Regulated expression of endothelial lipase by porcine brain capillary endothelial cells constituting the blood-brain barrier

Normal neurological function depends on a constant supply of polyunsaturated fatty acids to the brain. A considerable proportion of essential fatty acids originates from lipoprotein-associated lipids that undergo uptake and/or catabolism at the blood-brain barrier (BBB). This study aimed at identifying expression and regulation of endothelial lipase (EL) in brain capillary endothelial cells (BCEC), major constituents of the BBB. Our results revealed that BCEC are capable of EL synthesis and secretion. Overexpression of EL resulted in enhanced hydrolysis of extracellular high-density lipoprotein (HDL)-associated sn-2-labeled [(14)C]20 : 4 phosphatidylcholine. [(14)C]20 : 4 was recovered in cellular lipids, indicating re-uptake and intracellular re-esterification. To investigate local regulation of EL in the cerebrovasculature, BCEC were cultured in the presence of peroxisome-proliferator activated receptor (PPAR)- and liver X receptor (LXR)-agonists, known to regulate HDL levels. These experiments revealed that 24(S)OH-cholesterol (a LXR agonist), bezafibrate (a PPARalpha agonist), or pioglitazone (a PPARgamma agonist) resulted in down-regulation of EL mRNA and protein levels. Our findings implicate that EL could generate fatty acids at the BBB for transport to deeper regions of the brain as building blocks for membrane phospholipids. In addition PPAR and LXR agonists appear to contribute to HDL homeostasis at the BBB by regulating EL expression.     

Exogenous expression of claudin-5 induces barrier properties in cultured rat brain capillary endothelial cells

Claudins are thought to be major components of tight junctions (TJs), and claudin-5 and -12 are localized at TJs of the blood-brain barrier (BBB). Claudin-5-deficient mice exhibit size-selective (<800 Da) opening of the BBB. The purpose of this study was to clarify the expression levels of claudin-5 and -12 in rat brain capillary endothelial cells, and to examine the ability of claudin-5 to form TJs in cultured rat brain capillary endothelial cells (TR-BBB). Expression of claudin-5 mRNA in rat brain capillary fraction was 751-fold greater than that of claudin-12. The level of claudin-5 mRNA in the rat brain capillary fraction (per total mRNA) was 35.6-fold greater than that in whole brain, while the level of claudin-12 mRNA was only 13.9% of that in whole brain, suggesting that expression of claudin-12 mRNA is not restricted to brain capillaries. Transfection of TR-BBB cells with the claudin-5 gene afforded TR-BBB/CLD5 cells, which showed no change in expression of claudin-12 or ZO-1, while the expressed claudin-5 was detected at the cell-cell boundaries. The permeability surface product of [(14)C]inulin at a TR-BBB/CLD5 cell monolayer was significantly smaller (P < 0.01) than that for the parental TR-BBB cells, and the values of the permeability coefficient (Pe) were 1.14 x 10(-3) and 11.6 x 10(-3) cm/min, respectively. These results indicate that claudin-5, but not claudin-12, is predominantly expressed in brain capillaries, and plays a key role in the appearance of barrier properties of brain capillary endothelial cells.     

Immortalization of brain capillary endothelial cells with maintenance of structural characteristics of the blood-brain barrier endothelium

Summary Early passage bovine brain capillary endothelial cells were immortalized by transfection with the plasmid pSV3 neo. Cells from one clone, SV-BEC, expressed nuclear SV 40 large T antigen, displayed a contact-inhibited and anchorage-dependent proliferation, and a high sensitivity to the addition of exogenous basic fibroblast growth factor. SV-BEC cells are morphologically unaltered and express typical markers of endothelial cells: Factor VIII-related antigen, angiotensin-converting enzyme andGriffonia simplicifolia agglutinin binding site. Endothelium like immunoreactivity was detected in the conditioned medium from these cells. Moreover, SV-BECs present numerous intercellular tight junctions characteristic of the blood-brain barrier and possess functionalβ1- andβ2-adrenergic receptors, as observed on isolated bovine brain capillaries.     

Uptake mechanism of ApoE-modified nanoparticles on brain capillary endothelial cells as a blood-brain barrier model

Background

The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood.

Methodology/Principal Findings

In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor.

Conclusions/Significance

This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.     

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.     

Specific AHNAK expression in brain endothelial cells with barrier properties

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and low permeability. Because disruption of the BBB may contribute to many brain disorders, they are of considerable interests in the identification of the molecular mechanisms of BBB development and integrity. We here report that the giant protein AHNAK is expressed at the plasma membrane of endothelial cells (ECs) forming specific blood-tissue barriers, but is absent from the endothelium of capillaries characterized by extensive molecular exchanges between blood and extracellular fluid. In the brain, AHNAK is widely distributed in ECs with BBB properties, where it co-localizes with the tight junction protein ZO-1. AHNAK is absent from the permeable brain ECs of the choroid plexus and is down-regulated in permeable angiogenic ECs of brain tumors. In the choroid plexus, AHNAK accumulates at the tight junctions of the choroid epithelial cells that form the blood-cerebrospinal fluid (CSF) barrier. In EC cultures, the regulation of AHNAK expression and its localization corresponds to general criteria of a protein involved in barrier organization. AHNAK is up-regulated by angiopoietin-1 (Ang-1), a morphogenic factor that regulates brain EC permeability. In bovine cerebral ECs co-cultured with glial cells, AHNAK relocates from the cytosol to the plasma membrane when endothelial cells acquire BBB properties. Our results identify AHNAK as a protein marker of endothelial cells with barrier properties.     

Astrocytes enhance radical defence in capillary endothelial cells constituting the blood-brain barrier.

Astrocytes (AC) induce blood-brain barrier (BBB) properties in brain endothelial cells (EC). As antioxidative activity (AOA) is assumed to be a BBB characteristic, we tested whether AC improve AOA of EC. Monocultivated AC showed higher AOA [manganese superoxide dismutase (SOD), catalase (Cat), glutathione peroxidase (GPx)] than EC. Cocultivation elevated AOA in EC (MnSOD, CuZnSOD, Cat, GPx), and AC (MnSOD, CuZnSOD, GPx). Hypoxia increased radical-induced membrane lipid peroxidation in monocultivated, but not in cocultivated EC. Thus, EC/AC cocultivation intensifies AOA in both cell types, protects the EC, and therefore, the BBB against oxidative stress. The high AOA is regarded as an essential property of the BBB, which is induced by AC.     

A new astrocytic cell line which is able to induce a blood-brain barrier property in cultured brain capillary endothelial cells

Astrocytes, a member of the glial cell family in the central nervous system, are assumed to play a crucial role in the formation of the blood-brain barrier (BBB) in vertebrates. It was shown that astrocytes induce BBB-properties in brain capillary endothelial cells (BCEC) in vitro. We now established an astroglial cell line of non-tumoral origin. The cloned cell line (A7) shows a highly increased proliferation rate and expresses the astrocytic marker glial fibrillary acidic protein. Furthermore, the clone A7 expresses S-100-protein and vimentin, which are also expressed by primary cultured astrocytes. This cell line therefore shows general astrocytic features. In addition, we were able to show that A7 cells re-induce the BBB-related marker enzyme alkaline phosphatase in BCEC, when these two cell types are co-cultured. Thus we have a cell line which can be readily cultured in large quantities, shows common astrocyte properties and is able to influence BCEC with respect to a BBB-related feature. This revised version was published online in July 2006 with corrections to the Cover Date.     

Upregulation of the low density lipoprotein receptor at the blood-brain barrier: intercommunications between brain capillary endothelial cells and astrocytes

In contrast to the endothelial cells in large vessels where LDL receptors are downregulated, brain capillary endothelial cells in vivo express an LDL receptor. Using a cell culture model of the blood-brain barrier consisting of a coculture of brain capillary endothelial cells and astrocytes, we observed that the capacity of endothelial cells to bind LDL is enhanced threefold when cocultured with astrocytes. We next investigated the ability of astrocytes to modulate endothelial cell LDL receptor expression. We have shown that the lipid requirement of astrocytes increases the expression of endothelial cell LDL receptors. Experiments with dialysis membranes of different pore size showed that this effect is mediated by a soluble factor(s) with relative molecular mass somewhere between 3,500 and 14,000. Substituting astrocytes with smooth muscle cells or brain endothelium with endothelium from the aorta or the adrenal cortex did not enhance the luminal LDL receptor expression on endothelial cells, demonstrating the specificity of the interactions. This factor(s) is exclusively secreted by astrocytes cocultured with brain capillary endothelial cells, but it also upregulates the LDL receptor on other cell types. This study confirms the notion that the final fine tuning of cell differentiation is under local control.     

Use of isolated brain capillaries and cultured endothelial cells to study the blood-brain barrier

Brain capillary endothelial cells are responsible for forming the blood-brain barrier (BBB). Methods are now available to isolate microvessels from brain and study their biochemical and transport characteristics. From these investigations, new ideas have been proposed concerning the role of endothelial cells in the function of the BBB. More recently, success in culturing endothelial cells from brain microvessels has opened the way for novel approaches to the study of the regulation of endothelial cell permeability. We anticipate continued rapid progress in this area and expect that this will lead to a better understanding of the mechanisms involved in the regulation of BBB permeability and brain capillary function.     

Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood-brain barrier-specific properties

One of the main difficulties with primary rat brain endothelial cell (RBEC) cultures is obtaining pure cultures. The variation in purity limits the achievement of in vitro models of the rat blood-brain barrier. As P-glycoprotein expression is known to be much higher in RBECs than in any contaminating cells, we have tested the effect of five P-glycoprotein substrates (vincristine, vinblastine, colchicine, puromycin and doxorubicin) on RBEC cultures, assuming that RBECs would resist the treatment with these toxic compounds whereas contaminating cells would not. Treatment with either 4 microg/mL puromycin for the first 2 days of culture or 3 microg/mL puromycin for the first 3 days showed the best results without causing toxicity to the cells. Transendothelial electrical resistance was significantly increased in cell monolayers treated with puromycin compared with untreated cell monolayers. When cocultured with astrocytes in the presence of cAMP, the puromycin-treated RBEC monolayer showed a highly reduced permeability to sodium fluorescein (down to 0.75 x 10(-6) cm/s) and a high electrical resistance (up to 500 Omega x cm(2)). In conclusion, this method of RBEC purification will allow the production of in vitro models of the rat blood-brain barrier for cellular and molecular biology studies as well as pharmacological investigations.     

Specific binding of vasculotropin to bovine brain capillary endothelial cells

Recently, a new growth factor was purified to homogeneity; its biological activity appeared to be restricted to vascular endothelial derived cells. As it was also angiogenic in vivo, it was provisionally named vasculotropin. An iodination procedure used to label vasculotropin did not damage the molecule; it was thus possible to undertake binding studies. The binding of iodinated vasculotropin to bovine brain capillary endothelial cells reached saturation at 7 ng/ml and half maximal binding occurred at 1.5 ng/ml. Scatchard analysis of the data demonstrated 2 classes of binding sites with apparent dissociation constants of 4 and 41 pM and 600 and 4,100 sites per cell respectively. The interaction was specific since an excess of unlabelled vasculotropin, but no Fibroblast Growth Factor or Transforming Growth Factor Beta almost totally abolished the binding of the tracer. A sensitive radioreceptor-assay convenient for measuring vasculotropin in biological samples is described.     

Catecholamines stimulate the IFN-gamma-induced class II MHC expression on bovine brain capillary endothelial cells.

The brain has been considered for a long time as an immunologically privileged site because of the lack of a true lymphatic system and the existence of several barriers that isolate it from the periphery. In the last few years, it became evident that cells in the central nervous system (astrocytes, microglial cells, and brain capillary endothelial cells) can be induced to express class II MHC and present Ag to T lymphocytes. The brain capillary endothelial cells, which are strategically located at the interface between blood and brain, could be involved in the initiation of immune responses within the brain parenchyma. We have previously characterized bovine brain capillary endothelial cells in culture and shown that they maintain in vitro a fully differentiated phenotype associated with the blood-brain barrier endothelium. In order to assess the role of these cells in the development of immune responses in the brain, we initiated the present study on the regulation of their class II MHC surface expression. Our data indicate that this expression on bovine brain capillary endothelial cells is inducible by IFN-gamma and further stimulated by catecholamines through activation of beta-adrenergic receptors. However, this latter effect is not mimicked by forskolin, theophylline, or dibutyryl-cAMP, suggesting the involvement of a cAMP-independent mechanism.     

n-3 Fatty acids modulate brain glucose transport in endothelial cells of the blood-brain barrier

We have previously shown that glucose utilization and glucose transport were impaired in the brain of rats made deficient in n-3 polyunsaturated fatty acids (PUFA). The present study examines whether n-3 PUFA affect the expression of glucose transporter GLUT1 and glucose transport activity in the endothelial cells of the blood-brain barrier. GLUT1 expression in the cerebral cortex microvessels of rats fed different amounts of n-3 PUFA (low vs. adequate vs. high) was studied. In parallel, the glucose uptake was measured in primary cultures of rat brain endothelial cells (RBEC) exposed to supplemental long chain n-3 PUFA, docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, or to arachidonic acid (AA). Western immunoblotting analysis showed that endothelial GLUT1 significantly decreased (-23%) in the n-3 PUFA-deficient microvessels compared to control ones, whereas it increased (+35%) in the microvessels of rats fed the high n-3 PUFA diet. In addition, binding of cytochalasin B indicated that the maximum binding to GLUT1 (Bmax) was reduced in deficient rats. Incubation of RBEC with 15 microM DHA induced the membrane DHA to increase at a level approaching that of cerebral microvessels isolated from rats fed the high n-3 diet. Supplementation of RBEC with DHA or EPA increased the [(3)H]-3-O-methylglucose uptake (reflecting the basal glucose transport) by 35% and 50%, respectively, while AA had no effect. In conclusion, we suggest that n-3 PUFA can modulate the brain glucose transport in endothelial cells of the blood-brain barrier, possibly via changes in GLUT1 protein expression and activity.     

Microsphere embolism-induced endothelial nitric oxide synthase expression mediates disruption of the blood-brain barrier in rat brain

Microsphere embolism (ME)-induced up-regulation of endothelial nitric oxide synthase (eNOS) in endothelial cells of brain microvessels was observed 2-48 h after ischemia. eNOS induction preceded disruption of the blood-brain barrier (BBB) observed 6-72 h after ischemia. In vascular endothelial cells, ME-induced eNOS expression was closely associated with protein tyrosine nitration, which is a marker of generation of peroxynitrite. Leakage of rabbit IgG from microvessels was also evident around protein tyrosine nitration-immunoreactive microvessels. To determine whether eNOS expression and protein tyrosine nitration in vascular endothelial cells mediates BBB disruption in the ME brain, we tested the effect of a novel calmodulin-dependent NOS inhibitor, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), which inhibits eNOS activity and, in turn, protein tyrosine nitration. Concomitant with inhibition of protein tyrosine nitration in vascular endothelial cells, DY-9760e significantly inhibited BBB disruption as assessed by Evans blue (EB) excretion. DY-9760e also inhibited cleavage of poly (ADP-ribose) polymerase as a marker of the apoptotic pathway in vascular endothelial cells. Taken together with previous evidence in which DY-9760e inhibited brain edema, ME-induced eNOS expression in vascular endothelial cells likely mediates BBB disruption and, in turn, brain edema.     

Semliki Forest virus infects mouse brain endothelial cells and causes blood-brain barrier damage.

Induction of experimental allergic encephalomyelitis is facilitated in a genetically resistant BALB/c mouse strain by a nonpathogenic strain of a neurotropic alphavirus, Semliki Forest virus (SFV-A7). One possible explanation for this enhancement is virus infection of endothelial cells (EC), causing increased permeability of the blood-brain barrier. We have now sought evidence for virus infection of EC in vivo by immunocytochemistry and in situ hybridization. SFV-A7 antigens and RNA were detected in vascular EC and perivascular neurons in cerebellar and spinal cord white matter. Expression of viral antigens was followed by fibrinogen leakage from the blood vessels into brain parenchyma. This was shown by immunoperoxidase staining detecting fibrinogen extravascularly in central nervous system sections of infected mice. Simultaneously, expression of ICAM-1 (intercellular adhesion molecule 1) was induced on brain EC. SFV-A7 replicated in mouse brain microvascular EC in vitro and caused lysis of the cells. SFV-A7 did not induce ICAM-1 expression of mouse brain microvascular EC in vitro, while ICAM-1 was readily induced by gamma interferon and interleukin 1 beta. The observed increase of ICAM-1 expression on EC is immune mediated and not a direct effect of the virus infection. We conclude that SFV-A7 infection causes cerebral microvascular damage which contributes to the facilitation of experimental allergic encephalomyelitis in BALB/c mice.     

Dominant expression of androgen receptors and their functional regulation of organic anion transporter 3 in rat brain capillary endothelial cells; comparison of gene expression between the blood-brain and -retinal barriers

Brain and retinal capillary endothelial cells (BCECs and RCECs, respectively) exhibit a barrier structure and function. Comparison of gene expression in these cells could clarify the selective function of each barrier. The purpose of this study was to identify the genes selectively expressed at the blood-brain barrier (BBB) and to clarify the function of the selective gene, androgen receptor (AR). Gene expression was compared by a differential display using conditionally immortalized rat BCECs and RCECs (TR-BBB and TR-iBRB, respectively). A total of 12 gene fragments were identified as the selective genes dominantly expressed in TR-BBB cells. The most selective fragment in TR-BBB cells had the highest homology with the 3'-UTR of human and mouse AR. Rat AR mRNA was detected in TR-BBB cells and the brain capillary rich fraction, but not in TR-iBRB cells. Expression of organic anion transporter 3 (OAT3) mRNA in TR-BBB cells was induced by treatment with dihydrotestosterone (DHT), an AR ligand, and this induction was suppressed by flutamide. Moreover, uptake of benzylpenicillin by TR-BBB cells was also induced by DHT treatment. In contrast, OAT3 mRNA expression in TR-iBRB cells was not affected by DHT treatment. The brain-to-blood efflux rate of benzylpenicillin was not affected by gender. These results suggest that AR is involved in the functional regulation of OAT3 at the BBB, but not at the inner blood-retinal barrier (iBRB), and this regulation is not affected by gender. The BBB function will be affected by the androgen levels in the brain and/or plasma via AR.     

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