The mechanisms by which the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) induces neurological complications remain to be elucidated. We aimed to identify possible effects of hypoxia on the expression of SARS-CoV-2 cell entry mediators, angiotensin-converting enzyme 2 (ACE2) receptor and transmembrane protease serine 2 (TMPRSS2) protein, in human brain endothelial cells, in vitro . hCMEC/D3 cells were exposed to different oxygen tensions: 20% (Control group), 8% or 2% O 2 (Hypoxia groups). Cells were harvested 6-, 24- and 48 h following hypoxic challenge for assessment of mRNA and protein, using qPCR and Western Blot. The response of the brain endothelial cells to hypoxia was replicated using modular incubator chambers. We observed an acute increase (6 h, p < 0.05), followed by a longer-term decrease (48 h, p < 0.05) in ACE2 mRNA and protein expression, accompanied by reduced expression of TMPRSS2 protein levels (48 h, p < 0.05) under the more severe hypoxic condition (2% O 2 ). No changes in levels of von Willebrand Factor (vWF – an endothelial cell damage marker) or interleukin 6 (IL-6 – a pro-inflammatory cytokine) mRNA were observed. We conclude that hypoxia regulates brain endothelial cell ACE2 and TMPRSS2 expression in vitro , which may indicate human brain endothelial susceptibility to SARS-CoV-2 infection and subsequent brain sequelae.

P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) are two multidrug resistance (MDR) transporters expressed at the blood-brain barrier (BBB). They confer protection against entry of harmful molecules into the fetal brain. The fetus develops under relatively low oxygen concentrations; however, pregnancy disorders (including pre-eclampsia) may lead to even lower intrauterine oxygen levels. We investigated the effects of hypoxia on transporter expression and activity in human fetal brain endothelial cells (hfBECs) isolated in early and mid-gestation. Results indicate decreased BCRP protein and activity under hypoxia in early-gestation hfBECs. Mid-gestation hfBECs exhibited an increase in P-gp and BCRP activity following hypoxia. Results suggest a hypoxia-induced reduction in fetal brain protection in early-pregnancy, but a potential increase in transporter-mediated protection at the BBB during mid-gestation. This would modify accumulation of various key P-gp and BCRP physiological and pharmacological substrates in the developing fetal brain and may play a role in the pathogenesis of neurodevelopmental disorders commonly associated with in utero hypoxia.

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