Deficiency of the Deubiquitinase UCHL1 Attenuates Pulmonary Arterial Hypertension.

BACKGROUND: The ubiquitin-proteasome system regulates protein degradation and the development of pulmonary arterial hypertension (PAH), but knowledge about the role of deubiquitinating enzymes in this process is limited. UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), a deubiquitinase, has been shown to reduce AKT1 (AKT serine/threonine kinase 1) degradation, resulting in higher levels. Given that AKT1 is pathological in pulmonary hypertension, we hypothesized that UCHL1 deficiency attenuates PAH development by means of reductions in AKT1. METHODS: Tissues from animal pulmonary hypertension models as well as human pulmonary artery endothelial cells from patients with PAH exhibited increased vascular UCHL1 staining and protein expression. Exposure to LDN57444, a UCHL1-specific inhibitor, reduced human pulmonary artery endothelial cell and smooth muscle cell proliferation. Across 3 preclinical PAH models, LDN57444-exposed animals, Uchl1 knockout rats (Uchl1-/-), and conditional Uchl1 knockout mice (Tie2Cre-Uchl1fl/fl) demonstrated reduced right ventricular hypertrophy, right ventricular systolic pressures, and obliterative vascular remodeling. Lungs and pulmonary artery endothelial cells isolated from Uchl1-/- animals exhibited reduced total and activated Akt with increased ubiquitinated Akt levels. UCHL1-silenced human pulmonary artery endothelial cells displayed reduced lysine(K)63-linked and increased K48-linked AKT1 levels. RESULTS: Supporting experimental data, we found that rs9321, a variant in a GC-enriched region of the UCHL1 gene, is associated with reduced methylation (n=5133), increased UCHL1 gene expression in lungs (n=815), and reduced cardiac index in patients (n=796). In addition, Gadd45α (an established demethylating gene) knockout mice (Gadd45α-/-) exhibited reduced lung vascular UCHL1 and AKT1 expression along with attenuated hypoxic pulmonary hypertension. CONCLUSIONS: Our findings suggest that UCHL1 deficiency results in PAH attenuation by means of reduced AKT1, highlighting a novel therapeutic pathway in PAH.

Challenges and Opportunities for Commercializing Technologies in the Pulmonary Arena: An Official American Thoracic Society Report.

"Translational medicine" has been a buzzword for over two decades. The concept was intended to be lofty, to reflect a new "bench-to-bedside" approach to basic and clinical research that would bridge fields, close gaps, accelerate innovation, and shorten the time and effort it takes to bring novel technologies from basic discovery to clinical application. Has this approach been successful and lived up to its promise? Despite incredible scientific advances and innovations developed within academia, successful clinical translation into real-world solutions has been difficult. This has been particularly challenging within the pulmonary field, because there have been fewer U.S. Food and Drug Administration-approved drugs and higher failure rates for pulmonary therapies than with other common disease areas. The American Thoracic Society convened a working group with the goal of identifying major challenges related to the commercialization of technologies within the pulmonary space and opportunities to enhance this process. A survey was developed and administered to 164 participants within the pulmonary arena. This report provides a summary of these survey results. Importantly, this report identifies a number of poorly recognized challenges that exist in pulmonary academic settings, which likely contribute to diminished efficiency of commercialization efforts, ultimately hindering the rate of successful clinical translation. Because many innovations are initially developed in academic settings, this is a global public health issue that impacts the entire American Thoracic Society community. This report also summarizes key resources and opportunities and provides recommendations to enhance successful commercialization of pulmonary technologies.

Lung-Targeted Delivery of Dimethyl Fumarate Promotes the Reversal of Age-Dependent Established Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF), a severe and deadly form of lung fibrosis, is widely regarded as a disease of aging. We previously demonstrated that aged mice with persistent lung fibrosis and IPF lung myofibroblasts exhibit deficient Nrf2-mediated antioxidant responses. Tecfidera is an orally administered FDA-approved drug for the treatment of multiple sclerosis, where the active pharmaceutical ingredient is dimethyl fumarate (DMF), an active Nrf2 activator. However, no studies have evaluated the efficacy of DMF for age-associated persistent lung fibrosis. Here, we demonstrate that in IPF lung fibroblasts, DMF treatment inhibited both TGF-ß-mediated pro-fibrotic phenotypes and led to a reversal of established pro-fibrotic phenotypes. We also evaluated the pre-clinical efficacy of lung-targeted (inhaled) vs. systemic (oral) delivery of DMF in an aging murine model of bleomycin-induced persistent lung fibrosis. DMF or vehicle was administered daily to aged mice by oral gavage or intranasal delivery from 3-6 weeks post-injury when mice exhibited non-resolving lung fibrosis. In contrast to systemic (oral) delivery, only lung-targeted (inhaled) delivery of DMF restored lung Nrf2 expression levels, reduced lung oxidative stress, and promoted the resolution of age-dependent established fibrosis. This is the first study to demonstrate the efficacy of lung-targeted DMF delivery to promote the resolution of age-dependent established lung fibrosis.

Contracting scars from fibrin drops.

This paper describes a microscale fibroplasia and contraction model that is based on fibrin-embedded lung fibroblasts and provides a convenient visual readout of fibrosis. Cell-laden fibrin microgel drops are formed by aqueous two-phase microprinting. The cells deposit extracellular matrix (ECM) molecules such as collagen while fibrin is gradually degraded. Ultimately, the cells contract the collagen-rich matrix to form a compact cell-ECM spheroid. The size of the spheroid provides the visual readout of the extent of fibroplasia. Stimulation of this wound-healing model with the profibrotic cytokine TGF-ß1 leads to an excessive scar formation response that manifests as increased collagen production and larger cell-ECM spheroids. Addition of drugs also shifted the scarring profile: the FDA-approved fibrosis drugs (nintedanib and pirfenidone) and a PAI-1 inhibitor (TM5275) significantly reduced cell-ECM spheroid size. Not only is the assay useful for evaluation of antifibrotic drug effects, it is relatively sensitive; one of the few in vitro fibroplasia assays that can detect pirfenidone effects at submillimolar concentrations. Although this paper focuses on lung fibrosis, the approach opens opportunities for studying a broad range of fibrotic diseases and for evaluating antifibrotic therapeutics.

Aqueous two-phase deposition and fibrinolysis of fibroblast-laden fibrin micro-scaffolds.

This paper describes printing of microscale fibroblast-laden matrices using an aqueous two-phase approach that controls thrombin-mediated enzymatic crosslinking of fibrin. Optimization of aqueous two-phase formulations enabled polymerization of consistent sub-microliter volumes of cell-laden fibrin. When plasminogen was added to these micro-scaffolds, the primary normal human lung fibroblasts converted it to plasmin, triggering gradual degradation of the fibrin. Time-lapse live-cell imaging and automated image analysis provided readouts of time to degradation of 50% of the scaffold as well as maximum degradation rate. The time required for degradation decreased linearly with cell number while it increased in a dose-dependent manner upon addition of TGF-ß1. Fibroblasts isolated from idiopathic pulmonary fibrosis patients showed similar trends with regards to response to TGF-ß1 stimulation. Addition of reactive oxygen species (ROS) slowed fibrinolysis but only in the absence of TGF-ß1, consistent with published studies demonstrating that pro-fibrotic cellular phenotypes induced by TGF-ß1 are mediated, at least in part, through increased production of ROS. FDA-approved and experimental anti-fibrosis drugs were also tested for their effects on fibrinolysis rates. Given the central role of fibrinolysis in both normal and pathogenic wound healing of various tissues, the high-throughput cell-mediated fibrinolysis assay described has broad applicability in the study of many different cell types and diseases. Furthermore, aqueous two-phase printing of fibrin addresses several current limitations of fibrin bio-inks, potentially enabling future applications in tissue engineering andin vitromodels.

MUC1 contributes to goblet cell metaplasia and MUC5AC expression in response to cigarette smoke in vivo.

Goblet cell metaplasia (GCM) and mucin overproduction are a hallmark of chronic rhinosinusitis (CRS) and chronic obstructive pulmonary disease (COPD). In the airways, cigarette smoke (CS) induces activation of the epidermal growth factor receptor (EGFR) leading to GCM and overexpression of the gel-forming mucin MUC5AC. Although previous studies have demonstrated that a membrane-bound mucin, MUC1, modulates the activation of CS-induced EGFR, the role of MUC1 in CS-induced GCM and mucin overproduction has not been explored. In response to CS exposure, wild-type (WT) rats displayed Muc1 translocation from the apical surface of airway epithelium to the intracellular compartment of hyperplastic intermediate cells, EGFR phosphorylation, GCM, and Muc5ac overproduction. Similarly, human CRS sinonasal tissues demonstrated hyperplasia of intermediate cells enriched with MUC1 in the intracellular compartment, which was accompanied by GCM and increased MUC5AC expression. To further evaluate the role of Muc1 in vivo, a Muc1 knockout (KO) rat (MUC in humans and Muc in animals) was developed. In contrast to WT littermates, Muc1-KO rats exhibited no activation of EGFR, and were protected from GCM and Muc5ac overproduction. Genetic knockdown of MUC1 in human lung or Muc1 knockout in primary rat airway epithelial cells led to significantly diminished EGF-induced MUC5AC production. Together, these findings suggest that MUC1-dependent EGFR activation mediates CS-induced GCM and mucin overproduction. Strategies designed to suppress MUC1-dependent EGFR activation may provide a novel therapeutic approach for treating mucin hypersecretion in CRS and COPD.

NADPH oxidases: Pathophysiology and therapeutic potential in age-associated pulmonary fibrosis.

Oxidative stress has been associated with a number of human fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Although oxidative stress is associated with both fibrosis and aging, the precise cellular sources(s) of reactive oxygen species (ROS) that contribute to the disease pathogenesis remain poorly understood. NADPH oxidase (Nox) enzymes are an evolutionarily conserved family, where their only known function is the production of ROS. A growing body of evidence supports a link between excessive Nox-derived ROS and numerous chronic diseases (including fibrotic disease), which is most prevalent among the elderly population. In this review, we examine the evidence for Nox isoforms in the pathogenesis of IPF, and the potential to target this enzyme family for the treatment of IPF and related fibrotic disorders. A better understanding of the Nox-mediated redox imbalance in aging may be critical to the development of more effective therapeutic strategies for age-associated fibrotic disorders. Strategies aimed at specifically blocking the source(s) of ROS through Nox inhibition may prove to be more effective as anti-fibrotic therapies, as compared to antioxidant approaches. This review also discusses the potential of Nox-targeting therapeutics currently in development.

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.

Microphysiological systems modeling acute respiratory distress syndrome that capture mechanical force-induced injury-inflammation-repair.

Complex in vitro models of the tissue microenvironment, termed microphysiological systems, have enormous potential to transform the process of discovering drugs and disease mechanisms. Such a paradigm shift is urgently needed in acute respiratory distress syndrome (ARDS), an acute lung condition with no successful therapies and a 40% mortality rate. Here, we consider how microphysiological systems could improve understanding of biological mechanisms driving ARDS and ultimately improve the success of therapies in clinical trials. We first discuss how microphysiological systems could explain the biological mechanisms underlying the segregation of ARDS patients into two clinically distinct phenotypes. Then, we contend that ARDS-mimetic microphysiological systems should recapitulate three critical aspects of the distal airway microenvironment, namely, mechanical force, inflammation, and fibrosis, and we review models that incorporate each of these aspects. Finally, we recognize the substantial challenges associated with combining inflammation, fibrosis, and/or mechanical force in microphysiological systems. Nevertheless, complex in vitro models are a novel paradigm for studying ARDS, and they could ultimately improve patient care.

Mechanisms and consequences of oxidative stress in lung disease: therapeutic implications for an aging populace.

The rapid expansion of the elderly population has led to the recent epidemic of age-related diseases, including increased incidence and mortality of chronic and acute lung diseases. Numerous studies have implicated aging and oxidative stress in the pathogenesis of various pulmonary diseases; however, despite recent advances in these fields, the specific contributions of aging and oxidative stress remain elusive. This review will discuss the consequences of aging on lung morphology and physiology, and how redox imbalance with aging contributes to lung disease susceptibility. Here, we focus on three lung diseases for which aging is a significant risk factor: acute respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF). Preclinical and clinical development for redox- and senescence-altering therapeutic strategies are discussed, as well as scientific advancements that may direct current and future therapeutic development. A deeper understanding of how aging impacts normal lung function, redox balance, and injury-repair processes will inspire the development of new therapies to prevent and/or reverse age-associated pulmonary diseases, and ultimately increase health span and longevity. This review is intended to encourage basic, clinical, and translational research that will bridge knowledge gaps at the intersection of aging, oxidative stress, and lung disease to fuel the development of more effective therapeutic strategies for lung diseases that disproportionately afflict the elderly.

Survival benefits of remote ischemic conditioning in sepsis.

BACKGROUND: Sepsis remains the leading cause of death in the surgical intensive care unit. Prior studies have demonstrated a survival benefit of remote ischemic conditioning (RIC) in many disease states. The aim of this study was to determine the effects of RIC on survival in sepsis in an animal model and to assess alterations in inflammatory biochemical profiles. We hypothesized that RIC alters inflammatory biochemical profiles resulting in decreased mortality in a septic mouse model. MATERIALS AND METHODS: Eight to 12 week C57BL/6 mice received intra-peritoneal injection of 12.5-mg/kg lipopolysaccharide (LPS). Septic animals in the experimental group underwent RIC at 0, 2, and 6 h after LPS by surgical exploration and alternate clamping of the femoral artery. Six 4-min cycles of ischemia-reperfusion were performed. Primary outcome was survival at 5-d after LPS injection. Secondary outcome was to assess the following serum cytokine levels: interferon-γ (IFN-γ), interleukin (IL)-10, IL-1ß, and tumor necrosis factoralpha (TNFα) at the baseline before LPS injection, 0 hour after LPS injection, and at 2, 4, 24 hours after induction of sepsis (RIC was performed at 2 h after LPS injection). Kaplan-Meier survival analysis and log-rank test were used. ANOVA test was used to compare cytokine measurements. RESULTS: We performed experiments on 44 mice: 14 sham and 30 RIC mice (10 at each time point). Overall survival was higher in the experimental group compared to the sham group (57% versus 21%; P = 0.02), with the highest survival rate observed in the 2-hour post-RIC group (70%). On Kaplan-Meier analysis, 2-h post-RIC group had increased survival at 5 days after LPS (P = 0.04) with hazard ratio of 0.3 (95% confidence interval = 0.09-0.98). In the RIC group, serum concentrations of IFN-γ, IL-10, IL-1ß, and TNFα peaked at 2 h after LPS and then decreased significantly over 24 hours (P < 0.0001) compared to the baseline. CONCLUSIONS: RIC improves survival in sepsis and has the potential for implementation in the clinical practice. Early implementation of RIC may play an immune-modulatory role in sepsis. Further studies are necessary to refine understanding of the observed survival benefits and its implications in sepsis management.

Focal adhesion kinase signaling determines the fate of lung epithelial cells in response to TGF-ß.

Alveolar epithelial cell (AEC) injury and apoptosis are prominent pathological features of idiopathic pulmonary fibrosis (IPF). There is evidence of AEC plasticity in lung injury repair response and in IPF. In this report, we explore the role of focal adhesion kinase (FAK) signaling in determining the fate of lung epithelial cells in response to transforming growth factor-ß1 (TGF-ß1). Rat type II alveolar epithelial cells (RLE-6TN) were treated with or without TGF-ß1, and the expressions of mesenchymal markers, phenotype, and function were analyzed. Pharmacological protein kinase inhibitors were utilized to screen for SMAD-dependent and -independent pathways. SMAD and FAK signaling was analyzed using siRNA knockdown, inhibitors, and expression of a mutant construct of FAK. Apoptosis was measured using cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. TGF-ß1 induced the acquisition of mesenchymal markers, including α-smooth muscle actin, in RLE-6TN cells and enhanced the contraction of three-dimensional collagen gels. This phenotypical transition or plasticity, epithelial-myofibroblast plasticity (EMP), is dependent on SMAD3 and FAK signaling. FAK activation was found to be dependent on ALK5/SMAD3 signaling. We observed that TGF-ß1 induces both EMP and apoptosis in the same cell culture system but not in the same cell. While blockade of SMAD signaling inhibited EMP, it had a minimal effect on apoptosis; in contrast, inhibition of FAK signaling markedly shifted to an apoptotic fate. The data support that FAK activation determines whether AECs undergo EMP vs. apoptosis in response to TGF-ß1 stimulation. TGF-ß1-induced EMP is FAK- dependent, whereas TGF-ß1-induced apoptosis is favored when FAK signaling is inhibited.

Dysregulated Nox4 ubiquitination contributes to redox imbalance and age-related severity of acute lung injury.

Acute respiratory distress syndrome (ARDS) is a devastating critical illness disproportionately affecting the elderly population, with both higher incidence and mortality. The integrity of the lung endothelial cell (EC) monolayer is critical for preservation of lung function. However, mechanisms mediating EC barrier regulation in the context of aging remain unclear. We assessed the severity of acute lung injury (ALI) in young (2 mo) and aged (18 mo) mice using a two-hit preclinical model. Compared with young cohorts, aged mice exhibited increased ALI severity, with greater vascular permeability characterized by elevated albumin influx and levels of bronchoalveolar lavage (BAL) cells (neutrophils) and protein. Aged/injured mice also demonstrated elevated levels of reactive oxygen species (ROS) in the BAL, which was associated with upregulation of the ROS-generating enzyme, Nox4. We evaluated the role of aging in human lung EC barrier regulation utilizing a cellular model of replicative senescence. Senescent EC populations were defined by increases in ß-galactosidase activity and p16 levels. In response to lipopolysaccharide (LPS) challenge, senescent ECs demonstrate exacerbated permeability responses compared with control "young" ECs. LPS challenge led to a rapid induction of Nox4 expression in both control and senescent ECs, which was posttranslationally mediated via the proteasome/ubiquitin system. However, senescent ECs demonstrated deficient Nox4 ubiquitination, resulting in sustained expression of Nox4 and alterations in cellular redox homeostasis. Pharmacological inhibition of Nox4 in senescent ECs reduced LPS-induced alterations in permeability. These studies provide insight into the roles of Nox4/senescence in EC barrier responses and offer a mechanistic link to the increased incidence and mortality of ARDS associated with aging.

Reactive oxygen species-associated molecular signature predicts survival in patients with sepsis.

Sepsis-related multiple organ dysfunction syndrome is a leading cause of death in intensive care units. There is overwhelming evidence that oxidative stress plays a significant role in the pathogenesis of sepsis-associated multiple organ failure; however, reactive oxygen species (ROS)-associated biomarkers and/or diagnostics that define mortality or predict survival in sepsis are lacking. Lung or peripheral blood gene expression analysis has gained increasing recognition as a potential prognostic and/or diagnostic tool. The objective of this study was to identify ROS-associated biomarkers predictive of survival in patients with sepsis. In-silico analyses of expression profiles allowed the identification of a 21-gene ROS-associated molecular signature that predicts survival in sepsis patients. Importantly, this signature performed well in a validation cohort consisting of sepsis patients aggregated from distinct patient populations recruited from different sites. Our signature outperforms randomly generated signatures of the same signature gene size. Our findings further validate the critical role of ROSs in the pathogenesis of sepsis and provide a novel gene signature that predicts survival in sepsis patients. These results also highlight the utility of peripheral blood molecular signatures as biomarkers for predicting mortality risk in patients with sepsis, which could facilitate the development of personalized therapies.

Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury.

Radiotherapy as a primary treatment for thoracic malignancies induces deleterious effects, such as acute or subacute radiation-induced lung injury (RILI). Although the molecular etiology of RILI is controversial and likely multifactorial, a potentially important cellular target is the lung endothelial cytoskeleton that regulates paracellular gap formation and the influx of macromolecules and fluid to the alveolar space. Here we investigate the central role of a key endothelial cytoskeletal regulatory protein, the nonmuscle isoform of myosin light chain kinase (nmMLCK), in an established murine RILI model. Our results indicate that thoracic irradiation significantly augmented nmMLCK protein expression and enzymatic activity in murine lungs. Furthermore, genetically engineered mice harboring a deletion of the nmMLCK gene (nmMLCK(-/-) mice) exhibited protection from RILI, as assessed by attenuated vascular leakage and leukocyte infiltration. In addition, irradiated wild-type mice treated with two distinct MLCK enzymatic inhibitors, ML-7 and PIK (peptide inhibitor of kinase), also demonstrated attenuated RILI. Taken together, these data suggests a key role for nmMLCK in vascular barrier regulation in RILI and warrants further examination of RILI strategies that target nmMLCK.

Nuclear factor, erythroid 2-like 2-associated molecular signature predicts lung cancer survival.

Nuclear factor, erythroid 2-like 2 (NFE2L2), a transcription factor also known as NF-E2-related factor 2 (Nrf2), is a key cytoprotective gene that regulates critical antioxidant and stress-responsive genes. Nrf2 has been demonstrated to be a promising therapeutic target and useful biomarker in malignant disease. We hypothesized that NFE2L2-mediated gene expression would reflect cancer severity and progression. We conducted a meta-analysis of microarray data for 240 NFE2L2-mediated genes that were enriched in tumor tissues. We then developed a risk scoring system based on NFE2L2 gene expression profiling and designated 50 tumor-associated genes as the NFE2L2-associated molecular signature (NAMS). We tested the relationship between this gene expression signature and both recurrence-free survival and overall survival in lung cancer patients. We find that NAMS predicts clinical outcome in the training cohort and in 12 out of 20 validation cohorts. Cox proportional hazard regressions indicate that NAMS is a robust prognostic gene signature, independent of other clinical and pathological factors including patient age, gender, smoking, gene alteration, MYC level, and cancer stage. NAMS is an excellent predictor of recurrence-free survival and overall survival in human lung cancer. This gene signature represents a promising prognostic biomarker in human lung cancer.

Reversal of persistent fibrosis in aging by targeting Nox4-Nrf2 redox imbalance.

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.