Mitochondrial DNA and TLR9 activation contribute to SARS-CoV-2-induced endothelial cell damage

Tiago J. Costa, Simone R. Potje, Thais F.C. Fraga-Silva, Júlio A. da Silva-Neto, Paula R. Barros, Daniel Rodrigues, Mirele R. Machado, Ronaldo B. Martins, Rosangela A. Santos-Eichler, Maira N. Benatti, Keyla S.G. de Sá, Carlos Eduardo L. Almado, Ítalo A. Castro, Marjorie C. Pontelli, Leonardo La Serra, Fernando S. Carneiro, Christiane Becari, Paulo Louzada-Junior, Rene D.R. Oliveira, Dario S. ZamboniEurico Arruda, Maria Auxiliadora-Martins, Fernanda R.C. Giachini, Vânia L.D. Bonato, Natasha E. Zachara, Gisele F. Bomfim, Rita C. Tostes

Research output: Contribution to journalArticlepeer-review

Abstract

Background and purpose: Mitochondria play a central role in the host response to viral infection and immunity, being key to antiviral signaling and exacerbating inflammatory processes. Mitochondria and Toll-like receptor (TLR) have been suggested as potential targets in SARS-CoV-2 infection. However, the involvement of TLR9 in SARS-Cov-2-induced endothelial dysfunction and potential contribution to cardiovascular complications in COVID-19 have not been demonstrated. This study determined whether infection of endothelial cells by SARS-CoV-2 affects mitochondrial function and induces mitochondrial DNA (mtDNA) release. We also questioned whether TLR9 signaling mediates the inflammatory responses induced by SARS-CoV-2 in endothelial cells. Experimental approach: Human umbilical vein endothelial cells (HUVECs) were infected by SARS-CoV-2 and immunofluorescence was used to confirm the infection. Mitochondrial function was analyzed by specific probes and mtDNA levels by real-time polymerase chain reaction (RT-PCR). Inflammatory markers were measured by ELISA, protein expression by western blot, intracellular calcium (Ca2+) by FLUOR-4, and vascular reactivity with a myography. Key results: SARS-CoV-2 infected HUVECs, which express ACE2 and TMPRSS2 proteins, and promoted mitochondrial dysfunction, i.e. it increased mitochondria-derived superoxide anion, mitochondrial membrane potential, and mtDNA release, leading to activation of TLR9 and NF-kB, and release of cytokines. SARS-CoV-2 also decreased nitric oxide synthase (eNOS) expression and inhibited Ca2+ responses in endothelial cells. TLR9 blockade reduced SARS-CoV-2-induced IL-6 release and prevented decreased eNOS expression. mtDNA increased vascular reactivity to endothelin-1 (ET-1) in arteries from wild type, but not TLR9 knockout mice. These events were recapitulated in serum samples from COVID-19 patients, that exhibited increased levels of mtDNA compared to sex- and age-matched healthy subjects and patients with comorbidities. Conclusion and applications: SARS-CoV-2 infection impairs mitochondrial function and activates TLR9 signaling in endothelial cells. TLR9 triggers inflammatory responses that lead to endothelial cell dysfunction, potentially contributing to the severity of symptoms in COVID-19. Targeting mitochondrial metabolic pathways may help to define novel therapeutic strategies for COVID-19.

Original languageEnglish (US)
Article number106946
JournalVascular Pharmacology
Volume142
DOIs
StatePublished - Feb 2022

Keywords

  • Endothelial dysfunction
  • Mitochondria
  • SARS-CoV-2
  • Toll like receptor 9

ASJC Scopus subject areas

  • Molecular Medicine
  • Physiology
  • Pharmacology

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