SARS-CoV-2 spike protein downregulates ACE2 expression

In a recent study published in the Journal of Infection, researchers observed the downregulation of angiotensin-converting enzyme 2 (ACE2) by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).  

ACE2 is the cellular entry receptor for SARS-CoV-2 and plays a vital role in the renin-angiotensin system (RAS) homeostasis. It has been reported that SARS-CoV-2 infection might activate RAS and damage endothelial cells inducing thrombosis, oxidative stress, and cardiovascular dysfunction. Some studies have observed that myocardial damage was prevalent among patients with coronavirus disease 2019 (COVID-19).

Another study revealed that serum from COVID-19 exhibited higher levels of angiotensin II (Ang II), which ACE2 cleaves to generate Ang 1 – 7, than in healthy controls. As such, it has been speculated that infection with SARS-CoV-2 might cause cardiovascular diseases (CVD), albeit the underlying mechanism remains poorly understood.

The study and findings

The present study investigated the role of ACE2 in the cardiovascular pathology of COVID-19. ACE2 expression declines substantially after SARS-CoV-2 infection, and the authors confirmed this by infecting Vero E6 cells and human ACE2 (hACE2)-expressing HeLa cells. Twenty-six SARS-CoV-2 proteins were transfected (using expression vectors) in hACE2-HeLa cells to identify proteins that reduced ACE2 levels.

Cell lysates were examined by western blotting analysis. ACE2 expression was significantly reduced only in cells that expressed the viral spike protein, and the decrease in ACE2 levels was dose-dependent. Further, ACE2 expression was lower in bronchial epithelial cells collected from COVID-19 patients than in those from healthy controls. Additional investigations in mice revealed that ACE2 expression in bronchial epithelial cells from virus-infected animals was significantly decreased.

Moreover, they co-transfected ACE2 and spike subunit 1 (S1) or 2 (S2) in HEK-293T cells and observed a significant reduction of ACE2 levels in S1-expressing cells while S2 did not affect ACE2 expression. hACE2-HeLa cells were infected with pseudoviruses coated with SARS-CoV-2 spike. Intriguingly, pseudoviral infection did not reduce ACE2 expression in the cells. Notably, incubating hACE2-HeLa cells with spike protein in vitro showed no significant decline in ACE2 levels.

Next, hACE2-HeLa cells were infected with live SARS-CoV-2 and observed for protein levels two hours and 24 hours after infection. At 2h, ACE2 levels were unaffected but reduced significantly by 24h. This implied that internalized virion (spike protein) significantly downregulated ACE2 expression.

The team investigated if the spike-mediated ACE2 decline was by protein degradation and treated HEK-293T cells, after co-transfection of ACE2 and spike, with MG132 (proteasome inhibitor) and E64D (lysosome inhibitor) with dimethyl sulfoxide (DMSO) as the control. Decreased ACE2 expression was noted in cells treated with either inhibitor.

Similar results were observed with infection of hACE2-Hela cells. These results indicated that ACE2 reduction was independent of protein degradation. Vero E6 cells were infected with SARS-CoV-2, and total RNA was extracted for quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Interestingly, ACE2 mRNA levels were significantly lower in infected cells, and the same was observed in SARS-CoV-2-infected hACE2-HeLa cells.

Lastly, they assessed if RAS was dysregulated in COVID-19 patients. Serum samples from 30 patients and 11 healthy controls were obtained. Ang II levels were significantly elevated in the sera of COVID-19 patients than in healthy controls. Treatment of human pulmonary artery endothelial cells (HPAEC) with high levels of Ang II resulted in cell death. Ang II type 1 receptor (AT1R) expression was induced upon treatment of Ang II in HPAEC.

Lung tissues from one COVID-19 patient and an age-matched control were analyzed for evidence of apoptosis. Endothelial cells positive for cleaved caspase 3 were detected only in the patient specimen and not the control specimen. Higher von Willebrand factor (VWF) expression in COVID-19 specimen suggested vascular damage.

Conclusions

The study showed that SARS-CoV-2 spike protein downregulated ACE2 expression. Of note, ACE2 downregulation was not observed upon infection of HeLa cells with spike-coated pseudoviruses or when cells were treated in vitro with the spike protein. mRNA levels of ACE2 were also reduced, which might be due to modifications affecting its stability, albeit more investigation is required in the future to delineate the underlying mechanism(s).

The decline in ACE2 levels was accompanied by an increase in the secretion of Ang II, affecting the endothelial cells and aggravating the risk of cardiovascular complications. Endothelial cell death in the lung tissue of a COVID-19 patient was also observed. Overall, the study demonstrated that ACE2 expression decreases at protein and mRNA levels during SARS-CoV-2 infection.

Written by

Tarun Sai Lomte

Tarun is a writer based in Hyderabad, India. He has a Master’s degree in Biotechnology from the University of Hyderabad and is enthusiastic about scientific research. He enjoys reading research papers and literature reviews and is passionate about writing.