Culture-Induced Changes in Blood—Brain Barrier Transcriptome: Implications for Amino-Acid Transporters in vivo

Tight homeostatic control of brain amino acids (AA) depends on transport by solute carrier family proteins expressed by the blood—brain barrier (BBB) microvascular endothelial cells (BMEC). To characterize the mouse BMEC transcriptome and probe culture-induced changes, microarray analyses of platelet endothelial cell adhesion molecule-1-positive (PECAM1+) endothelial cells (ppMBMECs) were compared with primary MBMECs (pMBMEC) cultured in the presence or absence of glial cells and with b.End5 endothelioma cell line. Selected cell marker and AA transporter mRNA levels were further verified by reverse transcription real-time PCR. Regardless of glial coculture, expression of a large subset of genes was strongly altered by a brief culture step. This is consistent with the known dependence of BMECs on in vivo interactions to maintain physiologic functions, for example, tight barrier formation, and their consequent dedifferentiation in culture. Seven ( 4F2hc, Lat1, Taut, Snat3, Snat5, Xpct, and Cat1) of nine AA transporter mRNAs highly expressed in freshly isolated ppMBMECs were strongly downregulated for all cultures and two ( Snat2 and Eaat3) were variably regulated. In contrast, five AA transporter mRNAs with low expression in ppMBMECs, including y+ Lat2, xCT, and Snat1, were upregulated by culture. We hypothesized that the AA transporters highly expressed in ppMBMECs and downregulated in culture have a major in vivo function for BBB transendothelial transport.

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Cerebromicrovascular endothelial cells are resistant to l-glutamate

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.90430.2008 ◽

2008 ◽

Vol 295 (4) ◽

pp. R1099-R1108 ◽

Cited By ~ 16

Author(s):

Ferenc Domoki ◽

Béla Kis ◽

Tamás Gáspár ◽

Ferenc Bari ◽

David W. Busija

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Primary Cultures ◽

Glucose Deprivation ◽

Brain Barrier ◽

Cranial Window ◽

Blood Brain ◽

Microvascular Cells

Cerebral microvascular endothelial cells (CMVECs) have recently been implicated as targets of excitotoxic injury by l-glutamate (l-glut) or N-methyl-d-aspartate (NMDA) in vitro. However, high levels of l-glut do not compromise the function of the blood-brain barrier in vivo. We sought to determine whether primary cultures of rat and piglet CMVECs or cerebral microvascular pericytes (CMVPCs) are indeed sensitive to l-glut or NMDA. Viability was unaffected by 8-h exposure to 1–10 mM l-glut or NMDA in CMVECs or CMVPCs isolated from both species. Furthermore, neither 1 mM l-glut nor NMDA augmented cell death induced by 12-h oxygen-glucose deprivation in rat CMVECs or by 8-h medium withdrawal in CMVPCs. Additionally, transendothelial electrical resistance of rat CMVEC-astrocyte cocultures or piglet CMVEC cultures were not compromised by up to 24-h exposure to 1 mM l-glut or NMDA. The Ca2+ ionophore calcimycin (5 μM), but not l-glut (1 mM), increased intracellular Ca2+ levels in rat CMVECs and CMVPCs assessed with fluo-4 AM fluorescence and confocal microscopy. CMVEC-dependent pial arteriolar vasodilation to hypercapnia and bradykinin was unaffected by intracarotid infusion of l-glut in anesthetized piglets by closed cranial window/intravital microscopy. We conclude that cerebral microvascular cells are insensitive and resistant to glutamatergic stimuli in accordance with their in vivo role as regulators of potentially neurotoxic amino acids across the blood-brain barrier.

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Mechanisms of Glutamate Efflux at the Blood—Brain Barrier: Involvement of Glial Cells

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2011.121 ◽

2011 ◽

Vol 32 (1) ◽

pp. 177-189 ◽

Cited By ~ 34

Author(s):

Katayun Cohen-Kashi-Malina ◽

Itzik Cooper ◽

Vivian I Teichberg

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Glial Cells ◽

Excitatory Amino Acid ◽

Active Role ◽

In Vitro Models ◽

Brain Barrier ◽

Amino Acid Transporters ◽

Blood Brain

At high concentrations, glutamate (Glu) exerts potent neurotoxic properties, leading to irreversible brain damages found in numerous neurological disorders. The accepted notion that Glu homeostasis in brain interstitial fluid is maintained primarily through the activity of Glu transporters present on glial cells does not take into account the possible contribution of endothelial cells constituting the blood-brain barrier (BBB) to this process. Here, we present evidence for the presence of the Glu transporters, excitatory amino-acid transporters (EAATs) 1 to 3, in porcine brain endothelial cells (PBECs) and show their participation in Glu uptake into PBECs. Moreover, transport of Glu across three in vitro models of the BBB is investigated for the first time, and evidence for Glu transport across the BBB in both directions is presented. Our results provide evidence that the BBB can function in the efflux mode to selectively remove Glu, via specific transporters, from the abluminal side (brain) into the luminal compartment (blood). Furthermore, we found that glial cells lining the BBB have an active role in the efflux process by taking up Glu and releasing it, through hemichannels, anion channels, and possibly the reversal of its EAATs, in close proximity to ECs, which in turn take up Glu and release it to the blood.

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Cationic amino acid transport across the blood-brain barrier is mediated exclusively by system y+

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00007.2006 ◽

2006 ◽

Vol 291 (2) ◽

pp. E412-E419 ◽

Cited By ~ 61

Author(s):

Robyn L. O’Kane ◽

Juan R. Viña ◽

Ian Simpson ◽

Rosa Zaragozá ◽

Ashwini Mokashi ◽

...

Keyword(s):

Endothelial Cells ◽

Amino Acid ◽

Blood Brain Barrier ◽

Plasma Membranes ◽

Plasma Concentrations ◽

Membrane System ◽

Brain Barrier ◽

Cationic Amino Acid ◽

Blood Brain

Cationic amino acid (CAA) transport is brought about by two families of proteins that are found in various tissues: Cat (CAA transporter), referred to as system y+, and Bat [broad-scope amino acid (AA) transporter], which comprises systems b0,+, B0,+, and y+L. CAA traverse the blood-brain barrier (BBB), but experiments done in vivo have only been able to examine the BBB from the luminal (blood-facing) side. In the present study, plasma membranes isolated from bovine brain microvessels were used to identify and characterize the CAA transporter(s) on both sides of the BBB. From these studies, it was concluded that system y+ was the only transporter present, with a prevalence of activity on the abluminal membrane. System y+ was voltage dependent and had a Km of 470 ± 106 μM (SE) for lysine, a Ki of 34 μM for arginine, and a Ki of 290 μM for ornithine. In the presence of Na+, system y+ was inhibited by several essential neutral AAs. The Ki values were 3–10 times the plasma concentrations, suggesting that system y+ was not as important a point of access for these AAs as system L1. Several small nonessential AAs (serine, glutamine, alanine,and glycine) inhibited system y+ with Ki values similar to their plasma concentrations, suggesting that system y+ may account for the permeability of the BBB to these AAs. System y+ may be important in the provision of arginine for NO synthesis. Real-time PCR and Western blotting techniques established the presence of the three known nitric oxide synthases in cerebral endothelial cells: NOS-1 (neuronal), NOS-2 (inducible), and NOS-3 (endothelial). These results confirm that system y+ is the only CAA transporter in the BBB and suggest that NO can be produced in brain endothelial cells.

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Nitric Oxide Isoenzymes Regulate Lipopolysaccharide-Enhanced Insulin Transport across the Blood-Brain Barrier

Endocrinology ◽

10.1210/en.2007-1091 ◽

2008 ◽

Vol 149 (4) ◽

pp. 1514-1523 ◽

Cited By ~ 36

Author(s):

William A. Banks ◽

Shinya Dohgu ◽

Jessica L. Lynch ◽

Melissa A. Fleegal-DeMotta ◽

Michelle A. Erickson ◽

...

Keyword(s):

Nitric Oxide ◽

Blood Brain Barrier ◽

Neurovascular Unit ◽

Brain Barrier ◽

Mrna Levels ◽

Insulin Transport ◽

Blood Brain ◽

Inducible Nos

Insulin transported across the blood-brain barrier (BBB) has many effects within the central nervous system. Insulin transport is not static but altered by obesity and inflammation. Lipopolysaccharide (LPS), derived from the cell walls of Gram-negative bacteria, enhances insulin transport across the BBB but also releases nitric oxide (NO), which opposes LPS-enhanced insulin transport. Here we determined the role of NO synthase (NOS) in mediating the effects of LPS on insulin BBB transport. The activity of all three NOS isoenzymes was stimulated in vivo by LPS. Endothelial NOS and inducible NOS together mediated the LPS-enhanced transport of insulin, whereas neuronal NOS (nNOS) opposed LPS-enhanced insulin transport. This dual pattern of NOS action was found in most brain regions with the exception of the striatum, which did not respond to LPS, and the parietal cortex, hippocampus, and pons medulla, which did not respond to nNOS inhibition. In vitro studies of a brain endothelial cell (BEC) monolayer BBB model showed that LPS did not directly affect insulin transport, whereas NO inhibited insulin transport. This suggests that the stimulatory effect of LPS and NOS on insulin transport is mediated through cells of the neurovascular unit other than BECs. Protein and mRNA levels of the isoenzymes indicated that the effects of LPS are mainly posttranslational. In conclusion, LPS affects insulin transport across the BBB by modulating NOS isoenzyme activity. NO released by endothelial NOS and inducible NOS acts indirectly to stimulate insulin transport, whereas NO released by nNOS acts directly on BECs to inhibit insulin transport.

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Blood-brain barrier formation of grafted human umbilical vein endothelial cells in athymic mouse brain

Brain Research ◽

10.1016/s0006-8993(99)02471-3 ◽

2000 ◽

Vol 858 (1) ◽

pp. 172-176 ◽

Cited By ~ 21

Author(s):

Hideyuki Akiyama ◽

Takeshi Kondoh ◽

Takashi Kokunai ◽

Tatsuya Nagashima ◽

Naoaki Saito ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Mouse Brain ◽

Umbilical Vein ◽

Brain Barrier ◽

Athymic Mouse ◽

Human Umbilical Vein ◽

Blood Brain ◽

Barrier Formation ◽

Umbilical Vein Endothelial Cells

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Human Blood Vessel Organoids Penetrate Human Cerebral Organoids and Form a Vessel-Like System

Cells ◽

10.3390/cells10082036 ◽

2021 ◽

Vol 10 (8) ◽

pp. 2036

Author(s):

Yujin Ahn ◽

Ju-Hyun An ◽

Hae-Jun Yang ◽

Dong Gil Lee ◽

Jieun Kim ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Vessel ◽

Blood Brain Barrier ◽

Human Blood ◽

Cell Types ◽

Brain Barrier ◽

Vascular Cells ◽

Blood Brain ◽

Vessel Networks

Vascularization of tissues, organoids and organ-on-chip models has been attempted using endothelial cells. However, the cultured endothelial cells lack the capacity to interact with other somatic cell types, which is distinct from developing vascular cells in vivo. Recently, it was demonstrated that blood vessel organoids (BVOs) recreate the structure and functions of developing human blood vessels. However, the tissue-specific adaptability of BVOs had not been assessed in somatic tissues. Herein, we investigated whether BVOs infiltrate human cerebral organoids and form a blood–brain barrier. As a result, vascular cells arising from BVOs penetrated the cerebral organoids and developed a vessel-like architecture composed of CD31+ endothelial tubes coated with SMA+ or PDGFR+ mural cells. Molecular markers of the blood-brain barrier were detected in the vascularized cerebral organoids. We revealed that BVOs can form neural-specific blood-vessel networks that can be maintained for over 50 days.

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The Capsule Supports Survival but Not Traversal ofEscherichia coli K1 across the Blood-Brain Barrier

Infection and Immunity ◽

10.1128/iai.67.7.3566-3570.1999 ◽

1999 ◽

Vol 67 (7) ◽

pp. 3566-3570 ◽

Cited By ~ 46

Author(s):

Jill A. Hoffman ◽

Carol Wass ◽

Monique F. Stins ◽

Kwang Sik Kim

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Brain Barrier ◽

In Vivo Model ◽

E Coli ◽

Blood Brain ◽

Vitro Model ◽

K1 Capsule

ABSTRACT The vast majority of cases of gram-negative meningitis in neonates are caused by K1-encapsulated Escherichia coli. The role of the K1 capsule in the pathogenesis of E. coli meningitis was examined with an in vivo model of experimental hematogenousE. coli K1 meningitis and an in vitro model of the blood-brain barrier. Bacteremia was induced in neonatal rats with theE. coli K1 strain C5 (O18:K1) or its K1−derivative, C5ME. Subsequently, blood and cerebrospinal fluid (CSF) were obtained for culture. Viable bacteria were recovered from the CSF of animals infected with E. coli K1 strains only; none of the animals infected with K1− strains had positive CSF cultures. However, despite the fact that their cultures were sterile, the presence of O18 E. coli was demonstrated immunocytochemically in the brains of animals infected with K1− strains and was seen by staining of CSF samples. In vitro, brain microvascular endothelial cells (BMEC) were incubated with K1+ and K1− E. coli strains. The recovery of viable intracellular organisms of the K1+strain was significantly higher than that for the K1−strain (P = 0.0005). The recovery of viable intracellular K1− E. coli bacteria was increased by cycloheximide treatment of BMEC (P = 0.0059) but was not affected by nitric oxide synthase inhibitors or oxygen radical scavengers. We conclude that the K1 capsule is not necessary for the invasion of bacteria into brain endothelial cells but is responsible for helping to maintain bacterial viability during invasion of the blood-brain barrier.

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Escherichia coli K1 aslAContributes to Invasion of Brain Microvascular Endothelial Cells In Vitro and In Vivo

Infection and Immunity ◽

10.1128/iai.68.9.5062-5067.2000 ◽

2000 ◽

Vol 68 (9) ◽

pp. 5062-5067 ◽

Cited By ~ 57

Author(s):

Jill A. Hoffman ◽

Julie L. Badger ◽

Yan Zhang ◽

Sheng-He Huang ◽

Kwang Sik Kim

Keyword(s):

Escherichia Coli ◽

Endothelial Cells ◽

Blood Brain Barrier ◽

Brain Barrier ◽

Microvascular Endothelial Cells ◽

Brain Microvascular Endothelial Cells ◽

E Coli ◽

Blood Brain ◽

Microvascular Endothelial

ABSTRACT Neonatal Escherichia coli meningitis remains a devastating disease, with unacceptably high morbidity and mortality despite advances in supportive care measures and bactericidal antibiotics. To further our ability to improve the outcome of affected neonates, a better understanding of the pathogenesis of the disease is necessary. To identify potential bacterial genes which contribute toE. coli invasion of the blood-brain barrier, a cerebrospinal fluid isolate of E. coli K1 was mutagenized with TnphoA. TnphoA mutant 27A-6 was found to have a significantly decreased ability to invade brain microvascular endothelial cells compared to the wild type. In vivo, 32% of the animals infected with mutant 27A-6 developed meningitis, compared to 82% of those infected with the parent strain, despite similar levels of bacteremia. The DNA flanking the TnphoA insertion in 27A-6 was cloned and sequenced and determined to be homologous toE. coli K-12 aslA (arylsulfatase-like gene). The deduced amino acid sequence of the E. coli K1aslA gene product shows homology to a well-characterized arylsulfatase family of enzymes found in eukaryotes, as well as prokaryotes. Two additional aslA mutants were constructed by targeted gene disruption and internal gene deletion. Both of these mutants demonstrated decreased invasion phenotypes, similar to that of TnphoA mutant 27A-6. Complementation of the decreased-invasion phenotypes of these mutants was achieved whenaslA was supplied in trans. This is the first demonstration that this locus contributes to invasion of the blood-brain barrier by E. coli K1.

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Protein S controls hypoxic/ischemic blood-brain barrier disruption through the TAM receptor Tyro3 and sphingosine 1-phosphate receptor

Blood ◽

10.1182/blood-2010-01-262386 ◽

2010 ◽

Vol 115 (23) ◽

pp. 4963-4972 ◽

Cited By ~ 66

Author(s):

Donghui Zhu ◽

Yaoming Wang ◽

Itender Singh ◽

Robert D. Bell ◽

Rashid Deane ◽

...

Keyword(s):

Endothelial Cells ◽

Blood Brain Barrier ◽

Protein S ◽

Glucose Deprivation ◽

Brain Barrier ◽

Brain Endothelial Cells ◽

Blood Brain ◽

Mouse Brain Endothelial Cells ◽

Sphingosine 1 Phosphate

Abstract The anticoagulant factor protein S (PS) has direct cellular activities. Lack of PS in mice causes lethal coagulopathy, ischemic/thrombotic injuries, vascular dysgenesis, and blood-brain barrier (BBB) disruption with intracerebral hemorrhages. Thus, we hypothesized that PS maintains and/or enhances the BBB integrity. Using a BBB model with human brain endothelial cells, we show PS inhibits time- and dose-dependently (half maximal effective concentration [EC50] = 27 ± 3 nM) oxygen/glucose deprivation-induced BBB breakdown, as demonstrated by measurements of the transmonolayer electrical resistance, permeability of endothelial monolayers to dextran (40 kDa), and rearrangement of F-actin toward the cortical cytoskeletal ring. Using Tyro-3, Axl, and Mer (TAM) receptor, tyrosine kinase silencing through RNA interference, specific N-terminus–blocking antibodies, Tyro3 phosphorylation, and Tyro3-, Axl- and Mer-deficient mouse brain endothelial cells, we show that Tyro3 mediates PS vasculoprotection. After Tyro3 ligation, PS activated sphingosine 1-phosphate receptor (S1P1), resulting in Rac1-dependent BBB protection. Using 2-photon in vivo imaging, we show that PS blocks postischemic BBB disruption in Tyro3+/+, Axl−/−, and Mer−/− mice, but not in Tyro3−/− mice or Tyro3+/+ mice receiving low-dose W146, a S1P1-specific antagonist. Our findings indicate that PS protects the BBB integrity via Tyro3 and S1P1, suggesting potentially novel treatments for neurovascular dysfunction resulting from hypoxic/ischemic BBB damage.

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Human induced pluripotent stem cell-derived neuroectodermal epithelial cells mistaken for blood-brain barrier-forming endothelial cells

10.1101/699173 ◽

2019 ◽

Cited By ~ 6

Author(s):

Tyler M. Lu ◽

David Redmond ◽

Tarig Magdeldin ◽

Duc-Huy T. Nguyen ◽

Amanda Snead ◽

...

Keyword(s):

Endothelial Cells ◽

Epithelial Cells ◽

Blood Brain Barrier ◽

Neurological Diseases ◽

Brain Barrier ◽

Brain Functions ◽

Blood Brain ◽

Induced Pluripotent

AbstractBrain microvascular endothelial cells (BMECs) possess unique properties underlying the blood-brain-barrier (BBB), that are crucial for homeostatic brain functions and interactions with the immune system. Modulation of BBB function is essential for treatment of neurological diseases and effective tumor targeting. Studies to-date have been hampered by the lack of physiological models using cultivated human BMECs that sustain BBB properties. Recently, differentiation of induced pluripotent stem cells (iPSCs) into cells with BBB-like properties has been reported, providing a robust in vitro model for drug screening and mechanistic understanding of neurological diseases. However, the precise identity of these iBMECs remains unclear. Employing single-cell RNA sequencing, bioinformatic analysis and immunofluorescence for several pathways, transcription factors (TFs), and surface markers, we examined the molecular and functional properties of iBMECs differentiated either in the absence or presence of retinoic acid. We found that iBMECs lack both endothelial-lineage genes and ETS TFs that are essential for the establishment and maintenance of EC identity. Moreover, iBMECs fail to respond to angiogenic stimuli and form lumenized vessels in vivo. We demonstrate that human iBMECs are not barrier-forming ECs but rather EpCAM+ neuroectodermal epithelial cells (NE-EpiCs) that form tight junctions resembling those present in BBB-forming BMECs. Finally, overexpression of ETS TFs (ETV2, FLI1, and ERG) reprograms NE-EpiCs to become more like the BBB-forming ECs. Thus, although directed differentiation of human iBMECs primarily gives rise to epithelial cells, overexpression of several ETS TFs can divert them toward a vascular BBB in vitro.

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