Hedgehog and Platelet-derived Growth Factor Signaling Intersect during Postnatal Lung Development.

Normal lung development critically depends on HH (Hedgehog) and PDGF (platelet-derived growth factor) signaling, which coordinate mesenchymal differentiation and proliferation. PDGF signaling is required for postnatal alveolar septum formation by myofibroblasts. Recently, we demonstrated a requirement for HH in postnatal lung development involving alveolar myofibroblast differentiation. Given shared features of HH signaling and PDGF signaling and their impact on this key cell type, we sought to clarify their relationship during murine postnatal lung development. Timed experiments revealed that HH inhibition phenocopies the key lung myofibroblast phenotypes of Pdgfa (platelet-derived growth factor subunit A) and Pdgfra (platelet-derived growth factor receptor alpha) knockouts during secondary alveolar septation. Using a dual signaling reporter, Gli1lZ;PdgfraEGFP, we show that HH and PDGF pathway intermediates are concurrently expressed during alveolar septal myofibroblast accumulation, suggesting pathway convergence in the generation of lung myofibroblasts. Consistent with this hypothesis, HH inhibition reduces Pdgfra expression and diminishes the number of Pdgfra-positive and Pdgfra-lineage cells in postnatal lungs. Bulk RNA sequencing data of Pdgfra-expressing cells from Postnatal Day 8 (P8) lungs show that HH inhibition alters the expression not only of well-established HH targets but also of several putative PDGF target genes. This, together with the presence of Gli-binding sites in PDGF target genes, suggests HH input into PDGF signaling. We identified these HH/PDGF targets in several postnatal lung mesenchymal cell populations, including myofibroblasts, using single-cell transcriptomic analysis. Collectively, our data indicate that HH signaling and PDGF signaling intersect to support myofibroblast/fibroblast function during secondary alveolar septum formation. Moreover, they provide a molecular foundation relevant to perinatal lung diseases associated with impaired alveolarization.

A novel Vascular Leak Index identifies sepsis patients with a higher risk for in-hospital death and fluid accumulation.

PURPOSE: Sepsis is a leading cause of morbidity and mortality worldwide and is characterized by vascular leak. Treatment for sepsis, specifically intravenous fluids, may worsen deterioration in the context of vascular leak. We therefore sought to quantify vascular leak in sepsis patients to guide fluid resuscitation. METHODS: We performed a retrospective cohort study of sepsis patients in four ICU databases in North America, Europe, and Asia. We developed an intuitive vascular leak index (VLI) and explored the relationship between VLI and in-hospital death and fluid balance using generalized additive models (GAM). RESULTS: Using a GAM, we found that increased VLI is associated with an increased risk of in-hospital death. Patients with a VLI in the highest quartile (Q4), across the four datasets, had a 1.61-2.31 times increased odds of dying in the hospital compared to patients with a VLI in the lowest quartile (Q1). VLI Q2 and Q3 were also associated with increased odds of dying. The relationship between VLI, treated as a continuous variable, and in-hospital death and fluid balance was statistically significant in the three datasets with large sample sizes. Specifically, we observed that as VLI increased, there was increase in the risk for in-hospital death and 36-84 h fluid balance. CONCLUSIONS: Our VLI identifies groups of patients who may be at higher risk for in-hospital death or for fluid accumulation. This relationship persisted in models developed to control for severity of illness and chronic comorbidities.

COVID-19 and Respiratory System Disorders: Current Knowledge, Future Clinical and Translational Research Questions.

The severe acute respiratory syndrome coronavirus-2 emerged as a serious human pathogen in late 2019, causing the disease coronavirus disease 2019 (COVID-19). The most common clinical presentation of severe COVID-19 is acute respiratory failure consistent with the acute respiratory distress syndrome. Airway, lung parenchymal, pulmonary vascular, and respiratory neuromuscular disorders all feature in COVID-19. This article reviews what is known about the effects of severe acute respiratory syndrome coronavirus-2 infection on different parts of the respiratory system, clues to understanding the underlying biology of respiratory disease, and highlights current and future translation and clinical research questions.

Advances in Targeted Therapy for Progressive Fibrosing Interstitial Lung Disease.

Progressive fibrosing interstitial lung disease (PF-ILD) has been redefined as a new clinical syndrome that shares similar genetics, pathophysiology, and natural history to idiopathic pulmonary fibrosis (IPF). IPF is the most common form of idiopathic interstitial pneumonias, which is progressive in nature and is associated with significant mortality. Therapies targeting an inflammatory and/or immune response have not been consistently effective or well tolerated in patients with IPF. The two antifibrotic drugs approved for IPF treatment, nintedanib and pirfenidone, have been shown to reduce lung function decline in PF-ILD. Novel uses of antifibrotic therapy are emerging due to a paucity of evidence-based treatments for multiple ILD subtypes. In this review, we describe the current body of knowledge on antifibrotic therapy and immunomodulators in PF-ILD, drawing from experience in IPF where appropriate.

The Hedgehog target Gli1 is not required for bleomycin-induced lung fibrosis.

Sonic Hedgehog (SHH) signaling, a developmental pathway promoting lung mesenchymal expansion and differentiation during embryogenesis, has been increasingly recognized as a profibrotic factor in mature lung, where it might contribute to the pathogenesis of lung fibrosis. Pathway inhibition at the level of the downstream Gli transcription factors Gli1 and Gli2 (by GANT61) ameliorates lung fibrosis in the bleomycin model, whereas inhibition proximally at the level of HH ligand (by anti Hh antibody 5E1) or Smo (by GDC-0449) of the canonical pathway does not, implicating Gli1 and/or Gli2 as a key target. The fact that both the Gli1-labelled cell lineage and Gli1 expressing cells expand during fibrosis formation and contribute significantly to the pool of myofibroblasts in the fibrosis scars suggests a fibrogenic role for Gli1. Therefore to further dissect the roles of Gli1 and Gli2 in lung fibrosis we evaluated Gli1 KO and control mice in the bleomycin model. Monitoring of Gli1+/+ (n = 12), Gli1lZ/+ (n = 37) and Gli1lZ/lZ (n = 18) mice did not reveal differences in weight loss or survival. Lung evaluation at the 21-day endpoint did not show differences in lung fibrosis formation (as judged by morphology and trichrome staining), Ashcroft score, lung collagen content, lung weight, BAL protein content or BAL cell differential count. Our data suggest that Gli1 is not required for bleomycin-induced lung fibrosis.

Identification of autoantibodies to ECH1 and HNRNPA2B1 as potential biomarkers in the early detection of lung cancer.

Identification of biomarkers for early detection of lung cancer (LC) is important, in turn leading to more effective treatment and reduction of mortality. Serological proteome analysis (SERPA) was used to identify proteins around 34 kD as ECH1 and HNRNPA2B1, which had been recognized by serum autoantibody from 25 LC patients. In the validation study, including 90 sera from LC patients and 89 sera from normal individuals, autoantibody to ECH1 achieved an area under the curve (AUC) of 0.799 with sensitivity of 62.2% and specificity of 95.5% in discriminating LC from normal individuals, and showed negative correlation with tumor size (rs = -0.256, p = 0.023). Autoantibody to HNRNPA2B1 performed an AUC of 0.874 with sensitivity of 72.2% and specificity of 95.5%, and showed negative correlation with lymph node metastasis (rs = -0.279, p = 0.012). By using longitudinal preclinical samples, autoantibody to ECH1 showed an AUC of 0.763 with sensitivity of 60.0% and specificity of 89.3% in distinguishing early stage LC from matched normal controls, and elevated autoantibody levels could be detected greater than 2 y before LC diagnosis. ECH1 and HNRNPA2B1 are autoantigens that elicit autoimmune responses in LC and their autoantibody can be the potential biomarkers for the early detection of LC.

Sonic Hedgehog Signaling Regulates Myofibroblast Function during Alveolar Septum Formation in Murine Postnatal Lung.

Sonic Hedgehog (Shh) signaling regulates mesenchymal proliferation and differentiation during embryonic lung development. In the adult lung, Shh signaling maintains mesenchymal quiescence and is dysregulated in diseases such as idiopathic pulmonary fibrosis and chronic obstructive pulmonary disease. Our previous data implicated a role for Shh in postnatal lung development. Here, we report a detailed analysis of Shh signaling during murine postnatal lung development. We show that Shh pathway expression and activity during alveolarization (postnatal day [P] 0-P14) are distinct from those during maturation (P14-P24). This biphasic pattern is paralleled by the transient presence of Gli1+;α-smooth muscle actin (α-SMA)+ myofibroblasts in the growing alveolar septal tips. Carefully timed inhibition of Hedgehog (Hh) signaling during alveolarization defined mechanisms by which Shh influences the mesenchymal compartment. First, interruption of Hh signaling at earlier time points results in increased lung compliance and wall structure defects of increasing severity, ranging from moderately enlarged alveolar airspaces to markedly enlarged airspaces and fewer secondary septa. Second, Shh signaling is required for myofibroblast differentiation: Hh inhibition during early alveolarization almost completely eliminates Gli1+;α-SMA+ cells at the septal tips, and Gli1-lineage tracing revealed that Gli1+ cells do not undergo apoptosis after Hh inhibition but remain in the alveolar septa and are unable to express α-SMA. Third, Shh signaling is vital to mesenchymal proliferation during alveolarization, as Hh inhibition decreased proliferation of Gli1+ cells and their progeny. Our study establishes Shh as a new alveolarization-promoting factor that might be affected in perinatal lung diseases that are associated with impaired alveolarization.

Autoantibodies against tumor-associated antigens in the early detection of lung cancer.

OBJECTIVES: Autoantibodies against tumor-associated antigens (TAAs) identified in patients with advanced lung cancer may be detected in subjects with early lung cancer or even predate the diagnosis. The purpose of this study is to address the temporal relationship between lung cancer development and serum autoantibody response. MATERIALS AND METHODS: Two cohorts of patients with newly diagnosed lung cancer were included. The first cohort included 90 sera from patients with lung cancer (Stages I-III) and 89 normal control sera. In the second cohort, 93 serial serum samples from 25 patients with CT-scan screen-detected stage I lung cancer were collected before the diagnosis of lung cancer (average 32 months) and 56 controls were matched on age, gender, and smoking. Autoantibody levels were measured by immunoassay. RESULTS: Measurement of autoantibodies against seven TAAs (14-3-3ζ, c-Myc, MDM2, NPM1, p16, p53 and cyclin B1) individually could discriminate lung cancer patients from normal individuals in the first cohort and the area under curve (AUC) was 0.863 based on a panel of seven autoantibodies, with sensitivity of 68.9% and specificity of 79.5%. Autoantibodies in serial pre-diagnostic serum samples against the same panel of seven TAAs were detected prior to lung cancer diagnosis with sensitivity of 76.0% and specificity of 73.2% (AUC) (95%CI): 0.885 (0.797-0.973)). Elevated autoantibody levels could be detected greater than four years prior to lung cancer diagnosis. CONCLUSION: A panel of seven TAAs may enhance the early detection of lung cancer, consistent with a humoral immune response to TAAs that can be detected months to years prior to the diagnosis.

Reduced lung function in smokers in a lung cancer screening cohort with asbestos exposure and pleural plaques.

BACKGROUND: While low dose computed tomography (LDCT) screening for lung cancer is recommended for high-risk smokers, ages 55-74 years, information about asbestos exposure may not be routinely elicited. Asbestos exposure is associated with declining respiratory function over time; however, the effect of a history of asbestos exposure in LDCT screening cohorts is limited. We report the relationship between asbestos exposure and pulmonary function in a cohort of heavy smokers with a history of occupational asbestos exposure, hypothesizing that these subjects will have additional decreased pulmonary function. We also examined relationships between spirometric measurements and the presence of isolated pleural plaques. METHODS: A cross-sectional study was performed using data from the NYU Lung Cancer Biomarker Center cohort to compare study subjects with a history asbestos exposure primarily in the period since 1970 when tighter federal standards were in place (n = 359) to those without asbestos exposure (n = 1038) with respect to pulmonary function, LDCT lung imaging findings, and clinical symptoms. We further classified individuals with asbestos exposure by length of exposure time to examine the effect of duration of exposure on pulmonary function. Lastly, for asbestos-exposed participants, we examined the association of spirometric measurements with the presence of absence of isolated pleural plaques. RESULTS: Individuals with asbestos exposure had decreased FVC % predicted compared to those with no asbestos exposure (76% vs. 85% predicted, P < 0.01) and FEV1 % predicted (64% vs. 67% predicted, P < 0.01). Since there was no change in FEV1 /FVC ratio, the findings are consistent with restrictive impairment. Those with ≥20 years of exposure had a lower mean FVC % predicted compared to those with less than 20 years of exposure (74% vs. 78% predicted, P = 0.017). Individuals with asbestos exposure were more likely to have pleural plaques (P < 0.001) on CT. Those with isolated pleural plaques had lower mean % predicted FEV1 (P = 0.005) and FVC (P = 0.001) compared to those without pleural plaques. CONCLUSIONS: Occupational asbestos exposure in a cohort of heavy smokers was associated with a significant restrictive decline in pulmonary function, with longer duration of exposure associated with greater decline. The presence of isolated pleural plaques was also associated with reduced lung function.

Sonic hedgehog signaling in the lung. From development to disease.

Over the past two decades, the secreted protein sonic hedgehog (SHH) has emerged as a critical morphogen during embryonic lung development, regulating the interaction between epithelial and mesenchymal cell populations in the airway and alveolar compartments. There is increasing evidence that the SHH pathway is active in adult lung diseases such as pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, and lung cancer, which raises two questions: (1) What role does SHH signaling play in these diseases? and (2) Is it a primary driver of the disease or a response (perhaps beneficial) to the primary disturbance? In this review we aim to fill the gap between the well-studied period of embryonic lung development and the adult diseased lung by reviewing the hedgehog (HH) pathway during the postnatal period and in adult uninjured and injured lungs. We elucidate the similarities and differences in the epithelial-mesenchymal interplay during the fibrosis response to injury in lung compared with other organs and present a critical appraisal of tools and agents available to evaluate HH signaling.

Hedgehog signaling in neonatal and adult lung.

Sonic Hedgehog (Shh) signaling is essential during embryonic lung development, but its role in postnatal lung development and adult lung are not known. Using Gli1(nlacZ) reporter mice to identify cells with active Hh signaling, we found that Gli1(nlacZ)-positive mesenchymal cells are densely and diffusely present up to 2 weeks after birth and decline in number thereafter. In adult mice, Gli1(nlacZ)-positive cells are present around large airways and vessels and are sparse in alveolar septa. Hh-stimulated cells are mostly fibroblasts; only 10% of Gli1(nlacZ)-positive cells are smooth muscle cells, and most smooth muscle cells do not have activation of Hh signaling. To assess its functional relevance, we influenced Hh signaling in the developing postnatal lung and adult injured lung. Inhibition of Hh signaling during early postnatal lung development causes airspace enlargement without diminished alveolar septation. After bleomycin injury in the adult lung, there are abundant Gli1(nlacZ)-positive mesenchymal cells in fibrotic lesions and increased numbers of Gli1(nlacZ)-positive cells in preserved alveolar septa. Inhibition of Hh signaling with an antibody against all Hedgehog isoforms does not reduce bleomycin-induced fibrosis, but adenovirus-mediated overexpression of Shh increases collagen production in this model. Our data provide strong evidence that Hh signaling can regulate lung stromal cell function in two critical scenarios: normal development in postnatal lung and lung fibrosis in adult lung.

Cross talk among TGF-ß signaling pathways, integrins, and the extracellular matrix.

The growth factor TGF-ß is secreted in a latent complex consisting of three proteins: TGF-ß, an inhibitor (latency-associated protein, LAP, which is derived from the TGF-ß propeptide) and an ECM-binding protein (one of the latent TGF-ß binding proteins, or LTBPs). LTBPs interact with fibrillins and other ECM components and thus function to localize latent TGF-ß in the ECM. LAP contains an integrin-binding site (RGD), and several RGD-binding integrins are able to activate latent TGF-ß through binding this site. Mutant mice defective in integrin-mediated activators, and humans and mice with fibrillin gene mutations, show the critical role of ECM and integrins in regulating TGF-ß signaling.

Mice that lack activity of alphavbeta6- and alphavbeta8-integrins reproduce the abnormalities of Tgfb1- and Tgfb3-null mice.

The arginine-glycine-aspartate (RGD)-binding integrins alphavbeta6 and alphavbeta8 activate latent TGFbeta1 and TGFbeta3 in vivo, but it is uncertain whether other RGD-binding integrins such as integrins alphavbeta5 and alphavbeta3 activate these TGFbeta isoforms. To define the combined role of alphavbeta6- and alphavbeta8-integrin in TGFbeta activation, we analyzed mice lacking function of both integrins by means of gene deletion and/or pharmacologic inhibition. Most Itgb6-/-;Itgb8-/- embryos die at mid-gestation; those that survive develop cleft palate-as observed in Tgfb3-/- mice. Itgb8-/- mice treated with an anti-alphavbeta6-integrin antibody develop severe autoimmunity and lack Langerhans cells-similar to Tgfb1-null mice. These results support a model in which TGFbeta3-mediated palate fusion and TGFbeta1-mediated suppression of autoimmunity and generation of Langerhans cells require integrins alphavbeta6 and alphavbeta8 but not other RGD-binding integrins as TGFbeta activators.

TGFbeta1 and TGFbeta3 are partially redundant effectors in brain vascular morphogenesis.

Gene deletion experiments have shown that the three TGFbeta isoforms regulate distinct developmental processes. Recent work by our group and others showed that the integrins alphavbeta6 and alphavbeta8 activate latent forms of TGFbeta1 and TGFbeta3. This raises the possibility that TGFbeta1 and TGFbeta3 act redundantly in developmental processes where both isoforms are expressed and activation is by integrins. To investigate this issue, we generated mice with defective integrin-mediated TGFbeta1 activation (Tgfb1(RGE/RGE)) that were also homozygous for a null mutation in the TGFbeta3 gene. Tgfb1(RGE/RGE); Tgfb3(-/-) mice have severely perturbed development of the brain vasculature that is highly similar to that in mice lacking alphavbeta8. Some Tgfb1(RGE/RGE); Tgfb3(+/-) and Tgfb1(RGE/RGE); Tgfb3(+/+) mice have milder, background-dependent versions of the phenotype. In addition, we found that Tgfb3 gene status influences embryonic lethality due to TGFbeta1 deficiency after limited backcrossing to the BALB/c background. Conversely, Tgfb1 gene status modifies the extent of palate fusion in Tgfb3(-/-) mice after limited backcrossing to the ICR background. Our results are consistent with a functional connection between TGFbeta1 and TGFbeta3 during development based on a shared mechanism of activation.

Inhibition of integrin alpha(v)beta6, an activator of latent transforming growth factor-beta, prevents radiation-induced lung fibrosis.

RATIONALE: In experimental models, lung fibrosis is dependent on transforming growth factor (TGF)-beta signaling. TGF-beta is secreted in a latent complex with its propeptide, and TGF-beta activators release TGF-beta from this complex. Because the integrin alpha(v)beta6 is a major TGF-beta activator in the lung, inhibition of alpha(v)beta6-mediated TGF-beta activation is a logical strategy to treat lung fibrosis. OBJECTIVES: To determine, by genetic and pharmacologic approaches, whether murine radiation-induced lung fibrosis is dependent on alpha(v)beta6. METHODS: Wild-type mice, alpha(v)beta6-deficient (Itgb6-/-) mice, and mice heterozygous for a Tgfb1 mutation that eliminates integrin-mediated activation (Tgfb1(+/RGE)) were exposed to 14 Gy thoracic radiation. Some mice were treated with an anti-alpha(v)beta6 monoclonal antibody or a soluble TGF-beta receptor fusion protein. Alpha(v)beta6 expression was determined by immunohistochemistry. Fibrosis, inflammation, and gene expression patterns were assessed 20-32 weeks postirradiation. MEASUREMENTS AND MAIN RESULTS: Beta6 integrin expression increased within the alveolar epithelium 18 weeks postirradiation, just before onset of fibrosis. Itgb6-/- mice were completely protected from fibrosis, but not from late radiation-induced mortality. Anti-alpha(v)beta6 therapy (1-10 mg/kg/wk) prevented fibrosis, but only higher doses (6-10 mg/kg/wk) caused lung inflammation similar to that in Itgb6-/- mice. Tgfb1-haploinsufficient mice were also protected from fibrosis. CONCLUSIONS: Alpha(v)beta6-mediated TGF-beta activation is required for radiation-induced lung fibrosis. Together with previous data, our results demonstrate a robust requirement for alpha(v)beta6 in distinct fibrosis models. Inhibition of alphavbeta6-mediated TGF-beta activation is a promising new approach for antifibrosis therapy.

Absence of integrin-mediated TGFbeta1 activation in vivo recapitulates the phenotype of TGFbeta1-null mice.

The multifunctional cytokine transforming growth factor (TGF) beta1 is secreted in a latent complex with its processed propeptide (latency-associated peptide [LAP]). TGFbeta1 must be functionally released from this complex before it can engage TGFbeta receptors. One mechanism of latent TGFbeta1 activation involves interaction of the integrins alpha v beta6 and alpha v beta8 with an RGD sequence in LAP; other putative latent TGFbeta1 activators include thrombospondin-1, oxidants, and various proteases. To assess the contribution of RGD-binding integrins to TGFbeta1 activation in vivo, we created a mutation in Tgfb1 encoding a nonfunctional variant of the RGD sequence (RGE). Mice with this mutation (Tgfb1(RGE/RGE)) display the major features of Tgfb1(-/-) mice (vasculogenesis defects, multiorgan inflammation, and lack of Langerhans cells) despite production of normal levels of latent TGFbeta1. These findings indicate that RGD-binding integrins are requisite latent TGFbeta1 activators during development and in the immune system.

Integrin alphaVbeta6-mediated activation of latent TGF-beta requires the latent TGF-beta binding protein-1.

Transforming growth factor-betas (TGF-beta) are secreted as inactive complexes containing the TGF-beta, the TGF-beta propeptide, also called the latency-associated protein (LAP), and the latent TGF-beta binding protein (LTBP). Extracellular activation of this complex is a critical but incompletely understood step in TGF-beta regulation. We have investigated the role of LTBP in modulating TGF-beta generation by the integrin alphaVbeta6. We show that even though alphavbeta6 recognizes an RGD on LAP, LTBP-1 is required for alphaVbeta6-mediated latent TGF-beta activation. The domains of LTBP-1 necessary for activation include the TGF-beta propeptide-binding domain and a basic amino acid sequence (hinge domain) with ECM targeting properties. Our results demonstrate an LTBP-1 isoform-specific function in alphaVbeta6-mediated latent TGF-beta activation; LTBP-3 is unable to substitute for LTBP-1 in this assay. The results reveal a functional role for LTBP-1 in latent TGF-beta activation and suggest that activation of specific latent complexes is regulated by distinct mechanisms that may be determined by the LTBP isoform and its potential interaction with the matrix.

A genetic screen to identify latent transforming growth factor beta activators.

The mechanisms by which latent transforming growth factor beta (TGFbeta) is converted to the active cytokine are largely unknown. Here we present a genetic screen that combines retroviral mutagenesis and cDNA expression cloning to reveal proteins involved in the extracellular regulation of latent TGFbeta activation. The screen employs a cell line engineered to express green fluorescent protein (GFP) in response to TGFbeta. The cells produce their own latent TGFbeta. Therefore, after transduction with a retroviral cDNA library that contains an insert for an activator of latent TGFbeta, cells expressing the activator are GFP-bright. These cells are enriched by fluorescence-activated cell sorting and grown as individual clones. The isolated clones are cocultured with a second TGFbeta reporter cell line that produces luciferase in response to TGFbeta. Cells that have acquired the ability to activate latent TGFbeta induce luciferase expression in the absence but not in the presence of neutralizing antibodies to TGFbeta. The activator expressed by the positive clones can be identified by retrieval of the retrovirus cDNA insert.