Researchers from Canada and the United Kingdom investigated whether binding of the SARS-CoV-2 spike (S) protein to the integral membrane glycoprotein angiotensin converting enzyme 2 (ACE2) induces inflammation in endothelial cells and determined the role of ACE2 in this process. The results showed that the S1 subunit of the SARS-CoV-2 S protein induces endothelial cell inflammation via ACE2 through processes that are independent of ACE2 enzymatic activity and viral replication.
ACE2 is a key enzyme in the homeostasis of the renin–angiotensin–aldosterone system, where it regulates the degradation of the vasoconstrictor angiotensin (Ang) II and formation of the vasodilator Ang(1–7). SARS-CoV-2 enters the cell by binding to ACE2, which is mediated by the S protein on the viral coat. The S protein consists of two subunits: the S1 subunit, which contains the receptor binding domain (RBD) to ACE2, and the S2 subunit, which is responsible for membrane fusion. The virus binds to ACE2 via the S1 subunit, thereby enabling the S2 subunit to undergo cleavage by the host transmembrane serine protease 2 (TMPRSS2). This results in the fusion of the viral envelope and cell membrane, and subsequent internalisation of the virus.
It should be noted that the American scientists Suzuki Y and Gyschka SG in collaboration with other authors, have previously shown in their in vitro studies that the S1 subunit, without the rest of the virus strongly activates the MEK/ERK pathway in cultured human pulmonary artery endothelial cells. They have shown that the S1 subunit of the recombinant S protein alone is sufficient to promote cell signaling, without the rest of the viral components in the human endothelial cells. https://discovermednews.com/the-s1-subunit-of-the-spike-protein-by-itself-may-promote-cell-signaling-in-the-vasculature/
About the study
At the beginning of the study, the authors investigated the global effect of the SARS-CoV-2 S protein on endothelial cell protein expression, by assessing the proteome of human endothelial cells (hMEC) after 24 hours of incubation with the S1 subunit. The expression profiles and production of proinflammatory mediators were used to characterize proinflammatory responses in hMECs, exposed to the S1 subunit. The effect of the S1 subunit was also evaluated in other endothelial cells from different vascular beds, such as human lymphatic endothelial cells, human aortic endothelial cells and human pulmonary artery endothelial cells.
The authors also assessed the generation of microparticles, a functional marker of endothelial injury, and examined whether the SARS-CoV2 virus replicated in hMECs. To better understand the putative role of ACE2 in inflammatory effects of the S1 subunit, they investigated whether ACE2 was regulated by the S1 subunit in hMECs.
After 24 h incubation with the S1 subunit, an analysis of the proteome of hMECs revealed a total of 1150 proteins from major signalling pathways and cellular processes, including virus transcription, cell–cell adhesion, nuclear factor-kB (NF-κB) signalling, oxidation–reduction process, mitogen activated protein (MAPK) cascade, and T-cell receptor signalling. This suggests that the S1 subunit induced human endothelial cell inflammation and proliferation.
A comprehensive analysis of proinflammatory responses in hMECs showed that the S1 subunit induced an increase in mRNA expression and release of the cytokine interleukin (IL)-6 and the chemokine monocyte chemoattractant protein-1 (MCP-1). Furthermore, hMECs treated with the S1 subunit showed higher gene levels of tumor necrosis factor aplha (TNFα), vascular cell adhesion molecule 1 (VCAM-1), and plasminogen activator inhibitor 1 (PAI-1). Intercellular adhesion molecule 1 (ICAM-1), a cell adhesion molecule involved in inflammation, was also upregulated in hMECs treated with the S1 subunit. These results show that the S1 subunit increased the production of IL-6, MCP-1, ICAM-1 and PAI-1, and induced NFkB activation via ACE2 in endothelial cells.
There were no changes observed in the gene expression of tranforming growth factor beta (TGFβ) and preproendothelin 1 (preproET-1), whereas the mRNA expression of thrombin and angiopoietin-2 decreased.
The authors then investigated whether inflammation of hMECs was associated with replication of SARS-CoV2 in hMECs. They found that endothelial cells exposed to SARS-CoV-2 virus did not exhibit viral replication. According to the authors, ACE2 acts as a plasma membrane receptor in the S1 subunit-induced inflammation and injury of hMECs, but these effects are independent of virus replication in human endothelial cells.
The observed effects in hMECs were associated with the S1 subunit-induced increase in microparticle formation, which is an important biomarker of endothelial cell injury and apoptosis.
The S1 subunit-induced endothelial cell inflammation and injury were not specific to human microvascular cells. A similar proinflammatory response was observed in other endothelial cells from different vascular beds. The S1 subunit increased IL-6 mRNA and MCP-1 mRNA levels and production in human lymphatic endothelial cells, human aortic endothelial cells, and human pulmonary artery endothelial cells. The formation of microparticles was increased by the S1 subunit only in human lymphatic endothelial cells, but not in human aortic endothelial cells or human pulmonary artery endothelial cells.
To evaluate whether ACE2 inhibition potentiates or blocks inflammation induced by the S1 subunit, the scientists used MLN-4760, an ACE2 inhibitor. ACE2 inhibition reduced ACE2 activity to the same extent in cells that were exposed to the S1 subunit or not, but it did not change the expression of ACE2 protein.
Importantly, MLN-4760, an ACE2 inhibitor, did not inhibit the increase in the mRNA expression of IL-6 and MCP-1 and the production of IL-6 and MCP-1, which were induced by the S1 subunit. The activation of ACE2 enzymatic function by diminazine aceturate (DIZE) did not influence the S1 subunit-induced increase in IL-6, MCP-1, and TNFα in hMECs.
However, ACE2 inhibition blocked the increase in the expression of ICAM-1 and PAI-1 proteins, but it did not change effects of the S1 subunit on levels of PAI-1 mRNA. The increase in VCAM-1 gene expression induced by S1 subunit in hMECs was also inhibited by MLN-4760.
The authors stated that both, the S1 subunit and MLN-4760 compete for the same ligand-binding domain on the ACE2 structure, and MLN-4760 therefore blocks spike protein-mediated activation of ACE2-dependent pathways. However, the findings that inhibition or downregulation of ACE2 did not alter S1 subunit-induced production of MCP-1 or IL-6 suggest that other SARS-CoV-2 receptors are involved in endothelial cells.
Endotheliitis has been described in COVID-19 patients, and the molecular mechanisms underlying it remain controversial. In addition, endotheliitis is considered an important component of the long COVID syndrome. The investigators concluded that this study shows that S1 subunit of the S protein causes endothelial inflammation via ACE2 through processes that are independent of the enzymatic activity of ACE2 and viral replication. The investigators emphasized the relevance of these findings to the cardiovascular complications associated with COVID-19 and long COVID syndrome.
This article was published in Scientific Reports.
Montezano, AC, et al. SARS-CoV-2 spike protein induces endothelial inflammation via ACE2 independently of viral replication. Sci Rep 13, 14086 (2023). (Open Access). https://doi.org/10.1038/s41598-023-41115-3