Karen Bernard

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.

Inhibition of mechanosensitive signaling in myofibroblasts ameliorates experimental pulmonary fibrosis

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.

Curcumin Opens Cystic Fibrosis Transmembrane Conductance Regulator Channels by a Novel Mechanism That Requires neither ATP Binding nor Dimerization of the Nucleotide-binding Domains

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are essential mediators of salt transport across epithelia. Channel opening normally requires ATP binding to both nucleotide-binding domains (NBDs), probable dimerization of the two NBDs, and phosphorylation of the R domain. How phosphorylation controls channel gating is unknown. Loss-of-function mutations in the CFTR gene cause cystic fibrosis; thus, there is considerable interest in compounds that improve mutant CFTR function. Here we investigated the mechanism by which CFTR is activated by curcumin, a natural compound found in turmeric. Curcumin opened CFTR channels by a novel mechanism that required neither ATP nor the second nucleotide-binding domain (NBD2). Consequently, this compound potently activated CF mutant channels that are defective for the normal ATP-dependent mode of gating (e.g. G551D and W1282X), including channels that lack NBD2. The stimulation of NBD2 deletion mutants by curcumin was strongly inhibited by ATP binding to NBD1, which implicates NBD1 as a plausible activation site. Curcumin activation became irreversible during prolonged exposure to this compound following which persistently activated channels gated dynamically in the absence of any agonist. Although CFTR activation by curcumin required neither ATP binding nor heterodimerization of the two NBDs, it was strongly dependent on prior channel phosphorylation by protein kinase A. Curcumin is a useful functional probe of CFTR gating that opens mutant channels by circumventing the normal requirements for ATP binding and NBD heterodimerization. The phosphorylation dependence of curcumin activation indicates that the R domain can modulate channel opening without affecting ATP binding to the NBDs or their heterodimerization.

Glycolytic Reprogramming in Myofibroblast Differentiation and Lung Fibrosis.

RATIONALE Dysregulation of cellular metabolism has been shown to participate in several pathologic processes. However, the role of metabolic reprogramming is not well appreciated in the pathogenesis of organ fibrosis. OBJECTIVES To determine if glycolytic reprogramming participates in the pathogenesis of lung fibrosis and assess the therapeutic potential of glycolytic inhibition in treating lung fibrosis. METHODS A cell metabolism assay was performed to determine glycolytic flux and mitochondrial respiration. Lactate levels were measured to assess glycolysis in fibroblasts and lungs. Glycolytic inhibition by genetic and pharmacologic approaches was used to demonstrate the critical role of glycolysis in lung fibrosis. MEASUREMENTS AND MAIN RESULTS Augmentation of glycolysis is an early and sustained event during myofibroblast differentiation, which is dependent on the increased expression of critical glycolytic enzymes, in particular, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Augmented glycolysis contributes to the stabilization of hypoxia-inducible factor 1-α, a master regulator of glycolytic enzymes implicated in organ fibrosis, by increasing cellular levels of tricarboxylic acid cycle intermediate succinate in lung myofibroblasts. Inhibition of glycolysis by the PFKFB3 inhibitor 3PO or genomic disruption of the PFKFB3 gene blunted the differentiation of lung fibroblasts into myofibroblasts, and attenuated profibrotic phenotypes in myofibroblasts isolated from the lungs of patients with idiopathic pulmonary fibrosis. Inhibition of glycolysis by 3PO demonstrates therapeutic benefit in bleomycin-induced and transforming growth factor-β1-induced lung fibrosis in mice. CONCLUSIONS Our data support the novel concept of glycolytic reprogramming in the pathogenesis of lung fibrosis and provide proof-of-concept that targeting this pathway may be efficacious in treating fibrotic disorders, such as idiopathic pulmonary fibrosis.

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.

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.

NADPH oxidases in lung health and disease.

SIGNIFICANCE The evolution of the lungs and circulatory systems in vertebrates ensured the availability of molecular oxygen (O2; dioxygen) for aerobic cellular metabolism of internal organs in large animals. O2 serves as the physiologic terminal acceptor of mitochondrial electron transfer and of the NADPH oxidase (Nox) family of oxidoreductases to generate primarily water and reactive oxygen species (ROS), respectively. RECENT ADVANCES The purposeful generation of ROS by Nox family enzymes suggests important roles in normal physiology and adaptation, most notably in host defense against invading pathogens and in cellular signaling. CRITICAL ISSUES However, there is emerging evidence that, in the context of chronic stress and/or aging, Nox enzymes contribute to the pathogenesis of a number of lung diseases. FUTURE DIRECTIONS Here, we review evolving functions of Nox enzymes in normal lung physiology and emerging pathophysiologic roles in lung disease.

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation

Background: Myofibroblasts, by virtue of their functions, are highly energy-dependent. Results: TGF-β1-induced myofibroblast differentiation is associated with a metabolic reprogramming. This metabolic adaptation is essential to the expression of myofibroblast-related genes. Conclusion: Metabolic reprogramming is a hallmark of myofibroblast differentiation and is critical for its contractile function. Significance: This is the first report that links bioenergetics to myofibroblast activation. Contraction is crucial in maintaining the differentiated phenotype of myofibroblasts. Contraction is an energy-dependent mechanism that relies on the production of ATP by mitochondria and/or glycolysis. Although the role of mitochondrial biogenesis in the adaptive responses of skeletal muscle to exercise is well appreciated, mechanisms governing energetic adaptation of myofibroblasts are not well understood. Our study demonstrates induction of mitochondrial biogenesis and aerobic glycolysis in response to the differentiation-inducing factor transforming growth factor β1 (TGF-β1). This metabolic reprogramming is linked to the activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Inhibition of p38 MAPK decreased accumulation of active peroxisome proliferator-activated receptor γ coactivator 1α in the nucleus and altered the translocation of mitochondrial transcription factor A to the mitochondria. Genetic or pharmacologic approaches that block mitochondrial biogenesis or glycolysis resulted in decreased contraction and reduced expression of TGF-β1-induced α-smooth muscle actin and collagen α-2(I) but not of fibronectin or collagen α-1(I). These data indicate a critical role for TGF-β1-induced metabolic reprogramming in regulating myofibroblast-specific contractile signaling and support the concept of integrating bioenergetics with cellular differentiation.

NADPH Oxidase 4 (Nox4) Suppresses Mitochondrial Biogenesis and Bioenergetics in Lung Fibroblasts via a Nuclear Factor Erythroid-Derived 2-like 2 (Nrf2)-Dependent Pathway

Mitochondrial bioenergetics are critical for cellular homeostasis and stress responses. The reactive oxygen species-generating enzyme, NADPH oxidase 4 (Nox4), regulates a number of physiological and pathological processes, including cellular differentiation, host defense, and tissue fibrosis. In this study we explored the role of constitutive Nox4 activity in regulating mitochondrial function. An increase in mitochondrial oxygen consumption and reserve capacity was observed in murine and human lung fibroblasts with genetic deficiency (or silencing) of Nox4. Inhibition of Nox4 expression/activity by genetic or pharmacological approaches resulted in stimulation of mitochondrial biogenesis, as evidenced by elevated mitochondrial-to-nuclear DNA ratio and increased expression of the mitochondrial markers transcription factor A (TFAM), citrate synthase, voltage-dependent anion channel (VDAC), and cytochrome c oxidase subunit 4 (COX IV). Induction of mitochondrial biogenesis was dependent on TFAM up-regulation but was independent of the activation of the peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α). The enhancement of mitochondrial bioenergetics as well as the increase in mitochondrial proteins in Nox4-deficient lung fibroblasts is inhibited by silencing of nuclear factor erythroid-derived 2-like 2 (Nrf2), supporting a key role for Nrf2 in control of mitochondrial biogenesis. Together, these results indicate a critical role for both Nox4 and Nrf2 in counter-regulation of mitochondrial biogenesis and metabolism.

Developmental Reprogramming in Mesenchymal Stromal Cells of Human Subjects with Idiopathic Pulmonary Fibrosis

Cellular plasticity and de-differentiation are hallmarks of tissue/organ regenerative capacity in diverse species. Despite a more restricted capacity for regeneration, humans with age-related chronic diseases, such as cancer and fibrosis, show evidence of a recapitulation of developmental gene programs. We have previously identified a resident population of mesenchymal stromal cells (MSCs) in the terminal airways-alveoli by bronchoalveolar lavage (BAL) of human adult lungs. In this study, we characterized MSCs from BAL of patients with stable and progressive idiopathic pulmonary fibrosis (IPF), defined as <5% and ≥10% decline, respectively, in forced vital capacity over the preceding 6-month period. Gene expression profiles of MSCs from IPF subjects with progressive disease were enriched for genes regulating lung development. Most notably, genes regulating early tissue patterning and branching morphogenesis were differentially regulated. Network interactive modeling of a set of these genes indicated central roles for TGF-β and SHH signaling. Importantly, fibroblast growth factor-10 (FGF-10) was markedly suppressed in IPF subjects with progressive disease, and both TGF-β1 and SHH signaling were identified as critical mediators of this effect in MSCs. These findings support the concept of developmental gene re-activation in IPF, and FGF-10 deficiency as a potentially critical factor in disease progression.