Divergent effects of zinc depletion in brain vs non-brain endothelial cells

Dietary zinc deficiency is common in developing as well as developed countries. Endothelial cells (EC) lining the inner surface of peripheral blood vessels are sensitive to zinc deficiency and lose structural integrity when exposed to culture media low in zinc or to zinc chelators. In contrast, we demonstrate here that human brain microvascular EC (HBMEC), which constitute the blood-brain barrier (BBB), resist zinc depletion and respond by enhancing their barrier function. This response was specific for HBMEC and did not occur in non-brain EC, such as human umbilical vein endothelial cells, human aortic endothelial cells, and human iliac vein endothelial cells. Our results suggest the presence of specific mechanisms to counteract zinc deficiency at the BBB, likely involving HBMEC junctional complexes. Understanding the mechanisms involved in this unique response might provide means to modulate the BBB dysfunction associated with neurological disorders such as stroke, multiple sclerosis, and Alzheimer's disease.     

Human brain endothelial cells are responsive to adenosine receptor activation

The blood–brain barrier (BBB) of the central nervous system (CNS) consists of a unique subset of endothelial cells that possess tight junctions which form a relatively impervious physical barrier to a large variety of blood components. Until recently, there have been no good in vitro models for studying the human BBB without the co-culture of feeder cells. The hCMEC/D3 cell line is the first stable, well-differentiated human brain endothelial cell line that grows independently in culture with characteristics that closely resemble those of resident human brain endothelial cells. As our previously published findings demonstrated the importance of adenosine receptor (AR) signaling for lymphocyte entry into the CNS, we wanted to determine if human brain endothelial cells possess the capacity to generate and respond to extracellular adenosine. Utilizing the hCMEC/D3 cell line, we determined that these cells express CD73, the cell surface enzyme that converts extracellular AMP to adenosine. When grown under normal conditions, these cells also express the A₁, A_2A, and A_2B AR subtypes. Additionally, hCMEC/D3 cells are responsive to extracellular AR signaling, as cAMP levels increase following the addition of the broad spectrum AR agonist 5′-N-ethylcarboxamidoadenosine (NECA). Overall, these results indicate that human brain endothelial cells, and most likely the human BBB, have the capacity to synthesize and respond to extracellular adenosine.     

Growth of brain microvessel endothelial cells on collagen gels: Applications to the study of blood-brain barrier physiology and CNS inflammation

Summary Brain microvessel endothelial cells (BMEC) exhibit the tendency to migrate through 3.0-vm pore semipermeable inserts and establish monolayers on both apical and basal filter surfaces. This can potentially lead to complications in accurately assessing a wide variety of physiologic parameters uniquely associated with these cells. To avoid this problem, we have explored growing BMEC on Transwell filters coated with hydrated collagen gels. BMEC seeded on such gels grow as a monolayer until confluency, but do not invade the subendothelial collagen matrix or the underlying support filter. Furthermore, BMEC grown in this manner exhibit biochemical, morphologic, and electrophysiologic properties reflective of the endothelial cells that comprise the blood-brain barrier in vivo. Although the collagen gel acts as an impenetrable barrier to BMEC, and thus ensures the growth of only a single layer of cells, it nevertheless can be infiltrated by monocytes that have been stimulated by a chemotaxin to undergo diapedesis. Thus, growing BMEC on collagen gel-coated Transwells has broad applications for the in vitro study of both blood-brain barrier physiology as well as the mechanisms underlying central nervous system inflammation.     

Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain

1. Download : Download high-res image (311KB)
2. Download : Download full-size image

     

Differences in multidrug resistance phenotype and matrix metalloproteinases activity between endothelial cells from normal brain and glioma

Endothelial cells (ECs) are new targets for tumor therapy. In this work, we purified endothelial cells from intracerebral and subcutaneous experimental gliomas as well as from normal brain in order to define some of the phenotypical differences between angiogenic and quiescent brain vasculature. We show that the multidrug resistance genes encoding drug efflux pumps at the brain endothelium are expressed differently in normal and tumoral vasculature. We also show that ECs from gliomas present increased activity of gelatinase B (MMP9), key enzyme in the angiogenic process. Importantly, we observe a different phenotype between ECs in the intracerebral and subcutaneous models. Our results provide molecular evidence of phenotypic distinction between tumoral and normal brain vasculature and indicate that the EC phenotype depends on interactions both with tumor cells and also with the microenvironment.     

GSH Transport in Immortalized Mouse Brain Endothelial Cells

We have previously shown GSH transport across the blood-brain barrier in vivo and expression of transport in Xenopus laevis oocytes injected with bovine brain capillary mRNA. In the present study, we have used MBEC-4, an immortalized mouse brain endothelial cell line, to establish the presence of Na+-dependent and Na+-independent GSH transport and have localized the Na+-dependent transporter using domain-enriched plasma membrane vesicles. In cells depleted of GSH with buthionine sulfoximine, a significant increase of intracellular GSH could be demonstrated only in the presence of Na+. Partial but significant Na+ dependency of [35S]GSH uptake was observed for two GSH concentrations in MBEC-4 cells in which gamma-glutamyltranspeptidase and gamma-glutamylcysteine synthetase were inhibited to ensure absence of breakdown and resynthesis of GSH. Uniqueness of Na+-dependent uptake in MBEC-4 cells was confirmed with parallel uptake studies with Cos-7 cells that did not show this activity. Molecular form of uptake was verified as predominantly GSH, and very little conversion of [35S]cysteine to GSH occurred under the same incubation conditions. Poly(A)+ RNA from MBEC expressed GSH uptake with significant (approximately 40-70%) Na+ dependency, whereas uptake expressed by poly(A)+ RNA from HepG2 and Cos-1 cells was Na+ independent. Plasma membrane vesicles from MBEC were separated into three fractions (30, 34, and 38% sucrose, by wt) by density gradient centrifugation. Na+-dependent glucose transport, reported to be localized to the abluminal membrane, was found to be associated with the 38% fraction (abluminal). Na+-dependent GSH transport was present in the 30% fraction, which was identified as the apical (luminal) membrane by localization of P-glycoprotein 170 by western blot analysis. Localization of Na+-dependent GSH transport to the luminal membrane and its ability to drive up intracellular GSH may find application in the delivery of supplemented GSH to the brain in vivo.     

Transcellular transport of CCL2 across brain microvascular endothelial cells

The means by which the chemokine CCL2 produced in the brain parenchyma can recruit leukocytes lying behind the highly impervious endothelium of the blood–brain barrier (BBB) has remained a paradox. As other chemokines have been evidenced to stimulate their own synthesis and release by peripheral microvascular endothelial cells, and/or undergo transcytosis in the abluminal-to-luminal direction, we determined whether CCL2 experiences similar fates across brain microvascular endothelial cells (BMEC). Using cultured BMEC as a paradigm of the BBB, it was observed that exogenous unlabeled CCL2 actually depressed the release of endogenous CCL2, and further caused diminished CCL2 mRNA levels in these cells. On the other hand, exogenous ¹²⁵I-labeled CCL2 exhibited transport across BMEC in a manner that was sensitive to temperature, competition by excess unlabeled CCL2 but not unlabeled CCL3, knockdown of caveolin-1/caveolae, and elimination of the cognate CCL2 receptor CCR2. These results implied a facet of CCL2 transport by a transcellular mechanism partly involving binding of CCL2 to CCR2, and subsequent transfer to caveolae vesicles for transcytosis. This notion was supported by double-label immuno-electronmicroscopy, which revealed co-localization of caveolin-1 with exogenous CCL2, during this chemokine's transit across BMEC. Collectively, these findings provide a rationale by which CCL2, deposited on the abluminal side of the brain microvasculature during inflammatory episodes, can be relayed across the BBB to foster leukocyte recruitment.     

Intercommunications between brain capillary endothelial cells and glial cells increase the transcellular permeability of the blood-brain barrier during ischaemia

Increased cerebrovascular permeability is an important factor in the development of cerebral oedema after stroke, implicating the blood-brain barrier (BBB). To investigate the effect of hypoxia on the permeability changes, we used a cell culture model of the BBB consisting of a co-culture of brain capillary endothelial cells and glial cells. When endothelial cells from this co-culture model were submitted alone to hypoxic conditions, long exposures (48 h) were necessary to result in an increase in endothelial cell monolayer permeability to [3H]inulin. When endothelial cells were incubated in presence of glial cells, a huge increase in permeability occurred after 9 h of hypoxic conditions. Oxygen glucose deprivation (OGD) resulted in a much shorter time (i.e. 2 h) required for an increase in permeability. We have demonstrated that this OGD-induced permeability increase involves a transcellular rather than a paracellular pathway. Conditioned medium experiments showed that glial cells secrete soluble permeability factors during OGD. However, endothelial cells have to be made sensitive by OGD in order to respond to these glial soluble factors. This work shows that an early cross-talk between glial and endothelial cells occurs during ischaemic stroke and alters BBB transcellular transport by means of glial factor secretions.     

人骨髓基质细胞体外分离及定向培养内皮细胞

用Ficoll(比重1.077 g/ml)从正常成人骨髓中分离骨髓基质细胞(BMSCs),DMEM-HG 培养基内含20?S、GM-CSF(100 u/ml)、VEGF(10 ng/ml)、FGF(5 ng/ml)、L-谷氨酰胺(2mmol/ L)、肝素(90 u/ml),以及抗生素液进行定向培养和扩增其中的内皮细胞(ECs),Ⅷ因子相关抗原的免疫组化法和透射电镜观察(TEM)鉴定其细胞的性质。结果5.0×105个BMSCs在体外经定向ECs 培养和扩增8代后,获得了6.0×109个ECs,扩增了约1.2×104倍。70%-80%的细胞对Ⅷ因子相关抗原免疫组化呈阳性反应;光镜下细胞呈典型的“鹅卵石”样;TEM下可观察到胞浆内有Weible- palade小体,证实为内皮细胞。实验表明,BMSCs在体外分离和定向培养的ECs,经扩增后可能是心血管组织工程所需种子细胞的又一个重要来源。     

Brain endothelial cells and the glio-vascular complex

We present and discuss the role of endothelial and astroglial cells in managing the blood-brain barrier (BBB) and aspects of pathological alterations in the BBB. The impact of astrocytes, pericytes, and perivascular cells on the induction and maintenance of the gliovascular unit is largely unidentified so far. An understanding of the signaling pathways that lie between these cell types and the endothelium and that possibly are mediated by components of the basal lamina is just beginning to emerge. The metabolism for the maintenance of the endothelial barrier is intimately linked to and dependent on the microenvironment of the brain parenchyma. We report the structure and function of the endothelial cells of brain capillaries by describing structures involved in the regulation of permeability, including transporter systems, caveolae, and tight junctions. There is increasing evidence that caveolae are not only vehicles for endo- and transcytosis, but also important regulators of tight-junction-based permeability. Tight junctions separate the luminal from the abluminal membrane domains of the endothelial cell (“fence function”) and control the paracellular pathway (“gate function”) thus representing the most significant structure of the BBB. In addition, the extracellular matrix between astrocytes/pericytes and endothelial cells contains numerous molecules with inherent signaling properties that have to be considered if we are to improve our knowledge of the complex and closely regulated BBB. Any work of our own cited in this review was supported by grants from the Deutsche Krebshilfe (to H.W.), the Deutsche Forschungsgemeinschaft (to H.W.), and the Hertie-Foundation (to H.W. and to Britta Engelhardt, Bern, Switzerland).     

骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖的促进作用

本文通过制备小鼠骨髓内皮细胞无血清条件培养液(serum-free murine bone marrow endothelial cell conditioned medium, mBMEC-CM),经超滤分为分子量>10 kDa组分和<10 kDa组分,分别观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞集落生成的影响。用Wright’S Giemsa染色计数内皮细胞集落及检测骨髓内皮细胞的vWF,通过[3H]- TdR掺入量,观察mBMEC-CM原液及其组分以及外源性细胞因子对小鼠骨髓内皮细胞增殖的影响,并用分子杂交方法检测内皮细胞表达的细胞因子,从几个方面来研究mBMEC-CM对骨髓内皮细胞增殖的作用。结果显示,骨髓内皮细胞vWF 检测阳性。mBMEC-CM原液及其分子量>10 kDa组分能刺激骨髓内皮细胞集落增殖,且能明显增加骨髓内皮细胞[3H]-TdR 掺入量;分子量<10 kDa组分对骨髓内皮细胞集落增殖无明显刺激作用,也不能增加骨髓内皮细胞[3H]-TdR掺入量。外源加入IL-6、IL-11、SCF、GM-CSF、VEGF、bFGF 6种细胞因子能明显刺激骨髓内皮细胞集落增殖,SCF、VEGF、bFGF能明显增加骨髓内皮细胞[3H]-TdR掺入量。Atlas array膜杂交实验显示骨髓内皮细胞内源性表达GM-CSF、SCF、MSP-1、endothelin-2、thymosin β10、connective tissue GF、PDGF-A chain、MIP-2α、PlGF、neutrophil activating protein ENA-78、INF-γ、IL-1、IL-6、IL-13、IL-11、inhibin-α等细胞因子的mRNA。上述结果提示,骨髓内皮细胞无血清条件培养液对骨髓内皮细胞增殖具有促进作用。     

On the mechanism of oleate transport across human brain microvessel endothelial cells

The blood–brain barrier formed by the brain capillary endothelial cells provides a protective barrier between the systemic blood and the extracellular environment of the CNS. As most fatty acids in the brain enter from the blood, we examined the mechanism of oleate (C18:1) transport across primary human brain microvessel endothelial cells (HBMEC). The permeability of [1-¹⁴C]oleate was determined using confluent cells grown on Transwell® inserts in both the absence or presence of bovine serum albumin in the basolateral media, and following inhibition of various fatty acid transporters. The passage of [1-¹⁴C]oleate across confluent HBMEC monolayers was significantly enhanced when fatty acid free albumin was present in the basolateral media. The presence of the non-specific fatty acid uptake inhibitor phloretin significantly decreased [1-¹⁴C]oleate uptake by HBMEC and the subsequent release of [1-¹⁴C]oleate into the basolateral medium. Knockdown of fatty acid transport protein-1 or fatty acid translocase/CD36 significantly decreased [1-¹⁴C]oleate transport across the HBMEC monolayer from either apical as well as basolateral sides. The findings indicate that a fatty acid acceptor is a requirement for oleate transport across HBMEC monolayers. In addition, transport of oleate across HBMEC is, in part, a transcellular process mediated by fatty acid transport proteins.     

低温冻存对骨髓基质细胞生物学特性的影响

目的：探讨低温冻存对骨髓细胞和贴壁基质细胞生物学特性的影响。方法：取新鲜骨髓和经Dexter法培养14d的骨髓贴壁基质细胞(称“基质细胞”),经-196℃液氮冻存(前者称“冻存骨髓”,后者称“冻存基质细胞”)2周,复温,再用Dexter法培养这些细胞,检测细胞增殖、细胞形态、细胞化学染色、细胞表面抗原及基质细胞支持另一骨髓造血细胞形成的鹅卵石造血区(CAFC),长期培养起始细胞(LTCIC)的变化,比较冻存对骨髓细胞和基质细胞生物学特性的影响。结果：生长特性：冻存骨髓比新鲜骨髓、冻存基质细胞比新鲜基质细胞培养后融合成片的时间延迟,细胞增殖数比也有减低。细胞成分：冻存骨髓比新鲜骨髓形成的成纤维细胞、内皮细胞比率下降,而巨噬细胞和脂肪细胞比率升高,冻存基质细胞上述现象更明显：冻存后含凋亡小体的细胞在骨髓细胞和基质细胞内均有增加。细胞表面抗原：冻存骨髓、冻存基质细胞CD14、HLA-DR抗原表达百分率比新鲜骨髓、新鲜基质细胞高,CD45、CD33反之。支持造血：冻存前后骨髓和基质细胞支持形成的CAFC和LTC-IC,生长良好,无显著差异。结论：骨髓细胞和经培养生成的贴壁基质细胞,经冻存和复温,生物学特性有一定变化,但仍可以保留良好的支持造血重建功能。     

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.     

Regulation of P-glycoprotein by orphan nuclear receptors in human brain microvessel endothelial cells

In mammalian systems, pregnane X receptor (PXR) and constitutive androstane receptor (CAR) have been recognized as xenobiotic-sensors which can up-regulate the functional expression of drug transporters, such as P-glycoprotein (P-gp). In the brain, an increase in P-gp expression can further limit drug permeability across the blood-brain barrier (BBB) and potentially reduce CNS pharmacotherapy efficacy. At present, the involvement of human PXR (hPXR) and CAR (hCAR) in the regulation of P-gp expression at the human BBB is unknown. In this study, we investigate the role of hPXR and hCAR in the regulation of P-gp expression using a human cerebral microvessel endothelial cell culture system. We demonstrate that activation of hPXR and hCAR by their respective ligands leads to P-gp induction at both mRNA and protein levels, while pharmacological inhibitors of hPXR and hCAR prevent ligand-mediated P-gp induction. Ligand-induced nuclear translocation of hPXR is observed, although such effect could not be demonstrated for hCAR. Furthermore, down-regulation of hPXR and hCAR proteins using small-interfering RNA decreased P-gp expression. Our findings provide first evidence for P-gp regulation by hPXR and hCAR at the human BBB and suggest insights on how to achieve selective P-gp regulation at this site.