Dear Editor,
The Coronavirus disease 2019 (COVID-19) pandemic has caused over 670 million cases and 6.7 million deaths worldwide, many of which are attributed to cardiovascular complications. Virus-induced endothelial damage, endothelial barrier dysfunction, thrombosis, and cytokine storm are implicated in heart and multi-organ failure. The prognosis is worsened by comorbidities, including diabetes and arterial hypertension, characterized by an inflammatory and pro-thrombotic milieu and upregulation of total and glycosylated Angiotensin-Converting Enzyme 2 (ACE2) in cardiomyocytes.1,2
Cardiac pericytes represent a preferential target of SARS-CoV-2 infection.3,4 These perivascular cells preserve vascular integrity through physical and paracrine crosstalk with capillary endothelial cells. Pericyte dysfunction and detachment favor the SARS-CoV-2 to spread from the bloodstream and damage the myocardium.5
SARS-CoV-2 infection starts with the engagement of the Spike (S)-protein with its cellular ACE-2 and CD147 receptors. Due to the homology with human proteins, the S-protein also acts as a natural ligand activating the ERK1/2 MAPK signaling pathway in cardiac pericytes.6,7 Some evidence suggests that the S-protein, CD147, cyclophilin, and MAPK axis are essential in triggering the cytokine storm.7 However, an in vivo demonstration of the S-protein’s direct damaging effect on cardiac pericytes is lacking.
The present study investigated the acute effects of intravenously injected S-protein on the heart microvasculature of otherwise healthy mice. Moreover, we analyzed the expressional changes caused by the S-protein in primary cultures of human cardiac pericytes using bulk RNA-Sequencing. Finally, the RNA-Sequencing data were cross-referenced with single-nuclei (sn)-RNA-Sequencing datasets of COVID-19 patients’ hearts to determine how expressional changes after SARS-CoV-2 infection overlap with those caused by the S-protein alone.
Nine-week-old healthy CD1 mice (6 male, 6 female) were randomized to receive either 10 µg endotoxin-free S-protein resuspended in 100 µL sterile PBS or PBS only, intravenously. They were culled three days later for molecular and histological analyses (Fig. 1a). S-protein immunoreactive levels in the circulation were like those reported in COVID-19 patients early after infection and before seroconversion (26.8 ± 7.9 ng/mL).7 Immunohistochemistry of the hearts demonstrated that the S-protein, although not altering the capillary density, increased the fraction that expresses ICAM-1, an adhesion molecule implicated in leucocyte-endothelial interactions (Fig. 1b) and remarkably reduced the pericyte density, coverage, and viability (Fig. 1c–e). SARS-CoV-2 can trigger direct or indirect activation of all three complement pathways.8 Here, we show that the in vivo administration of S-protein increased complement-activated C5a protein levels in peripheral blood and the heart (Fig. 1f, g). Moreover, the S-protein increased the heart’s abundance of CD45+ immune cells (23.9 ± 2.2 cells/mm2 vs. 9.2 ± 1.9 cell/mm2 in PBS-treated mice), specifically Ly6G/6C+ neutrophils/monocytes (Fig. 1h) and F4/80+ macrophages (Fig. 1i). Leucocytes can crawl along pericyte processes to enlarged gaps between adjacent pericytes in an ICAM-1-dependent manner during inflammation. Controls for immunohistochemistry stainings are provided in Supplementary Fig. S1.
To further validate the theory of the S-protein acting as a direct transcriptomic influencer, we added it or the PBS vehicle to human primary cardiac pericytes in vitro for 48 h. RNA-Sequencing analysis indicated the differential modulation of 309 RNA transcripts, with 201 genes being up-regulated and 108 genes down-regulated by the S-protein at FDR < 0.1 (Fig. 1j). KEGG pathway analysis showed an overrepresentation of inflammatory pathways, for example, TNF, IL-17, and NF-kappa B signaling pathways, cytokine-cytokine receptor interaction, and cell adhesion molecules (CAMs). Moreover, there was an enrichment for pathways associated with infectious diseases, including Legionellosis, Pertussis, Malaria, Herpes virus, and Epstein-Barr virus infection (Fig. 1k). An overview of the pathway analysis based on the Reactome database further pinpointed the transcriptional induction of cytokine signaling pathways, such as IL-10, IL-4, and IL-13 signaling and Toll-like receptor cascade (Fig. 1l and Supplementary Fig. S2), and the downregulation of pathways implicated in histone deacetylation and methylation and chromatin modification, and RNA polymerase-related mechanisms controlling promoter opening and clearance, transcription, and chain elongation (Fig. 1l and Supplementary Fig. S2). The analysis of modulated biological processes confirmed the upregulation of cellular responses to stress and the downregulation of homeostatic responses associated with healing and angiogenesis processes (Supplementary Fig. S3). A comprehensive list of regulated pathways is provided in Supplementary Dataset 1.
Aiming to dissect clinically relevant targets further, we cross-interrogated the transcriptional landscape of pericytes exposed in vitro to the recombinant S-protein and pericytes from the hearts of COVID-19 patients. Additionally, we employed a pseudo-time inference approach to probe individual genes’ expression dynamics along with the progression of the disease. To this aim, we extracted pericytes from the integrated Seurat, R object (downloaded from Delorey et al., 2021)9 using marker genes followed by a pseudo-time analysis of pericytes collected from the heart of COVID-19 patients (Fig. 1m). The pseudo-time analysis allowed the identification of pericyte genes that are differential and co-expressed along the trajectory. This resulted in the recognition of 37 gene clusters (Fig. 1n). Next, to identify common signals between ex vivo and in vivo datasets, we tested for the overrepresentation of expressional changes in pericytes exposed to S-protein and gene clusters in the human heart. We observed that seven gene clusters (1, 2, 6, 13, 15, 20, and 27, FDR < 0.05) significantly overlapped with the expressional changes observed in pericytes exposed to the S-protein experiment (Fig. 1o). Cluster 15 was enriched for cytokine-related pathways, metallothioneins, and regulation of histone acetylation, while clusters 1, 6 and 27 were overrepresented for extracellular matrix organization, elastic fibre formation, and integrin cell surface interactions (Fig. 1p and Supplementary Dataset 2). Studies have reported that COVID-19 can cause cardiovascular complications due to impaired extracellular matrix organisation and reduced elastic fibre levels, potentially leading to blood clots.10 These findings suggest a convergence of signals that proteins of the virion envelope mediate at least part of the transcriptional changes induced by the virus in the hearts of infected people. Therefore, some of the in vivo effects of SARS-CoV-2 on human cardiac pericytes may be attributable to the interaction between the S-protein and the host’s transcriptomic program modulating inflammatory and epigenetic pathways.
Finally, we performed drug target enrichment analysis using the LINCS L1000CDS and DrugBank databases. This analysis allowed us to identify drugs that reverse the expressional changes induced by the S-protein in pericytes (Supplementary Dataset 3 and 4). Among the top fifty compounds, we found a prevalence of anti-tumoral, pro-apoptotic, anti-viral, anti-inflammatory and anti-thrombotic drugs, some of which have already been trialed in COVID-19 patients. Although more research is needed to determine if pharmacological interference with the signaling emanating from the S-protein can alleviate COVID-19 outcomes, these data suggest a competitive effect of anti-inflammatory and anti-tumoral drugs. In addition, several compounds like Quercetin or ubiquitin-conjugating enzyme inhibitors may help moderate inflammation by eliminating S-Protein-induced senescent cells.
Findings summarized in Fig. 1q provide novel evidence of the SARS-CoV-2 S-protein’s direct pathogenic action on cardiac pericytes and the heart’s microvasculature. It is plausible that the harmful effects observed in healthy mice three days after a single systemic injection of the S-protein might be intensified in the presence of cardiovascular risk factors and prolonged exposure. These possibilities merit further investigation. Moreover, we showed that the S-protein modifies the transcriptional program of human cells to the virus’ advantage. This new information could have significant implications for the treatment of COVID-19, for instance, using anti-S-protein engineering approaches to protect vascular cells.
Data availability
The article’s data can be obtained as reasonably required from the corresponding author. The main datasets underlying transcriptomic analyses are provided as supplementary datasets (Dataset 1–4). The bulk RNA-Seq raw data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE218644.
References
D’Onofrio, N. et al. Glycated ACE2 receptor in diabetes: open door for SARS-COV-2 entry in cardiomyocyte. Cardiovasc. Diabetol. 20, 99 (2021).
Sardu, C. et al. Could Anti-Hypertensive Drug Therapy Affect the Clinical Prognosis of Hypertensive Patients With COVID-19 Infection? Data From Centers of Southern Italy. J. Am. Heart Assoc. 9, e016948 (2020).
Tucker, N. R. et al. Myocyte-Specific Upregulation of ACE2 in Cardiovascular Disease: Implications for SARS-CoV-2-Mediated Myocarditis. Circulation 142, 708–710 (2020).
Muus, C. et al. Single-cell meta-analysis of SARS-CoV-2 entry genes across tissues and demographics. Nat. Med. 27, 546–559 (2021).
Daems, M. et al. SARS-CoV-2 infection causes prolonged cardiomyocyte swelling and inhibition of HIF1alpha translocation in an animal model COVID-19. Front. Cardiovasc. Med. 9, 964512 (2022).
Khan, A. O. et al. Preferential uptake of SARS-CoV-2 by pericytes potentiates vascular damage and permeability in an organoid model of the microvasculature. Cardiovasc. Res. 118, 3085–3096 (2022).
Avolio, E. et al. The SARS-CoV-2 Spike protein disrupts human cardiac pericytes function through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease. Clin. Sci. 135, 2667–2689 (2021).
Afzali, B., Noris, M., Lambrecht, B. N. & Kemper, C. The state of complement in COVID-19. Nat. Rev. Immunol. 22, 77–84 (2022).
Delorey, T. M. et al. COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets. Nature 595, 107–113 (2021).
Shi, S. et al. Association of Cardiac Injury With Mortality in Hospitalized Patients With COVID-19 in Wuhan, China. JAMA Cardiol. 5, 802–810 (2020).
Acknowledgements
The authors wish to acknowledge the members of the University of Bristol COVID-19 Emergency Research Group (UNCOVER) for their scientific support. Drawings were generated with BioRender.com.
Funding
This work was supported by the British Heart Foundation (BHF) project grant “Targeting the SARS-CoV-2 S-protein binding to the ACE2 receptor to preserve human cardiac pericytes function in COVID-19” (PG/20/10285) (to P.M. and E.A.); European Commission H2020 CORDIS project COVIRNA (project/id/101016072) (to C.E. and P.K.S.) and BHF Chair award (CH/15/1/31199) (to C.E). In addition, it was supported by a grant from the National Institute for Health Research (NIHR) Biomedical Research Centre at University Hospitals Bristol NHS Foundation Trust and the University of Bristol. E.A. is a postdoctoral researcher supported by the Heart Research UK translational project grant “Targeting pericytes for halting pulmonary hypertension in infants with congenital heart disease” (RG2697/21/23) (to P.M. and E.A.). I.B. is an investigator of the Wellcome Trust (106115/Z/14/Z).
Author information
Authors and Affiliations
Contributions
E.A., P.K.S., C.E., P.M.: research conception and design. E.A., P.K.S., P.M.: manuscript writing. E.A., M.C., C.T.W.T.: histological analyses of mice hearts. E.A.: cellular and molecular biology experiments. P.K.S., J.J.: transcriptomic analyses in pericytes. Y.G., A.C.T.: in vivo procedures with mice. K.G., I.B.: production and provision of Spike protein. C.E.: funding, supervision of transcriptomic studies, and manuscript editing. P.M.: funding provision. P.M.: study supervision. All authors: data interpretation and manuscript revision. All authors approved the authorship and the final version of the manuscript for publication.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Ethics declarations
The animal study was covered by a license from the British Home Office (PPL 1377882) and complied with EU Directive 2010/63/EU. Procedures were carried out according to the principles in the Guide for the Care and Use of Laboratory Animals (The Institute of Laboratory Animal Resources, 1996). Termination was conducted according to humane methods outlined in the Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 Home Office (2014). The collection of human patients’ cardiac waste tissue was covered by the ethical approval number 15/LO/1064 from the North Somerset and South Bristol Research Ethics Committee. Patients gave informed written consent.
Supplementary information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Avolio, E., Srivastava, P.K., Ji, J. et al. Murine studies and expressional analyses of human cardiac pericytes reveal novel trajectories of SARS-CoV-2 Spike protein-induced microvascular damage. Sig Transduct Target Ther 8, 232 (2023). https://doi.org/10.1038/s41392-023-01489-2
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41392-023-01489-2