key: cord-0760528-heyzwu0p authors: Imperio, Guinever E.; Lye, Phetcharawan; Mughis, Hafsah; Hamada, Hirotaka; Bloise, Enrrico; Lye, Stephen J.; Matthews, Stephen G. title: Hypoxia alters the expression of ACE2 and TMPRSS2 SARS-CoV-2 cell entry mediators in hCMEC/D3 cells date: 2021-08-18 journal: Microvasc Res DOI: 10.1016/j.mvr.2021.104232 sha: c5e07ab22296f3e7a9515096e8c91d3ed94e344a doc_id: 760528 cord_uid: heyzwu0p 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. The coronavirus disease 2019 , caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), emerged in Wuhan, China, in December 2019 (17) as a primary respiratory disease. Since then, however, several reports have shown neurological manifestations associated with Covid-19, including meningoencephalitis, encephalopathy, and ischaemic stroke (5, 6, 27, 31) . The routes of infection and the mechanisms underlying SARS-CoV-2-induced central nervous system (CNS) damage are not fully understood. Viral infections can impact the brain both directly (by infecting neurons) or indirectly (by inducing a hyper-inflammatory state/cytokine storm), both of which can induce brain disease and lead to relatively long-lasting sequelae, as observed in Covid-19 patients (32). Emerging evidence linking SARS-CoV-2 infection to coagulopathies and vascular damage has directed interest towards the brain endothelium (32, 35). The angiotensin-converting enzyme 2 (ACE2) receptor and transmembrane protease serine 2 (TMPRSS2) are postulated to be the two main facilitators of SARS-CoV-2 penetration within host cells: ACE2 binds to the coronavirus spike (S) glycoproteins, while TMPRSS2 induces the proteolytic cleavage of both ACE2 and S, resulting in viral uptake (15, 16) . ACE2 and TMPRSS2 are expressed by brain endothelial cells (BECs) (3, 7, 11) . Considering that systemic hypoxia is common in cases of Covid-19 clinical reports and that oxygen tension is an established regulator of ACE2 and other components of the renin-angiotensin-aldosterone system in some cells and somatic tissues (18, 20, 22, 26, 37) , we sought to elucidate the in vitro effects of moderate (8% O 2 ) and severe (2% O 2 ) hypoxic conditions on ACE2 and TMPRSS2 in the J o u r n a l P r e -p r o o f brain endothelium. This information will contribute to our understanding of how SARS-CoV-2 might penetrate the brain, as well as, a number of the endothelial pathologies that have recently been associated with Covid-19. The human cerebral microvascular endothelial cell line (hCMEC/D3, Cedarlane Labs #CLU512, Burlington, Canada) was cultured at 37°C, in 5% CO 2 and 20% O 2 with EndoGRO TM -MV Complete Culture Media Kit® (Millipore, Canada, #SCME004). Human basic fibroblast growth factor (1 ng/mL; Sigma, #F0291) and 1% penicillin-streptomycin (10000 units-10000 μg/mL, Life Technologies, #15140-122) were supplemented to the media. All procedures, treatments, and analyses were performed with the cells at passage 30. hCMEC/D3 cells were plated in clear flat bottom 6-well TC-treated polystyrene culture plates (Costar, Kennebunk, USA, #3516) pre-coated with type 1 collagen (Gibco, #A1048301) at a density of 25000 cells/cm 2 . 24h after seeding, cells were challenged with different oxygen tensions: 20% (normoxia, the control group), 8%, or 2% O 2 (hypoxia groups). The plates and wells were randomly assigned to the different groups. Cells were collected for protein or mRNA analysis after 6-, 24-and 48-hours of challenge. In order to confirm the brain endothelial cell response to hypoxia, we replicated the experiment in independent cultures, exposing hCMEC/D3 cells to 2% and 1% O 2 using modular incubator chambers (Billups-Rothenberg, Del Mar, CA, J o u r n a l P r e -p r o o f USA) for 6, 24 and 48h. The different oxygen tensions did not affect cell viability (ranged from 100% to 86.49%) at any time point during the study (Supplementary Figure 3) . Total RNA was extracted using the RNeasy Mini Kit (Qiagen #74104, Toronto, Canada), following the manufacturer's instructions. NanoDrop1000 Spectrophotometer (Thermo Scientific, Wilmington, USA) was used to determine the RNA purity/concentration, and total RNA (1 μg) was reverse transcribed to cDNA using iScript TM Reverse Transcription Supermix (Bio-Rad #1708840, Mississauga, Canada). SYBR Green (Sigma #S9194) was used to run the quantitative polymerase chain reaction (qPCR) using the CFX 380 Real-Time system C1000 TM Thermal Cycler (Bio-Rad), with the following parameters: 1 cycle of 95°C for 2 mins, and 40 cycles of 95°C for 5s and 60°C for 20s. 40ng or 10ng of RNA was used per reaction depending on the gene of interest, and samples were run in triplicate. In some cases, not all replicates amplified, generating some variation in the number of samples per group. The specific number of samples used in each group per target is displayed under each column in the graphs. Geometric mean of beta actin (ACTB) and DNA topoisomerase type 1 (TOP1) was used to normalize the expression of the target genes. Table 1 shows the sequences and origin of the primers used in this study. The 2 -ΔΔCT Method (24) was used to calculate the relative mRNA expression, and the control group was normalized to 1. Cells were collected in lysis buffer (1 mol/L Tris-HCL pH 6.8, 10% SDS, and 10% glycerol) with protease and phosphatase inhibitor (Thermo Scientific, Mississauga, Canada, J o u r n a l P r e -p r o o f #78440), and the protein was extracted by sonication. Protein concentration was determined using the Pierce BCA Protein assay kit (Thermo Scientific). Total protein (20 µg) was boiled (5 min) and loaded on 8% SDS polyacrylamide gels for electrophoretic separation (100V, 1h). Proteins were transferred from gels to polyvinylidene difluoride membranes (Bio-Rad, #1704157) using the Bio-Rad Trans-Blot® Turbo™ Transfer System. Membranes were blocked for 1h with 5% skim milk in Tris-Buffered Saline containing 0.1% Tween (TBS-T), followed by overnight incubation at 4°C with primary antibodies anti-ACE2 (Abcam #ab15348, 1:1000), anti-TMPRSS2 (Abcam #ab92323, 1:1000) or anti-beta actin (Sigma, #2066, 1:20000) as the loading control. Membranes were then washed (3x) with TBS-T and incubated (1h) with HRPlinked anti-rabbit secondary antibody (1:10000; GE Healthcare Bio-Science, Baie d'Urfe, Canada). Chemiluminescence was assessed using the SuperSignal™ West Femto (Thermo Scientific, #34095, for ACE2 and TMPRSS2) or the Luminata™ Crescendo Western HRP Substrate (Millipore, #WBLUR0100, for beta actin) for 5 min, and detected under UV using a ChemiDoc™ MP Imaging system (Bio-Rad). ACE2 and TMPRSS2 protein bands were quantified by densitometric analysis using the Image Lab™ software and normalized against beta-actin signal for total protein assessment. Statistical analyses were performed using GraphPad Prism (Inc., San Diego, USA) software version 7. Normal distribution was assessed using the Shapiro-Wilk test. Grubbs' test was used to exclude outliers. Gene and protein expression were analyzed using Two-way ANOVA, followed by Tukey's multiple comparisons test comparing between different oxygen tensions at each time point. The number of replicates (n) varied between 3 to 6 per group. In this study, we used the vascular endothelial growth factor (VEGF) to confirm the response of hCMEC/D3 cells to different levels of hypoxia (19, 25) . VEGF mRNA levels were upregulated under 8% hypoxia at 6h (p<0.05), and under 2% hypoxia at 6-(p<0.001), 24- vWF and IL-6 were investigated as markers of endothelial dysfunction (4) . No alterations at the mRNA level were found in vWF or IL-6 expression in hypoxic BECs in this study ( Figure 1D and E). ACE2 relative protein expression was increased under 8% hypoxia at 24h (p<0.05), and under 2% hypoxia at 48h (p<0.001) (Figure 2A and B) . In contrast, TMPRSS2 expression was decreased under 2% hypoxia after 48h (p<0.05) of exposure ( Figure 2C and D) . Using the modular incubator chambers, we found a trend towards an increased ACE2 expression in the 1% O 2 group (p<0.06) at 6 hours, and reduced expression of ACE2 in the 1% O 2 group (p<0.05) at 48 hours, compared to the control groups ( Supplementary Figure 2A and B) . Interestingly, TMPRSS2 protein expression was also elevated acutely (6 hours) after exposure to 2% (p<0.01) and 1% (p<0.05) O 2 , compared to control. No changes were observed in the 2% O 2 group, but decreased TMPRSS2 expression was observed in the 1% (p<0.001) O 2 group at 48 hours (Supplementary Figure 2C and D) . This study demonstrates, for the first time, that hypoxic conditions modulate SARS-Cov-2 cell entry-mediators ACE2 and TMPRSS2 in human BECs. An acute increase of ACE2 protein and mRNA expression following 6h of severe hypoxic exposure (2% O 2 ) may indicate an J o u r n a l P r e -p r o o f increased BEC susceptibility to SARS-CoV-2 infection acutely, with this susceptibility decreasing over time. This indicates a time-dependent regulatory mechanism and is consistent with previous findings in lung epithelial cells (37). TMPRSS2 mRNA expression was not changed in BECs under acute or longer-term (48h) hypoxic conditions, contrasting with two reports of decreased TMPRSS2 mRNA expression under hypoxia in human prostate and breast cancer cell lines (9, 10). We observed high variability between same-sample replicates, which is characteristic of low expressed genes of interest. Therefore, cell-specific characteristics and response of TMPRSS2 under hypoxia may explain the divergent observations found in mRNA expression. At the protein level, however, we found TMPRSS2 at the expected molecular weight using a well-established antibody. In vitro, TMPRSS2 appears to be higher and more consistently expressed when the cells reach confluence (48h). To our knowledge, this is the first demonstration of the effect of hypoxia on TMPRSS2 protein expression in any cell-type. We found decreased TMPRSS2 protein expression after 48h of severe hypoxia in BECs. Little is known about the physiological function of TMPRSS2 in BECs, and our results highlight the need for further studies on TMPRSS2 participation in the cellular adaptations to hypoxic conditions, and its possible relevance for altered susceptibility to SARS-CoV-2 infection in hypoxic brain endothelium. In addition to the numerous cases of stroke associated with SARS-CoV-2 infection (2, 14) , emerging evidence is suggesting that Covid-19 is predominantly a vascular pathology/disease. This includes the occurrence of cerebral microbleeds (32), the correlation between brain MRI hemorrhagic findings with clinical indicators of Covid-19 severity (21), as well as the association of abnormal coagulation parameters combined with a prothrombotic state with poor outcome of Covid-19 patients (Tang et al., 2020) . Thus, it is plausible to assume an It is imperative to understand how the brain could be affected in the context of altered BEC function. In this study, we investigated the expression of vWF and IL-6 as indicators of potential endothelial dysfunction induced by hypoxia. vWF is produced and secreted by endothelial cells following disturbance or damage (23). It has been implicated in the development of coagulopathies and systemic inflammatory response (33), and was found increased in severe Covid-19 patients (8). IL-6, a pro-inflammatory cytokine, was found to contribute to Covid-19-related hyperinflammatory syndrome (28) and to correlate with Covid-19 severity (12, 13) . Further, some Covid-19 patients responded positively to IL-6 inhibition (1, 34). In other model systems, both vWF and IL-6 have been shown to respond to hypoxic conditions (29, 30) . However, in the current study, we found no statistical changes in vWF or IL-6 mRNA levels after acute or sustained hypoxia. Although not significant, a trend towards an increase in vWF mRNA expression was observed in cells between 24 and 48h of culture. Since vWF is an endothelial cell marker, it is possible that this effect may result from increased cell numbers across the course of the experiment. Taken together, our data suggest that altered ACE2 and TMPRSS2 expression induced by hypoxia occurs independently of compromised endothelial function and/or enhanced inflammatory state, at least over the time frame that we investigated. Although modest, the effect hypoxia on ACE2 mRNA and TMPRSS2 mRNA is consistent and it is reproducible using different hypoxic systems. We recognize that using only one brain endothelial cell line is a limitation of the study, as it may introduce a bias towards this specific cell line. Nonetheless, we believe this study is extremely relevant because it is the first to J o u r n a l P r e -p r o o f describe the effects of hypoxia on SARS-CoV-2 receptors in brain endothelial cells. Other studies using primary cells or other cell lines will be important to confirm these effects and elucidate the mechanisms of hypoxia leading to ACE2 and TMPRSS2 regulation in brain endothelial cells. In conclusion, this study has clearly demonstrated that SARS-CoV-2 cell entry mediators qPCR, in hCMEC/D3 cells 6-, 24-or 48h after exposure to 20% (Control), 2% or 1% oxygen using the modular incubator chambers. N=3-6/group, specific n is presented under each column. Statistical analysis: two-way ANOVA followed by Tukey's multiple comparisons test. Mean ± SEM. *p<0.05 and **p<0.01. Figure 2: ACE2 (A and B) and TMPRSS2 (C and D) protein levels, measured by Western Blot, in hCMEC/D3 cells 6-, 24-or 48h after exposure to 20% (Control), 2% or 1% oxygen using the modular incubator chambers. A and C show representative blots of each group, C and D present the results of densitometry. Beta actin was used as the loading control. N=4-6/group. Statistical analysis: two-way ANOVA followed by Tukey's multiple comparisons test. Mean ± SEM. *p<0.05, **p<0.01, and ***p<0.001. Figure 3 : Trypan Blue cell viability analysis of the two subsets of hCMEC/D3 cells exposed for 6, 24 and 48h to hypoxic conditions. The first batch (A) was exposed to 20% (Control group), 8% and 2% O2 using regular incubators, while the second one (B) includes cells exposed to 20% (Control group), 2% and 1% O2 using modular incubator chambers. IL-6 Inhibitors in the Treatment of Serious COVID-19: A Promising Therapy COVID-19 presenting as stroke Evidence of the COVID-19 Virus Targeting the CNS: Tissue Distribution, Host-Virus Interaction, and Proposed Neurotropic Mechanisms Endothelial dysfunction and inflammatory markers of vascular disease Characteristics of ischaemic stroke associated with COVID-19 Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study Human Coronaviruses and Other Respiratory Viruses: Underestimated Opportunistic Pathogens of the Central Nervous System? Severe COVID-19 infection associated with endothelial activation Dual targeting of the androgen receptor and hypoxia-inducible factor 1alpha pathways synergistically inhibits castration-resistant prostate cancer cells Expression profiling metaanalysis of ACE2 and TMPRSS2, the putative anti-inflammatory receptor and priming protease of SARS-CoV-2 in human cells, and identification of putative modulators Profiling serum cytokines in COVID-19 patients reveals IL-6 and IL-10 are disease severity predictors TMPRSS2 and ADAM17 cleave ACE2 differentially and only proteolysis by TMPRSS2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Clinical features of patients infected with 2019 novel coronavirus in Wuhan Possible activation of the renin-angiotensin system in the feto-placental unit in preeclampsia Effect of oxygen on multidrug resistance in term human placenta Hypoxic regulation of angiotensin-converting enzyme 2 and Mas receptor in human CD34(+) cells Brain MRI Findings in Severe COVID-19: A Retrospective Observational Study A Human Long Non-Coding RNA ALT1 Controls the Cell Cycle of Vascular Endothelial Cells Via ACE2 and Cyclin D1 Pathway Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method Changes of renal AT1/AT2 receptors and structures in ovine fetuses following exposure to long-term hypoxia Neurologic Manifestations of Hospitalized Patients With Coronavirus Disease Across Speciality Collaboration UK. COVID-19: consider cytokine storm syndromes and immunosuppression Hypoxia results in upregulation and de novo activation of von Willebrand factor expression in lung endothelial cells The emerging spectrum of COVID-19 neurology: clinical, radiological and laboratory findings Von Willebrand factor as a thrombotic and inflammatory mediator in critical illness The authors declare no conflict of interest.