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

BACKGROUND: It is not known whether human fetal brain endothelial cells that form the blood-brain barrier express angiotensin-converting enzyme 2, transmembrane serine protease 2, and furin, which are SARS-CoV-2 cell entry proteins. Moreover, it is unclear whether hypoxia, commonly observed during severe maternal COVID-19, can modify their level of expression. We hypothesized that human fetal brain endothelial cells isolated from early- and midpregnancy brain microvessels express angiotensin-converting enzyme 2, transmembrane serine protease 2, and furin. Furthermore, we hypothesized that hypoxia modifies their expression levels in a gestational age- and time-of-exposure-dependent manner. OBJECTIVE: This study aimed to investigate whether early- and midpregnancy human fetal brain endothelial cells express angiotensin-converting enzyme 2, transmembrane serine protease 2, and furin SARS-CoV-2-associated cell entry proteins and to determine the effects of hypoxia on angiotensin-converting enzyme 2, transmembrane serine protease 2, and furin expression levels in human fetal brain endothelial cells. STUDY DESIGN: This was a prospective study where human fetal brain endothelial cells isolated from early-pregnancy (12.4±0.7 weeks of gestation) and midpregnancy (17.9±0.5 weeks of gestation) fetal brain microvessels (6 per group) were exposed to different oxygen tensions (20%, 5%, and 1% oxygen) for 6, 24, and 48 hours. Angiotensin-converting enzyme 2, transmembrane serine protease 2, and furin messenger RNA and protein levels and localization were assessed using quantitative polymerase chain reaction, Western blot testing, and immunofluorescence. RESULTS: Angiotensin-converting enzyme 2, transmembrane serine protease 2, and furin co-localize with the endothelial cell marker von Willebrand factor in human fetal brain endothelial cells isolated from early pregnancy and midpregnancy. In early pregnancy, TMPRSS2 messenger RNA expression was decreased by 5% oxygen compared with 20% oxygen after 6 hours of exposure (P<.05). In midpregnancy, 5% oxygen down-regulated ACE2 messenger RNA compared with 20% oxygen after 24 hours (P<.05). Furin messenger RNA expression was decreased under 5% and 1% oxygen compared with 20% oxygen (P<.05) after 24 hours. In midpregnancy, angiotensin-converting enzyme 2 protein levels were decreased under 5% and 1% oxygen (P<.001) after 24 hours. In contrast, furin protein levels were increased under 1% oxygen compared with 20% oxygen after 24 hours (P<.05). At 48 hours, 1% oxygen increased angiotensin-converting enzyme 2 protein levels compared with 20% oxygen (P<.01). CONCLUSION: Hypoxia modifies the expression of selected SARS-CoV-2 cell entry proteins in human fetal brain endothelial cells in a gestational age- and time-of-exposure-dependent manner. As severe COVID-19 may lead to maternal hypoxia, an altered expression of these proteins in the developing human blood-brain barrier could potentially lead to altered SARS-CoV-2 brain invasion and neurologic sequelae in neonates born to pregnancies complicated by SARS-CoV-2 infection.