Complement and endothelial cell activation in COVID-19 patients compared to controls with suspected SARS-CoV-2 infection: A prospective cohort study.

Background: Thromboinflammation may influence disease outcome in COVID-19. We aimed to evaluate complement and endothelial cell activation in patients with confirmed COVID-19 compared to controls with clinically suspected but excluded SARS-CoV-2 infection. Methods: In a prospective, observational, single-center study, patients presenting with clinically suspected COVID-19 were recruited in the emergency department. Blood samples on presentation were obtained for analysis of C5a, sC5b-9, E-selectin, Galectin-3, ICAM-1 and VCAM-1. Results: 153 cases and 166 controls (suffering mainly from non-SARS-CoV-2 respiratory viral infections, non-infectious inflammatory conditions and bacterial pneumonia) were included. Hospital admission occurred in 62% and 45% of cases and controls, respectively. C5a and VCAM-1 concentrations were significantly elevated and E-selectin concentrations decreased in COVID-19 out- and inpatients compared to the respective controls. However, relative differences in outpatients vs. inpatients in most biomarkers were comparable between cases and controls. Elevated concentrations of C5a, Galectin-3, ICAM-1 and VCAM-1 on presentation were associated with the composite outcome of ICU- admission or 30-day mortality in COVID-19 and controls, yet more pronounced in COVID-19. C5a and sC5b-9 concentrations were significantly higher in COVID-19 males vs. females, which was not observed in the control group. Conclusions: Our data indicate an activation of the complement cascade and endothelium in COVID-19 beyond a nonspecific inflammatory trigger as observed in controls (i.e., "over"-activation).

Mediators of Obesity Do Not Influence SARS-CoV-2 Infection or Activation of Primary Human Lung Microvascular Endothelial Cells In Vitro.

Clinical observations have shown that obesity is associated with the severe outcome of SARS-CoV-2 infection hallmarked by microvascular dysfunction in the lungs and other organs. Excess visceral fat and high systemic levels of adipose tissue (AT) derived mediators such as leptin and other adipokines have also been linked to endothelial dysfunction. Consequently, we hypothesized that AT-derived mediators may exacerbate microvascular dysfunction during of SARS-CoV-2 infection and tested this in a primary human lung microvascular endothelial (HLMVEC) cell model. Our results indicate that HLMVEC are not susceptible to SARS-CoV-2 infection since no expression of viral proteins and no newly produced virus was detected. In addition, exposure to the virus did not induce endothelial activation as evidenced by a lack of adhesion molecule, E-selectin, VCAM-1, ICAM-1, and inflammatory cytokine IL-6 induction. Incubation of endothelial cells with the pro-inflammatory AT-derived mediator, leptin, prior to virus inoculation, did not alter the expression of endothelial SARS-CoV-2 entry receptors and did not alter their susceptibility to infection. Furthermore, it did not induce inflammatory activation of endothelial cells. To verify if the lack of activated phenotype in the presence of adipokines was not leptin-specific, we exposed endothelial cells to plasma obtained from critically ill obese COVID-19 patients. Plasma exposure did not result in E-selectin, VCAM-1, ICAM-1, or IL-6 induction. Together our results strongly suggest that aberrant inflammatory endothelial responses are not mounted by direct SARS-CoV-2 infection of endothelial cells, even in the presence of leptin and other mediators of obesity. Instead, endothelial activation associated with COVID-19 is likely a result of inflammatory responses initiated by other cells. Further studies are required to investigate the mechanisms regulating endothelial behavior in COVID-19 and the mechanisms driving severe disease in obese individuals.

P- and E- selectin in venous thrombosis and non-venous pathologies.

Venous thromboembolism is a very common and costly health problem worldwide. Anticoagulant treatment for VTE is imperfect: all have the potential for significant bleeding, and none prevent the development of post thrombotic syndrome after deep vein thrombosis or chronic thromboembolic pulmonary hypertension after pulmonary embolism. For these reasons, alternate forms of therapy with improved efficacy and decreased bleeding are needed. Selectins are a family (P-selectin, E-selectin, L-selectin) of glycoproteins that facilitate and augment thrombosis, modulating neutrophil, monocyte, and platelet activity. P- and E-selectin have been investigated as potential biomarkers for thrombosis. Inhibition of P-selectin and E-selectin decrease thrombosis and vein wall fibrosis, with no increase in bleeding. Selectin inhibition is a promising avenue of future study as either a stand-alone treatment for VTE or as an adjunct to standard anticoagulation therapies.

Whole-genome sequencing reveals host factors underlying critical COVID-19.

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2-4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes-including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)-in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease.

Endothelial Cell-Activating Antibodies in COVID-19.

OBJECTIVE: While endothelial dysfunction has been implicated in the widespread thromboinflammatory complications of COVID-19, the upstream mediators of endotheliopathy remain, for the most part, unknown. This study was undertaken to identify circulating factors contributing to endothelial cell activation and dysfunction in COVID-19. METHODS: Human endothelial cells were cultured in the presence of serum or plasma from 244 patients hospitalized with COVID-19 and plasma from 100 patients with non-COVID-19-related sepsis. Cell adhesion molecules (E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 [ICAM-1]) were quantified using in-cell enzyme-linked immunosorbent assay. RESULTS: Serum and plasma from COVID-19 patients increased surface expression of cell adhesion molecules. Furthermore, levels of soluble ICAM-1 and E-selectin were elevated in patient serum and correlated with disease severity. The presence of circulating antiphospholipid antibodies was a strong marker of the ability of COVID-19 serum to activate endothelium. Depletion of total IgG from antiphospholipid antibody-positive serum markedly reduced the up-regulation of cell adhesion molecules. Conversely, supplementation of control serum with patient IgG was sufficient to trigger endothelial activation. CONCLUSION: These data are the first to indicate that some COVID-19 patients have potentially diverse antibodies that drive endotheliopathy, providing important context regarding thromboinflammatory effects of autoantibodies in severe COVID-19.

Evaluation of admission levels of P, E and L selectins as predictors for thrombosis in hospitalized COVID-19 patients.

Thromboembolic complications are the most reported cause of death in coronavirus disease-2019 (COVID-19). Hypercoagulability, platelets activation and endotheliopathy are well-recognized features in COVID-19 patients. The aim of this work was to evaluate circulating soluble selectins P, E and L at the time of hospital admission as predictors for upcoming thrombosis. This retrospective study included 103 hospitalized COVID-19 patients and 50 healthy volunteer controls. COVID-19 patients were categorized into two groups; group 1 who developed thrombosis during hospitalization and group 2 who did not. Soluble selectins were quantitated using ELISA technique. Higher levels of sP-selectin, sE-selectin and sL-selectin were detected in COVID-19 patients compared to controls. Furthermore, significantly higher levels were found in group 1 compared to group 2. Their means were [5.86 ± 1.72 ng/mL vs. 2.51 ± 0.81 ng/mL]; [50 ± 8.57 ng/mL vs. 23.96 ± 6.31 ng/mL] and [4.66 ± 0.83 ng/mL vs. 2.95 ± 0.66 ng/mL] for sP-selectin, sE-selectin and sL-selectin respectively. The elevated selectins correlated with the currently used laboratory biomarkers of disease severity. After adjustment of other factors, sP-selectin, sE-selectin and sL-selectin were independent predictors for thrombosis. At sP-selectin ≥ 3.2 ng/mL, sE-selectin ≥ 32.5 ng/mL and sL-selectin ≥ 3.6 ng/mL thrombosis could be predicted with 97.1%, 97.6% and 96.5% sensitivity. A panel of the three selectins provided 100% clinical sensitivity. Admission levels of circulating soluble selectins P, E and L can predict thrombosis in COVID-19 patients and could be used to identify patients who need prophylactic anticoagulants. E-selectin showed a superior clinical performance, as thrombo-inflammation biomarker, to the most commonly studied P-selectin.

Endothelial injury in COVID-19 and septic patients.

Systemic inflammatory response, as observed in sepsis and severe COVID-19, may lead to endothelial damage. Therefore, we aim to compare the extent of endothelial injury and its relationship to inflammation in both diseases. We included patients diagnosed with sepsis (SEPSIS group, n = 21), mild COVID-19 (MILD group, n = 31), and severe COVID-19 (SEVERE group, n = 24). Clinical and routine laboratory data were obtained, circulating cytokines (INF-γ, TNF-α, and IL-10) and endothelial injury markers (E-Selectin, Tissue Factor (TF) and von Willebrand factor (vWF)) were measured. Compared to the SEPSIS group, patients with severe COVID-19 present similar clinical and laboratory data, except for lower circulating IL-10 and E-Selectin levels. Compared to the MILD group, patients in the SEVERE group showed higher levels of TNF-α, IL-10, and TF. There was no clear relationship between cytokines and endothelial injury markers among the three studied groups; however, in SEVERE COVID-19 patients, there is a positive relationship between INF-γ with TF and a negative relationship between IL-10 and vWF. In conclusion, COVID-19 and septic patients have a similar pattern of cytokines and endothelial dysfunction markers. These findings highlight the importance of endothelium dysfunction in COVID-19 and suggest that endothelium should be better evaluated as a therapeutic target for the disease.

The Role of Endothelial Related Circulating Biomarkers in COVID-19. A Systematic Review and Meta-analysis.

BACKGROUND: Several studies have revealed the link between Coronavirus Disease 2019 (COVID-19) and endothelial dysfunction. To better understand the global pattern of this relationship, we conducted a meta-analysis on endothelial biomarkers related to COVID-19 severity. METHODS: We systematically searched the literature up to March 10, 2021, for studies investigating the association between COVID-19 severity and the following endothelial biomarkers: Intercellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion Molecule 1 (VCAM-1), E-selectin, P-selectin, Von Willebrand Factor Antigen (VWFAg), soluble Thrombomodulin (sTM), Mid-regional pro-adrenomedullin (MR-proADM), and Angiopoietin-2 (Ang-2). Pooled estimates and mean differences (PMD) for each biomarker were reported. RESULTS: A total of 27 studies (n=2213 patients) were included. Critically ill patients presented with higher levels of MR-proADM (PMD: 0.71 nmol/L, 95% CI: 0.22 to 1.20 nmol/L, p=0.02), E-selectin (PMD: 13,32 pg/ml, 95% CI: 4,89 to 21,75 pg/ml, p=0.008), VCAM-1 (PMD: 479 ng/ml, 95% CI: 64 to 896 ng/ml, p=0.03), VWF-Ag (PMD: 110.5 IU/dl, 95% CI: 44.8 to 176.1 IU/dl, p=0.04) and Ang-2 (PMD: 2388 pg/ml, 95% CI: 1121 to 3655 pg/ml, p=0.003), as compared to non-critically ill ones. ICAM-1, P-selectin and thrombomodulin did not differ between the two groups (p>0.05). CONCLUSION: Endothelial biomarkers display significant heterogeneity in COVID-19 patients, with higher MR-proADM, E-selectin, VCAM-1, VWF-Ag, and Ang-2 levels being associated with increased severity. These findings strengthen the evidence on the key role of endothelial dysfunction in disease progress.

Further Findings Concerning Endothelial Damage in COVID-19 Patients.

Systemic vascular damage with micro/macro-thrombosis is a typical feature of severe COVID-19. However, the pathogenesis of this damage and its predictive biomarkers remain poorly defined. For this reason, in this study, serum monocyte chemotactic protein (MCP)-2 and P- and E-selectin levels were analyzed in 204 patients with COVID-19. Serum MCP-2 and P-selectin were significantly higher in hospitalized patients compared with asymptomatic patients. Furthermore, MCP-2 increased with the WHO stage in hospitalized patients. After 1 week of hospitalization, MCP-2 levels were significantly reduced, while P-selectin increased in patients in WHO stage 3 and decreased in patients in WHO stages 5-7. Serum E-selectin was not significantly different between asymptomatic and hospitalized patients. The lower MCP-2 levels after 1 week suggest that endothelial damage triggered by monocytes occurs early in COVID-19 disease progression. MCP-2 may also predict COVID-19 severity. The increase in P-selectin levels, which further increased in mild patients and reduced in severe patients after 1 week of hospitalization, suggests that the inactive form of the protein produced by the cleavage of the active protein from the platelet membrane is present. This may be used to identify a subset of patients that would benefit from targeted therapies. The unchanged levels of E-selectin in these patients suggest that endothelial damage is less relevant.

Markers of endothelial and epithelial pulmonary injury in mechanically ventilated COVID-19 ICU patients.

BACKGROUND: Biomarkers can be used to detect the presence of endothelial and/or alveolar epithelial injuries in case of ARDS. Angiopoietin-2 (Ang-2), soluble intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion protein-1 (VCAM-1), P-selectin and E-selectin are biomarkers of endothelial injury, whereas the receptor for advanced glycation end-products (RAGE) reflects alveolar epithelial injury. The aims of this study were to evaluate whether the plasma concentration of the above-mentioned biomarkers was different 1) in survivors and non-survivors of COVID-19-related ARDS and 2) in COVID-19-related and classical ARDS. METHODS: This prospective study was performed in two COVID-19-dedicated Intensive Care Units (ICU) and one non-COVID-19 ICU at Ferrara University Hospital. A cohort of 31 mechanically ventilated patients with COVID-19 ARDS and a cohort of 11 patients with classical ARDS were enrolled. Ang-2, ICAM-1, VCAM-1, P-selectin, E-selectin and RAGE were determined with a bead-based multiplex immunoassay at three time points: inclusion in the study (T1), after 7 ± 2 days (T2) and 14 ± 2 days (T3). The primary outcome was to evaluate the plasma trend of the biomarker levels in survivors and non-survivors. The secondary outcome was to evaluate the differences in respiratory mechanics variables and gas exchanges between survivors and non-survivors. Furthermore, we compared the plasma levels of the biomarkers at T1 in patients with COVID-19-related ARDS and classical ARDS. RESULTS: In COVID-19-related ARDS, the plasma levels of Ang-2 and ICAM-1 at T1 were statistically higher in non-survivors than survivors, (p = 0.04 and p = 0.03, respectively), whereas those of P-selectin, E-selectin and RAGE did not differ. Ang-2 and ICAM-1 at T1 were predictors of mortality (AUROC 0.650 and 0.717, respectively). At T1, RAGE and P-selectin levels were higher in classical ARDS than in COVID-19-related ARDS. Ang-2, ICAM-1 and E-selectin were lower in classical ARDS than in COVID-19-related ARDS (all p < 0.001). CONCLUSIONS: COVID-19 ARDS is characterized by an early pulmonary endothelial injury, as detected by Ang-2 and ICAM-1. COVID-19 ARDS and classical ARDS exhibited a different expression of biomarkers, suggesting different pathological pathways. Trial registration NCT04343053 , Date of registration: April 13, 2020.

Angiopoietin-2 as a marker of endothelial activation is a good predictor factor for intensive care unit admission of COVID-19 patients.

BACKGROUND: Coronavirus disease-2019 (COVID-19), a respiratory disease has been associated with ischemic complications, coagulation disorders, and an endotheliitis. OBJECTIVES: To explore endothelial damage and activation-related biomarkers in COVID-19 patients with criteria of hospitalization for referral to intensive care unit (ICU) and/or respiratory worsening. METHODS: Analysis of endothelial and angiogenic soluble markers in plasma from patients at admission. RESULTS: Study enrolled 40 consecutive COVID-19 patients admitted to emergency department that fulfilled criteria for hospitalization. Half of them were admitted in conventional wards without any ICU transfer during hospitalization; whereas the 20 others were directly transferred to ICU. Patients transferred in ICU were more likely to have lymphopenia, decreased SpO2 and increased D-dimer, CRP and creatinine levels. In those patients, soluble E-selectin and angiopoietin-2 were significantly increased (p value at 0.009 and 0.003, respectively). Increase in SELE gene expression (gene coding for E-selectin protein) was confirmed in an independent cohort of 32 patients using a whole blood gene expression profile analysis. In plasma, we found a strong association between angiopoetin-2 and CRP, creatinine and D-dimers (with p value at 0.001, 0.001 and 0.003, respectively). ROC curve analysis identified an Angiopoietin-2 cut-off of 5000 pg/mL as the best predictor for ICU outcome (Se = 80.1%, Sp = 70%, PPV = 72.7%, NPV = 77%), further confirmed in multivariate analysis after adjustment for creatinine, CRP or D-dimers. CONCLUSION: Angiopoietin-2 is a relevant predictive factor for ICU direct admission in COVID-19 patients. This result showing an endothelial activation reinforces the hypothesis of a COVID-19-associated microvascular dysfunction.