Victor J. Thannickal

Recent developments in myofibroblast biology: paradigms for connective tissue remodeling.

The discovery of the myofibroblast has opened new perspectives for the comprehension of the biological mechanisms involved in wound healing and fibrotic diseases. In recent years, many advances have been made in understanding important aspects of myofibroblast basic biological characteristics. This review summarizes such advances in several fields, such as the following: i) force production by the myofibroblast and mechanisms of connective tissue remodeling; ii) factors controlling the expression of α-smooth muscle actin, the most used marker of myofibroblastic phenotype and, more important, involved in force generation by the myofibroblast; and iii) factors affecting genesis of the myofibroblast and its differentiation from precursor cells, in particular epigenetic factors, such as DNA methylation, microRNAs, and histone modification. We also review the origin and the specific features of the myofibroblast in diverse fibrotic lesions, such as systemic sclerosis; kidney, liver, and lung fibrosis; and the stromal reaction to certain epithelial tumors. Finally, we summarize the emerging strategies for influencing myofibroblast behavior in vitro and in vivo, with the ultimate goal of an effective therapeutic approach for myofibroblast-dependent diseases.

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.

Myofibroblast Differentiation by Transforming Growth Factor-β1 Is Dependent on Cell Adhesion and Integrin Signaling via Focal Adhesion Kinase

Myofibroblast differentiation and activation by transforming growth factor-β1 (TGF-β1) is a critical event in the pathogenesis of human fibrotic diseases, but regulatory mechanisms for this effect are unclear. In this report, we demonstrate that stable expression of the myofibroblast phenotype requires both TGF-β1and adhesion-dependent signals. TGF-β1-induced myofibroblast differentiation of lung fibroblasts is blocked in non-adherent cells despite the preservation of TGF-β receptor(s)-mediated signaling of Smad2 phosphorylation. TGF-β1 induces tyrosine phosphorylation of focal adhesion kinase (FAK) including that of its autophosphorylation site, Tyr-397, an effect that is dependent on cell adhesion and is delayed relative to early Smad signaling. Pharmacologic inhibition of FAK or expression of kinase-deficient FAK, mutated by substituting Tyr-397 with Phe, inhibit TGF-β1-induced α-smooth muscle actin expression, stress fiber formation, and cellular hypertrophy. Basal expression of α-smooth muscle actin is elevated in cells grown on fibronectin-coated dishes but is decreased on laminin and poly-d-lysine, a non-integrin binding polypeptide. TGF-β1 up-regulates expression of integrins and fibronectin, an effect that is associated with autophosphorylation/activation of FAK. Thus, a safer and more effective therapeutic strategy for fibrotic diseases characterized by persistent myofibroblast activation may be to target this integrin/FAK pathway while not interfering with tumor-suppressive functions of TGF-β1/Smad signaling.

CCR2-Mediated Recruitment of Fibrocytes to the Alveolar Space after Fibrotic Injury

Bone marrow-derived cells are known to play important roles in repair/regeneration of injured tissues, but their roles in pathological fibrosis are less clear. Here, we report a critical role for the chemokine receptor CCR2 in the recruitment and activation of lung fibrocytes (CD45(+), CD13(+), collagen 1(+), CD34(-)). Lung fibrocytes were isolated in significantly greater numbers from airspaces of fluorescein isothiocyanate-injured CCR2(+/+) mice than from CCR2(-/-) mice. Transplant of CCR2(+/+) bone marrow into CCR2(-/-) recipients restored recruitment of lung fibrocytes and susceptibility to fibrosis. Ex vivo PKH-26-labeled CCR2(+/+) lung fibrocytes also migrated to injured airspaces of CCR2(-/-) recipients in vivo. Isolated lung fibrocytes expressed CCR2 and migrated to CCL2, and CCL2 stimulated collagen secretion by lung fibrocytes. Fibrocytes could transition into fibroblasts in vitro, and this transition was associated with loss of CCR2 expression and enhanced production of collagen 1. This is the first report describing expression of CCR2 on lung fibrocytes and demonstrating that CCR2 regulates both recruitment and activation of these cells after respiratory injury.

miR-29 Is a Major Regulator of Genes Associated with Pulmonary Fibrosis

MicroRNAs (miRNA) are small regulatory RNAs that control gene expression by translational suppression and destabilization of target mRNAs. There is increasing evidence that miRNAs regulate genes associated with fibrosis in organs, such as the heart, kidney, liver, and the lung. In a large-scale screening for miRNAs potentially involved in bleomycin-induced fibrosis, we found expression of miR-29 family members significantly reduced in fibrotic lungs. Analysis of normal lungs showed the presence of miR-29 in subsets of interstitial cells of the alveolar wall, pleura, and at the entrance of the alveolar duct, known sites of pulmonary fibrosis. miR-29 levels inversely correlated with the expression levels of profibrotic target genes and the severity of the fibrosis. To study the impact of miR-29 down-regulation in the lung interstitium, we characterized gene expression profiles of human fetal lung fibroblast IMR-90 cells in which endogenous miR-29 was knocked down. This confirmed the derepression of reported miR-29 targets, including several collagens, but also revealed up-regulation of a large number of previously unrecognized extracellular matrix-associated and remodeling genes. Moreover, we found that miR-29 is suppressed by transforming growth factor (TGF)-β1 in these cells, and that many fibrosis-associated genes up-regulated by TGF-β1 are derepressed by miR-29 knockdown. Interestingly, a comparison of TGF-β1 and miR-29 targets revealed that miR-29 controls an additional subset of fibrosis-related genes, including laminins and integrins, independent of TGF-β1. Together, these strongly suggest a role of miR-29 in the pathogenesis of pulmonary fibrosis. miR-29 may be a potential new therapeutic target for this disease.

Targeted Injury of Type II Alveolar Epithelial Cells Induces Pulmonary Fibrosis

RATIONALE Ineffective repair of a damaged alveolar epithelium has been postulated to cause pulmonary fibrosis. In support of this theory, epithelial cell abnormalities, including hyperplasia, apoptosis, and persistent denudation of the alveolar basement membrane, are found in the lungs of humans with idiopathic pulmonary fibrosis and in animal models of fibrotic lung disease. Furthermore, mutations in genes that affect regenerative capacity or that cause injury/apoptosis of type II alveolar epithelial cells have been identified in familial forms of pulmonary fibrosis. Although these findings are compelling, there are no studies that demonstrate a direct role for the alveolar epithelium or, more specifically, type II cells in the scarring process. OBJECTIVES To determine if a targeted injury to type II cells would result in pulmonary fibrosis. METHODS A transgenic mouse was generated to express the human diphtheria toxin receptor on type II alveolar epithelial cells. Diphtheria toxin was administered to these animals to specifically target the type II epithelium for injury. Lung fibrosis was assessed by histology and hydroxyproline measurement. MEASUREMENTS AND MAIN RESULTS Transgenic mice treated with diphtheria toxin developed an approximately twofold increase in their lung hydroxyproline content on Days 21 and 28 after diphtheria toxin treatment. The fibrosis developed in conjunction with type II cell injury. Histological evaluation revealed diffuse collagen deposition with patchy areas of more confluent scarring and associated alveolar contraction. CONCLUSIONS The development of lung fibrosis in the setting of type II cell injury in our model provides evidence for a causal link between the epithelial defects seen in idiopathic pulmonary fibrosis and the corresponding areas of scarring.

Evidence for tissue-resident mesenchymal stem cells in human adult lung from studies of transplanted allografts

The origin and turnover of connective tissue cells in adult human organs, including the lung, are not well understood. Here, studies of cells derived from human lung allografts demonstrate the presence of a multipotent mesenchymal cell population, which is locally resident in the human adult lung and has extended life span in vivo. Examination of plastic-adherent cell populations in bronchoalveolar lavage samples obtained from 76 human lung transplant recipients revealed clonal proliferation of fibroblast-like cells in 62% (106 of 172) of samples. Immunophenotyping of these isolated cells demonstrated expression of vimentin and prolyl-4-hydroxylase, indicating a mesenchymal phenotype. Multiparametric flow cytometric analyses revealed expression of cell-surface proteins, CD73, CD90, and CD105, commonly found on mesenchymal stem cells (MSCs). Hematopoietic lineage markers CD14, CD34, and CD45 were absent. Multipotency of these cells was demonstrated by their capacity to differentiate into adipocytes, chondrocytes, and osteocytes. Cytogenetic analysis of cells from 7 sex-mismatched lung transplant recipients harvested up to 11 years after transplant revealed that 97.2% +/- 2.1% expressed the sex genotype of the donor. The presence of MSCs of donor sex identity in lung allografts even years after transplantation provides what we believe to be the first evidence for connective tissue cell progenitors that reside locally within a postnatal, nonhematopoietic organ.

Matrix Stiffness–Induced Myofibroblast Differentiation Is Mediated by Intrinsic Mechanotransduction

The mechanical properties of the extracellular matrix have recently been shown to promote myofibroblast differentiation and lung fibrosis. Mechanisms by which matrix stiffness regulates myofibroblast differentiation are not fully understood. The goal of this study was to determine the intrinsic mechanisms of mechanotransduction in the regulation of matrix stiffness-induced myofibroblast differentiation. A well established polyacrylamide gel system with tunable substrate stiffness was used in this study. Megakaryoblastic leukemia factor-1 (MKL1) nuclear translocation was imaged by confocal immunofluorescent microscopy. The binding of MKL1 to the α-smooth muscle actin (α-SMA) gene promoter was quantified by quantitative chromatin immunoprecipitation assay. Normal human lung fibroblasts responded to matrix stiffening with changes in actin dynamics that favor filamentous actin polymerization. Actin polymerization resulted in nuclear translocation of MKL1, a serum response factor coactivator that plays a central role in regulating the expression of fibrotic genes, including α-SMA, a marker for myofibroblast differentiation. Mouse lung fibroblasts deficient in Mkl1 did not respond to matrix stiffening with increased α-SMA expression, whereas ectopic expression of human MKL1 cDNA restored the ability of Mkl1 null lung fibroblasts to express α-SMA. Furthermore, matrix stiffening promoted production and activation of the small GTPase RhoA, increased Rho kinase (ROCK) activity, and enhanced fibroblast contractility. Inhibition of RhoA/ROCK abrogated stiff matrix-induced actin cytoskeletal reorganization, MKL1 nuclear translocation, and myofibroblast differentiation. This study indicates that actin cytoskeletal remodeling-mediated activation of MKL1 transduces mechanical stimuli from the extracellular matrix to a fibrogenic program that promotes myofibroblast differentiation, suggesting an intrinsic mechanotransduction mechanism.

Idiopathic pulmonary fibrosis - Pathogenesis and therapeutic approaches

Idiopathic pulmonary fibrosis (IPF), also termed cryptogenic fibrosing alveolitis, is a clinicopathological syndrome characterised by cough, exertional dyspneoa, basilar crackles, a restrictive defect on pulmonary function tests, honeycombing on high-resolution, thin-section computed tomographic scans and the histological diagnosis of usual interstitial pneumonia on lung biopsy. The course is usually indolent but inexorable. Most patients die of progressive respiratory failure within 3–8 years of the onset of symptoms. Current therapies are of unproven benefit. Although the pathogenesis of IPF has not been elucidated, early concepts focused on lung injury leading to a cycle of chronic alveolar inflammation eventuating in fibrosis and destruction of the lung architecture. Anti-inflammatory therapies employing corticosteroids or immunosuppressive or cytotoxic agents have been disappointing. More recent hypotheses acknowledge that sequential alveolar epithelial cell injury is likely to be a key event in the pathogenesis of IPF, but the cardinal event is an aberrant host response to wound healing. In this context, abnormal epithelial-mesenchymal interactions, altered fibroblast phenotypes, exaggerated fibroblast proliferation, and excessive deposition of collagen and extracellular matrix are pivotal to the fibrotic process.Several clinical trials are currently underway or in the planning stages, and include drugs such as interferon-γ 1b, pirfenidone, acetylcysteine, etanercept (a tumor necrosis factor-α antagonist), bosentan (an endothelin-1 receptor antagonist) and zileuton (a 5-lypoxygenase inhibitor). Future therapeutic strategies should be focused on alveolar epithelial cells aimed at enhancing re-epithelialisation and on fibroblastic/myofibroblastic foci, which play an essential role in the development of IPF. Stem cell progenitors of the alveolar epithelial cells and genetic and epigenetic therapies are attractive future approaches for this and other fibrotic lung disorders.

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.