Yan Y. Sanders

Reversal of Persistent Fibrosis in Aging by Targeting Nox4-Nrf2 Redox Imbalance

Fibrosis resolution is impaired by aging and is mediated by altered cellular redox homeostasis because of a Nox4-Nrf2 imbalance that promotes an apoptosis-resistant myofibroblast phenotype. Scarred for Life? Fibrosis or “scarring” of vital internal organs is an increasing cause of debilitation and death worldwide. The risk of organ fibrosis increases with age, accounting for a growing “epidemic” of fibrotic disorders in aging populations such as in the United States. A study by Hecker et al. provides new insights into how the aging process may lead to a predisposition to fibrosis. In a mouse model of injury-induced lung fibrosis, these investigators found that the ability to resolve fibrosis was impaired in aged mice compared to young cohorts. Resolution of fibrosis is normally dependent on a process known as “apoptosis” (or programmed cell death) of myofibroblasts in injured tissues; this normal wound-healing response was found to be less efficient in aged mice. Myofibroblasts from aged mice acquired a prolonged senescent and apoptosis-resistant phenotype, which was attributed to an imbalance between the oxidant-generating enzyme Nox4 [reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-4] and the antioxidant response factor Nrf2 (NFE2-related factor 2). Genetic or pharmacologic approaches to suppress the expression or activation of Nox4 in aged mice with persistent fibrosis enhanced the capacity for fibrosis resolution. There was evidence for Nox4-Nrf2 imbalance and apoptosis-resistant behavior of myofibroblasts in the lungs of human subjects with the progressive and fatal fibrotic disorder idiopathic pulmonary fibrosis. The results of these studies improve our understanding of how and why elderly patients become susceptible to progressive fibrotic disorders, such as idiopathic pulmonary fibrosis. Additionally, this study uncovers new approaches for treating fibrotic disorders by targeting the “stubborn” and apoptosis-resistant myofibroblast. The incidence and prevalence of pathological fibrosis increase with advancing age, although mechanisms for this association are unclear. We assessed the capacity for repair of lung injury in young (2 months) and aged (18 months) mice. Whereas the severity of fibrosis was not different between these groups, aged mice demonstrated an impaired capacity for fibrosis resolution. Persistent fibrosis in lungs of aged mice was characterized by the accumulation of senescent and apoptosis-resistant myofibroblasts. These cellular phenotypes were sustained by alterations in cellular redox homeostasis resulting from elevated expression of the reactive oxygen species–generating enzyme Nox4 [NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase-4] and an impaired capacity to induce the Nrf2 (NFE2-related factor 2) antioxidant response. Lung tissues from human subjects with idiopathic pulmonary fibrosis (IPF), a progressive and fatal lung disease, also demonstrated this Nox4-Nrf2 imbalance. Nox4 mediated senescence and apoptosis resistance in IPF fibroblasts. Genetic and pharmacological targeting of Nox4 in aged mice with established fibrosis attenuated the senescent, antiapoptotic myofibroblast phenotype and led to a reversal of persistent fibrosis. These studies suggest that loss of cellular redox homeostasis promotes profibrotic myofibroblast phenotypes that result in persistent fibrosis associated with aging. Our studies suggest that restoration of Nox4-Nrf2 redox balance in myofibroblasts may be a therapeutic strategy in age-associated fibrotic disorders, potentially able to resolve persistent fibrosis or even reverse its progression.

Thy-1 Promoter Hypermethylation: A Novel Epigenetic Pathogenic Mechanism in Pulmonary Fibrosis

Mechanisms regulating myofibroblastic differentiation of fibroblasts within fibroblastic foci in idiopathic pulmonary fibrosis (IPF) remain unclear. Epigenetic processes, including DNA methylation, produce heritable but potentially reversible changes in DNA or its associated proteins and are prominent in development and oncogenesis. We have shown that Thy-1 suppresses myofibroblastic differentiation of lung fibroblasts and that fibroblasts in fibroblastic foci are Thy-1(-). Epigenetic down-regulation of Thy-1 has been demonstrated in cellular transformation and clinical cancer. We hypothesized that epigenetic regulation of Thy-1 affects the lung fibroblast fibrogenic phenotype. RT-PCR, methylation-specific PCR (MSP), and bisulfite genomic sequencing were used to determine the methylation status of the Thy-1 promoter in Thy-1(+) and Thy-1(-) lung fibroblasts, and MSP-in situ hybridization (MSPISH) was performed on fibrotic tissue. Thy-1 gene expression is absent in Thy-1(-) human and rat fibroblasts despite intact Thy-1 genomic DNA. Cytosine-guanine islands in the Thy-1 gene promoter are hypermethylated in Thy-1(-), but not Thy-1(+), fibroblasts. RT-PCR and MSP demonstrate that, in IPF samples in which Thy-1 expression is absent, the Thy-1 promoter region is methylated, whereas in lung samples retaining Thy-1 expression, the promoter region is unmethylated. MSPISH confirms methylation of the Thy-1 promoter in fibroblastic foci in IPF. Treatment with DNA methyltransferase inhibitors restores Thy-1 expression in Thy-1(-) fibroblasts. Epigenetic regulation of Thy-1 is a novel and potentially reversible mechanism in fibrosis that may offer the possibility of new therapeutic options.

Participation of miR-200 in Pulmonary Fibrosis

Excessive extracellular matrix production by fibroblasts in response to tissue injury contributes to fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF). Epithelial-mesenchymal transition, involving transition of alveolar epithelial cells (AECs) to pulmonary fibroblasts, appears to be an important contributory process to lung fibrosis. Although aberrant expression of microRNAs (miRs) is involved in a variety of pathophysiologic processes, the role of miRs in fibrotic lung diseases is less well understood. In the present study, we found that miR-200a, miR-200b, and miR-200c are significantly down-regulated in the lungs of mice with experimental lung fibrosis. Levels of miR-200a and miR-200c were reduced in the lungs of patients with IPF. miR-200 had greater expression in AECs than in lung fibroblasts, and AECs from mice with experimental pulmonary fibrosis had diminished expression of miR-200. We found that the miR-200 family members inhibit transforming growth factor-β1-induced epithelial-mesenchymal transition of AECs. miR-200 family members can reverse the fibrogenic activity of pulmonary fibroblasts from mice with experimental pulmonary fibrosis and from patients with IPF. Indeed, the introduction of miR-200c diminishes experimental pulmonary fibrosis in mice. Thus, the miR-200 family members participate importantly in fibrotic lung diseases and suggest that restoring miR-200 expression in the lungs may represent a novel therapeutic approach in treating pulmonary fibrotic diseases.

Altered DNA Methylation Profile in Idiopathic Pulmonary Fibrosis

RATIONALE DNA methylation is an important epigenetic mechanism, which often occurs in response to environmental stimuli and is crucial in regulating gene expression. It is likely that epigenetic alterations contribute to pathogenesis in idiopathic pulmonary fibrosis (IPF). OBJECTIVES To determine the DNA methylation changes in IPF and their effects on gene expression. METHODS Total DNA methylation and DNA methyltransferase expression were compared in IPF and normal control lung tissues. IPF and normal tissues were subjected to comparative analysis of genome-wide DNA methylation and RNA expression using DNA hybridization to the Illumina HumanMethylation27 BeadChip and RNA hybridization to Illumina HumanHT-12 BeadChip. Functional analyses of differentially expressed and differentially methylated genes were done. Selected genes were validated at DNA, RNA, and protein levels. MEASUREMENTS AND MAIN RESULTS DNA methylation status was altered in IPF. IPF samples demonstrated higher DNA methyltransferase expression without observed alterations in global DNA methylation. Genome-wide differences in DNA methylation status and RNA expression were demonstrated by array hybridization. Among the genes whose DNA methylation status and RNA expression were both significantly altered, 16 genes were hypermethylated in DNA associated with decreased mRNA expression or vice versa. We validated CLDN5, ZNF467, TP53INP1, and DDAH1 genes at the level of DNA methylation status, RNA, and protein-level expression. CONCLUSIONS Changes in DNA methylation correspond to altered mRNA expression of a number of genes, some with known and others with previously uncharacterized roles in IPF, suggesting that DNA methylation is important in the pathogenesis of IPF.

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. HDACi SAHA induces IPF fibroblast apoptosis, modulates Bcl-2 family genes and ameliorates mice lung fibrosis http://ow.ly/sPLMI

Histone Modifications in Senescence-Associated Resistance to Apoptosis by Oxidative Stress

Aging and age-related diseases are associated with cellular senescence that results in variable apoptosis susceptibility to oxidative stress. Although fibroblast senescence has been associated with apoptosis resistance, mechanisms for this have not been well defined. In this report, we studied epigenetic mechanisms involving histone modifications that confer apoptosis resistance to senescent human diploid fibroblasts (HDFs). HDFs that undergo replicative senescence display typical morphological features, express senescence-associated β-galactosidase, and increased levels of the tumor suppressor genes, p16, p21, and caveolin-1. Senescent HDFs are more resistant to oxidative stress (exogenous H2O2)-induced apoptosis in comparison to non-senescent (control) HDFs; this is associated with constitutively high levels of the anti-apoptotic gene, Bcl-2, and low expression of the pro-apoptotic gene, Bax. Cellular senescence is characterized by global increases in H4K20 trimethylation and decreases in H4K16 acetylation in association with increased activity of Suv420h2 histone methyltransferase (which targets H4K20), decreased activity of the histone acetyltransferase, Mof (which targets H4K16), as well as decreased total histone acetyltransferase activity. In contrast to Bax gene, chromatin immunoprecipitation studies demonstrate marked enrichment of the Bcl-2 gene with H4K16Ac, and depletion with H4K20Me3, predicting active transcription of this gene in senescent HDFs. These data indicate that both global and locus-specific histone modifications of chromatin regulate altered Bcl-2:Bax gene expression in senescent fibroblasts, contributing to its apoptosis-resistant phenotype.

Absence of Thy-1 results in TGF-β induced MMP-9 expression and confers a profibrotic phenotype to human lung fibroblasts.

Fibroblasts differ in a variety of phenotypic features, including the expression of Thy-1 a glycophosphatidylinositol-linked glycoprotein. Fibroblasts in idiopathic pulmonary fibrosis (IPF) are Thy-1 negative, whereas most fibroblasts from normal lungs are Thy-1 positive. However, the functional consequences of the absence of Thy-1 are not fully understood. We analyzed the expression of Thy-1 in several primary fibroblasts lines derived from IPF, hypersensitivity pneumonitis (HP), and normal human lungs. We found that a high proportion, independently of their origin, expressed Thy-1 in vitro. We identified a primary culture of HP fibroblasts, which did not express Thy-1, and compared several functional activities between Thy-1 (−) and Thy-1 (+) fibroblasts. Thy-1 (−) fibroblasts were smaller (length: 41.3±20.8 μ versus 83.1±40 μ), showed increased proliferative capacity and enhanced PDGF-induced transmigration through collagen I (59.9% versus 42.2% over control under basal conditions, P<0.01). Likewise, Thy-1 (−) fibroblasts either spontaneously or after TGF-β stimulation demonstrated stronger contraction of collagen matrices (eg, 0.17±0.03 versus 0.6±0.05 cm2 after TGF-β stimulation at 24 h; P<0.01). Thy-1 (−) lung fibroblasts stimulated with TGF-β1 expressed MMP-9, an enzyme that is usually not produced by lung fibroblasts. TGFβ-induced MMP-9 expression was reversible upon re-expression of Thy-1 after transfection with full-length Thy-1. β-glycan, a TGF-β receptor antagonist abolished MMP-9 expression. TGF-β1-induced MMP-9 in Thy-1 (−) fibroblasts depended on the activation of ERK1/2 signaling pathway. Finally, we demonstrated that fibroblasts from IPF fibroblastic foci, which do not express Thy-1 exhibit strong staining for immunoreactive MMP-9 protein in vivo. These findings indicate that loss of Thy-1 in human lung fibroblasts induces a fibrogenic phenotype.

Novel Mechanisms for the Antifibrotic Action of Nintedanib

Idiopathic pulmonary fibrosis (IPF) is a disease with relentless course and limited therapeutic options. Nintedanib (BIBF-1120) is a multiple tyrosine kinase inhibitor recently approved by the U.S. Food and Drug Administration for the treatment of IPF. The precise antifibrotic mechanism(s) of action of nintedanib, however, is not known. Therefore, we studied the effects of nintedanib on fibroblasts isolated from the lungs of patients with IPF. Protein and gene expression of profibrotic markers were assessed by Western immunoblotting and real-time PCR. Autophagy markers and signaling events were monitored by biochemical assays, Western immunoblotting, microscopy, and immunofluorescence staining. Silencing of autophagy effector proteins was achieved with small interfering RNAs. Nintedanib down-regulated protein and mRNA expression of extracellular matrix (ECM) proteins, fibronectin, and collagen 1a1 while inhibiting transforming growth factor (TGF)-β1-induced myofibroblast differentiation. Nintedanib also induced beclin-1-dependent, ATG7-independent autophagy. Nintedanibs ECM-suppressive actions were not mediated by canonical autophagy. Nintedanib inhibited early events in TGF-β signaling, specifically tyrosine phosphorylation of the type II TGF-β receptor, activation of SMAD3, and p38 mitogen-activated protein kinase. Nintedanib down-regulates ECM production and induces noncanonical autophagy in IPF fibroblasts while inhibiting TGF-β signaling. These mechanisms appear to be uncoupled and function independently to mediate its putative antifibrotic effects.

Epigenetic mechanisms regulate NADPH oxidase-4 expression in cellular senescence.

Aging is a well-known risk factor for a large number of chronic diseases, including those of the lung. Cellular senescence is one of the hallmarks of aging, and contributes to the pathogenesis of age-related diseases. Recent studies implicate the reactive oxygen species (ROS)-generating enzyme, NADPH oxidase 4 (Nox4) in cellular senescence. In this study, we investigated potential mechanisms for epigenetic regulation of Nox4. We observed constitutively high levels of Nox4 gene/protein and activity in a model of replication-induced cellular senescence of lung fibroblasts. In replicative senescent fibroblasts, the Nox4 gene is enriched with the activation histone mark, H4K16Ac, and inversely associated with the repressive histone mark, H4K20Me3, supporting an active transcriptional chromatin conformation. Silencing of the histone acetyltransferase Mof, which specifically acetylates H4K16, down-regulates Nox4 gene/protein expression. The Nox4 gene promoter is rich in CpG sites; mixed copies of methylated and unmethylated Nox4 DNA were detected in both nonsenescent and senescent cells. Interestingly, the Nox4 gene is variably associated with specific DNA methyltransferases and methyl binding proteins in these two cell populations. These results indicate a critical role for histone modifications involving H4K16Ac in epigenetic activation of the Nox4 gene, while the role of DNA methylation may be contextual. Defining mechanisms for the epigenetic regulation of Nox4 will aid in the development of novel therapeutic strategies for age-related diseases in which this gene is overexpressed, in particular idiopathic pulmonary fibrosis and cancer.

Tgf-beta downregulation of distinct chloride channels in cystic fibrosis-affected epithelia.

Rationale The cystic fibrosis transmembrane conductance regulator (CFTR) and Calcium-activated Chloride Conductance (CaCC) each play critical roles in maintaining normal hydration of epithelial surfaces including the airways and colon. TGF-beta is a genetic modifier of cystic fibrosis (CF), but how it influences the CF phenotype is not understood. Objectives We tested the hypothesis that TGF-beta potently downregulates chloride-channel function and expression in two CF-affected epithelia (T84 colonocytes and primary human airway epithelia) compared with proteins known to be regulated by TGF-beta. Measurements and Main Results TGF-beta reduced CaCC and CFTR-dependent chloride currents in both epithelia accompanied by reduced levels of TMEM16A and CFTR protein and transcripts. TGF-beta treatment disrupted normal regulation of airway-surface liquid volume in polarized primary human airway epithelia, and reversed F508del CFTR correction produced by VX-809. TGF-beta effects on the expression and activity of TMEM16A, wtCFTR and corrected F508del CFTR were seen at 10-fold lower concentrations relative to TGF-beta effects on e-cadherin (epithelial marker) and vimentin (mesenchymal marker) expression. TGF-beta downregulation of TMEM16A and CFTR expression were partially reversed by Smad3 and p38 MAPK inhibition, respectively. Conclusions TGF-beta is sufficient to downregulate two critical chloride transporters in two CF-affected tissues that precedes expression changes of two distinct TGF-beta regulated proteins. Our results provide a plausible mechanism for CF-disease modification by TGF-beta through effects on CaCC.