Robert Matthew Kottmann

The Role of PPARs in Lung Fibrosis

Pulmonary fibrosis is a group of disorders characterized by accumulation of scar tissue in the lung interstitium, resulting in loss of alveolar function, destruction of normal lung architecture, and respiratory distress. Some types of fibrosis respond to corticosteroids, but for many there are no effective treatments. Prognosis varies but can be poor. For example, patients with idiopathic pulmonary fibrosis (IPF) have a median survival of only 2.9 years. Prognosis may be better in patients with some other types of pulmonary fibrosis, and there is variability in survival even among individuals with biopsy-proven IPF. Evidence is accumulating that the peroxisome proliferator-activated receptors (PPARs) play important roles in regulating processes related to fibrogenesis, including cellular differentiation, inflammation, and wound healing. PPARα agonists, including the hypolidipemic fibrate drugs, inhibit the production of collagen by hepatic stellate cells and inhibit liver, kidney, and cardiac fibrosis in animal models. In the mouse model of lung fibrosis induced by bleomycin, a PPARα agonist significantly inhibited the fibrotic response, while PPARα knockout mice developed more serious fibrosis. PPARβ/δ appears to play a critical role in regulating the transition from inflammation to wound healing. PPARβ/δ agonists inhibit lung fibroblast proliferation and enhance the antifibrotic properties of PPARγ agonists. PPARγ ligands oppose the profibrotic effect of TGF-β, which induces differentiation of fibroblasts to myofibroblasts, a critical effector cell in fibrosis. PPARγ ligands, including the thiazolidinedione class of antidiabetic drugs, effectively inhibit lung fibrosis in vitro and in animal models. The clinical availability of potent and selective PPARα and PPARγ agonists should facilitate rapid development of successful treatment strategies based on current and ongoing research.

Determinants of initiation and progression of idiopathic pulmonary fibrosis

IPF is a devastating disease with few therapeutic options. The precise aetiology of IPF remains elusive. However, our understanding of the pathologic processes involved in the initiation and progression of this disease is improving. Data on the mechanisms underlying IPF have been generated from epidemiologic investigations as well as cellular and molecular studies of human tissues. Although no perfect animal model of human IPF exists, pre‐clinical animal studies have helped define pathways which are likely important in human disease. Epithelial injury, fibroblast activation and repetitive cycles of injury and abnormal repair are almost certainly key events. Factors which have been associated with initiation and/or progression of IPF include viral infections, abnormal cytokine, chemokine and growth factor production, oxidant stress, autoimmunity, inhalational of toxicants and gastro‐oesophageal reflux disease. Furthermore, recent evidence identifies a role for a variety of genetic and epigenetic abnormalities ranging from mutations in surfactant protein C to abnormalities in telomere length and telomerase activity. The challenge remains to identify additional inciting agents and key dysregulated pathways that lead to disease progression so that we can develop targeted therapies to treat or prevent this serious disease.

The Triterpenoid CDDO-Me Inhibits Bleomycin-Induced Lung Inflammation and Fibrosis

Pulmonary Fibrosis (PF) is a devastating progressive disease in which normal lung structure and function is compromised by scarring. Lung fibrosis can be caused by thoracic radiation, injury from chemotherapy and systemic diseases such as rheumatoid arthritis that involve inflammatory responses. CDDO-Me (Methyl 2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate, Bardoxolone methyl) is a novel triterpenoid with anti-fibrotic and anti-inflammatory properties as shown by our in vitro studies. Based on this evidence, we hypothesized that CDDO-Me would reduce lung inflammation, fibrosis and lung function impairment in a bleomycin model of lung injury and fibrosis. To test this hypothesis, mice received bleomycin via oropharyngeal aspiration (OA) on day zero and CDDO-Me during the inflammatory phase from days -1 to 9 every other day. Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested on day 7 to evaluate inflammation, while fibrosis and lung function were evaluated on day 21. On day 7, CDDO-Me reduced total BALF protein by 50%, alveolar macrophage infiltration by 40%, neutrophil infiltration by 90% (p≤0.01), inhibited production of the inflammatory cytokines KC and IL-6 by over 90% (p≤0.001), and excess production of the pro-fibrotic cytokine TGFβ by 50%. CDDO-Me also inhibited α-smooth muscle actin and fibronectin mRNA by 50% (p≤0.05). On day 21, CDDO-Me treatment reduced histological fibrosis, collagen deposition and αSMA production. Lung function was significantly improved at day 21 by treatment with CDDO-Me, as demonstrated by respiratory rate and dynamic compliance. These new findings reveal that CDDO-Me exhibits potent anti-fibrotic and anti-inflammatory properties in vivo. CDDO-Me is a potential new class of drugs to arrest inflammation and ameliorate fibrosis in patients who are predisposed to lung injury and fibrosis incited by cancer treatments (e.g. chemotherapy and radiation) and by systemic autoimmune diseases.

Normal Human Lung Epithelial Cells Inhibit Transforming Growth Factor-β Induced Myofibroblast Differentiation via Prostaglandin E2

Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease with very few effective treatments. The key effector cells in fibrosis are believed to be fibroblasts, which differentiate to a contractile myofibroblast phenotype with enhanced capacity to proliferate and produce extracellular matrix. The role of the lung epithelium in fibrosis is unclear. While there is evidence that the epithelium is disrupted in IPF, it is not known whether this is a cause or a result of the fibroblast pathology. We hypothesized that healthy epithelial cells are required to maintain normal lung homeostasis and can inhibit the activation and differentiation of lung fibroblasts to the myofibroblast phenotype. To investigate this hypothesis, we employed a novel co-culture model with primary human lung epithelial cells and fibroblasts to investigate whether epithelial cells inhibit myofibroblast differentiation. Measurements and Main Results In the presence of transforming growth factor (TGF)-β, fibroblasts co-cultured with epithelial cells expressed significantly less α-smooth muscle actin and collagen and showed marked reduction in cell migration, collagen gel contraction, and cell proliferation compared to fibroblasts grown without epithelial cells. Epithelial cells from non-matching tissue origins were capable of inhibiting TGF-β induced myofibroblast differentiation in lung, keloid and Graves’ orbital fibroblasts. TGF-β promoted production of prostaglandin (PG) E2 in lung epithelial cells, and a PGE2 neutralizing antibody blocked the protective effect of epithelial cell co-culture. Conclusions We provide the first direct experimental evidence that lung epithelial cells inhibit TGF-β induced myofibroblast differentiation and pro-fibrotic phenotypes in fibroblasts. This effect is not restricted by tissue origin, and is mediated, at least in part, by PGE2. Our data support the hypothesis that the epithelium plays a crucial role in maintaining lung homeostasis, and that damaged and/ or dysfunctional epithelium contributes to the development of fibrosis.

Second harmonic generation microscopy reveals altered collagen microstructure in usual interstitial pneumonia versus healthy lung

BackgroundIt is not understood why some pulmonary fibroses such as cryptogenic organizing pneumonia (COP) respond well to treatment, while others like usual interstitial pneumonia (UIP) do not. Increased understanding of the structure and function of the matrix in this area is critical to improving our understanding of the biology of these diseases and developing novel therapies. The objectives herein are to provide new insights into the underlying collagen- and matrix-related biological mechanisms driving COP versus UIP.MethodsTwo-photon second harmonic generation (SHG) and excitation fluorescence microscopies were used to interrogate and quantify differences between intrinsic fibrillar collagen and elastin matrix signals in healthy, COP, and UIP lung.ResultsCollagen microstructure was different in UIP versus healthy lung, but not in COP versus healthy, as indicated by the ratio of forward-to-backward propagating SHG signal (FSHG/BSHG). This collagen microstructure as assessed by FSHG/BSHG was also different in areas with preserved alveolar architecture adjacent to UIP fibroblastic foci or honeycomb areas versus healthy lung. Fibrosis was evidenced by increased col1 and col3 content in COP and UIP versus healthy, with highest col1:col3 ratio in UIP. Evidence of elastin breakdown (i.e. reduced mature elastin fiber content), and increased collagen:mature elastin ratios, were seen in COP and UIP versus healthy.ConclusionsFibrillar collagen’s subresolution structure (i.e. “microstructure”) is altered in UIP versus COP and healthy lung, which may provide novel insights into the biological reasons why unlike COP, UIP is resistant to therapies, and demonstrates the ability of SHG microscopy to potentially distinguish treatable versus intractable pulmonary fibroses.

Bronchoscopy with bronchoalveolar lavage: determinants of yield and impact on management in immunosuppressed patients

Fibreoptic bronchoscopy with bronchoalveolar lavage (FOB/BAL) is a common modality for the evaluation of pulmonary infiltrates.1 2 We recognised there are limitations of comparison between subgroups of immunosuppressed patients, non-uniform definitions of a positive yield, suboptimal description of the impact of concurrent antimicrobial use at the time of the bronchoscopy and sometimes insufficient assessment of management decisions surrounding FOB/BAL.3–5 To address these issues, we performed a retrospective analysis of 190 immunosuppressed patients who underwent FOB/BAL for a pulmonary abnormality (clinical or radiographic) at the University of Rochester Medical Center from 2005 to 2008. A positive yield was defined as one of the following: (1) positive culture—bacterial, viral or fungal (not including Candida albicans alone); (2) positive finding on cytopathology or fungal stain; or (3) diffuse alveolar haemorrhage. Antimicrobial and corticosteroid treatment changes …

Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase

Pulmonary fibrosis is a common and dose-limiting side-effect of ionizing radiation used to treat cancers of the thoracic region. Few effective therapies are available for this disease. Pulmonary fibrosis is characterized by an accumulation of myofibroblasts and excess deposition of extracellular matrix proteins. Although prior studies have reported that ionizing radiation induces fibroblast to myofibroblast differentiation and collagen production, the mechanism remains unclear. Transforming growth factor-β (TGF-β) is a key profibrotic cytokine that drives myofibroblast differentiation and extracellular matrix production. However, its activation and precise role in radiation-induced fibrosis are poorly understood. Recently, we reported that lactate activates latent TGF-β through a pH-dependent mechanism. Here, we wanted to test the hypothesis that ionizing radiation leads to excessive lactate production via expression of the enzyme lactate dehydrogenase-A (LDHA) to promote myofibroblast differentiation. We found that LDHA expression is increased in human and animal lung tissue exposed to ionizing radiation. We demonstrate that ionizing radiation induces LDHA, lactate production, and extracellular acidification in primary human lung fibroblasts in a dose-dependent manner. We also demonstrate that genetic and pharmacologic inhibition of LDHA protects against radiation-induced myofibroblast differentiation. Furthermore, LDHA inhibition protects from radiation-induced activation of TGF-β. We propose a profibrotic feed forward loop, in which radiation induces LDHA expression and lactate production, which can lead to further activation of TGF-β to drive the fibrotic process. These studies support the concept of LDHA as an important therapeutic target in radiation-induced pulmonary fibrosis.

Reply: from idiopathic pulmonary fibrosis to cystic fibrosis: got lactate?

From the Authors: We thank Worlitzsch and colleagues for their thoughtful response and are pleased that they read our article with such enthusiasm. We echo their thoughts on the potential pathogenic role of lactic acid in lung disease. In our article, we described the impact of lactate on fibroblasts, notably its induction of myofibroblast differentiation via pH-dependent activation of transforming growth factor (TGF)-β (1). Although we demonstrated that fibroblasts produce excess lactic acid in the presence of TGF-β and that fibroblasts obtained from patients with idiopathic pulmonary fibrosis (IPF) produced more lactic acid than normal fibroblasts, we do acknowledge that fibroblasts are not the only cells in the lung responsible for the generation of lactic acid. Neutrophils, although present in the lung tissue of patients with IPF, are not abundant, and therefore, at least in the case of IPF, are less likely to be the predominant source of lactic acid. We suspect that the epithelium, particularly damaged epithelium in areas of fibrosis, may also be a source of lactic acid. Regardless of the cell type of origin, we anticipate that lactic acid may eventually be shown to be part of the pathogenesis of a variety of lung diseases, including cystic fibrosis (CF) and IPF. The current paradigm proposed for the initiation of pulmonary fibrosis includes an insult to the epithelium, a subsequent aberrant subepithelial fibroblast cellular response resulting in exaggerated scar formation, and an inability of the epithelial cells to restore a functional barrier (2). Worlitzsch and colleagues’ response to our article detailing their experience with lactic acid release by neutrophils raises important questions regarding the role of lactic acid in the development of lung disease. First, is lactic acid an important component of normal wound healing in the lung and elsewhere? There are reports of elevated lactic acid concentrations in skin wounds that are associated with the normal, healthy healing response to dermal injury (3). The normal wound healing response in the lung is less well characterized but likely involves many cell types, including neutrophils, epithelial cells, and/or fibroblasts. The exact role of each cell type in the wound healing response is still being investigated. One potentially important mechanism in wound repair is the cross talk between fibroblasts and neutrophils. Ling and colleagues demonstrated that fibroblasts enhance the proinflammatory properties of neutrophils and promote neutrophil survival (4). This raises the possibility of a potential prohomeostatic mechanism for the promotion of wound healing. Neutrophil influx in damaged lung tissue could result in an increase in lactic acid and a decrease in the extracellular pH. This in turn could result in a pro–wound-healing milieu. The decrease in pH could directly create a hostile environment for microbial organisms, and the activation of TGF-β and induction of myofibroblast differentiation could promote wound contracture and enhance neutrophil activity. Although neutrophils are not abundant in IPF lung tissue, they may be important in the initiation of the disease and may certainly play a more central role in CF. Second, if lactic acid is part of a normal wound-healing process, what factors lead to its excess production and how can this process be mitigated? In skin wound healing, lactic acid concentrations exceeding the 10 to 15 mM range have been shown to be associated with poor wound healing (5). Therefore, we hypothesize that an important problem in both CF and IPF is not the mere elevation of lactate, but the degree of elevation. Equally important are the cell type(s) responsible for the generation of lactic acid and the resulting local tissue concentrations of lactic acid. For CF, this may be the characteristic neutrophil infiltrates. For IPF, we suspect that fibroblasts and epithelial cells may be responsible. The exact mechanisms regulating each cell type and its metabolic contribution to lactic acid production and/or lung disease require additional investigation.