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

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.

The homeostatic role of hydrogen peroxide, superoxide anion and nitric oxide in the vasculature.

Reactive oxygen and nitrogen species are produced in a wide range of physiological reactions that, at low concentrations, play essential roles in living organisms. There is a delicate equilibrium between formation and degradation of these mediators in a healthy vascular system, which contributes to maintaining these species under non-pathological levels to preserve normal vascular functions. Antioxidants scavenge reactive oxygen and nitrogen species to prevent or reduce damage caused by excessive oxidation. However, an excessive reductive environment induced by exogenous antioxidants may disrupt redox balance and lead to vascular pathology. This review summarizes the main aspects of free radical biochemistry (formation, sources and elimination) and the crucial actions of some of the most biologically relevant and well-characterized reactive oxygen and nitrogen species (hydrogen peroxide, superoxide anion and nitric oxide) in the physiological regulation of vascular function, structure and angiogenesis. Furthermore, current preclinical and clinical evidence is discussed on how excessive removal of these crucial responses by exogenous antioxidants (vitamins and related compounds, polyphenols) may perturb vascular homeostasis. The aim of this review is to provide information of the crucial physiological roles of oxidation in the endothelium, vascular smooth muscle cells and perivascular adipose tissue for developing safer and more effective vascular interventions with antioxidants.