MicroRNA-377-3p exacerbates chronic obstructive pulmonary disease through suppressing ZFP36L1 expression and inducing lung fibroblast senescence.

Chronic obstructive pulmonary disease (COPD) is a leading aging related cause of global mortality. Small airway narrowing is recognized as an early and significant factor for COPD development. Senescent fibroblasts were observed to accumulate in lung of COPD patients and promote COPD progression through aberrant extracellular matrix (ECM) deposition and senescence-associated secretory phenotype (SASP). On the basis of our previous study, we further investigated the the causes for the increased levels of miR-377-3p in the blood of COPD patients, as well as its regulatory function in the pathological progression of COPD. We found that the majority of up-regulated miR-377-3p was localized in lung fibroblasts. Inhibition of miR-377-3p improved chronic smoking-induced COPD in mice. Mechanistically, miR-377-3p promoted senescence of lung fibroblasts, while knockdown of miR-377-3p attenuated bleomycin-induced senescence in lung fibroblasts. We also identified ZFP36L1 as a direct target for miR-377-3p that likely mediated its pro senescence activity in lung fibroblasts. Our data reveal that miR-377-3p is crucial for COPD pathogenesis, and may serve as a potential target for COPD therapy.

Vistusertib improves pulmonary inflammation and fibrosis by modulating inflammatory/oxidative stress mediators via suppressing the mTOR signalling.

INTRODUCTION: Inflammation and oxidative stress are key factors in the development of pulmonary fibrosis (PF) by promoting the differentiation of fibroblasts through modulating various pathways including Wnt/ß-catenin, TGF-ß and mTOR signalling. OBJECTIVE AND METHODS: This study aimed to evaluate the effects and elucidate the mechanisms of vistusertib (VSB) in treating pulmonary inflammation/fibrosis, specifically by targeting the mTOR pathway using various in vitro and in vivo models. RESULTS: Lipopolysaccharide (LPS)-induced inflammation model in macrophages (RAW 264.7), epithelial (BEAS-2B) and endothelial (HMVEC-L) cells revealed that treatment with VSB significantly reduced the IL-6, TNF-α, CCL2, and CCL7 expression. TGF-ß induced differentiation was also significantly reduced upon VSB treatment in fibrotic cells (LL29 and DHLF). Further, bleomycin-induced inflammation and fibrosis models demonstrated that treatment with VSB significantly ameliorated the severe inflammation, and lung architectural distortion, by reducing the inflammatory markers expression/levels, inflammatory cells and oxidative stress indicators. Further, fibrosis model results exhibited that, VSB treatment significantly reduced the α-SMA, collagen and TGF-ß expressions, improved the lung architecture and restored lung functions. CONCLUSION: Overall, this study uncovers the anti-inflammatory/anti-fibrotic effects of VSB by modulating the mTOR activation. Although VSB was tested for lung fibrosis, it can be tested for other fibrotic disorders to improve the patient's survival and quality of life.

Cargo-eliminated osteosarcoma-derived small extracellular vesicles mediating competitive cellular uptake for inhibiting pulmonary metastasis of osteosarcoma.

Osteosarcoma (OS) derived small extracellular vesicles (OS-sEVs) have been shown to induce the formation of cancer-associated fibroblasts (CAFs), characterized by elevated pro-inflammatory factor expression and enhanced migratory and contractile abilities. These CAFs play a crucial role in priming lung metastasis by orchestrating the pre-metastatic niche (PMN) in the lung. Disrupting the communication between OS-sEVs and lung fibroblasts (LFs) emerges as a potent strategy to hinder OS pulmonary metastasis. Our previously established saponin-mediated cargo-elimination strategy effectively reduces the cancer-promoting ability of tumor-derived small extracellular vesicles (TsEVs) while preserving their inherent targeting capability. In this study, we observed that cargo-eliminated OS-sEVs (CE-sEVs) display minimal pro-tumoral and LFs activation potential, yet retain their ability to target LFs. The uptake of OS-sEVs by LFs can be concentration-dependently suppressed by CE-sEVs, preventing the conversion of LFs into CAFs and thus inhibiting PMN formation and pulmonary metastasis of OS. In summary, this study proposes a potential strategy to prevent LFs activation, PMN formation in the lung, and OS pulmonary metastasis through competitive inhibition of OS-sEVs' function by CE-sEVs.

Ferulic acid grafted into ß-cyclodextrin nanosponges ameliorates Paraquat-induced human MRC-5 fibroblast injury.

Paraquat (PQ) is a commercially important and effective herbicide in the world. Nevertheless, it has higher toxicity causing acute organ damage and different complications, mainly in the lungs and kidneys. Ferulic acid (FA), 4-hydroxy-3-methoxycinnamic acid imposes multiple pharmacological impacts. No protective effect of FA on PQ poisoning-caused human embryonic lung fibroblast damage has not been reported. Despite their many beneficial effects, FA is characterized by poor water solubility, low bioavailability, and phytochemical instability. To solve the problem, ß-cyclodextrin nanosponge (ß-CD NSs) was utilized to increase the solubility of FA so that it was grafted into ß-CD NSs to establish ß-CD@FA NSs. The purpose of this work was to examine for the first time the protective effect of ß-CD@FA NS on MRC-5 human lung cells damages induced by PQ poisoning. MTS assay was performed to investigate the viability of MRC-5 cells at different concentrations of FA/ß-CD@FA NSs when cells were co-cultured with 0.2 µg/mL PQ. The flow cytometry study was carried out to determine apoptosis. Malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) levels were detected using appropriate biochemistry kits. Compared with the PQ group, the cell activity, CAT, and SOD levels were significantly increased in the FA and chiefly in ß-CD@FA NSs intervention groups, whereas apoptosis and MDA levels were markedly decreased. The inflammatory factors tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and interleukin 22 (IL-22) were detected. The results demonstrate that ß-CD@FA NSs can inhibit PQ-induced cell damage by enhancing antioxidant stress capacity and regulation of inflammatory responses.

Expression of Stress-Induced Genes in Bronchoalveolar Lavage Cells and Lung Fibroblasts from Healthy and COPD Subjects.

Chronic obstructive pulmonary disease (COPD) is commonly caused from smoking cigarettes that induce biological stress responses. Previously we found disorganized endoplasmic reticulum (ER) in fibroblasts from COPD with different responses to chemical stressors compared to healthy subjects. Here, we aimed to investigate differences in stress-related gene expressions within lung cells from COPD and healthy subjects. Bronchoalveolar lavage (BAL) cells were collected from seven COPD and 35 healthy subjects. Lung fibroblasts were derived from 19 COPD and 24 healthy subjects and exposed to cigarette smoke extract (CSE). Gene and protein expression and cell proliferation were investigated. Compared to healthy subjects, we found lower gene expression of CHOP in lung fibroblasts from COPD subjects. Exposure to CSE caused inhibition of lung fibroblast proliferation in both groups, though the changes in ER stress-related gene expressions (ATF6, IRE1, PERK, ATF4, CHOP, BCL2L1) and genes relating to proteasomal subunits mostly occurred in healthy lung fibroblasts. No differences were found in BAL cells. In this study, we have found that lung fibroblasts from COPD subjects have an atypical ER stress gene response to CSE, particularly in genes related to apoptosis. This difference in response to CSE may be a contributing factor to COPD progression.

IQGAP1 Regulates Actin Polymerization and Contributes to Bleomycin-Induced Lung Fibrosis.

We previously found IQ motif containing GTPase activating protein (IQGAP1) to be consistently elevated in lung fibroblasts (LF) isolated from patients with scleroderma (systemic sclerosis, SSc)-associated interstitial lung disease (ILD) and reported that IQGAP1 contributed to SSc by regulating expression and organization of α-smooth muscle actin (SMA) in LF. The aim of this study was to compare the development of ILD in the presence and absence of IQGAP1. Pulmonary fibrosis was induced in IQGAP1 knockout (KO) and wild-type (WT) mice by a single-intratracheal instillation of bleomycin. Two and three weeks later, mice were euthanized and investigated. We observed that the IQGAP1 KO mouse was characterized by a reduced rate of actin polymerization with reduced accumulation of actin in the lung compared to the WT mouse. After exposure to bleomycin, the IQGAP1 KO mouse demonstrated decreased contractile activity of LF, reduced expression of SMA, TGFß, and collagen, and lowered overall fibrosis scores compared to the WT mouse. The numbers of inflammatory cells and expression of pro-inflammatory cytokines in lung tissue were not significantly different between IQGAP1 KO and WT mice. We conclude that IQGAP1 plays an important role in the development of lung fibrosis induced by bleomycin, and the absence of IQGAP1 reduces the contractile activity of lung fibroblast and bleomycin-induced pulmonary fibrosis. Thus, IQGAP1 may be a potential target for novel anti-fibrotic therapies for lung fibrosis.

Tumor-derived exosomal linc00881 induces lung fibroblast activation and promotes osteosarcoma lung migration.

Osteosarcoma (OS) commonly metastasizes to the lung, yet the underlying molecular mechanisms remain poorly understood. Exosomes play a crucial role in tumor migration, including OS lung migration. However, the underlying mechanism by which exosome-derived long non-coding RNAs (lncRNAs) contribute to lung migration in osteosarcoma (OS) remains unclear. This study presents a newly discovered lncRNA, linc00881, derived from OS exosomes. Our study shows that linc00881 promotes the migration of OS cells to the lung and induces the conversion of normal lung fibroblasts into cancer-associated fibroblasts (CAFs). Subsequently, we found that exosomal linc00881 secreted by OS cells can regulate the expression of matrix metalloproteinase 2 (MMP2) in HFL-1 cells by sponging miR-29c-3p, thereby activating the NF-κB signaling in lung fibroblasts. Finally, we discovered that pro-inflammatory cytokines, namely IL-1ß, IL-6, and IL-8, were secreted through the linc00881/miR-29c-3p/MMP2 axis. These results suggest that OS-derived exosomes can mediate the intercellular crosstalk between OS cells and lung fibroblasts, ultimately impacting OS lung migration. Our study provides a potential target for the treatment of OS lung migration.

Screening for Inhibitors of YAP Nuclear Localization Identifies Aurora Kinase A as a Modulator of Lung Fibrosis.

Idiopathic pulmonary fibrosis is a progressive lung disease with limited therapeutic options that is characterized by pathological fibroblast activation and aberrant lung remodeling with scar formation. YAP (Yes-associated protein) is a transcriptional coactivator that mediates mechanical and biochemical signals controlling fibroblast activation. We previously identified HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors (statins) as YAP inhibitors based on a high-throughput small-molecule screen in primary human lung fibroblasts. Here we report that several Aurora kinase inhibitors were also identified from the top hits of this screen. MK-5108, a highly selective inhibitor for AURKA (Aurora kinase A), induced YAP phosphorylation and cytoplasmic retention and significantly reduced profibrotic gene expression in human lung fibroblasts. The inhibitory effect on YAP nuclear translocation and profibrotic gene expression is specific to inhibition of AURKA, but not Aurora kinase B or C, and is independent of the Hippo pathway kinases LATS1 and LATS2 (Large Tumor Suppressor 1 and 2). Further characterization of the effects of MK-5108 demonstrate that it inhibits YAP nuclear localization indirectly via effects on actin polymerization and TGFß (Transforming Growth Factor ß) signaling. In addition, MK-5108 treatment reduced lung collagen deposition in the bleomycin mouse model of pulmonary fibrosis. Our results reveal a novel role for AURKA in YAP-mediated profibrotic activity in fibroblasts and highlight the potential of small-molecule screens for YAP inhibitors for identification of novel agents with antifibrotic activity.

The profibrotic and senescence phenotype of old lung fibroblasts is reversed or ameliorated by genetic and pharmacological manipulation of Slc7a11 expression.

Increased senescence and expression of profibrotic genes in old lung fibroblasts contribute to disrepair responses. We reported that primary lung fibroblasts from old mice have lower expression and activity of the cystine transporter Slc7a11/xCT than cells from young mice, resulting in changes in both the intracellular and extracellular redox environments. This study examines the hypothesis that low Slc7a11 expression in old lung fibroblasts promotes senescence and profibrotic gene expression. The levels of mRNA and protein of Slc7a11, senescence markers, and profibrotic genes were measured in primary fibroblasts from the lungs of old (24 mo) and young (3 mo) mice. In addition, the effects of genetic and pharmacological manipulation of Slc7a11 were investigated. We found that decreased expression of Slc7a11 in old cells was associated with elevated markers of senescence (p21, p16, p53, and ß-galactosidase) and increased expression of profibrotic genes (Tgfb1, Smad3, Acta2, Fn1, Col1a1, and Col5a1). Silencing of Slc7a11 in young cells replicated the aging phenotype, whereas overexpression of Slc7a11 in old cells decreased expression of senescence and profibrotic genes. Young cells were induced to express the senescence and profibrotic phenotype by sulfasalazine, a Slc7a11 inhibitor, whereas treatment of old cells with sulforaphane, a Slc7a11 inducer, decreased senescence without affecting profibrotic genes. Like aging cells, idiopathic pulmonary fibrosis fibroblasts show decreased Slc7a11 expression and increased profibrotic markers. In short, old lung fibroblasts manifest a profibrotic and senescence phenotype that is modulated by genetic or pharmacological manipulation of Slc7a11.

Electronic cigarette liquids impair metabolic cooperation and alter proteomic profiles in V79 cells.

BACKGROUND: Although still considered a safer alternative to classical cigarettes, growing body of work points to harmful effects of electronic cigarettes (e-cigarettes) affecting a range of cellular processes. The biological effect of e-cigarettes needs to be investigated in more detail considering their widespread use. METHODS: In this study, we treated V79 lung fibroblasts with sub-cytotoxic concentration of e-cigarette liquids, with and without nicotine. Mutagenicity was evaluated by HPRT assay, genotoxicity by comet assay and the effect on cellular communication by metabolic cooperation assay. Additionally, comprehensive proteome analysis was performed via high resolution, parallel accumulation serial fragmentation-PASEF mass spectrometry. RESULTS: E-cigarette liquid concentration used in this study showed no mutagenic or genotoxic effect, however it negatively impacted metabolic cooperation between V79 cells. Both e-cigarette liquids induced significant depletion in total number of proteins and impairment of mitochondrial function in treated cells. The focal adhesion proteins were upregulated, which is in accordance with the results of metabolic cooperation assay. Increased presence of posttranslational modifications (PTMs), including carbonylation and direct oxidative modifications, was observed. Data are available via ProteomeXchange with identifier PXD032071. CONCLUSIONS: Our study revealed impairment of metabolic cooperation as well as significant proteome and PTMs alterations in V79 cells treated with e-cigarette liquid warranting future studies on e-cigarettes health impact.

Invadosome Formation by Lung Fibroblasts in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by abnormal fibroblast accumulation in the lung leading to extracellular matrix deposition and remodeling that compromise lung function. However, the mechanisms of interstitial invasion and remodeling by lung fibroblasts remain poorly understood. The invadosomes, initially described in cancer cells, consist of actin-based adhesive structures that coordinate with numerous other proteins to form a membrane protrusion capable of degrading the extracellular matrix to promote their invasive phenotype. In this regard, we hypothesized that invadosome formation may be increased in lung fibroblasts from patients with IPF. Public RNAseq datasets from control and IPF lung tissues were used to identify differentially expressed genes associated with invadosomes. Lung fibroblasts isolated from bleomycin-exposed mice and IPF patients were seeded with and without the two approved drugs for treating IPF, nintedanib or pirfenidone on fluorescent gelatin-coated coverslips for invadosome assays. Several matrix and invadosome-associated genes were increased in IPF tissues and in IPF fibroblastic foci. Invadosome formation was significantly increased in lung fibroblasts isolated from bleomycin-exposed mice and IPF patients. The degree of lung fibrosis found in IPF tissues correlated strongly with invadosome production by neighboring cells. Nintedanib suppressed IPF and PDGF-activated lung fibroblast invadosome formation, an event associated with inhibition of the PDGFR/PI3K/Akt pathway and TKS5 expression. Fibroblasts derived from IPF lung tissues express a pro-invadosomal phenotype, which correlates with the severity of fibrosis and is responsive to antifibrotic treatment.

Bmi-1 alleviates adventitial fibroblast senescence by eliminating ROS in pulmonary hypertension.

OBJECTIVES: Pulmonary hypertension (PH) is a life-threatening progressive disease with high mortality in the elderly. However, the pathogenesis of PH has not been fully understood and there is no effective therapy to reverse the disease process. This study aims to determine whether cellular senescence is involved in the development of PH. METHODS: The rat PH model was established by intraperitoneal injection of monocrotaline and evaluated by pulmonary arteriole wall thickness and right ventricular hypertrophy index. Human lung fibroblasts (HLFs) were treated with CoCl2 or hypoxia to induce cellular senescence in vitro. SA-ß-gal staining and the changes of senescent markers were used to examine cellular senescence. The molecular mechanism of cellular senescence was further explored by detecting reactive oxygen species (ROS) levels and culturing cells with a conditioned medium. RESULTS: We revealed the cellular senescence of pulmonary adventitial fibroblasts in vivo in the rat PH model. The expression of Bmi-1, an important regulator of senescence, was decreased in the lungs of PH rats and localized in adventitial fibroblasts. The in vitro experiments showed that p16 expression was increased while Bmi-1 expression was decreased after CoCl2 treatment in HLFs. Mechanistically, Bmi-1 could alleviate CoCl2-induced HLFs senescence by eliminating ROS which further promoted the proliferation of pulmonary artery smooth muscle cells by paracrine mode of action of HLFs. CONCLUSION: Bmi-1 alleviates the cellular senescence of pulmonary fibroblasts in PH, which expands the pathogenesis of PH and provides a theoretical basis for targeting senescent cells in the treatment of PH.

Biological and epigenetic alterations of mitochondria involved in cellular replicative and hydrogen peroxide-induced premature senescence of human embryonic lung fibroblasts.

The mitoepigenetic modifications may be closely related to cellular fate. Both the replicative and hydrogen peroxide (H2O2)-induced premature senescence models were used to detect the mitochondrial biological characteristics and the epigenetic factors during senescence of human embryonic lung fibroblasts. The mitochondrial quantity was decreased in two senescence stages, while the mitochondrial DNA (mtDNA) copy number was increased significantly and the methyltransferases activity likewise. And the acute mtROS accumulation could launch premature senescence. Later, the persistent premature senescence owned the higher level of adenosine triphosphate (ATP) and mitochondrial 5-methylcytosine (mt-5-mC), and the less level of 8-hydroxydeoxyguanosine (8-OHdG) than those of replicative senescence. The mtDNA methylation-related enzymes, binding protein and the mitochondrial transcription regulators presented the differentially expressed profiles in both senescent states. Interestingly, the hypermethylation in the CpG region of mitochondrial transcription factor B2 (TFB2M) contributed to its downregulation of mRNA level in replicative senescence. The alterations of the mitochondrial biological functions and mtDNA features would be novel candidate biomarkers involved in cellular senescence. The specific methylation status of mtDNA may also have a crosstalk with oxidative stress to the mitochondrial function, contributing to cellular senescence.

LncRNA-PVT1 activates lung fibroblasts via miR-497-5p and is facilitated by FOXM1.

It is little known about the lncRNA-PVT1 effect on occupational pulmonary fibrosis, although researches show it plays an essential role in cancer. Studies reveal that lung fibroblast activation is one of the key events in silica-induced fibrosis. Here, we found that lncRNA-PVT1 promoted the proliferation, activation, and migration of lung fibroblasts. The isolation of cytoplasmic and nuclear RNA assay and fluorescence in situ hybridization experiment showed that lncRNA-PVT1 was abundantly expressed in the cytoplasm. Luciferase reporter gene assay and RNA pull-down experiment indicated that the cytoplasmic-localized lncRNA-PVT1 could competitively bind miR-497-5p. MiR-497-5p was further observed to attenuate silica-induced pulmonary fibrosis by targeting Smad3 and Bcl2. Moreover, the transcription factor FOXM1 acted as a profibrotic factor by elevating lncRNA-PVT1 transcription in lung fibroblasts. Inhibition of FOXM1 expression with thiostrepton alleviated silica-induced pulmonary fibrosis in vivo. Collectively, we revealed that FOXM1-facilitated lncRNA-PVT1 activates lung fibroblasts via miR-497-5p during silica-induced pulmonary fibrosis, which may provide potential therapeutic targets for pulmonary fibrosis.

Mercury Chloride but Not Lead Acetate Causes Apoptotic Cell Death in Human Lung Fibroblast MRC5 Cells via Regulation of Cell Cycle Progression.

Heavy metals are important for various biological systems, but, in excess, they pose a serious risk to human health. Heavy metals are commonly used in consumer and industrial products. Despite the increasing evidence on the adverse effects of heavy metals, the detailed mechanisms underlying their action on lung cancer progression are still poorly understood. In the present study, we investigated whether heavy metals (mercury chloride and lead acetate) affect cell viability, cell cycle, and apoptotic cell death in human lung fibroblast MRC5 cells. The results showed that mercury chloride arrested the sub-G1 and G2/M phases by inducing cyclin B1 expression. In addition, the exposure to mercury chloride increased apoptosis through the activation of caspase-3. However, lead had no cytotoxic effects on human lung fibroblast MRC5 cells at low concentration. These findings demonstrated that mercury chloride affects the cytotoxicity of MRC5 cells by increasing cell cycle progression and apoptotic cell death.

Neutrophil extracellular traps activate lung fibroblast to induce polymyositis-related interstitial lung diseases via TLR9-miR-7-Smad2 pathway.

Excessive neutrophil extracellular trap (NET) formation may contribute to polymyositis (PM)-associated interstitial lung diseases (ILD), but the underlying mechanism is not fully revealed. In this study, we found that NET accelerated the progression of ILD and promoted pulmonary fibrosis (PF) in vivo. miR-7 expression was down-regulated in lung tissue of PM group than control group, and NETs further decreased miR-7 expression. TLR9 and Smad2 were up-regulated in lung tissue of PM group than control group, and NETs further increased TLR9 and Smad2 expressions. In vitro experiments showed that PMA-treated NETs accelerated the proliferation of LF and their differentiation into myofibroblast (MF), whereas DNase I decreased the promotion effect of NETs. Neutrophil extracellular trap components myeloperoxidase (MPO) and histone 3 also promoted the proliferation and differentiation of LF. In addition, we demonstrated that TLR9 involved in the regulation of NETs on LF proliferation and differentiation, and confirmed the interaction between miR-7 and Smad2 in LF. Finally, miR-7-Smad2 pathway was confirmed to be involved in the regulation of TLR9 on LF proliferation and differentiation. Therefore, NETs promote PM-related ILD, and TLR9-miR-7-Smad2 signalling pathway is involved in the proliferation of LFs and their differentiation into MFs.

MeCP2 epigenetically regulates alpha-smooth muscle actin in human lung fibroblasts.

BACKGROUND: A critical feature for fibroblasts differentiation into myofibroblasts is the expression of alpha-smooth muscle actin (α-SMA) during the tissue injury and repair process. The epigenetic mechanism, DNA methylation, is involved in regulating α-SMA expression. It is not clear how methyl-CpG-binding protein 2 (MeCP2) interacts with CpG-rich region in α-SMA, and if the CpG methylation status would affect MeCP2 binding and regulation of α-SMA expression. METHODS: The association of MeCP2 with α-SMA CpG rich region were examined by chromatin immunoprecipitation (ChIP) assays in primary fibroblasts from idiopathic pulmonary fibrosis (IPF) and non-IPF control individuals, and in the lung fibroblasts treated with profibrotic cytokine transforming growth factor ß1 (TGF-ß1). The regulation of α-SMA by MeCP2 was examined by knocking down MeCP2 with small interfering RNA (siRNA). To explore the effects of the DNA methylation status of the CpG rich region on α-SMA expression, the cells were treated with DNA methyltransferase inhibitor, 5'-azacytidine (5'-aza). The expression of α-SMA was examined by Western blot and quantitative polymerase chain reaction, the association with MeCP2 was assessed by ChIP assays, and the methylation status was checked by bisulfate sequencing. RESULTS: The human lung fibroblasts with increased α-SMA showed an enriched association of MeCP2, while knockdown MeCP2 by siRNA reduced α-SMA upregulation by TGF-ß1. The 5'-Aza-treated cells have decreased α-SMA expression with reduced MeCP2 association. However, bisulfite sequencing revealed that most CpG sites are unmethylated despite the different expression levels of α-SMA after being treated by TGF-ß1 or 5'-aza. CONCLUSION: Our data indicate that the methyl-binding protein MeCP2 is critical for α-SMA expression in human lung myofibroblast, and the DNA methylation status at the CpG rich region of α-SMA is not a determinative factor for its inducible expression.

Discovery of cell-internalizing artificial nucleic acid aptamers for lung fibroblasts and targeted drug delivery.

Lung fibroblasts play major roles in the lung repair/fibrosis process through synthesis and remodeling of extracellular matrix. Those aberrant activations and elevated proliferations are associated with several fibrotic lung diseases, such as idiopathic pulmonary fibrosis (IPF). Targeting fibroblasts is a promising approach for preventing aberrant remodeling of lung architecture and protect irreversible pulmonary fibrosis. In this study, we developed an aptamer that can target lung fibroblasts and explored its potential as a delivery vehicle of cytotoxic agents intracellularly. The aptamer was discovered from artificial nucleic acid libraries through cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX). This indole-modified aptamer can bind to LL97A cells, a fibroblast cell line derived from IPF patients, with high affinity (Kd = 70 nM). It also showed affinity to other lung fibroblasts, while cross-reactivity to epithelial cells was minimal. An aptamer-monomethyl auristatin F (MMAF) conjugate was generated by hybridizing with complementary DNA linked to MMAF. The resulting aptamer-MMAF conjugate inhibited proliferation of fibroblasts but appeared non-toxic to non-targeted epithelial cells. Our results show that artificial nucleic acid aptamer may potentially be used for fibroblast-specific therapy and diagnostic applications.

Sphingosine Kinase 1/S1P Signaling Contributes to Pulmonary Fibrosis by Activating Hippo/YAP Pathway and Mitochondrial Reactive Oxygen Species in Lung Fibroblasts.

The sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling axis is emerging as a key player in the development of idiopathic pulmonary fibrosis (IPF) and bleomycin (BLM)-induced lung fibrosis in mice. Recent evidence implicates the involvement of the Hippo/Yes-associated protein (YAP) 1 pathway in lung diseases, including IPF, but its plausible link to the SPHK1/S1P signaling pathway is unclear. Herein, we demonstrate the increased co-localization of YAP1 with the fibroblast marker FSP1 in the lung fibroblasts of BLM-challenged mice, and the genetic deletion of Sphk1 in mouse lung fibroblasts (MLFs) reduced YAP1 localization in fibrotic foci. The PF543 inhibition of SPHK1 activity in mice attenuated YAP1 co-localization with FSP1 in lung fibroblasts. In vitro, TGF-ß stimulated YAP1 translocation to the nucleus in primary MLFs, and the deletion of Sphk1 or inhibition with PF543 attenuated TGF-ß-mediated YAP1 nuclear localization. Moreover, the PF543 inhibition of SPHK1, or the verteporfin inhibition of YAP1, decreased the TGF-ß- or BLM-induced mitochondrial reactive oxygen species (mtROS) in human lung fibroblasts (HLFs) and the expression of fibronectin (FN) and alpha-smooth muscle actin (α-SMA). Furthermore, scavenging mtROS with MitoTEMPO attenuated the TGF-ß-induced expression of FN and α-SMA. The addition of the S1P antibody to HLFs reduced TGF-ß- or S1P-mediated YAP1 activation, mtROS, and the expression of FN and α-SMA. These results suggest a role for SPHK1/S1P signaling in TGF-ß-induced YAP1 activation and mtROS generation, resulting in fibroblast activation, a critical driver of pulmonary fibrosis.

Activated Human Lung Fibroblasts Produce Extracellular Vesicles with Antifibrotic Prostaglandins.

The differentiation of interstitial lung fibroblasts into contractile myofibroblasts that proliferate and secrete excessive extracellular matrix is critical for the pathogenesis of pulmonary fibrosis. Certain lipid signaling molecules, such as prostaglandins (PGs), can inhibit myofibroblast differentiation. However, the sources and delivery mechanisms of endogenous PGs are undefined. Activated primary human lung fibroblasts (HLFs) produce PGs such as PGE2. We report that activation of primary HLFs with IL-1ß inhibited transforming growth factor ß-induced myofibroblast differentiation in both the IL-1ß-treated cells themselves (autocrine signal) and adjacent naive HLFs in cocultures (paracrine signal). Additionally, we demonstrate for the first time that at least some of the antifibrotic effect of activated fibroblasts on nearby naive fibroblasts is carried by exosomes and other extracellular vesicles that contain several PGs, including high levels of the antifibrotic PGE2. Thus, activated fibroblasts communicate with surrounding cells to limit myofibroblast differentiation and maintain homeostasis. This work opens the way for future research into extracellular vesicle-mediated intercellular signaling in the lung and may inform the development of novel therapies for fibrotic lung diseases.