Re-evaluation of the role of P-glycoprotein inin vitrodrug permeability studies with the bovine brain microvessel endothelial cells

Abstract

1. Currently available in vitro blood-brain barrier models all have recognized restrictions. In addition to leakiness, inconsistent data about P-glycoprotein mediated efflux limit the attractiveness of the primary bovine brain microvessel endothelial cells (BBMECs). Therefore, we re-evaluated the role of P-glycoprotein mediated efflux with two culture conditions in BBMECs for prediction of drug permeability of potential P-glycoprotein substrates. 2. BBMECs were monocultured on filters on petri dishes and on filter inserts, and expression and localization of P-glycoprotein were compared by using western blot and confocal microscopy, respectively. The functionality of P-glycoprotein was assessed by using cellular uptake, calcein-AM and bidirectional transport assays. 3. P-glycoprotein expression was higher in BBMECs cultured on filter inserts decreasing the permeability of digoxin and paclitaxel, but not the permeability of vinblastine. However, the monocultured BBMECs were not able to demonstrate efflux in the bidirectional transport assays. Under certain culture conditions, occludin may not be correctly located, perhaps explaining in part the leakiness of BBMECs. 4. In conclusion, BBMECs, despite possessing a functional P-glycoprotein, under certain culture conditions may not be a suitable in vitro model for the bidirectional transport assays and for predicting the permeability of drugs and xenobiotics that are potential P-glycoprotein substrates.

An Electrically Tight In Vitro Blood–Brain Barrier Model Displays Net Brain-to-Blood Efflux of Substrates for the ABC Transporters, P-gp, Bcrp and Mrp-1

“…A recent study by Hakkarainen et al demonstrated P-gp expression in primary bovine endothelial cells with western blotting and immunofluorescence, as well as P-gp function as evaluated by the calcein-AM assay, but did not observe a net transendothelial P-gp-mediated efflux. The authors also reported low TEER values and high sucrose permeabilities, indicating that the paracellular flux component may have masked the transcellular efflux [20]. We found high TEER values in this study, although they decreased gradually during the transport experiments.…”

“…Immunocytochemical stainings further confirmed the presence of P-gp, Bcrp and Mrp-1 in the endothelial cells as well as in freshly isolated capillaries. P-gp and Bcrp expression have previously been shown in BBB models [6,19,20,30], whereas Mrp expression is more controversial. Mrp-1 has been shown on the mRNA level in bovine brain capillaries [9] and in bovine and porcine brain endothelial cells [9,68] and on the protein level in rat brain endothelial cells [30], but also Mrp-4 and -5 mRNA have been shown in bovine and porcine endothelial cells [9,10,68].…”

“…Other studies have demonstrated expression and function of P-gp in the bovine brain endothelial cells [19][20][21], but vectorial transport studies have shown efflux ratios below 2 [19][20][21][22][23], which is the generally accepted threshold for concluding active efflux transporter involvement [24]. However, with the exception of the Cecchelli et al [21], these cell culture models displayed transendothelial electrical resistance (TEER) values in the range of 100-300 Ω·cm 2 [20,22,23], which are relatively low compared to the estimated in vivo barrier TEER of 1,000-2,000 Ω·cm 2 [25,26]. The apparently low functional expression of P-gp observed in these studies could be due to insufficient differentiation of the endothelial cells into a BBB-like phenotype, or alternatively, an active efflux could have been masked by high paracellular fluxes in the low-resistance monolayers [27][28][29].…”

Organization of Endothelial Cells, Pericytes, and Astrocytes into a 3D Microfluidic in Vitro Model of the Blood–Brain Barrier

“…Since the expression of P-glycoprotein alone cannot positively determine the functionality of the P-gp efflux pump, − a quantitative approach using bidirectional permeability was used, instead of methods that only evaluate the level of expression of proteins such as a Western blot or qPCR, to evaluate its functional expression. We measured the apical-to-basolateral (AB) and basoalteral-to-apical (BA) permeability of [ 3 H]-dexamethasone, which is a substrate for the P-gp efflux pump across b.End3 cell monolayers in the bi- and triculture models.…”

In vitro models of the blood–brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use

“…For instance, many reports have shown bovine BBB models with TEER values in the range of 30-150 cm 2 and/or lack of functional activity of both ABC and SLC-transporters known to be present at the BBB in vivo. 149,166,173,198,199 Intra-laboratory variations are evident in a series of publications by the group of de Boer where TEER varies from high values around 800 cm 2 to around 150-300 cm 2,19,187,192,193 using the same model in the same laboratory. This is even clearer in a study by Helms et al, 53 where TEER averages varied from 327 AE 30 cm 2 to 2555 AE399 cm 2 across model batches within the same study.…”