Screening for Inhibitors of YAP Nuclear Localization Identifies Aurora Kinase A as a Modulator of Lung Fibrosis.

Idiopathic pulmonary fibrosis is a progressive lung disease with limited therapeutic options that is characterized by pathological fibroblast activation and aberrant lung remodeling with scar formation. YAP (Yes-associated protein) is a transcriptional coactivator that mediates mechanical and biochemical signals controlling fibroblast activation. We previously identified HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors (statins) as YAP inhibitors based on a high-throughput small-molecule screen in primary human lung fibroblasts. Here we report that several Aurora kinase inhibitors were also identified from the top hits of this screen. MK-5108, a highly selective inhibitor for AURKA (Aurora kinase A), induced YAP phosphorylation and cytoplasmic retention and significantly reduced profibrotic gene expression in human lung fibroblasts. The inhibitory effect on YAP nuclear translocation and profibrotic gene expression is specific to inhibition of AURKA, but not Aurora kinase B or C, and is independent of the Hippo pathway kinases LATS1 and LATS2 (Large Tumor Suppressor 1 and 2). Further characterization of the effects of MK-5108 demonstrate that it inhibits YAP nuclear localization indirectly via effects on actin polymerization and TGFß (Transforming Growth Factor ß) signaling. In addition, MK-5108 treatment reduced lung collagen deposition in the bleomycin mouse model of pulmonary fibrosis. Our results reveal a novel role for AURKA in YAP-mediated profibrotic activity in fibroblasts and highlight the potential of small-molecule screens for YAP inhibitors for identification of novel agents with antifibrotic activity.

Endothelial-Specific Loss of Sphingosine-1-Phosphate Receptor 1 Increases Vascular Permeability and Exacerbates Bleomycin-induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease which leads to significant morbidity and mortality from respiratory failure. The two drugs currently approved for clinical use slow the rate of decline in lung function but have not been shown to halt disease progression or reverse established fibrosis. Thus, new therapeutic targets are needed. Endothelial injury and the resultant vascular permeability are critical components in the response to tissue injury and are present in patients with IPF. However, it remains unclear how vascular permeability affects lung repair and fibrosis following injury. Lipid mediators such as sphingosine-1-phosphate (S1P) are known to regulate multiple homeostatic processes in the lung including vascular permeability. We demonstrate that endothelial cell-(EC) specific deletion of the S1P receptor 1 (S1PR1) in mice (EC-S1pr1-/-) results in increased lung vascular permeability at baseline. Following a low-dose intratracheal bleomycin challenge, EC-S1pr1-/- mice had increased and persistent vascular permeability compared with wild-type mice, which was strongly correlated with the amount and localization of resulting pulmonary fibrosis. EC-S1pr1-/- mice also had increased immune cell infiltration and activation of the coagulation cascade within the lung. However, increased circulating S1P ligand in ApoM-overexpressing mice was insufficient to protect against bleomycin-induced pulmonary fibrosis. Overall, these data demonstrate that endothelial cell S1PR1 controls vascular permeability in the lung, is associated with changes in immune cell infiltration and extravascular coagulation, and modulates the fibrotic response to lung injury.

Circulating Biomarkers of Endothelial Dysfunction Associated With Ventilatory Ratio and Mortality in ARDS Resulting From SARS-CoV-2 Infection Treated With Antiinflammatory Therapies.

BACKGROUND: The association of plasma biomarkers and clinical outcomes in ARDS resulting from SARS-CoV-2 infection predate the evidence-based use of immunomodulators. RESEARCH QUESTION: Which plasma biomarkers are associated with clinical outcomes in patients with ARDS resulting from SARS-CoV-2 infection treated routinely with immunomodulators? STUDY DESIGN AND METHODS: We collected plasma from patients with ARDS resulting from SARS-CoV-2 infection within 24 h of admission to the ICU between December 2020 and March 2021 (N = 69). We associated 16 total biomarkers of inflammation (eg, IL-6), coagulation (eg, D-dimer), epithelial injury (eg, surfactant protein D), and endothelial injury (eg, angiopoietin-2) with the primary outcome of in-hospital mortality and secondary outcome of ventilatory ratio (at baseline and day 3). RESULTS: Thirty patients (43.5%) died within 60 days. All patients received corticosteroids and 6% also received tocilizumab. Compared with survivors, nonsurvivors demonstrated a higher baseline modified Sequential Organ Failure Assessment score (median, 8.5 [interquartile range (IQR), 7-9] vs 7 [IQR, 5-8]); P = .004), lower Pao2 to Fio2 ratio (median, 153 [IQR, 118-182] vs 184 [IQR, 142-247]; P = .04), and higher ventilatory ratio (median, 2.0 [IQR, 1.9-2.3] vs 1.5 [IQR, 1.4-1.9]; P < .001). No difference was found in inflammatory, coagulation, or epithelial biomarkers between groups. Nonsurvivors showed higher median neural precursor cell expressed, developmentally down-regulated 9 (NEDD9) levels (median, 8.4 ng/mL [IQR, 7.0-11.2 ng/mL] vs 6.9 ng/mL [IQR, 5.5-8.0 ng/mL]; P = .0025), von Willebrand factor domain A2 levels (8.7 ng/mL [IQR, 7.9-9.7 ng/mL] vs 6.5 ng/mL [IQR, 5.7-8.7 ng/mL]; P = .007), angiopoietin-2 levels (9.0 ng/mL [IQR, 7.9-14.1 ng/mL] vs 7.0 ng/mL [IQR, 5.6-10.6 ng/mL]; P = .01), and syndecan-1 levels (15.9 ng/mL [IQR, 14.5-17.5 ng/mL] vs 12.6 ng/mL [IQR, 10.5-16.1 ng/mL]; P = .01). Only NEDD9 level met the adjusted threshold for significance (P < .003). Plasma NEDD9 level was associated with 60-day mortality (adjusted OR, 9.7; 95% CI, 1.6-60.4; P = .015). Syndecan-1 level correlated with both baseline (ρ = 0.4; P = .001) and day 3 ventilatory ratio (ρ = 0.5; P < .001). INTERPRETATION: Biomarkers of inflammation, coagulation, and epithelial injury were not associated with clinical outcomes in a small cohort of patients with ARDS uniformly treated with immunomodulators. However, endothelial biomarkers, including plasma NEDD9, were associated with 60-day mortality.

Plasma NEDD9 is increased following SARS-CoV-2 infection and associates with indices of pulmonary vascular dysfunction.

Compared to healthy volunteers, participants with post-acute sequelae of SARS-CoV-2 infection (PASC) demonstrated increased plasma levels of the prothrombotic protein NEDD9, which associated inversely with indices of pulmonary vascular function. This suggests persistent pulmonary vascular dysfunction may play a role in the pathobiology of PASC.

Serial laboratory biomarkers are associated with ICU outcomes in patients hospitalized with COVID-19.

BACKGROUND: Clinical utility of routinely measured serial biomarkers in predicting escalation of inpatient care intensity and mortality among hospitalized patients with COVID-19 remains unknown. METHODS: This retrospective cohort study included patients with COVID-19 who admitted to the Massachusetts General Hospital between March and June 2020 and January to March 2021. White blood cell (WBC) count, platelet count, C-reactive protein (CRP), and D-dimer values were measured on days 1, 3, and 7 of admission. Clinical outcomes include 30- and 60-day morality, ICU transfer, and overall survival (OS) over a follow-up period of 90 days. The association between serial biomarkers and outcomes were assessed using multivariable logistic regression and Cox proportional hazards models. MEASUREMENTS AND MAIN RESULTS: Of the 456 patients hospitalized with COVID-19, 199 (43.6%) were ICU, 179 (39.3%) were medical floor, and 78 (17.1%) were initially admitted to the medical floor and then transferred to the ICU. In adjusted analyses, each unit increase in the slope of CRP was associated with a 42% higher odds of ICU transfer after controlling for the initial admission level (OR = 1.42, 95% CI: 1.25-1.65, P < 0.001). Including serial change in CRP levels from initial level on admission achieved the greatest predictive accuracy for ICU transfer (AUC = 0.72, 95% CI: 0.64-0.79). CONCLUSIONS: Serial change in CRP levels from admission is associated with escalations of inpatient care intensity and mortality among hospitalized patients with COVID-19.

A rapid microglial metabolic response controls metabolism and improves memory.

Chronic high-fat feeding triggers widespread metabolic dysfunction including obesity, insulin resistance, and diabetes. While these ultimate pathological states are relatively well understood, we have a limited understanding of how high-fat intake first triggers physiological changes. Here, we identify an acute microglial metabolic response that rapidly translates intake of high-fat diet (HFD) to a surprisingly beneficial effect on spatial and learning memory. Acute high-fat intake increases palmitate levels in cerebrospinal fluid and triggers a wave of microglial metabolic activation characterized by mitochondrial membrane activation, fission and metabolic skewing towards aerobic glycolysis. These effects are generalized, detectable in the hypothalamus, hippocampus, and cortex all within 1-3 days of HFD exposure. In vivo microglial ablation and conditional DRP1 deletion experiments show that the microglial metabolic response is necessary for the acute effects of HFD. 13C-tracing experiments reveal that in addition to processing via ß-oxidation, microglia shunt a substantial fraction of palmitate towards anaplerosis and re-release of bioenergetic carbons into the extracellular milieu in the form of lactate, glutamate, succinate, and intriguingly, the neuro-protective metabolite itaconate. Together, these data identify microglial cells as a critical nutrient regulatory node in the brain, metabolizing away harmful fatty acids and liberating the same carbons instead as alternate bioenergetic and protective substrates. The data identify a surprisingly beneficial effect of short-term HFD on learning and memory.

A human model of asthma exacerbation reveals transcriptional programs and cell circuits specific to allergic asthma.

Asthma is a chronic disease most commonly associated with allergy and type 2 inflammation. However, the mechanisms that link airway inflammation to the structural changes that define asthma are incompletely understood. Using a human model of allergen-induced asthma exacerbation, we compared the lower airway mucosa in allergic asthmatics and allergic non-asthmatic controls using single-cell RNA sequencing. In response to allergen, the asthmatic airway epithelium was highly dynamic and up-regulated genes involved in matrix degradation, mucus metaplasia, and glycolysis while failing to induce injury-repair and antioxidant pathways observed in controls. IL9-expressing pathogenic TH2 cells were specific to asthmatic airways and were only observed after allergen challenge. Additionally, conventional type 2 dendritic cells (DC2 that express CD1C) and CCR2-expressing monocyte-derived cells (MCs) were uniquely enriched in asthmatics after allergen, with up-regulation of genes that sustain type 2 inflammation and promote pathologic airway remodeling. In contrast, allergic controls were enriched for macrophage-like MCs that up-regulated tissue repair programs after allergen challenge, suggesting that these populations may protect against asthmatic airway remodeling. Cellular interaction analyses revealed a TH2-mononuclear phagocyte-basal cell interactome unique to asthmatics. These pathogenic cellular circuits were characterized by type 2 programming of immune and structural cells and additional pathways that may sustain and amplify type 2 signals, including TNF family signaling, altered cellular metabolism, failure to engage antioxidant responses, and loss of growth factor signaling. Our findings therefore suggest that pathogenic effector circuits and the absence of proresolution programs drive structural airway disease in response to type 2 inflammation.

Soluble Suppression of Tumorigenicity-2 Associates With Ventilator Dependence in Coronavirus Disease 2019 Respiratory Failure.

OBJECTIVES: We hypothesize that elevated soluble suppression of tumorigenicity-2 concentrations, a marker of pulmonary epithelial injury, reflect ongoing lung injury in acute hypoxemic respiratory failure due to coronavirus disease 2019 and associate with continued ventilator dependence. DESIGN: We associated serial plasma soluble suppression of tumorigenicity-2 levels and markers of systemic inflammation including d-dimer, C-reactive protein, and erythrocyte sedimentation rate with 30-day mortality and ventilator dependence. SETTING: Adult medical ICUs and general medicine wards at an academic teaching hospital in Boston, MA. PATIENTS: Adult patients with severe acute respiratory syndrome coronavirus 2 infection and acute hypoxemic respiratory failure admitted to the ICU (n = 72) and non-ICU patients managed with supplemental oxygen (n = 77). INTERVENTIONS: Observational study from April 25 to June 25, 2020. MEASUREMENTS AND MAIN RESULTS: ICU patients had a higher baseline body mass index and median soluble suppression of tumorigenicity-2, d-dimer, and C-reactive protein concentrations compared with non-ICU patients. Among ICU patients, elevated baseline modified Sequential Organ Failure Assessment score and log (soluble suppression of tumorigenicity-2) were associated with 30-day mortality, whereas initial Pao2/Fio2 and markers of systemic inflammation were similar between groups. Only log (soluble suppression of tumorigenicity-2) associated with ventilator dependence over time, with the last measured log (soluble suppression of tumorigenicity-2) concentration obtained on ICU day 11.5 (interquartile range [7-17]) higher in patients who required reintubation or tracheostomy placement compared with patients who were successfully extubated (2.10 [1.89-2.26] vs 1.87 ng/mL [1.72-2.13 ng/mL]; p = 0.03). Last measured systemic inflammatory markers, modified Sequential Organ Failure Assessment score, and Pao2/Fio2 were not different between patients who were successfully extubated compared with those with continued ventilator dependence. CONCLUSIONS: Plasma soluble suppression of tumorigenicity-2 is a biomarker readily measured in blood that can provide dynamic information about the degree of a patient's lung injury and real-time assessment of the likelihood of extubation success. Measures of systemic inflammation, illness severity, and oxygenation did not associate with ventilator outcomes.