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 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.

Microscopic computed tomography with AI-CNN powered image analysis: the path to phenotype the bleomycin-induced pulmonary injury.

Bleomycin (BLM)-induced lung injury in mice is a valuable model for investigating the molecular mechanisms that drive inflammation and fibrosis and for evaluating potential therapeutic approaches to treat the disease. Given high variability in the BLM model, it is critical to accurately phenotype the animals in the course of an experiment. In the current study, we aimed to demonstrate the utility of microscopic computed tomography (µCT) imaging combined with an artificial intelligence (AI) convolutional neural network (CNN)-powered lung segmentation for rapid phenotyping of BLM mice. µCT was performed in freely breathing C57Bl/6J mice under isoflurane anaesthesia on days 7 and 21 post BLM administration. Terminal invasive lung function measurement and histological assessment of the left lung collagen content were conducted as well. µCT image analysis demonstrated gradual and time-dependent development of lung injury as evident by alterations in the lung density, air-to-tissue volume ratio, and lung aeration in mice treated with BLM. The right and left lung were unequally affected. µCT-derived parameters such as lung density, air-to-tissue volume ratio, and non-aerated lung volume correlated well with the invasive lung function measurement and left lung collagen content. Our study demonstrates the utility of AI-CNN-powered µCT image analysis for rapid and accurate phenotyping of BLM mice in the course of the disease development and progression.

Alveolar macrophage-expressed Plet1 is a driver of lung epithelial repair after viral pneumonia.

Influenza A virus (IAV) infection mobilizes bone marrow-derived macrophages (BMDM) that gradually undergo transition to tissue-resident alveolar macrophages (TR-AM) in the inflamed lung. Combining high-dimensional single-cell transcriptomics with complex lung organoid modeling, in vivo adoptive cell transfer, and BMDM-specific gene targeting, we found that transitioning ("regenerative") BMDM and TR-AM highly express Placenta-expressed transcript 1 (Plet1). We reveal that Plet1 is released from alveolar macrophages, and acts as important mediator of macrophage-epithelial cross-talk during lung repair by inducing proliferation of alveolar epithelial cells and re-sealing of the epithelial barrier. Intratracheal administration of recombinant Plet1 early in the disease course attenuated viral lung injury and rescued mice from otherwise fatal disease, highlighting its therapeutic potential.

Pulmonary siRNA Delivery with Sophisticated Amphiphilic Poly(Spermine Acrylamides) for the Treatment of Lung Fibrosis.

RNA interference (RNAi) is an efficient strategy to post-transcriptionally silence gene expression. While all siRNA drugs on the market target the liver, the lung offers a variety of currently undruggable targets, which can potentially be treated with RNA therapeutics. To achieve this goal, the synthesis of poly(spermine acrylamides) (P(SpAA) is reported herein. Polymers are prepared via polymerization of N-acryloxysuccinimide (NAS) and afterward this active ester is converted into spermine-based pendant groups. Copolymerizations with decylacrylamide are employed to increase the hydrophobicity of the polymers. After deprotection, polymers show excellent siRNA encapsulation to obtain perfectly sized polyplexes at very low polymer/RNA ratios. In vitro 2D and 3D cell culture, ex vivo and in vivo experiments reveal superior properties of amphiphilic spermine-copolymers with respect to delivery of siRNA to lung cells in comparison to commonly used lipid-based transfection agents. In line with the in vitro results, siRNA delivery to human lung explants confirm more efficient gene silencing of protease-activated receptor 2 (PAR2), a G protein-coupled receptor involved in fibrosis. This study reveals the importance of the balance between efficient polyplex formation, cellular uptake, gene knockdown, and toxicity for efficient siRNA delivery in vitro, in vivo, and in fibrotic human lung tissue ex vivo.

Decoding the role of fatty acids and their metabolites in lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and life­threatening interstitial lung disease of familial or sporadic onset. The incidence and prevalence of IPF range from 0.09 to 1.3 and from 0.33 to 4.51 per 10 000 people, respectively. IPF has a poor prognosis, and death usually occurs within 2 to 5 years following the diagnosis due to secondary respiratory failure. Currently, there are 2 drugs available to treat IPF, pirfenidone and nintedanib. Both only slow the disease progression and, in addition, have unfavorable safety profiles. IPF bears the histology of usual interstitial pneumonia, which is characterized by bronchiolization of distal airspaces, honeycombing, fibroblastic foci, and abnormal epithelial hyperplasia. In the last years, alterations in metabolic pathways, in particular those associated with fatty acid (FA) metabolism have been linked with the pathogenesis of lung fibrosis. Changes in FA profiles have been reported in lung tissue, plasma, and bronchoalveolar lavage fluid of IPF patients, and have been found to correlate with the disease progression and outcome. In addition, they have been associated with the development of a profibrotic phenotype of epithelial cells, macrophages, and fibroblasts / myofibroblasts contributing to their (trans)differentiation and production of the disease­relevant mediators. Furthermore, strategies focusing on the correction of FA profiles in experimental models of lung fibrosis brought advances in understanding tissue scarring processes and contributed to the transition of new molecules into clinical development. This review highlights the role of FAs and their metabolites in IPF and provides evidence for therapeutic potential of lipidome manipulations in the treatment of this disease.

Diverse functions of apolipoprotein A-I in lung fibrosis.

Apolipoprotein A-I (apoA-I) mediates reverse cholesterol transport (RCT) out of cells. In addition to its important role in the RTC, apoA-I also possesses anti-inflammatory and antioxidative functions including the ability to activate inflammasome and signal via toll-like receptors. Dysfunctional apoA-I or its low abundance may cause accumulation of cholesterol mass in alveolar macrophages, leading to the formation of foam cells. Increased numbers of foam cells have been noted in the lungs of mice after experimental exposure to cigarette smoke, silica, or bleomycin and in the lungs of patients suffering from different types of lung fibrosis, including idiopathic pulmonary fibrosis (IPF). This suggests that dysregulation of lipid metabolism may be a common event in the pathogenesis of interstitial lung diseases. Recognition of the emerging role of cholesterol in the regulation of lung inflammation and remodeling provides a challenging concept for understanding lung diseases and offers novel and exciting avenues for therapeutic development. Accordingly, a number of preclinical studies demonstrated decreased expression of inflammatory and profibrotic mediators and preserved lung tissue structure following the administration of the apoA-I or its mimetic peptides. This review highlights the role of apoA-I in lung fibrosis and provides evidence for its potential use in the treatment of this pathological condition.

Circulating hyaluronic acid signature in CAP and ARDS - the role of pneumolysin in hyaluronic acid shedding.

Shedding of hyaluronan (HA), the component of endothelial cell (EC) glycocalyx, has been associated with acute lung injury. HA degradation allows plasma proteins and fluid to penetrate across the vascular wall leading to lung edema formation and leukocyte recruitment. Here, we analyzed sHA levels and size in patients with community-acquired pneumonia (CAP) and acute respiratory distress syndrome (ARDS), correlated them to disease severity, and evaluated the impact of pneumolysin (PLY), the Streptococcus pneumoniae (S.p.) exotoxin, on HA shedding from human pulmonary microvascular EC (HPMVEC). sHA levels were elevated in CAP and ARDS and correlated with the CRB65 severity score and with markers of inflammation (interleukin-6), EC activation (E-selectin), and basement membrane destruction (collagen IV). Furthermore, sHA levels were associated with an increase in 28-day mortality. Small and large sHA fragments were detected in plasma of most severe CAP or ARDS patients, and the presence of large sHA fragments was accompanied by the elevated levels of circulating collagen IV. In vitro, PLY induced sHA release from HPMVEC. This effect was dependent on reactive oxygen species (ROS) production and was not associated with endothelial barrier dysfunction. Conversely, HA shedding was impaired following HPMVEC infection with a S.p. PLY-deficient mutant. Our study identifies association between the severity of CAP and ARDS and the levels and size of sHA in plasma. It links sHA levels with, inflammation, EC activation status and basement membrane disassembly in ARDS and provides insights into the mechanism of HA shedding during infection.

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.

Transcriptional Profiling of Insulin-like Growth Factor Signaling Components in Embryonic Lung Development and Idiopathic Pulmonary Fibrosis.

Insulin-like growth factor (IGF) signaling controls the development and growth of many organs, including the lung. Loss of function of Igf1 or its receptor Igf1r impairs lung development and leads to neonatal respiratory distress in mice. Although many components of the IGF signaling pathway have shown to be dysregulated in idiopathic pulmonary fibrosis (IPF), the expression pattern of such components in different cellular compartments of the developing and/or fibrotic lung has been elusive. In this study, we provide a comprehensive transcriptional profile for such signaling components during embryonic lung development in mice, bleomycin-induced pulmonary fibrosis in mice and in human IPF lung explants. During late gestation, we found that Igf1 is upregulated in parallel to Igf1r downregulation in the lung mesenchyme. Lung tissues derived from bleomycin-treated mice and explanted IPF lungs revealed upregulation of IGF1 in parallel to downregulation of IGF1R, in addition to upregulation of several IGF binding proteins (IGFBPs) in lung fibrosis. Finally, treatment of IPF lung fibroblasts with recombinant IGF1 led to myogenic differentiation. Our data serve as a resource for the transcriptional profile of IGF signaling components and warrant further research on the involvement of this pathway in both lung development and pulmonary disease.

A fluorescence microscopy-based protocol for volumetric measurement of lysolecithin lesion-associated de- and re-myelination in mouse brain.

Lysolecithin injections into the white matter tracts of the central nervous system are a valuable tool to study remyelination, but evaluating the resulting demyelinating lesion size is challenging. Here, we present a protocol to consistently measure the volume of demyelination and remyelination in mice following brain lysolecithin injections. We describe serial sectioning of the lesion, followed by the evaluation of the demyelinated area in two-dimensional images. We then detail the computation of the volume using our own automated iPython script. For complete details on the use and execution of this profile, please refer to Bosch-Queralt et al. (2021).

QUAREP-LiMi: A community-driven initiative to establish guidelines for quality assessment and reproducibility for instruments and images in light microscopy.

A modern day light microscope has evolved from a tool devoted to making primarily empirical observations to what is now a sophisticated , quantitative device that is an integral part of both physical and life science research. Nowadays, microscopes are found in nearly every experimental laboratory. However, despite their prevalent use in capturing and quantifying scientific phenomena, neither a thorough understanding of the principles underlying quantitative imaging techniques nor appropriate knowledge of how to calibrate, operate and maintain microscopes can be taken for granted. This is clearly demonstrated by the well-documented and widespread difficulties that are routinely encountered in evaluating acquired data and reproducing scientific experiments. Indeed, studies have shown that more than 70% of researchers have tried and failed to repeat another scientist's experiments, while more than half have even failed to reproduce their own experiments. One factor behind the reproducibility crisis of experiments published in scientific journals is the frequent underreporting of imaging methods caused by a lack of awareness and/or a lack of knowledge of the applied technique. Whereas quality control procedures for some methods used in biomedical research, such as genomics (e.g. DNA sequencing, RNA-seq) or cytometry, have been introduced (e.g. ENCODE), this issue has not been tackled for optical microscopy instrumentation and images. Although many calibration standards and protocols have been published, there is a lack of awareness and agreement on common standards and guidelines for quality assessment and reproducibility. In April 2020, the QUality Assessment and REProducibility for instruments and images in Light Microscopy (QUAREP-LiMi) initiative was formed. This initiative comprises imaging scientists from academia and industry who share a common interest in achieving a better understanding of the performance and limitations of microscopes and improved quality control (QC) in light microscopy. The ultimate goal of the QUAREP-LiMi initiative is to establish a set of common QC standards, guidelines, metadata models and tools, including detailed protocols, with the ultimate aim of improving reproducible advances in scientific research. This White Paper (1) summarizes the major obstacles identified in the field that motivated the launch of the QUAREP-LiMi initiative; (2) identifies the urgent need to address these obstacles in a grassroots manner, through a community of stakeholders including, researchers, imaging scientists, bioimage analysts, bioimage informatics developers, corporate partners, funding agencies, standards organizations, scientific publishers and observers of such; (3) outlines the current actions of the QUAREP-LiMi initiative and (4) proposes future steps that can be taken to improve the dissemination and acceptance of the proposed guidelines to manage QC. To summarize, the principal goal of the QUAREP-LiMi initiative is to improve the overall quality and reproducibility of light microscope image data by introducing broadly accepted standard practices and accurately captured image data metrics.

Diet-dependent regulation of TGFß impairs reparative innate immune responses after demyelination.

Proregenerative responses are required for the restoration of nervous-system functionality in demyelinating diseases such as multiple sclerosis (MS). Yet, the limiting factors responsible for poor CNS repair are only partially understood. Here, we test the impact of a Western diet (WD) on phagocyte function in a mouse model of demyelinating injury that requires microglial innate immune function for a regenerative response to occur. We find that WD feeding triggers an ageing-related, dysfunctional metabolic response that is associated with impaired myelin-debris clearance in microglia, thereby impairing lesion recovery after demyelination. Mechanistically, we detect enhanced transforming growth factor beta (TGFß) signalling, which suppresses the activation of the liver X receptor (LXR)-regulated genes involved in cholesterol efflux, thereby inhibiting phagocytic clearance of myelin and cholesterol. Blocking TGFß or promoting triggering receptor expressed on myeloid cells 2 (TREM2) activity restores microglia responsiveness and myelin-debris clearance after demyelinating injury. Thus, we have identified a druggable microglial immune checkpoint mechanism regulating the microglial response to injury that promotes remyelination.

The Drosophila FUS ortholog cabeza promotes adult founder myoblast selection by Xrp1-dependent regulation of FGF signaling.

The number of adult myofibers in Drosophila is determined by the number of founder myoblasts selected from a myoblast pool, a process governed by fibroblast growth factor (FGF) signaling. Here, we show that loss of cabeza (caz) function results in a reduced number of adult founder myoblasts, leading to a reduced number and misorientation of adult dorsal abdominal muscles. Genetic experiments revealed that loss of caz function in both adult myoblasts and neurons contributes to caz mutant muscle phenotypes. Selective overexpression of the FGF receptor Htl or the FGF receptor-specific signaling molecule Stumps in adult myoblasts partially rescued caz mutant muscle phenotypes, and Stumps levels were reduced in caz mutant founder myoblasts, indicating FGF pathway deregulation. In both adult myoblasts and neurons, caz mutant muscle phenotypes were mediated by increased expression levels of Xrp1, a DNA-binding protein involved in gene expression regulation. Xrp1-induced phenotypes were dependent on the DNA-binding capacity of its AT-hook motif, and increased Xrp1 levels in founder myoblasts reduced Stumps expression. Thus, control of Xrp1 expression by Caz is required for regulation of Stumps expression in founder myoblasts, resulting in correct founder myoblast selection.

Pro-inflammatory activation following demyelination is required for myelin clearance and oligodendrogenesis.

Remyelination requires innate immune system function, but how exactly microglia and macrophages clear myelin debris after injury and tailor a specific regenerative response is unclear. Here, we asked whether pro-inflammatory microglial/macrophage activation is required for this process. We established a novel toxin-based spinal cord model of de- and remyelination in zebrafish and showed that pro-inflammatory NF-κB-dependent activation in phagocytes occurs rapidly after myelin injury. We found that the pro-inflammatory response depends on myeloid differentiation primary response 88 (MyD88). MyD88-deficient mice and zebrafish were not only impaired in the degradation of myelin debris, but also in initiating the generation of new oligodendrocytes for myelin repair. We identified reduced generation of TNF-α in lesions of MyD88-deficient animals, a pro-inflammatory molecule that was able to induce the generation of new premyelinating oligodendrocytes. Our study shows that pro-inflammatory phagocytic signaling is required for myelin debris degradation, for inflammation resolution, and for initiating the generation of new oligodendrocytes.

Two adhesive systems cooperatively regulate axon ensheathment and myelin growth in the CNS.

Central nervous system myelin is a multilayered membrane produced by oligodendrocytes to increase neural processing speed and efficiency, but the molecular mechanisms underlying axonal selection and myelin wrapping are unknown. Here, using combined morphological and molecular analyses in mice and zebrafish, we show that adhesion molecules of the paranodal and the internodal segment work synergistically using overlapping functions to regulate axonal interaction and myelin wrapping. In the absence of these adhesive systems, axonal recognition by myelin is impaired with myelin growing on top of previously myelinated fibers, around neuronal cell bodies and above nodes of Ranvier. In addition, myelin wrapping is disturbed with the leading edge moving away from the axon and in between previously formed layers. These data show how two adhesive systems function together to guide axonal ensheathment and myelin wrapping, and provide a mechanistic understanding of how the spatial organization of myelin is achieved.

Actin filament turnover drives leading edge growth during myelin sheath formation in the central nervous system.

During CNS development, oligodendrocytes wrap their plasma membrane around axons to generate multilamellar myelin sheaths. To drive growth at the leading edge of myelin at the interface with the axon, mechanical forces are necessary, but the underlying mechanisms are not known. Using an interdisciplinary approach that combines morphological, genetic, and biophysical analyses, we identified a key role for actin filament network turnover in myelin growth. At the onset of myelin biogenesis, F-actin is redistributed to the leading edge, where its polymerization-based forces push out non-adhesive and motile protrusions. F-actin disassembly converts protrusions into sheets by reducing surface tension and in turn inducing membrane spreading and adhesion. We identified the actin depolymerizing factor ADF/cofilin1, which mediates high F-actin turnover rates, as an essential factor in this process. We propose that F-actin turnover is the driving force in myelin wrapping by regulating repetitive cycles of leading edge protrusion and spreading.

KV10.1 K(+)-channel plasma membrane discrete domain partitioning and its functional correlation in neurons.

KV10.1 potassium channels are implicated in a variety of cellular processes including cell proliferation and tumour progression. Their expression in over 70% of human tumours makes them an attractive diagnostic and therapeutic target. Although their physiological role in the central nervous system is not yet fully understood, advances in their precise cell localization will contribute to the understanding of their interactions and function. We have determined the plasma membrane (PM) distribution of the KV10.1 protein in an enriched mouse brain PM fraction and its association with cholesterol- and sphingolipid-rich domains. We show that the KV10.1 channel has two different populations in a 3:2 ratio, one associated to and another excluded from Detergent Resistant Membranes (DRMs). This distribution of KV10.1 in isolated PM is cholesterol- and cytoskeleton-dependent since alteration of those factors changes the relationship to 1:4. In transfected HEK-293 cells with a mutant unable to bind Ca(2+)/CaM to KV10.1 protein, Kv10.1 distribution in DRM/non-DRM is 1:4. Mean current density was doubled in the cholesterol-depleted cells, without any noticeable effects on other parameters. These results demonstrate that recruitment of the KV10.1 channel to the DRM fractions involves its functional regulation.