42 research outputs found
Effects of oxygen-glucose deprivation (OGD) on barrier properties and mRNA transcript levels of selected marker proteins in brain endothelial cells/astrocyte co-cultures
Ischemic stroke has been shown to induce breakdown of the blood-brain barrier, although these changes are not fully characterized. Oxygen-glucose deprivation (OGD) has been used to investigate the effects of ischemia in cultured brain capillary endothelial cells, however this involves a change of medium which in itself may affect the cells. The aim of the present study was to investigate the effect of OGD and simple medium exchange followed by 48 h of reperfusion on barrier properties of primary bovine endothelial cells co-cultured with rat astrocytes. Barrier properties were evaluated by transendothelial electrical resistance measurements, passive permeability of flux markers, RT-qPCR and immunocytochemistry. Both OGD and simple medium exchange caused an increase in endothelial monolayer permeability. This correlated with reduced transcript levels of a number of tight junction and tight junction-associated proteins (claudin-1, claudin-5, occludin, ZO-1, tricellulin, marveld3 and PECAM-1), as well as with altered transcript level of several transporters and receptors (GLUT-1, HB-EGF, InsR, TfR, two members of the low density lipoprotein receptor family, LDLR and LRP-1, and the efflux transporter BCRP). In contrast, effects induced specifically by OGD were transient de-localization of claudin-5 from the junction zone, increased InsR localization at the plasma membrane and transient downregulation of MRP-1 and P-gp transcript levels. In conclusion, OGD caused changes in claudin-5 and InsR localization, as well as in MRP-1 and P-gp transcript levels. Our results however also indicated that medium exchange alone caused changes in functional barrier properties and expression levels of wide range of proteins
Endothelial cell phenotype differs strongly between in viro BCEC monocultures and BCEC/rat astrocyte co-cultures, a phenomenon which may contribute to the differences in barrier tightness
Culture of brain capillary pericytes for cytosolic calcium measurements and calcium imaging studies
Glutamate Efflux at the Blood-Brain Barrier:Cellular Mechanisms and Potential Clinical Relevance
Generation of a rat in-vitro blood-brain barrier model and initial insights on excitatory amino acid transporter expression
Bovine endothelial/rat astrocyte co-cultures cultured in highly buffered media functionally express MDR1 and BCRP
Hypoxia increases expression of selected blood–brain barrier transporters GLUT-1, P-gp, SLC7A5 and TFRC, while maintaining barrier integrity, in brain capillary endothelial monolayers
BACKGROUND: Brain capillary endothelial cells (BCECs) experience hypoxic conditions during early brain development. The newly formed capillaries are tight and functional before astrocytes and pericytes join the capillaries and establish the neurovascular unit. Brain endothelial cell phenotype markers P-gp (ABCB1), LAT-1(SLC7A5), GLUT-1(SLC2A1), and TFR(TFRC) have all been described to be hypoxia sensitive. Therefore, we hypothesized that monolayers of BCECs, cultured under hypoxic conditions, would show an increase in LAT-1, GLUT-1 and TFR expression and display tight endothelial barriers. METHODS AND RESULTS: Primary bovine BCECs were cultured under normoxic and hypoxic conditions. Chronic hypoxia induced HIF-1α stabilization and translocation to the nucleus, as judged by immunocytochemistry and confocal laser scanning imaging. Endothelial cell morphology, claudin-5 and ZO-1 localization and barrier integrity were unaffected by hypoxia, indicating that the tight junctions in the BBB model were not compromised. SLC7A5, SLC2A1, and TFRC-mRNA levels were increased in hypoxic cultures, while ABCB1 remained unchanged as shown by real-time qPCR. P-gp, TfR and GLUT-1 were found to be significantly increased at protein levels. An increase in uptake of [(3)H]-glucose was demonstrated, while a non-significant increase in the efflux ratio of the P-gp substrate [3H]-digoxin was observed in hypoxic cells. No changes were observed in functional LAT-1 as judged by uptake studies of [(3)H]-leucine. Stabilization of HIF-1α under normoxic conditions with desferrioxamine (DFO) mimicked the effects of hypoxia on endothelial cells. Furthermore, low concentrations of DFO caused an increase in transendothelial electrical resistance (TEER), suggesting that a slight activation of the HIF-1α system may actually increase brain endothelial monolayer tightness. Moreover, exposure of confluent monolayers to hypoxia resulted in markedly increase in TEER after 24 and 48 h, which corresponded to a higher transcript level of CLDN5. CONCLUSIONS: Our findings collectively suggest that hypoxic conditions increase some BBB transporters' expression via HIF-1α stabilization, without compromising monolayer integrity. This may in part explain why brain capillaries show early maturation, in terms of barrier tightness and protein expression, during embryogenesis, and provides a novel methodological tool for optimal brain endothelial culture. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12987-021-00297-6
Generation of transient and tunable oxygen gradients in microfluidic channels utilizing the oxygen scavenging properties of thiol-ene polymers
IPEC-J2 MDR1, a Novel High-Resistance Cell Line with Functional Expression of Human P‑glycoprotein (ABCB1) for Drug Screening Studies
The P-glycoprotein (P-gp) efflux
pump has been shown to affect
drug distribution and absorption in various organs and to cause drug
resistance in cancer therapy. The aim of this work was to develop
a cell line to serve as a screening system for potential substrates
of P-gp. This requires a cell line with high paracellular tightness,
low expression of nonhuman ABC transporters, and high expression of
functional human P-gp (ABCB1). The porcine intestinal epithelial cell
line, IPEC-J2, was selected as a transfection host, due to its ability
to form extremely high-resistance monolayers (>10 000 Ω·cm<sup>2</sup>) and its low endogenous expression of ABC-type efflux transporters.
The IPEC-J2 cells were transfected with a plasmid that contained the
sequence of the human <i>MDR1</i> gene, which encodes P-gp,
followed by a selection of successfully transfected cells with geneticin
and puromycin. The resulting cell line, IPEC-J2 MDR1, retained its
high transepithelilal resistance (>15 000 Ω·cm<sup>2</sup>), which translated into low permeability of the small hydrophilic
tracer, mannitol (<i>P</i> < 10<sup>–7</sup> cm·s<sup>–1</sup>). The lipophilic compound, diazepam, displayed high
permeability resulting in a dynamic range of 1500 (<i>P</i><sub>Diazepam</sub>/<i>P</i><sub>mannitol</sub>) to separate
high and low permeability compounds. Human P-gp was expressed predominantly
in the apical membrane, as demonstrated by immunocytochemistry, Western
blots, and a high efflux ratios (<i>P</i><sub>basolateral‑apical</sub>/<i>P</i><sub>apical‑basolateral</sub>) of known
P-gp substrates. P-gp was demonstrated to be responsible for the efflux
transport by substrate profiling, combined with application of P-gp
and BCRP inhibitors. Furthermore, the compounds atenolol, citalopram,
and mitoxantrone were identified as P-gp substrates. Functional P-gp
expression was shown to be stable through at least 10 cell passages.
In conclusion, the IPEC-J2 MDR1 cell line displays high paracellular
tightness combined with high expression of human P-gp and low expression
of porcine ABC transporters, and it may serve as a useful tool in
drug development studies