scholarly journals Search and Characterization of ITM2A as a New Potential Target for Brain Delivery

2021 ◽  
Author(s):  
Céline Cegarra ◽  
Catarina Chaves ◽  
Catherine Déon ◽  
Tuan Minh Do ◽  
Bruno Dumas ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Transmembrane Protein ◽  
Brain Barrier ◽  
Hek293 Cells ◽  
Phage Library ◽  
Brain Delivery ◽  
Blood Brain

Abstract Background Integral membrane protein 2A (ITM2A) is a transmembrane protein whose function is not well described. This target was identified as highly enriched in human brain vs peripheral endothelial cells by transcriptomic and proteomic studies during the European Collaboration on the Optimization of Macromolecular Pharmaceutical Innovative Medicines Initiative (COMPACT IMI) consortium. The object of the present paper is to report the work we have undertaken to characterize this protein as a potential brain delivery target. Methods A series of ITM2A constructs, cell lines and specific anti-human and mouse ITM2A antibodies were generated. Binding and internalization in Human Embryonic Kidney 293 (HEK293) cells overexpressing ITM2A and in brain microvascular endothelial cells from mouse and non-human primate (NHP) were performed with these tools. The best antibody was evaluated in an in vitro human blood brain barrier (BBB) model and in a mouse in vivo pharmacokinetic study to validate blood brain barrier crossing. Results Antibodies specifically recognizing extracellular parts of ITM2A or tags inserted in its extracellular domain showed selective binding and uptake in ITM2A-expressing cells. However, despite high RNA expression in mouse and human microvessels, this protein, is rapidly downregulated upon endothelial cells culture, probably explaining why transcytosis could not be evidenced in vitro. An attempt to directly demonstrate in vivo transcytosis in mice turned out unconvincing, using a cross reactive anti ITM2A antibody on one hand and in vivo phage panning of an anti ITM2A phage library on the other hand. Conclusions The present article describes the work we have undertaken to explore the potential of ITM2A as target to mediate transcytosis through BBB. This work highlights the multiple challenges linked to the identification of new brain delivery targets.

scholarly journals Cerebromicrovascular endothelial cells are resistant to l-glutamate

2008 ◽  
Vol 295 (4) ◽  
pp. R1099-R1108 ◽  
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.

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

1999 ◽  
Vol 67 (7) ◽  
pp. 3566-3570 ◽  
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.

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

2019 ◽  
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.

Adsorptive endocytosis mediates the passage of HIV-1 across the blood-brain barrier: evidence for a post-internalization coreceptor

1998 ◽  
Vol 111 (4) ◽  
pp. 533-540 ◽  
Author(s):  
W.A. Banks ◽  
V. Akerstrom ◽  
A.J. Kastin
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Aids Dementia Complex ◽  
Blood Brain ◽  
Wheatgerm Agglutinin ◽  
Adsorptive Endocytosis ◽  
Hiv 1

HIV-1 induces the AIDS dementia complex and infects brain endothelial and glial cells. Because the endothelial cells comprising the blood-brain barrier (BBB) do not possess CD4 receptors or galactosylceramide binding sites, it is unclear how HIV-1 negotiates the BBB. Previous work has suggested that gp120, the glycoprotein viral coat of HIV-1, is capable of inducing adsorptive endocytosis. Glycoprotein lectins like wheatgerm agglutinin induce adsorptive endocytosis and greatly potentiate the uptake by and passage across mouse endothelial cells in vivo and in vitro. We show here that the wheatgerm agglutinin-induced binding of gp120 is dose-dependent and involves components of the cytoskeleton. The uptake is partially dependent on temperature and energy and is modestly enhanced by potassium depletion. Glycosylation of gp120 is critical for its uptake by adsorptive endocytosis since the non-glycosylated form of gp120 is unaffected by wheatgerm agglutinin. Evidence is presented for the existence of a coreceptor sensitive to protamine sulfate that is primarily involved in membrane fusion after 125I-gp120 has bound to the cell membrane and is probably activated after internalization. This coreceptor probably contains a negatively charged heparin sulfate group and could be a member of the chemokine receptor family.

The First Step into the Brain: Uptake of NIO-PBCA Nanoparticles by Endothelial Cells in vitro and in vivo, and Direct Evidence for their Blood-Brain Barrier Permeation

ChemMedChem ◽  
2008 ◽  
Vol 3 (9) ◽  
pp. 1395-1403 ◽  
Author(s):  
Clemens K. Weiss ◽  
Maria-Verena Kohnle ◽  
Katharina Landfester ◽  
Thomas Hauk ◽  
Dietmar Fischer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Direct Evidence ◽  
Brain Barrier ◽  
Brain Uptake ◽  
Blood Brain ◽  
The Brain

scholarly journals Connexin Channels Provide a Target to Manipulate Brain Endothelial Calcium Dynamics and Blood—Brain Barrier Permeability

2011 ◽  
Vol 31 (9) ◽  
pp. 1942-1957 ◽  
Author(s):  
Marijke De Bock ◽  
Maxime Culot ◽  
Nan Wang ◽  
Mélissa Bol ◽  
Elke Decrock ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Purinergic Signaling ◽  
Endothelial Permeability ◽  
Brain Barrier ◽  
In Vivo Experiments ◽  
Blood Brain ◽  
Novel Approach

The cytoplasmic Ca2+ concentration ([Ca2+]i) is an important factor determining the functional state of blood-brain barrier (BBB) endothelial cells but little is known on the effect of dynamic [Ca2+]i changes on BBB function. We applied different agonists that trigger [Ca2+]i oscillations and determined the involvement of connexin channels and subsequent effects on endothelial permeability in immortalized and primary brain endothelial cells. The inflammatory peptide bradykinin (BK) triggered [Ca2+]i oscillations and increased endothelial permeability. The latter was prevented by buffering [Ca2+]i with BAPTA, indicating that [Ca2+]i oscillations are crucial in the permeability changes. Bradykinin-triggered [Ca2+]i oscillations were inhibited by interfering with connexin channels, making use of carbenoxolone, Gap27, a peptide blocker of connexin channels, and Cx37/43 knockdown. Gap27 inhibition of the oscillations was rapid (within minutes) and work with connexin hemichannel-permeable dyes indicated hemichannel opening and purinergic signaling in response to stimulation with BK. Moreover, Gap27 inhibited the BK-triggered endothelial permeability increase in in vitro and in vivo experiments. By contrast, [Ca2+]i oscillations provoked by exposure to adenosine 5’ triphosphate (ATP) were not affected by carbenoxolone or Gap27 and ATP did not disturb endothelial permeability. We conclude that interfering with endothelial connexin hemichannels is a novel approach to limiting BBB-permeability alterations.

Activation of PKC modulates blood-brain barrier endothelial cell permeability changes induced by hypoxia and posthypoxic reoxygenation

2005 ◽  
Vol 289 (5) ◽  
pp. H2012-H2019 ◽  
Author(s):  
Melissa A. Fleegal ◽  
Sharon Hom ◽  
Lindsay K. Borg ◽  
Thomas P. Davis
Keyword(s):  
Blood Brain Barrier ◽  
Paracellular Permeability ◽  
Cell Permeability ◽  
Brain Barrier ◽  
Cerebral Microvessels ◽  
Permeability Changes ◽  
Blood Brain ◽  
Brain Microvessel

The blood-brain barrier (BBB) is a metabolic and physiological barrier important for maintaining brain homeostasis. The aim of this study was to determine the role of PKC activation in BBB paracellular permeability changes induced by hypoxia and posthypoxic reoxygenation using in vitro and in vivo BBB models. In rat brain microvessel endothelial cells (RMECs) exposed to hypoxia (1% O2-99% N2; 24 h), a significant increase in total PKC activity was observed, and this was reduced by posthypoxic reoxygenation (95% room air-5% CO2) for 2 h. The expression of PKC-βII, PKC-γ, PKC-η, PKC-μ, and PKC-λ also increased following hypoxia (1% O2-99% N2; 24 h), and these protein levels remained elevated following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Increases in the expression of PKC-ε and PKC-ζ were also observed following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Moreover, inhibition of PKC with chelerythrine chloride (10 μM) attenuated the hypoxia-induced increases in [14C]sucrose permeability. Similar to what was observed in RMECs, total PKC activity was also stimulated in cerebral microvessels isolated from rats exposed to hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min). In contrast, hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min) significantly increased the expression levels of only PKC-γ and PKC-θ in the in vivo hypoxia model. These data demonstrate that hypoxia-induced BBB paracellular permeability changes occur via a PKC-dependent mechanism, possibly by differentially regulating the protein expression of the 11 PKC isozymes.

scholarly journals Intravenous injection of cyclophilin A realizes the transient and reversible opening of barrier of neural vasculature through basigin in endothelial cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Narumi Nakada-Honda ◽  
Dan Cui ◽  
Satoshi Matsuda ◽  
Eiji Ikeda
Keyword(s):  
Endothelial Cells ◽  
Single Dose ◽  
Blood Brain Barrier ◽  
Intravenous Injection ◽  
Cyclophilin A ◽  
Brain Barrier ◽  
Blood Brain ◽  
Neural Tissues ◽  
Neural Diseases

AbstractNeural vasculature forms the blood–brain barrier against the delivery of systemically administered therapeutic drugs into parenchyma of neural tissues. Therefore, procedures to open the blood–brain barrier with minimal damage to tissues would lead to the great progress in therapeutic strategy for intractable neural diseases. In this study, through analyses with mouse in vitro brain microvascular endothelial cells and in vivo neural vasculature, we demonstrate that the administration of cyclophilin A (CypA), a ligand of basigin which is expressed in barrier-forming endothelial cells, realizes the artificial opening of blood–brain barrier. Monolayers of endothelial cells lost their barrier properties through the disappearance of claudin-5, an integral tight junction molecule, from cell membranes in a transient and reversible manner. Furthermore, the intravenous injection of a single dose of CypA into mice resulted in the opening of blood–brain barrier for a certain period which enabled the enhanced delivery of systemically administered doxorubicin into the parenchyma of neural tissues. These findings that the pre-injection of a single dose of CypA realizes an artificial, transient as well as reversible opening of blood–brain barrier are considered to be a great step toward the establishment of therapeutic protocols to overcome the intractability of neural diseases.

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

2009 ◽  
Vol 29 (9) ◽  
pp. 1491-1502 ◽  
Author(s):  
Ruth Lyck ◽  
Nadine Ruderisch ◽  
Anton G Moll ◽  
Oliver Steiner ◽  
Clemens D Cohen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Amino Acid Transporters ◽  
Mrna Levels ◽  
Blood Brain ◽  
Barrier Formation ◽  
Platelet Endothelial Cell Adhesion ◽  
Induced Changes

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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