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 μM 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 [³H]-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.     

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 μM 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 [³H]-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.     

Iron transport kinetics through blood-brain barrier endothelial cells

Background

Transferrin and its receptors play an important role during the uptake and transcytosis of iron through blood-brain barrier (BBB) endothelial cells (ECs) to maintain iron homeostasis in BBB endothelium and brain. Any disruptions in the cell environment may change the distribution of transferrin receptors on the cell surface, which eventually alter the homeostasis and initiate neurodegenerative disorders. In this paper, we developed a comprehensive mathematical model that considers the necessary kinetics for holo-transferrin internalization and acidification, apo-transferrin recycling, and exocytosis of free iron and transferrin-bound iron through basolateral side of BBB ECs.

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.     

Kinetic analysis of blood-brain barrier transport of amino acids

The Michaelis-Menten kinetics of blood-brain barrier transport of fourteen amino acids was investigated with a tissue-sampling, single-injection technique in the anesthetized rat. Tracer quantities of ¹⁴C-labelled amino acids and ³H₂O, used as a freely diffusible internal reference, were mixed in 0.2 ml of buffered Ringer's solution and injected rapidly into a common carotid artery. Circulation was terminated by decapitation at 15 s following injection. A brain uptake index (I_b) was determined from the ratio of ¹⁴C dpm in the brain tissue and the injection mixture divided by the same ratio for the ³H₂O reference. Brain clearance of tracer concentration of amino acid was saturable when various concentrations of unlabeled amino acid were added to the injection solution. Double reciprocal plots of the saturation data yielded K_m (mM) values that ranged from a low of 0.09 mM for arginine to a high of 0.75 mM for cycloleucine. Transport V values were determined from the relationship $\text{P =}\text{V}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ where P is the blood-brain barrier permeability constant (ml/g per min): P was calculated from the I_b for each amino acid based on a cerebral blood flow of 0.56 ml/g per min and a fractional extraction of 0.75 for the ³H₂O reference 15 s following carotid injection. The V values ranged from a low of 6.2 nmol/g per min for lysine to a high of 64 nmol/g per min for l-DOPA. Efflux of the tracer amino acid during the 15-s period after injection was assumed to be slow, since the rate constant of cycloleucine from brain to blood was low, 0.11 min^?1.     

The blood-brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells

Antibodies against receptors that undergo transcytosis across the blood-brain barrier (BBB) have been used as vectors to target drugs or therapeutic peptides into the brain. We have recently discovered a novel single domain antibody, FC5, which transmigrates across human cerebral endothelial cells in vitro and the BBB in vivo. The purpose of this study was to characterize mechanisms of FC5 endocytosis and transcytosis across the BBB and its putative receptor on human brain endothelial cells. The transport of FC5 across human brain endothelial cells was polarized, charge independent and temperature dependent, suggesting a receptor-mediated process. FC5 taken up by human brain endothelial cells co-localized with clathrin but not with caveolin-1 by immunochemistry and was detected in clathrin-enriched subcellular fractions by western blot. The transendothelial migration of FC5 was reduced by inhibitors of clathrin-mediated endocytosis, K+ depletion and chlorpromazine, but was insensitive to caveolae inhibitors, filipin, nystatin or methyl-beta-cyclodextrin. Following internalization, FC5 was targeted to early endosomes, bypassed late endosomes/lysosomes and remained intact after transcytosis. The transcytosis process was inhibited by agents that affect actin cytoskeleton or intracellular signaling through PI3-kinase. Pretreatment of human brain endothelial cells with wheatgerm agglutinin, sialic acid, alpha(2,3)-neuraminidase or Maackia amurensis agglutinin that recognizes alpha(2,3)-, but not with Sambucus nigra agglutinin that recognizes alpha(2,6) sialylgalactosyl residues, significantly reduced FC5 transcytosis. FC5 failed to recognize brain endothelial cells-derived lipids, suggesting that it binds luminal alpha(2,3)-sialoglycoprotein receptor which triggers clathrin-mediated endocytosis. This putative receptor may be a new target for developing brain-targeting drug delivery vectors.     

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.     

Kinetic analysis of blood-brain barrier transport of amino acids.

The Michaelis-Menten kinetics of blood-brain barrier transport of fourteen amino acids was investigated with a tissue-sampling, single-injection technique in the anesthetized rat. Tracer quantities of 14C-labelled amino acids and 3H2O, used as a freely diffusible internal reference, were mixed in 0.2 ml of buffered Ringer's solution and injected rapidly into a common carotid artery. Circulation was terminated by decapitation at 15s following injection. A brain uptake index (Ib) was determined from the ratio of 14C dpm in the brain tissue and the injection mixture divided by the same ratio for the 3H2O reference. Brain clearance of tracer concentration of amino acid was saturable when various concentrations of unlabeled amino acid were added to the injection solution. Double reciprocal plots of the saturation data yielded Km (mM) values that ranged from a low of 0.09 mM for arginine to a high of 0.75 mM for cycloleucine. Transport V values were determined from the relationship P = V/Km where P is the blood-brain barrier permeability constant (ml/g per min): P was calculated from the Ib for each amino acid based on a cerebral blood flow of 0.56 ml/g per min and a fractional extraction of 0.75 for the 3H2O reference 15s following carotid injection. The V values ranged from a low of 6.2 nmol/g per min for lysine to a high of 64 nmol/g per min for l-DOPA. Efflux of the tracer amino acid during the 15-s period after injection was assumed to be slow, since the rate constant of cycloleucine from brain to blood was low, 0.11 min-1.     

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.     

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.     

Fatty acids and signalling in endothelial cells

The endothelium is critical for the maintenance of a proper vessel function. Disturbances of endothelial function, called endothelial dysfunction, have serious implications, and lead to the development of atherosclerosis. It is well established that the risk for atherosclerosis development is influenced by nutritional factors such as the intake of certain fatty acids. Due to the fundamental role of the endothelium for atherosclerosis development, it is, therefore, likely that fatty acids directly influence the function of endothelial cells. The present review aims to explain the divergent effects of different types of fatty acids on cardiovascular disease risk by summarizing in vitro-data on the effects of fatty acids on (1) important signalling pathways involved in the modulation of endothelial cell function, and (2) endothelial cell functional properties, namely vasoactive mediator release and mononuclear cell recruitment, both of which are typically dysregulated during endothelial dysfunction.     

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.     

Hypoxanthine transport through the blood-brain barrier

The unidirectional influx of hypoxanthine across cerebral capillaries, the anatomical locus of the blood=brain barrier, was measured with an in situ rat brain perfusion technique employing [³H]hypoxanthine. Hypoxanthine was transported across the blood-brain barrier by a saturable system with a one-half saturation concentration of approximately 0.4 mM. The permeability-surface area product was 3×10^–4 sec^–1 with a hypoxanthine concentration of 0.02 M in the perfusate. Adenine (4 mM) and uracil and theophylline (both 10 mM), but not inosine (10 mM) or leucine (1 mM), inhibited hypoxanthine transfer through the blood-brain barrier. Thus, hypoxanthine is transported through the blood-brain barrier by a high-capacity, saturable transport system with a half-saturation concentration about 100 times the plasma hypoxanthine concentration. Although involved in the transport hypoxanthine from blood into brain, this system is not powerful enough to transfer important quantities of hypoxanthine from blood into brain.     

Myo-inositol transport through the blood-brain barrier

The unidirectional transport of [³H]myo-inositol across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured using an in situ rat brain perfusion technique. Myo-inositol was transported across the blood-brain barrier by a low capacity, saturable system with a one-half saturation concentration of 0.1 mM. The permeability surface-area product was 6.2×10^–5S^–1 with a myo-inositol concentration of 0.02 mM in the perfusate. The myo-inositol stereoisomer scyllo-inositol but not (+)-chiro-inositol (both 1 mM) inhibited myo-inositol transfer through the blood-brain barrier. These observations provide evidence that myo-inositol is transferred through the blood-brain barrier by simple diffusion and a stereospecific, saturable transport system.     

Niacinamide transport through the blood-brain barrier

The unidirectional influx of niacinamide across cerebral capillaries, the anatomical locus of the blood-brain barrier, was measured with an in situ rat brain perfusion technique employing [¹⁴C]niacinamide. Niacinamide was transported rapidly across the blood-brain barrier by a system that was not saturable with 10 mM niacinamide in the perfusate. However, with periods of perfusion longer than 30 seconds, there was substantial backflow of [¹⁴C]niacinamide into the perfusate. Niacinamide (1.7 M) transport through the blood-brain barrier was not significantly inhibited by 3-acetylpyridine. Thus, niacinamide is transported rapidly and bidirectionally through the blood-brain barrier by a high capacity transport system. Although involved in the transfer of niacinamide between blood and brain, this transport system does not play an important regulatory role in the synthesis of NMN, NAD, and NADP from niacinamide in brain.     

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.     

RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain

Amyloid-beta peptide (Abeta) interacts with the vasculature to influence Abeta levels in the brain and cerebral blood flow, providing a means of amplifying the Abeta-induced cellular stress underlying neuronal dysfunction and dementia. Systemic Abeta infusion and studies in genetically manipulated mice show that Abeta interaction with receptor for advanced glycation end products (RAGE)-bearing cells in the vessel wall results in transport of Abeta across the blood-brain barrier (BBB) and expression of proinflammatory cytokines and endothelin-1 (ET-1), the latter mediating Abeta-induced vasoconstriction. Inhibition of RAGE-ligand interaction suppresses accumulation of Abeta in brain parenchyma in a mouse transgenic model. These findings suggest that vascular RAGE is a target for inhibiting pathogenic consequences of Abeta-vascular interactions, including development of cerebral amyloidosis.     

n-6 and n-3 polyunsaturated fatty acids differentially modulate oncogenic Ras activation in colonocytes

Ras proteins are critical regulators of cell function, including growth, differentiation, and apoptosis, with membrane localization of the protein being a prerequisite for malignant transformation. We have recently demonstrated that feeding fish oil, compared with corn oil, decreases colonic Ras membrane localization and reduces tumor formation in rats injected with a colon carcinogen. Because the biological activity of Ras is regulated by posttranslational lipid attachment and its interaction with stimulatory lipids, we investigated whether docosahexaenoic acid (DHA), found in fish oil, compared with linoleic acid (LA), found in corn oil, alters Ras posttranslational processing, activation, and effector protein function in young adult mouse colon cells overexpressing H-ras (YAMC-ras). We show here that the major n-3 polyunsaturated fatty acid (PUFA) constituent of fish oil, DHA, compared with LA (an n-6 PUFA), reduces Ras localization to the plasma membrane without affecting posttranslational lipidation and lowers GTP binding and downstream p42/44(ERK)-dependent signaling. In view of the central role of oncogenic Ras in the development of colon cancer, the finding that n-3 and n-6 PUFA differentially modulate Ras activation may partly explain why dietary fish oil protects against colon cancer development.