ZMPSTE24 Regulates SARS-CoV-2 Spike Protein-enhanced Expression of Endothelial PAI-1.

Endothelial dysfunction is implicated in the thrombotic events reported in patients with coronavirus disease (COVID-19), but the underlying molecular mechanisms are unknown. Circulating levels of the coagulation cascade activator PAI-1 are substantially higher in patients with COVID-19 with severe respiratory dysfunction than in patients with bacterial sepsis and acute respiratory distress syndrome. Indeed, the elevation of PAI-1 is recognized as an early marker of endothelial dysfunction. Here, we report that the rSARS-CoV-2-S1 (recombinant severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] viral envelope spike) glycoprotein stimulated robust production of PAI-1 by human pulmonary microvascular endothelial cells (HPMECs). We examined the role of protein degradation in this SARS-CoV-2-S1 induction of PAI-1 and found that the proteasomal degradation inhibitor bortezomib inhibited SARS-CoV-2-S1-mediated changes in PAI-1. Our data further show that bortezomib upregulated KLF2, a shear-stress-regulated transcription factor that suppresses PAI-1 expression. Aging and metabolic disorders are known to increase mortality and morbidity in patients with COVID-19. We therefore examined the role of ZMPSTE24 (zinc metallopeptidase STE24), a metalloprotease with a demonstrated role in host defense against RNA viruses that is decreased in older individuals and in metabolic syndrome, in the induction of PAI-1 in HPMECs by SARS-CoV-2-S1. Indeed, overexpression of ZMPSTE24 blunted enhancement of PAI-1 production in spike protein-exposed HPMECs. In addition, we found that membrane expression of the SARS-CoV-2 entry receptor ACE2 was reduced by ZMPSTE24-mediated cleavage and shedding of the ACE2 ectodomain, leading to accumulation of ACE2 decoy fragments that may bind SARS-CoV-2. These data indicate that decreases in ZMPSTE24 with age and comorbidities may increase vulnerability to vascular endothelial injury by SARS-CoV-2 viruses and that enhanced production of endothelial PAI-1 might play role in prothrombotic events in patients with COVID-19.

Development of an Image-Based HCS-Compatible Method for Endothelial Barrier Function Assessment.

The recent renascence of phenotypic drug discovery (PDD) is catalyzed by its ability to identify first-in-class drugs and deliver results when the exact molecular mechanism is partially obscure. Acute respiratory distress syndrome (ARDS) is a severe, life-threatening condition with a high mortality rate that has increased in frequency due to the COVID-19 pandemic. Despite decades of laboratory and clinical study, no efficient pharmacological therapy for ARDS has been found. An increase in endothelial permeability is the primary event in ARDS onset, causing the development of pulmonary edema that leads to respiratory failure. Currently, the detailed molecular mechanisms regulating endothelial permeability are poorly understood. Therefore, the use of the PDD approach in the search for efficient ARDS treatment can be more productive than classic target-based drug discovery (TDD), but its use requires a new cell-based assay compatible with high-throughput (HTS) and high-content (HCS) screening. Here we report the development of a new plate-based image cytometry method to measure endothelial barrier function. The incorporation of image cytometry in combination with digital image analysis substantially decreases assay variability and increases the signal window. This new method simultaneously allows for rapid measurement of cell monolayer permeability and cytological analysis. The time-course of permeability increase in human pulmonary artery endothelial cells (HPAECs) in response to the thrombin and tumor necrosis factor α treatment correlates with previously published data obtained by transendothelial resistance (TER) measurements. Furthermore, the proposed image cytometry method can be easily adapted for HTS/HCS applications.

Pulmonary Artery Filling Defects in COVID-19 Patients Revealed Using CT Pulmonary Angiography: A Predictable Complication?

PURPOSE: This study is aimed at assessing the prevalence of pulmonary artery filling defects (PAFDs) consistent with pulmonary artery embolism (PAE) in patients with SARS-CoV-2 infection and at investigating possible radiological or clinical predictors. MATERIALS AND METHODS: Computed Tomography Pulmonary Angiographies (CTPAs) from 43 consecutive patients with a confirmed COVID-19 infection were retrospectively reviewed, taking into consideration the revised Geneva score and the D-dimer value for each patient. Filling defects within the pulmonary arteries were recorded along with pleural and parenchymal findings such as ground glass opacities, consolidation, crazy paving, linear consolidation, and pleural effusion. All these variables were compared between patients with and without PAFD. The predictive performance of statistically different parameters was investigated using the receiver operating characteristics (ROC). RESULTS: Pulmonary embolism was diagnosed in 15/43 patients (35%), whereas CTPA and parenchymal changes related to pulmonary COVID-19 disease were evident in 39/43 patients (91%). The revised Geneva score and the mean D-dimer value obtained using two consecutive measurements were significantly higher in patients with PAFD. The ROC analysis demonstrated that a mean D-dimer value is the parameter with the higher predictivity (AUC 0.831) that is a cut-off value > 1800 µg/l which predicts the probability of PAFD with a sensitivity and specificity of 70% and 78%, respectively. CONCLUSIONS: This single centre retrospective report shows a high prevalence of pulmonary artery filling defects revealed using CTPA in COVID-19 patients and demonstrates that the mean value of multiple D-dimer measurements may represent a predicting factor of this complication.

SARS-CoV-2 spike protein-mediated cell signaling in lung vascular cells.

Currently, the world is suffering from the pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses angiotensin-converting enzyme 2 (ACE2) as a receptor to enter the host cells. So far, 60 million people have been infected with SARS-CoV-2, and 1.4 million people have died because of COVID-19 worldwide, causing serious health, economical, and sociological problems. However, the mechanism of the effect of SARS-CoV-2 on human host cells has not been defined. The present study reports that the SARS-CoV-2 spike protein alone without the rest of the viral components is sufficient to elicit cell signaling in lung vascular cells. The treatment of human pulmonary artery smooth muscle cells or human pulmonary artery endothelial cells with recombinant SARS-CoV-2 spike protein S1 subunit (Val16 - Gln690) at 10 ng/ml (0.13 nM) caused an activation of MEK phosphorylation. The activation kinetics was transient with a peak at 10 min. The recombinant protein that contains only the ACE2 receptor-binding domain of the SARS-CoV-2 spike protein S1 subunit (Arg319 - Phe541), on the other hand, did not cause this activation. Consistent with the activation of cell growth signaling in lung vascular cells by the SARS-CoV-2 spike protein, pulmonary vascular walls were found to be thickened in COVID-19 patients. Thus, SARS-CoV-2 spike protein-mediated cell growth signaling may participate in adverse cardiovascular/pulmonary outcomes, and this mechanism may provide new therapeutic targets to combat COVID-19.

Characteristics of Acute Pulmonary Embolism in Patients With COVID-19 Associated Pneumonia From the City of Wuhan.

The aim of this study was to describe clinical, imaging, and laboratory features of acute pulmonary embolism (APE) in patients with COVID-19 associated pneumonia. Patients with COVID-19 associated pneumonia who underwent a computed tomography pulmonary artery (CTPA) scan for suspected APE were retrospectively studied. Laboratory data and CTPA images were collected. Imaging characteristics were analyzed descriptively. Laboratory data were analyzed and compared between patients with and without APE. A series of 25 COVID-19 patients who underwent CTPA between January 2020 and February 2020 were enrolled. The median D-dimer level founded in these 25 patients was 6.06 µg/mL (interquartile range [IQR] 1.90-14.31 µg/mL). Ten (40%) patients with APE had a significantly higher level of D-dimer (median, 11.07 µg/mL; IQR, 7.12-21.66 vs median, 2.44 µg/mL; IQR, 1.68-8.34, respectively, P = .003), compared with the 15 (60%) patients without APE. No significant differences in other laboratory data were found between patients with and without APE. Among the 10 patients with APE, 6 (60%) had a bilateral pulmonary embolism, while 4 had a unilateral embolism. The thrombus-prone sites were the right lower lobe (70%), the left upper lobe (60%), both upper lobe (40%) and the right middle lobe (20%). The thrombus was partially or completely absorbed after anticoagulant therapy in 3 patients who underwent a follow-up CTPA. Patients with COVID-19 associated pneumonia have a risk of developing APE during the disease. When the D-dimer level abnormally increases in patients with COVID-19 pneumonia, CTPA should be performed to detect and assess the severity of APE.