TY - JOUR
T1 - Mitochondrial DNA and TLR9 activation contribute to SARS-CoV-2-induced endothelial cell damage
AU - Costa, Tiago J.
AU - Potje, Simone R.
AU - Fraga-Silva, Thais F.C.
AU - da Silva-Neto, Júlio A.
AU - Barros, Paula R.
AU - Rodrigues, Daniel
AU - Machado, Mirele R.
AU - Martins, Ronaldo B.
AU - Santos-Eichler, Rosangela A.
AU - Benatti, Maira N.
AU - de Sá, Keyla S.G.
AU - Almado, Carlos Eduardo L.
AU - Castro, Ítalo A.
AU - Pontelli, Marjorie C.
AU - Serra, Leonardo La
AU - Carneiro, Fernando S.
AU - Becari, Christiane
AU - Louzada-Junior, Paulo
AU - Oliveira, Rene D.R.
AU - Zamboni, Dario S.
AU - Arruda, Eurico
AU - Auxiliadora-Martins, Maria
AU - Giachini, Fernanda R.C.
AU - Bonato, Vânia L.D.
AU - Zachara, Natasha E.
AU - Bomfim, Gisele F.
AU - Tostes, Rita C.
N1 - Funding Information:
This study was supported by The São Paulo State Research Foundation (FAPESP; Grant 2013/08216–2 – Research Center in Inflammatory Diseases), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
Funding Information:
Tiago J. Costa is a FAPESP post-doctoral fellow (2017/25116–2 and 2019/26376–0). Simone R. Potje received a FAPESP post-doctoral fellowship (2016/21239–0).
Publisher Copyright:
© 2021
PY - 2022/2
Y1 - 2022/2
N2 - 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.
AB - 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.
KW - Endothelial dysfunction
KW - Mitochondria
KW - SARS-CoV-2
KW - Toll like receptor 9
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U2 - 10.1016/j.vph.2021.106946
DO - 10.1016/j.vph.2021.106946
M3 - Article
C2 - 34838735
AN - SCOPUS:85120912061
SN - 1537-1891
VL - 142
JO - Vascular Pharmacology
JF - Vascular Pharmacology
M1 - 106946
ER -