The NADPH oxidase (NOX) family of enzymes, which catalyze the reduction of O2 to form reactive oxygen species (ROS), have increased in number during eukaryotic evolution1,2. Seven isoforms of the NOX gene family have been identified in mammals; however, specific roles of NOX enzymes in mammalian physiology and pathophysiology have not been fully elucidated3,4. The best established physiological role of NOX enzymes is in host defense against pathogen invasion in diverse species, including plants5,6. The prototypical member of this family, NOX2 (gp91phox), is expressed in phagocytic cells and mediates microbicidal activities7,8. Here, we report a role for the NOX4 isoform in tissue repair functions of myofibroblasts and fibrogenesis. Transforming growth factor-β1 (TGF-β1) induces NOX4 expression in lung mesenchymal cells by a SMAD3-dependent mechanism. NOX4-dependent generation of hydrogen peroxide (H2O2) is required for TGF-β1-induced myofibroblast differentiation, extracellular matrix (ECM) production, and contractility. NOX4 is upregulated in lungs of mice subjected to non-infectious injury and in human idiopathic pulmonary fibrosis (IPF). Genetic or pharmacologic targeting of NOX4 abrogates fibrogenesis in two different murine models of lung injury. These studies support a novel function for NOX4 in tissue fibrogenesis and provide proof-of-concept for therapeutic targeting of NOX4 in recalcitrant fibrotic disorders.

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. While the severity of fibrosis was not significantly different between these groups, aged mice demonstrated an impaired capacity for fibrosis resolution. Persistent fibrosis in lungs of aged mice is characterized by the accumulation of senescent and apoptosis-resistant myofibroblasts. These cellular phenotypes are sustained by alterations in cellular redox homeostasis resulting from elevated expression of the reactive oxygen species (ROS)-generating enzyme, NADPH oxidase-4 (Nox4), and an impaired capacity to induce the NFE2-related factor 2 (Nrf2) antioxidant response. Lung tissues from human subjects with idiopathic pulmonary fibrosis (IPF), a progressive and fatal lung disease, also demonstrate this Nox4-Nrf2 imbalance. Nox4 mediates senescence and apoptosis resistance in IPF fibroblasts. Genetic and pharmacologic targeting of Nox4 in aged mice with established fibrosis attenuated the senescent, anti-apoptotic myofibroblast phenotype and led to a reversal of persistent fibrosis. These studies support the concept that loss of cellular redox homeostasis promotes pro-fibrotic myofibroblast phenotypes that result in persistent fibrosis associated with aging. Importantly, our studies suggest that restoration of Nox4-Nrf2 redox balance in myofibroblasts may be an effective therapeutic strategy in age-associated fibrotic disorders, potentially to resolve persistent fibrosis or even reverse its progression.

Matrix stiffening and myofibroblast resistance to apoptosis are cardinal features of chronic fibrotic diseases involving diverse organ systems. The interactions between altered tissue biomechanics and cellular signaling that sustain progressive fibrosis are not well defined. In this study, we used ex vivo and in vivo approaches to define a mechanotransduction pathway involving Rho/Rho kinase (Rho/ROCK), actin cytoskeletal remodeling, and a mechanosensitive transcription factor, megakaryoblastic leukemia 1 (MKL1), that coordinately regulate myofibroblast differentiation and survival. Both in an experimental mouse model of lung fibrosis and in human subjects with idiopathic pulmonary fibrosis (IPF), we observed activation of the Rho/ROCK pathway, enhanced actin cytoskeletal polymerization, and MKL1 cytoplasmic-nuclear shuttling. Pharmacologic disruption of this mechanotransduction pathway with the ROCK inhibitor fasudil induced myofibroblast apoptosis through a mechanism involving downregulation of BCL-2 and activation of the intrinsic mitochondrial apoptotic pathway. Treatment with fasudil during the postinflammatory fibrotic phase of lung injury or genetic ablation of Mkl1 protected mice from experimental lung fibrosis. These studies indicate that targeting mechanosensitive signaling in myofibroblasts to trigger the intrinsic apoptosis pathway may be an effective approach for treatment of fibrotic disorders.

The primary function of the lung is to facilitate the transfer of molecular oxygen (O 2 ; dioxygen) from the atmosphere to the systemic circulation. In addition to its essential role in aerobic metabolism, O 2 serves as the physiologic terminal acceptor of electron transfer catalyzed by the NADPH oxidase (NOX) family of oxidoreductases. The evolution of the lungs and circulatory systems in vertebrates was accompanied by increasing diversification of NOX family enzymes, suggesting adaptive roles for NOX-derived reactive oxygen species in normal physiology. However, this adaptation may paradoxically carry detrimental consequences in the setting of overwhelming=persistent environmental stressors, both infectious and noninfectious, and during the process of aging. Here, we review current understanding of NOX enzymes in normal lung physiology and their pathophysiologic roles in a number of pulmonary diseases, including lung infections, acute lung injury, pulmonary arterial hypertension, obstructive lung disorders, fibrotic lung disease, and lung cancer. Antioxid. Redox Signal. 11, 2505-2516.

Myofibroblasts participate in tissue repair processes in diverse mammalian organ systems. The deactivation of myofibroblasts is critical for termination of the reparative response and restoration of tissue structure and function. The current paradigm on normal tissue repair is the apoptotic clearance of terminally differentiated myofibroblasts; while, the accumulation of activated myofibroblasts is associated with progressive human fibrotic disorders. The capacity of myofibroblasts to undergo de-differentiation as a potential mechanism for myofibroblast deactivation has not been examined. In this report, we have uncovered a role for MyoD in the induction of myofibroblast differentiation by transforming growth factor-β1 (TGF-β1). Myofibroblasts demonstrate the capacity for de-differentiation and proliferation by modulation of endogenous levels of MyoD. We propose a model of reciprocal signaling between TGF-β1/ALK5/MyoD and mitogen(s)/ERK-MAPK/CDKs that regulate myofibroblast differentiation and dedifferentiation, respectively. Our studies provide the first evidence for MyoD in controlling myofibroblast activation and deactivation. Restricted capacity for dedifferentiation of myofibroblasts may underlie the progressive nature of recalcitrant human fibrotic disorders.

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.

Here, we review evolving functions of Nox enzymes in normal lung physiology and emerging pathophysiologic roles in lung disease.

Oxidative stress has been associated with a number of human fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Oxidative stress is most often defined as an imbalance between the generation of reactive oxygen species (ROS) in excess of the capacity of cells/tissues to detoxify or scavenge them. Additionally, the regulated production of ROS participates in cellular signaling. Therapeutic strategies to treat IPF have, thus far, focused on augmenting anti-oxidant capacity. Recent studies have demonstrated a critical role for ROS-generating enzymatic systems, specifically, NADPH oxidase (NOX) family oxidoreductases in fibrotic processes. In this review, we examine the evidence for NOX isoforms in the generation and perpetuation of fibrosis, and the potential to target this gene family for the treatment of IPF and related fibrotic disorders.