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      Endothelial cell infection and endotheliitis in COVID-19

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          Abstract

          Cardiovascular complications are rapidly emerging as a key threat in coronavirus disease 2019 (COVID-19) in addition to respiratory disease. The mechanisms underlying the disproportionate effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on patients with cardiovascular comorbidities, however, remain incompletely understood.1, 2 SARS-CoV-2 infects the host using the angiotensin converting enzyme 2 (ACE2) receptor, which is expressed in several organs, including the lung, heart, kidney, and intestine. ACE2 receptors are also expressed by endothelial cells. 3 Whether vascular derangements in COVID-19 are due to endothelial cell involvement by the virus is currently unknown. Intriguingly, SARS-CoV-2 can directly infect engineered human blood vessel organoids in vitro. 4 Here we demonstrate endothelial cell involvement across vascular beds of different organs in a series of patients with COVID-19 (further case details are provided in the appendix). Patient 1 was a male renal transplant recipient, aged 71 years, with coronary artery disease and arterial hypertension. The patient's condition deteriorated following COVID-19 diagnosis, and he required mechanical ventilation. Multisystem organ failure occurred, and the patient died on day 8. Post-mortem analysis of the transplanted kidney by electron microscopy revealed viral inclusion structures in endothelial cells (figure A, B ). In histological analyses, we found an accumulation of inflammatory cells associated with endothelium, as well as apoptotic bodies, in the heart, the small bowel (figure C) and lung (figure D). An accumulation of mononuclear cells was found in the lung, and most small lung vessels appeared congested. Figure Pathology of endothelial cell dysfunction in COVID-19 (A, B) Electron microscopy of kidney tissue shows viral inclusion bodies in a peritubular space and viral particles in endothelial cells of the glomerular capillary loops. Aggregates of viral particles (arrow) appear with dense circular surface and lucid centre. The asterisk in panel B marks peritubular space consistent with capillary containing viral particles. The inset in panel B shows the glomerular basement membrane with endothelial cell and a viral particle (arrow; about 150 nm in diameter). (C) Small bowel resection specimen of patient 3, stained with haematoxylin and eosin. Arrows point to dominant mononuclear cell infiltrates within the intima along the lumen of many vessels. The inset of panel C shows an immunohistochemical staining of caspase 3 in small bowel specimens from serial section of tissue described in panel D. Staining patterns were consistent with apoptosis of endothelial cells and mononuclear cells observed in the haematoxylin-eosin-stained sections, indicating that apoptosis is induced in a substantial proportion of these cells. (D) Post-mortem lung specimen stained with haematoxylin and eosin showed thickened lung septa, including a large arterial vessel with mononuclear and neutrophilic infiltration (arrow in upper inset). The lower inset shows an immunohistochemical staining of caspase 3 on the same lung specimen; these staining patterns were consistent with apoptosis of endothelial cells and mononuclear cells observed in the haematoxylin-eosin-stained sections. COVID-19=coronavirus disease 2019. Patient 2 was a woman, aged 58 years, with diabetes, arterial hypertension, and obesity. She developed progressive respiratory failure due to COVID-19 and subsequently developed multi-organ failure and needed renal replacement therapy. On day 16, mesenteric ischaemia prompted removal of necrotic small intestine. Circulatory failure occurred in the setting of right heart failure consequent to an ST-segment elevation myocardial infarction, and cardiac arrest resulted in death. Post-mortem histology revealed lymphocytic endotheliitis in lung, heart, kidney, and liver as well as liver cell necrosis. We found histological evidence of myocardial infarction but no sign of lymphocytic myocarditis. Histology of the small intestine showed endotheliitis (endothelialitis) of the submucosal vessels. Patient 3 was a man, aged 69 years, with hypertension who developed respiratory failure as a result of COVID-19 and required mechanical ventilation. Echocardiography showed reduced left ventricular ejection fraction. Circulatory collapse ensued with mesenteric ischaemia, and small intestine resection was performed, but the patient survived. Histology of the small intestine resection revealed prominent endotheliitis of the submucosal vessels and apoptotic bodies (figure C). We found evidence of direct viral infection of the endothelial cell and diffuse endothelial inflammation. Although the virus uses ACE2 receptor expressed by pneumocytes in the epithelial alveolar lining to infect the host, thereby causing lung injury, the ACE2 receptor is also widely expressed on endothelial cells, which traverse multiple organs. 3 Recruitment of immune cells, either by direct viral infection of the endothelium or immune-mediated, can result in widespread endothelial dysfunction associated with apoptosis (figure D). The vascular endothelium is an active paracrine, endocrine, and autocrine organ that is indispensable for the regulation of vascular tone and the maintenance of vascular homoeostasis. 5 Endothelial dysfunction is a principal determinant of microvascular dysfunction by shifting the vascular equilibrium towards more vasoconstriction with subsequent organ ischaemia, inflammation with associated tissue oedema, and a pro-coagulant state. 6 Our findings show the presence of viral elements within endothelial cells and an accumulation of inflammatory cells, with evidence of endothelial and inflammatory cell death. These findings suggest that SARS-CoV-2 infection facilitates the induction of endotheliitis in several organs as a direct consequence of viral involvement (as noted with presence of viral bodies) and of the host inflammatory response. In addition, induction of apoptosis and pyroptosis might have an important role in endothelial cell injury in patients with COVID-19. COVID-19-endotheliitis could explain the systemic impaired microcirculatory function in different vascular beds and their clinical sequelae in patients with COVID-19. This hypothesis provides a rationale for therapies to stabilise the endothelium while tackling viral replication, particularly with anti-inflammatory anti-cytokine drugs, ACE inhibitors, and statins.7, 8, 9, 10, 11 This strategy could be particularly relevant for vulnerable patients with pre-existing endothelial dysfunction, which is associated with male sex, smoking, hypertension, diabetes, obesity, and established cardiovascular disease, all of which are associated with adverse outcomes in COVID-19.

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          Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

          Summary Background Since December, 2019, Wuhan, China, has experienced an outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Epidemiological and clinical characteristics of patients with COVID-19 have been reported but risk factors for mortality and a detailed clinical course of illness, including viral shedding, have not been well described. Methods In this retrospective, multicentre cohort study, we included all adult inpatients (≥18 years old) with laboratory-confirmed COVID-19 from Jinyintan Hospital and Wuhan Pulmonary Hospital (Wuhan, China) who had been discharged or had died by Jan 31, 2020. Demographic, clinical, treatment, and laboratory data, including serial samples for viral RNA detection, were extracted from electronic medical records and compared between survivors and non-survivors. We used univariable and multivariable logistic regression methods to explore the risk factors associated with in-hospital death. Findings 191 patients (135 from Jinyintan Hospital and 56 from Wuhan Pulmonary Hospital) were included in this study, of whom 137 were discharged and 54 died in hospital. 91 (48%) patients had a comorbidity, with hypertension being the most common (58 [30%] patients), followed by diabetes (36 [19%] patients) and coronary heart disease (15 [8%] patients). Multivariable regression showed increasing odds of in-hospital death associated with older age (odds ratio 1·10, 95% CI 1·03–1·17, per year increase; p=0·0043), higher Sequential Organ Failure Assessment (SOFA) score (5·65, 2·61–12·23; p<0·0001), and d-dimer greater than 1 μg/mL (18·42, 2·64–128·55; p=0·0033) on admission. Median duration of viral shedding was 20·0 days (IQR 17·0–24·0) in survivors, but SARS-CoV-2 was detectable until death in non-survivors. The longest observed duration of viral shedding in survivors was 37 days. Interpretation The potential risk factors of older age, high SOFA score, and d-dimer greater than 1 μg/mL could help clinicians to identify patients with poor prognosis at an early stage. Prolonged viral shedding provides the rationale for a strategy of isolation of infected patients and optimal antiviral interventions in the future. Funding Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences; National Science Grant for Distinguished Young Scholars; National Key Research and Development Program of China; The Beijing Science and Technology Project; and Major Projects of National Science and Technology on New Drug Creation and Development.
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            Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2

            Summary We have previously provided the first genetic evidence that angiotensin converting enzyme 2 (ACE2) is the critical receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), and ACE2 protects the lung from injury, providing a molecular explanation for the severe lung failure and death due to SARS-CoV infections. ACE2 has now also been identified as a key receptor for SARS-CoV-2 infections, and it has been proposed that inhibiting this interaction might be used in treating patients with COVID-19. However, it is not known whether human recombinant soluble ACE2 (hrsACE2) blocks growth of SARS-CoV-2. Here, we show that clinical grade hrsACE2 reduced SARS-CoV-2 recovery from Vero cells by a factor of 1,000–5,000. An equivalent mouse rsACE2 had no effect. We also show that SARS-CoV-2 can directly infect engineered human blood vessel organoids and human kidney organoids, which can be inhibited by hrsACE2. These data demonstrate that hrsACE2 can significantly block early stages of SARS-CoV-2 infections.
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              Trials of anti-tumour necrosis factor therapy for COVID-19 are urgently needed

              With more than 81 000 deaths worldwide from coronavirus disease 2019 (COVID-19) by April 8, 2020, 1 it is incumbent on researchers to accelerate clinical trials of any readily available and potentially acceptably safe therapies that could reduce the rising death toll. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gains access to host cells via angiotensin-converting enzyme 2, which is expressed in the type II surfactant-secreting alveolar cells of the lungs. 2 Severe COVID-19 is associated with a major immune inflammatory response with abundant neutrophils, lymphocytes, macrophages, and immune mediators. Which mediators are most important in driving the immune pathology remains to be elucidated. Deaths from COVID-19 are chiefly due to diffuse alveolar damage with pulmonary oedema, hyaline membrane formation, and interstitial mononuclear inflammatory infiltrate compatible with early-phase adult respiratory distress syndrome (ARDS). 3 Prevention of ARDS and death in patients with COVID-19 is a pressing health emergency. Anti-tumour necrosis factor (TNF) antibodies have been used for more than 20 years in severe cases of autoimmune inflammatory disease such as rheumatoid arthritis, inflammatory bowel disease, or ankylosing spondylitis. There are ten (as reported on Sept 29, 2019) US Food and Drug Administration approved and four off-label indications for anti-TNF therapy, 4 indicating that TNF is a valid target in many inflammatory diseases. TNF is present in blood and disease tissues of patients with COVID-19 5 and TNF is important in nearly all acute inflammatory reactions, acting as an amplifier of inflammation. We propose that anti-TNF therapy should be evaluated in patients with COVID-19 on hospital admission to prevent progression to needing intensive care support. There is evidence of an inflammatory excess in patients with COVID-19. Lung pathology in COVID-19 is characterised by capillary leakage of fluid and recruitment of immune-inflammatory lymphocytes, neutrophils, and macrophages, 6 implying a role for adhesion molecules, chemokines, and cytokines targeting vascular endothelium. Cytokine upregulation is documented in COVID-19. In patients with COVID-19, there is upregulation of pro-inflammatory cytokines in the blood, including interleukin (IL)-1, IL-6, TNF, and interferon γ,7, 8 and patients in intensive care units have increased concentrations of many cytokines. Preliminary data from Salford Royal Hospital and the University of Manchester in the UK document the presence of proliferating excess monocytes expressing TNF by intracellular staining in patients with COVID-19 in intensive care (Hussell T, Grainger J, Menon M, Mann E, University of Manchester, Manchester, UK, personal communication). Available cytokine data on immunology and inflammation in COVID-19 are summarised in the appendix. Initial reports comprising a trial of 21 severe and critical COVID-19 patients in China (ChiCTR2000029765) and a case study from France 9 of clinical benefit with the anti-IL6 receptor antibody 10 tocilizumab in COVID-19 suggest that cytokines are of importance in the “cytokine storm” and further controlled clinical trials are in progress. Although there are many potential drug candidates for reducing inflammation in COVID-19, only a few drugs such as the anti-TNF antibodies infliximab or adalimumab are potentially effective, widely available, and have a well established safety profile. The potential role of anti-TNF therapy thus warrants consideration. Preclinical studies suggest that the response to severe respiratory syncytial virus (RSV) and influenza in mice is ameliorated by anti-TNF therapy, which reduces weight loss, disease duration, and cell and fluid infiltrate. 11 This research suggests a potential rationale for use of anti-TNF therapy in viral pneumonia, especially given the known mechanism of action of TNF and the reversal of TNF-induced immunopathology by TNF blockade in multiple diseases. It is known TNF is produced in most types of inflammation, especially in the acute phase, and is important in the coordination and development of the inflammatory response. However, too much production of TNF for too long becomes immune suppressive. 12 Blockade of TNF alone is clinically effective in many circumstances and diseases, despite the presence of many other pro-inflammatory cytokines and mediators. There is evidence of a “TNF dependent cytokine cascade” in rheumatoid arthritis tissue and upon bacterial challenge in baboons.13, 14 Thus, if TNF is blocked, there is a rapid (ie, <12 h) decrease of IL-6 and IL-1 concentrations in patients with active rheumatoid arthritis 15 and, importantly, a reduction of adhesion molecules and vascular endothelial growth factor, which is also known as vascular permeability factor, denoting its importance in capillary leak.15, 16, 17, 18, 19 Furthermore, a reduction in leucocyte trafficking occurs in inflamed tissues of joints due to reduction in adhesion molecules and chemokines 20 with reduction in cell content and exudate. Finally, after anti-TNF infusion tissue TNF is reduced as it passes into the blood bound to the anti-TNF antibody. Blood concentrations of immunoreactive, but biologically inactive, TNF increase more than ten times after infusion. 15 For these reasons it is possible that a single infusion of anti-TNF antibody might reduce some of the processes that occur during COVID-19 lung inflammation, reducing TNF and other inflammatory mediators, cellularity, and exudate. © 2020 Marco Mantovani/Getty Images 2020 Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. What would be the best time for intervention with anti-TNF therapy in patients with COVID-19? We postulate that the earlier the better after hospital admission might be the answer because patients will already have initiated anti-viral immunity for several days. There is a balance to be struck between stage of intervention and ensuring patients are at sufficient risk of a poor outcome and can be appropriately monitored. We propose that initial assessment of anti-TNF therapy in clinical trials should be in patients with moderate disease admitted to hospital and who require oxygen support but not intensive care. If this treatment approach proved beneficial with a good safety profile, treatment in the community for people identified as being at high risk of progressing to hospital admission might be considered. The range of available formulations and administration routes of anti-TNF products could facilitate this treatment approach. Is there a trade-off between immunity and virus clearance? The use of powerful anti-inflammatory drugs in acute viral diseases has to be approached with caution because of the risk of increasing viral replication or bacterial infections. For lung viral infections, the higher the infectious dose, the greater the tissue damage from viral replication and the ensuing immune response. In animal models that resemble lung viral infection in humans, the immune response to the virus is so great that even a moderate reduction in inflammation is beneficial—eg, mice with severe pneumonia from RSV or influenza benefit from anti-TNF treatment without compromising viral clearance 11 because more of the lung architecture is preserved. However, concerns about safety are important when considering new therapy. Would anti-TNF therapy increase the risk of bacterial or fungal super-infections? After respiratory viral infection, superinfections with other organisms occur at the most severe end of the disease spectrum. Many research groups have elucidated the mechanisms responsible 21 and anecdotal evidence suggests that bacteria might have a role in in COVID-19,5, 22 although this remains to be confirmed. Bacteria gain a foothold faster in a lung that is damaged. Experimental studies suggest that if the duration of inflammation is limited, with its associated collateral lung damage, then bacterial superinfection is reduced. 23 There is concern that anti-TNF therapy might increase the risk of bacterial infection. 24 Yet two randomised studies in critically unwell patients with septic shock25, 26 showed that monoclonal anti-TNF therapy had good safety data with no evidence of increased secondary bacterial infections in the anti-TNF treated group. In an observational trial in rheumatoid arthritis patients with serious infections, the risk of sepsis and death was reduced in patients on TNF inhibitors compared with those on synthetic disease-modifying anti-rheumatic drugs (DMARDS). 27 46 (11%) of 399 patients on TNF inhibitors developed sepsis after serious infection, of whom 20 (43%) died, compared with 74 (17%) of 444 patients on DMARDS who developed sepsis, of whom 54 (74%) died. 27 Paradoxically, another class of TNF inhibitor, a TNF-R2 Ig-Fc fusion protein, etanercept, was associated with moderately increased mortality in a randomised trial of this treatment for sepsis, 28 possibly due to its faster off-rate for TNF potentially resulting in some redistribution and bioavailability of pathogenic TNF rather than its clearance. There has been interest as to whether the safety of anti-TNF therapy in patients with COVID-19 might be gleaned from analysis of the course of COVID-19 in patients with inflammatory bowel disease (IBD) or rheumatoid arthritis who are already on anti-TNF treatment. As of April 6, 2020, on SECURE-IBD, a coronavirus and IBD reporting database with a register of outcomes of IBD patients with COVID-19, there were 116 patients on anti-TNF therapy alone, 99 of whom recovered without hospitalisation and one patient died. By contrast, about half of 71 patients on sulfasalazine/mesalamine recovered without hospital admission and six patients died. Thus IBD patients with COVID-19 on anti-TNF therapy do not fare worse than those treated with other drugs, but there are insufficient data to make conclusions about a better outcome. We believe there is sufficient evidence to support clinical trials of anti-TNF therapy in patients with COVID-19. With an average of 2 days between hospital admission and ARDS, 7 we propose anti-TNF therapy should be initiated as early as is practicable. If there is preliminary evidence of benefit and safety of anti-TNF therapy in hospitalised patients, we suggest consideration should be given to out of hospital treatment for patients with COVID-19 at high risk, such as older people and those with pre-existing conditions, and who can be monitored appropriately.
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                Author and article information

                Contributors
                Journal
                Lancet
                Lancet
                Lancet (London, England)
                Elsevier Ltd.
                0140-6736
                1474-547X
                21 April 2020
                21 April 2020
                Affiliations
                [a ]Department of Pathology and Molecular Pathology, University Hospital Zurich, CH-8091 Zurich, Switzerland
                [b ]Department of Cardiology, University Heart Center, University Hospital Zurich, CH-8091 Zurich, Switzerland
                [c ]Institute for Intensive Care Medicine, University Hospital Zurich, CH-8091 Zurich, Switzerland
                [d ]Division of Infectious Diseases, University Hospital Zurich, CH-8091 Zurich, Switzerland
                [e ]Department of Internal Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA
                Article
                S0140-6736(20)30937-5
                10.1016/S0140-6736(20)30937-5
                7172722
                32325026
                98fad0be-e8bf-4a5b-813f-14cd56d85e49
                © 2020 Elsevier Ltd. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

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