Endothelial barrier (EB) disruption contributes to acute lung injury in COVID-19, and levels of both VEGF-A and Ang-2, which are mediators of EB integrity, have been associated with COVID-19 severity. Here we explored the participation of additional mediators of barrier integrity in this process, as well as the potential of serum from COVID-19 patients to induce EB disruption in cell monolayers. In a cohort from a clinical trial consisting of thirty patients with COVID-19 that required hospital admission due to hypoxia we demonstrate that i) levels of soluble Tie2 were increase, and of soluble VE-cadherin were decreased when compared to healthy individuals; ii) sera from these patients induce barrier disruption in monolayers of endothelial cells; and iii) that the magnitude of this effect is proportional to disease severity and to circulating levels of VEGF-A and Ang-2. Our study confirms and extends previous findings on the pathogenesis of acute lung injury in COVID-19, reinforcing the concept that EB is a relevant component of this disease. Our results pave the way for future studies that can refine our understanding of the pathogenesis of acute lung injury in viral respiratory disorders, and contribute to the identification of new biomarkers and therapeutic targets for these conditions.

Background: the pathogenesis of severe COVID-19 involves the deregulated activation of different compartments of immunothrombosis, which are otherwise important for pathogen eradication and tissue repair. Coagulation activation, angiogenesis and alterations of endothelial barrier (EB) are elements of immunothrombosis that have been shown to be involved in the pathogenesis of COVID-19. Angiopoietins (Ang) 1 and 2 and their receptor Tie2 and VEGF-A are well-known pro-angiogenic mediators that, during inflammation also mediate EB disruption. Recently, it has also been demonstrated that the Ang/Tie2 pathway is involved in coagulation activation. Here we explored whether increased levels of angiogenesis/EB regulators (which have been previously associated with disease severity in COVID-19) are also associated with both EB disruption and coagulation activation in this condition. Methods: the study population consisted of 30 patients with COVID-19 confirmed by RT-PCR and presenting typical CT findings admitted due to hypoxemia. Thirty sex- and age-matched healthy individuals were recruited at the same time, from the same geographic region. Patients were part of a clinical trial (REBEC: U1111-1250-1843) but samples were obtained before any study intervention, within 24 hours from diagnosis confirmation. Circulating levels of angiogenesis/EB regulation mediators and coagulation biomarkers were measured by commercial assays (immunological or functional). Monolayers of endothelial cells from umbilical veins (HUVECs) or lung (HULECs) were used for measurement of EB integrity using an impedance sensor system (ECIS, Electric Cell-substrate Impedance Sensing System). Cells were stimulated with serum from patients or healthy individuals and EB integrity was continuously monitored for 36 hours. Clinical outcomes were obtained from the digital medical records. Results: mean length of hospital stay (LOS) was 12.9 ± 9.8 days. Twelve patients (40%) required intensive care (ICU) and 28/30 patients survived. Mean D-dimer was 3,609 ± 14,440 ng/mL. Circulating levels of Ang1, Ang2, sTie2 and VEGF-A were all significantly increased in patients compared to healthy individuals (Ang1: 463.2 ± 194.6 vs 237.4 ± 104.9 pg/mL, p<0.0001; Ang2: 1,926 (1,275 - 3,134) vs 1,215 (9 - 1,440 pg/mL), p<0.0001; Tie2: 10,753 ± 2,377 vs 8,603 ± 1,851 pg/mL, p<0.0001 and VEGF-A: 94.7 (73.4 - 116.0) vs 45.9 (39.7 - 57.0 pg/mL), p<0.0001.). In contrast, soluble VE-cadherin levels were decreased in patients compared to healthy individuals (1,234 ± 318 vs 1,539 ± 363 ng/mL, P=0.001). Serum from COVID-19 patients induced decreases of EB integrity in monolayers of both HUVECs and HULECs as early as 15 minutes, lasting up to 5 hours after stimulation (figure 1). The magnitude of EB disruption was correlated with clinically relevant outcomes such as time of ICU stay and LOS (figure 1). Interestingly, levels of Ang1, Ang2 and soluble VE-cadherin levels were also significantly correlated with the magnitude of EB disruption, as well as with biomarkers of coagulation activation such as fibrinogen, Von Willebrand Factor antigen levels, PAI-1, P-selectin and urokinase receptor (uPAR). Conclusions: Ang-1/Ang-2 mediated Tie2 signaling has been shown to be important for the fine regulation of barrier integrity and coagulation activation at the endothelial level, which are two critical elements of immunothrombosis. Our results provide evidence supporting that the interplay between these processes can play a role in the mechanisms driving COVID-19 severity, and suggest that targeting the Ang/Tie2 and VEGF-A pathways could be attractive strategies to modulate not only changes of the alveolar-capillary barrier, but also of coagulation activation in COVID-19. Figure 1. In (a), endothelial barrier (EB) integrity of HUVEC monolayers upon stimulation by serum from COVID-19 patients and healthy individuals (n=27-30 per group). The lower the normalized resistance, the higher the magnitude of EB disruption. Significant differences (* to ****) are evident from 15 min to 5 hours (Anova corrected for multiple comparisons). In the lower panels, the correlation of EB disruption with clinically relevant outcomes such as length of hospital stay (b) and days of intensive care (c) are shown. Negative correlations (Spearman test) indicate that the magnitude of EB disruption is associated with worse outcomes. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: Hemolytic diseases such as Sickle Cell Disease (SCD) are characterized by a natural propensity for both arterial and venous thrombosis. Evidence showing that heme can induce tissue factor (TF) expression in endothelial cells and TF-dependent coagulation activation in animal models of SCD suggest that heme can contribute to hypercoagulability in this condition. We recently demonstrated that heme can induce coagulation activation in whole blood of healthy volunteers in a TF-dependent fashion. Methods: Herein, we aimed to evaluate whether this heme-induced coagulation activity was dependent on the expression and/or activation of hematopoietic TF in human mononuclear cells. TF mRNA expression was evaluated by qPCR and TF procoagulant activity was evaluated using a 2-stage assay based on the generation of FXa. Results: Heme was capable of inducing TF expression and activation in a TLR4-dependent pathway. This activity was further amplified after TNF-α-priming. Conclusion: Our results provide additional evidences on the mechanisms by which heme is involved in the pathogenesis of hypercoagulability in hemolytic diseases.

Heme-oxygenase 1 (HO-1) is an enzyme with well-known anti-inflammatory and antioxidant properties, whose levels have been previously associated with disease severity in the context of sterile and infectious diseases. Moreover, the heme/HO-1 pathway has been associated with prothrombotic changes in other diseases. Accordingly, the potential of modulating HO-1 levels for the treatment of COVID-19 was extensively speculated during the COVID-19 pandemic, but very few actual data were generated. The aim of our study was to explore the association of HO-1, heme, and hemopexin (HPX) levels with COVID-19 severity and with markers of inflammation and coagulation activation. The study was conducted in 30 consecutive patients with COVID-19 admitted due to hypoxemia, and 30 healthy volunteers matched by sex, age, and geographic region. HO-1 and HPX levels were measured by enzyme immunoassay (ELISA) and heme levels were measured by a colorimetric method. A comprehensive panel of coagulation and fibrinolysis activation was also used. Patients with COVID-19 presented increased levels of HO-1 when compared to controls (5741 ± 2696 vs 1953 ± 612 pg/mL, respectively, P < 0.0001), as well as a trend toward increased levels of HPX (3.724 ± 0.880 vs 3.254 ± 1.022 mg/mL, respectively; P = 0.06). In addition, HO-1 and HPX levels reduced from admission to day + 4. HO-1 levels were associated with duration of intensive care unit stay and with several markers of coagulation activation. In conclusion, modulation of HO-1 could be associated with the prothrombotic state observed in COVID-19, and HO-1 could also represent a relevant biomarker for COVID-19. New independent studies are warranted to explore and expand these findings.

Hemolytic diseases such as Sickle Cell Disease (SCD) are characterized by a natural propensity for both arterial and venous thrombosis. The ability of heme to induce tissue factor (TF) activation has been shown both in animal models of SCD, and in human endothelial cells and monocytes. Moreover, it was recently demonstrated that heme can induce coagulation activation in the whole blood of healthy volunteers in a TF-dependent fashion. Herein, we aim to further explore the cellular mechanisms by which heme induces TF-coagulation activation, using human mononuclear cells, which have been shown to be relevant to in vivo hemostasis. TF mRNA expression was evaluated by qPCR and TF procoagulant activity was evaluated using a 2-stage assay based on the generation of activated factor X (FXa). Heme was capable of inducing both TF expression and activation in a TLR4-dependent pathway. This activity was further amplified after TNF-α-priming. Our results provide additional details on the mechanisms by which heme is involved in the pathogenesis of hypercoagulability in hemolytic diseases.