key: cord-0928525-2sj751am
authors: McCracken, Ian; Saginc, Gaye; He, Liqun; Huseynov, Alik; Daniels, Alison; Fletcher, Sarah; Peghaire, Claire; Kalna, Viktoria; Andaloussi-Mäe, Maarja; Muhl, Lars; Craig, Nicky M.; Griffiths, Samantha J.; Haas, Jürgen G.; Tait-Burkard, Christine; Lendahl, Urban; Birdsey, Graeme M.; Betsholtz, Christer; Noseda, Michela; Baker, Andrew; Randi, Anna M.
title: Lack of evidence of ACE2 expression and replicative infection by SARS-CoV-2 in human endothelial cells
date: 2020-12-02
journal: bioRxiv
DOI: 10.1101/2020.12.02.391664
sha: 5bc4a67fc52c34f09963e89629dbd4b4be6782e3
doc_id: 928525
cord_uid: 2sj751am

A striking feature of severe COVID-19 is thrombosis in large as well as small vessels of multiple organs. This has led to the assumption that SARS-CoV-2 virus directly infects and damages the vascular endothelium. However, endothelial expression of ACE2, the cellular receptor for SARS-CoV-2, has not been convincingly demonstrated. Interrogating human bulk and single-cell transcriptomic data, we found ACE2 expression in endothelial cells to be extremely low or absent in vivo and not upregulated by exposure to inflammatory agents in vitro. Also, the endothelial chromatin landscape at the ACE2 locus showed presence of repressive and absence of activation marks, suggesting that the gene is inactive in endothelial cells. Finally, we failed to achieve infection and replication of SARS-CoV-2 in cultured human endothelial cells, which were permissive to productive infection by coronavirus 229E that uses CD13 as the receptor. Our data suggest that SARS-Cov-2 is unlikely to infect endothelial cells directly; these findings are consistent with a scenario where endothelial injury is indirectly caused by the infection of neighbouring epithelial cells and/or due to systemic effects mediated by immune cells, platelets, complement activation, and/or proinflammatory cytokines.

A striking feature of severe forms of coronavirus disease 2019 (COVID-19), the current pandemic caused by the coronavirus SARS-CoV-2, is severe endothelial injury with micro-and macro-thrombotic disease in the lung and other organs, including the heart. This has led to speculation that viral infection may damage the endothelium through two mechanisms: indirectly, via neighbourhood effects, circulating mediators and immune mechanisms, or directly by viral infection of endothelial cells (EC).

To support the hypothesis of direct viral damage of EC via virus-induced infection, the cells should express the main receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), a metalloproteinase component of the renin-angiotensin hormone system and a critical regulator of cardiovascular homeostasis 1 . Indeed, several recent review articles propose that SARS-CoV-2 binding to ACE2 on EC is the mechanism through which the virus may cause direct endothelial damage and endothelialitis 1 . However, expression of ACE2 in EC has not been convincingly demonstrated to support this assumption, nor has there been sufficient evidence to support a direct infection of EC by SARS-CoV-2.

To address the questions of ACE2 expression in human EC and of the ability of SARS-CoV-2 to infect the endothelium, we interrogated transcriptomic and epigenomic data on human EC and studied the interaction and replication of SARS-Cov-2 and its viral proteins with EC in vitro. Analysis of RNA-seq was carried out on ENCODE # data from EC from arterial, venous and microvascular beds, in comparison with epithelial cells from respiratory, gastrointestinal and skin sources. Very low or no basal ACE2 expression was found in EC, compared to epithelial cells ( Figure A-B) . Moreover, in vitro exposure of EC to inflammatory cytokines reported as elevated in the plasma of patients with severe COVID-19 failed to upregulate ACE2 expression ( Figure C ).

Single-cell RNA-sequencing (scRNAseq) of human organ donor hearts 2 showed that while ACE2 sequence reads are abundant in pericytes (PC), they are rare in EC ( Figure D) . Out of 100,579 EC, only 468 (0,47%) were ACE2 + , and in the majority (424) only a single ACE2 transcript was detected. This could reflect true low and rare endothelial ACE2 expression, but also contamination from adherent PC fragments, a common confounder in vascular scRNAseq data 3 . If such fragments contributed the ACE2 transcripts observed in certain EC, we would expect to detect other pericyte transcripts in the same cells. Indeed, among the top-50 gene transcripts enriched in ACE2 + vs. ACE2 -EC, we noticed several known pericyte markers, including PDGFRB, ABCC9, KCNJ8 and RGS5 ( Figure E) . Comparison of transcript abundance across the three major vascular and mesothelial cells showed that the top-50 gene transcripts were expressed at the highest levels in PC ( Figure E ). This suggests that the rare occurrence of ACE2 transcripts in human heart EC is likely caused by pericyte contamination. Similar conclusions have previously been reached in mouse tissues 3 .

Analysis of the chromatin landscape at the ACE2 gene locus in human umbilical vein EC (HUVEC) using data from ENCODE further supports this concept. The histone modification mark H3K27me3, which indicates repressed chromatin, was enriched at the ACE2 transcription start site (TSS); conversely, promoter, enhancer and gene body activation marks (H3K27ac, H3K4me1, H3K4me2, H3K4me3, H3K36me3), RNA polymerase-II and DNase I hypersensitivity were absent or low, suggesting that ACE2 is inactive in EC. In marked contrast, the adjacent gene BMX, an endothelial-restricted non-receptor tyrosine kinase displays an epigenetic profile consistent with active endothelial expression ( Figure F ). Thus, transcriptomic and epigenomic data indicate that ACE2 is not expressed in human EC.

Other cell surface molecules have been suggested as possible receptors for the virus, but their role in supporting SARS-CoV-2 cell infection remains to be demonstrated. We therefore tested directly whether EC could be capable of supporting coronavirus replication in vitro. Productive levels of replication in primary human cardiac and pulmonary EC were observed for the human coronavirus 229E GFP reporter virus 4 , which utilises CD13 as its receptor, demonstrating directly that human EC can support coronavirus replication in principle ( Figure G) . However, when cells were exposed to SARS-CoV-2, replication levels were extremely low for EC, even following exposure to very high concentrations of virus compared to more permissive VeroE6 cells ( Figure H ). The observed low levels of SARS-CoV-2 replication in EC are likely explained by viral entry via a non-ACE2 dependent route, due to exposure to supraphysiological concentrations of virus in these experiments (MOI 10 and 100).

These data indicate that direct endothelial infection by SARS-Cov-2 is not likely to occur. The endothelial damage reported in severely ill COVID19 patients is more likely secondary to infection of neighbouring cells and/or other mechanisms, including immune cells, platelets and complement activation, and circulating proinflammatory cytokines. Our hypothesis is corroborated by recent evidence that plasma from critically ill and convalescent patients with COVID-19 causes endothelial cell cytotoxicity 5 . These finding have implications for the therapeutic approaches to tackle vascular damage in severe COVID19 disease.

RNA-seq data files with gene quantifications for primary human epithelial and endothelial cells were downloaded from the ENCODE database (www.encodeproject.org) 6 , using Bioconductor package "ENCODEExplorer" and R. For consistency, only total RNAseq data generated by the same source, "Thomas Gingeras, CSHL", and aligned to GrCh38 reference genome were included in the analysis. Cell types with one replicate were excluded. Raw counts were converted to counts per million (CPM), filtered based on expression, log2 transformed and normalized with Trimmed Mean of M-value (TMM) method using Bioconductor package "EdgeR". Only genes with more than 0.3 counts per million (CPM) in at least three samples were kept for the analysis (19360 genes).

The accession numbers for the ENCODE files are ENCFF592BLV, ENCFF788JHJ, ENCFF235TGN, ENCFF401NRN, ENCFF207VFL, ENCFF623ZJJ, ENCFF620THF, ENCFF699QEJ, ENCFF797YZO, ENCFF985PDI, ENCFF110UGQ, ENCFF764AOQ, ENCFF325DPM, ENCFF564EGR, ENCFF233LYV, ENCFF378BDR, ENCFF555QVG, ENCFF972UYG, ENCFF511TST, ENCFF711SNV, ENCFF037WEH, ENCFF498AYE, ENCFF577VBY, ENCFF604VVJ, ENCFF177SUW, ENCFF456RET, ENCFF145DRX, ENCFF224ZRP, ENCFF176IZX, ENCFF674SRB, ENCFF580CHD, ENCFF709VCC, ENCFF747XTG, ENCFF756RKP, ENCFF060LPA, ENCFF262OBL, ENCFF091SWU, ENCFF224YSC, ENCFF819IDA, ENCFF894GLT, ENCFF231GYQ, ENCFF836KPM, ENCFF361WEZ, ENCFF592KDP.

Pooled human umbilical vein endothelial cells (HUVEC; Lonza C2519A) were cultured in Endothelial Cell Growth Medium-2 media (EGM-2) (Lonza). HUVEC were plated at a density of 10,000 cells/96-well plate in EGM-2 supplemented with recombinant human TNF-α (300-01A, PeproTech EC Ltd), IL1-β (200-01B, PeproTech EC Ltd), IL8 (72aa, monocytes derived, 200-08M, PeproTech EC Ltd) and IL-6/IL-6R Alpha Protein Chimera (8954-SR, R&D Biotechne) at 0, 0.01, 0.1 or 1.0 ng/ml for 4 or 24 hours. Total RNA was isolated by using the RNeasy kit (Qiagen) and reverse transcribed into cDNA using Superscript III Reverse Transcriptase (Invitrogen). Quantitative real-time PCR was performed using PerfeCTa SYBR Green Fastmix (Quanta Biosciences) on a Bio-Rad CFX96 system. Gene expression values were normalized to GAPDH expression. Sequences of the human primers used in this study are listed below: GAPDH forward: 5'-CAAGGTCATCCATGACAACTTTG-3' and GAPDH reverse: 5'-GGGCCATCCACAGTCTTCTG-3'; ACE2 forward: 5'-ACAGTCCACACTTGCCCAAAT-3' and ACE2 reverse: 5'-TGAGAGCACTGAAGACCCATT-3'.

Human heart single cell raw counts data and the cell annotation meta data were obtained from a recent published study 2 . The data were processed using Seurat package (version: 3.1.1) in R. The endothelial cells which contain ACE2 reads were grouped as ACE2 + EC cells, and the rest as ACE2 -EC cells. The genes enriched in ACE2 + EC cells were identified using FindMarkers function in Seurat. It applies a Wilcoxon Rank Sum test and then performs Bonferroni-correction using all genes in the dataset. The corrected p value < 0.05 was used as cutoff for significance. To visualize the top 50 genes among different cell groups, the DotPlot function was applied. The size of the dot represents percentage of cells expressing the gene and color represents the scaled average expression levels.

Genome-wide ChIP-seq data for histone modifications and DNase I hypersensitivity in HUVEC were obtained from the ENCODE 6 /Broad Institute under GEO accession number GSE29611. ChIP-seq reads were mapped to the human reference genome GRCh37/hg19. Tracks were visualized using the UCSC Genome Browser database (https://genome.ucsc.edu). HUVEC chromatin-state discovery and genome annotation was obtained using ChromHMM 7 from ENCODE.

Stocks of HCoV-229E GFP reporter virus described by Cervantes-Barragan et al 4 were generated following cultivation on the HUH7 cell line. HPAEC (Lonza) and HCAEC (Promocell) were seeded in 96 well plates (2.1x10 4 cells/cm 2 ) 24 hours prior to infection with HCoV-229E-GFP at MOI = 0.6. Virus inoculum was then replaced 1 hour later with 100ul fresh media before incubation at 34°C/5%CO2. GFP fluorescence was measured every 2 hours from 20 to 58 hours post inoculation using the ClarioStar plate reader (BMG).

A sample from a confirmed COVID-19 patient was collected by a trained healthcare professional using combined nose-and-throat swabbing. The sample was stored in virus transport medium prior to cultivation and isolation on Vero E6 (ATCC CRL-1586) cells. Samples were obtained anonymised by coding, compliant with Tissue Governance for the South East Scotland Scottish 279 Academic Health Sciences Collaboration Human Annotated BioResource (reference no. SR1452). Virus sequence was confirmed by Nanopore sequencing according to the ARCTIC network protocol (https://artic.network/ncov-2019), amplicon set V3, and validated against the patient isolate sequence. For the virus isolate used in this project is EDB-2 (Scotland/EDB1827/2020, UK lineage 109, B 1.5) genetic stability was observed up to 5 passages on Vero E6 cells, with particular attention to the S1/S2 furin cleavage site. HCAEC (Promocell) and HPAEC (Lonza) were seeded in a 12-well plate (2.0x10 4 cells/cm 2 ) one day prior to infection with SARS-CoV-2 EDB-2, passage 2, at MOI = 100 or 10 (assessed by endpoint titration on Vero E6 cells). At 0, 24 and 48 hours post infection (hpi) 10 µl of supernatant was lysed in VL buffer (20mM Tris-HCl pH 7.5, 300mM NaCl, 2.5% IGEPAL CA-630, 1:200 RNAsin Plus). The lysate was analysed by RT-qPCR, using GoTaq 1-Step RT-qPCR (Promega) with the SARS-CoV-2 CDC N3 primers (F -GGGAGCCTTGAATACACCAAAA, R -TGTAGCACGATTGCAGCATTG) at 350nM each to determine viral copy number. To assess viral copy numbers, resulting Cts were analysed against a standard curve of lysate with known TCID50/ml value as well as an RNA template. 

infection of neighbouring epithelial cells and/or due to systemic effects mediated by immune cells, platelets, complement activation, and/or proinflammatory cytokines

Endothelial dysfunction in COVID-19: a position paper of the ESC Working Group for Atherosclerosis and Vascular Biology, and the ESC Council of Basic Cardiovascular Science

Cells of the adult human heart

Pericyte-specific vascular expression of SARS-CoV-2 receptor ACE2 -implications for microvascular inflammation and hypercoagulopathy in COVID-19

Dendritic cell-specific antigen delivery by coronavirus vaccine vectors induces long-lasting protective antiviral and antitumor immunity

Lille CRN and Members of the LSC. Endotheliopathy Is Induced by Plasma From Critically Ill Patients and Associated With Organ Failure in Severe COVID-19

An integrated encyclopedia of DNA elements in the human genome

Chromatin-state discovery and genome annotation with ChromHMM

The data, analytic methods, and study materials will be maintained by the corresponding author and made available to other researchers on reasonable request.