Direct Activation of Endothelial Cells by SARS-CoV-2 Nucleocapsid Protein Is Blocked by Simvastatin

Abstract

Emerging evidence suggests that endothelial activation plays a central role in the pathogenesis of acute respiratory distress syndrome (ARDS) and multiorgan failure in patients with coronavirus disease 2019 (COVID-19). However, the molecular mechanisms underlying endothelial activation in COVID-19 patients remain unclear. In this study, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteins that potently activate human endothelial cells were screened to elucidate the molecular mechanisms involved in endothelial activation. It was found that nucleocapsid protein (NP) of SARS-CoV-2 significantly activated human endothelial cells through Toll-like receptor 2 (TLR2)/NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Remarkably, though the protein sequences of N proteins from coronaviruses are highly conserved, only NP from SARS-CoV-2 induced endothelial activation. The NPs from other coronaviruses such as SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), HUB1-CoV, and influenza virus H1N1 did not activate endothelial cells. These findings are consistent with the results from clinical investigations showing broad endotheliitis and organ injury in severe COVID-19 patients. In conclusion, the study provides insights on SARS-CoV-2-induced vasculopathy and coagulopathy and suggests that simvastatin, an FDA-approved lipid-lowering drug, may help prevent the pathogenesis and improve the outcome of COVID-19 patients. IMPORTANCE Coronavirus disease 2019 (COVID-19), caused by the betacoronavirus SARS-CoV-2, is a worldwide challenge for health care systems. The leading cause of mortality in patients with COVID-19 is hypoxic respiratory failure from acute respiratory distress syndrome (ARDS). To date, pulmonary endothelial cells (ECs) have been largely overlooked as a therapeutic target in COVID-19, yet emerging evidence suggests that these cells contribute to the initiation and propagation of ARDS by altering vessel barrier integrity, promoting a procoagulative state, inducing vascular inflammation and mediating inflammatory cell infiltration. Therefore, a better mechanistic understanding of the vasculature is of utmost importance. In this study, we screened the SARS-CoV-2 viral proteins that potently activate human endothelial cells and found that nucleocapsid protein (NP) significantly activated human endothelial cells through TLR2/NF-κB and MAPK signaling pathways. Moreover, by screening a natural microbial compound library containing 154 natural compounds, simvastatin was identified as a potent inhibitor of NP-induced endothelial activation. Our results provide insights on SARS-CoV-2-induced vasculopathy and coagulopathy, and suggests that simvastatin, an FDA-approved lipid-lowering drug, may benefit to prevent the pathogenesis and improve the outcome of COVID-19 patients.

Keywords: COVID-19; SARS-CoV-2; endothelial activation; endothelial cells; nucleocapsid protein; simvastatin.

Figures

FIG 1

SARS-CoV2 nucleocapsid protein (NP) is a potent inducer of human endothelial cell activation. (a) HLMECs were incubated with SARS-CoV2 structural proteins (S, N, and E proteins; 1 μg/ml) and five nonstructural proteins (NSP1, NSP3, NSP5, NSP7, and NSP8; 1 μg/ml) for 8 h. (b) HLMECs were treated with 1 μg/ml of NP or 10 ng/ml of TNF-α for different incubation periods as indicated. (c) HLMECs were incubated with indicated concentrations of NP for 8 h. TNF-α at 10 ng/ml served as a positive control. (d) Different cultured cells, including mouse lung vascular endothelial cells (MECs), A549 cells, 293T cells, HUVECs, HAECs, HCAECs, HDMECs, and HLMECs, were treated with NP (1 μg/ml) for 8 h. The expression of ICAM-1, VCAM-1, and VE-cadherin was detected by Western blotting. β-Actin served as a loading control. (e) HLMECs were treated with PBS, NP (1 μg/ml), TNF-α (10 ng/ml), or lipopolysaccharide (LPS) (1 μg/ml) for 8 h. The total RNA was isolated and qPCR was performed for measuring the mRNA levels of TNF-α, ICAM-1, VCAM-1, MCP-1, and IL-6. (f) HLMECs were treated with PBS, NP (1 μg/ml), or TNF-α (10 ng/ml) for 8 h and cocultured with Zombie Red-labeled THP-1 cells for 1 h. After being washed, the adherent cells were imaged and quantitatively analyzed.

FIG 2

N protein activated NF-κB and MAPK signaling pathways in human endothelial cells. HLMECs were incubated with NP (1 μg/ml), SP (1 μg/ml), TNF-α (10 ng/ml), and LPS (1 μg/ml) for the indicated times. The phosphorylation of IKKs, p65, IκBα, ERK, JNK, and p38, as well as IκBα degradation, was detected by Western blotting. GAPDH served as a loading control.

FIG 3

N protein induced endothelial cell activation via the TLR2-mediated signaling pathway. (a) HLMECs were pretreated with inhibitors of endocytosis (Pitstop2 [12.5 μM] and Dynasore hydrate [12.5 μM]) and antagonists of TLR4 (LPS-RS [10 μg/ml]), TLR2 (CU-CPT22 [20 μM]), IL-1R (IL-1R antagonist [20 μM]), inhibitors of IKK (IKK16 [20 μM]), ERK (U0126 [20 μM]), JNK (JNK inhibitor V [20 μM]), and p38 (SB203580 [20 μM]) for 1 h followed by treatment with NP (1 μg/ml) for 8 h. (b) The Flag control and Flag-NP expression plasmids were transfected into HLMECs by electroporation. The whole-cell lysate was harvested after 48 h transfection. The expression of ICAM-1, VCAM-1, NP, and Flag was detected by Western blotting. (c) HLMECs were incubated with NP (1 μg/ml) for different time as indicated. After being washed, the cells were harvested, and NP was detected by Western blotting. (d) HLMECs were treated with the indicated concentrations of CU-CPT22 for 1 h followed by treatment with NP (1 μg/ml) for 8 h. (e) Wild-type (left) and TLR2-overexpressing 293T cells were treated with or without NP (1 μg/ml) for 15 min. pJNK and pP38 were detected by Western blotting. GAPDH served as a loading control. All experiments were repeated at least once.

FIG 4

Screening of chemicals for inhibition of N protein-induced endothelial activation. A total of 155 chemicals from microbial natural product library were added to HLMECs at 30 μM 1 h before the induction of N protein (1 μg/ml). The effect of chemicals on endothelial activation was measured by Western blotting with anti-ICAM-1 antibody. β-Actin served as a loading control. Arrows indicate effective compounds; stars indicate toxic compounds causing cell death. The experiments were repeated once.

FIG 5

Simvastatin is an effective inhibitor of N protein-induced endothelial activation in vitro. (a) HLMECs were pretreated with simvastatin, lovastatin, atorvastatin, mevastatin, and rosuvastatin at 30 μM for 1 h followed by treatment with NP (1 μg/ml) for 8 h. (b) HLMECs were pretreated with indicated concentrations of lovastatin and simvastatin for 1 h followed by treatment with NP (1 μg/ml) for 8 h. The expression of ICAM-1 and VCAM-1 was detected by Western blotting. (c) HLMECs were pretreated with indicated concentrations of lovastatin and simvastatin for 1 h by treatment with NP (1 μg/ml) for 15 min. The activation of NF-κB and MAPK signal pathways was detected by Western blotting. (d) HLMECs were pretreated with or without lovastatin (Lova) or simvastatin (Simva) followed by treatment with NP (1 μg/ml) for 8 h and then cocultured with Zombie Red-labeled THP-1 cells for 1 h. After washing, the adherent cells were imaged.

FIG 6

Chemical structures of simvastatin, lovastatin, mevastatin, atorvastatin, and rosuvastatin. Structures were adapted from Wikipedia (simvastatin, https://commons.wikimedia.org/w/index.php?curid=4486633; lovastatin, https://commons.wikimedia.org/wiki/File:Lovastatin.svg; mevastatin, https://commons.wikimedia.org/wiki/File:Mevastatin.svg; atorvastatin, https://commons.wikimedia.org/wiki/File:Atorvastatin.svg; rosuvastatin, https://commons.wikimedia.org/wiki/File:Rosuvastatin_structure.svg; all structures are available under open access licenses or are in the public domain).

FIG 7

The N protein from SARS-CoV2 but not the other coronaviruses potently induced endothelial cell activation. HLMECs were treated with or without five different recombinant viral N proteins (1 μg/ml), including SARS-CoV2, SARS-CoV, MERS-CoV, H7N9, and HKU1-CoV, for 8 h. The expression of ICAM-1 and VCAM-1 was detected by Western blotting. TNF-α (10 ng/ml) served as a positive control. GAPDH served as the loading control. The experiments were repeated at least once.