Tracy R. Luckhardt

NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury

Members of the NADPH oxidase (NOX) family of enzymes, which catalyze the reduction of O2 to reactive oxygen species, have increased in number during eukaryotic evolution. 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 elucidated. The best established physiological role of NOX enzymes is in host defense against pathogen invasion in diverse species, including plants. The prototypical member of this family, NOX-2 (gp91phox), is expressed in phagocytic cells and mediates microbicidal activities. Here we report a role for the NOX4 isoform in tissue repair functions of myofibroblasts and fibrogenesis. Transforming growth factor-β1 (TGF-β1) induces NOX-4 expression in lung mesenchymal cells via SMAD-3, a receptor-regulated protein that modulates gene transcription. NOX-4–dependent generation of hydrogen peroxide (H2O2) is required for TGF-β1–induced myofibroblast differentiation, extracellular matrix (ECM) production and contractility. NOX-4 is upregulated in lungs of mice subjected to noninfectious injury and in cases of human idiopathic pulmonary fibrosis (IPF). Genetic or pharmacologic targeting of NOX-4 abrogates fibrogenesis in two murine models of lung injury. These studies support a function for NOX4 in tissue fibrogenesis and provide proof of concept for therapeutic targeting of NOX-4 in recalcitrant fibrotic disorders.

Latent Herpesvirus Infection Augments Experimental Pulmonary Fibrosis

RATIONALE No effective treatment exists for idiopathic pulmonary fibrosis, and its pathogenesis remains unclear. Accumulating evidence implicates herpesviruses as cofactors (either initiating or exacerbating agents) of fibrotic lung disease, but a role for latent herpesvirus infection has not been studied. OBJECTIVES To develop a murine model to determine whether latent herpesvirus infection can augment fibrotic responses and to gain insight into potential mechanisms of enhanced fibrogenesis. METHODS Mice were infected with murine gammaherpesvirus 14 to 70 days before a fibrotic challenge with fluorescein isothiocyanate or bleomycin so that the virus was latent at the time of fibrotic challenge. Measurements were made after viral infection alone or after the establishment of fibrosis. MEASUREMENTS AND MAIN RESULTS gammaHerpesvirus is latent by 14 days post infection, and infection 14 to 70 days before fibrotic challenge augmented fibrosis. Fibrotic augmentation was not dependent on reactivation of the latent virus to a lytic state. Total cell numbers and fibrocyte numbers were increased in the lungs of latently infected mice administered fibrotic challenge compared with mock-infected mice that received fibrotic challenge. Latent infection up-regulates expression of proinflammatory chemokines, transforming growth factor-beta1, and cysteinyl leukotrienes in alveolar epithelial cells. CONCLUSIONS Latent gammaherpesvirus infection augments subsequent fibrotic responses in mice. Enhanced fibrosis is associated with the induction of profibrotic factors and the recruitment of fibrocytes. Our data complement existing human and animal data supporting the hypothesis that gammaherpesviruses can serve as initiating cofactors in the pathogenesis of pulmonary fibrosis.

New Insights into the Pathogenesis and Treatment of Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common and lethal of the idiopathic interstitial pneumonias. There are currently no effective pharmacological therapies approved for the treatment of IPF. Despite the focus on targeting fibrogenic pathways, recent clinical trials have been largely disappointing. Progress is being made in elucidating key cellular processes and molecular pathways critical to IPF pathogenesis, and this should facilitate the development of more effective therapeutics for this recalcitrant disease. Emerging pathobiological concepts include the role of aging and cellular senescence, oxidative stress, endoplasmic reticulum stress, cellular plasticity, microRNAs and mechanotransduction. Therapeutic approaches that target molecular pathways to modulate aberrant cellular phenotypes and promote tissue homeostasis in the lung must be developed. Heterogeneity in biological and clinical phenotypes of IPF warrants a personalized medicine approach to diagnosis and treatment of this lung disorder.

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.

Therapeutic Targeting of Src Kinase in Myofibroblast Differentiation and Pulmonary Fibrosis

Myofibroblasts are effector cells in fibrotic disorders that synthesize and remodel the extracellular matrix (ECM). This study investigated the role of the Src kinase pathway in myofibroblast activation in vitro and fibrogenesis in vivo. The profibrotic cytokine, transforming growth factor β1 (TGF-β1), induced rapid activation of Src kinase, which led to myofibroblast differentiation of human lung fibroblasts. The Src kinase inhibitor AZD0530 (saracatinib) blocked TGF-β1–induced Src kinase activation in a dose-dependent manner. Inhibition of Src kinase significantly reduced α-smooth muscle actin (α-SMA) expression, a marker of myofibroblast differentiation, in TGF-β1–treated lung fibroblasts. In addition, the induced expression of collagen and fibronectin and three-dimensional collagen gel contraction were also significantly inhibited in AZD0530-treated fibroblasts. The therapeutic efficiency of Src kinase inhibition in vivo was tested in the bleomycin murine lung fibrosis model. Src kinase activation and collagen accumulation were significantly reduced in the lungs of AZD0530-treated mice when compared with controls. Furthermore, the total fibrotic area and expression of α-SMA and ECM proteins were significantly decreased in lungs of AZD0530-treated mice. These results indicate that Src kinase promotes myofibroblast differentiation and activation of lung fibroblasts. Additionally, these studies provide proof-of-concept for targeting the noncanonical TGF-β signaling pathway involving Src kinase as an effective therapeutic strategy for lung fibrosis.

Neuronal Wiskott-Aldrich syndrome protein (N-WASP) is critical for formation of α-smooth muscle actin filaments during myofibroblast differentiation

Myofibroblasts are implicated in pathological stromal responses associated with lung fibrosis. One prominent phenotypic marker of fully differentiated myofibroblasts is the polymerized, thick cytoplasmic filaments containing newly synthesized α-smooth muscle actin (α-SMA). These α-SMA-containing cytoplasmic filaments are important for myofibroblast contractility during tissue remodeling. However, the molecular mechanisms regulating the formation and maturation of α-SMA-containing filaments have not been defined. This study demonstrates a critical role for neuronal Wiskott-Aldrich syndrome protein (N-WASP) in regulating the formation of α-SMA-containing cytoplasmic filaments during myofibroblast differentiation and in myofibroblast contractility. Focal adhesion kinase (FAK) is activated by transforming growth factor-β1 (TGF-β1) and is required for phosphorylation of tyrosine residue 256 (Y256) of N-WASP. Phosphorylation of Y256 of N-WASP is essential for TGF-β1-induced formation of α-SMA-containing cytoplasmic filaments in primary human lung fibroblasts. In addition, we demonstrate that actin-related protein (Arp) 2/3 complex is downstream of N-WASP and mediates the maturation of α-SMA-containing cytoplasmic filaments. Together, this study supports a critical role of N-WASP in integrating FAK and Arp2/3 signaling to mediate formation of α-SMA-containing cytoplasmic filaments during myofibroblast differentiation and maturation.

TLR9-induced interferon β is associated with protection from gammaherpesvirus-induced exacerbation of lung fibrosis

BackgroundWe have shown previously that murine gammaherpesvirus 68 (γHV68) infection exacerbates established pulmonary fibrosis. Because Toll-like receptor (TLR)-9 may be important in controlling the immune response to γHV68 infection, we examined how TLR-9 signaling effects exacerbation of fibrosis in response to viral infection, using models of bleomycin- and fluorescein isothiocyanate-induced pulmonary fibrosis in wild-type (Balb/c) and TLR-9-/- mice.ResultsWe found that in the absence of TLR-9 signaling, there was a significant increase in collagen deposition following viral exacerbation of fibrosis. This was not associated with increased viral load in TLR-9-/- mice or with major alterations in T helper (Th)1 and Th2 cytokines. We examined alveolar epithelial-cell apoptosis in both strains, but this could not explain the altered fibrotic outcomes. As expected, TLR-9-/- mice had a defect in the production of interferon (IFN)-β after viral infection. Balb/c fibroblasts infected with γHV68 in vitro produced more IFN-β than did infected TLR-9-/- fibroblasts. Accordingly, in vitro infection of Balb/c fibroblasts resulted in reduced proliferation rates whereas infection of TLR-9-/- fibroblasts did not. Finally, therapeutic administration of CpG oligodeoxynucleotides ameliorated bleomycin-induced fibrosis in wild-type mice.ConclusionsThese results show a protective role for TLR-9 signaling in murine models of lung fibrosis, and highlight differences in the biology of TLR-9 between mice and humans.

Macrophage Migration Inhibitory Factor Promotes Tumor Growth in the Context of Lung Injury and Repair

RATIONALE Tissue injury and repair involve highly conserved processes governed by mechanisms that can be co-opted in tumors. We hypothesized that soluble factors released during the repair response to lung injury would promote orthotopic tumor growth. OBJECTIVES To determine whether lung injury promoted growth of orthotopic lung tumors and to study the molecular mechanisms. METHODS We initiated lung injury in C57Bl6 mice using different stimuli, then injected Lewis lung carcinoma cells during the repair phase. We assessed tumor growth 14 days later. We measured tumor angiogenesis, cytokine expression, proliferation, and apoptosis. MEASUREMENTS AND MAIN RESULTS Regardless of the mechanism, injured lungs contained more numerous and larger tumors than sham-injured lungs. Tumors from injured lungs were no more vascular, but had higher levels of proliferation and reduced rates of apoptosis. The cytokine macrophage migration inhibitory factor (MIF) was highly expressed in both models of tissue injury. We observed no increase in tumor growth after lung injury in MIF knockout mice. We induced lung-specific overexpression of MIF in a double-transgenic mouse, and observed that MIF overexpression by itself was sufficient to accelerate the growth of orthotopic Lewis lung carcinoma tumors. CONCLUSIONS Lung injury leads to increased expression of the cytokine MIF, which results in protection from apoptosis and increased proliferation in orthotopic tumors injected after the acute phase of injury.

Alveolar epithelial disintegrity in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by progressive decline in lung function, resulting in significant morbidity and mortality. Current concepts of the pathogenesis of IPF primarily center on dysregulated epithelial cell repair and altered epithelial-mesenchymal communication and extracellular matrix deposition following chronic exposure to cigarette smoke or environmental toxins. In recent years, increasing attention has been directed toward the role of the intercellular junctional complex in determining the specific properties of epithelia in pulmonary diseases. Additionally, recent genomewide association studies suggest that specific genetic variants predictive of epithelial cell dysfunction may confer susceptibility to the development of sporadic idiopathic pulmonary fibrosis. A number of genetic disorders linked to pulmonary fibrosis and familial interstitial pneumonias are associated with loss of epithelial integrity. However, the potential links between extrapulmonary clinical syndromes associated with defects in epithelial cells and the development of pulmonary fibrosis are not well understood. Here, we report a case of hereditary mucoepithelial dysplasia that presented with pulmonary fibrosis and emphysema on high-resolution computed tomography. This case illustrates a more generalizable concept of epithelial disintegrity in the development of fibrotic lung diseases, which is explored in greater detail in this review article.

Systemic sclerosis-associated fibrosis: an accelerated aging phenotype?

Purpose of reviewSystemic sclerosis (SSc) is an autoimmune disease with fibrosis seen in multiple organs. Although not traditionally regarded as a disease of aging, SSc-associated fibrosis shares many of the hallmarks of aging seen in other age-related fibrotic disorders. Here, we review the current literature of the potential role of aging and age-related cellular processes in the development of SSc and fibrosis. Recent findingsAccumulating evidence supports a role for immune dysregulation, epigenetic modifications, cellular senescence, mitochondrial dysregulation and impaired autophagy in fibrosis that occurs in aging and SSc. SummaryCellular alterations linked to aging may promote the development and/or progression of SSc-associated fibrosis.