key: cord-0821710-d5d55kr4
authors: Ahmetaj-Shala, Blerina; Vaja, Ricky; Atanur, Santosh S.; George, Peter M.; Kirkby, Nicholas S.; Mitchell, Jane A.
title: Cardiorenal tissues express SARS-CoV-2 entry genes and basigin (BSG/CD147) increases with age in endothelial cells
date: 2020-10-09
journal: JACC Basic Transl Sci
DOI: 10.1016/j.jacbts.2020.09.010
sha: 1f7cb8a6a839538f44e11febed05b20b8ee74298
doc_id: 821710
cord_uid: d5d55kr4

Vascular/cardiovascular inflammation and thrombosis occur in severe COVID-19. Advancing age is the most significant risk factor for severe COVID-19. Using transcriptomic databases, we found: (i) cardiovascular tissues/endothelial cells express putative genes for SARS-CoV-2 infection including ACE2 and BSG, (ii) SARS-CoV-2 receptor pathways, ACE2TMPRSS2 and BSG/PPIB(A) polarise to lung/epithelium and vessel/endothelium respectively, (iii) expression of host genes are relatively stable with age and (iv) notable exceptions are ACE2 which decreases with age in some tissues and BSG which increases with age in endothelial cells, suggesting that BSG expression in the vasculature may explain the heightened risk of severe disease with age.

• In light of the cardiovascular sequalae associated with severe COVID-19 disease and the increased risk with age, our aim was to obtain mechanistic insight by quantifying the two putative receptors for SARS-CoV-2, ACE2 and BSG and a selected range of genes thought to be involved in virus binding/processing. • We have made four important observations: (i) cardiovascular tissues and/or endothelial cells express the required genes for SARS-CoV-2 infection, including ACE2 and BSG (ii) SARS-CoV-2 receptor pathways, ACE2/TMPRSS2 and BSG/PPIB(A) somewhat polarise to lung/epithelium and vessel/endothelium respectively, (iii) expression of SARS-CoV-2 host genes are mainly relatively stable with age and (iv) notable exceptions were ACE2 which decreases with age in some tissues and BSG which increases with age in endothelial cells.

SUMMARY: Vascular/cardiovascular inflammation and thrombosis occur in severe COVID-19.

Advancing age is the most significant risk factor for severe COVID-19. Using transcriptomic databases, we found: (i) cardiovascular tissues/endothelial cells express putative genes for SARS-CoV-2 infection including ACE2 and BSG, (ii) SARS-CoV-2 receptor pathways, ACE2/TMPRSS2 and BSG/PPIB(A) polarise to lung/epithelium and vessel/endothelium respectively, (iii) expression of host genes are relatively stable with age and (iv) notable exceptions are ACE2 which decreases with age in some tissues and BSG which increases with age in endothelial cells, suggesting that BSG expression in the vasculature may explain the heightened risk of severe disease with age. (1). In addition, BSG (also known as Basigin, CD147 or EMMPRIN) is a second but putative receptor by which SARS-CoV-2 may enter cells (2, 3) . For viral entry by ACE2, it is thought that the SARS-CoV-2 spike protein is primed and ACE2 cleaved, by the cellular serine proteases TMPRSS2(1) and

ADAM17. Intracellular processing of SARS-CoV-2 spike protein is thought to involve the lysosomal cysteine proteases cathepsin B/L (CTSL, CTSB) which, can also substitute for TMPRSS2 in some cells(1). FURIN cleaves viral enveloping proteins providing another putative priming step for the spike protein of SARS-COV-2 (4) . For viral entry via BSG, less is known regarding specific receptor/viral processing partners for SARS-CoV-2. Indeed, 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 (5) . However, for SARS-CoV (6) , HIV(7) and the measles virus (8) , respectively, peptidylprolyl isomerase A (PPIA; also knowns as cyclophilin A) and peptidylprolyl isomerase B (PPIB; also known as cyclophilin B), which are natural ligands for BSG, incorporate into virus and facilitate binding to BSG. Similarly, cyclophilins are required for infection via BSG in malaria. In this case PPIB forms a complex with the malaria pathogen (Plasmodium falciparum merozoites) and BSG to facilitate infection of red blood cells (9) .

Initial infection with SARS-CoV-2 occurs via the respiratory epithelium; high gene expression of ACE2 and TMPRSS2 in nasal epithelium (10, 11) have been taken to imply that J o u r n a l P r e -p r o o f the nose is a primary entry point for the virus (10) . ACE2 and TMPRSS2 are also coexpressed in bronchial epithelium (10) (11) (12) . However, where COVID-19 progresses to severe disease the lung and other organs are also affected. The emerging pattern of severe and fatal COVID-19 disease includes pneumonia with acute respiratory distress syndrome, cytokine storm, widespread vasculopathy, thrombosis, renal failure, hypertension and endothelial dysregulation seen across multiple vascular beds and organ systems (13, 14) . While hypertension and thrombosis are common features after COVID-19 (13, 15) , the important question as to whether COVID-19 is an independent risk factor for cardiovascular disease in the acute setting and during the recovery period, is a concern and remains to be established.

This secondary thrombotic/vascular clinical syndrome of severe COVID-19 suggests that SARS-CoV-2 infects not only respiratory epithelium but also the endothelium disrupting barrier function and allowing access to cardiovascular tissues and other organs of the body (16) . This idea is supported by reports showing that SARS-COV-2 can infect endothelial cells in vitro (17) and that coronaviruses including SARS-CoV-2 can progress to a systemic infection (18, 19) with some patients showing detectable viral RNA in blood samples (20) (21) (22) .

The reasons that underpin progression of mild to severe or fatal COVID-19 disease remain incompletely understood but risk factors have been defined (23) While some studies report expression profiles of ACE2 and TMPRSS2 in epithelial cells (10, 12) and immune cells (11, 12) , expression patterns of a wider range of host SARS-CoV-2 entry and processing genes in these cells was recently reported (12) . However, the relative expression levels of SARS-CoV-2 entry and processing genes in vessels and in endothelial cells has not been fully established. Finally, the impact of age on the expression of these genes in a cardiovascular setting is incompletely understood.

Here we have used publicly available gene expression data to determine the relative expression of key SARS-CoV-2 host entry/ processing genes in human cardiorenal tissues including aorta, coronary artery, heart (atria and left ventricle), whole blood and the kidney and for comparison the colon, spleen and lung. We went on to investigate gene expression in endothelial cells and, for comparison, airway (nasal and bronchial) epithelium and leukocytes (peripheral blood mononuclear cells; PBMCs). We used blood outgrowth endothelial cells as a model because, since they are obtained from blood samples of living donors, data sets across age ranges have been created. Furthermore, blood outgrowth endothelial cells are an accepted model for application in personalised medicine since they retain elements of disease phenotype across a number of cardiovascular and other conditions (25) (26) (27) . After mapping gene expression across our target tissues and cells, our primary objective was to determine how age, as the single most dominant risk factor for severe COVID-19, impacts on J o u r n a l P r e -p r o o f expression of SARS-CoV-2 entry and processing genes in human cardiorenal and other tissues.

All data is derived from publicly available open access data bases and so did not require ethical approval.

The Genotype-Tissue Expression (GTEx) project (28) is an ongoing effort to build a comprehensive public resource to study tissue-specific gene expression and. We downloaded gene expression data from GTEx version 8 (https://www.gtexportal.org/home/datasets) which contain expression data from 54 tissues from 948 donors. We identified tissues of interest based on organ systems affected by severe COVID-19 disease and extracted expression data specifically from those tissues. Tissues were split into two categories; (i) cardiorenal tissues including aorta, coronary artery, heart (atrial and appendage), left ventricle, kidney (cortex)

and whole blood and (ii) 'other tissues' including lung, colon and spleen. We performed principle component analysis (PCA) on gene expression data from each tissue of interest. We observed that the major variation in gene expression was due to type of death (Hardy Score;

Supplementary Figure 1 ) and so corrected for this. We normalised the gene expression data for each tissue separately using ComBat-seq (29) with Hardy score as a batch. After and CTSL were more highly expressed in arteries than kidney, heart or blood. Intracellular proteases CTSB and CTSL were both also enriched in endothelial cells followed by airway epithelium (CTSB, nasal>bronchial; CTSL, bronchial>nasal) and low levels in

PBMCs.

Next, in line with recent Public Health England's review of disparities in risks and outcomes for COVID-19 (11), we grouped data into two age categories, <40 and >40 years to determine differences in gene expression. Where differences were found and based on clinical evidence

showing that the risk of death from COVID-19 directly correlates with age (13), we performed follow-on correlation analysis.

Arteries:

In the aorta, FURIN and PPIB were increased while ACE2 was deceased in samples from adults >40 years of age ( Figure 3 ). Reductions of ACE2 or increases of PPIB linearly correlated with age ( Figure 4; Supplementary Figure 2 ). FURIN expression did not linearly correlate with age (Supplementary Figure 2) . None of our selected genes were affected by age in the coronary artery ( Figure 3 ).

Heart and Kidney:

In the heart (atria and left ventricle) or kidney none of our selected genes were affected by age ( Figure 3; Supplementary Figure 2 ). Spleen:

None of our studied genes were altered with age in the spleen (Figure 3 ).

In endothelial cells BSG, but not other genes, was increased in samples from adults >40 years ( Figure 3 and Figure 5 ) and levels showed a positive linear correlation with age ( Figure 5 ).

By contrast, only ACE was fractionally (but statistically significantly) reduced in nasal epithelium between age categories ( Figure 3 ) and this did not linearly correlate with age (Supplementary Figure 4) . No genes were altered in bronchial epithelium and PBMCs with age ( Figure 3) .

Whilst not the primary outcome of our study, which was age, we also investigated the effect Here, we report that SARS-CoV-2 receptors and processing genes are expressed across all cardiorenal target tissues and/or in endothelial cells, supporting the idea that systemic organs contain the required machinery to be infected by the virus. In regard to the two SARS-CoV-2 receptor pathways; expression levels of ACE2 were higher in cardiovascular tissues than the lung. By contrast, TMPRSS2, thought to be required for SARS-CoV-2 infection in epithelial cells, was present in lung colon and kidney but essentially absent in other cardiovascular tissues (heart, vessels and whole blood). Our findings describing the relative levels of these genes in human tissues are in line with others using similar approaches for ACE2 (33) (34) (35) (36) and

TMPRSS2 (35, 37) . Moreover, we found that both ACE2 and TMPRSS2 were enriched in nasal epithelium with low levels in bronchial epithelium and PBMCs which is in agreement J o u r n a l P r e -p r o o f with recent work from others (nasal versus bronchial epithelium (10, 11) and versus

PBMCs (11)). Radzikowsa et al., profiled a wider range of SARS-CoV-2 entry genes in immune cells and differentiated primary bronchial epithelial cells and also reported relatively high levels of expression of PPIA, BSG, PPIB with much lower levels of TMPRSS2 followed by ACE2 in airway cells (12) . However, our focus was on the cardiovascular system and kidney. Importantly we confirm that endothelial cells express ACE2 and TMPRSS2 although at lower levels than nasal epithelium but higher levels than bronchial epithelial cells (ACE2)

and PBMCs (ACE2 and TMPRSS2) . These findings suggest that SARS-COV-2 could infect endothelial cells via the ACE2 pathway. In cells where TMPRSS2 is low, SARS-CoV-2 can gain access by utilising CTSL and/or CTSB(1). In our study both CTSL and CTSB were found to be enriched in endothelial cells. Furthermore, in the setting of SARS-CoV-2 infection, this pathway may well be facilitated by the release of lysosomal proteases during inflammation (38) .

Nevertheless, in contrast to the ACE:TMPRSS2 pathway which, based on TMPRSS2 expression levels, was better represented in lung and nasal epithelium, the BSG:PPIB/PPIA pathway was, as a whole, better represented in vessels and endothelial cells than in the lung and airway epithelial cells. Our findings suggest that SARS-CoV-2 and other relevant viruses may, in addition to ACE2, exploit BSG as receptor pathway in the vasculature. Our findings align with recent work by Ganier and colleagues (39) and add evidence to the recent 'proposed mechanism' explaining how SARS-CoV-2 accesses endothelial cells, presented by Acosta Saltos and Acosta Saltos (40) .

Severe COVID-19 is exceptionally rare in children. In adults the strongest risk factor for severe disease and death is age, with those under 40 years being at very low risk; the risk of J o u r n a l P r e -p r o o f severe COVID-19 disease and death increases proportionally after the age of 40(24) . Of the genes that we studied, several candidates, including ACE2, were affected by age but with the exception of BSG in endothelial cells and PPIB and FURIN in aorta, expression was reduced in those >40. We found consistent age-related reductions in ACE2 in whole blood, aorta and in the colon. Our findings are in line with those published by Chen and co-workers who also reported a negative correlation between ACE2 and age in a range of tissues including colon and blood (33) . Moreover, our work corroborates earlier studies showing that ACE2 (protein) declines with age in mouse aorta (41) . Other studies in rats also showed that ACE2 declines with age in the lung and kidney (42, 43) . It should be noted, however, that Li and colleagues found no effect of age on ACE2 expression across a similar selection of tissues (34) and that

Santesmasses and colleagues found that ACE2 expression increased with age in the lung (44) .

We also found a trend for ACE2 to increase in the lung but this did not reach statistical significance in our study. Key differences between the studies include the analytical approaches applied, the number of tissues selected, and the age groups used.

Since ACE2 is a receptor for SARS-CoV-2, which declines with age in some settings (this study) (34, (41) (42) (43) and because age is the strongest predictor for fatal COVID-19 disease a paradox has emerged (45) The data were aligned and analysed using PartekFlow® and corrected for batch effects using

ComBat-seq and expressed as individual data points and mean +/-SEM. Cells were ranked in order of expression each gene. A heat map showing expression of ACE2 and BSG pathways and viral processing proteases in each cell type was generated (K). Data were coloured by gene, whereby black is the lowest expressing cell type and red is highest expressing cell type. 

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