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.

Senescence of lung mesenchymal stem cells of preterm infants by cyclic stretch and hyperoxia via p21.

Phenotype distortion of lung resident mesenchymal stem cells (MSC) in preterm infants is a hallmark event in the pathogenesis of bronchopulmonary dysplasia (BPD). Here, we evaluated the impact of cyclic mechanical stretch (CMS) and hyperoxia (HOX). The negative action of HOX on proliferation and cell death was more pronounced at 80% than at 40%. Although the impact of CMS alone was modest, CMS plus HOX displayed the strongest effect sizes. Exposure to CMS and/or HOX induced the downregulation of PDGFRα, and cellular senescence preceded by p21 accumulation. p21 interference interfered with cellular senescence and resulted in aggravated cell death, arguing for a prosurvival mechanism. HOX 40% and limited exposure to HOX 80% prevailed in a reversible phenotype with reuptake of proliferation, while prolonged exposure to HOX 80% resulted in definite MSC growth arrest. Our mechanistic data explain how HOX and CMS induce the effects on MSC phenotype disruption. The results are congruent with the clinical observation that preterm infants requiring supplemental oxygen plus mechanical ventilation are at particular risk for BPD. Although inhibiting p21 is not a feasible approach, limiting the duration and magnitude of the exposures is promising.NEW & NOTEWORTHY Rarefication of lung mesenchymal stem cells (MSC) due to exposure to cyclic mechanical stretch (CMS) during mechanical ventilation with oxygen-rich gas is a hallmark of bronchopulmonary dysplasia in preterm infants, but the pathomechanistic understanding is incomplete. Our studies identify a common signaling mechanism mediated by p21 accumulation, leading to cellular senescence and cell death, most pronounced during the combined exposure with in principle reversible phenotype change depending on strength and duration of exposures.

Multilineage murine stem cells generate complex organoids to model distal lung development and disease.

Organoids derived from mouse and human stem cells have recently emerged as a powerful tool to study organ development and disease. We here established a three-dimensional (3D) murine bronchioalveolar lung organoid (BALO) model that allows clonal expansion and self-organization of FACS-sorted bronchioalveolar stem cells (BASCs) upon co-culture with lung-resident mesenchymal cells. BALOs yield a highly branched 3D structure within 21 days of culture, mimicking the cellular composition of the bronchioalveolar compartment as defined by single-cell RNA sequencing and fluorescence as well as electron microscopic phenotyping. Additionally, BALOs support engraftment and maintenance of the cellular phenotype of injected tissue-resident macrophages. We also demonstrate that BALOs recapitulate lung developmental defects after knockdown of a critical regulatory gene, and permit modeling of viral infection. We conclude that the BALO model enables reconstruction of the epithelial-mesenchymal-myeloid unit of the distal lung, thereby opening numerous new avenues to study lung development, infection, and regenerative processes in vitro.

Evidence for a lipofibroblast-to- Cthrc1 + myofibroblast reversible switch during the development and resolution of lung fibrosis in young mice.

BACKGROUND: Fibrosis is often associated with aberrant repair mechanisms that ultimately lead to organ failure. In the lung, idiopathic pulmonary fibrosis (IPF) is a fatal form of interstitial lung disease (ILD) to which there is currently no curative therapy. From the cell biology point of view, the cell of origin and eventual fate of activated myofibroblasts (aMYFs) have taken center stage as these cells are believed to drive structural remodeling and lung function impairment. While aMYFs are now widely believed to originate from resident fibroblasts, the heterogeneity of aMYFs and ultimate fate during fibrosis resolution remain elusive. We have previously shown that aMYFs dedifferentiation and acquisition of a lipofibroblast (LIF)-like phenotype represent a route of fibrosis resolution. METHODS: In this study, we combined genetic lineage tracing and single-cell transcriptomics in mice, and data mining of human IPF datasets to decipher the heterogeneity of aMYFs and investigate differentiation trajectories during fibrosis resolution. Furthermore, organoid cultures were utilized as a functional readout for the alveolar mesenchymal niche activity during various phases of injury and repair in mice. RESULTS: Our data demonstrate that aMYFs consist of four subclusters displaying unique pro-alveologenic versus profibrotic profiles. Alveolar fibroblasts displaying a high LIF-like signature largely constitute both the origin and fate of aMYFs during fibrogenesis and resolution respectively. The heterogeneity of aMYFs is conserved in humans and a significant proportion of human aMYFs displays a high LIF signature. CONCLUSION: Our work identifies a subcluster of aMYFs that is potentially relevant for future management of IPF.

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.

Mesenchyme-derived vertebrate lonesome kinase controls lung organogenesis by altering the matrisome.

Vertebrate lonesome kinase (VLK) is the only known secreted tyrosine kinase and responsible for the phosphorylation of a broad range of secretory pathway-resident and extracellular matrix proteins. However, its cell-type specific functions in vivo are still largely unknown. Therefore, we generated mice lacking the VLK gene (protein kinase domain containing, cytoplasmic (Pkdcc)) in mesenchymal cells. Most of the homozygous mice died shortly after birth, most likely as a consequence of their lung abnormalities and consequent respiratory failure. E18.5 embryonic lungs showed a reduction of alveolar type II cells, smaller bronchi, and an increased lung tissue density. Global mass spectrometry-based quantitative proteomics identified 97 proteins with significantly and at least 1.5-fold differential abundance between genotypes. Twenty-five of these had been assigned to the extracellular region and 15 to the mouse matrisome. Specifically, fibromodulin and matrilin-4, which are involved in extracellular matrix organization, were significantly more abundant in lungs from Pkdcc knockout embryos. These results support a role for mesenchyme-derived VLK in lung development through regulation of matrix dynamics and the resulting modulation of alveolar epithelial cell differentiation.

Notch1 Induces Defective Epithelial Surfactant Processing and Pulmonary Fibrosis.

Rationale: Although type II alveolar epithelial cells (AEC2s) are chronically injured in idiopathic pulmonary fibrosis (IPF), they contribute to epithelial regeneration in IPF. Objectives: We hypothesized that Notch signaling may contribute to AEC2 proliferation, dedifferentiation characterized by loss of surfactant processing machinery, and lung fibrosis in IPF. Methods: We applied microarray analysis, kinome profiling, flow cytometry, immunofluorescence analysis, western blotting, quantitative PCR, and proliferation and surface activity analysis to study epithelial differentiation, proliferation, and matrix deposition in vitro (AEC2 lines, primary murine/human AEC2s), ex vivo (human IPF-derived precision-cut lung slices), and in vivo (bleomycin and pepstatin application, Notch1 [Notch receptor 1] intracellular domain overexpression). Measurements and Main Results: We document here extensive SP-B and -C (surfactant protein-B and -C) processing defects in IPF AEC2s, due to loss of Napsin A, resulting in increased intra-alveolar surface tension and alveolar collapse and induction of endoplasmic reticulum stress in AEC2s. In vivo pharmacological inhibition of Napsin A results in the development of AEC2 injury and overt lung fibrosis. We also demonstrate that Notch1 signaling is already activated early in IPF and determines AEC2 fate by inhibiting differentiation (reduced lamellar body compartment, reduced capacity to process hydrophobic SP) and by causing increased epithelial proliferation and development of lung fibrosis, putatively via altered JAK (Janus kinase)/Stat (signal transducer and activator of transcription) signaling in AEC2s. Conversely, inhibition of Notch signaling in IPF-derived precision-cut lung slices improved the surfactant processing capacity of AEC2s and reversed fibrosis. Conclusions: Notch1 is a central regulator of AEC2 fate in IPF. It induces alveolar epithelial proliferation and loss of Napsin A and of surfactant proprotein processing, and it contributes to fibroproliferation.

Complex urban atmosphere alters alveolar stem cells niche properties and drives lung fibrosis.

There is growing evidence suggesting that urban pollution has adverse effects on lung health. However, how urban pollution affects alveolar mesenchymal and epithelial stem cell niches remains unknown. This study aimed to determine how complex representative urban atmospheres alter alveolar stem cell niche properties. Mice were placed in an innovative chamber realistically simulating the atmosphere of a megalopolis, or "clean air," for 7 days. Lungs were collected, and fibroblasts and epithelial cells (EpCAM+) were isolated. Proliferative capacities of fibroblasts were tested by population doubling levels (PDL), and microarray analyses were performed. Fibroblasts and EpCAM+ cells from exposed, nonexposed, or naive mice were cocultured in organoid assays to assess the stem cell properties. Collagen deposition (Sirius red), lipofibroblasts (ADRP, COL1A1), myofibroblasts (αSMA), alveolar type 2 cells (AT2, SFTPC+), and alveolar differentiation intermediate cell [ADI, keratin-8-positive (KRT8+)/claudin-4-positive (CLDN4+)] markers were quantified in the lungs. Fibroblasts obtained from mice exposed to urban atmosphere had lower PDL and survival and produced fewer and smaller organoids. Microarray analysis showed a decrease of adipogenesis and an increase of genes associated with fibrosis, suggesting a lipofibroblast to myofibroblast transition. Collagen deposition and myofibroblast number increased in the lungs of urban atmosphere-exposed mice. AT2 number was reduced and associated with an increase in ADI cells KRT8+/CLDN4+. Furthermore, EpCAM+ cells from exposed mice also produced fewer and smaller organoids. In conclusion, urban atmosphere alters alveolar mesenchymal stem cell niche properties by inducing a lipofibroblast to myofibroblast shift. It also results in alveolar epithelial dysfunction and a fibrotic-like phenotype.NEW & NOTEWORTHY Urban pollution is known to have major adverse effects on lung health. To assess the effect of pollution on alveolar regeneration, we exposed adult mice to a simulated high-pollution urban atmosphere, using an innovative CESAM simulation chamber (Multiphase Atmospheric Experimental Simulation Chamber, https://cesam.cnrs.fr/). We demonstrated that urban atmosphere alters alveolar mesenchymal stem cell niche properties by inducing a lipofibroblast to myofibroblast shift and induces alveolar epithelial dysfunction.

miR-142-3p regulates cortical oligodendrocyte gene co-expression networks associated with tauopathy.

Oligodendrocytes exist in a heterogenous state and are implicated in multiple neuropsychiatric diseases including dementia. Cortical oligodendrocytes are a glial population uniquely positioned to play a key role in neurodegeneration by synchronizing circuit connectivity but molecular pathways specific to this role are lacking. We utilized oligodendrocyte-specific translating ribosome affinity purification and RNA-seq (TRAP-seq) to transcriptionally profile adult mature oligodendrocytes from different regions of the central nervous system. Weighted gene co-expression network analysis reveals distinct region-specific gene networks. Two of these mature myelinating oligodendrocyte gene networks uniquely define cortical oligodendrocytes and differentially regulate cortical myelination (M8) and synaptic signaling (M4). These two cortical oligodendrocyte gene networks are enriched for genes associated with dementia including MAPT and include multiple gene targets of the regulatory microRNA, miR-142-3p. Using a combination of TRAP-qPCR, miR-142-3p overexpression in vitro, and miR-142-null mice, we show that miR-142-3p negatively regulates cortical myelination. In rTg4510 tau-overexpressing mice, cortical myelination is compromised, and tau-mediated neurodegeneration is associated with gene co-expression networks that recapitulate both the M8 and M4 cortical oligodendrocyte gene networks identified from normal cortex. We further demonstrate overlapping gene networks in mature oligodendrocytes present in normal cortex, rTg4510 and miR-142-null mice, and existing datasets from human tauopathies to provide evidence for a critical role of miR-142-3p-regulated cortical myelination and oligodendrocyte-mediated synaptic signaling in neurodegeneration.

Fibroblast growth factor 10 is a negative regulator of postnatal neurogenesis in the mouse hypothalamus.

New neurons are generated in the postnatal rodent hypothalamus, with a subset of tanycytes in the third ventricular (3V) wall serving as neural stem/progenitor cells. However, the precise stem cell niche organization, the intermediate steps and the endogenous regulators of postnatal hypothalamic neurogenesis remain elusive. Quantitative lineage-tracing in vivo revealed that conditional deletion of fibroblast growth factor 10 (Fgf10) from Fgf10-expressing ß-tanycytes at postnatal days (P)4-5 results in the generation of significantly more parenchymal cells by P28, composed mostly of ventromedial and dorsomedial neurons and some glial cells, which persist into adulthood. A closer scrutiny in vivo and ex vivo revealed that the 3V wall is not static and is amenable to cell movements. Furthermore, normally ß-tanycytes give rise to parenchymal cells via an intermediate population of α-tanycytes with transient amplifying cell characteristics. Loss of Fgf10 temporarily attenuates the amplification of ß-tanycytes but also appears to delay the exit of their α-tanycyte descendants from the germinal 3V wall. Our findings suggest that transience of cells through the α-tanycyte domain is a key feature, and Fgf10 is a negative regulator of postnatal hypothalamic neurogenesis.

Fibroblast growth factor 10 reverses cigarette smoke- and elastase-induced emphysema and pulmonary hypertension in mice.

BACKGROUND: COPD is an incurable disease and a leading cause of death worldwide. In mice, fibroblast growth factor (FGF)10 is essential for lung morphogenesis, and in humans, polymorphisms in the human FGF10 gene correlate with an increased susceptibility to develop COPD. METHODS: We analysed FGF10 signalling in human lung sections and isolated cells from healthy donor, smoker and COPD lungs. The development of emphysema and PH was investigated in Fgf10+/- and Fgfr2b+/- (FGF receptor 2b) mice upon chronic exposure to cigarette smoke. In addition, we overexpressed FGF10 in mice following elastase- or cigarette smoke-induced emphysema and pulmonary hypertension (PH). RESULTS: We found impaired FGF10 expression in human lung alveolar walls and in primary interstitial COPD lung fibroblasts. In contrast, FGF10 expression was increased in large pulmonary vessels in COPD lungs. Consequently, we identified impaired FGF10 signalling in alveolar walls as an integral part of the pathomechanism that leads to emphysema and PH development: mice with impaired FGF10 signalling (Fgf10+/- and Fgfr2b+/- ) spontaneously developed lung emphysema, PH and other typical pathomechanistic features that generally arise in response to cigarette smoke exposure. CONCLUSION: In a therapeutic approach, FGF10 overexpression successfully restored lung alveolar and vascular structure in mice with established cigarette smoke- and elastase-induced emphysema and PH. FGF10 treatment triggered an initial increase in the number of alveolar type 2 cells that gradually returned to the basal level when the FGF10-mediated repair process progressed. Therefore, the application of recombinant FGF10 or stimulation of the downstream signalling cascade might represent a novel therapeutic strategy in the future.

Wnt5a Promotes AT1 and Represses AT2 Lineage-Specific Gene Expression in a Cell-Context-Dependent Manner.

Lung maturation is not limited to proper structural development but also includes differentiation and functionality of various highly specialized alveolar cell types. Alveolar type 1 (AT1s) cells occupy nearly 95% of the alveolar surface and are critical for establishing efficient gas exchange in the mature lung. AT1 cells arise from progenitors specified during the embryonic stage as well as alveolar epithelial progenitors expressing surfactant protein C (Sftpcpos cells) during postnatal and adult stages. Previously, we found that Wnt5a, a non-canonical Wnt ligand, is required for differentiation of AT1 cells during the saccular phase of lung development. To further investigate the role of Wnt5a in AT1 cell differentiation, we generated and characterized a conditional Wnt5a gain-of-function mouse model. Neonatal Wnt5a gain-of-function disrupted alveologenesis through inhibition of cell proliferation. In this setting Wnt5a downregulated ß-catenin-dependent canonical Wnt signaling, repressed AT2 (anti-AT2) and promoted AT1 (pro-AT1) lineage-specific gene expression. In addition, we identified 2 subpopulations of Sftpchigh and Sftpclow alveolar epithelial cells. In Sftpclow cells, Wnt5a exhibits pro-AT1 and anti-AT2 effects, concurrent with inhibition of canonical Wnt signaling. Interestingly, in the Sftpchigh subpopulation, although increasing AT1 lineage-specific gene expression, Wnt5a gain-of-function did not change AT2 gene expression, nor inhibit canonical Wnt signaling. Using primary epithelial cells isolated from human fetal lungs, we demonstrate that this property of Wnt5a is evolutionarily conserved. Wnt5a therefore serves as a selective regulator that ensures proper AT1/AT2 balance in the developing lung.

FGF10 Triggers De Novo Alveologenesis in a Bronchopulmonary Dysplasia Model: Impact on Resident Mesenchymal Niche Cells.

Bronchopulmonary dysplasia (BPD) is a neonatal lung disease developing in premature babies characterized by arrested alveologenesis and associated with decreased Fibroblast growth factor 10 (FGF10) expression. One-week hyperoxia (HYX) exposure of newborn mice leads to a permanent arrest in alveologenesis. To test the role of Fgf10 signaling to promote de novo alveologenesis following hyperoxia, we used transgenic mice allowing inducible expression of Fgf10 and recombinant FGF10 (rFGF10) protein delivered intraperitoneally. We carried out morphometry analysis, and IF on day 45. Alveolospheres assays were performed co-culturing AT2s from normoxia (NOX) with FACS-isolated Sca1Pos resident mesenchymal cells (rMC) from animals exposed to NOX, HYX-PBS, or HYX-FGF10. scRNAseq between rMC-Sca1Pos isolated from NOX and HYX-PBS was also carried out. Transgenic overexpression of Fgf10 and rFGF10 administration rescued the alveologenesis defects following HYX. Alveolosphere assays indicate that the activity of rMC-Sca1Pos is negatively impacted by HYX and partially rescued by rFGF10 treatment. Analysis by IF demonstrates a significant impact of rFGF10 on the activity of resident mesenchymal cells. scRNAseq results identified clusters expressing Fgf10, Fgf7, Pdgfra, and Axin2, which could represent the rMC niche cells for the AT2 stem cells. In conclusion, we demonstrate that rFGF10 administration is able to induce de novo alveologenesis in a BPD mouse model and identified subpopulations of rMC-Sca1Pos niche cells potentially representing its cellular target.

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.

A dominant negative mutation uncovers cooperative control of caudal Wolffian duct development by Sprouty genes.

The Wolffian ducts (WD) are paired epithelial tubules central to the development of the mammalian genitourinary tract. Outgrowths from the WD known as the ureteric buds (UB) generate the collecting ducts of the kidney. Later during development, the caudal portion of the WD will form the vas deferens, epididymis and seminal vesicle in males, and will degenerate in females. While the genetic pathways controlling the development of the UB are firmly established, less is known about those governing development of WD portions caudal to the UB. Sprouty proteins are inhibitors of receptor tyrosine kinase (RTK) signaling in vivo. We have recently shown that homozygous mutation of a conserved tyrosine (Tyr53) of Spry1 results in UB defects indistinguishable from that of Spry1 null mice. Here, we show that heterozygosity for the Spry1 Y53A allele causes caudal WD developmental defects consisting of ectopically branched seminal vesicles in males and persistent WD in females, without affecting kidney development. Detailed analysis reveals that this phenotype also occurs in Spry1+/- mice but with a much lower penetrance, indicating that removal of tyrosine 53 generates a dominant negative mutation in vivo. Supporting this notion, concomitant deletion of one allele of Spry1 and Spry2 also recapitulates the genital phenotype of Spry1Y53A/+ mice with high penetrance. Mechanistically, we show that unlike the effects of Spry1 in kidney development, these caudal WD defects are independent of Ret signaling, but can be completely rescued by lowering the genetic dosage of Fgf10. In conclusion, mutation of tyrosine 53 of Spry1 generates a dominant negative allele that uncovers fine-tuning of caudal WD development by Sprouty genes.

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.

Stem/Progenitor Cells and Related Therapy in Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease commonly seen in preterm infants, and is triggered by infection, mechanical ventilation, and oxygen toxicity. Among other problems, lifelong limitations in lung function and impaired psychomotor development may result. Despite major advances in understanding the disease pathologies, successful interventions are still limited to only a few drug therapies with a restricted therapeutic benefit, and which sometimes have significant side effects. As a more promising therapeutic option, mesenchymal stem cells (MSCs) have been in focus for several years due to their anti-inflammatory effects and their secretion of growth and development promoting factors. Preclinical studies provide evidence in that MSCs have the potential to contribute to the repair of lung injuries. This review provides an overview of MSCs, and other stem/progenitor cells present in the lung, their identifying characteristics, and their differentiation potential, including cytokine/growth factor involvement. Furthermore, animal studies and clinical trials using stem cells or their secretome are reviewed. To bring MSC-based therapeutic options further to clinical use, standardized protocols are needed, and upcoming side effects must be critically evaluated. To fill these gaps of knowledge, the MSCs' behavior and the effects of their secretome have to be examined in more (pre-) clinical studies, from which only few have been designed to date.