GLI1+ Cells Contribute to Vascular Remodeling in Pulmonary Hypertension.

BACKGROUND: The precise origin of newly formed ACTA2+ (alpha smooth muscle actin-positive) cells appearing in nonmuscularized vessels in the context of pulmonary hypertension is still debatable although it is believed that they predominantly derive from preexisting vascular smooth muscle cells (VSMCs). METHODS: Gli1Cre-ERT2; tdTomatoflox mice were used to lineage trace GLI1+ (glioma-associated oncogene homolog 1-positive) cells in the context of pulmonary hypertension using 2 independent models of vascular remodeling and reverse remodeling: hypoxia and cigarette smoke exposure. Hemodynamic measurements, right ventricular hypertrophy assessment, flow cytometry, and histological analysis of thick lung sections followed by state-of-the-art 3-dimensional reconstruction and quantification using Imaris software were used to investigate the contribution of GLI1+ cells to neomuscularization of the pulmonary vasculature. RESULTS: The data show that GLI1+ cells are abundant around distal, nonmuscularized vessels during steady state, and this lineage contributes to around 50% of newly formed ACTA2+ cells around these normally nonmuscularized vessels. During reverse remodeling, cells derived from the GLI1+ lineage are largely cleared in parallel to the reversal of muscularization. Partial ablation of GLI1+ cells greatly prevented vascular remodeling in response to hypoxia and attenuated the increase in right ventricular systolic pressure and right heart hypertrophy. Single-cell RNA sequencing on sorted lineage-labeled GLI1+ cells revealed an Acta2high fraction of cells with pathways in cancer and MAPK (mitogen-activated protein kinase) signaling as potential players in reprogramming these cells during vascular remodeling. Analysis of human lung-derived material suggests that GLI1 signaling is overactivated in both group 1 and group 3 pulmonary hypertension and can promote proliferation and myogenic differentiation. CONCLUSIONS: Our data highlight GLI1+ cells as an alternative cellular source of VSMCs in pulmonary hypertension and suggest that these cells and the associated signaling pathways represent an important therapeutic target for further studies.

WSB1, a Hypoxia-Inducible E3 Ligase, Promotes Myofibroblast Accumulation and Attenuates Alveolar Epithelial Regeneration in Mouse Lung Fibrosis.

Idiopathic pulmonary fibrosis is a progressive interstitial lung disease for which there is no curative therapy available. Repetitive alveolar epithelial injury repair, myofibroblast accumulation, and excessive collagen deposition are key pathologic features of idiopathic pulmonary fibrosis, eventually leading to cellular hypoxia and respiratory failure. The precise mechanism driving this complex maladaptive process remains inadequately understood. WD repeat and suppressor of cytokine signaling box containing 1 (WSB1) is an E3 ubiquitin ligase, the expression of which is associated strongly with hypoxia, and forms a positive feedback loop with hypoxia-inducible factor 1α (HIF-1α) under anoxic condition. This study explored the expression, cellular distribution, and function of WSB1 in bleomycin (BLM)-induced mouse lung injury and fibrosis. WSB1 expression was highly induced by BLM injury and correlated with the progression of lung fibrosis. Significantly, conditional deletion of Wsb1 in adult mice ameliorated BLM-induced pulmonary fibrosis. Phenotypically, Wsb1-deficient mice showed reduced lipofibroblast to myofibroblast transition, but enhanced alveolar type 2 proliferation and differentiation into alveolar type 1 after BLM injury. Proteomic analysis of mouse lung tissues identified caveolin 2 as a potential downstream target of WSB1, contributing to BLM-induced epithelial injury repair and fibrosis. These findings unravel a vital role for WSB1 induction in lung injury repair, thus highlighting it as a potential therapeutic target for pulmonary fibrosis.

Regulation of the Hippo-YAP Pathway by Glucose Sensor O-GlcNAcylation.

The Hippo pathway is crucial in organ size control and tissue homeostasis, with deregulation leading to cancer. An extracellular nutrition signal, such as glucose, regulates the Hippo pathway activation. However, the mechanisms are still not clear. Here, we found that the Hippo pathway is directly regulated by the hexosamine biosynthesis pathway (HBP) in response to metabolic nutrients. Mechanistically, the core component of Hippo pathway (YAP) is O-GlcNAcylated by O-GlcNAc transferase (OGT) at serine 109. YAP O-GlcNAcylation disrupts its interaction with upstream kinase LATS1, prevents its phosphorylation, and activates its transcriptional activity. And this activation is not dependent on AMPK. We also identified OGT as a YAP-regulated gene that forms a feedback loop. Finally, we confirmed that glucose-induced YAP O-GlcNAcylation and activation promoted tumorigenesis. Together, our data establish a molecular mechanism and functional significance of the HBP in directly linking extracellular glucose signal to the Hippo-YAP pathway and tumorigenesis.

FGF1 ameliorates obesity-associated hepatic steatosis by reversing IGFBP2 hypermethylation.

Obesity is a major contributing factor for metabolic-associated fatty liver disease (MAFLD). Fibroblast growth factor (FGF) 1 is the first paracrine FGF family member identified to exhibit promising metabolic regulatory properties capable of conferring glucose-lowering and insulin-sensitizing effect. This study explores the role and molecular underpinnings of FGF1 in obesity-associated hepatic steatosis. In a mouse high-fat diet (HFD)-induced MAFLD model, chronic treatment with recombinant FGF1(rFGF1) was found to effectively reduce the severity of insulin resistance, hyperlipidemia, and inflammation. FGF1 treatment decreased lipid accumulation in the mouse liver and palmitic acid-treated AML12 cells. These effects were associated with decreased mature form SREBF1 expression and its target genes FASN and SCD1. Interestingly, we uncovered that rFGF1 significantly induced IGFBP2 expression at both mRNA and protein levels in HFD-fed mouse livers and cultured hepatocytes treated with palmitic acid. Adeno-associated virus-mediated IGFBP2 suppression significantly diminished the therapeutic benefit of rFGF1 on MAFLD-associated phenotypes, indicating that IGFBP2 plays a crucial role in the FGF1-mediated reduction of hepatic steatosis. Further analysis revealed that rFGF1 treatment reduces the recruitment of DNA methyltransferase 3 alpha to the IGFBP2 genomic locus, leading to decreased IGFBP2 gene methylation and increased mRNA and protein expression. Collectively, our findings reveal FGF1 modulation of lipid metabolism via epigenetic regulation of IGFBP2 expression, and unravel the therapeutic potential of the FGF1-IGFBP2 axis in metabolic diseases associated with obesity.

Injury activated alveolar progenitors (IAAPs): the underdog of lung repair.

Alveolar epithelial type II cells (AT2s) together with AT1s constitute the epithelial lining of lung alveoli. In contrast to the large flat AT1s, AT2s are cuboidal and smaller. In addition to surfactant production, AT2s also serve as prime alveolar progenitors in homeostasis and play an important role during regeneration/repair. Based on different lineage tracing strategies in mice and single-cell transcriptomic analysis, recent reports highlight the heterogeneous nature of AT2s. These studies present compelling evidence for the presence of stable or transitory AT2 subpopulations with distinct marker expression, signaling pathway activation and functional properties. Despite demonstrated progenitor potentials of AT2s in maintaining homeostasis, through self-renewal and differentiation to AT1s, the exact identity, full progenitor potential and regulation of these progenitor cells, especially in the context of human diseases remain unclear. We recently identified a novel subset of AT2 progenitors named "Injury-Activated Alveolar Progenitors" (IAAPs), which express low levels of Sftpc, Sftpb, Sftpa1, Fgfr2b and Etv5, but are highly enriched for the expression of the surface receptor programmed cell death-ligand 1 (Pd-l1). IAAPs are quiescent during lung homeostasis but activated upon injury with the potential to proliferate and differentiate into AT2s. Significantly, a similar population of PD-L1 positive cells expressing intermediate levels of SFTPC are found to be expanded in human IPF lungs. We summarize here the current understanding of this newly discovered AT2 progenitor subpopulation and also try to reconcile the relationship between different AT2 stem cell subpopulations regarding their progenitor potential, regulation, and relevance to disease pathogenesis and therapeutic interventions.

Metformin alleviates benzo[a]pyrene-induced alveolar injury by inhibiting necroptosis and protecting AT2 cells.

Exposure to benzo[a]pyrene (B[a]P) has been linked to lung injury and carcinogenesis. Airway epithelial cells express the B[a]P receptor AHR, so B[a]P is considered to mainly target airway epithelial cells, whereas its potential impact on alveolar cells remains inadequately explored. Metformin, a first-line drug for diabetes, has been shown to exert anti-inflammatory and tissue repair-promoting effects under various injurious conditions. Here, we explored the effect of chronic B[a]P exposure on alveolar cells and the impact of metformin on B[a]P-induced lung injury by examining the various parameters including lung histopathology, inflammation, fibrosis, and related signal pathway activation. MLKL knockout (Mlkl-/-) and AT2-lineage tracing mice (SftpcCre-ERT2;LSL-tdTomatoflox+/-) were used to delineate the role of necroptosis in B[a]P-induced alveolar epithelial injury and repair. Mice receiving weekly administration of B[a]P for 6 weeks developed a significant alveolar damaging phenotype associated with pulmonary inflammation, fibrosis, and activation of the necroptotic cell death pathway. These effects were significantly relieved in MLKL null mice. Furthermore, metformin treatment, which were found to promote AMPK phosphorylation and inhibit RIPK3, as well as MLKL phosphorylation, also significantly alleviated B[a]P-induced necroptosis and lung injury phenotype. However, the protective efficacy of metformin was rendered much less effective in Mlkl null mice or by blocking the necroptotic pathway with RIPK3 inhibitor. Our findings unravel a potential protective efficacy of metformin in mitigating the detrimental effects of B[a]P exposure on lung health by inhibiting necroptosis and protecting AT2 cells.

FGF10 mitigates doxorubicin-induced myocardial toxicity in mice via activation of FGFR2b/PHLDA1/AKT axis.

Doxorubicin is a common chemotherapeutic agent in clinic, but myocardial toxicity limits its use. Fibroblast growth factor (FGF) 10, a multifunctional paracrine growth factor, plays diverse roles in embryonic and postnatal heart development as well as in cardiac regeneration and repair. In this study we investigated the role of FGF10 as a potential modulator of doxorubicin-induced cardiac cytotoxicity and the underlying molecular mechanisms. Fgf10+/- mice and an inducible dominant negative FGFR2b transgenic mouse model (Rosa26rtTA; tet(O)sFgfr2b) were used to determine the effect of Fgf10 hypomorph or blocking of endogenous FGFR2b ligands activity on doxorubicin-induced myocardial injury. Acute myocardial injury was induced by a single injection of doxorubicin (25 mg/kg, i.p.). Then cardiac function was evaluated using echocardiography, and DNA damage, oxidative stress and apoptosis in cardiac tissue were assessed. We showed that doxorubicin treatment markedly decreased the expression of FGFR2b ligands including FGF10 in cardiac tissue of wild type mice, whereas Fgf10+/- mice exhibited a greater degree of oxidative stress, DNA damage and apoptosis as compared with the Fgf10+/+ control. Pre-treatment with recombinant FGF10 protein significantly attenuated doxorubicin-induced oxidative stress, DNA damage and apoptosis both in doxorubicin-treated mice and in doxorubicin-treated HL-1 cells and NRCMs. We demonstrated that FGF10 protected against doxorubicin-induced myocardial toxicity via activation of FGFR2/Pleckstrin homology-like domain family A member 1 (PHLDA1)/Akt axis. Overall, our results unveil a potent protective effect of FGF10 against doxorubicin-induced myocardial injury and identify FGFR2b/PHLDA1/Akt axis as a potential therapeutic target for patients receiving doxorubicin treatment.

Fgfr2b signaling is essential for the maintenance of the alveolar epithelial type 2 lineage during lung homeostasis in mice.

Fibroblast growth factor receptor 2b (Fgfr2b) signaling is essential throughout lung development to form the alveolar epithelial lineage. However, its role in alveolar epithelial type 2 cells (AT2s) homeostasis was recently considered dispensable. SftpcCreERT2; Fgfr2bflox/flox; tdTomatoflox/flox mice were used to delete Fgfr2b expression in cells belonging to the AT2 lineage, which contains mature AT2s and a novel SftpcLow lineage-traced population called "injury activated alveolar progenitors" or IAAPs. Upon continuous tamoxifen exposure for either 1 or 2 weeks to delete Fgfr2b, a shrinking of the AT2 population is observed. Mature AT2s exit the cell cycle, undergo apoptosis and fail to form alveolospheres in vitro. However, the lung morphometry appears normal, suggesting the involvement of compensatory mechanisms. In mutant lungs, IAAPs which escaped Fgfr2b deletion expand, display enhanced alveolosphere formation in vitro and increase drastically their AT2 signature, suggesting differentiation towards mature AT2s. Interestingly, a significant increase in AT2s and decrease in IAPPs occurs after a 1-week tamoxifen exposure followed by an 8-week chase period. Although mature AT2s partially recover their alveolosphere formation capabilities, the IAAPs no longer display this property. Single-cell RNA seq analysis confirms that AT2s and IAAPs represent stable and distinct cell populations and recapitulate some of their characteristics observed in vivo. Our results underscore the essential role played by Fgfr2b signaling in the maintenance of the AT2 lineage in the adult lung during homeostasis and suggest that the IAAPs could represent a new population of AT2 progenitors.

FGFR2b signalling restricts lineage-flexible alveolar progenitors during mouse lung development and converges in mature alveolar type 2 cells.

The specification, characterization, and fate of alveolar type 1 and type 2 (AT1 and AT2) progenitors during embryonic lung development are poorly defined. Current models of distal epithelial lineage formation fail to capture the heterogeneity and dynamic contribution of progenitor pools present during early development. Furthermore, few studies explore the pathways involved in alveolar progenitor specification and fate. In this paper, we build upon our previously published work on the regulation of airway epithelial progenitors by fibroblast growth factor receptor 2b (FGFR2b) signalling during early (E12.5) and mid (E14.5) pseudoglandular stage lung development. Our results suggest that a significant proportion of AT2 and AT1 progenitors are lineage-flexible during late pseudoglandular stage development, and that lineage commitment is regulated in part by FGFR2b signalling. We have characterized a set of direct FGFR2b targets at E16.5 which are likely involved in alveolar lineage formation. These signature genes converge on a subpopulation of AT2 cells later in development and are downregulated in AT2 cells transitioning to the AT1 lineage during repair after injury in adults. Our findings highlight the extensive heterogeneity of pneumocytes by elucidating the role of FGFR2b signalling in these cells during early airway epithelial lineage formation, as well as during repair after injury.

GLI1+ cells are a source of repair-supportive mesenchymal cells (RSMCs) during airway epithelial regeneration.

Repair-supportive mesenchymal cells (RSMCs) have been recently reported in the context of naphthalene (NA)-induced airway injury and regeneration. These cells transiently express smooth muscle actin (Acta2) and are enriched with platelet-derived growth factor receptor alpha (Pdgfra) and fibroblast growth factor 10 (Fgf10) expression. Genetic deletion of Ctnnb1 (gene coding for beta catenin) or Fgf10 in these cells using the Acta2-Cre-ERT2 driver line after injury (defined as NA-Tam condition; Tam refers to tamoxifen) led to impaired repair of the airway epithelium. In this study, we demonstrate that RSMCs are mostly captured using the Acta2-Cre-ERT2 driver when labeling occurs after (NA-Tam condition) rather than before injury (Tam-NA condition), and that their expansion occurs mostly between days 3 and 7 following NA treatment. Previous studies have shown that lineage-traced peribronchial GLI1+ cells are transiently amplified after NA injury. Here, we report that Gli1 expression is enriched in RSMCs. Using lineage tracing with Gli1Cre-ERT2 mice combined with genetic inactivation of Fgf10, we show that GLI1+ cells with Fgf10 deletion fail to amplify around the injured airways, thus resulting in impaired airway epithelial repair. Interestingly, Fgf10 expression is not upregulated in GLI1+ cells following NA treatment, suggesting that epithelial repair is mostly due to the increased number of Fgf10-expressing GLI1+ cells. Co-culture of SCGB1A1+ cells with GLI1+ cells isolated from non-injured or injured lungs showed that GLI1+ cells from these two conditions are similarly capable of supporting bronchiolar organoid (or bronchiolosphere) formation. Single-cell RNA sequencing on sorted lineage-labeled cells showed that the RSMC signature resembles that of alveolar fibroblasts. Altogether, our study provides strong evidence for the involvement of mesenchymal progenitors in airway epithelial regeneration and highlights the critical role played by Fgf10-expressing GLI1+ cells in this context.

Enhanced nuclear localization of YAP1-2 contributes to EGF-induced EMT in NSCLC.

YAP1, a key mediator of the Hippo pathway, plays an important role in tumorigenesis. Alternative splicing of human YAP1 mRNA results in two major isoforms: YAP1-1, which contains a single WW domain, and YAP1-2, which contains two WW domains, respectively. We here investigated the functions and the underlying regulatory mechanisms of the two YAP1 isoforms in the context of EGF-induced epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC). Human NSCLC cell lines express both YAP1-1 and YAP1-2 isoforms-although when compared to YAP1-1, YAP1-2 mRNA levels are higher while its protein expression levels are lower. EGF treatment significantly promoted YAP1 expression as well as EMT process in NSCLCs, whereas EGF-induced EMT phenotype was significantly alleviated upon YAP1 knockdown. Under normal culture condition, YAP1-1 stable expression cells exhibited a stronger migration ability than YAP1-2 expressing cells. However, upon EGF treatment, YAP1-2 stable cells showed more robust migration than YAP1-1 expressing cells. The protein stability and nuclear localization of YAP1-2 were preferentially enhanced with EGF treatment. Moreover, EGF-induced EMT and YAP1-2 activity were suppressed by inhibitor of AKT. Our results suggest that YAP1-2 is the main isoform that is functionally relevant in promoting EGF-induced EMT and ultimately NSCLC progression.

P38 initiates degeneration of midbrain GABAergic and glutamatergic neurons in diabetes models.

Diabetes mellitus may cause tau protein hyperphosphorylation and neurodegeneration, but the exact mechanism by which diabetic conditions induce tau pathology remains unclear. Tau protein hyperphosphorylation is considered a major pathological hallmark of neurodegeneration and can be triggered by diabetes. Various tau-directed kinases, including P38, can be activated upon diabetic stress and induce tau hyperphosphorylation. Despite extensive research efforts, the exact tau specie(s) and kinases driving neurodegeneration in diabetes mellitus have not been clearly elucidated. We herein employed different techniques to determine the exact molecular mechanism of tau pathology triggered by diabetes in in vivo and in vitro models. We showed that diabetes-related stresses and glucose metabolism deficiency could induce cis P-tau (an early driver of the tau pathology) accumulation in the midbrain and corpus callosum of the diabetic mice models and cells treated with 2-deoxy-D-glucose, respectively. We found that the active phosphorylated level of P38 was increased in the treated cells and diabetic mice models. We observed that oxidative stress activated P38, which directly and indirectly drove tau pathology in the GABAergic and glutamatergic neurons of the midbrain of the diabetic mice after 96 h, which accumulated in the other neighboring brain areas after 2 months. Notably, P38 inhibition suppressed tau pathogenicity and risk-taking behaviors in the animal models after 96 h. The data establish P38 as a central mediator of diabetes mellitus-induced tau pathology. Our findings provide mechanistic insight into the consequences of this metabolic disorder on the nervous system.

FGF1 alleviates LPS-induced acute lung injury via suppression of inflammation and oxidative stress.

BACKGROUND: Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are devastating clinical disorders with high mortality, and for which more effective therapies are urgently needed. FGF1, the prototype member of the FGF family, is shown to exert protective effects against injurious stimuli in multiple disease models. Here we aimed to evaluate whether FGF1 pretreatment is protective against LPS-induced ALI and elucidate the potential underlying mechanisms. METHODS: For drug-treated groups, C57B/6 mice received a single i.p. injection of FGF1 (1 mg/kg) 1 h before the LPS challenge or not. To induce the ALI model, the mice were treated by intratracheal instillation of LPS (5 mg/kg). Then, histopathological changes in lung tissues were assessed by hematoxylin and eosin staining and transmission electron microscopy. ELISA and qPCR assays were used to detect pro-inflammatory cytokine levels in BALF and lung tissues, respectively. The total number of inflammatory cells (neutrophils and macrophages) in BALF were counted using the Wright-Giemsa method. The expressions of reactive oxygen species (ROS) and malondialdehyde (MDA) were measured using their respective kits. Western blot and immunostaining were used to evaluate the expressions of antioxidants (Nrf-2, HO-1, SOD2, GPX4, and Catalase), as well as the inflammatory and/or apoptosis-related factors (TLR4, NF-κB, and Cleaved- caspase 3). RESULTS: FGF1 pretreatment significantly ameliorated the LPS-induced histopathological changes, reduced lung wet/dry ratios, ROS and MDA levels, total BALF protein, inflammatory cell infiltration, proinflammatory cytokine levels, and significantly increased the expression of antioxidant proteins (Nrf-2, HO-1, Catalase, and SOD2). In addition, FGF1 pretreatment significantly reduced the expression of TLR4 and cleaved- caspase 3, inhibited NF-κB activation, and reduced LPS-induced cell apoptosis. CONCLUSIONS: Altogether, our results suggest that FGF1 pretreatment is protective against LPS-induced ALI through mediating anti-inflammatory and antioxidant effects, which may be attributed to the downregulation of TLR4 expression and inhibition of NF-κB activation, as well as promotion of antioxidant defenses. Therefore, FGF1 administration may prove beneficial in preventative strategies for ALI/ARDS.

Hippo signaling promotes lung epithelial lineage commitment by curbing Fgf10 and ß-catenin signaling.

Organ growth and tissue homeostasis rely on the proliferation and differentiation of progenitor cell populations. In the developing lung, localized Fgf10 expression maintains distal Sox9-expressing epithelial progenitors and promotes basal cell differentiation in the cartilaginous airways. Mesenchymal Fgf10 expression is induced by Wnt signaling but inhibited by Shh signaling, and epithelial Fgf10 signaling activates ß-catenin signaling. The Hippo pathway is a well-conserved signaling cascade that regulates organ size and stem/progenitor cell behavior. Here, we show that Hippo signaling promotes lineage commitment of lung epithelial progenitors by curbing Fgf10 and ß-catenin signaling. Our findings show that both inactivation of the Hippo pathway (nuclear Yap) or ablation of Yap result in increased ß-catenin and Fgf10 signaling, suggesting a cytoplasmic role for Yap in epithelial lineage commitment. We further demonstrate redundant and non-redundant functions for the two nuclear effectors of the Hippo pathway, Yap and Taz, during lung development.

Characterization in mice of the resident mesenchymal niche maintaining AT2 stem cell proliferation in homeostasis and disease.

Resident mesenchymal cells (rMCs defined as Cd31Neg Cd45Neg EpcamNeg ) control the proliferation and differentiation of alveolar epithelial type 2 (AT2) stem cells in vitro. The identity of these rMCs is still elusive. Among them, Axin2Pos mesenchymal alveolar niche cells (MANCs), which are expressing Fgf7, have been previously described. We propose that an additional population of rMCs, expressing Fgf10 (called rMC-Sca1Pos Fgf10Pos ) are equally important to maintain AT2 stem cell proliferation. The alveolosphere model, based on the AT2-rMC co-culture in growth factor-reduced Matrigel, was used to test the efficiency of different rMC subpopulations isolated by FACS from adult murine lung to sustain the proliferation and differentiation of AT2 stem cells. We demonstrate that rMC-Sca1Pos Fgf10Pos cells are efficient to promote the proliferation and differentiation of AT2 stem cells. Co-staining of adult lung for Fgf10 mRNA and Sftpc protein respectively, indicate that 28% of Fgf10Pos cells are located close to AT2 cells. Co-ISH for Fgf7 and Fgf10 indicate that these two populations do not significantly overlap. Gene arrays comparing rMC-Sca1Pos Axin2Pos and rMC-Sca1Pos Fgf10Pos support that these two cell subsets express differential markers. In addition, rMC function is decreased in obese ob/ob mutant compared to WT mice with a much stronger loss of function in males compared to females. In conclusion, rMC-Sca1Pos Fgf10Pos cells play important role in supporting AT2 stem cells proliferation and differentiation. This result sheds a new light on the subpopulations of rMCs contributing to the AT2 stem cell niche in homeostasis and in the context of pre-existing metabolic diseases.

Impact of Fgf10 deficiency on pulmonary vasculature formation in a mouse model of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD), characterized by alveoli simplification and dysmorphic pulmonary microvasculature, is a chronic lung disease affecting prematurely born infants. Pulmonary hypertension (PH) is an important BPD feature associated with morbidity and mortality. In human BPD, inflammation leads to decreased fibroblast growth factor 10 (FGF10) expression but the impact on the vasculature is so far unknown. We used lungs from Fgf10+/- versus Fgf10+/+ pups to investigate the effect of Fgf10 deficiency on vascular development in normoxia (NOX) and hyperoxia (HOX, BPD mouse model). To assess the role of fibroblast growth factor receptor 2b (Fgfr2b) ligands independently of early developmentaldefects, we used an inducible double transgenic system in mice allowing inhibition of Fgfr2b ligands activity. Using vascular morphometry, we quantified the pathological changes. Finally, we evaluated changes in FGF10, surfactant protein C (SFTPC), platelet endothelial cell adhesion molecule (PECAM) and alpha-smooth muscle actin 2 (α-SMA) expression in human lung samples from patients suffering from BPD. In NOX, no major difference in the lung vasculature between Fgf10+/- and control pups was detected. In HOX, a greater loss of blood vessels in Fgf10+/- lungs is associated with an increase of poorly muscularized vessels. Fgfr2b ligands inhibition postnatally in HOX is sufficient to decrease the number of blood vessels while increasing the level of muscularization, suggesting a PH phenotype. BPD lungs exhibited decreased FGF10, SFTPC and PECAM but increased α-SMA. Fgf10 deficiency-associated vascular defects are enhanced in HOX and could represent an additional cause of morbidity in human patients with BPD.

Identification of a novel subset of alveolar type 2 cells enriched in PD-L1 and expanded following pneumonectomy.

Alveolar type 2 (AT2) cells are heterogeneous cells, with specialised AT2 subpopulations within this lineage exhibiting stem cell properties. However, the existence of quiescent, immature cells within the AT2 lineage that are activated during lung regeneration is unknown.SftpcCreERT2/+;tdTomatoflox/flox mice were used for the labelling of AT2 cells and labelled subpopulations were analysed by flow cytometry, quantitative PCR, assay for transposase-accessible chromatin using sequencing (ATAC-seq), gene arrays, pneumonectomy and culture of precision-cut lung slices. Single-cell RNA-sequencing (scRNA-seq) data from human lungs were analysed.In mice, we detected two distinct AT2 subpopulations, with low tdTomato level (TomLow) and high tdTomato level (TomHigh). TomLow cells express lower levels of the AT2 differentiation markers Fgfr2b and Etv5, while TomHigh, as bona fide mature AT2 cells, show higher levels of Sftpc, Sftpb, Sftpa1, Fgfr2b and Etv5 expression. ATAC-seq analysis indicates that TomLow and TomHigh cells constitute two distinct cell populations, with specific silencing of Sftpc, Rosa26 and cell cycle gene loci in the TomLow population. Upon pneumonectomy, the number of TomLow but not TomHigh cells increases and TomLow cells show upregulated expression of Fgfr2b, Etv5, Sftpc, Ccnd1 and Ccnd2 compared to Sham. TomLow cells overexpress programmed cell death 1 ligand 1 (PD-L1), an immune inhibitory membrane receptor ligand, which is used by flow cytometry to differentially isolate these two subpopulations. In the human lung, data mining of a recent scRNA-seq AT2 data set demonstrates the existence of a PD-L1 Pos population. Therefore, we have identified a novel population of AT2 quiescent, immature progenitor cells in mouse that expand upon pneumonectomy and we have provided evidence for the existence of such cells in human.

Polygenic Risk Scores have high diagnostic capacity in ankylosing spondylitis.

OBJECTIVE: We sought to test the hypothesis that Polygenic Risk Scores (PRSs) have strong capacity to discriminate cases of ankylosing spondylitis (AS) from healthy controls and individuals in the community with chronic back pain. METHODS: PRSs were developed and validated in individuals of European and East Asian ethnicity, using data from genome-wide association studies in 15 585 AS cases and 20 452 controls. The discriminatory values of PRSs in these populations were compared with other widely used diagnostic tests, including C-reactive protein (CRP), HLA-B27 and sacroiliac MRI. RESULTS: In people of European descent, PRS had high discriminatory capacity with area under the curve (AUC) in receiver operator characteristic analysis of 0.924. This was significantly better than for HLA-B27 testing alone (AUC=0.869), MRI (AUC=0.885) or C-reactive protein (AUC=0.700). PRS developed and validated in individuals of East Asian descent performed similarly (AUC=0.948). Assuming a prior probability of AS of 10% such as in patients with chronic back pain under 45 years of age, compared with HLA-B27 testing alone, PRS provides higher positive values for 35% of patients and negative predictive values for 67.5% of patients. For PRS, in people of European descent, the maximum positive predictive value was 78.2% and negative predictive value was 100%, whereas for HLA-B27, these values were 51.9% and 97.9%, respectively. CONCLUSIONS: PRS have higher discriminatory capacity for AS than CRP, sacroiliac MRI or HLA-B27 status alone. For optimal performance, PRS should be developed for use in the specific ethnic groups to which they are to be applied.

A critical role for miR-142 in alveolar epithelial lineage formation in mouse lung development.

The respiratory epithelium arises from alveolar epithelial progenitors which differentiate into alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. AT2 cells are stem cells in the lung critical for the repair process after injury. Mechanisms regulating AT1 and AT2 cell maturation are poorly defined. We report that the activation of the glucocorticoid pathway in an in vitro alveolar epithelial lineage differentiation assay led to increased AT2 marker Sftpc and decreased miR-142 expression. Using miR-142 KO mice, we demonstrate an increase in the AT2/AT1 cell number ratio. Overexpression of miR-142 in alveolar progenitor cells in vivo led to the opposite effect. Examination of the KO lungs at E18.5 revealed enhanced expression of miR-142 targets Apc, Ep300 and Kras associated with increased ß-catenin and p-Erk signaling. Silencing of miR-142 expression in lung explants grown in vitro triggers enhanced Sftpc expression as well as increased AT2/AT1 cell number ratio. Pharmacological inhibition of Ep300-ß-catenin but not Erk in vitro prevented the increase in Sftpc expression triggered by loss of miR-142. These results suggest that the glucocorticoid-miR-142-Ep300-ß-catenin signaling axis controls pneumocyte maturation.

Antithetical NFATc1-Sox2 and p53-miR200 signaling networks govern pancreatic cancer cell plasticity.

In adaptation to oncogenic signals, pancreatic ductal adenocarcinoma (PDAC) cells undergo epithelial-mesenchymal transition (EMT), a process combining tumor cell dedifferentiation with acquisition of stemness features. However, the mechanisms linking oncogene-induced signaling pathways with EMT and stemness remain largely elusive. Here, we uncover the inflammation-induced transcription factor NFATc1 as a central regulator of pancreatic cancer cell plasticity. In particular, we show that NFATc1 drives EMT reprogramming and maintains pancreatic cancer cells in a stem cell-like state through Sox2-dependent transcription of EMT and stemness factors. Intriguingly, NFATc1-Sox2 complex-mediated PDAC dedifferentiation and progression is opposed by antithetical p53-miR200c signaling, and inactivation of the tumor suppressor pathway is essential for tumor dedifferentiation and dissemination both in genetically engineered mouse models (GEMM) and human PDAC. Based on these findings, we propose the existence of a hierarchical signaling network regulating PDAC cell plasticity and suggest that the molecular decision between epithelial cell preservation and conversion into a dedifferentiated cancer stem cell-like phenotype depends on opposing levels of p53 and NFATc1 signaling activities.