Resistance of endothelial cells to SARS-CoV-2 infection in vitro

Rationale The secondary thrombotic/vascular clinical syndrome of COVID-19 suggests that SARS-CoV-2 infects not only respiratory epithelium but also the endothelium activating thrombotic pathways, disrupting barrier function and allowing access of the virus to other organs of the body. However, a direct test of susceptibility to SARS-CoV-2 of authentic endothelial cell lines has not been performed. Objective To determine infectibility of primary endothelial cell lines with live SARS-CoV-2 and pseudoviruses expressing SARS-CoV-2 spike protein. Methods and Results Expression of ACE2 and BSG pathways genes was determined in three types of endothelial cells; blood outgrowth, lung microvascular and aortic endothelial cells. For comparison nasal epithelial cells, Vero E6 cells (primate kidney fibroblast cell line) and HEK 293T cells (human embryonic kidney cells) transfected with either ACE2 or BSG were used as controls. Endothelial and Vero E6 cells were treated with live SARS-CoV-2 virus for 1 hour and imaged at 24 and 72 hours post infection. Pseudoviruses containing SARS-CoV-2, Ebola and Vesicular Stomatis Virus glycoproteins were generated and added to endothelial cells and HEK 239Ts for 2 hours and infection measured using luminescence at 48 hours post infection. Compared to nasal epithelial cells, endothelial cells expressed low or undetectable levels of ACE2 and TMPRSS2 but comparable levels of BSG, PPIA and PPIB. Endothelial cells showed no susceptibility to live SARS-CoV-2 or SARS-CoV-2 pseudovirus (but showed susceptibility to Ebola and Vesicular Stomatitis Virus). Overexpression of ACE2 but not BSG in HEK 239T cells conferred SARS-CoV-2 pseudovirus entry. Endothelial cells primed with IL-1ß remained resistant to SARS-CoV-2. Conclusion Endothelial cells are resistant to infection with SARS-CoV-2 virus, in line with relatively low levels of ACE2 and TMPRSS2, suggesting that the vascular dysfunction and thrombosis seen in severe COVID-19 is a result of factors released by adjacent infected cells (e.g. epithelial cells) and/or circulating, systemic inflammatory mediators.


Introduction:
COVID-19 represents one of the most important clinical challenges the scientific community has faced in recent memory. In the absence of an effective vaccine and no clear evidence that prior infection confers long-term immunity 1 , there is an urgent unmet need to understand disease pathology and to establish therapeutics that mitigate COVID-19 severity and reduce associated lethality 2 . SARS-CoV-2 spike protein binds to host cells via ACE2 3 and viral entry is facilitated by the cell-surface protease TMPRSS2 3 or lysosomal cysteine proteases cathepsin B/L (CTSB, CTSL) 3 . It has also been suggested that, as an alternative pathway, SARS-CoV-2 binds to cells via BSG (Basigin; also known as CD147 or EMMPRIN) 4, 5 , although firm evidence for BSG as a standalone receptor for SARS-CoV-2 remains the subject of investigation with a recent study noting no 'direct' binding of SARS-CoV-2 spike protein to BSG 6 .
Initial infection with SARS-CoV-2 occurs via the respiratory epithelium 7 . In most people symptoms are mild but in a significant minority COVID-19 progresses to severe disease and in those that 'recover' symptoms can persist leading to a syndrome recently defined as 'long-COVID' 8 . In severe COVID-19 and in long-COVID multiple organs including the cardiovascular system are affected 9,10 . This secondary thrombotic/vascular clinical syndrome of COVID-19 suggests that SARS-CoV-2 infects not only respiratory epithelium but also the endothelium disrupting barrier function and allowing dissemination to other organs of the body 11 . This notion is supported by early reports showing that SARS-CoV-2 infects endothelial cells in vitro 12 and in vivo 13 . However, for the in vitro studies performed to date, nonphysiological cell lines were used and for in vivo studies serious concerns have been raised regarding the validity of the interpretation of data and the resulting conclusions 14 . Currently, therefore, it is not clear if endothelial cells are permissive to SARS-CoV-2 or not. Only by addressing this can biomarkers, mechanisms and therapeutic targets directed at the vasculopathy and thrombosis associated with COVID-19 be established.
We have recently shown that blood outgrowth endothelial cells express relatively low levels of ACE2 and TMPRSS2 but high levels of BSG and speculated that if endothelial cells are infected by SARS-CoV-2, BSG, rather than ACE2, would act as the receptor for viral entry 15 .
However, in our previous work and that of others, a direct test of susceptibility to SARS-CoV-2 of authentic endothelial cell lines has not been performed.

Cells
Primary human endothelial cell lines including blood outgrowth endothelial cells (obtained in house 16 ), lung microvascular endothelial cells and aortic endothelial cells (obtained from Lonza; UK) were used in this study. In each case cells from 3 separate donors/ per endothelial cell type were used except for qPCR studies using blood outgrowth endothelial cells where cells from 2 donors were used. Nasal epithelial cells were obtained from Promocell (Germany) and grown in submersed culture and differentiated nasal epithelial cells (MucilAir™) were obtained from Epithelix (Switzerland) and grown in air-liquid interface culture.

RT-qPCR
Endothelial and nasal epithelial cells were plated in duplicate wells on uncoated 6 well plates in their cell-specific media (see above) and grown to confluence. The day before RNA extraction media was replaced with the treatment media. After 24 hours cells were washed with phosphate-buffered saline (PBS), duplicate wells combined and RNA extracted using RNeasy Extraction kit (Qiagen, UK). RNA was converted to cDNA using the iScript cDNA synthesis kit (BioRad, CA, USA). Gene expression levels were determined using a TaqMan expression assay, with the following primers (ThermoScientific, UK); ACE2 (Hs01085333_m1), TMPRSS2 (Hs00237175_m1), BSG (Hs00936295_m1), PPIA (Hs04194521_s1) and PPIB (Hs00168719_m1). Genes were quantified relative to housekeeping genes (GAPDH and 18S) by the comparative Ct method.

SARS-CoV-2 live virus infection studies
For infection studies with Mucilair human airway epithelial cells, SARS-CoV-2/England/IC19/202 (IC19) 18 was diluted in serum-free DMEM, 1% NEAA, 1% P/S to a multiplicity of infection (MOI) of 0.1. Inoculum was added to the exposed apical face of the cells and incubated at 37°C for 1 hour. Inoculum was then removed and cells maintained as described above. At each timepoint in the infection, virus was collected from the apical surface of the cultures by washing with pre-warmed PBS for 10 minutes at 37°C and quantified by plaque assay on Vero E6 cells. Briefly, cells were washed with PBS then serial dilutions of inoculum, diluted in serum-free DMEM, 1% NEAA, 1% P/S, were overlayed onto cells for one hour at 37°C. Inoculum was then removed and replaced with SARS-CoV-2 overlay media (1x minimal essential media (MEM), 0.2% w/v bovine serum albumin, 0.16% w/v NaHCO3, 10mM Hepes, 2mM L-Glutamine, 1x P/S, 0.6% w/v agarose). Plates were incubated for 3 days at 37°C before overlay was removed and cells were stained for 1 hour at RT in crystal violet solution.
For infection studies using endothelial cells, cells were plated on sterile round 16mm diameter coverslips in 12 well plates (5x10 4 cells/well) without coating. Their usual media (see above) was added and cells allowed to settle overnight. Control Vero E6 cells were plated to achieve approximately 80% confluency. The following day, media was removed and cells washed in PBS. For endothelial cells, treatment media was added either alone (untreated/ control) or with IL-1b (10ng/ml) for 3 hours. Meanwhile IC19 18 was diluted in serum free DMEM, 1%NEAA, 1% P/S, to a multiplicity of infection (MOI) of 0.1. After 3 hours treatment with either media alone or IL-1b, media was replaced with IC19 containing inoculum and incubated at 37°C for 1 hour. Inoculum was then removed and replaced with treatment media and cells were maintained until 24, 48 or 72 hours post-infection. At the appropriate timepoint, treatment media was removed and cells were fixed in 4% paraformaldehyde (PFA) for 30 minutes. PFA was removed with three washes of PBS. Cover slips were dehydrated in an ethanol series and stored in 100% ethanol at -20°C until further processing.

Fluorescent imaging
For fluorescent imaging, infected cells on cover slips were first rehydrated in an ethanol series.
Cells were permeabilised in PBS with 0.5% triton-X for 10 minutes, washed 3x in PBS, then blocked in PBS with 2% bovine serum albumin (BSA) and 0.1% tween (blocking buffer).
Primary antibodies were diluted 1:1000 in blocking buffer and incubated on cells at room temperature for 1 hour. Primary antibodies used were against spike protein (S) (Mouse monoclonal, Gene tex (1A9)) or nucleoprotein (N) (Rabbit monoclonal, Sino Biological).
Following 3x PBS washes, cells were incubated with secondary antibodies (anti-rabbit 488, anti-mouse 594) diluted 1:500 and DAPI (diluted 1 in 1000) in PBS with 2% BSA at room temperature for 1 hour in the dark. Stained cover slips were mounted on glass slides in ProLong gold antifade mounting medium (Invitrogen). Images were acquired using either a Zeiss Axiovert 135 TV microscope ( Figure 2B) or a Zeiss Cell Observer widefield microscope ( Figure 2C). All images were analysed and prepared using FIJI software 19 . For each cell type/condition slides were first reviewed by eye before representative fields of view were captured. Images were captured and processed in an identical manner across each experiment to ensure fair comparison either as a single plane of focus ( Figure 2B) or as Z stacks presented as maximum intensity projections ( Figure 2C). For quantitative analysis, all images were blinded and independently scored between 1-5, where 1= 0-2, 2= 3-5, 3=6-8, 4= 9-10 and 5= >10 nucleocapsid stained cells.

IL-6 and IL-8 ELISA
IL-6 and IL-8 was measured using duo set ELISAs from R&D Systems according to manufactures instructions.

Data analysis:
All data were analysed on GraphPad Prism v8 and are shown as individual data points and/or mean +/-standard error of the mean (SEM) for samples as described in the figure legends.

Results:
In our recent study, where we performed a systematic analysis of online transcriptomic databases in endothelial cells and nasal and bronchial epithelium, we found endothelial cells express relatively low levels of ACE2 and TMPRSS2 but high levels of BSG and PPIA/PPIB (also known as cyclophilin A/B; CypA/B) 15 Figure 4A and B).

Discussion:
There is mounting speculation that the vascular and thrombotic sequela associated with severe COVID-19 is a result of endothelial cell infection with SARS-CoV-2. However, there is currently no direct evidence to support this idea. In response to this, we have used standard While we have worked to the highest standards with empirical virology protocols, there are limitations in our approach, and we cannot definitively conclude that endothelial cells are nonsusceptible to infection by SARS-CoV-2 in some individuals or in some highly specific conditions in vivo. This is because the assay systems we used did not take account of critical factors present in at risk populations and/or at the site of inflammation. In an attempt to take account of basic inflammatory conditions we performed experiments in cells primed with IL-1ß, which did not confer infectability to any of our endothelial lines. However, as we find more about the complex mix of inflammatory mediators present in the lung and circulation in COVID-19 and the specific biological factors that predispose certain groups of individuals to severe disease, these can be recapitulated within in vitro assay systems. Nonetheless what our study does prove is that if endothelial cells are susceptible to SARS-CoV-2 at some distant point in the natural history of COVID-19, the pathways of viral entry are more complex than for airway epithelium.

Acknowledgements:
We thank the FILM Facility at Imperial College London for the use of the microscopes and their technical support.

Disclosures:
The authors have no disclosures to declare.

cells (NEC) and endothelial cells (aortic, microvascular and blood outgrowth).
Expression levels for the genes ACE2, TMPRSS2, BSG, PPIA and PPIB were obtained from aortic (AoEC), microvascular (HMVEC) and blood outgrowth (BOEC) endothelial cells and nasal epithelial cells (NEC). Data for each donor were normalised using the average of the housekeepers (18S and Gapdh) and analysed using a comparative Ct method (2DDCt). Data are shown as the mean +/-SEM fold change compared to nasal epithelium (n=3 wells using cells from 2 donors) for AoEC (n=3 wells using cells of 3 separate donors), HMVEC ((n=3 wells using cells of 3 separate donors and BOECs (n=2 wells using cells of 2 separate donors).