key: cord-1034501-ug4s7ob7 authors: Violi, Francesco; Oliva, Alessandra; Cangemi, Roberto; Ceccarelli, Giancarlo; Pignatelli, Pasquale; Carnevale, Roberto; Cammisotto, Vittoria; Lichtner, Miriam; Alessandri, Francesco; De Angelis, Massimiliano; Miele, Maria Claudia; D’Ettorre, Gabriella; Ruberto, Franco; Venditti, Mario; Pugliese, Francesco; Mastroianni, Claudio Maria title: Nox2 activation in Covid-19 date: 2020-07-25 journal: Redox Biol DOI: 10.1016/j.redox.2020.101655 sha: 3a91910ecf2daf7ccbf1b16c2d5888a8555c3f2c doc_id: 1034501 cord_uid: ug4s7ob7 Nox2 is responsible for artery dysfunction via production of reactive oxidant species. RNA viruses may activate Nox2, but it is unknown if this occurs in coronavirus 2019(Covid-19). Nox2 activation by soluble Nox2-derived peptide(sNox2-dp) was measured in patients hospitalized for Covid-19 (n=182) and controls (n=91). sNox2-dp values were higher in Covid-19 patients versus controls and in severe versus non severe Covid-19. Patients with thrombotic events(n=35,19%) had higher sNox2-dp than thrombotic event-free ones. A logistic regression analysis showed that sNox2 and coronary heart disease predicted thrombotic events. Oxidative stress by Nox2 activation is associated severe disease and thrombotic events in Covid-19 patients. There is a growing body of evidence that Coronavirus 2019 poor outcome is related not only to pulmonary pneumonia but also to ischemic episodes occurring in the artery and venous circulation. 1 Among the putative mechanisms accounting for vascular disease, Nox2, one the most important cellular producers of reactive oxidant species (ROS), could play a role. Nox2 encompass, indeed, pro-inflammatory and vasoconstriction properties which may favor vascular occlusion 2, 3 . These properties have been deduced by experiments in patients with chronic granulomatous disease, who are characterized by hereditary deficiency of Nox2 and display enhanced artery dilatation and reduced atherosclerotic burden 4 . Experimental studies in Nox2 knock-out models showed impaired thrombus formation in small but not in large arteries 5, 6 . As a previous study from our group reported that Nox2 is overactivated in patients with community-acquired pneumonia 7 , we speculated that a similar behavior could be detected in patients hospitalized for Covid-19, which are essentially complicated by several pneumonia read-outs 8 . Thus, the aim of the present study was to assess the behavior of Nox2 in Covid-19 patients with different degrees of disease severity and its relationship with thrombosis complications. Covid-19 cohort was prospectively recruited and followed-up by 3 divisions at University hospital located in Rome and Latina, (Italy). We included in the study adult (≥18 years) patients with laboratory-confirmed Covid-19 and severe acute respiratory syndrome coronavirus-2(SARS-CoV2)-related pneumonia, consecutively hospitalized in March 2020. Covid-19 was diagnosed on the basis of the WHO interim guidance. 9 Severe disease was defined as the need of intensive care unit (ICU) admission. Patients matched for demographic and clinical characteristics but without acute infections were used as controls. Nox2 activation was measured as soluble Nox2-derived peptide (sNox2-dp) with an ELISA method as previously reported. 10 The clinical course of the disease and its evolution were monitored during hospitalization. The appearance of new ischemic/embolic events was diagnosed as follows: 1) pulmonary thrombo-embolism by lung CT scan; 11 2) myocardial infarction by EKG changes associated with enhanced markers of cell necrosis ; 12 3) acute brain ischemia by onset of new focal neurological signs and symptoms and confirmed, whenever possible, by NMR or CT imaging; 13 4) acute limb ischemia diagnosed according to AHA guidelines 14 . Clinical characteristics of Covid-19 patients and controls are reported in the table 1. No significant differences were present for age, sex, body mass index, smoking habit and for the prevalence of arterial hypertension, diabetes, peripheral artery disease (PAD) and atrial fibrillation between patients and controls; coronary heart disease (CAD), heart failure and chronic obstructive pulmonary disease(COPD) were more prevalent among Covid-19 patients. Serum sNox2-dp was higher in Covid-19 patients than in control subjects (figure Patients experiencing thrombotic events were older, had a higher prevalence of CAD, PAD, and were more frequently admitted to ICU than thrombotic event -free ones (43 vs 12%). Patients with thrombotic events showed higher levels of sNox2-dp (Table 2; figure 1C ) and D-dimer(Tab. 2) than thrombosis-free ones; no correlation was found between sNOX2-dp and D-dimer levels (Spearman's rho=0.083; p=0.336). A logistic regression analysis showed that sNox2-dp (OR: 1.028; 95% CI: 1.001-1.055; p=0.041) and CAD (OR: 3.481; 95%CI: 1.524-7.048; p=0.003) predicted thrombotic events, after adjusting for age and PAD. The study provides evidence that, compared to controls, Covid-19 patients displayed overactivation of Nox2, which was more marked in patients admitted to ICU. Covid-19 patients with thrombotic complications had higher Nox2 activation compared to event-free patients suggesting a role for Nox2 as mechanism favoring thrombotic-related ischemic events. and may enhance angiotensin II levels and eventually Nox2 activation. 15 Covid-19 patients with thrombotic complications had higher Nox2 activation compared to event-free ones, independently upon confounders such as coexistent atherosclerotic burden including coronary artery disease and peripheral artery disease, that are associated to Nox2 activation 19, 20 and age. We must acknowledge, however, the overlap of Nox2 activation in the Covid-19 population with and without vascular disease, which may entail that Nox2 activation is not the only factor concurring to ischemic complications. We have recently demonstrated, for instance, that serum albumin, which an important blood antioxidant, is reduced in Covid-19 and associated with a higher rate of thrombotic complications; 1 this would suggest that more than one component of redox The study has implications and limitations. Analysis of Nox2 in serum reflects essentially the enzyme activation by leucocytes and platelets, 10 thereby inference regarding Nox2 activation at level of vascular cells , where Nox2 is also expressed, 22 cannot be drawn. We did not investigate if angiotensin II, Toll-like 7 or both are trigger of Nox2 activation. Our data provide a rationale to assess if downregulation of Nox2 improves Covid-19 morbidity and mortality. In conclusion, we report for the first time that Covid-19 is associated with Nox2-derived oxidative stress so providing a novel insight in the pathophysiology of Covid-19 and a clue to explore future therapeutic approach to fight SARS-CoV-2. Legend: ACE: angiotensin converting enzyme, ARBs: angiotensin receptor blockers, BMI: body mass index, CAD: coronary heart disease, COPD: chronic obstructive pulmonary disease, hs-CRP: high sensitivity C reactive protein, P/F: PaO 2 /Fi0 2 ratio, sNox2-dp: serum Nox2-derived peptide, SpO 2 : oxygen saturation. Differences Coagulopathy and Vascular Disease in Covid-19. 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Fourth Universal Definition of American Heart Association Stroke Council CoC, Stroke Nursing CoCC and Council on Peripheral Vascular D. Guidelines for the prevention of stroke in patients with stroke and transient ischemic attack: a guideline for healthcare professionals from the Acute Limb Ischemia and Outcomes With Vorapaxar in Patients With Peripheral Artery Disease: Results From the Trial to Assess the Effects of Vorapaxar in Preventing Heart Attack and Stroke in Patients With Atherosclerosis-Thrombolysis in Myocardial Infarction 50 (TRA2 degrees P-TIMI 50) Nox2+ Myeloid cells drive vascular inflammation and endothelial dysfunction in heart failure after myocardial infarction via angiotensin II receptor type 1 SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin System: Celebrating the 20th Anniversary of the Discovery of ACE2 Angiotensin Converting Enzyme 2: A Double-Edged Sword Clinical and biochemical indexes from 2019-nCoV infected patients linked to viral loads and lung injury NOX2 up-regulation is associated with artery dysfunction in patients with peripheral artery disease Serum NOX2 and urinary isoprostanes predict vascular events in patients with atrial fibrillation between percentages were assessed by Fisher exact tests. All continuous variables were tested for normality with the Shapiro-Wilk test. Student unpaired t-test was used for normally distributed continuous variables (expressed as mean±SD). Mann-Whitney U test was used for not-normally distributed continuous variables • Nox2 is responsible for artery dysfunction via production of reactive oxidant species and RNA viruses may activate Nox2, but it is unknown if this occurs in coronavirus 2019(Covid-19).• sNox2-dp values were higher in Covid-19 patients versus controls and in severe versus non severe Covid-19.• Patients with thrombotic events had higher sNox2-dp than thrombotic event-free ones. A logistic regression analysis showed that sNox2 and coronary heart disease predicted thrombotic events.• Oxidative stress by Nox2 activation is associated severe disease and thrombotic events in Covid-19 patients. The short communication you find enclosed is submitted to the journal for publication.The authors involved in this study declared that they do not possess any conflict of interest concerning the study and the issue evaluated in it.All the Authors read and approved the last version of the manuscript, that has not been submitted for publication elsewhere.On behalf of the entire group of authors Rome