Revisiting the Hayflick Limit: Insights from an Integrated Analysis of Changing Transcripts, Proteins, Metabolites and Chromatin

Replicative senescence (RS) as a model has become the central focus of research into cellular aging in vitro. Despite decades of study, this process through which cells cease dividing is not fully understood in culture, and even much less so in vivo during development and with aging. Here, we revisit Hayflicks original observation of RS in WI-38 human fetal lung fibroblasts equipped with a battery of high dimensional modern techniques and analytical methods to deeply profile the process of RS across each aspect of the central dogma and beyond. We applied and integrated RNA-seq, proteomics, metabolomics, and ATAC-seq to a high resolution RS time course. We found that the transcriptional changes that underlie RS manifest early, gradually increase, and correspond to a concomitant global increase in accessibility in nucleolar and lamin associated domains. During RS WI-38 fibroblast gene expression patterns acquire a striking resemblance to those of myofibroblasts in a process similar to the epithelial to mesenchymal transition (EMT). This observation is supported at the transcriptional, proteomic, and metabolomic levels of cellular biology. In addition, we provide evidence suggesting that this conversion is regulated by the transcription factors YAP1/TEAD1 and the signaling molecule TGFB-2.

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Matrix GLA protein modulates branching morphogenesis in fetal rat lung

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00188.2003 ◽

2004 ◽

Vol 286 (6) ◽

pp. L1179-L1187 ◽

Cited By ~ 18

Author(s):

Kirk A. Gilbert ◽

Stephen R. Rannels

Keyword(s):

Mrna Expression ◽

Time Course ◽

Immunohistochemical Analysis ◽

Fetal Lung ◽

Branching Morphogenesis ◽

Matrix Gla Protein ◽

Antibody Treatment ◽

Developmentally Regulated

The regulation of matrix γ-carboxyglutamic acid protein (MGP) expression during the process of lung branching morphogenesis and development was investigated. MGP mRNA expression was determined over an embryonic and postnatal time course and shown to be developmentally regulated. Immunohistochemical analysis revealed increased staining for MGP in peripheral mesenchyme surrounding distal epithelial tubules. Fetal lung explants were used as an in vitro growth model to examine expression and regulation of MGP during branching morphogenesis. MGP mRNA expression over the culture interval mimicked the in vivo time course. Explants cultured in the presence of antibodies against MGP showed gross dilation and reduced terminal lung bud counts, accompanied by changes in MGP, sonic hedgehog, and patched mRNA expression. Similarly, antifibronectin antibody treatment resulted in explant dilation and reduced MGP expression, providing evidence for an interaction with MGP and fibronectin. Conversely, intraluminal microinjection of anti-MGP antibodies had no effect either on explant growth or MGP expression, supporting the hypothesis that MGP exerts its effects through the mesenchyme. Taken together, the results suggest that MGP plays a role in lung growth and development, likely via temporally and spatially specific interactions with other branching morphogenesis-related proteins to influence growth processes.

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Human Cytomegalovirus Replication and Infection-Induced Syncytia Formation in Labial, Foreskin, and Fetal Lung Fibroblasts

Viruses ◽

10.3390/v13122355 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2355

Author(s):

Alexis Aguiar ◽

Melissa Galinato ◽

Maite’ Bradley Silva ◽

Bryant Toth ◽

Michael A. McVoy ◽

...

Keyword(s):

Endothelial Cells ◽

Human Cytomegalovirus ◽

Fetal Lung ◽

Lung Fibroblasts ◽

Anatomical Site ◽

Cmv Infection ◽

Viral Spread ◽

Syncytia Formation

Only a handful of cell types, including fibroblasts, epithelial, and endothelial cells, can support human cytomegalovirus (CMV) replication in vitro, in striking contrast to the situation in vivo. While the susceptibility of epithelial and endothelial cells to CMV infection is strongly modulated by their anatomical site of origin, multiple CMV strains have been successfully isolated and propagated on fibroblasts derived from different organs. As oral mucosal cells are likely involved in CMV acquisition, we sought to evaluate the ability of infant labial fibroblasts to support CMV replication, compared to that of commonly used foreskin and fetal lung fibroblasts. No differences were found in the proportion of cells initiating infection, or in the amounts of viral progeny produced after exposure to the fibroblast-adapted CMV strain AD169 or to the endothelial cell-adapted strain TB40/E. Syncytia formation was, however, significantly enhanced in infected labial and lung fibroblasts compared to foreskin-derived cells, and did not occur after infection with AD169. Together, these data indicate that fibroblast populations derived from different tissues are uniformly permissive to CMV infection but retain phenotypic differences of potential importance for infection-induced cell–cell fusion, and ensuing viral spread and pathogenesis in different organs.

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Protective Effect of Remdesivir Against Pulmonary Fibrosis in Mice

Frontiers in Pharmacology ◽

10.3389/fphar.2021.692346 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xiaohe Li ◽

Rui Liu ◽

Yunyao Cui ◽

Jingjing Liang ◽

Zhun Bi ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Antiviral Drug ◽

Alveolar Epithelial Cells ◽

Lung Damage ◽

Lung Fibroblasts ◽

Mesenchymal Transition ◽

Alveolar Epithelial ◽

Tgf Β1

Pulmonary fibrosis is a known sequela of severe or persistent lung damage. Existing clinical, imaging and autopsy studies have shown that the lungs exhibit a pathological pulmonary fibrosis phenotype after infection with coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Pulmonary fibrosis may be one of the most serious sequelae associated with coronavirus disease 2019 (COVID-19). In this study, we aimed to examine the preventative effects of the antiviral drug remdesivir on pulmonary fibrosis. We used a mouse model of bleomycin-induced pulmonary fibrosis to evaluate the effects of remdesivir on pulmonary fibrosis in vivo and further explored the potential pharmacological mechanisms of remdesivir in lung fibroblasts and alveolar epithelial cells in vitro. The preventive remdesivir treatment was started on the day of bleomycin installation, and the results showed that remdesivir significantly alleviated bleomycin-induced collagen deposition and improved pulmonary function. In vitro experiments showed that remdesivir dose-dependently suppressed TGF-β1-induced lung fibroblast activation and improved TGF-β1-induced alveolar epithelial to mesenchymal transition. Our results indicate that remdesivir can preventatively alleviate the severity of pulmonary fibrosis and provide some reference for the prevention of pulmonary fibrosis in patients with COVID-19.

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In vivo expression patterns of MyoD, p21, and Rb proteins in myonuclei and satellite cells of denervated rat skeletal muscle

AJP Cell Physiology ◽

10.1152/ajpcell.00080.2004 ◽

2004 ◽

Vol 287 (2) ◽

pp. C484-C493 ◽

Cited By ~ 65

Author(s):

Minenori Ishido ◽

Katsuya Kami ◽

Mitsuhiko Masuhara

Keyword(s):

Skeletal Muscle ◽

Satellite Cell ◽

Time Course ◽

Kinase Inhibitor ◽

Expression Patterns ◽

Cell Types ◽

Cell Nuclei ◽

Myogenic Regulatory Factor

MyoD, a myogenic regulatory factor, is rapidly expressed in adult skeletal muscles in response to denervation. However, the function(s) of MyoD expressed in denervated muscle has not been adequately elucidated. In vitro, it directly transactivates cyclin-dependent kinase inhibitor p21 (p21) and retinoblastoma protein (Rb), a downstream target of p21. These factors then act to regulate cell cycle withdrawal and antiapoptotic cell death. Using immunohistochemical approaches, we characterized cell types expressing MyoD, p21, and Rb and the relationship among these factors in the myonucleus of denervated muscles. In addition, we quantitatively examined the time course changes and expression patterns among distinct myofiber types of MyoD, p21, and Rb during denervation. Denervation induced MyoD expression in myonuclei and satellite cell nuclei, whereas p21 and Rb were found only in myonuclei. Furthermore, coexpression of MyoD, p21, and Rb was induced in the myonucleus, and quantitative analysis of these factors determined that there was no difference among the three myofiber types. These observations suggest that MyoD may function in myonuclei in response to denervation to protect against denervation-induced apoptosis via perhaps the activation of p21 and Rb, and function of MyoD expressed in satellite cell nuclei may be negatively regulated. The present study provides a molecular basis to further understand the function of MyoD expressed in the myonuclei and satellite cell nuclei of denervated skeletal muscle.

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FGF-18 is upregulated in the postnatal rat lung and enhances elastogenesis in myofibroblasts

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00096.2004 ◽

2005 ◽

Vol 288 (1) ◽

pp. L43-L51 ◽

Cited By ~ 43

Author(s):

Bernadette Chailley-Heu ◽

Olivier Boucherat ◽

Anne-Marie Barlier-Mur ◽

Jacques R. Bourbon

Keyword(s):

Lung Development ◽

Lysyl Oxidase ◽

Smooth Muscle Actin ◽

Fold Increase ◽

Fetal Lung ◽

Lung Fibroblasts ◽

Rat Lung ◽

Fetal Fibroblasts

The fibroblast growth factors (FGFs) are key players in fetal lung development, but little is known about their status in postnatal lung. Here, we investigated the expression pattern of FGF-18 transcripts through the perinatal period and evidenced a sevenfold increase after birth that paralleled changes in elastin expression. In vitro, recombinant human (rh)FGF-18 had a mitogenic activity on day 21 fetal rat lung fibroblasts and stimulated its own expression in the latter, whereas FGF-2 inhibited it. At 50 or 100 ng/ml, rhFGF-18 increased the expression of α-smooth muscle actin (α-SMA; 2.5-fold), a characteristic marker of myofibroblasts, of tropoelastin (6.5-fold), of lysyl oxidase (2-fold), and of fibulins 1 and 5 (8- and 2.2-fold) in confluent fibroblasts isolated from fetal day 21 lung; similar results were obtained with fibroblasts from day 3 postnatal lungs. Elastin protein expression was also slightly increased in fetal fibroblasts. Lung analysis on day 4 in rat pups that had received rhFGF-18 (3 μg) on days 0 and 1 showed a 1.7-fold increase of tropoelastin transcripts, whereas α-SMA transcripts were unchanged. In contrast, rhFGF-2 markedly decreased expression of elastin in vitro and in vivo and of fibulin 5 in vitro. In addition, vitamin A, which is known to enhance alveolar development, elevated FGF-18 and elastin expressions in day 2 lungs, thus advancing the biological increase. We postulate that FGF-18 is involved in postnatal lung development through stimulating myofibroblast proliferation and differentiation.

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Let-7d microRNA affects mesenchymal phenotypic properties of lung fibroblasts

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00149.2013 ◽

2014 ◽

Vol 306 (6) ◽

pp. L534-L542 ◽

Cited By ~ 57

Author(s):

Luai Huleihel ◽

Ahmi Ben-Yehudah ◽

Jadranka Milosevic ◽

Guoying Yu ◽

Kusum Pandit ◽

...

Keyword(s):

Epithelial To Mesenchymal Transition ◽

Smooth Muscle Actin ◽

High Mobility ◽

Lung Fibroblasts ◽

Phenotypic Properties ◽

Mesenchymal Transition ◽

Junction Protein ◽

Primary Fibroblasts

MicroRNAs are small noncoding RNAs that inhibit protein expression. We have previously shown that the inhibition of the microRNA let-7d in epithelial cells caused changes consistent with epithelial-to-mesenchymal transition (EMT) both in vitro and in vivo. The aim of this study was to determine whether the introduction of let-7d into fibroblasts alters their mesenchymal properties. Transfection of primary fibroblasts with let-7d caused a decrease in expression of the mesenchymal markers α-smooth muscle actin, N-cadherin, fibroblast-specific protein-1, and fibronectin, as well as an increase in the epithelial markers tight junction protein-1 and keratin 19. Phenotypic changes were also present, including a delay in wound healing, reduced motility, and proliferation of fibroblasts following transfection. In addition, we examined the effects of transfection on fibroblast responsiveness to TGF-β, an important factor in many fibrotic processes such as lung fibrosis and found that let-7d transfection significantly attenuated high-mobility group-A2 protein induction by TGF-β. Our results indicate that administration of the epithelial microRNA let-7d can significantly alter the phenotype of primary fibroblasts.

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LINC00152 upregulates ZEB1 expression and enhances epithelial-mesenchymal transition and oxaliplatin resistance in esophageal cancer by interacting with EZH2

Cancer Cell International ◽

10.1186/s12935-020-01620-1 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Shuyao Zhang ◽

Wei Liao ◽

Qinshui Wu ◽

Xiaoshan Huang ◽

Zhen Pan ◽

...

Keyword(s):

Esophageal Cancer ◽

Epithelial Mesenchymal Transition ◽

Expression Patterns ◽

Special Focus ◽

Loss Of Function ◽

Mesenchymal Transition ◽

Ec Cells ◽

Zeb1 Expression

Abstract Background Expression of the long non-coding mRNA LINC00152 has been reported to correlate with cancer cell resistance to oxaliplatin (L-OHP). However, little is known regarding the molecular mechanism of LINC00152 in esophageal cancer (EC). Hence, we intended to characterize the role of LINC00152 in EC, with a special focus on epithelial-mesenchymal transition (EMT) and L-OHP resistance. Methods We collected EC tissues and identified EC cell lines with higher L-OHP resistance, and then characterized expression patterns of LINC00152, Zeste Homologue 2 (EZH2), Zinc finger e-box binding homeobox (ZEB1) and EMT-related genes using RT-qPCR and Western blot analysis. Furthermore, their functional significance was identified by gain and loss-of-function experiments. The relationship among LINC00152, EZH2 and ZEB1 was examined using RIP, RNA pull-down and ChIP assays. Additionally, resistance of EC cells to L-OHP was reflected by CCK-8 assay to detect cell viability. Animal experiments were also conducted to detect the effects of the LINC00152/EZH2/ZEB1 on EMT and L-OHP resistance. Results LINC00152, EZH2 and ZEB1 were highly expressed in EC tissues and Kyse−150/TE-1 cells. As revealed by assays in vitro and in vivo, LINC00152 positively regulated ZEB1 expression through interaction with EZH2 to enhance EMT and L-OHP resistance in EC cells. In contrast, silencing of LINC00152 contributed to attenuated EMT and drug resistance of EC cells to L-OHP. Conclusions Our study demonstrates that LINC00152/EZH2/ZEB1 axis can regulate EMT and resistance of EC cells to L-OHP, thus presenting a potential therapeutic target for EC treatment.

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In Vivo Analysis of Staphylococcus aureus-Infected Mice Reveals Differential Temporal and Spatial Expression Patterns of fhuD2

Infection and Immunity ◽

10.1128/iai.00270-17 ◽

2017 ◽

Vol 85 (10) ◽

Cited By ~ 6

Author(s):

Marta Bacconi ◽

Andreas F. Haag ◽

Emiliano Chiarot ◽

Paolo Donato ◽

Fabio Bagnoli ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Time Course ◽

Protective Efficacy ◽

Expression Patterns ◽

Vaccine Antigen ◽

Pcr Analysis ◽

Content Type ◽

Iron Deprivation

ABSTRACT Staphylococcus aureus is an opportunistic human pathogen and a major cause of invasive infections such as bacteremia, endocarditis, pneumonia, and wound infections. FhuD2 is a staphylococcal lipoprotein involved in the uptake of iron-hydroxymate and is under the control of the iron uptake regulator Fur. This protein is part of an investigational multicomponent vaccine formulation that has shown protective efficacy in several murine models of infection. Even though fhuD2 expression has been shown to be upregulated in murine kidneys infected with S. aureus, it is not known whether the bacterium undergoes increased iron deprivation during prolonged infection. Furthermore, different S. aureus infection niches might provide different environments and levels of iron availability, resulting in different fhuD2 expression patterns among organs of the same host. To address these questions, we characterized the in vitro expression of the fhuD2 gene and confirmed Fur-dependent regulation of its expression. We further investigated its expression in mice infected with a bioluminescent reporter strain of S. aureus expressing the luciferase operon under the control of the fhuD2 promoter. The emission of bioluminescence in different organs was followed over a 7-day time course, and quantitative real-time PCR analysis of the RNA transcribed from the endogenous fhuD2 gene was performed. Using this approach, we were able to show that fhuD2 expression was induced during infection in all organs analyzed and that differences in expression were observed at different time points and in different infected organs. Our data suggest that S. aureus undergoes increased iron deprivation during the progression of infection in diverse host organs and accordingly induces dedicated iron acquisition mechanisms. Since FhuD2 plays a central role in providing the pathogen with the required iron, further knowledge of the patterns of fhuD2 expression in vivo during infection will be instrumental in better defining the role of this antigen in S. aureus pathogenesis and as a vaccine antigen.

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Ginsenoside Rg3 Inhibits Pulmonary Fibrosis by Preventing HIF-1α Nuclear Localisation

10.21203/rs.3.rs-67612/v1 ◽

2020 ◽

Author(s):

Zhuo Fu ◽

Yong-sheng Xu ◽

Chun-quan Cai

Keyword(s):

Pulmonary Fibrosis ◽

Cell Injury ◽

Epithelial Mesenchymal Transition ◽

Lung Fibroblasts ◽

Cell Phenotype ◽

Ginsenoside Rg3 ◽

Nuclear Localisation ◽

Mesenchymal Transition

Abstract Introduction: Excessive fibroblast proliferation during pulmonary fibrosis leads to structural abnormalities in lung tissue and causes hypoxia and cell injury. However, the mechanisms and effective treatment are still limited.Methods: In vivo, we used bleomycin to induce pulmonary fibrosis in mice. IHC and Masson staining were used to evaluate the inhibitory effect of ginsenoside Rg3. In vitro, scanning electron microscopy, transwell and wound healing were used to evaluate the cell phenotype of LL 29 cells. In addition, biacore was used to detect the binding of ginsenoside Rg3 and HIF-1α.Results: Here, we find that bleomycin induces the activation of the HIF-1α/TGFβ1 signalling pathway and further enhances the migration and proliferation of fibroblasts through the epithelial mesenchymal transition (EMT). Ginsenoside Rg3 can slow down the progression of pulmonary fibrosis by inhibiting the nuclear localisation of HIF-1α. In addition, molecular docking and biacore experiments indicated that ginsenoside Rg3 can bind HIF-1α and restrict the progression of pulmonary fibrosis in animals. Hypoxia can lead to excessive proliferation of lung fibroblasts and further accelerate lung fibrosis.Conclusions: This finding suggests that early targeted treatment of hypoxia may have potential value in the treatment of pulmonary fibrosis.

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Nifuroxazide Ameliorates Pulmonary Fibrosis By Blocking Myofibroblast Genesis And Stat3 Activation: A Drug Repurposing Study

10.21203/rs.3.rs-557091/v1 ◽

2021 ◽

Author(s):

Cailing Gan ◽

Qianyu Zhang ◽

Hongyao Liu ◽

Guan Wang ◽

Liqun Wang ◽

...

Keyword(s):

Epithelial Cells ◽

Pulmonary Fibrosis ◽

Western Blot ◽

Immune Cells ◽

Transforming Growth Factor ◽

Lung Fibroblasts ◽

Mesenchymal Transition ◽

Tgf Β1

Abstract Background: Idiopathic pulmonary fibrosis (IPF) is a lung disease with complex pathogenesis, high mortality. The development of new drugs is time-consuming and laborious, and the research on new use of old drugs can save time and clinical costs and even avoid serious side effects. Nifuroxazide (NIF) was originally used to treat diarrhoea, but in recent years it has been found to have other pharmacological effects such as anti-tumor and inhibiting inflammatory diseases related to diabetic nephropathy. However, there are no reports about its role in pulmonary fibrosis.Methods: The therapeutic effect of NIF on bleomycin (BLM)-induced pulmonary fibrosis in vivo was measured by ELISA, hydroxyproline content, H&E and Masson staining, IHC and Western blot. The content of immune cells in lung tissue was analyzed by flow cytometry. NIF cytotoxicity were evaluated in NIH/3T3, Human pulmonary fibroblasts (HPF), A549 and Rat primary lung fibroblasts (RPLF) using MTT assay. Finally, a cell model induced by transforming growth factor-β1 (TGF-β1) stimulation and different in vitro experiments (Immunofluorescence, Western blot, Wound migration assay) were conducted to determine the effect of NIF on the activation of fibroblasts and the epithelial-mesenchymal transition (EMT) and migration of epithelial cells.Results: In vivo, intraperitoneal injection of NIF relieved and reversed pulmonary fibrosis caused by BLM bronchial instillation. In addition, nifuroxazide inhibited the expression of a variety of cellular inflammatory factors and immune cells. Furthermore, nifuroxazide suppressed the activation of fibroblasts and the EMT of epithelial cells induced by TGF-β1. Most importantly, we used an analytical docking experiment and thermal shift assay to further verify that nifuroxazide worked in conjunction with Stat3. Moreover, nifuroxazide decreased the expression of p-Stat3 in vitro and in vivo.Conclusion: These results suggest that NIF inhibits and reverses pulmonary fibrosis and support NIF as a viable treatment option that may bring benefits to patients with IPF.

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