Wnt5a/ß-catenin axis is involved in the downregulation of AT2 lineage by PAI-1.

Failure to regenerate injured alveoli functionally and promptly causes a high incidence of fatality in coronavirus disease 2019 (COVID-19). How elevated plasminogen activator inhibitor-1 (PAI-1) regulates the lineage of alveolar type 2 (AT2) cells for re-alveolarization has not been studied. This study aimed to examine the role of PAI-1-Wnt5a-ß catenin cascades in AT2 fate. Dramatic reduction in AT2 yield was observed in Serpine1Tg mice. Elevated PAI-1 level suppressed organoid number, development efficiency, and total surface area in vitro. Anti-PAI-1 neutralizing antibody restored organoid number, proliferation and differentiation of AT2 cells, and ß-catenin level in organoids. Both Wnt family member 5A (Wnt5a) and Wnt5a-derived N-butyloxycarbonyl hexapeptide (Box5) altered the lineage of AT2 cells. This study demonstrates that elevated PAI-1 regulates AT2 proliferation and differentiation via the Wnt5a/ß catenin cascades. PAI-1 could serve as autocrine signaling for lung injury repair.

COVID-19-associated Lung Microvascular Endotheliopathy: A "From the Bench" Perspective.

Rationale: Autopsy and biomarker studies suggest that endotheliopathy contributes to coronavirus disease (COVID-19)-associated acute respiratory distress syndrome. However, the effects of COVID-19 on the lung endothelium are not well defined. We hypothesized that the lung endotheliopathy of COVID-19 is caused by circulating host factors and direct endothelial infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Objectives: We aimed to determine the effects of SARS-CoV-2 or sera from patients with COVID-19 on the permeability and inflammatory activation of lung microvascular endothelial cells. Methods: Human lung microvascular endothelial cells were treated with live SARS-CoV-2; inactivated viral particles; or sera from patients with COVID-19, patients without COVID-19, and healthy volunteers. Permeability was determined by measuring transendothelial resistance to electrical current flow, where decreased resistance signifies increased permeability. Inflammatory mediators were quantified in culture supernatants. Endothelial biomarkers were quantified in patient sera. Measurements and Main Results: Viral PCR confirmed that SARS-CoV-2 enters and replicates in endothelial cells. Live SARS-CoV-2, but not dead virus or spike protein, induces endothelial permeability and secretion of plasminogen activator inhibitor 1 and vascular endothelial growth factor. There was substantial variability in the effects of SARS-CoV-2 on endothelial cells from different donors. Sera from patients with COVID-19 induced endothelial permeability, which correlated with disease severity. Serum levels of endothelial activation and injury biomarkers were increased in patients with COVID-19 and correlated with severity of illness. Conclusions: SARS-CoV-2 infects and dysregulates endothelial cell functions. Circulating factors in patients with COVID-19 also induce endothelial cell dysfunction. Our data point to roles for both systemic factors acting on lung endothelial cells and viral infection of endothelial cells in COVID-19-associated endotheliopathy.

The Role of Fibrinolytic System in Health and Disease.

The fibrinolytic system is composed of the protease plasmin, its precursor plasminogen and their respective activators, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), counteracted by their inhibitors, plasminogen activator inhibitor type 1 (PAI-1), plasminogen activator inhibitor type 2 (PAI-2), protein C inhibitor (PCI), thrombin activable fibrinolysis inhibitor (TAFI), protease nexin 1 (PN-1) and neuroserpin. The action of plasmin is counteracted by α2-antiplasmin, α2-macroglobulin, TAFI, and other serine protease inhibitors (antithrombin and α2-antitrypsin) and PN-1 (protease nexin 1). These components are essential regulators of many physiologic processes. They are also involved in the pathogenesis of many disorders. Recent advancements in our understanding of these processes enable the opportunity of drug development in treating many of these disorders.

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.

Dysregulation of Pulmonary Responses in Severe COVID-19.

Patients with coronavirus disease 2019 (COVID-19) predominantly have a respiratory tract infection with various symptoms and high mortality is associated with respiratory failure second to severe disease. The risk factors leading to severe disease remain unclear. Here, we reanalyzed a published single-cell RNA-Seq (scRNA-Seq) dataset and found that bronchoalveolar lavage fluid (BALF) of patients with severe disease compared to those with mild disease contained decreased TH17-type cells, decreased IFNA1-expressing cells with lower expression of toll-like receptor 7 (TLR7) and TLR8, increased IgA-expressing B cells, and increased hyperactive epithelial cells (and/or macrophages) expressing matrix metalloproteinases (MMPs), hyaluronan synthase 2 (HAS2), and plasminogen activator inhibitor-1 (PAI-1), which may together contribute to the pulmonary pathology in severe COVID-19. We propose IFN-I (and TLR7/TLR8) and PAI-1 as potential biomarkers to predict the susceptibility to severe COVID-19.

Endothelial cell dysfunction, coagulation, and angiogenesis in coronavirus disease 2019 (COVID-19).

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been led to a pandemic emergency. So far, different pathological pathways for SARS-CoV-2 infection have been introduced in which the excess release of pro-inflammatory cytokines (such as interleukin 1 ß [IL-1ß], IL-6, and tumor necrosis factor α [TNFα]) has earned most of the attentions. However, recent studies have identified new pathways with at least the same level of importance as cytokine storm in which endothelial cell (EC) dysfunction is one of them. In COVID-19, two main pathologic phenomena have been seen as a result of EC dysfunction: hyper-coagulation state and pathologic angiogenesis. The EC dysfunction-induced hypercoagulation state seems to be caused by alteration in the levels of different factors such as plasminogen activator inhibitor 1 (PAI-1), von Willebrand factor (vWF) antigen, soluble thrombomodulin, and tissue factor pathway inhibitor (TFPI). As data have shown, these thromboembolic events are associated with severity of disease severity or even death in COVID-19 patients. Other than thromboembolic events, pathologic angiogenesis is among the recent findings. Furthermore, over-expression/higher levels of different proangiogenic factors such as vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1 α (HIF-1α), IL-6, TNF receptor super family 1A and 12, and angiotensin-converting enzyme 2 (ACE2) have been found in the lung biopsies/sera of both survived and non-survived COVID-19 patients. Also, there are some hypotheses regarding the role of nitric oxide in EC dysfunction and acute respiratory distress syndrome (ARDS) in SARS-CoV-2 infection. It has been demonstrated that different pathways involved in inflammation are generally common with EC dysfunction and angiogenesis. Altogether, considering the common possible upstream pathways in cytokine storm, pathologic angiogenesis, and EC dysfunction, it seems that targeting these molecules (such as nuclear factor κB) could be more effective in the management of patients with COVID-19.

Dissemination of extreme levels of extracellular vesicles: tissue factor activity in patients with severe COVID-19.

Coronavirus disease 2019 (COVID-19) has become one of the biggest public health challenges of this century. Severe forms of the disease are associated with a thrombo-inflammatory state that can turn into thrombosis. Because tissue factor (TF) conveyed by extracellular vesicles (EVs) has been implicated in thrombosis, we quantified the EV-TF activity in a cohort of hospitalized patients with COVID-19 (n = 111) and evaluated its link with inflammation, disease severity, and thrombotic events. Patients with severe disease were compared with those who had moderate disease and with patients who had septic shock not related to COVID-19 (n = 218). The EV-TF activity was notably increased in patients with severe COVID-19 compared with that observed in patients with moderate COVID-19 (median, 231 [25th to 75th percentile, 39-761] vs median, 25 [25th to 75th percentile, 12-59] fM; P < .0001); EV-TF was correlated with leukocytes, D-dimer, and inflammation parameters. High EV-TF values were associated with an increased thrombotic risk in multivariable models. Compared with patients who had septic shock, those with COVID-19 were characterized by a distinct coagulopathy profile with significantly higher EV-TF and EV-fibrinolytic activities that were not counterbalanced by an increase in plasminogen activator inhibitor-1 (PAI-1). Thus, this article is the first to describe the dissemination of extreme levels of EV-TF in patients with severe COVID-19, which supports the international recommendations of systematic preventive anticoagulation in hospitalized patients and potential intensification of anticoagulation in patients with severe disease.

IL-6 trans-signaling induces plasminogen activator inhibitor-1 from vascular endothelial cells in cytokine release syndrome.

Cytokine release syndrome (CRS) is a life-threatening complication induced by systemic inflammatory responses to infections, including bacteria and chimeric antigen receptor T cell therapy. There are currently no immunotherapies with proven clinical efficacy and understanding of the molecular mechanisms of CRS pathogenesis is limited. Here, we found that patients diagnosed with CRS from sepsis, acute respiratory distress syndrome (ARDS), or burns showed common manifestations: strikingly elevated levels of the four proinflammatory cytokines interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), and IL-10 and the coagulation cascade activator plasminogen activator inhibitor-1 (PAI-1). Our in vitro data indicate that endothelial IL-6 trans-signaling formed an inflammation circuit for robust IL-6, IL-8, and MCP-1 production and promoted PAI-1 production; additionally, an IL-6 signaling blockade by the human monoclonal antibody tocilizumab blunted endothelial cell activation. Plasma from severe COVID-19 patients similarly exhibited increased IL-6, IL-10, and MCP-1 levels, but these levels were not as high as those in patients with CRS from other causes. In contrast, the PAI-1 levels in COVID-19 patients were as highly elevated as those in patients with bacterial sepsis or ARDS. Tocilizumab treatment decreased the PAI-1 levels and alleviated critical illness in severe COVID-19 patients. Our findings suggest that distinct levels of cytokine production are associated with CRS induced by bacterial infection and COVID-19, but both CRS types are accompanied by endotheliopathy through IL-6 trans-signaling. Thus, the present study highlights the crucial role of IL-6 signaling in endothelial dysfunction during bacterial infection and COVID-19.