Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress 2023 EHA Congress 2023 ASCO Annual Meeting
Clinical Collections
Advances in Alzheimer’s

At a glance: The ONWARDS insulin icodec trials

A round-up of the ONWARDS phase 3 clinical trials evaluating the novel weekly insulin icodec in people with diabetes.
ADVANCED SEARCH
Log in
Top
Fluids and Barriers of the CNS
Published in: Fluids and Barriers of the CNS 1/2016

Open Access 01-12-2016 | Research

Cerebral hypoxia/ischemia selectively disrupts tight junctions complexes in stem cell-derived human brain microvascular endothelial cells

Authors: Shyanne Page, Alli Munsell, Abraham J. Al-Ahmad

Published in: Fluids and Barriers of the CNS | Issue 1/2016

Login to get access

Abstract

Background

Cerebral hypoxia/ischemia (H/I) is an important stress factor involved in the disruption of the blood–brain barrier (BBB) following stroke injury, yet the cellular and molecular mechanisms on how the human BBB responds to such injury remains unclear. In this study, we investigated the cellular response of the human BBB to chemical and environmental H/I in vitro.

Methods

In this study, we used immortalized hCMEC/D3 and IMR90 stem-cell derived human brain microvascular endothelial cell lines (IMR90-derived BMECs). Hypoxic stress was achieved by exposure to cobalt chloride (CoCl2) or by exposure to 1 % hypoxia and oxygen/glucose deprivation (OGD) was used to model ischemic injury. We assessed barrier function using both transendothelial electrical resistance (TEER) and sodium fluorescein permeability. Changes in cell junction integrity were assessed by immunocytochemistry and cell viability was assessed by trypan-blue exclusion and by MTS assays. Statistical analysis was performed using one-way analysis of variance (ANOVA).

Results

CoCl2 selectively disrupted the barrier function in IMR90-derived BMECs but not in hCMEC/D3 monolayers and cytotoxic effects did not drive such disruption. In addition, hypoxia/OGD stress significantly disrupted the barrier function by selectively disrupting tight junctions (TJs) complexes. In addition, we noted an uncoupling between cell metabolic activity and barrier integrity.

Conclusions

In this study, we demonstrated the ability of IMR90-derived BMECs to respond to hypoxic/ischemic injury triggered by both chemical and environmental stress by showing a disruption of the barrier function. Such disruption was selectively targeting TJ complexes and was not driven by cellular apoptosis. In conclusion, this study suggests the suitability of stem cell-derived human BMECs monolayers as a model of cerebral hypoxia/ischemia in vitro.
Appendix
Available only for authorised users
Literature
1.
Lopes Pinheiro MA, Kooij G, Mizee MR, Kamermans A, Enzmann G, Lyck R, et al. Immune cell trafficking across the barriers of the central nervous system in multiple sclerosis and stroke. Biochim Biophys Acta. 2015;1862(3):461–71.CrossRefPubMed
2.
Duits FH, Hernandez-Guillamon M, Montaner J, Goos JD, Montanola A, Wattjes MP, et al. Matrix metalloproteinases in Alzheimer’s disease and concurrent cerebral microbleeds. J Alzheimers Dis. 2015;48(3):711–20.CrossRefPubMed
3.
Drouin-Ouellet J, Sawiak SJ, Cisbani G, Lagace M, Kuan WL, Saint-Pierre M, et al. Cerebrovascular and blood-brain barrier impairments in Huntington’s disease: potential implications for its pathophysiology. Ann Neurol. 2015;78(2):160–77.CrossRefPubMed
4.
Qosa H, Abuasal BS, Romero IA, Weksler B, Couraud PO, Keller JN, et al. Differences in amyloid-beta clearance across mouse and human blood-brain barrier models: kinetic analysis and mechanistic modeling. Neuropharmacology. 2014;79:668–78.CrossRefPubMedPubMedCentral
5.
6.
Shah K, Abbruscato T. The role of blood-brain barrier transporters in pathophysiology and pharmacotherapy of stroke. Curr Pharm Des. 2014;20(10):1510–22.CrossRefPubMed
7.
Jickling GC, Liu D, Stamova B, Ander BP, Zhan X, Lu A, et al. Hemorrhagic transformation after ischemic stroke in animals and humans. J Cereb Blood Flow Metab. 2014;34:185–99.CrossRefPubMed
8.
Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Blaha MJ, et al. Heart disease and stroke statistics–2014 update: a report from the American Heart Association. Circulation. 2014;129:e28–292.CrossRefPubMed
9.
Engelhardt S, Al-Ahmad AJ, Gassmann M, Ogunshola OO. Hypoxia selectively disrupts brain microvascular endothelial tight junction complexes through a hypoxia-inducible factor-1 (HIF-1) dependent mechanism. J Cell Physiol. 2014;229(8):1096–105.CrossRefPubMed
10.
Engelhardt S, Huang SF, Patkar S, Gassmann M, Ogunshola OO. Differential responses of blood-brain barrier associated cells to hypoxia and ischemia: a comparative study. Fluids Barriers CNS. 2015;12:4.CrossRefPubMedPubMedCentral
11.
Al Ahmad A, Gassmann M, Ogunshola OO. Involvement of oxidative stress in hypoxia-induced blood-brain barrier breakdown. Microvasc Res. 2012;84(2):222–5.CrossRefPubMed
12.
Lee B, Clarke D, Al Ahmad A, Kahle M, Parham C, Auckland L, et al. Perlecan domain V is neuroprotective and proangiogenic following ischemic stroke in rodents. J Clin Investig. 2011;121(8):3005–23.CrossRefPubMedPubMedCentral
13.
Al Ahmad A, Gassmann M, Ogunshola OO. Maintaining blood-brain barrier integrity: pericytes perform better than astrocytes during prolonged oxygen deprivation. J Cell Physiol. 2009;218(3):612–22.CrossRefPubMed
14.
Abbruscato TJ, Davis TP. Combination of hypoxia/aglycemia compromises in vitro blood-brain barrier integrity. J Pharmacol Exp Ther. 1999;289:668–75.PubMed
15.
Yamagata K, Tagami M, Takenaga F, Yamori Y, Itoh S. Hypoxia-induced changes in tight junction permeability of brain capillary endothelial cells are associated with IL-1beta and nitric oxide. Neurobiol Dis. 2004;17:491–9.CrossRefPubMed
16.
Brown RC, Davis TP. Hypoxia/aglycemia alters expression of occludin and actin in brain endothelial cells. Biochem Biophys Res Commun. 2005;327:1114–23.CrossRefPubMed
17.
Culot M, Mysiorek C, Renftel M, Roussel BD, Hommet Y, Vivien D, et al. Cerebrovascular protection as a possible mechanism for the protective effects of NXY-059 in preclinical models: an in vitro study. Brain Res. 2009;1294:144–52.CrossRefPubMed
18.
Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318(5858):1917–20.CrossRefPubMed
19.
Wilson HK, Canfield SG, Hjortness MK, Palecek SP, Shusta EV. Exploring the effects of cell seeding density on the differentiation of human pluripotent stem cells to brain microvascular endothelial cells. Fluids Barriers CNS. 2015;12:13.CrossRefPubMedPubMedCentral
20.
Lippmann ES, Al-Ahmad A, Azarin SM, Palecek SP, Shusta EV. A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources. Sci Rep. 2014;4:4160.CrossRefPubMedPubMedCentral
21.
Lippmann ES, Azarin SM, Kay JE, Nessler RA, Wilson HK, Al-Ahmad A, et al. Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells. Nat Biotechnol. 2012;30(8):783–91.CrossRefPubMedPubMedCentral
22.
Weksler BB, Subileau EA, Perriere N, Charneau P, Holloway K, Leveque M, et al. Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J. 2005;19:1872–4.PubMed
23.
Perriere N, Demeuse P, Garcia E, Regina A, Debray M, Andreux JP, et al. Puromycin-based purification of rat brain capillary endothelial cell cultures. Effect on the expression of blood-brain barrier-specific properties. J Neurochem. 2005;93(2):279–89.CrossRefPubMed
24.
Guo L, Lan J, Lin Y, Guo P, Nie Q, Mao Q, et al. Hypoxia/ischemia up-regulates Id2 expression in neuronal cells in vivo and in vitro. Neurosci Lett. 2013;554:88–93.CrossRefPubMed
25.
Gotoh M, Sano-Maeda K, Murofushi H, Murakami-Murofushi K. Protection of neuroblastoma Neuro2A cells from hypoxia-induced apoptosis by cyclic phosphatidic acid (cPA). PLoS ONE. 2012;7(12):e51093.CrossRefPubMedPubMedCentral
26.
Wang B, Zou Y, Yuan ZL, Xiao JG. Genistein suppressed upregulation of vascular endothelial growth factor expression by cobalt chloride and hypoxia in rabbit retinal pigment epithelium cells. J Ocul Pharmacol Ther. 2003;19(5):457–64.CrossRefPubMed
27.
Bernaudin M, Bellail A, Marti HH, Yvon A, Vivien D, Duchatelle I, et al. Neurons and astrocytes express EPO mRNA: oxygen-sensing mechanisms that involve the redox-state of the brain. Glia. 2000;30:271–8.CrossRefPubMed
28.
Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA. 1995;92:5510–4.CrossRefPubMedPubMedCentral
29.
Minchenko A, Bauer T, Salceda S, Caro J. Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo. Lab Invest. 1994;71:374–9.PubMed
30.
Naik P, Cucullo L. In vitro blood-brain barrier models: current and perspective technologies. J Pharm Sci. 2012;101:1337–54.CrossRefPubMed
31.
Poller B, Gutmann H, Krahenbuhl S, Weksler B, Romero I, Couraud PO, et al. The human brain endothelial cell line hCMEC/D3 as a human blood-brain barrier model for drug transport studies. J Neurochem. 2008;107:1358–68.CrossRefPubMed
32.
Cucullo L, Couraud PO, Weksler B, Romero IA, Hossain M, Rapp E, et al. Immortalized human brain endothelial cells and flow-based vascular modeling: a marriage of convenience for rational neurovascular studies. J Cereb Blood Flow Metab. 2008;28(2):312–28.CrossRefPubMed
33.
Hartwig K, Fackler V, Jaksch-Bogensperger H, Winter S, Furtner T, Couillard-Despres S, et al. Cerebrolysin protects PC12 cells from CoCl2-induced hypoxia employing GSK3 beta signaling. Int J Dev Neurosci. 2014;38:52–8.CrossRefPubMed
34.
Guan D, Su Y, Li Y, Wu C, Meng Y, Peng X, et al. Tetramethylpyrazine inhibits CoCl2 -induced neurotoxicity through enhancement of Nrf2/GCLc/GSH and suppression of HIF1alpha/NOX2/ROS pathways. J Neurochem. 2015;134(3):551–65.CrossRefPubMed
35.
Huang CY, Hsieh YL, Ju DT, Lin CC, Kuo CH, Liou YF, et al. Attenuation of magnesium sulfate on CoCl(2)-induced cell death by activating ERK1/2/MAPK and inhibiting HIF-1alpha via mitochondrial apoptotic signaling suppression in a neuronal cell line. Chin J Physiol. 2015;58(4):244–53.CrossRefPubMed
36.
Mo SJ, Hong J, Chen X, Han F, Ni Y, Zheng Y, et al. VEGF-mediated NF-kappaB activation protects PC12 cells from damage induced by hypoxia. Neurosci Lett. 2016;610:54–9.CrossRefPubMed
37.
38.
Ogunshola OO, Al Ahmad A. HIF-1 at the blood-brain barrier: a mediator of permeability? Special topic: the many faces of hypoxia HIF-1 at the blood-brain barrier. High Alt Med Biol. 2012;13:153–61.CrossRefPubMed
39.
Zhu H, Wang Z, Xing Y, Gao Y, Ma T, Lou L, et al. Baicalin reduces the permeability of the blood-brain barrier during hypoxia in vitro by increasing the expression of tight junction proteins in brain microvascular endothelial cells. J Ethnopharmacol. 2011;141:714–20.CrossRefPubMed
40.
Yan J, Zhou B, Taheri S, Shi H. Differential effects of HIF-1 inhibition by YC-1 on the overall outcome and blood-brain barrier damage in a rat model of ischemic stroke. PLoS ONE. 2011;6:e27798.CrossRefPubMedPubMedCentral
41.
Kimura K, Teranishi S, Kawamoto K, Nishida T. Protective effect of dexamethasone against hypoxia-induced disruption of barrier function in human corneal epithelial cells. Exp Eye Res. 2011;92:388–93.CrossRefPubMed
42.
Lochhead JJ, McCaffrey G, Quigley CE, Finch J, DeMarco KM, Nametz N, et al. Oxidative stress increases blood-brain barrier permeability and induces alterations in occludin during hypoxia-reoxygenation. J Cereb Blood Flow Metab. 2010;30:1625–36.CrossRefPubMedPubMedCentral
43.
Natah SS, Srinivasan S, Pittman QJ, Zhao Z, Dunn JF. Effects of acute hypoxia and hyperthermia on the permeability of the blood brain barrier in adult rats. J Appl Physiol. 2009;107(4):1348–56.CrossRefPubMedPubMedCentral
44.
McCaffrey G, Willis CL, Staatz WD, Nametz N, Quigley CA, Hom S, et al. Occludin oligomeric assemblies at tight junctions of the blood-brain barrier are altered by hypoxia and reoxygenation stress. J Neurochem. 2009;110:58–71.CrossRefPubMedPubMedCentral
45.
Semenza GL. Dynamic regulation of stem cell specification and maintenance by hypoxia-inducible factors. Mol Aspects Med. 2015;47:15–23.PubMed
46.
Patak P, Jin F, Schafer ST, Metzen E, Hermann DM. The ATP-binding cassette transporters ABCB1 and ABCC1 are not regulated by hypoxia in immortalised human brain microvascular endothelial cells. Exp Transl Stroke Med. 2011;3:12.CrossRefPubMedPubMedCentral
47.
Cunningham LA, Wetzel M, Rosenberg GA. Multiple roles for MMPs and TIMPs in cerebral ischemia. Glia. 2005;50:329–39.CrossRefPubMed
48.
Yang T, Roder KE, Abbruscato TJ. Evaluation of bEnd5 cell line as an in vitro model for the blood-brain barrier under normal and hypoxic/aglycemic conditions. J Pharm Sci. 2007;96:3196–213.CrossRefPubMed
49.
50.
Vandekeere S, Dewerchin M, Carmeliet P. Angiogenesis revisited: an overlooked role of endothelial cell metabolism in vessel sprouting. Microcirculation. 2015;22(7):509–17.CrossRefPubMed
51.
Berg K, Hansen MB, Nielsen SE. A new sensitive bioassay for precise quantification of interferon activity as measured via the mitochondrial dehydrogenase function in cells (MTT-method). APMIS. 1990;98(2):156–62.CrossRefPubMed
Metadata
Title
Cerebral hypoxia/ischemia selectively disrupts tight junctions complexes in stem cell-derived human brain microvascular endothelial cells
Authors
Shyanne Page
Alli Munsell
Abraham J. Al-Ahmad
Publication date
01-12-2016
Publisher
BioMed Central
Published in
Fluids and Barriers of the CNS / Issue 1/2016
Electronic ISSN: 2045-8118
DOI
https://doi.org/10.1186/s12987-016-0042-1

Other articles of this Issue 1/2016

Fluids and Barriers of the CNS 1/2016 Go to the issue

Research

A comparison between the pathophysiology of multiple sclerosis and normal pressure hydrocephalus: is pulse wave encephalopathy a component of MS?

Research

Improving the clinical management of traumatic brain injury through the pharmacokinetic modeling of peripheral blood biomarkers

Review

Evolutionary development of embryonic cerebrospinal fluid composition and regulation: an open research field with implications for brain development and function

Research paper

MicroRNA-155 contributes to shear-resistant leukocyte adhesion to human brain endothelium in vitro

Review

Fluid and ion transfer across the blood–brain and blood–cerebrospinal fluid barriers; a comparative account of mechanisms and roles

Research

Increased CSF aquaporin-4, and interleukin-6 levels in dogs with idiopathic communicating internal hydrocephalus and a decrease after ventriculo-peritoneal shunting