Sfrp1 inhibits lung fibroblast invasion during transition to injury-induced myofibroblasts.

BACKGROUND: Fibroblast-to-myofibroblast conversion is a major driver of tissue remodelling in organ fibrosis. Distinct lineages of fibroblasts support homeostatic tissue niche functions, yet their specific activation states and phenotypic trajectories during injury and repair have remained unclear. METHODS: We combined spatial transcriptomics, multiplexed immunostainings, longitudinal single-cell RNA-sequencing and genetic lineage tracing to study fibroblast fates during mouse lung regeneration. Our findings were validated in idiopathic pulmonary fibrosis patient tissues in situ as well as in cell differentiation and invasion assays using patient lung fibroblasts. Cell differentiation and invasion assays established a function of SFRP1 in regulating human lung fibroblast invasion in response to transforming growth factor (TGF)ß1. MEASUREMENTS AND MAIN RESULTS: We discovered a transitional fibroblast state characterised by high Sfrp1 expression, derived from both Tcf21-Cre lineage positive and negative cells. Sfrp1 + cells appeared early after injury in peribronchiolar, adventitial and alveolar locations and preceded the emergence of myofibroblasts. We identified lineage-specific paracrine signals and inferred converging transcriptional trajectories towards Sfrp1 + transitional fibroblasts and Cthrc1 + myofibroblasts. TGFß1 downregulated SFRP1 in noninvasive transitional cells and induced their switch to an invasive CTHRC1+ myofibroblast identity. Finally, using loss-of-function studies we showed that SFRP1 modulates TGFß1-induced fibroblast invasion and RHOA pathway activity. CONCLUSIONS: Our study reveals the convergence of spatially and transcriptionally distinct fibroblast lineages into transcriptionally uniform myofibroblasts and identifies SFRP1 as a modulator of TGFß1-driven fibroblast phenotypes in fibrogenesis. These findings are relevant in the context of therapeutic interventions that aim at limiting or reversing fibroblast foci formation.

Targeting miR-34a/Pdgfra interactions partially corrects alveologenesis in experimental bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a common complication of preterm birth characterized by arrested lung alveolarization, which generates lungs that are incompetent for effective gas exchange. We report here deregulated expression of miR-34a in a hyperoxia-based mouse model of BPD, where miR-34a expression was markedly increased in platelet-derived growth factor receptor (PDGFR)α-expressing myofibroblasts, a cell type critical for proper lung alveolarization. Global deletion of miR-34a; and inducible, conditional deletion of miR-34a in PDGFRα+ cells afforded partial protection to the developing lung against hyperoxia-induced perturbations to lung architecture. Pdgfra mRNA was identified as the relevant miR-34a target, and using a target site blocker in vivo, the miR-34a/Pdgfra interaction was validated as a causal actor in arrested lung development. An antimiR directed against miR-34a partially restored PDGFRα+ myofibroblast abundance and improved lung alveolarization in newborn mice in an experimental BPD model. We present here the first identification of a pathology-relevant microRNA/mRNA target interaction in aberrant lung alveolarization and highlight the translational potential of targeting the miR-34a/Pdgfra interaction to manage arrested lung development associated with preterm birth.

Understanding alveolarization to induce lung regeneration.

BACKGROUND: Gas exchange represents the key physiological function of the lung, and is dependent upon proper formation of the delicate alveolar structure. Malformation or destruction of the alveolar gas-exchange regions are key histopathological hallmarks of diseases such as bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis; all of which are characterized by perturbations to the alveolo-capillary barrier structure. Impaired gas-exchange is the primary initial consequence of these perturbations, resulting in severe clinical symptoms, reduced quality of life, and death. The pronounced morbidity and mortality associated with malformation or destruction of alveoli underscores a pressing need for new therapeutic concepts. The re-induction of alveolarization in diseased lungs is a new and exciting concept in a regenerative medicine approach to manage pulmonary diseases that are characterized by an absence of alveoli. MAIN TEXT: Mechanisms of alveolarization first need to be understood, to identify pathways and mediators that may be exploited to drive the induction of alveolarization in the diseased lung. With this in mind, a variety of candidate cell-types, pathways, and molecular mediators have recently been identified. Using lineage tracing approaches and lung injury models, new progenitor cells for epithelial and mesenchymal cell types - as well as cell lineages which are able to acquire stem cell properties - have been discovered. However, the underlying mechanisms that orchestrate the complex process of lung alveolar septation remain largely unknown. CONCLUSION: While important progress has been made, further characterization of the contributing cell-types, the cell type-specific molecular signatures, and the time-dependent chemical and mechanical processes in the developing, adult and diseased lung is needed in order to implement a regenerative therapeutic approach for pulmonary diseases.

Recent advances in our understanding of the mechanisms of late lung development and bronchopulmonary dysplasia.

The objective of lung development is to generate an organ of gas exchange that provides both a thin gas diffusion barrier and a large gas diffusion surface area, which concomitantly generates a steep gas diffusion concentration gradient. As such, the lung is perfectly structured to undertake the function of gas exchange: a large number of small alveoli provide extensive surface area within the limited volume of the lung, and a delicate alveolo-capillary barrier brings circulating blood into close proximity to the inspired air. Efficient movement of inspired air and circulating blood through the conducting airways and conducting vessels, respectively, generates steep oxygen and carbon dioxide concentration gradients across the alveolo-capillary barrier, providing ideal conditions for effective diffusion of both gases during breathing. The development of the gas exchange apparatus of the lung occurs during the second phase of lung development-namely, late lung development-which includes the canalicular, saccular, and alveolar stages of lung development. It is during these stages of lung development that preterm-born infants are delivered, when the lung is not yet competent for effective gas exchange. These infants may develop bronchopulmonary dysplasia (BPD), a syndrome complicated by disturbances to the development of the alveoli and the pulmonary vasculature. It is the objective of this review to update the reader about recent developments that further our understanding of the mechanisms of lung alveolarization and vascularization and the pathogenesis of BPD and other neonatal lung diseases that feature lung hypoplasia.

A novel mouse Cre-driver line targeting Perilipin 2-expressing cells in the neonatal lung.

Pulmonary diseases such as chronic obstructive pulmonary disease, lung fibrosis, and bronchopulmonary dysplasia are characterized by the destruction or malformation of the alveolar regions of the lung. The underlying pathomechanisms at play are an area of intense interest since these mechanisms may reveal pathways suitable for interventions to drive reparative processes. Lipid-laden fibroblasts (lipofibroblasts) express the Perilipin 2 (Plin2) gene-product, PLIN2, commonly called adipose-differentiation related protein (ADRP). These cells are also thought to play a role in alveolarization and repair after injury to the alveolus. Progress in defining the functional contribution of lipofibroblasts to alveolar generation and repair is hampered by a lack of in vivo tools. The present study reports the generation of an inducible mouse Cre-driver line to target cells of the ADRP lineage. Robust Cre-mediated recombination in this mouse line was detected in mesenchymal cells of the postnatal lung, and in additional organs including the heart, liver, and spleen. The generation and validation of this valuable new tool to genetically target, manipulate, and trace cells of the ADRP lineage is critical for assessing the functional contribution of lipofibroblasts to lung development and repair.

Origin and characterization of alpha smooth muscle actin-positive cells during murine lung development.

ACTA2 expression identifies pulmonary airway and vascular smooth muscle cells (SMCs) as well as alveolar myofibroblasts (MYF). Mesenchymal progenitors expressing fibroblast growth factor 10 (Fgf10), Wilms tumor 1 (Wt1), or glioma-associated oncogene 1 (Gli1) contribute to SMC formation from early stages of lung development. However, their respective contribution and specificity to the SMC and/or alveolar MYF lineages remain controversial. In addition, the contribution of mesenchymal cells undergoing active WNT signaling remains unknown. Using Fgf10CreERT2 , Wt1CreERT2 , Gli1CreERT2 , and Axin2CreERT2 inducible driver lines in combination with a tdTomatoflox reporter line, the respective differentiation of each pool of labeled progenitor cells along the SMC and alveolar MYF lineages was quantified. The results revealed that while FGF10+ and WT1+ cells show a minor contribution to the SMC lineage, GLI1+ and AXIN2+ cells significantly contribute to both the SMC and alveolar MYF lineages, but with limited specificity. Lineage tracing using the Acta2-CreERT2 transgenic line showed that ACTA2+ cells labeled at embryonic day (E)11.5 do not expand significantly to give rise to new SMCs at E18.5. However, ACTA2+ cells labeled at E15.5 give rise to the majority (85%-97%) of the SMCs in the lung at E18.5 as well as alveolar MYF progenitors in the lung parenchyma. Fluorescence-activated cell sorting-based isolation of different subpopulations of ACTA2+ lineage-traced cells followed by gene arrays, identified transcriptomic signatures for alveolar MYF progenitors versus airway and vascular SMCs at E18.5. Our results establish a new transcriptional landscape for further experiments addressing the function of signaling pathways in the formation of different subpopulations of ACTA2+ cells. Stem Cells 2017;35:1566-1578.

Stereological monitoring of mouse lung alveolarization from the early postnatal period to adulthood.

Postnatal lung maturation generates a large number of small alveoli, with concomitant thinning of alveolar septal walls, generating a large gas exchange surface area but minimizing the distance traversed by the gases. This demand for a large and thin gas exchange surface area is not met in disorders of lung development, such as bronchopulmonary dysplasia (BPD) histopathologically characterized by fewer, larger alveoli and thickened alveolar septal walls. Diseases such as BPD are often modeled in the laboratory mouse to better understand disease pathogenesis or to develop new interventional approaches. To date, there have been no stereology-based longitudinal studies on postnatal mouse lung development that report dynamic changes in alveoli number or alveolar septal wall thickness during lung maturation. To this end, changes in lung structure were quantified over the first 22 mo of postnatal life of C57BL/6J mice. Alveolar density peaked at postnatal day (P)39 and remained unchanged at 9 mo (P274) but was reduced by 22 mo (P669). Alveoli continued to be generated, initially at an accelerated rate between P5 and P14, and at a slower rate thereafter. Between P274 and P669, loss of alveoli was noted, without any reduction in lung volume. A progressive thinning of the alveolar septal wall was noted between P5 and P28. Pronounced sex differences were observed in alveoli number in adult (but not juvenile) mice, when comparing male and female mouse lungs. This sex difference was attributed exclusively to the larger volume of male mouse lungs.

Fgf10 deficiency is causative for lethality in a mouse model of bronchopulmonary dysplasia.

Inflammation-induced FGF10 protein deficiency is associated with bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurely born infants characterized by arrested alveolar development. So far, experimental evidence for a direct role of FGF10 in lung disease is lacking. Using the hyperoxia-induced neonatal lung injury as a mouse model of BPD, the impact of Fgf10 deficiency in Fgf10+/- versus Fgf10+/+ pups was investigated. In normoxia, no lethality of Fgf10+/+ or Fgf10+/- pups was observed. By contrast, all Fgf10+/- pups died within 8 days of hyperoxic injury, with lethality starting at day 5, whereas Fgf10+/+ pups were all alive. Lungs of pups from the two genotypes were collected on postnatal day 3 following normoxia or hyperoxia exposure for further analysis. In hyperoxia, Fgf10+/- lungs exhibited increased hypoalveolarization. Analysis by FACS of the Fgf10+/- versus control lungs in normoxia revealed a decreased ratio of alveolar epithelial type II (AECII) cells over total Epcam-positive cells. In addition, gene array analysis indicated reduced AECII and increased AECI transcriptome signatures in isolated AECII cells from Fgf10+/- lungs. Such an imbalance in differentiation is also seen in hyperoxia and is associated with reduced mature surfactant protein B and C expression. Attenuation of the activity of Fgfr2b ligands postnatally in the context of hyperoxia also led to increased lethality with decreased surfactant expression. In summary, decreased Fgf10 mRNA levels lead to congenital lung defects, which are compatible with postnatal survival, but which compromise the ability of the lungs to cope with sub-lethal hyperoxic injury. Fgf10 deficiency affects quantitatively and qualitatively the formation of AECII cells. In addition, Fgfr2b ligands are also important for repair after hyperoxia exposure in neonates. Deficient AECII cells could be an additional complication for patients with BPD. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Tamoxifen dosing for Cre-mediated recombination in experimental bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is the most common complication of preterm birth characterized by blunted post-natal lung development. BPD can be modelled in mice by exposure of newborn mouse pups to elevated oxygen levels. Little is known about the mechanisms of perturbed lung development associated with BPD. The advent of transgenic mice, where genetic rearrangements can be induced in particular cell-types at particular time-points during organogenesis, have great potential to explore the pathogenic mechanisms at play during arrested lung development. Many inducible, conditional transgenic technologies available rely on the application of the estrogen-receptor modulator, tamoxifen. While tamoxifen is well-tolerated and has been widely employed in adult mice, or in healthy developing mice; tamoxifen is not well-tolerated in combination with hyperoxia, in the most widely-used mouse model of BPD. To address this, we set out to establish a safe and effective tamoxifen dosing regimen that can be used in newborn mouse pups subjected to injurious stimuli, such as exposure to elevated levels of environmental oxygen. Our data reveal that a single intraperitoneal dose of tamoxifen of 0.2 mg applied to newborn mouse pups in 10 µl Miglyol vehicle was adequate to successfully drive Cre recombinase-mediated genome rearrangements by the fifth day of life, in a murine model of BPD. The number of recombined cells was comparable to that observed in regular tamoxifen administration protocols. These findings will be useful to investigators where tamoxifen dosing is problematic in the background of injurious stimuli and mouse models of human and veterinary disease.

Two-Way Conversion between Lipogenic and Myogenic Fibroblastic Phenotypes Marks the Progression and Resolution of Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a form of progressive interstitial lung disease with unknown etiology. Due to a lack of effective treatment, IPF is associated with a high mortality rate. The hallmark feature of this disease is the accumulation of activated myofibroblasts that excessively deposit extracellular matrix proteins, thus compromising lung architecture and function and hindering gas exchange. Here we investigated the origin of activated myofibroblasts and the molecular mechanisms governing fibrosis formation and resolution. Genetic engineering in mice enables the time-controlled labeling and monitoring of lipogenic or myogenic populations of lung fibroblasts during fibrosis formation and resolution. Our data demonstrate a lipogenic-to-myogenic switch in fibroblastic phenotype during fibrosis formation. Conversely, we observed a myogenic-to-lipogenic switch during fibrosis resolution. Analysis of human lung tissues and primary human lung fibroblasts indicates that this fate switching is involved in IPF pathogenesis, opening potential therapeutic avenues to treat patients.

Characterization of the platelet-derived growth factor receptor-α-positive cell lineage during murine late lung development.

A reduced number of alveoli is the structural hallmark of diseases of the neonatal and adult lung, where alveoli either fail to develop (as in bronchopulmonary dysplasia), or are progressively destroyed (as in chronic obstructive pulmonary disease). To correct the loss of alveolar septa through therapeutic regeneration, the mechanisms of septa formation must first be understood. The present study characterized platelet-derived growth factor receptor-α-positive (PDGFRα(+)) cell populations during late lung development in mice. PDGFRα(+) cells (detected using a PDGFRα(GFP) reporter line) were noted around the proximal airways during the pseudoglandular stage. In the canalicular stage, PDGFRα(+) cells appeared in the more distal mesenchyme, and labeled α-smooth muscle actin-positive tip cells in the secondary crests and lipofibroblasts in the primary septa during alveolarization. Some PDGFRα(+) cells appeared in the mesenchyme of the adult lung. Over the course of late lung development, PDGFRα(+) cells consistently expressed collagen I, and transiently expressed markers of mesenchymal stem cells. With the use of both, a constitutive and a conditional PDGFRα(Cre) line, it was observed that PDGFRα(+) cells generated alveolar myofibroblasts including tip cells of the secondary crests, and lipofibroblasts. These lineages were committed before secondary septation. The present study provides new insights into the time-dependent commitment of the PDGFRα(+) cell lineage to lipofibroblasts and myofibroblasts during late lung development that is needed to better understand the cellular contribution to the process of alveolarization.

Phenotypical and ultrastructural features of Oct4-positive cells in the adult mouse lung.

Octamer binding trascription factor 4 (Oct4) is a transcription factor of POU family specifically expressed in embryonic stem cells (ESCs). A role for maintaining pluripotency and self-renewal of ESCs is assigned to Oct4 as a pluripotency marker. Oct4 can also be detected in adult stem cells such as bone marrow-derived mesenchymal stem cells. Several studies suggest a role for Oct4 in sustaining self-renewal capacity of adult stem cells. However, Oct4 gene ablation in adult stem cells revealed no abnormalities in tissue turnover or regenerative capacity. In the present study we have conspicuously found pulmonary Oct4-positive cells closely resembling the morphology of telocytes (TCs). These cells were found in the perivascular and peribronchial areas and their presence and location were confirmed by electron microscopy. Moreover, we have used Oct4-GFP transgenic mice which revealed a similar localization of the Oct4-GFP signal. We also found that Oct4 co-localized with several described TC markers such as vimentin, Sca-1, platelet-derived growth factor receptor-beta C-kit and VEGF. By flow cytometry analyses carried out with Oct4-GFP reporter mice, we described a population of EpCAM(neg) /CD45(neg) /Oct4-GFP(pos) that in culture displayed TC features. These results were supported by qRT-PCR with mRNA isolated from lungs by using laser capture microdissection. In addition, Oct4-positive cells were found to express Nanog and Klf4 mRNA. It is concluded for the first time that TCs in adult lung mouse tissue comprise Oct4-positive cells, which express pluripotency-related genes and represent therefore a population of adult stem cells which might contribute to lung regeneration.

Quantitative proteome analysis of alveolar type-II cells reveals a connection of integrin receptor subunits beta 2/6 and WNT signaling.

Alveolar type-II cells (ATII cells) are lung progenitor cells responsible for regeneration of alveolar epithelium during homeostatic turnover and in response to injury. Characterization of ATII cells will have a profound impact on our understanding and treatment of lung disease. The identification of novel ATII cell-surface proteins can be used for sorting and enrichment of these cells for further characterization. Here we combined a high-resolution mass spectrometry-based membrane proteomic approach using lungs of the SILAC mice with an Affymetrix microarray-based transcriptome analysis of ATII cells. We identified 16 proteins that are enriched in the membrane fraction of ATII cells and whose genes are highly expressed in these cells. Interestingly, we confirmed our data for two of these genes, integrin beta 2 and 6 (Itgb2 and Itgb6), by qRT-PCR expression analysis and Western blot analysis of protein extracts. Moreover, flow cytometry and immunohistochemistry in adult lung revealed that ITGB2 and ITGB6 are present in subpopulations of surfactant-associated-protein-C-positive cells, suggesting the existence of different types of ATII cells. Furthermore, analysis of the Itgb2(-/-) mice showed that Itgb2 is required for proper WNT signaling regulation in the lung.

Spatiotemporal expression of flk-1 in pulmonary epithelial cells during lung development.

Vascular endothelial growth factor-A (VEGF-A) responsive effects mediated via the receptors fetal liver kinase-1 (flk-1) and fms-like tyrosine kinase (flt-1), are key processes of pulmonary vascular development. Flk-1 has been shown to be involved in early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis. Recent reports suggested a role of VEGF-A in lung epithelial cell function. Based on these observations, we hypothesize that epithelial flk-1 has a unique function in pulmonary development. Thus, the aim of this study is to elucidate spatiotemporal expression of flk-1 during lung development with respect to the epithelial system. Embryonic lungs were screened for flk-1 messenger RNA and protein at daily intervals, including postnatal stages. From Embryonic Day (ED) 12.5 through ED 15.5, flk-1 expression was restricted to the early vascular primitive network, while from ED 16.5 on flk-1 was detectable in the epithelial system and persisted there postnatally. At postnatal stages, flk-1 expression was increasingly restricted to individual cells in the alveolar septa. Isolation and in vitro cultivation of alveolar epithelial cells confirmed flk-1 expression and showed VEGF secretion into the supernatant. To our knowledge, this is the first murine study characterizing epithelial flk-1 expression at different stages throughout lung organogenesis until birth and at postnatal stages. To confirm epithelial flk-1 expression, we performed reporter gene analysis of the flk-1 promoter in vivo. Investigations on transgenic mouse strains, containing either a complete or incomplete flk-1 promoter driving expression of the lacZ reporter gene, suggested differential flk-1 regulation in endothelial and epithelial cells.

Down-regulation of protein kinase Ceta by antisense oligonucleotides sensitises A549 lung cancer cells to vincristine and paclitaxel.

Previous studies point to protein kinase C (PKC) isozyme eta as a resistance factor in cancer cells. Therefore, we investigated whether down-regulation of PKCeta with second generation antisense oligonucleotides (ODNs) would sensitise A549 human lung carcinoma cells to cytostatics. The effects were compared to the outcome of Bcl-xL down-regulation. Upon treatment with antisense ODNs, PKCeta and Bcl-xL were both significantly reduced on mRNA and protein level. Down-regulation of either PKCeta or Bcl-xL in combination with vincristine or paclitaxel resulted in a significant increase in caspase-3 activity compared to that in the control oligonucleotide treated cells. In addition, PKCeta down-regulation augmented vincristine-induced dissipation of mitochondrial transmembrane potential. In conclusion, these results confirm that PKCeta might represent a considerable resistance factor and an interesting target to improve anticancer chemotherapy.