Extracellular vesicle‐mediated endothelial apoptosis and EV‐associated proteins correlate with COVID‐19 disease severity

Abstract

Coronavirus disease-2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has lead to a global pandemic with a rising toll in infections and deaths. Better understanding of its pathogenesis will greatly improve the outcomes and treatment of affected patients. Here we compared the inflammatory and cardiovascular disease-related protein cargo of circulating large and small extracellular vesicles (EVs) from 84 hospitalized patients infected with SARS-CoV-2 with different stages of disease severity. Our findings reveal significant enrichment of proinflammatory, procoagulation, immunoregulatory and tissueremodelling protein signatures in EVs, which remarkably distinguished symptomatic COVID-19 patients from uninfected controls with matched comorbidities and delineated those with moderate disease from those who were critically ill. Specifically, EN-RAGE, followed by TF and IL-18R1, showed the strongest correlation with disease severity and length of hospitalization. Importantly, EVs from COVID-19 patients induced apoptosis of pulmonary microvascular endothelial cells in the order of disease severity. In conclusion, our findings support a role for EVs in the pathogenesis of COVID-19 disease and underpin the development of EV-based approaches to predicting disease severity, determining need for patient hospitalization and identifying new therapeutic targets. K E Y WO R D S endothelial injury, inflammation, SARS-CoV-2, thrombosis  INTRODUCTIONThe coronavirus disease-2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (Dashboard WCDC-2020; Wu et al., 2020) is marked by endothelial dysfunction and dysregulated immune responses (Huertas et al., 2020). Like the SARS-CoV pathogen that led to epidemics in 2002 and 2003, SARS-CoV-2 enters cells via binding of its spike protein to angiotensin converting enzyme 2 (ACE2) receptors. ACE2 receptors are abundantly present in pulmonary alveolar type II and endothelial cells, thereby making the lungs and pulmonary vasculature susceptible to SARS-CoV-2-induced inflammation and injury (Zhao et al., 2020). Further, alveolar capillary micro-thrombi and endothelial damage with evidence of intracellular virus have been noted on post-mortem analysis of infected lungs (Ackermann et al., 2020). While there is growing evidence that endothelial injury, vascular remodelling and coagulopathy are key consequences of COVID-19 infection, it remains unclear how the virus induces these changes. Extracellular vesicles (EVs) carry proteins, coding and non-coding RNA, DNA fragments and lipids, which facilitate cross-talk between cells. The transfer of EV cargo plays aThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Platelet extracellular vesicles in COVID-19: Potential markers and makers

“…When stratifying COVID‐19 patients with severe disease by mortality, no differences in EV‐TF‐activity was noted, as the level of EV‐TF‐activity was approximately 50 pM in both survivors and nonsurvivors. 131 These data appear to contrast with Guervilly et al., 36 who reported far lower levels of EV‐associated TF‐activity in COVID‐19. While the discrepancy might be explained by an overestimation of TF activity measured with commercial assays in absence of blocking antibodies to ensure of the specificity of the measurements, 132 , 133 , 134 these studies suggest that high EV‐TF activity may be associated with increased severity and mortality in COVID‐19.…”

“…Given the high prevalence of thrombosis in COVID‐19, 30 , 31 , 32 TF activity associated with EVs and its involvement in COVID‐19 pathology is of high interest. 36 , 70 , 118 , 130 Several studies 36 , 70 , 118 , 131 report a significant increase in TF activity associated with EVs from COVID‐19 patients when compared with healthy controls. Moreover, EV‐TF activity was significantly higher in COVID‐19 than in sepsis.…”

“… 36 Krishnamachary et al. 131 reported enrichment of TF‐protein and TF‐activity associated with EVs isolated from COVID‐19 patients with severe disease. When stratifying COVID‐19 patients with severe disease by mortality, no differences in EV‐TF‐activity was noted, as the level of EV‐TF‐activity was approximately 50 pM in both survivors and nonsurvivors.…”

Extracellular Vesicles and Thrombosis: Update on the Clinical and Experimental Evidence

“…In addition to observations of elevated circulating EV numbers and their associations with disease severity, first studies reported a specific surface antigen signature [220] or protein cargo [222] in circulating EVs isolated from patients diagnosed with COVID-19. It also was speculated that changes in EV cargo in subgroups of COVID-19 patients, such as those with diabetes mellitus, obesity or hypertension, may contribute to the increased morbidity and mortality observed in these high-risk populations [223].…”

“…It also was speculated that changes in EV cargo in subgroups of COVID-19 patients, such as those with diabetes mellitus, obesity or hypertension, may contribute to the increased morbidity and mortality observed in these high-risk populations [223]. Importantly, addition of plasma from severely-ill patients with COVID-19 to endothelial cells ex vivo was found to transfer a prothrombotic phenotype and to induce apoptotic cell death [222] suggesting the presence of a vicious cycle between endothelial injury and EV release in COVID-19 associated thrombosis.…”