Mitochondria of seeds face the challenge of remaining quiescent over long periods and rapidly resuming their respiratory function once environmental conditions become favorable. Despite the fundamental importance of mitochondrial functions for germination, the composition and functionality of seed mitochondria remain poorly understood. Previous work has suggested that dry seeds contain immature promitochondria largely devoid of cristae or respiratory complexes and that key mitochondrial functions need to be re-established by rebuilding a functional proteome to support germination. Here, we examined the onset of respiration in the germination of several plant species and investigated the composition of Arabidopsis seed mitochondria using cryo-preservation-based transmission electron microscopy, affinity- and centrifugation-based mitochondrial isolations, native gel electrophoresis, and advanced proteomic and protein localization analyses. Our data reveal pre-formed cristae, the full set of respiratory complexes in the mitochondria of dry seeds, as well as the presence of proteins for all key functions that mitochondria fulfill in vegetative tissues. While the overall protein composition of Arabidopsis seed mitochondria is similar to that in other developmental stages and dark-grown cell cultures, it deviates particularly strongly in selected proteoforms and yet-unassigned candidate mitochondrial or mitochondria-associated proteins. Our findings establish that Arabidopsis seed mitochondria are preserved in a structurally and functionally competent state, accounting for the immediate activation of respiration that occurs upon seed rehydration. They further expand our understanding of mitochondrial plasticity by assignment of novel proteins to the plant mitochondrial proteome, laying the foundation for future investigations of their potential significance in desiccation tolerance and metabolic regulation.

Fused in sarcoma (FUS) is a highly conserved RNA-binding protein with essential roles in RNA processing and genomic stability. While extensively studied in the context of neurodegeneration, its involvement in fibrotic diseases, particularly idiopathic pulmonary fibrosis (IPF), remains largely unexplored. This study investigated the pathological role of FUS in IPF and assessed its viability as a therapeutic target. Specifically, we examine how FUS dysregulation contributes to fibrotic signaling and evaluate whether therapeutic silencing of FUS offers a rational strategy to modulate disease progression. To assess the effects of FUS overexpression and knockdown, functional assays were performed on primary lung fibroblasts derived from healthy donors and IPF patients. Precision-cut lung slices (PCLs) and 3D alveolosphere cultures from IPF patients were treated with a FUS-targeted antisense oligonucleotide (ASO;ION363). FUS-RNA interactions were mapped via CLIP-Seq, and global transcriptional changes following FUS inhibition were analyzed via RNA sequencing. FUS overexpression in healthy fibroblasts promoted proliferation, whereas FUS knockdown attenuated the hyperproliferative phenotype in IPF fibroblasts. IPF cells demonstrated aberrant cytoplasmic mislocalization of FUS. Standard-of-care treatments (pirfenidone, nintedanib) reduced FUS expression in PCLs. CLIP-Seq revealed that FUS binds to a distinct set of profibrotic RNAs in IPF. ION363 treatment downregulated fibrotic gene programs, including those linked to ECM remodeling, TGFβ signaling, and epithelial dysfunction. In contrast, ION363 promoted functional marker expression and improved morphology in patient-derived 3D alveolospheres. We conclude that FUS is a pivotal regulator of fibrotic signaling in IPF and that targeting FUS via ASO represents a promising therapeutic avenue for IPF.

Tissue repair after myocardial infarction entails a vigorous angiogenic response that mitigates scarring and worsening of heart function. Angiogenesis in the infarct wound is guided by incompletely defined myeloid cell-endothelial cell interactions. Here, we identify the 75-amino acid microprotein BRICK1 (BRK1) as an indispensable driver of postinfarction angiogenesis in a mouse model of reperfused myocardial infarction. We show that BRK1 is preferentially expressed by myeloid cells and translocates to the extracellular space after myocardial infarction in mice and humans. As a subunit of the intracellular actin-regulatory WAVE complex, BRK1 was not previously known to function outside the cell. We find that BRK1 is not actively secreted but released during myeloid cell death. Cre-loxP-driven myeloid cell-selective genetic deletion of Brk1 or antibody-mediated neutralization of extracellular BRK1 impaired microvessel formation in the infarct border zone and resulted in severe postinfarction heart failure in mice. Conversely, treatment with recombinant BRK1 preserved heart function in infarcted mice. Mechanistically, BRK1 induced an angiogenic phenotype in human cardiac endothelial cells by signaling via the small GTPase Ras-related protein Rap-1 and mitogen-activated protein kinases 1 and 3 to promote retinoblastoma protein hyperphosphorylation and E2F transcription factor activation. BRK1 thus emerges as an angiogenic factor linking myeloid cell death to ischemic tissue repair, potentially enabling a protein-based therapy for myocardial infarction.

The lysosomal storage disorder Fabry disease results from α-galactosidase A deficiency, leading to excessive glycosphingolipid substrate accumulation, primarily globotriaosylceramide (Gb3). While the underlying molecular mechanisms remain elusive, multi-systemic complications ultimately culminate in premature death, with heart failure being the leading cause of death. Current treatment options fail to treat Fabry disease adequately and only delay its progression. Preclinical studies on an alternative approach, systemic delivery of nucleoside-modified GLA mRNA (modGLA), suggest improved effectiveness over existing therapies in reducing glycosphingolipid levels in the heart. It remains unclear whether modGLA can rescue Fabry cardiomyopathy phenotypes at the cellular level, which are not faithfully recapitulated in current animal models. To address this, we investigated characteristic phenotypes in two new models of Fabry cardiomyopathy utilizing human iPSC-derived cardiomyocytes in transcriptomic and functional analyses. These human Fabry disease cardiomyocytes displayed broad transcriptional dysregulation, apoptosis, mitochondrial dysfunction, impaired reactive oxygen species handling, altered contractility, and enhanced calcium transient decay parameters. Mechanistically, phospholamban hyperphosphorylation may contribute to this calcium dysregulation. Consistently, modGLA therapy restored α-galactosidase A activity, reduced glycosphingolipid deposition, and normalized molecular alterations, including phospholamban hyperphosphorylation and calcium decay parameters, supporting modGLA as a promising therapeutic strategy for Fabry disease.

BACKGROUND:Cardiomyocyte mitochondria align with sarcomeres during heart development. Mitochondrial motility is controlled by RHOT (ras homolog family member T) 1 and RHOT2. RHOT1 and RHOT2 are atypical Rho-like small GTPases that are anchored to the outer mitochondrial membrane and couple mitochondria to kinesin and dynein motors. We hypothesized that RHOT protein expression and mitochondrial motility are required for mitochondrial positioning during cardiomyocyte development.METHODS:We generated mice with cardiomyocyte-selective deletion of Rhot1 and Rhot2 during embryogenesis (cRhot1/2-KO [constitutive and embryonic cardiomyocyte-selective Rhot1/2 knockout]) or tamoxifen-inducible deletion in the adult heart (iRhot1/2-KO [inducible cardiomyocyte-selective Rhot1/2 knockout]) to assess the importance of mitochondrial motility during and after cardiomyocyte maturation. Mitochondrial motility was determined by a motor protein-driven single mitochondria motility assay. Respiratory capacity was measured in isolated mitochondria. Intracellular mitochondrial localization and ATP production in isolated cardiomyocytes were assessed by confocal microscopy and after adenoviral expression of the fluorescence resonance energy transfer-based ATP biosensor ATeam. Cardiac ultrastructure was assessed by electron micrographs; mass spectrometry was used for proteome analysis.RESULTS:cRhot1/2-KO mice developed fatal cardiomyopathy associated with sarcomere disarray and perinuclear accumulation of mitochondria and ATP production. Mitochondria isolated from cRhot1/2-KO hearts exhibited impaired motility but preserved respiratory capacity. Mechanistically, proteome analysis identified that RHOT proteins bind mitochondria to contractile muscle fiber proteins. In contrast, inducible deletion of Rhot1 and Rhot2 in adult iRhot1/2-KO mice did not result in heart failure. Despite impaired motility of isolated mitochondria, intracellular mitochondrial localization, local ATP production, and sarcomere structure were preserved in adult iRhot1/2-KO hearts after cardiomyocyte maturation.CONCLUSIONS:RHOT proteins bind mitochondria to contractile muscle fiber proteins and are required for mitochondrial positioning in cardiomyocytes during development. Our study links mitochondrial motility and local ATP production to structural and functional maturation of the heart.

Pregnancy-associated hemodynamic overload and hormonal changes induce hypertrophy and metabolic remodeling of the maternal heart. Mitochondrial motility, mediated by ras homolog family member T (RHOT) 1 and RHOT2, is essential for cardiac adaptation to increased workload, cardiomyocyte hypertrophy, and sarcomere maturation. To test the hypothesis that Rhot1/2 expression is required for pregnancy- and postpartum-associated adaptations of the maternal heart, female mice with tamoxifen-inducible, cardiomyocyte-selective deletion of Rhot1 and Rhot2 (iRhot1/2-KO) were mated. Following gene deletion in adult mice, cardiac tissue and function were analyzed after three to five successive pregnancies and postpartum nursing periods. Age-matched nulliparous iRhot1/2-KO mice and age-matched mice expressing Rhot1 and Rhot2 served as controls. Motility of mitochondria isolated from iRhot1/2-KO hearts was impaired, as determined by the number of mobile mitochondria in an in vitro motor protein-driven single mitochondrion motility assay performed on surface-immobilized microtubules. Despite loss of Rhot1/2 expression, contractile function assessed by transthoracic echocardiography, mRNA expression of peripartum-associated heart failure markers, cardiac structure, mitochondrial morphology, mitochondrial enzymatic activity, and mitochondrial DNA content were all comparable to controls expressing Rhot1/2 at the investigated time points. RNA sequencing-based gene profiling identified a transcriptional program through which RHOT proteins preserve cardiac energetic and contraction gene expression during pregnancy and postpartum. Together, cardiomyocyte-selective loss of Rhot1/2 expression in the adult heart does not cause peripartum-associated heart failure, despite reduced cardiac energetic and contraction gene expression.

Pleuroparenchymal fibroelastosis (PPFE) is a progressive interstitial lung disease (ILD) with defining histology of intra-alveolar fibrosis with septal elastosis (AFE), suggesting unique cellular disease processes. Here, we present a binational single-nucleus RNA sequencing atlas of PPFE, based on explanted lungs from 40 patients. Immunofluorescence microscopy, RNA in situ hybridization, micro-computed tomography (CT), and hierarchical phase-contrast (HiP) synchrotron CT provided spatial context. We identify PPFE-associated adventitial and elastofibrotic fibroblasts as key drivers of elastotic remodeling within an inflammatory microenvironment, maintained by immune cells forming tertiary lymphoid structures. Spatial mapping reveals an intriguing zonation of AFE, maintained by intercellular circuits between PPFE-associated cell types. Comparative analysis with idiopathic pulmonary fibrosis highlights CTHRC1+ fibrotic fibroblasts and aberrant basaloid cells as conserved profibrotic cellular machinery mediating collagen deposition across ILDs. This integrative atlas defines the cellular landscape of PPFE and dissects elastotic from fibrotic remodeling, providing a molecular rationale for niche-specific therapeutic strategies.

ABSTRACT Biofilm-associated diseases like peri-implant mucositis (PIM) and peri-implantitis (PI) are significant clinical challenges affecting millions of dental implant patients globally. Although studies have described the role of microbial, host, or environmental factors in disease development, their complex interplay, particularly during dysbiosis, remains poorly understood. This cross-sectional study characterized the microbiome composition and metatranscriptomes of 125 peri-implant biofilms from 48 individuals, uncovering molecular signatures linked to peri-implant health (PIH), PIM, and PI. Distinct variations were observed in biofilm amount, microbial composition and activity, phage populations, and host response. Biofilms were categorized into four community types (CTs) based on the bacterial transcriptional activity: one linked to PIH, one to PI, and two to PIM. PIH and PIM were primarily characterized by aerotolerant taxa with increased anabolic processes, while PI was dominated by obligate anaerobes with complex biofilm morphology. PIM samples, relative to PIH, were characterized by biofilm expansion with minimal functional changes, except for the Neisseria-rich PIM subtype showing higher pyruvate and lipoic acid metabolism. The phagome mirrored the bacterial compositional variations across disease states. Furthermore, human transcriptome responses varied, indicating increased keratinization in PIH, enhanced expression of ribosome components in PIM, and inflammatory signaling and hypoxia in PI. Additionally, we identified complex species–enzyme, phage–bacterium, and host–microbe associations within the peri-implant ecosystem. Our integrative multi-omics approach provides a comprehensive view of microbial, biochemical, host, and ecological factors associated with dysbiosis, offering novel insights into peri-implant disease dynamics.IMPORTANCEPeri-implant mucositis and peri-implantitis are highly prevalent inflammatory conditions that compromise the long-term survival and success of dental implants, yet their underlying biological mechanisms are largely unresolved. The full-length 16S rRNA gene amplicon sequencing (full-16S) allows for high-resolution taxonomic profiling of peri-implant biofilms, thereby advancing our understanding of microbial composition across health and peri-implant diseases. The integration of metatranscriptomics, furthermore, captures actively transcribed genes within the biofilm and offers direct insights into microbial community functions and the broader molecular context of peri-implant dysbiosis. DNA- and RNA-derived abundances were strongly correlated, with only a few microbial classes showing moderate diagnosis-related differences after DNA-based normalization of transcriptional activity. In this study, we integrated full-16S with metatranscriptomic profiling to simultaneously assess microbial taxonomy, functional activity, phage dynamics, and host gene expression in peri-implant biofilms. Importantly, we provide a systems-level view and report previously undescribed associations between different molecular signatures in the peri-implant ecosystem.

BACKGROUND:Vessel-lining endothelial cells (ECs) rely on heparan sulfate (HS) proteoglycans to regulate vascular permeability and to maintain vascular homeostasis. Hpa2 (heparanase 2) is a little-known, nonenzymatic, HS-binding protein. We hypothesized major functions and thus characterized the role of endogenous Hpa2 in the vertebrate vascular system. METHODS:We use zebrafish larvae as our primary animal model. Hpa2 loss-of-function (LOF) was induced by CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) and morpholino antisense strategies. We assessed vascular permeability, blood vessel architecture, and EC morphology using transgenic zebrafish and transmission electron microscopy. rHpa2 (recombinant heparanase 2) was generated to study the functionality of Hpa2 in endothelial tissue cultures, zebrafish, and mice. RESULTS:We detected Hpa2 expression in hepatic tissue and localized Hpa2 protein in the vasculature of zebrafish and mammals. Hpa2 LOF increased zebrafish vascular permeability and altered EC and extracellular matrix morphology. rHpa2 rescued the Hpa2 LOF phenotype. Hpa2 LOF reduced HS levels and caused EC gene expression changes involved in signal transduction. rHpa2 competed with growth factors FGF2 (fibroblast growth factor-2) and VEGFA165 (vascular endothelial growth factor A165) for binding on the EC surface and consequently reduced the signal response these factors elicit. rHpa2 prevented VEGFA165-induced vascular permeability in murine ex vivo kidneys. Pharmacological inhibition of FGF2/VEGFR (VEGF receptor) signaling alleviated the Hpa2 LOF phenotype in zebrafish. CONCLUSIONS:We suggest that Hpa2 is a circulating molecule that maintains vascular integrity by regulating vascular HS-dependent growth factor signaling. Our model outlines Hpa2-related vascular function and could indicate therapeutic utilities.

The lysosomal storage disorder Fabry disease results from α-galactosidase A deficiency, leading to excessive glycosphingolipid substrate accumulation, primarily globotriaosylceramide (Gb3). While the underlying molecular mechanisms remain elusive, multi-systemic complications ultimately culminate in premature death, with heart failure being the leading cause of death. Current treatment options fail to treat Fabry disease adequately and only delay its progression. Preclinical studies on an alternative approach, systemic delivery of nucleoside-modified GLA mRNA (modGLA), suggest improved effectiveness over existing therapies in reducing glycosphingolipid levels in the heart. It remains unclear whether modGLA can rescue Fabry cardiomyopathy phenotypes at the cellular level, which are not faithfully recapitulated in current animal models. To address this, we investigated characteristic phenotypes in two new models of Fabry cardiomyopathy utilizing human iPSC-derived cardiomyocytes in transcriptomic and functional analyses. These human Fabry disease cardiomyocytes displayed broad transcriptional dysregulation, apoptosis, mitochondrial dysfunction, impaired reactive oxygen species handling, as well as enhanced decay parameters of calcium transients. Mechanistically, we identified hyperphosphorylated phospholamban as a major player in this calcium dysregulation. Strikingly, modGLA therapy of Fabry cardiomyocytes restored α-galactosidase A enzyme activity, reduced glycosphingolipid deposition, and normalized the observed molecular alterations, supporting modGLA therapy as a promising strategy for the treatment of Fabry disease.

Surfactant protein B (SP-B) is essential for surface tension reducing function of pulmonary surfactant and alveolar unfolding processes during inspiration. SP-B is reduced early in acute lung injury. Hence, we hypothesize that 1) reduced SP-B expression increases susceptibility to ventilation-induced lung injury (VILI), and 2) deep inflations (DI) are protective against VILI. Conditional SP-B knockout mice were randomized into OFF (reduced SP-B) and ON groups (normal SP-B) and subjected to mechanical ventilation at zero end-expiratory pressure. Over 4 h of ventilation, either 4 or 16 DI were administered. Lung mechanics were recorded, and pulmonary structure was quantified by design-based stereology. Inflammatory cells and bulk RNA sequencing were measured in bronchoalveolar lavage (BAL) and tissue, respectively. No differences in inflammatory cells in BAL were detected between ON and OFF groups. During ventilation, alveolar derecruitment-related increase in elastance was most pronounced in OFF-4DI but reversible by DI so that lung mechanics did not worsen. Finally, volumes of the alveolar liquid lining layer and the intracellular surfactant were largest, whereas the surface area of the apical plasma membrane of type II pneumocytes was smallest in OFF-4DI, suggesting impaired surfactant secretion. A higher frequency of DI prevented these abnormalities. Electron microscopy revealed disorganized tight junctions between alveolar epithelial cells in OFF-4DI, which was linked with decreased expression of genes relevant to the apical junctional complex. Reduced SP-B resulted in a progressive increase in surface tension and a disturbed fluid balance without triggering definite VILI. Maintenance of residual surfactant function is highly dependent on DI in conditions of reduced SP-B levels. NEW & NOTEWORTHY Surfactant protein B (SP-B) is critical for efficient surfactant function in the lung. Reduced SP-B levels occur at an early stage of acute lung injury and impair alveolar unfolding. In this study, we demonstrate that mechanical ventilation of lungs with reduced SP-B levels does not trigger ventilation-induced lung injury but results in disbalance of alveolar fluid volume and increase in surface tension due to failure of surfactant maintenance. Deep inflations prevent these ventilation-induced effects.

Ischemic heart disease is the leading cause of death worldwide. Reduced oxygen supply and myocardial hypoxia lead to tissue damage and impairment of the heart function. To the best of our knowledge, the primary functional effects of hypoxia in the multicellular model of living myocardial slices (LMSs) have not been investigated so far. In this study, we analyzed force generation, ultrastructure, gene expression, and proteome changes in rat LMS after 24 h of ex vivo culture in normal and reduced levels of oxygen (O2). We observed a significant reduction in absolute force and a slowdown of force kinetics as well as an increase in cardiomyocyte apoptosis and myofibrillar and mitochondrial damage, as well as transcriptomic changes. Proteome analysis revealed the deregulation of proteins involved in metabolic processes, hypoxic response, and neutralizing of reactive oxygen species. Our results indicate that hypoxia induces substantial primary changes in heart tissue, which are independent of perfusion and immune responses. Our new LMS model could serve as a screening system for drug development and new mechanistic insights.

Severely impaired mucociliary airway function is the primary pathomechanism in Cystic Fibrosis (CF) lung disease. Despite significant advances in CF therapy, there is still a critical need for alternative, individualized treatment options, especially for patients with untreatable CFTR mutations. Although intestinal organoids and primary airway cells are widely used as preclinical models of CF, both systems exhibit limitations with regard to the proper modelling of mucociliary clearance or the availability of sufficient cell quantities. Patient-specific human induced pluripotent stem cells (hiPSCs) are a promising alternative due to their unlimited expansion potential and capacity to differentiate into airway epithelia. However, cellular inhomogeneities in iPSC-derived airway cultures complicated conventional assays that determine CFTR function such as Ussing chamber measurements, and a comprehensive demonstration of CF pathophysiology in hiPSC-derived airway models has been largely lacking. This study provides comprehensive data demonstrating very similar gene expression, (ultra)structure and CFTR function in CF iPSC-derived airway (iALI) and primary airway (pALI) cultures. Addressing current limitations, we have implemented a sensitive, straightforward, and automatable ciliary beat frequency (CBF) assay, which is largely unaffected by inhomogeneities and directly reflects disturbed mucus viscosity and mucociliary transport in CF lung disease. Electron microscopy images confirmed the disease phenotype showing a highly dense and dehydrated mucus layer on top of CF iALI cultures. Furthermore, established CFTR modulator drugs partially rescued the disease phenotype in CF iALI cultures, which validated the utility of iALI cultures as a scalable, patient-specific platform for CF research and personalized drug development.

RATIONALE: RNA-binding proteins (RBPs) exert significant control over various aspects of mRNA biogenesis and metabolism by binding to target RNAs. Cellular stress condition stimulates stress granule (SG) formation which encapsulates RBPs along with untranslated or stalled RNA transcripts. RBPs and SGs play a crucial role in organs with intricate RNA metabolism, such as the brain and lungs. Our current study delves into the involvement of RBPs and SGs in idiopathic pulmonary fibrosis (IPF). METHODS: CLIP-Sequencing was performed to identify gene targets for FUS in IPF and Donor fibroblasts. Protein charcterisation and colocalization studies were performed using immunoblots and immunofluorescence respectively. Loss and gain of function studies were performed through RNA interference and plasmid overexpression. Especially, silencing of FUS was performed using antisence oligonucleotide (ASO) for FUS in IPF fibroblasts as well as in precision cut lung slices derived from IPF patients, followed by RNA-sequencing. RESULTS: The data revealed significant increased expression of RBPs-FUS, TDP43 and PABPC1 in interstitial fibroblasts of IPF patients as compared to those of healthy donors. Overexpression of FUS in healthy fibroblasts resulted in enhanced cell proliferation and on the contrary, knockdown of FUS in IPF fibroblasts resulted in a decreased cell proliferation. Mislocalization of FUS into the cytoplasm was also observed in IPF fibroblasts. Additionally, treatment of precision cut lung slices (PCLS) derived from IPF patients with the IPF standard of care drugs, pirfenidone and nintedanib resulted in reduced expression levels of FUS, suggesting a novel mechanism of action for pirfenidone/nintedanib. RNA-IP for FUS followed by RNA-seq analysis identified distinct fibrotic gene targets associated with FUS in IPF compared to healthy donor fibroblasts. RNA sequencing from IPF fibroblasts as well as PCLS derived from IPF patients that were treated with FUS-ASO (or non targeting ASO) revealed a down regulation of genes associated with IPF, that include several genes of the extracellular matrix, TGF-ß signaling as well as surfactants. CONCLUSION: In this study, we show a previously unidentified pathological role of the RNA-binding protein FUS in IPF. We conclude that ASO based silencing of FUS expression is a novel therapeutic approach for IPF.

Aims Iron deficiency (ID) is a frequent comorbidity in heart failure (HF) and contributes to exercise intolerance. Tissue iron levels are maintained by cellular iron uptake, sequestration, and release, processes that are tightly controlled by iron regulatory proteins (IRP). Our aim was to explore the role of IRP activity in skeletal muscle function and exercise capacity during HF.Methods and results We observed that skeletal muscle ID is associated with IRP1 and 2 inactivation 12 weeks after transverse aortic constriction (TAC) in mice with left ventricular (LV) dysfunction and cachexia. To understand the functional implications of IRP inactivation in skeletal muscle, we generated skeletal muscle-specific Irp1/2 knock-out mice (SkM-Irp1/2-KO). These mice developed muscle ID, along with lower transferrin receptor 1 (TFR1) levels and decreased non-haem iron content, within 5 weeks after birth. SkM-Irp1/2-KO mice exhibited shorter running distances and slower velocities during treadmill exercise. Transcriptomic analysis revealed up-regulation of gene clusters associated with endoplasmic reticulum stress, atrophy, mitochondrial dysfunction, and inflammation. Moreover, enhanced glycolysis, increased 18F-deoxyglucose uptake in quadriceps, and faster plasma glucose clearance were detected in SkM-Irp1/2-KO vs. control mice. In contrast, SkM-Irp1/2-KO mice had markedly reduced complex I and II expression, a change that confirmed defects in oxidative phosphorylation.Conclusion HF leads to IRP1/2 inactivation, ID, and metabolic dysfunction in skeletal muscle in mice. IRP1/2 inactivation in skeletal muscle causes ID, impairs oxidative energy production, and promotes exercise intolerance by reducing the capacity for effective energy utilization.

The urothelium is a stratified epithelium with an important barrier function in the urinary drainage system. The differentiation and maintenance of the three major urothelial cell types (basal, intermediate and superficial cells) is incompletely understood. Here, we show that mice with a conditional deletion of the transcription factor gene peroxisome proliferator activated receptor gamma (Pparg) in the ureteric epithelium have a dilated ureter at postnatal stages with a urothelium consisting of a layer of undifferentiated luminal cells and a layer of proliferating basal cells. Molecular analysis of fetal stages revealed that the expression of a large number of genes is not activated in superficial cells and that of a few genes, including Shh, is not activated in intermediate and basal cells. Pharmacological activation of SHH signaling in explant cultures of perinatal Pparg-deficient ureters reduced ureteral width and urothelial cell number to normal levels, increased the number of intermediate cells and slightly reduced basal cell proliferation. Our data suggest that PPARG independently activates the expression of structural genes in superficial cells and of Shh in basal and intermediate cells, and that both functions contribute to urothelial integrity.

RATIONALE: The entity pleuro-parenchymal fibroelastosis (PPFE) is a progressive interstitial lung disease histologically characterized by the pattern of alveolar fibroelastosis with distinct hallmarks in comparison to other fibrosing lung diseases. Driver cells in the pathogenetic cascade remain incompletely understood. METHODS: Fresh lung tissue from 3 PPFE explants and from 8 control patients within distinct non-PPFE interstitial lung disease (ILD) was collected. Correlative light- and electron microscopy (CLEM) was performed to characterize mesenchymal cells in the area of active fibrotic remodelling. Mesenchymal cell outgrow of PPFE and control tissues were cultured and analysed by scanning electron microscopy (SEM) and single-cell RNA sequencing (scRNAseq). Celltype annotation was performed by reference mapping to recently generated snRNAseq data from a larger PPFE-cohort. RESULTS: CLEM analysis of the active subpleural remodelling zone, revealed a network of numerous elongated myofibroblast-like cells characterized by significant cellular extensions in association with elastic fibres and collagen matrices. Furthermore CLEM identified light microscopic intra-fibrotic fibrils as infiltrating cell body extensions, phenotypically resembling adventitial fibroblasts. Cell outgrow experiments identified a PPFE-specific cell population showing overlap with features of adventitial fibroblast (MFAP5+, CEMIP+) and myofibroblasts (CTHRC1+). Morphology-wise, SEM revealed numerous elongated cell extensions within the outgrown PPFE mesenchyme. KNN mapping to a reference snRNAseq dataset of a larger multinational PPFE cohort identified the outgrown PPFE-specific cells as a subpopulation of adventitial fibroblasts and myofibroblasts. CONCLUSIONS: CLEM and scRNAseq analysis shed light on an invasive mesenchymal cell population in PPFE with transcriptomic and ultrastructural features of adventitial fibroblast, which may be a driver of fibroelastotic remodeling in PPFE.