Rewiring Endothelial Sphingolipid Metabolism to Favor S1P Over Ceramide Protects From Coronary Atherosclerosis.

BACKGROUND: Growing evidence correlated changes in bioactive sphingolipids, particularly S1P (sphingosine-1-phosphate) and ceramides, with coronary artery diseases. Furthermore, specific plasma ceramide species can predict major cardiovascular events. Dysfunction of the endothelium lining lesion-prone areas plays a pivotal role in atherosclerosis. Yet, how sphingolipid metabolism and signaling change and contribute to endothelial dysfunction and atherosclerosis remain poorly understood. METHODS: We used an established model of coronary atherosclerosis in mice, combined with sphingolipidomics, RNA-sequencing, flow cytometry, and immunostaining to investigate the contribution of sphingolipid metabolism and signaling to endothelial cell (EC) activation and dysfunction. RESULTS: We demonstrated that hemodynamic stress induced an early metabolic rewiring towards endothelial sphingolipid de novo biosynthesis, favoring S1P signaling over ceramides as a protective response. This finding is a paradigm shift from the current belief that ceramide accrual contributes to endothelial dysfunction. The enzyme SPT (serine palmitoyltransferase) commences de novo biosynthesis of sphingolipids and is inhibited by NOGO-B (reticulon-4B), an ER membrane protein. Here, we showed that NOGO-B is upregulated by hemodynamic stress in myocardial EC of ApoE-/- mice and is expressed in the endothelium lining coronary lesions in mice and humans. We demonstrated that mice lacking NOGO-B specifically in EC (Nogo-A/BECKOApoE-/-) were resistant to coronary atherosclerosis development and progression, and mortality. Fibrous cap thickness was significantly increased in Nogo-A/BECKOApoE-/- mice and correlated with reduced necrotic core and macrophage infiltration. Mechanistically, the deletion of NOGO-B in EC sustained the rewiring of sphingolipid metabolism towards S1P, imparting an atheroprotective endothelial transcriptional signature. CONCLUSIONS: These data demonstrated that hemodynamic stress induced a protective rewiring of sphingolipid metabolism, favoring S1P over ceramide. NOGO-B deletion sustained the rewiring of sphingolipid metabolism toward S1P protecting EC from activation under hemodynamic stress and refraining coronary atherosclerosis. These findings also set forth the foundation for sphingolipid-based therapeutics to limit atheroprogression.

A signature of enhanced proliferation associated with response and survival to anti-PD-L1 therapy in early-stage non-small cell lung cancer.

In early-stage non-small cell lung cancer, the combination of neoadjuvant anti-PD-L1 and subablative stereotactic body radiation therapy (SBRT) is associated with higher rates of major pathologic response compared to anti-PD-L1 alone. Here, we identify a 140-gene set, enriched in genes characteristic of highly proliferating cells, associated with response to the dual therapy. Analysis of on-treatment transcriptome data indicate roles for T and B cells in response. The 140-gene set is associated with disease-free survival when applied to the combined trial arms. This 140-gene set identifies a subclass of tumors in all 7 of The Cancer Genome Atlas tumor types examined. Worse survival is associated with the 140-gene signature in 5 of these tumor types. Collectively, our data support that this 140-gene set, discovered in association with response to combined anti-PD-L1 and SBRT, identifies a clinically aggressive subclass of solid tumors that may be more likely to respond to immunotherapies.

Pathogenesis of Pulmonary Long COVID-19.

COVID-19 is characterized by an acute respiratory illness that, in some patients, progresses to respiratory failure, largely demonstrating a pattern of acute respiratory distress syndrome. Excluding fatal cases, the outcome of this severe illness ranges from complete resolution to persistent respiratory dysfunction. This subacute-to-chronic respiratory illness has different manifestations and is collectively termed as "long COVID." The pathogenesis of organ dysfunction in acute injury stems from exaggerated innate immune response, complement activation, and monocyte influx, with a shift toward an organ injury state with abnormalities in cellular maturation. Although the increased rate of thrombosis observed in acute COVID-19 does not appear to persist, interestingly, ongoing symptomatic COVID-19 and post-COVID pathogeneses appear to reflect the persistence of immune and cellular disturbances triggered by the acute and subacute periods.

Neoadjuvant durvalumab plus radiation versus durvalumab alone in stages I-III non-small cell lung cancer: survival outcomes and molecular correlates of a randomized phase II trial.

We previously reported the results of a randomized phase II trial (NCT02904954) in patients with early-stage non-small cell lung cancer (NSCLC) who were treated with either two preoperative cycles of the anti-PD-L1 antibody durvalumab alone or combined with immunomodulatory doses of stereotactic radiation (DRT). The trial met its primary endpoint of major pathological response, which was significantly higher following DRT with no new safety signals. Here, we report on the prespecified secondary endpoint of disease-free survival (DFS) regardless of treatment assignment and the prespecified exploratory analysis of DFS in each arm of the trial. DFS at 2 and 3 years across patients in both arms of the trial were 73% (95% CI: 62.1-84.5) and 65% (95% CI: 52.5-76.9) respectively. For the exploratory endpoint of DFS in each arm of the trial, three-year DFS was 63% (95% CI: 46.0-80.4) in the durvalumab monotherapy arm compared to 67% (95% CI: 49.6-83.4) in the dual therapy arm. In addition, we report post hoc exploratory analysis of progression-free survival as well as molecular correlates of response and recurrence through high-plex immunophenotyping of sequentially collected peripheral blood and gene expression profiles from resected tumors in both treatment arms. Together, our results contribute to the evolving landscape of neoadjuvant treatment regimens for NSCLC and identify easily measurable potential biomarkers of response and recurrence.

International Association for the Study of Lung Cancer Study of Reproducibility in Assessment of Pathologic Response in Resected Lung Cancers After Neoadjuvant Therapy.

INTRODUCTION: Pathologic response has been proposed as an early clinical trial end point of survival after neoadjuvant treatment in clinical trials of NSCLC. The International Association for the Study of Lung Cancer (IASLC) published recommendations for pathologic evaluation of resected lung cancers after neoadjuvant therapy. The aim of this study was to assess pathologic response interobserver reproducibility using IASLC criteria. METHODS: An international panel of 11 pulmonary pathologists reviewed hematoxylin and eosin-stained slides from the lung tumors of resected NSCLC from 84 patients who received neoadjuvant immune checkpoint inhibitors in six clinical trials. Pathologic response was assessed for percent viable tumor, necrosis, and stroma. For each slide, tumor bed area was measured microscopically, and pre-embedded formulas calculated unweighted and weighted major pathologic response (MPR) averages to reflect variable tumor bed proportion. RESULTS: Unanimous agreement among pathologists for MPR was observed in 68 patients (81%), and inter-rater agreement (IRA) was 0.84 (95% confidence interval [CI]: 0.76-0.92) and 0.86 (95% CI: 0.79-0.93) for unweighted and weighted averages, respectively. Overall, unweighted and weighted methods did not reveal significant differences in the classification of MPR. The highest concordance by both methods was observed for cases with more than 95% viable tumor (IRA = 0.98, 95% CI: 0.96-1) and 0% viable tumor (IRA = 0.94, 95% CI: 0.89-0.98). The most common reasons for discrepancies included interpretations of tumor bed, presence of prominent stromal inflammation, distinction between reactive and neoplastic pneumocytes, and assessment of invasive mucinous adenocarcinoma. CONCLUSIONS: Our study revealed excellent reliability in cases with no residual viable tumor and good reliability for MPR with the IASLC recommended less than or equal to 10% cutoff for viable tumor after neoadjuvant therapy.

Epigenetic memory of coronavirus infection in innate immune cells and their progenitors.

Inflammation can trigger lasting phenotypes in immune and non-immune cells. Whether and how human infections and associated inflammation can form innate immune memory in hematopoietic stem and progenitor cells (HSPC) has remained unclear. We found that circulating HSPC, enriched from peripheral blood, captured the diversity of bone marrow HSPC, enabling investigation of their epigenomic reprogramming following coronavirus disease 2019 (COVID-19). Alterations in innate immune phenotypes and epigenetic programs of HSPC persisted for months to 1 year following severe COVID-19 and were associated with distinct transcription factor (TF) activities, altered regulation of inflammatory programs, and durable increases in myelopoiesis. HSPC epigenomic alterations were conveyed, through differentiation, to progeny innate immune cells. Early activity of IL-6 contributed to these persistent phenotypes in human COVID-19 and a mouse coronavirus infection model. Epigenetic reprogramming of HSPC may underlie altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors.

Malignant and Benign Tracheobronchial Neoplasms: Comprehensive Review with Radiologic, Bronchoscopic, and Pathologic Correlation.

Tracheobronchial neoplasms are much less common than lung parenchymal neoplasms but can be associated with significant morbidity and mortality. They include a broad differential of both malignant and benign entities, extending far beyond more commonly known pathologic conditions such as squamous cell carcinoma and carcinoid tumor. Airway lesions may be incidental findings at imaging or manifest with symptoms related to airway narrowing or mucosal irritation, invasion of adjacent structures, or distant metastatic disease. While there is considerable overlap in clinical manifestation, imaging features, and bronchoscopic appearances, an awareness of potential distinguishing factors may help narrow the differential diagnosis. The authors review the epidemiology, imaging characteristics, typical anatomic distributions, bronchoscopic appearances, and histopathologic findings of a wide range of neoplastic entities involving the tracheobronchial tree. Malignant neoplasms discussed include squamous cell carcinoma, malignant salivary gland tumors (adenoid cystic carcinoma and mucoepidermoid carcinoma), carcinoid tumor, sarcomas, primary tracheobronchial lymphoma, and inflammatory myofibroblastic tumor. Benign neoplasms discussed include hamartoma, chondroma, lipoma, papilloma, amyloidoma, leiomyoma, neurogenic lesions, and benign salivary gland tumors (pleomorphic adenoma and mucous gland adenoma). Familiarity with the range of potential entities and any distinguishing features should prove valuable to thoracic radiologists, pulmonologists, and cardiothoracic surgeons when encountering the myriad of tracheobronchial neoplasms in clinical practice. Attention is paid to any features that may help render a more specific diagnosis before pathologic confirmation. ©RSNA, 2023 Quiz questions for this article are available in the supplemental material.

Heterogeneous Expression of PD-L1, B7x, B7-H3, and HHLA2 in Pulmonary Sarcomatoid Carcinoma and the Related Regulatory Signaling Pathways.

Immunotherapy has transformed lung cancer management, but PSC remains an aggressive subtype with a poor prognosis. This study investigates the differential expression of PD-L1 and alternative immune checkpoints (ICs; B7x, B7-H3, and HHLA2), and genetic alterations in PSCs. Tumor specimens of 41 PSC patients were evaluated. PD-L1, B7x, B7-H3, and HHLA2 were positive in 75.0%, 67.6%, 73.0%, and 91.9% of tumors, respectively. PD-L1 expression was significantly higher in the epithelial compared to the sarcomatoid component (median TPS: 50% vs. 0%, p = 0.010). Expression of PD-L1 in both components was only seen in 32.1% of patients. However, at least one IC was expressed in 92.9% of epithelial and 100% of sarcomatoid components. Furthermore, METex14 was detected in 19.5% of patients and was associated with a higher sarcomatoid percentage. Our preclinical studies revealed that METex14 induced PD-L1 expression via MAPK or PI3K/Akt pathways, and MET inhibitors decreased PD-L1 expression. Our findings demonstrate distinct expressions of ICs in PSC subcomponents. Thus, combination IC inhibition as a therapeutic strategy in PSC warrants further exploration. A high percentage of METex14 in PSC and its role in regulating PD-L1 expression reveal different therapeutic targets in this aggressive NSCLC subtype.

Identification of Safe and Effective Intravenous Dose of AAVrh.10hFXN to Treat the Cardiac Manifestations of Friedreich's Ataxia.

Friedreich's ataxia (FA) is a life-threatening autosomal recessive disorder characterized by neurological and cardiac dysfunction. Arrhythmias and heart failure are the main cause of premature death. From prior studies in murine models of FA, adeno-associated virus encoding the normal human frataxin gene (AAVrh.10hFXN) effectively treated the cardiac manifestations of the disease. However, the therapeutic dose window is limited by high level of human frataxin (hFXN) gene expression associated with toxicity. As a therapeutic goal, since FA heterozygotes have no clinical manifestations of FA, we estimated the level of frataxin (FXN) necessary to convert the heart of a homozygote to that of a heterozygote. In noncardiac cells, FA heterozygotes have 30-80% of normal FXN levels (17.7-47.2 ng/mg, average 32.5 ng/mg) and FA homozygotes 2-30% normal levels (1.2-17.7 ng/mg, average 9.4 ng/mg). Therefore, an AAV vector would need to augment endogenous in an FA homozygote by >8.3 ng/mg. To determine the required dose of AAVrh.10hFXN, we administered 1.8 × 1011, 5.7 × 1011, or 1.8 × 1012 gc/kg of AAVrh.10hFXN intravenously (IV) to muscle creatine kinase (mck)-Cre conditional knockout Fxn mice, a cardiac and skeletal FXN knockout model. The minimally effective dose was 5.7 × 1011 gc/kg, resulting in cardiac hFXN levels of 6.1 ± 4.2 ng/mg and a mild (p < 0.01 compared with phosphate-buffered saline controls) improvement in mortality. A dose of 1.8 × 1012 gc/kg resulted in cardiac hFXN levels of 33.7 ± 6.4 ng/mg, a significant improvement in ejection fraction and fractional shortening (p < 0.05, both comparisons) and a 21.5% improvement in mortality (p < 0.001). To determine if the significantly effective dose of 1.8 × 1012 gc/kg could achieve human FA heterozygote levels in a large animal, this dose was administered IV to nonhuman primates. After 12 weeks, the vector-expressed FXN in the heart was 17.8 ± 4.9 ng/mg, comparable to the target human levels. These data identify both minimally and significantly effective therapeutic doses that are clinically relevant for the treatment of the cardiac manifestations of FA.

The mast cell exosome-fibroblast connection: A novel pro-fibrotic pathway.

Introduction: In addition to the traditional activation of resident receptors by release of local mediators, new evidence favors the existence of exosomes in cell-to-cell communication that mediates delivery of specific cargo to modulate recipient cell function. We report that mast cell exosomes are an additional source of pro-fibrotic substances and constitute a unique pathway for the generation of excess collagen. Methods: We use primary human lung fibroblasts (HLFs) to demonstrate the uptake of labeled exosomes isolated from the human mast cell line HMC-1 (MC-EXOs), previously shown to contain protein cargo in common with human mast cell exosomes. Results: The MC-EXO uptake by HLF is to the cytosol and increases both proline hydroxylation in HLF lysate and secreted collagen, within 24 h, which is sustained over 72 h, the same time required for transforming growth factor-ß (TGF-ß) to activate collagen synthesis in the HLFs. Unlike TGF-ß, MC-EXO uptake does not induce fibrillar gene activation or invoke the Smad-nuclear transcription pathway. We show that MC-EXO uptake and TGF-ß have an additive effect on collagen synthesis in HLF and postulate that MC-EXO uptake by HLFs is a contributing factor to excess collagen synthesis and represents a unique paradigm for understanding fibrosis. Discussion: It is known that, in the lungs, mast cells are more activated and increase in number with inflammation, injury and viral infection associated with fibrosis. With the reported increased incidence of post-COVID-pulmonary fibrosis (PCPF), data from patients with severe COVID-19 are presented that show an increase in the mast cell number in lung parenchyma, the site of PCPF. Our findings provide a rationale for targeting multiple fibrogenic pathways in the management of lung fibrosis and the use of mast cell exosomes as a biomarker for the prognostic and diagnostic management of evolving fibrotic lung disease.

Defining Morphologic Features of Invasion in Pulmonary Nonmucinous Adenocarcinoma With Lepidic Growth: A Proposal by the International Association for the Study of Lung Cancer Pathology Committee.

INTRODUCTION: Since the eight edition of the Union for International Cancer Control and American Joint Committee on Cancer TNM classification system, the primary tumor pT stage is determined on the basis of presence and size of the invasive components. The aim of this study was to identify histologic features in tumors with lepidic growth pattern which may be used to establish criteria for distinguishing invasive from noninvasive areas. METHODS: A Delphi approach was used with two rounds of blinded anonymized analysis of resected nonmucinous lung adenocarcinoma cases with presumed invasive and noninvasive components, followed by one round of reviewer de-anonymized and unblinded review of cases with known outcomes. A digital pathology platform was used for measuring total tumor size and invasive tumor size. RESULTS: The mean coefficient of variation for measuring total tumor size and tumor invasive size was 6.9% (range: 1.7%-22.3%) and 54% (range: 14.7%-155%), respectively, with substantial variations in interpretation of the size and location of invasion among pathologists. Following the presentation of the results and further discussion among members at large of the International Association for the Study of Lung Cancer Pathology Committee, extensive epithelial proliferation (EEP) in areas of collapsed lepidic growth pattern is recognized as a feature likely to be associated with invasive growth. The EEP is characterized by multilayered luminal epithelial cell growth, usually with high-grade cytologic features in several alveolar spaces. CONCLUSIONS: Collapsed alveoli and transition zones with EEP were identified by the Delphi process as morphologic features that were a source of interobserver variability. Definition criteria for collapse and EEP are proposed to improve reproducibility of invasion measurement.

Persistent alveolar type 2 dysfunction and lung structural derangement in post-acute COVID-19.

SARS-CoV-2 infection can manifest as a wide range of respiratory and systemic symptoms well after the acute phase of infection in over 50% of patients. Key questions remain on the long-term effects of infection on tissue pathology in recovered COVID-19 patients. To address these questions we performed multiplexed imaging of post-mortem lung tissue from 12 individuals who died post-acute COVID-19 (PC) and compare them to lung tissue from patients who died during the acute phase of COVID-19, or patients who died with idiopathic pulmonary fibrosis (IPF), and otherwise healthy lung tissue. We find evidence of viral presence in the lung up to 359 days after the acute phase of disease, including in patients with negative nasopharyngeal swab tests. The lung of PC patients are characterized by the accumulation of senescent alveolar type 2 cells, fibrosis with hypervascularization of peribronchial areas and alveolar septa, as the most pronounced pathophysiological features. At the cellular level, lung disease of PC patients, while distinct, shares pathological features with the chronic pulmonary disease of IPF. which may help rationalize interventions for PC patients. Altogether, this study provides an important foundation for the understanding of the long-term effects of SARS-CoV-2 pulmonary infection at the microanatomical, cellular, and molecular level.

The pathogenesis of coronavirus-19 disease.

Severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) is the causal agent of coronavirus disease-2019 (COVID-19), a systemic illness characterized by variably severe pulmonary symptoms, cardiac conduction abnormalities, diarrhea, and gastrointestinal bleeding, as well as neurologic deficits, renal insufficiency, myalgias, endocrine abnormalities, and other perturbations that reflect widespread microvascular injury and a pro-inflammatory state. The mechanisms underlying the various manifestations of viral infection are incompletely understood but most data suggest that severe COVID-19 results from virus-driven perturbations in the immune system and resultant tissue injury. Aberrant interferon-related responses lead to alterations in cytokine elaboration that deplete resident immune cells while simultaneously recruiting hyperactive macrophages and functionally altered neutrophils, thereby tipping the balance from adaptive immunity to innate immunity. Disproportionate activation of these macrophages and neutrophils further depletes normal activity of B-cells, T-cells, and natural killer (NK) cells. In addition, this pro-inflammatory state stimulates uncontrolled complement activation and development of neutrophil extracellular traps (NETS), both of which promote the coagulation cascade and induce a state of "thrombo-inflammation". These perturbations have similar manifestations in multiple organ systems, which frequently show pathologic findings related to microvascular injury and thrombosis of large and small vessels. However, the pulmonary findings in patients with severe COVID-19 are generally more pronounced than those of other organs. Not only do they feature inflammatory thromboses and endothelial injury, but much of the parenchymal damage stems from failed maturation of alveolar pneumocytes, interactions between type 2 pneumocytes and non-resident macrophages, and a greater degree of NET formation. The purpose of this review is to discuss the pathogenesis underlying organ damage that can occur in patients with SARS-CoV-2 infection. Understanding these mechanisms of injury is important to development of future therapies for patients with COVID-19, many of which will likely target specific components of the immune system, particularly NET induction, pro-inflammatory cytokines, and subpopulations of immune cells.

A Knock-In Mouse Model of Thymoma With the GTF2I L424H Mutation.

INTRODUCTION: The pathogenesis of thymic epithelial tumors remains largely unknown. We previously identified GTF2I L424H as the most frequently recurrent mutation in thymic epithelial tumors. Nevertheless, the precise role of this mutation in tumorigenesis of thymic epithelial cells is unclear. METHODS: To investigate the role of GTF2I L424H mutation in thymic epithelial cells in vivo, we generated and characterized a mouse model in which the Gtf2i L424H mutation was conditionally knocked-in in the Foxn1+ thymic epithelial cells. Digital spatial profiling was performed on thymomas and normal thymic tissues with GeoMx-mouse whole transcriptome atlas. Immunohistochemistry staining was performed using both mouse tissues and human thymic epithelial tumors. RESULTS: We observed that the Gtf2i mutation impairs development of the thymic medulla and maturation of medullary thymic epithelial cells in young mice and causes tumor formation in the thymus of aged mice. Cell cycle-related pathways, such as E2F targets and MYC targets, are enriched in the tumor epithelial cells. Results of gene set variation assay analysis revealed that gene signatures of cortical thymic epithelial cells and thymic epithelial progenitor cells are also enriched in the thymomas of the knock-in mice, which mirrors the human counterparts in The Cancer Genome Atlas database. Immunohistochemistry results revealed similar expression pattern of epithelial cell markers between mouse and human thymomas. CONCLUSIONS: We have developed and characterized a novel thymoma mouse model. This study improves knowledge of the molecular drivers in thymic epithelial cells and provides a tool for further study of the biology of thymic epithelial tumors and for development of novel therapies.

SARS-CoV-2 infection in hamsters and humans results in lasting and unique systemic perturbations after recovery.

The host response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in prolonged pathologies collectively referred to as post-acute sequalae of COVID-19 (PASC) or long COVID. To better understand the mechanism underlying long COVID biology, we compared the short- and long-term systemic responses in the golden hamster after either SARS-CoV-2 or influenza A virus (IAV) infection. Results demonstrated that SARS-CoV-2 exceeded IAV in its capacity to cause permanent injury to the lung and kidney and uniquely affected the olfactory bulb (OB) and olfactory epithelium (OE). Despite a lack of detectable infectious virus, the OB and OE demonstrated myeloid and T cell activation, proinflammatory cytokine production, and an interferon response that correlated with behavioral changes extending a month after viral clearance. These sustained transcriptional changes could also be corroborated from tissue isolated from individuals who recovered from COVID-19. These data highlight a molecular mechanism for persistent COVID-19 symptomology and provide a small animal model to explore future therapeutics.

SARS-CoV-2 infection produces chronic pulmonary epithelial and immune cell dysfunction with fibrosis in mice.

A subset of individuals who recover from coronavirus disease 2019 (COVID-19) develop post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal tissue samples. The mouse-adapted SARS-CoV-2 strain MA10 produces an acute respiratory distress syndrome in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute to clinical recovery phases. At 15 to 120 days after virus clearance, pulmonary histologic findings included subpleural lesions composed of collagen, proliferative fibroblasts, and chronic inflammation, including tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal up-regulation of profibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early antifibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

Prevalence and Mechanisms of Mucus Accumulation in COVID-19 Lung Disease.

Rationale: The incidence and sites of mucus accumulation and molecular regulation of mucin gene expression in coronavirus (COVID-19) lung disease have not been reported. Objectives: To characterize the incidence of mucus accumulation and the mechanisms mediating mucin hypersecretion in COVID-19 lung disease. Methods: Airway mucus and mucins were evaluated in COVID-19 autopsy lungs by Alcian blue and periodic acid-Schiff staining, immunohistochemical staining, RNA in situ hybridization, and spatial transcriptional profiling. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected human bronchial epithelial (HBE) cultures were used to investigate mechanisms of SARS-CoV-2-induced mucin expression and synthesis and test candidate countermeasures. Measurements and Main Results: MUC5B and variably MUC5AC RNA concentrations were increased throughout all airway regions of COVID-19 autopsy lungs, notably in the subacute/chronic disease phase after SARS-CoV-2 clearance. In the distal lung, MUC5B-dominated mucus plugging was observed in 90% of subjects with COVID-19 in both morphologically identified bronchioles and microcysts, and MUC5B accumulated in damaged alveolar spaces. SARS-CoV-2-infected HBE cultures exhibited peak titers 3 days after inoculation, whereas induction of MUC5B/MUC5AC peaked 7-14 days after inoculation. SARS-CoV-2 infection of HBE cultures induced expression of epidermal growth factor receptor (EGFR) ligands and inflammatory cytokines (e.g., IL-1α/ß) associated with mucin gene regulation. Inhibiting EGFR/IL-1R pathways or administration of dexamethasone reduced SARS-CoV-2-induced mucin expression. Conclusions: SARS-CoV-2 infection is associated with a high prevalence of distal airspace mucus accumulation and increased MUC5B expression in COVID-19 autopsy lungs. HBE culture studies identified roles for EGFR and IL-1R signaling in mucin gene regulation after SARS-CoV-2 infection. These data suggest that time-sensitive mucolytic agents, specific pathway inhibitors, or corticosteroid administration may be therapeutic for COVID-19 lung disease.

Angiopoietin 2 Is Associated with Vascular Necroptosis Induction in Coronavirus Disease 2019 Acute Respiratory Distress Syndrome.

Vascular injury is a well-established, disease-modifying factor in acute respiratory distress syndrome (ARDS) pathogenesis. Recently, coronavirus disease 2019 (COVID-19)-induced injury to the vascular compartment has been linked to complement activation, microvascular thrombosis, and dysregulated immune responses. This study sought to assess whether aberrant vascular activation in this prothrombotic context was associated with the induction of necroptotic vascular cell death. To achieve this, proteomic analysis was performed on blood samples from COVID-19 subjects at distinct time points during ARDS pathogenesis (hospitalized at risk, N = 59; ARDS, N = 31; and recovery, N = 12). Assessment of circulating vascular markers in the at-risk cohort revealed a signature of low vascular protein abundance that tracked with low platelet levels and increased mortality. This signature was replicated in the ARDS cohort and correlated with increased plasma angiopoietin 2 levels. COVID-19 ARDS lung autopsy immunostaining confirmed a link between vascular injury (angiopoietin 2) and platelet-rich microthrombi (CD61) and induction of necrotic cell death [phosphorylated mixed lineage kinase domain-like (pMLKL)]. Among recovery subjects, the vascular signature identified patients with poor functional outcomes. Taken together, this vascular injury signature was associated with low platelet levels and increased mortality and can be used to identify ARDS patients most likely to benefit from vascular targeted therapies.