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Diverse roles of SARS-CoV-2 Spike and Nucleocapsid proteins in EndMT stimulation through the TGF-β-MRTF axis inhibited by aspirin

Ciszewski et al., Cell Communication and Signaling, doi:10.1186/s12964-024-01665-z
May 2024  
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Aspirin for COVID-19
24th treatment shown to reduce risk in August 2021
 
*, now known with p = 0.000087 from 73 studies, recognized in 3 countries.
Lower risk for mortality and progression.
No treatment is 100% effective. Protocols combine complementary and synergistic treatments. * >10% efficacy in meta analysis with ≥3 clinical studies.
4,200+ studies for 70+ treatments. c19early.org
In Vitro study showing that SARS-CoV-2 spike and nucleocapsid proteins induce endothelial-mesenchymal transition (EndMT) in endothelial cells, with the nucleocapsid protein having a more potent effect. The spike protein stimulates TGF-β1 secretion through ACE2 downregulation, while the nucleocapsid protein stimulates TGF-β2 secretion through the TLR4-ROS pathway. TGF-β1 and TGF-β2 then induce EndMT through MRTF-B and MRTF-A/B nuclear translocation, respectively. Authors find that aspirin inhibits and reverses the SARS-CoV-2 protein-induced EndMT by blocking TGF-β secretion and MRTF nuclear translocation. The results suggest that aspirin may reduce the risk of COVID-19-associated fibrosis.
2 preclinical studies support the efficacy of aspirin for COVID-19:
Ciszewski et al., 28 May 2024, peer-reviewed, 3 authors. Contact: katarzyna.sobierajska@umed.lodz.pl.
In Vitro studies are an important part of preclinical research, however results may be very different in vivo.
This PaperAspirinAll
Diverse roles of SARS-CoV-2 Spike and Nucleocapsid proteins in EndMT stimulation through the TGF-β-MRTF axis inhibited by aspirin
Wojciech M Ciszewski, Lucyna A Woźniak, Katarzyna Sobierajska
Cell Communication and Signaling, doi:10.1186/s12964-024-01665-z
Background The SARS-CoV-2 virus causes severe COVID-19 in one-fifth of patients. In addition to high mortality, infection may induce respiratory failure and cardiovascular complications associated with inflammation. Acute or prolonged inflammation results in organ fibrosis, the cause of which might be endothelial disorders arising during the endothelial-mesenchymal transition (EndMT). Methods HUVECs and HMEC-1 cells were stimulated with SARS-CoV-2 S (Spike) and N (Nucleocapsid) proteins, and EndMT induction was evaluated by studying specific protein markers via Western blotting. Wound healing and tube formation assays were employed to assess the potential of SARS-CoV-2 to stimulate changes in cell behaviour. MRTF nuclear translocation, ROS generation, TLR4 inhibitors, TGF-β-neutralizing antibodies, and inhibitors of the TGF-βdependent pathway were used to investigate the role of the TGF-β-MRTF signalling axis in SARS-CoV-2-dependent EndMT stimulation. Results Both viral proteins stimulate myofibroblast trans-differentiation. However, the N protein is more effective at EndMT induction. The TGF-β-MRTF pathway plays a critical role in this process. The N protein preferentially favours action through TGF-β2, whose secretion is induced through TLR4-ROS action. TGF-β2 stimulates MRTF-A and MRTF-B nuclear translocation and strongly regulates EndMT. In contrast, the Spike protein stimulates TGF-β1 secretion as a result of ACE2 downregulation. TGF-β1 induces only MRTF-B, which, in turn, weakly regulates EndMT. Furthermore, aspirin, a common nonsteroidal anti-inflammatory drug, might prevent and reverse SARS-CoV-2-dependent EndMT induction through TGF-β-MRTF pathway deregulation. Conclusion The reported study revealed that SARS-CoV-2 infection induces EndMT. Moreover, it was demonstrated for the first time at the molecular level that the intensity of the EndMT triggered by SARS-CoV-2 infection may vary and depend on the viral protein involved. The N protein acts through TLR4-ROS-TGF-β2-MRTF-A/B, whereas the S protein acts through ACE2-TGF-β1-MRTF-B. Furthermore, we identified aspirin as a potential anti-fibrotic drug for treating patients with SARS-CoV-2 infection.
Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12964-024-01665-z. Supplementary Material 1 Author contributions WMC conducted the research, analysed and interpreted the data, and wrote the original manuscript. LAW analysed and interpreted the data, and made corrections to the original manuscript. KS designed the research, designed the experiments, analysed and interpreted the data, and contributed to writing the original manuscript. Declarations Competing interests The authors declare no competing interests. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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MRTF nuclear ' 'translocation, ROS generation, TLR4 inhibitors, TGF-β-neutralizing antibodies, and inhibitors ' 'of the TGF-β-dependent pathway were used to investigate the role of the TGF-β-MRTF signalling ' 'axis in SARS-CoV-2-dependent EndMT stimulation.</jats:p>\n' ' </jats:sec><jats:sec>\n' ' <jats:title>Results</jats:title>\n' ' <jats:p>Both viral proteins stimulate myofibroblast trans-differentiation. ' 'However, the N protein is more effective at EndMT induction. The TGF-β-MRTF pathway plays a ' 'critical role in this process. The N protein preferentially favours action through TGF-β2, ' 'whose secretion is induced through TLR4-ROS action. TGF-β2 stimulates MRTF-A and MRTF-B ' 'nuclear translocation and strongly regulates EndMT. In contrast, the Spike protein stimulates ' 'TGF-β1 secretion as a result of ACE2 downregulation. TGF-β1 induces only MRTF-B, which, in ' 'turn, weakly regulates EndMT. 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' '2021;11:3455.', 'journal-title': 'Sci Rep'}, { 'key': '1665_CR65', 'doi-asserted-by': 'publisher', 'first-page': '108720', 'DOI': '10.1016/j.clim.2021.108720', 'volume': '226', 'author': 'F Perna', 'year': '2021', 'unstructured': 'Perna F, Bruzzaniti S, Piemonte E, Maddaloni V, Atripaldi L, Sale S, ' 'Sanduzzi A, Nicastro C, Pepe N, Bifulco M, et al. Serum levels of ' 'SARS-CoV-2 nucleocapsid antigen associate with inflammatory status and ' 'disease severity in COVID-19 patients. Clin Immunol. 2021;226:108720.', 'journal-title': 'Clin Immunol'}, { 'key': '1665_CR66', 'doi-asserted-by': 'publisher', 'first-page': '254', 'DOI': '10.1089/vim.2021.0168', 'volume': '35', 'author': 'A Takamatsu', 'year': '2022', 'unstructured': 'Takamatsu A, Oshiro S, Mizutani N, Tada T, Tabe Y, Miida T, Kirikae T, ' 'Tagashira Y. Correlation of COVID-19 Severity and Immunoglobulin ' 'Presence against Spike and Nucleocapsid Proteins in SARS-CoV-2. Viral ' 'Immunol. 2022;35:254–8.', 'journal-title': 'Viral Immunol'}, { 'key': '1665_CR67', 'doi-asserted-by': 'publisher', 'first-page': '35', 'DOI': '10.1038/s41533-022-00300-z', 'volume': '32', 'author': 'P Kushner', 'year': '2022', 'unstructured': 'Kushner P, McCarberg BH, Grange L, Kolosov A, Haveric AL, Zucal V, ' 'Petruschke R, Bissonnette S. The use of non-steroidal anti-inflammatory ' 'drugs (NSAIDs) in COVID-19. NPJ Prim Care Respir Med. 2022;32:35.', 'journal-title': 'NPJ Prim Care Respir Med'}, { 'key': '1665_CR68', 'doi-asserted-by': 'publisher', 'first-page': '4899', 'DOI': '10.1111/bph.15206', 'volume': '177', 'author': 'CT Robb', 'year': '2020', 'unstructured': 'Robb CT, Goepp M, Rossi AG, Yao C. Non-steroidal anti-inflammatory ' 'drugs, prostaglandins, and COVID-19. 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' '2021;52:124–9.', 'journal-title': 'J Thromb Thrombolysis'}, { 'key': '1665_CR71', 'doi-asserted-by': 'crossref', 'unstructured': 'Russell B, Moss C, Rigg A, Van Hemelrijck M. COVID-19 and treatment with ' 'NSAIDs and corticosteroids: should we be limiting their use in the ' 'clinical setting? Ecancermedicalscience 2020, 14:1023.', 'DOI': '10.3332/ecancer.2020.1023'}, { 'key': '1665_CR72', 'first-page': '1351', 'volume': '45', 'author': 'Z Zhang', 'year': '2020', 'unstructured': 'Zhang Z, Li S, Deng J, Yang S, Xiang Z, Guo H, Xi H, Sang M, Zhang W. ' 'Aspirin inhibits endometrial fibrosis by suppressing the ' 'TGF–beta1–Smad2/Smad3 pathway in intrauterine adhesions. Int J Mol Med. ' '2020;45:1351–60.', 'journal-title': 'Int J Mol Med'}, { 'key': '1665_CR73', 'doi-asserted-by': 'crossref', 'unstructured': 'Sun Y, Liu B, Xie J, Jiang X, Xiao B, Hu X, Xiang J. Aspirin attenuates ' 'liver fibrosis by suppressing TGF–beta1/Smad signaling. 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