Modeling and rescue of defective blood-brain barrier function of induced brain microvascular endothelial cells from childhood cerebral adrenoleukodystrophy patients

Catherine A.A. Lee, Hannah S. Seo, Anibal G. Armien, Frank S. Bates, Jakub Tolar, Samira M. Azarin

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Background: X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene. 40% of X-ALD patients will convert to the deadly childhood cerebral form (ccALD) characterized by increased permeability of the brain endothelium that constitutes the blood-brain barrier (BBB). Mutation information and molecular markers investigated to date are not predictive of conversion. Prior reports have focused on toxic metabolic byproducts and reactive oxygen species as instigators of cerebral inflammation and subsequent immune cell invasion leading to BBB breakdown. This study focuses on the BBB itself and evaluates differences in brain endothelium integrity using cells from ccALD patients and wild-type (WT) controls. Methods: The blood-brain barrier of ccALD patients and WT controls was modeled using directed differentiation of induced pluripotent stem cells (iPSCs) into induced brain microvascular endothelial cells (iBMECs). Immunocytochemistry and PCR confirmed characteristic expression of brain microvascular endothelial cell (BMEC) markers. Barrier properties of iBMECs were measured via trans-endothelial electrical resistance (TEER), sodium fluorescein permeability, and frayed junction analysis. Electron microscopy and RNA-seq were used to further characterize disease-specific differences. Oil-Red-O staining was used to quantify differences in lipid accumulation. To evaluate whether treatment with block copolymers of poly(ethylene oxide) and poly(propylene oxide) (PEO-PPO) could mitigate defective properties, ccALD-iBMECs were treated with PEO-PPO block copolymers and their barrier properties and lipid accumulation levels were quantified. Results: iBMECs from patients with ccALD had significantly decreased TEER (2592 ± 110 Ω cm2) compared to WT controls (5001 ± 172 Ω cm2). They also accumulated lipid droplets to a greater extent than WT-iBMECs. Upon treatment with a PEO-PPO diblock copolymer during the differentiation process, an increase in TEER and a reduction in lipid accumulation were observed for the polymer treated ccALD-iBMECs compared to untreated controls. Conclusions: The finding that BBB integrity is decreased in ccALD and can be rescued with block copolymers opens the door for the discovery of BBB-specific molecular markers that can indicate the onset of ccALD and has therapeutic implications for preventing the conversion to ccALD.

Original languageEnglish (US)
Article number9
JournalFluids and Barriers of the CNS
Volume15
Issue number1
DOIs
StatePublished - Apr 4 2018

Fingerprint

Adrenoleukodystrophy
Blood-Brain Barrier
Endothelial Cells
Brain
Ethylene Oxide
Electric Impedance
Lipids
Endothelium
Permeability
Induced Pluripotent Stem Cells
Mutation
Poisons
Fluorescein
Reactive Oxygen Species
Electron Microscopy
Polymers
Therapeutics
Immunohistochemistry
RNA
Staining and Labeling

Keywords

  • Adrenoleukodystrophy
  • Amphiphilic block copolymers
  • Brain microvascular endothelial cells
  • Human induced pluripotent stem cells (hiPSC)
  • In vitro human blood-brain barrier (BBB) model
  • Trans-endothelial electrical resistance

Cite this

@article{8e9ee29db85746d9a0063dde5720c847,
title = "Modeling and rescue of defective blood-brain barrier function of induced brain microvascular endothelial cells from childhood cerebral adrenoleukodystrophy patients",
abstract = "Background: X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene. 40{\%} of X-ALD patients will convert to the deadly childhood cerebral form (ccALD) characterized by increased permeability of the brain endothelium that constitutes the blood-brain barrier (BBB). Mutation information and molecular markers investigated to date are not predictive of conversion. Prior reports have focused on toxic metabolic byproducts and reactive oxygen species as instigators of cerebral inflammation and subsequent immune cell invasion leading to BBB breakdown. This study focuses on the BBB itself and evaluates differences in brain endothelium integrity using cells from ccALD patients and wild-type (WT) controls. Methods: The blood-brain barrier of ccALD patients and WT controls was modeled using directed differentiation of induced pluripotent stem cells (iPSCs) into induced brain microvascular endothelial cells (iBMECs). Immunocytochemistry and PCR confirmed characteristic expression of brain microvascular endothelial cell (BMEC) markers. Barrier properties of iBMECs were measured via trans-endothelial electrical resistance (TEER), sodium fluorescein permeability, and frayed junction analysis. Electron microscopy and RNA-seq were used to further characterize disease-specific differences. Oil-Red-O staining was used to quantify differences in lipid accumulation. To evaluate whether treatment with block copolymers of poly(ethylene oxide) and poly(propylene oxide) (PEO-PPO) could mitigate defective properties, ccALD-iBMECs were treated with PEO-PPO block copolymers and their barrier properties and lipid accumulation levels were quantified. Results: iBMECs from patients with ccALD had significantly decreased TEER (2592 ± 110 Ω cm2) compared to WT controls (5001 ± 172 Ω cm2). They also accumulated lipid droplets to a greater extent than WT-iBMECs. Upon treatment with a PEO-PPO diblock copolymer during the differentiation process, an increase in TEER and a reduction in lipid accumulation were observed for the polymer treated ccALD-iBMECs compared to untreated controls. Conclusions: The finding that BBB integrity is decreased in ccALD and can be rescued with block copolymers opens the door for the discovery of BBB-specific molecular markers that can indicate the onset of ccALD and has therapeutic implications for preventing the conversion to ccALD.",
keywords = "Adrenoleukodystrophy, Amphiphilic block copolymers, Brain microvascular endothelial cells, Human induced pluripotent stem cells (hiPSC), In vitro human blood-brain barrier (BBB) model, Trans-endothelial electrical resistance",
author = "Lee, {Catherine A.A.} and Seo, {Hannah S.} and Armien, {Anibal G.} and Bates, {Frank S.} and Jakub Tolar and Azarin, {Samira M.}",
year = "2018",
month = "4",
day = "4",
doi = "10.1186/s12987-018-0094-5",
language = "English (US)",
volume = "15",
journal = "Fluids and Barriers of the CNS",
issn = "2045-8118",
publisher = "BioMed Central",
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}

TY - JOUR

T1 - Modeling and rescue of defective blood-brain barrier function of induced brain microvascular endothelial cells from childhood cerebral adrenoleukodystrophy patients

AU - Lee, Catherine A.A.

AU - Seo, Hannah S.

AU - Armien, Anibal G.

AU - Bates, Frank S.

AU - Tolar, Jakub

AU - Azarin, Samira M.

PY - 2018/4/4

Y1 - 2018/4/4

N2 - Background: X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene. 40% of X-ALD patients will convert to the deadly childhood cerebral form (ccALD) characterized by increased permeability of the brain endothelium that constitutes the blood-brain barrier (BBB). Mutation information and molecular markers investigated to date are not predictive of conversion. Prior reports have focused on toxic metabolic byproducts and reactive oxygen species as instigators of cerebral inflammation and subsequent immune cell invasion leading to BBB breakdown. This study focuses on the BBB itself and evaluates differences in brain endothelium integrity using cells from ccALD patients and wild-type (WT) controls. Methods: The blood-brain barrier of ccALD patients and WT controls was modeled using directed differentiation of induced pluripotent stem cells (iPSCs) into induced brain microvascular endothelial cells (iBMECs). Immunocytochemistry and PCR confirmed characteristic expression of brain microvascular endothelial cell (BMEC) markers. Barrier properties of iBMECs were measured via trans-endothelial electrical resistance (TEER), sodium fluorescein permeability, and frayed junction analysis. Electron microscopy and RNA-seq were used to further characterize disease-specific differences. Oil-Red-O staining was used to quantify differences in lipid accumulation. To evaluate whether treatment with block copolymers of poly(ethylene oxide) and poly(propylene oxide) (PEO-PPO) could mitigate defective properties, ccALD-iBMECs were treated with PEO-PPO block copolymers and their barrier properties and lipid accumulation levels were quantified. Results: iBMECs from patients with ccALD had significantly decreased TEER (2592 ± 110 Ω cm2) compared to WT controls (5001 ± 172 Ω cm2). They also accumulated lipid droplets to a greater extent than WT-iBMECs. Upon treatment with a PEO-PPO diblock copolymer during the differentiation process, an increase in TEER and a reduction in lipid accumulation were observed for the polymer treated ccALD-iBMECs compared to untreated controls. Conclusions: The finding that BBB integrity is decreased in ccALD and can be rescued with block copolymers opens the door for the discovery of BBB-specific molecular markers that can indicate the onset of ccALD and has therapeutic implications for preventing the conversion to ccALD.

AB - Background: X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene. 40% of X-ALD patients will convert to the deadly childhood cerebral form (ccALD) characterized by increased permeability of the brain endothelium that constitutes the blood-brain barrier (BBB). Mutation information and molecular markers investigated to date are not predictive of conversion. Prior reports have focused on toxic metabolic byproducts and reactive oxygen species as instigators of cerebral inflammation and subsequent immune cell invasion leading to BBB breakdown. This study focuses on the BBB itself and evaluates differences in brain endothelium integrity using cells from ccALD patients and wild-type (WT) controls. Methods: The blood-brain barrier of ccALD patients and WT controls was modeled using directed differentiation of induced pluripotent stem cells (iPSCs) into induced brain microvascular endothelial cells (iBMECs). Immunocytochemistry and PCR confirmed characteristic expression of brain microvascular endothelial cell (BMEC) markers. Barrier properties of iBMECs were measured via trans-endothelial electrical resistance (TEER), sodium fluorescein permeability, and frayed junction analysis. Electron microscopy and RNA-seq were used to further characterize disease-specific differences. Oil-Red-O staining was used to quantify differences in lipid accumulation. To evaluate whether treatment with block copolymers of poly(ethylene oxide) and poly(propylene oxide) (PEO-PPO) could mitigate defective properties, ccALD-iBMECs were treated with PEO-PPO block copolymers and their barrier properties and lipid accumulation levels were quantified. Results: iBMECs from patients with ccALD had significantly decreased TEER (2592 ± 110 Ω cm2) compared to WT controls (5001 ± 172 Ω cm2). They also accumulated lipid droplets to a greater extent than WT-iBMECs. Upon treatment with a PEO-PPO diblock copolymer during the differentiation process, an increase in TEER and a reduction in lipid accumulation were observed for the polymer treated ccALD-iBMECs compared to untreated controls. Conclusions: The finding that BBB integrity is decreased in ccALD and can be rescued with block copolymers opens the door for the discovery of BBB-specific molecular markers that can indicate the onset of ccALD and has therapeutic implications for preventing the conversion to ccALD.

KW - Adrenoleukodystrophy

KW - Amphiphilic block copolymers

KW - Brain microvascular endothelial cells

KW - Human induced pluripotent stem cells (hiPSC)

KW - In vitro human blood-brain barrier (BBB) model

KW - Trans-endothelial electrical resistance

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U2 - 10.1186/s12987-018-0094-5

DO - 10.1186/s12987-018-0094-5

M3 - Article

C2 - 29615068

AN - SCOPUS:85044872064

VL - 15

JO - Fluids and Barriers of the CNS

JF - Fluids and Barriers of the CNS

SN - 2045-8118

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