Idiopathic Pulmonary Fibrosis Myofibroblasts Research Articles

ASK1-mediated Apoptosis in Human Lung Fibroblasts

Idiopathic pulmonary fibrosis (IPF) is a chronic, devastating lung disease characterized by progressive fibrosis and dysregulated wound repair that negatively affect lung function. Although lung fibroblasts are important in repair mechanisms following lung injury, differentiation into myofibroblasts in the presence of TGF-β and resistance to apoptosis can promote the progression of pulmonary fibrosis. Apoptosis signal regulating kinase 1 (ASK1) is a member of the mitogen activated protein (MAP) kinase family that is known to promote apoptosis. The objective of this study was to test the hypothesis that ASK1 is an important mediator of apoptosis in lung fibroblasts. Normal human lung fibroblasts were obtained from five donors and cultured in the presence or absence of TGF-β (1ng/mL) for 72h. Apoptosis was assessed by a Caspase-Glo® 3/7 assay following treatment with a combination of cycloheximide and Fas ligand antibody with or without the ASK1 inhibitor, selonsertib. Expression of α-smooth muscle actin (α-SMA), collagen, ASK1, and survivin (a member of the inhibitor of apoptosis protein family) were determined by western blot. Apoptosis was induced in three of the five donor lines, and selonsertib treatment reduced caspase-3/7 activity. These donor lines expressed lower levels of survivin compared with the non-responding lines. TGF-β did not significantly affect these results. As expected, TGF-β stimulated expression of collagen and α-SMA in all donor lines, but this was reduced by treatment with selonsertib. Interestingly, TGF-β caused a reduction in the expression of ASK1 which is consistent with the idea that myofibroblasts in IPF are resistant to apoptosis. These results suggest that ASK1 can promote apoptosis in normal lung fibroblasts, but this is dependent upon the expression of survivin. Supported by VA Merit Award BX005889 (CMW) and NIH grant HL131526 (CMW). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Single-nucleus chromatin accessibility identifies a critical role for TWIST1 in idiopathic pulmonary fibrosis myofibroblast activity.

In idiopathic pulmonary fibrosis (IPF), myofibroblasts are key effectors of fibrosis and architectural distortion by excessive deposition of extracellular matrix and their acquired contractile capacity. Single-cell RNA-sequencing (scRNA-seq) has precisely defined the IPF myofibroblast transcriptome, but identifying critical transcription factor activity by this approach is imprecise. We performed single-nucleus assay for transposase-accessible chromatin sequencing on explanted lungs from patients with IPF (n=3) and donor controls (n=2) and integrated this with a larger scRNA-seq dataset (10 IPF, eight controls) to identify differentially accessible chromatin regions and enriched transcription factor motifs within lung cell populations. We performed RNA-sequencing on pulmonary fibroblasts of bleomycin-injured Twist1-overexpressing COL1A2 Cre-ER mice to examine alterations in fibrosis-relevant pathways following Twist1 overexpression in collagen-producing cells. TWIST1, and other E-box transcription factor motifs, were significantly enriched in open chromatin of IPF myofibroblasts compared to both IPF nonmyogenic (log2 fold change (FC) 8.909, adjusted p-value 1.82×10-35) and control fibroblasts (log2FC 8.975, adjusted p-value 3.72×10-28). TWIST1 expression was selectively upregulated in IPF myofibroblasts (log2FC 3.136, adjusted p-value 1.41×10- 24), with two regions of TWIST1 having significantly increased accessibility in IPF myofibroblasts. Overexpression of Twist1 in COL1A2-expressing fibroblasts of bleomycin-injured mice resulted in increased collagen synthesis and upregulation of genes with enriched chromatin accessibility in IPF myofibroblasts. Our studies utilising human multiomic single-cell analyses combined with in vivo murine disease models confirm a critical regulatory function for TWIST1 in IPF myofibroblast activity in the fibrotic lung. Understanding the global process of opening TWIST1 and other E-box transcription factor motifs that govern myofibroblast differentiation may identify new therapeutic interventions for fibrotic pulmonary diseases.

Central lung gene expression associates with myofibroblast features in idiopathic pulmonary fibrosis

RationaleContribution of central lung tissues to pathogenesis of idiopathic pulmonary fibrosis (IPF) remains unknown.ObjectiveTo ascertain the relationship between cell types of IPF-central and IPF-peripheral lung explants using RNA sequencing (RNA-seq)...

Inhibition of the SET8 Pathway Ameliorates Lung Fibrosis Even Through Fibroblast Dedifferentiation.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown etiopathogenesis. The activation of extracellular matrix (ECM)-producing myofibroblasts plays a key role in fibrotic tissue remodeling. The dedifferentiation of myofibroblasts has attracted considerable attention as a promising target for the development of effective therapeutic interventions against IPF. Here, we screened a small library of epigenetics-related inhibitors using dedifferentiation assay of lung myofibroblasts prepared from a patient at the terminal stages of IPF and chose UNC0379. The inhibition of SET8, a histone H4 lysine 20 (H4K20) monomethyltransferase, by UNC0379 markedly suppressed the expression of α-smooth muscle actin (SMA) and ED-A-fibronectin in myofibroblasts. In IPF myofibroblasts, SET8 expression and H4K20 monomethylation (H4K20me1) levels, which were significantly higher than those in normal human lung fibroblasts, were reduced upon treatment with UNC0379. Hence, the changes in the expression of the two fibrotic markers clearly correlated with those in SET8 expression and H4K20me1 level. Furthermore, in a mouse model of bleomycin (BLM)-induced lung fibrosis, the intratracheal administration of UNC0379 at an early fibrotic stage markedly ameliorated the histopathological changes associated with collagen deposition in the lungs. However, treatment with UNC0379 did not significantly affect the number of proinflammatory cells or cytokine production in the bronchoalveolar lavage fluids from mice treated with BLM. In the BLM-injured lung, SET8 was predominantly localized to the nuclei of α-SMA-positive cells, which colocalized with H4K20me1. Taken together, our results indicate that the inhibition of SET8 resulting in myofibroblast dedifferentiation may partly mitigate lung fibrosis without affecting the inflammatory responses.

Single-cell RNA-seq reveals ectopic and aberrant lung-resident cell populations in idiopathic pulmonary fibrosis.

We provide a single-cell atlas of idiopathic pulmonary fibrosis (IPF), a fatal interstitial lung disease, by profiling 312,928 cells from 32 IPF, 28 smoker and nonsmoker controls, and 18 chronic obstructive pulmonary disease (COPD) lungs. Among epithelial cells enriched in IPF, we identify a previously unidentified population of aberrant basaloid cells that coexpress basal epithelial, mesenchymal, senescence, and developmental markers and are located at the edge of myofibroblast foci in the IPF lung. Among vascular endothelial cells, we identify an ectopically expanded cell population transcriptomically identical to bronchial restricted vascular endothelial cells in IPF. We confirm the presence of both populations by immunohistochemistry and independent datasets. Among stromal cells, we identify IPF myofibroblasts and invasive fibroblasts with partially overlapping cells in control and COPD lungs. Last, we confirm previous findings of profibrotic macrophage populations in the IPF lung. Our comprehensive catalog reveals the complexity and diversity of aberrant cellular populations in IPF.

Transcriptomic changes involved in the dedifferentiation of myofibroblasts derived from the lung of a patient with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown etiology. Under pathological conditions in lungs with IPF, myofibroblasts serve a key role in fibrogenesis via the accumulation of an excessive amount of extracellular matrix. To develop effective therapeutic interventions against IPF, studies have recently focused on how to dedifferentiate established myofibroblasts. The present study revealed that JQ1, an inhibitor of bromodomain and extra-terminal proteins, markedly suppressed the expression levels of α-smooth muscle actin and ED-A-fibronectin in myofibroblasts prepared from the lung of a patient with end-stage IPF. Furthermore, these findings were supported by transcriptome analysis using RNA sequencing, in which differentially expressed genes (DEGs) downregulated by JQ1 treatment were significantly enriched in the fibrosis-related signaling pathway. On the other hand, the upregulated DEGs in response to JQ1 treatment were significantly enriched in glutathione metabolism, which may affect the cell status of fibroblast/myofibroblast. To the best of our knowledge, this was the first study to comprehensively analyze transcriptome profiles associated with dedifferentiation of IPF myofibroblasts.

Impaired Myofibroblast Dedifferentiation Contributes to Nonresolving Fibrosis in Aging.

Idiopathic pulmonary fibrosis (IPF) is a fatal age-associated disease with no cure. Although IPF is widely regarded as a disease of aging, the cellular mechanisms that contribute to this age-associated predilection remain elusive. In this study, we sought to evaluate the consequences of senescence on myofibroblast cell fate and fibrotic responses to lung injury in the context of aging. We demonstrated that nonsenescent lung myofibroblasts maintained the capacity for dedifferentiation, whereas senescent/IPF myofibroblasts exhibited an impaired capacity for dedifferentiation. We previously demonstrated that the transcription factor MyoD acts as a critical switch in the differentiation and dedifferentiation of myofibroblasts. Here, we demonstrate that decreased levels of MyoD preceded myofibroblast dedifferentiation and apoptosis susceptibility in nonsenescent cells, whereas MyoD expression remained elevated in senescent/IPF myofibroblasts, which failed to undergo dedifferentiation and demonstrated resistance to apoptosis. Genetic strategies to silence MyoD restored the susceptibility of IPF myofibroblasts to undergo apoptosis and led to a partial reversal of age-associated persistent fibrosis in vivo. The capacity for myofibroblast dedifferentiation and subsequent apoptosis may be critical for normal physiologic responses to tissue injury, whereas restricted dedifferentiation and apoptosis resistance in senescent cells may underlie the progressive nature of age-associated human fibrotic disorders. These studies support the concept that senescence may promote profibrotic effects via impaired myofibroblast dedifferentiation and apoptosis resistance, which contributes to myofibroblast accumulation and ultimately persistent fibrosis in aging.

MicroRNA let-7 Downregulates Ligand-Independent Estrogen Receptor-mediated Male-Predominant Pulmonary Fibrosis.

Rationale: The relevance of hormones in idiopathic pulmonary fibrosis (IPF), a predominantly male lung disease, is unknown.Objectives: To determine whether the ER (estrogen receptor) facilitates the development of pulmonary fibrosis and is mediated in part through microRNA regulation of ERα and ERα-activated profibrotic pathways.Methods: ER expression in male lung tissue and myofibroblasts from control subjects (n = 6) and patients with IPF (n = 6), aging bleomycin (BLM)-treated mice (n = 7), and BLM-treated AF2ERKI mice (n = 7) was determined. MicroRNAs that regulate ER and fibrotic pathways were assessed. Transfections with a reporter plasmid containing the 3' untranslated region of the gene encoding ERα (ESR1) with and without miRNA let-7 mimics or inhibitors or an estrogen response element-driven reporter construct (ERE) construct were conducted.Measurements and Main Results: ERα expression increased in IPF lung tissue, myofibroblasts, or BLM mice. In vitro treatment with let-7 mimic transfections in human myofibroblasts reduced ERα expression and associated fibrotic pathways. AF2ERKI mice developed BLM-induced lung fibrosis, suggesting a role for growth factors in stimulating ER and fibrosis. IGF-1 (insulin-like growth factor 1) expression was increased and induced a fourfold increase of an ERE construct.Conclusions: Our data show 1) a critical role for ER and let-7 in lung fibrosis, and 2) that IGF may stimulate ER in an E2-independent manner. These results underscore the role of sex steroid hormones and their receptors in diseases that demonstrate a sex prevalence, such as IPF.

Proliferating SPP1/MERTK-expressing macrophages in idiopathic pulmonary fibrosis.

A comprehensive understanding of the changes in gene expression in cell types involved in idiopathic pulmonary fibrosis (IPF) will shed light on the mechanisms underlying the loss of alveolar epithelial cells and development of honeycomb cysts and fibroblastic foci. We sought to understand changes in IPF lung cell transcriptomes and gain insight into innate immune aspects of pathogenesis.We investigated IPF pathogenesis using single-cell RNA-sequencing of fresh lung explants, comparing human IPF fibrotic lower lobes reflecting late disease, upper lobes reflecting early disease and normal lungs.IPF lower lobes showed increased fibroblasts, and basal, ciliated, goblet and club cells, but decreased alveolar epithelial cells, and marked alterations in inflammatory cells. We found three discrete macrophage subpopulations in normal and fibrotic lungs, one expressing monocyte markers, one highly expressing FABP4 and INHBA (FABP4hi), and one highly expressing SPP1 and MERTK (SPP1hi). SPP1hi macrophages in fibrotic lower lobes showed highly upregulated SPP1 and MERTK expression. Low-level local proliferation of SPP1hi macrophages in normal lungs was strikingly increased in IPF lungs.Co-localisation and causal modelling supported the role for these highly proliferative SPP1hi macrophages in activation of IPF myofibroblasts in lung fibrosis. These data suggest that SPP1hi macrophages contribute importantly to lung fibrosis in IPF, and that therapeutic strategies targeting MERTK and macrophage proliferation may show promise for treatment of this disease.

Epithelial-Mesenchymal Transition Contributes to Pulmonary Fibrosis via Aberrant Epithelial/Fibroblastic Cross-Talk

Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic interstitial lung disease. Median survival is only 3 years, and treatment options are limited. IPF is thought to be a result of a combination of genetic and environmental factors with repetitive micro-injuries to alveolar epithelial cells playing a central role. IPF is characterised by aberrant extra cellular matrix (ECM) deposition by activated myofibroblasts. Epithelial-mesenchymal transition (EMT) is a process where polarised epithelial cells undergo molecular changes allowing them to gain a mesenchymal phenotype, with a subsequent enhanced ability to produce ECM components and increased migration and/or invasion. The source of myofibroblasts in IPF has been debated for many years, and EMT has been proposed as a source of these cells. However, lineage tracing in transgenic mice suggests the contribution of epithelial cells, which have undergone EMT, to the fibroblast population may be negligible. Instead, recent findings suggest that alveolar epithelial type II (ATII) cells undergoing EMT promote a pro-fibrotic microenvironment through paracrine signalling activating local fibroblasts. This review paper explores the contribution of ATII cells, which have undergone EMT, in the context of pulmonary fibrosis.

Novel differences in gene expression and functional capabilities of myofibroblast populations in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF), a chronic progressive interstitial pneumonia, is characterized by excessive fibroproliferation. Key effector cells in IPF are myofibroblasts that are recruited from three potential sources: resident fibroblasts, fibrocytes, and epithelial cells. We hypothesized that IPF myofibroblasts from different sources display unique gene expression differences and distinct functional characteristics. Primary human pulmonary fibroblasts (normal and IPF), fibrocytes, and epithelial cells were activated using the profibrotic factors TGF-β and TNF-α. The resulting myofibroblasts were characterized using cell proliferation, soluble collagen, and contractility assays, ELISA, and human fibrosis PCR arrays. Genes of significance in human whole lung were validated by immunohistochemistry on human lung sections. Fibroblast-derived myofibroblasts exhibited the greatest increase in expression of profibrotic genes and genes involved in extracellular matrix remodeling and signal transduction. Functional studies demonstrated that myofibroblasts derived from fibrocytes expressed mostly soluble collagen and chemokine (C-C) motif ligand (CCL) 18 but were the least proliferative of the myofibroblast progeny. Activated IPF fibroblasts displayed the highest levels of contractility and CCL2 production. This study identified novel differences in gene expression and functional characteristics of different myofibroblast populations. Further investigation into the myofibroblast phenotype may lead to potential therapeutic targets in future IPF research.

LTBP2 is secreted from lung myofibroblasts and is a potential biomarker for idiopathic pulmonary fibrosis.

Although differentiation of lung fibroblasts into α-smooth muscle actin (αSMA)-positive myofibroblasts is important in the progression of idiopathic pulmonary fibrosis (IPF), few biomarkers reflecting the fibrotic process have been discovered. We performed microarray analyses between FACS-sorted steady-state fibroblasts (lineage (CD45, TER-119, CD324, CD31, LYVE-1, and CD146)-negative and PDGFRα-positive cells) from untreated mouse lungs and myofibroblasts (lineage-negative, Sca-1-negative, and CD49e-positive cells) from bleomycin-treated mouse lungs. Amongst several genes up-regulated in the FACS-sorted myofibroblasts, we focussed on Ltbp2, the gene encoding latent transforming growth factor-β (TGF-β) binding protein-2 (LTBP2), because of the signal similarity to Acta2, which encodes αSMA, in the clustering analysis. The up-regulation was reproduced at the mRNA and protein levels in human lung myofibroblasts induced by TGF-β1. LTBP2 staining in IPF lungs was broadly positive in the fibrotic interstitium, mainly as an extracellular matrix (ECM) protein; however, some of the αSMA-positive myofibroblasts were also stained. Serum LTBP2 concentrations, evaluated using ELISA, in IPF patients were significantly higher than those in healthy volunteers (mean: 21.4 compared with 12.4 ng/ml) and showed a negative correlation with % predicted forced vital capacity (r = −0.369). The Cox hazard model demonstrated that serum LTBP2 could predict the prognosis of IPF patients (hazard ratio for death by respiratory events: 1.040, 95% confidence interval: 1.026–1.054), which was validated using the bootstrap method with 1000-fold replication. LTBP2 is a potential prognostic blood biomarker that may reflect the level of differentiation of lung fibroblasts into myofibroblasts in IPF.

Epithelial contribution to the profibrotic stiff microenvironment and myofibroblast population in lung fibrosis.

The contribution of epithelial-to-mesenchymal transition (EMT) to the profibrotic stiff microenvironment and myofibroblast accumulation in pulmonary fibrosis remains unclear. We examined EMT-competent lung epithelial cells and lung fibroblasts from control (fibrosis-free) donors or patients with idiopathic pulmonary fibrosis (IPF), which is a very aggressive fibrotic disorder. Cells were cultured on profibrotic conditions including stiff substrata and TGF-β1, and analyzed in terms of morphology, stiffness, and expression of EMT/myofibroblast markers and fibrillar collagens. All fibroblasts acquired a robust myofibroblast phenotype on TGF-β1 stimulation. Yet IPF myofibroblasts exhibited higher stiffness and expression of fibrillar collagens than control fibroblasts, concomitantly with enhanced FAKY397 activity. FAK inhibition was sufficient to decrease fibroblast stiffness and collagen expression, supporting that FAKY397 hyperactivation may underlie the aberrant mechanobiology of IPF fibroblasts. In contrast, cells undergoing EMT failed to reach the values exhibited by IPF myofibroblasts in all parameters examined. Likewise, EMT could be distinguished from nonactivated control fibroblasts, suggesting that EMT does not elicit myofibroblast precursors either. Our data suggest that EMT does not contribute directly to the myofibroblast population, and may contribute to the stiff fibrotic microenvironment through their own stiffness but not their collagen expression. Our results also support that targeting FAKY397 may rescue normal mechanobiology in IPF.

Heterogeneity of Fibroblasts and Myofibroblasts in Pulmonary Fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is the most common form of interstitial lung diseases of unknown eathiopathogenesis, mean survival of 3-5 years and limited therapeutics. Characterized by a loss of alveolar type II epithelial cells and aberrant activation of stromal cells, considerable effort was undertaken to characterize the origin and activation mechanisms of fibroblasts and myofibroblasts in IPF lungs. In this review, the origin and contribution of fibroblast and myofibroblasts in lung fibrosis will be summarized. Lineage tracing experiments suggested that interstitial lung fibroblasts and lipofibroblasts, pericytes and mesothelial cells differentiate into myofibroblasts. However, epithelial and bone marrow derived cells may give rise to collagen expressing fibroblasts but do not differentiate into myofibroblasts. There is great heterogeneity in fibroblasts and myofibroblasts in fibrotic lungs. Further, there is evidence for the expansion of pericyte derived myofibroblasts and loss of lipofibroblasts and lipofibroblast derived myofibroblasts in IPF.

EZH2 enhances the differentiation of fibroblasts into myofibroblasts in idiopathic pulmonary fibrosis.

The accumulation of fibroblasts/myofibroblasts in fibrotic foci is one of the characteristics of idiopathic pulmonary fibrosis (IPF). Enhancer of zeste homolog 2 (EZH2) is the catalytic component of a multiprotein complex, polycomb repressive complex 2, which is involved in the trimethylation of histone H3 at lysine 27. In this study, we investigated the role and mechanisms of EZH2 in the differentiation of fibroblasts into myofibroblasts. We found that EZH2 was upregulated in the lungs of patients with IPF and in mice with bleomycin‐induced lung fibrosis. The upregulation of EZH2 occurred in myofibroblasts. The inhibition of EZH2 by its inhibitor 3‐deazaneplanocin A (DZNep) or an shRNA reduced the TGF‐β1‐induced differentiation of human lung fibroblasts into myofibroblasts, as demonstrated by the expression of the myofibroblast markers α‐smooth muscle actin and fibronectin, and contractility. DZNep inhibited Smad2/3 nuclear translocation without affecting Smad2/3 phosphorylation. DZNep treatment attenuated bleomycin‐induced pulmonary fibrosis in mice. We conclude that EZH2 induces the differentiation of fibroblasts to myofibroblasts by enhancing Smad2/3 nuclear translocation.

Pirfenidone and nintedanib modulate properties of fibroblasts and myofibroblasts in idiopathic pulmonary fibrosis.

BackgroundIdiopathic pulmonary fibrosis (IPF) is an incurable lung disease with a poor prognosis. Fibroblasts and myofibroblasts are the key cells in the fibrotic process. Recently two drugs, pirfenidone and nintedanib, were approved for clinical use as they are able to slow down the disease progression. The mechanisms by which these two drugs act in in vitro cell systems are not known. The aim of this study was therefore to examine the effects of pirfenidone and nintedanib on fibroblasts and myofibroblasts structure and function established from patients with or without IPF.MethodsStromal cells were collected and cultured from control lung (n = 4) or IPF (n = 7). The cells were treated with pirfenidone and/or nintedanib and the effect of treatment was evaluated by measuring cell proliferation, alpha smooth muscle actin (α-SMA) and fibronectin expression by Western analysis and/or immunoelectron microscopy, ultrastructural properties by transmission electron microscopy and functional properties by collagen gel contraction and invasion assays.ResultsBoth pirfenidone and nintedanib reduced in vitro proliferation of fibroblastic cells in a dose dependent manner. The number of cells from control lung was reduced to 47 % (p = 0.04) and of IPF cells to 42 % (p = 0.04) by 1 mM pirfenidone and correspondingly to 67 % (p = 0.04) and 68 % (p = 0.04), by 1 μM nintedanib. If both drugs were used together, a further reduced proliferation was observed. Both pirfenidone and nintedanib were able to reduce the amount of α-SMA and the myofibroblastic appearance although the level of reduction was cell line dependent. In functional assays, the effect of both drugs was also variable.ConclusionsWe conclude that the ultrastructure and function of fibroblasts and myofibroblasts are affected by pirfenidone and nintedanib. Combination of the drugs reduced cell proliferation more than either of them individually. Human lung derived cell culture systems represent a potential platform for screening and testing drugs for fibrotic diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-016-0328-5) contains supplementary material, which is available to authorized users.

Expression of WNT5A in Idiopathic Pulmonary Fibrosis and Its Control by TGF-β and WNT7B in Human Lung Fibroblasts.

The wingless (Wnt) family of signaling ligands contributes significantly to lung development and is highly expressed in patients with usual interstitial pneumonia (UIP). We sought to define the cellular distribution of Wnt5A in the lung tissue of patients with idiopathic pulmonary fibrosis (IPF) and the signaling ligands that control its expression in human lung fibroblasts and IPF myofibroblasts. Tissue sections from 40 patients diagnosed with IPF or UIP were probed for the immunolocalization of Wnt5A. Further, isolated lung fibroblasts from normal or IPF human lungs, adenovirally transduced for the overexpression or silencing of Wnt7B or treated with TGF-β1 or its inhibitor, were analyzed for Wnt5A protein expression. Wnt5A was expressed in IPF lungs by airway and alveolar epithelium, smooth muscle cells, endothelium, and myofibroblasts of fibroblastic foci and throughout the interstitium. Forced overexpression of Wnt7B with or without TGF-β1 treatment significantly increased Wnt5A protein expression in normal human smooth muscle cells and fibroblasts but not in IPF myofibroblasts where Wnt5A was already highly expressed. The results demonstrate a wide distribution of Wnt5A expression in cells of the IPF lung and reveal that it is significantly increased by Wnt7B and TGF-β1, which, in combination, could represent key signaling pathways that modulate the pathogenesis of IPF.

Increased constitutive αSMA and Smad2/3 expression in idiopathic pulmonary fibrosis myofibroblasts is KCa3.1-dependent.

BackgroundIdiopathic pulmonary fibrosis is a common and invariably fatal disease with limited therapeutic options. Ca2+-activated KCa3.1 potassium channels play a key role in promoting TGFβ1 and bFGF-dependent profibrotic responses in human lung myofibroblasts (HLMFs). We hypothesised that KCa3.1 channel-dependent cell processes regulate HLMF αSMA expression via Smad2/3 signalling pathways.MethodsIn this study we have compared the phenotype of HLMFs derived from non-fibrotic healthy control lungs (NFC) with cells derived from IPF lungs. HLMFs grown in vitro were examined for αSMA expression by immunofluorescence (IF), RT-PCR and flow cytommetry. Basal Smad2/3 signalling was examined by RT-PCR, western blot and immunofluorescence. Two specific and distinct KCa3.1 blockers (TRAM-34 200 nM and ICA-17043 [Senicapoc] 100 nM) were used to determine their effects on HLMF differentiation and the Smad2/3 signalling pathways.ResultsIPF-derived HLMFs demonstrated increased constitutive expression of both α-smooth muscle actin (αSMA) and actin stress fibres, indicative of greater myofibroblast differentiation. This was associated with increased constitutive Smad2/3 mRNA and protein expression, and increased Smad2/3 nuclear localisation. The increased Smad2/3 nuclear localisation was inhibited by removing extracellular Ca2+ or blocking KCa3.1 ion channels with selective KCa3.1 blockers (TRAM-34, ICA-17043). This was accompanied by de-differentiation of IPF-derived HLMFs towards a quiescent fibroblast phenotype as demonstrated by reduced αSMA expression and reduced actin stress fibre formation.ConclusionsTaken together, these data suggest that Ca2+- and KCa3.1-dependent processes facilitate “constitutive” Smad2/3 signalling in IPF-derived fibroblasts, and thus promote fibroblast to myofibroblast differentiation. Importantly, inhibiting KCa3.1 channels reverses this process. Targeting KCa3.1 may therefore provide a novel and effective approach for the treatment of IPF and there is the potential for the rapid translation of KCa3.1-directed therapy to the clinic.

Histone deacetylase inhibition promotes fibroblast apoptosis and ameliorates pulmonary fibrosis in mice

Idiopathic pulmonary fibrosis (IPF) is a fatal disease, and therapeutic agents have shown only modest efficacy. Epigenetic alterations contribute to the pathogenesis of IPF. The histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), has been approved for clinical use in cancer; however, its potential efficacy in modulating fibroblast survival and lung fibrosis has not been extensively investigated. We investigated the effects of SAHA on apoptosis of primary IPF myofibroblasts and on injury-induced lung fibrosis in a murine model. SAHA-induced apoptosis of IPF myofibroblasts, an effect that was mediated, at least in part, by upregulation of the pro-apoptotic gene Bak and downregulation of the anti-apoptotic gene Bcl-xL. Alterations in the expression of these apoptosis-related genes were associated with histone modifications and changes in DNA methylation. In addition to the expected higher levels of histone acetylation in treated cells, we also detected changes in other histone modifications, such as histone methylation. In a murine model of bleomycin-induced pulmonary fibrosis, SAHA-treated mice displayed decreased lung fibrosis and improved lung function compared to the bleomycin only group. These results suggest that histone deacetylase inhibitors may offer a new therapeutic strategy in IPF by modulating myofibroblast susceptibility to apoptosis.

Heterogeneity in fibroblast proliferation and survival in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common form of interstitial lung disease characterized by the persistence of activated myofibroblasts resulting in excessive deposition of extracellular matrix proteins and profound tissue remodeling. Myofibroblasts have been shown to arise from interstitial fibroblasts, epithelial to mesenchymal transition of type II alveolar epithelial cells, and the differentiation of recruited fibrocytes. There are many mechanisms that are utilized by these cells for survival, proliferation, and persistent activation including up-regulation of cytokines [i.e., Interleukin 6 (IL-6) and C-C motif chemokine ligand 21 (CCL21)], cytokine receptors [i.e., Interleukin 6 Receptor 1 (IL-6R1), Glycoprotein 130 (gp130) and C-C Chemokine Receptor type 7 (CCR7)], and innate pattern recognition receptors [(PRRs; i.e., Toll Like Receptor 9 (TLR9)]. In this review, we will discuss the role of the cytokines IL-6 and CCL21, their receptors and the PRR, TLR9, in fibroblast recruitment, activation, survival, and differentiation into myofibroblasts in IPF.