PO016 | TOLL-LIKE RECEPTOR 4-MEDIATED ENDOTHELIAL DYSFUNCTION VIA NOX2 ACTIVATION IN COVID-19

C. Nocella^1|2, V. Castellani², L. Loffredo¹, V. Cammisotto¹, S. Bartimoccia¹, P. Pignatelli¹, R. Carnevale^3|4, F. Violi¹ | ¹Dip di Scienze Mediche e Cardiovascolari, Sapienza Università di Roma, Italy; ²Fondazione Cenci Bolognetti, Istituto Pasteur Italia, Italy; ³IRCCS Neuromed, Italy; ⁴Dip di Scienze e Biotecnologie Medico-Chirurgiche, Sapienza Università di Roma, Italy

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, p47^phox, PKC, and ACE2. Markers of endothelial activation (NO bioavailability, endothelin-1) and oxidative stress (H₂O₂, sNOX2-dp, p47^phox 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 p47^phox inhibition, whereas ACE2 inhibition (DX600) had no impact. Mechanistically, S protein increased H₂O₂ production, sNOX2-dp levels, p47^phox, and PKC phosphorylation in a dose-dependent manner. NOX2 activation and p47^phox phosphorylation were reduced by TLR4 or p47^phox 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 p47^phox.
Conclusions. The Spike protein directly induces endothelial oxidative stress and dysfunction through a TLR4–NOX2–p47^phox 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.