PO016 | TOLL-LIKE RECEPTOR 4-MEDIATED ENDOTHELIAL DYSFUNCTION VIA NOX2 ACTIVATION IN COVID-19
Abstract
Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with endothelial dysfunction. Multiple pathophysiological mechanisms have been proposed, including direct endothelial infection and indirect injury mediated by systemic inflammation. However, alternative receptors involved in SARS-CoV-2–induced endothelial damage remain to be fully elucidated. We hypothesized that the SARS-CoV-2 Spike protein contributes to endothelial dysfunction via Toll-like receptor 4 (TLR4)-mediated oxidative stress. Methods. We studied 20 patients with SARS-CoV-2 infection. Endothelial function was assessed by flow-mediated dilation (FMD). Serum levels of Spike protein and TNF-α were measured. In vitro, human umbilical vein endothelial cells (HUVECs) were incubated with recombinant Spike protein, TNF-α, or both, in the presence or absence of specific inhibitors of TLR4, NOX2, p47phox, PKC, and ACE2. Markers of endothelial activation (NO bioavailability, endothelin-1) and oxidative stress (H₂O₂, sNOX2-dp, p47phox phosphorylation) were evaluated. Results. Compared with controls, COVID-19 patients exhibited significantly reduced FMD. Linear regression analysis in the overall population showed that FMD inversely correlated with serum Spike protein and TNF-α levels. A significant positive correlation between TNF-α and Spike protein was also observed. In vitro, S protein (250 and 500 ng/mL) reduced nitric oxide (NO) bioavailability and significantly increased endothelin-1 levels compared with unstimulated HUVECs. These effects were attenuated by TLR4 or p47phox inhibition, whereas ACE2 inhibition (DX600) had no impact. Mechanistically, S protein increased H₂O₂ production, sNOX2-dp levels, p47phox, and PKC phosphorylation in a dose-dependent manner. NOX2 activation and p47phox phosphorylation were reduced by TLR4 or p47phox inhibitors, while ACE2 inhibition did not affect S protein–induced oxidative stress. Exposure of HUVECs to TNF-α alone, at concentrations comparable to those observed in patients, or to S protein alone induced endothelial activation and oxidative stress. Co-incubation with TNF-α and S protein further amplified these effects, which were significantly reduced by inhibition of TLR4, S protein, TNF-α, or p47phox. Conclusions. The Spike protein directly induces endothelial oxidative stress and dysfunction through a TLR4–NOX2–p47phox signaling pathway, independently of ACE2. TNF-α synergistically enhances these effects. These findings identify TLR4-mediated signaling as a key mechanism underlying COVID-19–related vascular damage and suggest potential therapeutic targets to prevent thrombotic complications.
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BibTeX
@article{nocella2026po016,
title = {{PO016 | TOLL-LIKE RECEPTOR 4-MEDIATED ENDOTHELIAL DYSFUNCTION VIA NOX2 ACTIVATION IN COVID-19}},
author = {Cristina Nocella},
journal = {Bleeding, Thrombosis and Vascular Biology},
year = {2026},
month = oct,
url = {https://www.btvb.org/btvb/article/view/832},
note = {CC BY-NC},
}
RIS
TY - JOUR
TI - PO016 | TOLL-LIKE RECEPTOR 4-MEDIATED ENDOTHELIAL DYSFUNCTION VIA NOX2 ACTIVATION IN COVID-19
AU - Cristina Nocella
PY - 2026
DA - 2026/10/26
JO - Bleeding, Thrombosis and Vascular Biology
UR - https://www.btvb.org/btvb/article/view/832
AB - Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with endothelial dysfunction. Multiple pathophysiological mechanisms have been proposed, including direct endothelial infection and indirect injury mediated by systemic inflammation. However, alternative receptors involved in SARS-CoV-2–induced endothelial damage remain to be fully elucidated. We hypothesized that the SARS-CoV-2 Spike protein contributes to endothelial dysfunction via Toll-like receptor 4 (TLR4)-mediated oxidative stress. Methods. We studied 20 patients with SARS-CoV-2 infection. Endothelial function was assessed by flow-mediated dilation (FMD). Serum levels of Spike protein and TNF-α were measured. In vitro, human umbilical vein endothelial cells (HUVECs) were incubated with recombinant Spike protein, TNF-α, or both, in the presence or absence of specific inhibitors of TLR4, NOX2, p47phox, PKC, and ACE2. Markers of endothelial activation (NO bioavailability, endothelin-1) and oxidative stress (H₂O₂, sNOX2-dp, p47phox phosphorylation) were evaluated. Results. Compared with controls, COVID-19 patients exhibited significantly reduced FMD. Linear regression analysis in the overall population showed that FMD inversely correlated with serum Spike protein and TNF-α levels. A significant positive correlation between TNF-α and Spike protein was also observed. In vitro, S protein (250 and 500 ng/mL) reduced nitric oxide (NO) bioavailability and significantly increased endothelin-1 levels compared with unstimulated HUVECs. These effects were attenuated by TLR4 or p47phox inhibition, whereas ACE2 inhibition (DX600) had no impact. Mechanistically, S protein increased H₂O₂ production, sNOX2-dp levels, p47phox, and PKC phosphorylation in a dose-dependent manner. NOX2 activation and p47phox phosphorylation were reduced by TLR4 or p47phox inhibitors, while ACE2 inhibition did not affect S protein–induced oxidative stress. Exposure of HUVECs to TNF-α alone, at concentrations comparable to those observed in patients, or to S protein alone induced endothelial activation and oxidative stress. Co-incubation with TNF-α and S protein further amplified these effects, which were significantly reduced by inhibition of TLR4, S protein, TNF-α, or p47phox. Conclusions. The Spike protein directly induces endothelial oxidative stress and dysfunction through a TLR4–NOX2–p47phox signaling pathway, independently of ACE2. TNF-α synergistically enhances these effects. These findings identify TLR4-mediated signaling as a key mechanism underlying COVID-19–related vascular damage and suggest potential therapeutic targets to prevent thrombotic complications.
SN - 2785-5309
ER -