Project description:We studied the role of p16INK4a+ fibroblasts in lung fibrosis. We used single cell RNA seq (scRNA-seq) to characterize p16INK4a+ fibroblasts in fibrotic lung.
Project description:Pulmonary fibrosis develops as a consequence of environmentally induced lung injury and/or an inherent disease susceptibility causing fibroblast activation, proliferation and extracellular matrix deposition. The study was undertaken to characterise global gene expression in pulmonary fibroblasts to better understand the mechanisms underlying the fibrotic fibroblast phenotype. Gene expression was evaluated in lung fibroblasts derived from ten controls (normal periphery of resected tumor), open lung biopsies from eight patients with interstitial lung disease associated with systemic sclerosis (fibrotic non specific interstitial pneumonia pattern on biopsy), and from three patients with usual interstitial pneumonia. Lung fibroblasts were grown to confluence in DMEM with 10% fetal calf serum. At confluence, lung fibroblasts were serum-deprived for 44 hours in the presence of fibroblast growth medium with the addition of 0.1% bovine serum albumin (Sigma).
Project description:Pulmonary fibrosis develops as a consequence of environmentally induced lung injury and/or an inherent disease susceptibility causing fibroblast activation, proliferation and extracellular matrix deposition. The study was undertaken to characterise global gene expression in pulmonary fibroblasts to better understand the mechanisms underlying the fibrotic fibroblast phenotype.
Project description:Progressive tissue fibrosis is a major cause of morbidity, and idiopathic pulmonary fibrosis (IPF) is a terminal illness characterized by unremitting matrix deposition in the lung with very limited choice of therapies. The imcomplete understanding of the mechanisms of progressive fibrosis curbs the progress in therapeutics development. Of which, the origin of fibrotic fibroblasts has been poorly defined during the pathogenesis of tissue fibrosis. Here, we fate-mapped a early embryonic transcription factor T-box gene 4 (Tbx4)-derived mesenchymal progenitors in injured adult lung and found that Tbx4+ lineage cells are the major source of myofibroblasts. The ablation of Tbx4+ cells or disruption of Tbx4 signaling attenuated lung fibrosis in bleomycin injury model in mice in vivo. Furthermore, Tbx4+ fibroblasts are more invasive and the regulation of fibroblast invasiveness by Tbx4 is through mediating hyaluronan synthase 2 (HAS2). This study identified a major mesenchymal transcription factor driving the development of fibrotic fibroblasts during lung fibrosis. Understanding the origin, signaling, and functions of these fibroblasts would prove pivotal in the development of therapeutics for patients with progressive fibrotic diseases. We used microarrays to detail the gene expression of Tbx4 and non-Tbx4 cultured fibroblasts.
Project description:Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease characterized by impaired fibroblast clearance, accumulation, and excessive extracellular matrix (ECM) protein production. Wilms' Tumor 1 (WT1), a transcription factor, is selectively upregulated in IPF fibroblasts. However, the mechanisms by which WT1 contributes to fibroblast accumulation and ECM production remain unknown. Here, we investigated the heterogeneity of WT1-expressing fibroblasts using single-nucleus RNA sequencing on the distal lung tissues of IPF patients and healthy controls. WT1 was selectively upregulated in a subset of IPF fibroblasts that co-expressed several pro-survival genes. Both the loss-of-function and gain-of-function studies support the idea that WT1 functions as a positive regulator of multiple pro-survival genes to impair apoptotic clearance and promote ECM production. In support, fibroblast-specific overexpression of WT1 augmented fibroproliferation, myofibroblast accumulation, and ECM production during bleomycin-induced pulmonary fibrosis in both young and old mice. Together, these findings identify WT1 as a potential therapeutic target to attenuate fibroblast expansion, and ECM production in the distal areas of fibrosing lungs.
Project description:Skin fibrotic disease representsa major global healthcare burden, characterized by fibroblast hyperproliferation and excessive accumulation of extracellular matrix.Fibroblasts are found to be heterogeneous in multiple fibrotic diseases,but the fibroblast heterogeneity of skin fibrotic diseases remains unknown.In this study, we performed single-cell RNA-seq in keloid, a paradigm of skin fibrotic diseases, andnormal scardermis tissues.Our results indicate thatkeloid and normal scar fibroblasts could be divided into 4 subpopulations: secretory-papillary, secretory-reticular, mesenchymal and pro-inflammatory.The percentage of mesenchymal fibroblast subpopulationincreased significantly in keloid compared to normal scar. Interestingly, we also found increasing mesenchymal fibroblast subpopulation in scleroderma, another skin fibrotic disease.Function studies showed that the mesenchymal fibroblasts promoted collagen synthesis of the other fibroblasts in keloid partiallythrough secreting POSTN. These findings will help us understandskin fibroticpathogenesis in depth,and provided potential target cells for fibrotic diseases therapies.
Project description:p16INK4A inhibits the CDK4/6 kinases and is therefore an important cell cycle regulator. Accumulation of p16INK4A in response to oncogenic transformation leads to cellular senescence and it is therefore frequently lost in cancer. p16INK4A is also known to accumulate under conditions of cellular oxidative stress and therefore could potentially be regulated by redox signaling, which is a form of signal transduction that is mediated by the reversible oxidation of cysteine-thiol side chains in proteins. We found that oxidation of the single cysteine residue in p16INK4A in human cells occurs under relatively mild oxidizing conditions and that this leads to disulfide dependent dimerization. p16INK4A is a well-characterized all alpha-helical protein, but we find that upon cysteine-dependent dimerization, p16INK4A undergoes a dramatic structural rearrangement and forms aggregates that have the typical features of amyloid fibrils, including binding of diagnostic dyes, presence of cross-β sheet structure, and typical dimensions found in electron microscopy. We find that p16INK4A amyloid formation abolishes its function as a CDK4/6 inhibitor in human cells. Taken together, these observations mechanistically link the cellular redox state to the inactivation of p16INK4A through the formation of amyloid fibrils.
Project description:Systemic scleroderma (SSc) is an autoimmune disease which results in fibrotic production in the lung. Resultant SSC-pulmonary fibrosis is the main cause of mortality among SSc patients. From high throughput RNAi screening, we uncovered the ubiquitin E3 ligase KLHL42 as a potential pro-fibrotic mediator of TGFb-dependent fibrotic signaling in primary SSc lung fibroblasts. In this analysis, we sought to uncover putative substrates for KLHL42 by comparing SSc lung fibroblasts with control or KLHL42 siRNA prior to TGFb-treatment, lysis, and TUBE precipitation. The resultant pull-down was analyzed with LC-MS/MS.
Project description:Fibrosis is a hallmark feature of airway remodeling in asthma. However, the exact molecular mechanisms driving its development and progression remain inadequately understood. Interleukin-5 (IL-5) plays a potent role in the inflammatory response associated with eosinophils and asthma pathophysiology, but its direct impact on lung fibroblasts and contribution to fibrosis have yet to be fully explored. In our study, we investigated the pro-fibrotic effects of IL-5 on human lung fibroblasts derived from asthmatic and normal subjects. Following IL-5 stimulation, these fibroblasts were subjected to a series of experiments to assess IL-5’s impact on fibrosis. Our findings reveal that IL-5 triggers the release of several extracellular matrix (ECM) components, and disrupts the balance of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) . This imbalance further disrupts the MMP/TIMP ratio. Additionally, IL-5 stimulation promotes the release of pro-inflammatory and pro-fibrotic cytokines, Overall, these findings indicate a novel pro-fibrotic role of IL-5 through its direct action on lung fibroblasts, offering valuable insights into the therapeutic potential of targeting IL-5 to mitigate or possibly reverse airway remodeling in asthma.