Abstract
The blood–brain barrier (BBB) plays an important role in brain homeostasis. Hypoxia/ischemia constitutes an important stress factor involved in several neurological disorders by inducing the disruption of the BBB, ultimately leading to cerebral edema formation. Yet, our current understanding of the cellular and molecular mechanisms underlying the BBB disruption following cerebral hypoxia/ischemia remains limited. Stem cell-based models of the human BBB present some potentials to address such issues. Yet, such models have not been validated in regard of its ability to respond to hypoxia/ischemia as existing models. In this study, we investigated the cellular response of two iPSC-derived brain microvascular endothelial cell (BMEC) monolayers to respond to oxygen-glucose deprivation (OGD) stress, using two induced pluripotent stem cells (iPSC) lines. iPSC-derived BMECs responded to prolonged (24 h) and acute (6 h) OGD by showing a decrease in the barrier function and a decrease in tight junction complexes. Such iPSC-derived BMECs responded to OGD stress via a partial activation of the HIF-1 pathway, whereas treatment with anti-angiogenic pharmacological inhibitors (sorafenib, sunitinib) during reoxygenation worsened the barrier function. Taken together, our results suggest such models can respond to hypoxia/ischemia similarly to existing in vitro models and support the possible use of this model as a screening platform for identifying novel drug candidates capable to restore the barrier function following hypoxic/ischemic injury.
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Acknowledgements
S.P. and A.A. performed the experiments presented in this study. A.A. designed the study and wrote the manuscript.
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This study was funded by TTUHSC institutional funds and by a Laura W. Bush Institute for Women’s Health seed grant to A.A.
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Supplementary Figure 1: Effect of prolonged OGD stress on iPSC-derived neurons and astrocytes. iPSC-derived astrocytes and neurons were incubated in DMEM- for 24 hours. Cells incubated in DMEM+ for 24 hours served as controls. Cell metabolic activity was assessed by measuring changes in MTS in astrocytes (A) and neurons (B) cultures. Secreted VEGF levels in astrocytes (C) and neurons (D) monocultures. Note the higher basal VEGF levels in astrocytes compared to neurons, and the higher VEGF levels in CTR90F-astrocytes compared to CTR65M. (E) Representative micrograph pictures of iPSC-derived neurons following normoxic or prolonged OGD stress. Scale bar = 50µm. (F) Quantitative analysis of neurite cell density. N=3/group, * and ** denote P<0.05 and P<0.01 versus control. Supplementary Figure 2: Mannitol permeability profile in CTR90F and CTR65M monocultures following OGD stress. Changes in mannitol were directly assessed in CTR90F and CTR65M-BMECs after OGD treatment by adding [14C]-mannitol in the apical chamber, whereas sampling occurred for every 15 minutes for 60 minutes. Supplementary Figure 3: Effect of OGD/reoxygenation on astrocytes and neurons. (A) Cell metabolic activity in iPSC-derived astrocytes. Cells were exposed to OGD for 6 hours followed by reoxygenation for 18 hours. (B) HIF-1α protein levels in iPSC-derived astrocytes in normoxic and following 6 hours OGD stress. Note the lower basal expression and the mitigated increase in CTR65M-astrocytes (C) Secreted VEGF levels in iPSC-derived astrocytes in normoxic and OGD stress. (D) Cell metabolic activity in iPSC-derived neurons following OGD/reoxygenation stress. (E) Representative micrograph pictures of iPSC-derived neurons following exposure to OGD and reoxygenation stress. Note the alteration in the quality of neurites, with a severe degradation following reoxygenation stress. Scale bar = 50µm. (F) Quantitative analysis of neurite cell density. N=3/group, * and ** denote P<0.05 and P<0.01 versus control. Supplementary Figure 3: Influence of co-cultures on BMECs response to OGD/reoxygenation stress. iPSC-derived BMECs were co-cultured with their isogenic iPSC-derived astrocytes or neurons for 48 hours prior exposure to OGD/reoxygenation stress. (A) TEER values and (B) fluorescein permeability in BMECs/astrocytes co-cultures. Note the differential outcomes between CTR90F co-cultures versus CTR65M co-cultures. (C and D) TEER and fluorescein permeability values in BMECs/neurons co-cultures. Note the similar outcomes in terms of paracellular permeability in BMECs/neurons co-cultures versus BMECs/astrocytes co-cultures. N=3/group, * and ** denote P<0.05 and P<0.01 versus control. Supplementary material 1 (PDF 1399 KB)
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Page, S., Raut, S. & Al-Ahmad, A. Oxygen-Glucose Deprivation/Reoxygenation-Induced Barrier Disruption at the Human Blood–Brain Barrier is Partially Mediated Through the HIF-1 Pathway. Neuromol Med 21, 414–431 (2019). https://doi.org/10.1007/s12017-019-08531-z
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DOI: https://doi.org/10.1007/s12017-019-08531-z