Extracellular vesicles from post-COVID-19 patients alter endothelial function under protein restriction

The COVID-19 pandemic worsened global food insecurity and malnutrition. Protein restriction increases the risk of poor COVID-19 outcomes and cardiovascular disease. Post-COVID-19 syndrome remains a public health concern, although its underlying mechanisms are not yet fully understood. Extracellular vesicles (EVs), released by most cell types in response to infections, have been implicated in endothelial dysfunction during the post-COVID phase. We hypothesized that EV contribute to endothelial cell (EC) dysfunction in long-term COVID-19, particularly in the setting of protein malnutrition. Circulating EVs were isolated from patients at 1 and 6 months (mo) after hospital discharge due to severe COVID-19. Endothelial relaxation was assessed in mouse aortas after a 3-mo normoprotein or low-protein diet (LP). LP feeding reduced endothelium-dependent relaxation to acetylcholine, but EVs from post-COVID patients (1 and 6 mo) restored endothelium-dependent relaxation. This EV effect was abolished by catalase, but not by l-NAME (a nitric oxide synthase inhibitor) or indomethacin (a cyclooxygenase inhibitor). Aortas from LP mice incubated with post-COVID EVs exhibited reduced catalase expression and increased 4-hydroxynonenal (4-HNE) adducts. In vitro amino acid restriction increased EC death (Hoechst/Pi), and reduced nitric oxide (Diaminofluorescein-FM diacetato) and H₂O₂ (Amplex red) levels. Incubation with post-COVID EVs for 24 h increased H₂O₂ only in amino acid-restricted EC. EVs had no significant effect on acetylcholine-induced relaxation in normoprotein-fed mice or on EC parameters in vitro under control conditions. These findings suggest that EVs from patients 1 and 6 mo after severe COVID-19 impact aortic endothelial function by increasing H₂O₂ contribution under conditions of malnutrition.NEW & NOTEWORTHY Our study demonstrated that circulating extracellular vesicles (EVs) from patients 1 and 6 mo after severe COVID-19 altered endothelial function under protein restriction but not in healthy vessels. Post-COVID EVs enhanced the contribution of H₂O₂ to endothelium-dependent relaxation, associated with reduced catalase and increased 4-HNE-modified protein expression. These findings identified EVs from long COVID patients as potential mediators of endothelial dysfunction particularly under malnutrition-related conditions.