Functional Expression of Multidrug-Resistance (MDR) Transporters in Developing Human Fetal Brain Endothelial Cells

Lye, Phetcharawan; Bloise, Enrrico; Império, Güínever E.; Chitayat, David; Matthews, Stephen G.

doi:10.3390/cells11142259

Cited by 8 publications

(26 citation statements)

References 75 publications

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“…The angiogenesis of fetal BMECs in humans may be related to P-gp (Barakat et al, 2008 ). In vitro studies on human primary fetal BMECs detected P-gp in capillary-like microvessels, cytoplasm, and cell nuclei through immunolocalization (Lye et al, 2022 ). Tsai et al ( 2002 ) showed that in the mouse brain, P-gp expression is lower both during late embryogenesis and early neonatal period and increases gradually during brain maturation (P21), where P-gp protein levels approached adult levels.…”

Section: Resultsmentioning

confidence: 99%

“…Tsai et al ( 2002 ) showed that in the mouse brain, P-gp expression is lower both during late embryogenesis and early neonatal period and increases gradually during brain maturation (P21), where P-gp protein levels approached adult levels. Nevertheless, even though P-gp protein and ABCB1 gene expression are lower during early and mid-gestational periods, its function does not change, and it works enough to protect BBB (Lye et al, 2022 ). The lower expression seems to be conserved since it was observed in mice, rats, and humans (Schinkel et al, 1994 , 1995 ; Matsuoka et al, 1999 ; Watchko et al, 2001 ; Tsai et al, 2002 ; Daood et al, 2008 ).…”

Section: Resultsmentioning

confidence: 99%

“…BMECs have a mesodermal origin and invade the nervous system in the early stages of neural tube development, forming the blood–brain barrier's anatomical core (BBB). During these initial stages, BMECs display a non-specialized phenotype and lower gene expression of constitutive proteins, which may compromise BBB's integrity and permeability, exposing the brain to xenobiotics and making it vulnerable to inflammation (Strazielle and Ghersi-Egea, 2015 ; Sweeney et al, 2018 ; Ahmad et al, 2020 ; Yu et al, 2020 ; Eng et al, 2022 ; Lye et al, 2022 ). However, after the remodeling and maturation of brain regions, BMECs specialize and adapt to diverse phenotypes, such as veins, arteries, and capillaries (Hogan et al, 2004 ; Fantin et al, 2013 ; Engelhardt and Liebner, 2014 ; Dejana et al, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

“…However, BMECs malfunction may cause the progressive loss and degeneration of the NVU, a condition observed in many neurological disorders, such as stroke, brain injury, cancer, and neurodegenerative diseases such as Alzheimer's disease (De Luca et al, 2020 ; Schaeffer and Iadecola, 2021 ; Soto-Rojas et al, 2021 ). Xenobiotics can cause deleterious changes in brain maturation that can lead to the development of neurological disorders throughout life (Strazielle and Ghersi-Egea, 2015 ; Eng et al, 2022 ; Lye et al, 2022 ). Adult BMECs provide essential neuroprotection against environmental toxins through elevated levels of efflux transporters (P-gp/ABCB1, MRP1/ABCC1, BCRP/ABCG2) (Gomez-Zepeda et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

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Simple and efficient protocol to isolate and culture brain microvascular endothelial cells from newborn mice

Nicolicht-Amorim

Delgado-García

Nakamura

et al. 2022

Front. Cell. Neurosci.

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The neurovascular unit (NVU) is a multicellular structure comprising of neurons, glial cells, and non-neural cells, and it is supported by a specialized extracellular matrix, the basal lamina. Astrocytes, brain microvascular endothelial cells (BMECs), pericytes, and smooth muscle cells constitute the blood–brain barrier (BBB). BMECs have a mesodermal origin and invade the nervous system early in neural tube development, forming the BBB anatomical core. BMECs are connected by adherent junction complexes composed of integral membrane and cytoplasmic proteins. In vivo and in vitro studies have shown that, given the proximity and relationship with neural cells, BMECs acquire a unique gene expression profile, proteome, and specific mechanical and physical properties compared to endothelial cells from the general vasculature. BMECs are fundamental in maintaining brain homeostasis by regulating transcellular and paracellular transport of fluids, molecules, and cells. Therefore, it is essential to gain in-depth knowledge of the dynamic cellular structure of the cells in the NVU and their interactions with health and disease. Here we describe a significantly improved and simplified protocol using C57BL/6 newborn mice at postnatal day 1 (PND1) to isolate, purify, and culture BMECs monolayers in two different substrates (glass coverslips and transwell culture inserts). In vitro characterization and validation of the BMEC primary culture monolayers seeded on glass or insert included light microscopy, immunolabeling, and gene expression profile. Transendothelial electrical resistance (TEER) measurement and diffusion test were used as functional assays for adherent junction complexes and integrity and permeability of BMECs monolayers. The protocol presented here for the isolation and culture of BMECs is more straightforward than previously published protocols and yields a high number of purified cells. Finally, we tested BMECs function using the oxygen–glucose deprivation (OGD) model of hypoxia. This protocol may be suitable as a bioscaffold for secondary cell seeding allowing the study and better understanding of the NVU.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Simple and efficient protocol to isolate and culture brain microvascular endothelial cells from newborn mice

Nicolicht-Amorim

Delgado-García

Nakamura

et al. 2022

Front. Cell. Neurosci.

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show abstract

“…Functional expression of P-gp and BCRP has been demonstrated in isolated human fetal brain endothelial cells (hfBECs) derived from early and mid-gestation [ 9 ]. These results indicate that P-gp and BCRP at the BBB protect the developing CNS from early-gestation and throughout mid-pregnancy.…”

Section: Introductionmentioning

confidence: 99%

Effects of bacterial and viral pathogen-associated molecular patterns (PAMPs) on multidrug resistance (MDR) transporters in brain endothelial cells of the developing human blood–brain barrier

Lye

Bloise

Matthews

2023

Fluids Barriers CNS

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Background The multidrug resistance (MDR) transporters, P-glycoprotein (P-gp, encoded by ABCB1) and breast cancer resistance protein (BCRP/ABCG2) contribute to the blood–brain barrier (BBB), protecting the brain from drug exposure. The impact of infection on MDR in the developing human BBB remains to be determined. We hypothesized that exposure to bacterial and viral pathogen-associated molecular patterns (PAMPs) modify MDR expression and activity in human fetal brain endothelial cells (hfBECs) isolated from early and mid-gestation brain microvessels. Methods We modelled infection (4 h and 24 h) using the bacterial PAMP, lipopolysaccharide (LPS; a toll-like receptor [TLR]-4 ligand) or the viral PAMPs, polyinosinic polycytidylic acid (Poly I:C; TLR-3 ligand) and single-stranded RNA (ssRNA; TLR-7/8 ligand). mRNA expression was assessed by qPCR, whereas protein expression was assessed by Western blot or immunofluorescence. P-gp and BCRP activity was evaluated by Calcein-AM and Chlorin-6 assays. Results TLRs-3,4 and 8 were expressed by the isolated hfBECs. Infection mimics induced specific pro-inflammatory responses as well as changes in P-gp/ABCB1 or BCRP/ABCG2 expression (P < 0.05). LPS and ssRNA significantly decreased P-gp activity at 4 and 24 h in early and mid-gestation (P < 0.03-P < 0.001), but significantly increased BCRP activity in hfBECs in a dose-dependent pattern (P < 0.05-P < 0.002). In contrast, Poly-IC significantly decreased P-gp activity after 4 h in early (P < 0.01) and mid gestation (P < 0.04), but not 24 h, and had no overall effect on BCRP activity, though BCRP activity was increased with the highest dose at 24 h in mid-gestation (P < 0.05). Conclusions Infectious PAMPs significantly modify the expression and function of MDR transporters in hfBECs, though effects are PAMP-, time- and dose-specific. In conclusion, bacterial and viral infections during pregnancy likely have profound effects on exposure of the fetal brain to physiological and pharmacological substrates of P-gp and BCRP, potentially leading to altered trajectories of fetal brain development.

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Hypoxia modifies levels of the SARS-CoV-2 cell entry proteins, angiotensin-converting enzyme 2, and furin in fetal human brain endothelial cells

Mughis¹,

Lye²,

Matthews³

et al. 2023

American Journal of Obstetrics & Gynecology MFM

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Functional Expression of Multidrug-Resistance (MDR) Transporters in Developing Human Fetal Brain Endothelial Cells

Cited by 8 publications

References 75 publications

Simple and efficient protocol to isolate and culture brain microvascular endothelial cells from newborn mice

Simple and efficient protocol to isolate and culture brain microvascular endothelial cells from newborn mice

Effects of bacterial and viral pathogen-associated molecular patterns (PAMPs) on multidrug resistance (MDR) transporters in brain endothelial cells of the developing human blood–brain barrier

Hypoxia modifies levels of the SARS-CoV-2 cell entry proteins, angiotensin-converting enzyme 2, and furin in fetal human brain endothelial cells

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