brain.     

The Involvement of CXCL11 in Bone Marrow-Derived Mesenchymal Stem Cell Migration Through Human Brain Microvascular Endothelial Cells

Bone marrow-derived mesenchymal stem cells (MSCs) transplant into the brain, where they play a potential therapeutic role in neurological diseases. However, the blood–brain barrier (BBB) is a native obstacle for MSCs entry into the brain. Little is known about the mechanism behind MSCs migration across the BBB. In the present study, we modeled the interactions between human MSCs (hMSCs) and human brain microvascular endothelial cells (HBMECs) to mimic the BBB microenvironment. Real-time PCR analysis indicated that the chemokine CXCL11 is produced by hMSCs and the chemokine receptor CXCR3 is expressed on HBMECs. Further results indicate that CXCL11 secreted by hMSCs may interact with CXCR3 on HBMECs to induce the disassembly of tight junctions through the activation of ERK1/2 signaling in the endothelium, which promotes MSCs transendothelial migration. These findings are relevant for understanding the biological responses of MSCs in BBB environments and helpful for the application of MSCs in neurological diseases.     

大肠杆菌yijP侵袭蛋白诱导人脑微血管内皮细胞凋亡

为研究大肠杆菌的脑微血管内皮细胞侵袭基因yijP的功能,将yijP基因（1．04kb）克隆到pQE30表达载体,构建表达产物为N末端带有6个组氨酸（His）序列的yijP汇合蛋白,以M15（pREP4）为受体菌,大量表达（His）6-yijP汇合蛋白,利用Ni—NTA亲和层析纯化汇合蛋白,将经透析法复性的一定浓度的（His）6-yijP蛋白加入到体外培养的人脑微血管内皮细胞中,结果显示yijP蛋白对人脑微血管内皮细胞有较强的细胞毒作用：在相差显微镜下可观察到细胞皱缩、胞膜呈泡状膨出,随着时间延长细胞逐渐脱落;荧光显微镜下可见细胞核呈现为致密团块状或圆形浓染颗粒状,呈凋亡样改变：DNA琼脂糖凝胶电泳可见DNA阶梯状条带;流式细胞仪显示在正常二倍体峰之前出现一个亚二倍体峰;Western印迹可检测到caspase-3的活性片段。这些现象均出现在yijP蛋白作用于人脑微血管内皮细胞的16h之后,提示在大肠杆菌侵袭人脑微血管内皮细胞过程中,yijP蛋白可能起到诱导脑微血管内皮细胞迟发性凋亡的毒素作用。     

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.     

Human Alveolar Epithelial Cells Attenuate Pulmonary Microvascular Endothelial Cell Permeability under Septic Conditions

Acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), are characterised by high-protein pulmonary edema and severe hypoxaemic respiratory failure due to increased permeability of pulmonary microvascular endothelial cells (PMVEC). Alveolar epithelial cells (AEC) contribute importantly to normal alveolar function, and AEC dysfunction in ALI/ARDS is associated with worse outcomes. We hypothesized that AEC can modulate human PMVEC barrier function, and investigated the effects of AEC presence on human PMVEC barrier under septic conditions in vitro. PMVEC isolated from human lung were treated in vitro with septic stimulation (lipopolysaccharide [LPS], a mixture of clinically-relevant cytokines [cytomix], or plasma from patients with severe sepsis), and the trans-PMVEC leak of Evans Blue dye-labeled albumin assessed. PMVEC septic responses were compared in the presence/absence of co-cultured A549 epithelial cell line or primary human AEC. Septic stimulation with LPS, cytomix, or septic plasma induced marked PMVEC hyper-permeability (10.2±1.8, 8.9±2.2, and 3.7±0.2 fold-increase vs. control, respectively, p<0.01 for all). The presence of A549 cells or primary human AEC in a non-contact co-culture model attenuated septic PMVEC hyper-permeability by 39±4% to 100±3%, depending on the septic stimulation (p<0.05). Septic PMVEC hyper-permeability was also attenuated following treatment with culture medium conditioned by previous incubation with either naïve or cytomix-treated A549 cells (p<0.05), and this protective effect of A549 cell-conditioned medium was both heat-stable and transferable following lipid extraction. Cytomix-stimulated PMN-dependent PMVEC hyper-permeability and trans-PMVEC PMN migration were also inhibited in the presence of A549 cells or A549 cell-conditioned medium (p<0.05). Human AEC appear to protect human PMVEC barrier function under septic conditions in vitro, through release of a soluble mediator(s), which are at least partly lipid in nature. This study suggests a scientific and potential clinical therapeutic importance of epithelial-endothelial cross talk in maintaining alveolar integrity in ALI/ARDS.     

An Immortalized Human Blood-Nerve Barrier Endothelial Cell Line for In Vitro Permeability Studies

Solute and macromolecular transport studies may elucidate nutritional requirements and drug effects in healthy and diseased peripheral nerves. Endoneurial endothelial cells are specialized microvascular cells that form the restrictive blood-nerve barrier (BNB). Primary human endoneurial endothelial cells (pHEndECs) are difficult to isolate, limiting their widespread availability for biomedical research. We developed a simian virus-40 large T-antigen (SV40-LTA) immortalized human BNB cell line via stable transfection of low passage pHEndECs and observed continuous growth in culture for >45 population doublings. As observed with pHEndECs, the immortalized BNB endothelial cells were Ulex Europaeus agglutinin-1-positive and endocytosed low density lipoprotein, but lost von Willebrand factor expression. Glucose transporter-1, P-glycoprotein (P-gp), γ-glutamyl transpeptidase (γ-GT), large neutral amino acid transporter-1 (LAT-1), creatine transporter (CRT), and monocarboxylate transporter-1 (MCT-1) mRNA expression were retained at all passages with loss of alkaline phosphatase (AP) expression after passages 16–20. Compared with an SV40-LTA immortalized human blood-brain barrier endothelial cell line, there was increased γ-GT protein expression, equivalent expression of organic anion transporting polypeptide-C (OATP-C), organic anion transporter 3 (OAT-3), MCT-1, and LAT-1, and reduced expression of AP, CRT, and P-gp by the BNB cell line at passage 20. Further studies demonstrated lower transendothelial electrical resistance (~181 vs. 191 Ω cm²), equivalent permeability to fluoresceinated sodium (4.84 vs. 4.39 %), and lower permeability to fluoresceinated high molecular weight (70 kDa) dextran (0.39 vs. 0.52 %) by the BNB cell line. This cell line retained essential molecular and biophysical properties suitable for in vitro peripheral nerve permeability studies.     

Characterization of the Blood-Brain Barrier: Protein Composition of the Capillary Endothelial Cell Membrane

Microvessels were isolated from canine cerebral cortex, and the composition of the endothelial cell membrane was investigated. Endothelial cell membranes were separated from the surrounding basement membrane, solubilized, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 12% gels. Staining with Coomassie Blue revealed a characteristic banding pattern of at least 12 major proteins with apparent molecular weights between 14,000 and 250,000. When proteins from red blood cell ghosts were run simultaneously, no similarities were observed, except for proteins at apparent molecular weights of 43,000 (band 3) and 35,000 (band 4). These two proteins migrated exactly to the positions of the erythrocyte proteins actin and glyceraldehyde 3-phosphate dehydrogenase, respectively. Membrane glycoproteins in gels were also examined by the use of fluorescent lectins. Of the fluorescein isothiocyanate-conjugated (FITC) lectins tested, only FITC-concanavalin A had an affinity for any membrane components. Diazotized [125I]iodosulfanilic acid, a membrane-impermeable reagent, was used to label the internal (lumen) cell surface and the external (antilumen) cell surface. Autoradiography and determination of radioactivity levels in gel slices showed that several proteins were specifically labeled, and that major differences in radioactivity of proteins existed in internal and external labeling experiments. It is concluded that the protein composition of the luminal membrane is different from that of the antiluminal membrane.     

Epithelial Ovarian Cancer-Induced Angiogenic Phenotype of Human Omental Microvascular Endothelial Cells May Occur Independently of VEGF Signaling

Epithelial ovarian cancer (EOC) metastasizes transcoelomically to the peritoneum and omentum, and despite surgery and chemotherapy, recurrent disease is likely. Metastasis requires the induction of proangiogenic changes in the omental microenvironment and EOC-induced omental angiogenesis is currently a key therapeutic target. In particular, antiangiogenic therapies targeting the vascular endothelial growth factor A (VEGFA) pathway are commonly used, although, with limited effects. Here, using human omental microvascular endothelial cells (HOMECs) and ovarian cancer cell lines as an in vitro model, we show that factors secreted from EOC cells increased proliferation, migration, and tube-like structure formation in HOMECs. However, EOC-induced angiogenic tube-like formation and migration were unaffected by inhibition of tyrosine kinase activity of VEGF receptors 1 and 2 (Semaxanib; SU5416) or neutralization of VEGFA (neutralizing anti-VEGFA antibody), although VEGFA₁₆₅-induced HOMEC migration and tube-like structure formation were abolished. Proteomic investigation of the EOC secretome identified several alternative angiogenesis-related proteins. We screened these for their ability to induce an angiogenic phenotype in HOMECs, i.e., proliferation, migration, and tube-like structure formation. Hepatocyte growth factor (HGF) and insulin-like growth factor binding protein 7 (IGFBP-7) increased all three parameters, and cathepsin L (CL) increased migration and tubule formation. Further investigation confirmed expression of the HGF receptor c-Met in HOMECs. HGF- and EOC-induced proliferation and angiogenic tube structure formation were blocked by the c-Met inhibitor PF04217903. Our results highlight key alternative angiogenic mediators for metastatic EOC, namely, HGF, CL, and IGFBP-7, suggesting that effective antiangiogenic therapeutic strategies for this disease require inhibition of multiple angiogenic pathways.     

Peptide Derived from HIV-1 TAT Protein Destabilizes a Monolayer of Endothelial Cells in an in Vitro Model of the Blood-Brain Barrier and Allows Permeation of High Molecular Weight Proteins

Most chemotherapeutic agents are blood-brain barrier (BBB) impermeants. HIV-1-derived TAT protein variants contain a transmembrane domain, which may enable them to cross the BBB and reach the brain. Here we synthesized CAYGRKKRRQRRR, a peptide containing a cysteine moiety attached to the N terminus of the transmembrane domain (C-TAT peptide), and studied its effects in an in vitro BBB model, which we found to reflect penetration by a receptor-independent pathway. Incubation of the brain capillary endothelial cell monolayer with 0.3–0.6 μmol/ml of this C-TAT peptide, for a period of 1–2 h, destabilizes brain capillary endothelial cell monolayer and introduces the ability of impermeant therapeutic agents including high molecular weight proteins to penetrate it substantially. The cysteinyl moiety at position 1 of the C-TAT peptide contributes largely to the destabilizing potency and the penetration efficacy of impermeant substances. The destabilizing effect was reversed using heparin. In summary, experimental conditions allowing a significant increase in entry of impermeant low and high molecular weight substances from the luminal (blood) to the abluminal side (brain) were found in an in vitro BBB model reflecting in vivo protein penetrability by a receptor-independent pathway.