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Yildirim, A. O.

Publications and source records attributed to Yildirim, A. O..

10 recordsLinked to original sources

Carcinogen-induced preinvasive human squamous lung cancer is characterised by a novel atypical basal cell state and presents opportunities for chemoprevention

There are no adequate carcinogen-induced models of the earliest stages of human squamous lung cancer (LUSC), limiting the development of effective chemoprevention strategies. We developed a novel human model of early LUSC by exposing differentiated primary human airway cells to a chemical carcinogen known to cause murine LUSC. Carcinogen exposure induced a phenotype recapitulating the earliest stages of human LUSC and associated with the emergence of a novel population of atypical basal cells (ABCs). ABCs express key cancer hallmark markers but without evidence of either common LUSC-associated mutations or an emergent major clonal/subclonal population. We established the clinical relevance of this model by demonstrating enrichment of the ABC expression profile in independent clinical precancer specimens. We then show that capivasertib, a clinically approved kinase inhibitor, prevented the emergence of the abnormal phenotype and the accumulation of cells with an active DNA damage response. Further, capivasertib reversed the established phenotype; and remained effective when nebulised in vitro, mimicking therapeutic inhalation. Together, these findings establish a tractable human model of carcinogen-induced early LUSC and provide a proof-of-principle for repurposing targeted therapies for the prevention of lung cancer.

cancer biology↗

Single-cell and spatial transcriptomics resolve airway obliteration in bronchiolitis obliterans syndrome

Background: Chronic lung allograft dysfunction (CLAD) is the leading cause of death beyond the first year after lung transplantation, and its most frequent phenotype is bronchiolitis obliterans syndrome (BOS), a fibrotic small-airway disease. Mechanistic work has focused on the immune compartment, yet intensified immunosuppression does not alter established disease. Aim: To resolve which structural cell states populate the BOS graft and how they are spatially organized during airway obliteration. Methods: We profiled explanted lungs from 33 BOS patients undergoing re-transplantation and 33 controls, combining single-nucleus RNA sequencing (14 BOS, 13 controls) with targeted spatial transcriptomics of 108 regions (27 BOS, 24 controls) and multiplex immunofluorescence validation. Single-nucleus data were integrated with a published restrictive allograft syndrome (RAS) atlas. Results: Across 175,128 nuclei and 1.67 million spatially resolved cells, BOS lungs harbored a profibrotic circuit of Aberrant Basaloid cells and CTHRC1+ fibrotic fibroblasts previously described in fibrotic lung diseases, including RAS. Spatial mapping identified a CXCL14+TNC+ injury-associated basal cell state arising early in the obliterative cascade, identifying basal cells as their major reservoir. CTHRC1+ fibroblasts expanded subepithelially replacing resident peribronchial fibroblasts, alongside a peribronchial vascular shift toward systemic venous endothelium. The circuit extended beyond the airway wall to the alveolar interface, defining two convergent remodeling fronts. Conclusion: BOS engages structural-cell circuits largely shared with RAS and fibrotic lung diseases, but along an airway-centered rather than parenchyma-centered axis. CLAD thus emerges as a spatial rather than cellular spectrum, defined by anatomical distribution more than cell identity. Shared structural programs may therefore be targetable across CLAD phenotypes.

cell biology↗

Lifespan Analysis of the Lung Epithelium Reveals Inflammatory Reprogramming and Regenerative Decline

Aging is a major risk factor for chronic lung diseases, associated with chronic low-grade inflammation (inflammaging) and impaired epithelial regeneration. How epithelial-intrinsic aging intersects with inflammaging across the lifespan remains poorly understood. Here, we systematically analyzed lung epithelial cells from neonatal, young adult, and aged mice to define age-dependent changes in regenerative capacity. RNA sequencing revealed lifespan-associated shifts characterized by early repression of developmental and WNT/{beta}-catenin programs and progressive activation of DNA damage, inflammation, and senescence signatures. Functionally, neonatal epithelial cells exhibited markedly enhanced organoid-forming capacity compared with young and aged cells. Aged organoids maintained a pro-inflammatory secretory profile indicative of cell-intrinsic inflammaging, and transfer of the aged secretome or TNF- to young cultures significantly impaired regeneration. Comparison of freshly isolated cells and long-term organoid cultures revealed sustained repression of regenerative pathways with age, consistent with stable epigenetic imprinting. Pharmacological inhibition of DNA methylation and WNT signalling partially restored regenerative capacity in adult organoids. Together, these findings identify epigenetic reprogramming and epithelial-intrinsic inflammaging as key determinants of age-dependent regenerative decline.

cell biology↗

Alveolar macrophages initiate the spatially targeted neutrophil recruitment during nanoparticle inhalation

Exposure to air pollution, including nanoparticles (NPs), is a major health concern associated with various diseases, triggered by subtle inflammatory responses in the lung. To investigate the dynamic immune response in vivo, lung intravital microscopy (L-IVM), was used to analyze the behavior of alveolar macrophages (AMs) and neutrophils, combined with ventilator-assisted inhalation of nebulized NPs in mice. Inhalation of fluorescent quantum dot NPs (cQDs) and soot-like carbon black NPs (CNPs, ambient pollutants), led to rapid spatially focused recruitment of neutrophils near alveolar deposited NPs. Neutrophil recruitment was initiated by NPs uptake by AMs, dependent on AM motility and AM NP surface recognition. Prior airway application of neutralizing antibodies against alveolar ICAM-1 and LFA-1, leading to reduced AM motility, inhibition of C5aR1 and Fc{gamma}RI receptor mediated NPs uptake by AMs, as well as neutralizing of TNF and application of a cellular degranulation inhibitor, abolished the early immune response induced by NPs. Overall, our data demonstrates the crucial role of AM activity (migration, phagocytosis, cytokine release) in the rapid and site-specific recruitment of neutrophils during the early phase of particle inhalation, suggesting these processes to be key events in mounting the immune response upon NP inhalation in the lung. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=175 HEIGHT=200 SRC="FIGDIR/small/623349v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@d67e38org.highwire.dtl.DTLVardef@1f8c139org.highwire.dtl.DTLVardef@55be9dorg.highwire.dtl.DTLVardef@1555042_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

eATP/P2X7R axis drives nanoparticle induced neutrophil recruitment in the pulmonary microcirculation

Exposure to nanoparticles (NPs) is frequently associated with adverse cardiovascular effects. In contrast, NPs in nanomedicine hold great promise for precise lung-specific drug delivery, especially considering the extensive pulmonary capillary network that facilitates interactions with bloodstream-suspended particles. Therefore, exact knowledge about interactions and effects of engineered NPs with the pulmonary microcirculation are instrumental for future application of this technology in patients. To unravel the real-time dynamics of intravenously delivered NPs and their effects in the pulmonary microvasculature, we employed intravital microscopy of the mouse lung. PEG amine-modified quantum dots (aQDs) with a low potential for biomolecule and cell interactions and carboxyl-modified quantum dots (cQDs) with a high interaction potential were used, representing two different NP subtypes. Only aQDs triggered rapid neutrophil recruitment in microvessels and their subsequent recruitment to the alveolar space. Application of specific inhibitors revealed that the aQDs induced neutrophil recruitment was linked to cellular degranulation, TNF-, and DAMP release into the circulation, particularly extracellular ATP (eATP). Stimulation of the ATP-gated P2X7R induced the expression of E-selectin on microvascular endothelium with the subsequent E-selectin depended neutrophilic immune response. Leukocyte integrins (LFA-1 and MAC-1) mediated adhesion and reduction in neutrophil crawling velocity on the vascular surface. In summary, this study unravels the complex cascade of neutrophil recruitment during NP-induced sterile inflammation. Thereby we demonstrate novel adverse effects for NPs in the pulmonary microcirculation and provide critical insights for optimizing NP-based drug delivery and therapeutic intervention strategies, to ensure their efficacy and safety in clinical applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/584398v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@d10acborg.highwire.dtl.DTLVardef@1bbfe7org.highwire.dtl.DTLVardef@1d3e415org.highwire.dtl.DTLVardef@2328de_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

Cell circuits underlying nanomaterial specific respiratory toxicology

Nanomaterials emerged as boundless resource of innovation, but their shape and biopersistence related to respiratory toxicology raise longstanding concerns. The development of predictive safety tests for inhaled nanomaterials, however, is hampered by limited understanding of cell type-specific responses. To advance this knowledge, we used single-cell RNA-sequencing to longitudinally analyze cellular perturbations in mice, caused by three carbonaceous nanomaterials of different shape and toxicity upon pulmonary delivery. Focusing on nanomaterial-specific dynamics of lung inflammation, we found persistent depletion of alveolar macrophages by fiber-shaped nanotubes. While only little involvement was observed for alveolar macrophages during the initiation phase, they emerged, together with infiltrating monocyte-derived macrophages, as decisive factors in shifting inflammation towards resolution for spherical nanomaterials, or chronic inflammation for fibers. Fibroblasts, central for fibrosis, sensed macrophage and epithelial signals and emerged as orchestrators of nanomaterial-induced inflammation. Thus, the mode of actions identified in this study will significantly inspire the precision of future in vitro testing. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/579746v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1d6f061org.highwire.dtl.DTLVardef@3fabb1org.highwire.dtl.DTLVardef@21040eorg.highwire.dtl.DTLVardef@1915c3b_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

Primary human lung fibroblasts exhibit trigger- but not disease-specific cellular senescence and impair alveolar epithelial cell progenitor function

Aging is the main risk factor for chronic lung diseases including idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). Accordingly, hallmarks of aging such as cellular senescence are increased in different cell types such as fibroblasts in the lungs of these patients. However, whether the senescent phenotype of fibroblasts derived from IPF or COPD differs is still unknown. Therefore, we characterized senescence at baseline and after exposure to disease-relevant insults (H2O2, bleomycin, and TGF-{beta}1) in cultured primary human lung fibroblasts (phLF) from control donors, IPF, or COPD patients. We found that phLF from different disease-origins have a low baseline senescence. H2O2 and bleomycin treatment induced a senescent phenotype in phLF whereas TGF-{beta}1 only had a pro-fibrotic effect. Interestingly, we did not observe any differences in susceptibility to senescence induction in phLF based on disease origin. However, exposure to different stimuli resulted in different senescent programs in phLF. Moreover, senescent phLF reduced colony formation efficiency of alveolar epithelial progenitor cells. In conclusion, the senescent phenotype of phLF is mainly determined by the senescence inducer and impairs alveolar epithelial progenitor capacity in vitro.

cell biology↗

SARS-CoV-2 Spike Protein Accumulation in the Skull-Meninges-Brain Axis: Potential Implications for Long-Term Neurological Complications in post-COVID-19

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has been associated mainly with a range of neurological symptoms, including brain fog and brain tissue loss, raising concerns about the viruss acute and potential chronic impact on the central nervous system. In this study, we utilized mouse models and human post-mortem tissues to investigate the presence and distribution of the SARS-CoV-2 spike protein in the skull-meninges-brain axis. Our results revealed the accumulation of the spike protein in the skull marrow, brain meninges, and brain parenchyma. The injection of the spike protein alone caused cell death in the brain, highlighting a direct effect on brain tissue. Furthermore, we observed the presence of spike protein in the skull of deceased long after their COVID-19 infection, suggesting that the spikes persistence may contribute to long-term neurological symptoms. The spike protein was associated with neutrophil-related pathways and dysregulation of the proteins involved in the PI3K-AKT as well as complement and coagulation pathway. Overall, our findings suggest that SARS-CoV-2 spike protein trafficking from CNS borders into the brain parenchyma and identified differentially regulated pathways may present insights into mechanisms underlying immediate and long-term consequences of SARS-CoV-2 and present diagnostic and therapeutic opportunities. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/535604v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@b223eforg.highwire.dtl.DTLVardef@15539e9org.highwire.dtl.DTLVardef@4d17a9org.highwire.dtl.DTLVardef@14c63af_HPS_FORMAT_FIGEXP M_FIG C_FIG Short SummaryThe accumulation of SARS-CoV-2 spike protein in the skull-meninges-brain axis presents potential molecular mechanisms and therapeutic targets for neurological complications in long-COVID-19 patients.

neuroscience↗

Phosphoproteomics of cellular mechanosensing reveals NFATC4 as a regulator of myofibroblast activity

Feedback connections between tissue stiffness and cellular contractile forces can instruct cell identity and activity via a process referred to as mechanosensing. Specific phosphoproteome changes during mechanosensing are poorly characterized. In this work, we chart the global phosphoproteome dynamics of primary human lung fibroblasts sensing the stiffness of injury relevant fibronectin coated Poly(dimethylsiloxane) substrates. We discovered a key signaling threshold at a Youngs modulus of eight kPa stiffness, above which cells activated a large number of pathways including RhoA, CK2A1, PKA, AMPK, AKT1, and Hippo-YAP1/TAZ mediated signaling. Time-resolved phosphoproteomics of cell spreading on stiff substrates revealed the temporal dynamics of these stiffness-sensitive signaling pathways. ECM substrate stiffness above eight kPA induced fibroblast contractility, cytoskeletal rearrangements, ECM secretion, and a fibroblast to myofibroblast transition. Our data indicate that phosphorylation of the transcriptional regulator NFATC4 at S213/S217 enhances myofibroblast activity, which is the key hallmark of fibrotic diseases. NFATC4 knock down cells display reduced stiffness induced collagen secretion, cell contractility, nuclear deformation and invasion, suggesting NFATC4 as a novel target for antifibrotic therapy. SynopsisHow tissue stiffness regulates identity and activity of tissue fibroblasts is unclear. Mass spectrometry based analysis of tissue stiffness dependent phosphoproteome changes reveals how primary lung fibroblasts sense the mechanical properties of their environment and identifies NFATC4 as a novel regulator of the stiffness dependent transition of fibroblasts to ECM secreting myofibroblasts. O_LIMass spectrometry analysis reveals the signaling landscape of fibroblast mechanosensing C_LIO_LITime-resolved phosphoproteomic analysis of cell spreading on fibronectin C_LIO_LINFATC4 regulates myofibroblast collagen secretion, cell contractility and invasion C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=38 SRC="FIGDIR/small/528335v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@329c68org.highwire.dtl.DTLVardef@ba7513org.highwire.dtl.DTLVardef@15c32c4org.highwire.dtl.DTLVardef@90b96_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

Fibroblasts-derived extracellular vesicles contain SFRP1 and mediate pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a lethal and chronic lung disease characterized by aberrant intercellular communication, increased extracellular matrix (ECM) deposition, and destruction of functional lung tissue. Extracellular vesicles (EVs) accumulate within the lung in IPF, but their cargo and biological effects remain unclear. Here, we provide the entire the proteome of EV and non-EV fraction during pulmonary fibrosis, and functionally characterize their contribution to fibrosis. EVs were isolated by differential ultracentrifugation of bronchoalveolar lavage fluid (BALF) collected from mice challenged with bleomycin (or PBS as control) or culture supernatants from primary mouse lung fibroblasts. EVs were characterized by nanoparticle tracking analysis, Western Blotting, and quantitative mass spectrometry to define their proteome. EVs accumulation peaked at 14 days post-bleomycin instillation and correlated with decreased lung function. Label-free proteomics identified 107 proteins specific to fibrotic BALF-EVs. This signature was associated with wound healing, extracellular matrix organization, and cell motility. BALF-EVs from fibrotic lungs promoted fibrogenesis, including induction of ECM proteins in precision cut lung slices ex vivo and impaired alveolar epithelial cell stem cell function. Deconvolution using single cell RNA sequencing datasets revealed that fibroblasts are the major cellular source of BALF-EVs. EVs from fibroblasts were significantly enriched in Secreted Frizzled Related Protein 1 (SFRP1). In the lungs of patients with IPF, SFRP1 was significantly increased in mesenchymal cells. Sfrp1 deficiency reduced the ability of fibroblast-derived EVs to potentiate bleomycin-induced lung fibrosis in vivo and led to a reduction in fibrosis marker gene expression. In sum, EVs carry specific protein cargos, such as SFRP1, to contribute to organ remodeling during fibrosis. Our data identified EVs transporting SFRP1 as a potential therapeutic target for IPF.

cell biology↗