Functional expression of the P-glycoproteinmdr in primary cultures of bovine cerebral capillary endothelial cells

The P-glycoproteinmdr is expressed not only in tumoral cells, but also in nontransformed cells, including the specialized endothelial cells of brain capillaries which build up the blood-brain barrier. Since all previously identified blood-brain barrier markers are rapidly lost when cerebral capillary endothelial cells are maintained in primary culture, we have investigated whether P-glycoprotein (P-gp) would follow the same rule, in order to address the influence of the cerebral environment on the specific P-gp expression in the brain endothelium. As compared to freshly isolated purified cerebral capillaries, P-glycoprotein was detected by immunochemistry at a high level in 5–7 day primary cultures. In our culture conditions, P-glycoprotein was immunodetected at a lower molecular weight than that found in freshly isolated capillaries. Enzymatic deglycosylation led to the same 130 kDa protein for both fresh and cultured samples, suggesting that P-gp post-translational modifications were altered in primary cultures. However, studies on the uptake and efflux of the P-gp substrate [³H]vinblastine, and on the effect of variousmdr reversing agents on the uptake and efflux, clearly indicated that the efflux pump function of the P-glycoprotein was maintained in primary cultures of bovine cerebral capillary endothelial cells. P-Glycoprotein may thus represent the first blood-brain barrier marker which is maintained in cerebral endothelial cells cultured in the absence of factors originating from the brain parenchyma.Abbreviations BBB blood-brain barrier - BCEC brain capillary endothelial cells - -GT -glutamyltranspeptidase - HBSS Hank's balanced salt solution - Mab monoclonal antibody - mdr multidrug resistance - P-gp P-glycoprotein     

Effects of Dexamethasone In Vivo and In Vitro on Hexose Transport in Brain Microvasculature

Glucocorticoids induce hyperinsulinemia, hyperglycemia, and depress glucose transport by aortic endothelium. High glucocorticoid doses are used for many diseases, but with unknown effects on brain glucose transport or metabolism. This study tested the hypothesis that glucocorticoids affect glucose transport or metabolism by brain microvascular endothelium. Male rats received dexamethasone (DEX) sc with sucrose feeding for up to seven days. Cerebral microvessels from rats treated with DEX/sucrose demonstrated increased GLUT1 and brain glucose extraction compared to controls. Glucose transport in vivo correlated with hyperinsulinemia. Pre-treatment with low doses of strep-tozotocin blunted hyperinsulinemia and prevented increased glucose extraction induced by DEX. In contrast, isolated brain microvessels exposed to DEX in vitro demonstrated suppression of 2-deox-yglucose uptake and glucose oxidation. We conclude that DEX/sucrose treatment in vivo increases blood-brain glucose transport in a manner that requires the effects of chronic hyperinsulinemia. These effects override any direct inhibitory effects of either hyperglycemia or DEX.     

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.     

On the mechanism of cerebral accumulation of cholestanol in patients with cerebrotendinous xanthomatosis

The most serious consequence of sterol 27-hydroxylase deficiency in humans [cerebrotendinous xanthomatosis (CTX)] is the development of cholestanol-containing brain xanthomas. The cholestanol in the brain may be derived from the circulation or from 7alpha-hydroxylated intermediates in bile acid synthesis, present at 50- to 250-fold increased levels in plasma. Here, we demonstrate a transfer of 7alpha-hydroxy-4-cholesten-3-one across cultured porcine brain endothelial cells (a model for the blood-brain barrier) that is approximately 100-fold more efficient than the transfer of cholestanol. Furthermore, there was an efficient conversion of 7alpha-hydroxy-4-cholesten-3-one to cholestanol in cultured neuronal and glial cells as well as in monocyte-derived macrophages of human origin. It is concluded that the continuous intracellular production of cholestanol from a bile acid precursor capable of rapidly passing biomembranes, including the blood-brain barrier, is likely to be of major importance for the accumulation of cholestanol in patients with CTX. Such a mechanism also fits well with the observation that treatment with chenodeoxycholic acid, which normalizes the level of the bile acid precursor, results in a reduction of cholestanol-containing xanthomas even in the brain.     

mRNA expression levels of tight junction protein genes in mouse brain capillary endothelial cells highly purified by magnetic cell sorting

Tight junctions (TJs) are an important component of the blood-brain barrier, and claudin-1, -3, -5 and -12 have been reported to be localized at the TJs of brain capillary endothelial cells (BCECs). To understand the contribution of each claudin subtype to TJ formation, we have measured the mRNA expression levels of claudin subtypes (claudin-1 to -23) and other relevant proteins in highly purified mouse BCECs. Mouse BCECs were labeled with anti-platelet endothelial cellular adhesion molecule-1 antibody and 2.3 × 10⁶ cells were isolated from 15 mice by magnetic cell sorting. Expression of Tie-2, Mdr1a and GLUT1 mRNAs was concentrated in the isolated fraction, and contamination with neurons and astrocytes was substantially less than in the brain capillary fraction prepared by the standard glass-beads column method. Expression of occludin, junctional adhesion molecule and endothelial-specific adhesion molecule mRNAs was concentrated in the isolated fraction, suggesting that the corresponding proteins are selectively expressed in mouse BCECs. Among claudin subtypes, claudin-5 was most highly expressed, at a level which was at least 593-fold greater that that of claudin-1, -3 or -12. Expression of mRNAs of claudin-8, -10, -15, -17, -19, -20, -22 or -23 was also concentrated in the isolated fraction, suggesting these subtypes are expressed in mouse BCECs. The levels of claudin-10 and -22 mRNAs were comparable with that of occludin mRNA. These results indicate that claudin-5 is the most abundant claudin subtype in mouse BCECs, and are consistent with the idea that claudin-10 and -22 are involved in TJ formation at the blood-brain barrier in cooperation with claudin-5.     

Brain mast cell degranulation regulates blood-brain barrier

Mast cells synthesize vasoactive agents and a number of neurotransmitters. They are particularly numerous in the medial habenular region of the epithalamus, the attachment site of the choroid plexus. The present study examined whether degranulation of brain mast cells alters the permeability of the blood-brain barrier (BBB). To this end, doves were injected intramuscularly with the mast cell degranulator, compound 48/80 (C40/80), followed by intravenous injection of Evans blue. The distribution of the dye in the parenchyma was examined using digital imaging. Three brain areas were analyzed: the medial habenula (which also contains mast cells), the paraventricular nucleus (PVN, which abuts the third ventricle, but has no mast cells), and the lateral septal organ (LSO, a circumventricular organ with fenestrated capillaries). Significantly more Evans blue tracer and fewer toluidine blue-positive mast cells were detected in the medial habenula of subjects treated with C48/80 compared to saline controls. Evans blue did not enter the PVN in either the experimental or control group, while it entered the LSO equally in both. Degranulation of mast cells after C48/80 treatment was confirmed histochemically and ultrastructurally. The results support the hypothesis that brain mast cell degranulation locally alters BBB permeability. Activation of brain mast cells may provide a mechanism for regulated opening of the BBB. © 1996 John Wiley & Sons, Inc.     

The impact of glia-derived extracellular matrices on the barrier function of cerebral endothelial cells: an in vitro study

The blood-brain barrier (BBB) is composed of the cerebral microvascular endothelium, which, together with astrocytes, pericytes, and the extracellular matrix (ECM), contributes to a "neurovascular unit". It was our objective to clarify the impact of endogenous extracellular matrices on the barrier function of BBB microvascular endothelial cells cultured in vitro. The study was performed in two consecutive steps: (i) The ECM-donating cells (astrocytes, pericytes, endothelial cells) were grown to confluence and then removed from the growth substrate by a protocol that leaves the ECM behind. (ii) Suspensions of cerebral endothelial cells were seeded on the endogenous matrices and barrier formation was followed with time. In order to quantify the tightness of the cell junctions, all experiments were performed on planar gold-film electrodes that can be used to read the electrical resistance of the cell layers as a direct measure for endothelial barrier function (electric cell-substrate impedance sensing, ECIS). We observed that endogenously isolated ECM from both, astrocytes and pericytes, improved the tightness of cerebral endothelial cells significantly compared to ECM that was derived from the endothelial cells themselves as a control. Moreover, when cerebral endothelial cells were grown on extracellular matrices produced by non-brain endothelial cells (aorta), the electrical resistances were markedly reduced. Our observations indicate that glia-derived ECM - as an essential part of the BBB - is required to ensure proper barrier formation of cerebral endothelial cells.     

Qualitative and quantitative analysis of monocyte transendothelial migration by confocal microscopy and three-dimensional image reconstruction

A novel method for qualitative and quantitative analysis of monocyte transendothelial migration is described. By labeling monocytes and endothelial cells with different fluorophores, and utilizing confocal microscopy and three-dimensional image reconstruction, transmigrating monocytes were resolved and quantified within a subendothelial collagen gel. Comparison of monocyte migration across endothelial monolayers derived from human brain microvessels versus umbilical veins revealed diapedesis across brain endothelium to be significantly delayed. Inclusion of astrocytes within the subendothelial collagen gel resulted in the formation of an array of astrocytic processes that simulated the glia limitans surrounding brain microvessels in situ, thus yielding a more physiologic paradigm of the blood-brain barrier. By virtue of its unique capacity to provide information on the total number of migrating cells, this analytic approach overcomes significant caveats associated with sampling only aspects of the migration process. The potential adaptability of this method to computer-assisted analysis further enhances its prospective use in high-throughput screening.     

Puromycin selectively increases mdr1a expression in immortalized rat brain endothelial cell lines

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from blood to brain extracellular fluid. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain endothelium and contributes to the formation of a functional barrier to lipid-soluble drugs such as anticancer agents. The mdr1a P-gp-encoding gene is exclusively expressed in the rodent BBB. Primary cultures of rat brain endothelial cells and GP8.3 cells showed a dramatic decrease in mdr1a mRNA level and some expression of mdr1b mRNA. GPNT cells, derived from GP8.3 cells after transfection with a puromycin resistance gene, were chronically treated with 5 microg/mL puromycin, a P-gp substrate. Compared with rat brain endothelial cells and GP8.3 cells, GPNT cells exhibited a very high level of expression of mdr1a mRNA together with a moderate level of mdr1b mRNA expression. Accordingly, P-gp expression and activity were strongly increased. When GP8.3 and puromycin-starved GPNT cells were treated with puromycin, mdr1a expression was selectively increased. High expression of mdr1a mRNA in GPNT cells may thus be related to the chronic treatment with puromycin. We conclude that GPNT cells may be used as a valuable rat in vitro model for studying the regulation of mdr1a expression at the BBB level.     

Mesenchymal stem cell modification of endothelial matrix regulates their vascular differentiation

Mesenchymal stem cells (MSCs) respond to a variety of differentiation signal provided by their local environments. A large portion of these signals originate from the extracellular matrix (ECM). At the same time, MSCs secrete various matrix‐altering agents, including proteases, that alter ECM‐encoded differentiation signals. Here we investigated the interactions between MSC and ECM produced by endothelial cells (EC‐matrix), focusing not only on the differentiation signals provided by EC‐matrix, but also on MSC‐alteration of these signals and the resultant affects on MSC differentiation. MSCs were cultured on EC‐matrix modified in one of three distinct ways. First, MSCs cultured on native EC‐matrix underwent endothelial cell (EC) differentiation early during the culture period and smooth muscle cell (SMC) differentiation at later time points. Second, MSCs cultured on crosslinked EC‐matrix, which is resistant to MSC modification, differentiated towards an EC lineage only. Third, MSCs cultured on EC‐matrix pre‐modified by MSCs underwent SMC‐differentiation only. These MSC‐induced matrix alterations were found to deplete the factors responsible for EC‐differentiation, yet activate the SMC‐differentiation factors. In conclusion, our results demonstrate that the EC‐matrix contains factors that support MSC differentiation into both ECs and SMCs, and that these factors are modified by MSC‐secreted agents. By analyzing the framework by which EC‐matrix regulates differentiation in MSCs, we have uncovered evidence of a feedback system in which MSCs are able to alter the very matrix signals acting upon them. J. Cell. Biochem. 107: 706–713, 2009. Published 2009 Wiley‐Liss, Inc.     

Synergistic effects of neurons and astrocytes on the differentiation of brain capillary endothelial cells in culture

Brain capillary endothelial cells form a functional barrier between blood and brain, based on the existence of tight junctions that limit paracellular permeability. Occludin is one of the major transmembrane proteins of tight junctions and its peripheral localization gives indication of tight junction formation. We previously reported that RBE4.B cells (brain capillary endothelial cells), cultured on collagen IV, synthesize occludin and correctly localize it at the cell periphery only when cocultured with neurons. In the present study, we describe a three-cell type-culture system that allowed us to analyze the combined effects of neurons and astrocytes on differentiation of brain capillary endothelial cells in culture. In particular, we found that, in the presence of astrocytes, the neuron-induced synthesis and localization of occludin is precocious as compared to cells cocultured with neurons only.     

Growth of endothelial cells on microfabricated silicon nitride membranes for an<Emphasis Type="Italic">in vitro</Emphasis> model of the blood-brain barrier

The blood-brain barrier (BBB) is composed of the brain capillaries, which are lined by endothelial cells displaying extremely tight intercellular junctions. Several attempts at creating anin vitro model of the BBB have been met with moderate success as brain capillary endothelial cells lose their barrier properties when isolated in cell culture. This may be due to a lack of recreation of thein vivo endothelial cellular environment in these models, including nearly constant contact with astrocyte foot processes. This work is motivated by the hypothesis that growing endothelial cells on one side of an ultra-thin, highly porous membrane and differentiating astrocyte or astrogliomal cells on the opposite side will lead to a higher degree of interaction between the two cell types and therefore to an improved model. Here we describe our initial efforts towards testing this hypothesis including a procedure for membrane fabrication and methods for culturing endothelial cells on these membranes. We have fabricated a 1 μm thick, 2.0 μm pore size, and ∼55% porous membrane with a very narrow pore size distribution from low-stress silicon nitride (SiN) utilizing techniques from the microelectronics industry. We have developed a base, acid, autoclave routine that prepares the membranes for cell culture both by cleaning residual fabrication chemicals from the surface and by increasing the hydrophilicity of the membranes (confirmed by contact angle measurements). Gelatin, fibronectin, and a 50/50 mixture of the two proteins were evaluated as potential basement membrane protein treatments prior to membrane cell seeding. All three treatments support adequate attachment and growth on the membranes compared to the control.     

Stability of von Willebrand Factor secretion in different human endothelial hybrid cell lines

Endothelial cells form a highly differentiated tissue on the inner surface of blood vessels. One of the typical characteristics is the expression of von Willebrand Factor, a protein that participates in blood coagulation. Thein vitro cultivation of endothelial cells is limited by the fact that primary cells become senescent after 40 generation doublings. We have immortalized human endothelial cells by somatic cell hybridization. Primary cells were fused to different tumor cell lines of murine and human origin. The degree of differentiation of the resulting hybrids was analyzed by characterizing the expression of von Willebrand Factor. This protein was identified intracellularly and in the culture supernatant. During long-term cultivation the hybrid cells showed a tendency to lose this differentiated property even after several subcloning steps. However by fusing them with primary endothelial cells a second time, cell lines expressing von Willebrand Factor for more than 180 population doublings were generated.     

Class IIb HDAC6 regulates endothelial cell migration and angiogenesis by deacetylation of cortactin

Histone deacetylases (HDACs) deacetylate histones and non-histone proteins, thereby affecting protein activity and gene expression. The regulation and function of the cytoplasmic class IIb HDAC6 in endothelial cells (ECs) is largely unexplored. Here, we demonstrate that HDAC6 is upregulated by hypoxia and is essential for angiogenesis. Silencing of HDAC6 in ECs decreases sprouting and migration in vitro and formation of functional vascular networks in matrigel plugs in vivo. HDAC6 regulates zebrafish vessel formation, and HDAC6-deficient mice showed a reduced formation of perfused vessels in matrigel plugs. Consistently, overexpression of wild-type HDAC6 increases sprouting from spheroids. HDAC6 function requires the catalytic activity but is independent of ubiquitin binding and deacetylation of α-tubulin. Instead, we found that HDAC6 interacts with and deacetylates the actin-remodelling protein cortactin in ECs, which is essential for zebrafish vessel formation and which mediates the angiogenic effect of HDAC6. In summary, we show that HDAC6 is necessary for angiogenesis in vivo and in vitro, involving the interaction and deacetylation of cortactin that regulates EC migration and sprouting.     

Secretome derived from breast tumor cell lines alters the morphology of human umbilical vein endothelial cells

Metastases, responsible for most of the solid tumor associated deaths, require angiogenesis and changes in endothelial cells. In this work, the effect of the secretomes of three breast tumor cell lines (MCF-7, MDA-MB-231 and ZR-75-30) on human umbilical vein endothelial cells (HUVEC) morphology was investigated. HUVEC treated with secretomes from breast cells were analyzed by confocal and time-lapse microscopy. Secretomes from ZR-75-30 and MDA-MB-231 cells modify the morphology and adhesion of HUVEC. These changes may provoke the loss of endothelial monolayer integrity. In consequence, tumor cells could have an increased access to circulation, which would then enhance metastasis.     

Effect of nicotine and polyaromtic hydrocarbons on cerebral endothelial cells

The present study was designed to investigate the effect of nicotine and polyaromatic hydrocarbon compounds on cerebral endothelial cells (CECs). Nicotine treatments from 15 min to 5h did not cause any changes in the expression and localization of principal junctional proteins. One day of treatment with a relatively high concentration of nicotine induced a decrease in the expression of the tight junction protein ZO-1, occludin, and the adherens junction protein, cadherin. Treatment with 3 x 10(-5)M phenanthrene for 24h caused a redistribution of occludin from the Triton X-100 insoluble to the Triton X-100 soluble fraction. Transendothelial electrical resistance was not significantly affected by 24h treatments with nicotine, methylanthracene or phenanthrene. However, 24h nicotine treatment increased transendothelial permeability in CECs exposed to oxidative stress. Both nicotine and phenanthrene were able to regulate the expression of a large number of proteins as revealed by 2D electrophoresis. Our experiments suggest that tobacco smoking may affect the junctional complex of CECs, and that this effect is enhanced by oxidative stress.     

Variability in E-selectin expression, mRNA levels and sE-selectin release between endothelial cell lines and primary endothelial cells

Endothelial cell lines express markers and are assumed to exhibit other endothelial cell responses. We investigated E-selectin expression from human umbilical vein endothelial cells, the spontaneously transformed ECV304 line and the hybrid line EA.hy926 by flow cytometry and immunofluorescence, mRNA and soluble E-selectin release. In cells exposed to tumour necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), median (range) percentage of E-selectin-positive HUVECs increased from 1.6(0.9-6. 2)% to 91.4(83.0-96.1)%, (P=0.001) using flow cytometry. In contrast, E-selectin expression by ECV304 and EA.hy926 cell lines was 100-fold lower. E-selectin mRNA was detectable after 2 h, maximal at 6 h in HUVECs and undetectable in EA.hy926 and ECV304 cell lines after exposure to TNF-alpha/IL-1beta. sE-selectin accumulation increased (P=0.004) in HUVECs only. Neutrophil adherence to ECV304 and EA.hy926 cells was poor compared to HUVECs (P=0.004). The cell lines ECV304 and EA.hy926 do not exhibit normal endothelium expression of E-selectin, and may not be appropriate for studies of adhesion.     

Hyperosmotic stress induces Axl activation and cleavage in cerebral endothelial cells

Because of the relative impermeability of the blood‐brain barrier (BBB), many drugs are unable to reach the CNS in therapeutically relevant concentration. One method to deliver drugs to the CNS is the osmotic opening of the BBB using mannitol. Hyperosmotic mannitol induces a strong phosphorylation on tyrosine residues in a broad spectrum of proteins in cerebral endothelial cells, the principal components of the BBB. Previously, we have shown that among targets of tyrosine phosphorylation are β‐catenin, extracellular signal‐regulated kinase 1/2 and the non‐receptor tyrosine kinase Src. The aim of this study was to identify new signalling pathways activated by hypertonicity in cerebral endothelial cells. Using an antibody array and immunoprecipitation we identified the receptor tyrosine kinase Axl to become tyrosine phosphorylated in response to hyperosmotic mannitol. Besides activation, Axl was also cleaved in response to osmotic stress. Degradation of Axl proved to be metalloproteinase‐ and proteasome‐dependent and resulted in 50–55 kDa C‐terminal products which remained phosphorylated even after degradation. Specific knockdown of Axl increased the rate of apoptosis in hyperosmotic mannitol‐treated cells; therefore, we assume that activation of Axl may be a protective mechanism against hypertonicity‐induced apoptosis. Our results identify Axl as an important element of osmotic stress‐induced signalling.     

Quantitative detection of blood-brain barrier-associated enzymes in cultured endothelial cells of procine brain microvessels

Summary The present study deals with a rapid and convenient assay for blood-brain barrier (BBB)-associated enzymes, γ-glutamyl transpeptidase (γ-GTP) and alkaline phosphatase (ALP), in cultured endothelial cells and other cells. These enzyme activities in cultured cells could be efficiently measured by direct incubation of each substrate in the culture plates without pretreatment of the cells. This new direct in situ-in plate assay was more rapid and convenient than conventional ex-plate assays, and these assays gave similar values for specific enzyme activities. γ-GTP and ALP activities could be detected by this in situ method in primary-cultured endothelial cells of porcine brain microvessels, but their levels were lower than those before culture. The degree of loss due to culture differed, between γ-GTP and ALP; a relatively large amount of ALP remained but the γ-GTP level decreased greatly In this direct in situ-in plate assay, cultured porcine aortic endothelial cells exhibited negligibly small activities for both enzymes, whereas cultured astroglial cells of neonatal porcine brain showed moderate γ-GTP activity and a trace of ALP activity. This direct in situ-in plate assay can be used for microculture and automatic measurement and offers a convenient means for studying the possible regulatory mechanisms of the expression of the BBB-associated enzymes.