Lesions In Idiopathic Pulmonary Fibrosis Research Articles

Delineating associations of progressive pleuroparenchymal fibroelastosis in patients with pulmonary fibrosis.

Computer quantification of baseline computed tomography (CT) radiological pleuroparenchymal fibroelastosis (PPFE) associates with mortality in idiopathic pulmonary fibrosis (IPF). We examined mortality associations of longitudinal change in computer-quantified PPFE-like lesions in IPF and fibrotic hypersensitivity pneumonitis (FHP). Two CT scans 6-36 months apart were retrospectively examined in one IPF (n=414) and one FHP population (n=98). Annualised change in computerised upper-zone pleural surface area comprising radiological PPFE-like lesions (Δ-PPFE) was calculated. Δ-PPFE >1.25% defined progressive PPFE above scan noise. Mixed-effects models evaluated Δ-PPFE against change in visual CT interstitial lung disease (ILD) extent and annualised forced vital capacity (FVC) decline. Multivariable models were adjusted for age, sex, smoking history, baseline emphysema presence, antifibrotic use and diffusion capacity of the lung for carbon monoxide. Mortality analyses further adjusted for baseline presence of clinically important PPFE-like lesions and ILD change. Δ-PPFE associated weakly with ILD and FVC change. 22-26% of IPF and FHP cohorts demonstrated progressive PPFE-like lesions which independently associated with mortality in the IPF cohort (hazard ratio 1.25, 95% CI 1.16-1.34, p<0.0001) and the FHP cohort (hazard ratio 1.16, 95% CI 1.00-1.35, p=0.045). Progression of PPFE-like lesions independently associates with mortality in IPF and FHP but does not associate strongly with measures of fibrosis progression.

MiRNA Expression in Fibroblastic Foci within Idiopathic Pulmonary Fibrosis Lungs Reveals Novel Disease-Relevant Pathways

miRNAs are 22 nucleotides long and belong to a class of noncoding RNAs that plays an important role in regulating gene expression at a post-transcriptional level. Studies show aberrant levels of miRNAs to be associated with profibrotic processes in idiopathic pulmonary fibrosis (IPF). However, most of these studies used whole IPF tissue or invitro monocultures in which fibrosis was artificially induced. The current study used laser microdissection to collect fibroblastic foci (FF), the key pathologic lesion in IPF, isolated miRNAs, and compared their expression levels with those found in whole IPF lung tissue and/or invitro cultured fibroblast from IPF or normal lungs. Sequencing libraries were generated, and data generated were bioinformatically analyzed. A total of 18 miRNAs were significantly overexpressed in FF tissue when compared with whole IPF tissue. Of those, 15 were unique to FF. Comparison of FF with cultured IPF fibroblasts also revealed differences in miRNA composition that impacted several signaling pathways. The miRNA composition of FF is both overlapping and distinct from that of whole IPF tissue or cultured IPF fibroblasts and highlights the importance of characterizing FF biology as a phenotypically and functionally discrete tissue microenvironment.

Treatment of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrosing interstitial pneumonia of unknown cause, occurring in adults and limited to the lungs. In the past, treatment was aimed at minimizing inflammation and slowing the progression of inflammation to fibrosis. However, the underlying lesion in IPF may be more fibrotic than inflammatory, explaining why few patients respond to anti-inflammatory therapies and the prognosis remains poor.In this review of literature, we will be focusing on main lines of treatment including current medications, supportive care, lung transplantation evaluation, and potential future strategies of treatment.

Fibroblasts positive for meflin have anti-fibrotic properties in pulmonary fibrosis.

The prognosis of elderly individuals with idiopathic pulmonary fibrosis (IPF) remains poor. Fibroblastic foci, in which aggregates of proliferating fibroblasts and myofibroblasts are involved, are the pathological hallmark lesions in IPF to represent focal areas of active fibrogenesis. Fibroblast heterogeneity in fibrotic lesions hampers the discovery of the pathogenesis of pulmonary fibrosis. Therefore, to determine the pathogenesis of IPF, identification of functional fibroblasts is warranted. The aim of this study was to determine the role of fibroblasts positive for meflin, identified as a potential marker for mesenchymal stromal cells, during the development of pulmonary fibrosis.We characterised meflin-positive cells in a single-cell atlas established by single-cell RNA sequencing (scRNA-seq)-based profiling of 243 472 cells from 32 IPF lungs and 29 normal lung samples. We determined the role of fibroblasts positive for meflin using bleomycin (BLM)-induced pulmonary fibrosis.scRNA-seq combined with in situ RNA hybridisation identified proliferating fibroblasts positive for meflin in fibroblastic foci, not dense fibrosis, of fibrotic lungs in IPF patients. A BLM-induced lung fibrosis model for meflin-deficient mice showed that fibroblasts positive for meflin had anti-fibrotic properties to prevent pulmonary fibrosis. Although transforming growth factor-β-induced fibrogenesis and cell senescence with the senescence-associated secretory phenotype were exacerbated in fibroblasts via the repression or lack of meflin, these were inhibited in meflin-deficient fibroblasts with meflin reconstitution.These findings provide evidence to show the biological importance of meflin expression on fibroblasts and myofibroblasts in the active fibrotic region of pulmonary fibrosis.

MDM4: A Novel Molecular Target for Treating Idiopathic Pulmonary Fibrosis Associated with Aging

Idiopathic pulmonary fibrosis (IPF) is the end stage of many diffuse parenchymal lung diseases typified by excessive matrix deposition leading to destruction of normal lung architecture and function of unknown etiology. Aging is considered as a strong risk factor and an independent prognostic factor for progression of IPF. However, the exact mechanisms that link the IPF with aging remained unknown, but a number of changes associated with aging revealed in IPF lungs. The p53 gene is a tumour suppressor gene that played a vital role in cancer and it is also known to have activity in fibrosis. MDM2 and MDM4 are the two major inhibitors of p53 .They only differ in their intrinsic E3-ligase activity and promote degradation of p53. MDM4 is a matrix stiffness -regulated negative regulator of p53 highly expressed in fibrotic lesions of IPF. Some of the invitro studies reported that MDM4-p53 pathway promoted lung fibrosis resolution in aged mice, this suggests that MDM4 can be better target against persistent lung fibrosis associated with aging. Also, better knowledge of the pathophysiological mechanisms linking aging to IPF may provide new therapeutic windows to treat this devastating disease.

Transcriptome analysis of IPF fibroblastic foci identifies key pathways involved in fibrogenesis

IntroductionFibroblastic foci represent the cardinal pathogenic lesion in idiopathic pulmonary fibrosis (IPF) and comprise activated fibroblasts and myofibroblasts, the key effector cells responsible for dysregulated extracellular matrix deposition in multiple...

Inhibition of Aurora Kinase B attenuates fibroblast activation and pulmonary fibrosis

Fibroblast activation including proliferation, survival, and ECM production is central to initiation and maintenance of fibrotic lesions in idiopathic pulmonary fibrosis (IPF). However, druggable molecules that target fibroblast activation remain limited. In this study, we show that multiple pro‐fibrotic growth factors, including TGFα, CTGF, and IGF1, increase aurora kinase B (AURKB) expression and activity in fibroblasts. Mechanistically, we demonstrate that Wilms tumor 1 (WT1) is a key transcription factor that mediates TGFα‐driven AURKB upregulation in fibroblasts. Importantly, we found that inhibition of AURKB expression or activity is sufficient to attenuate fibroblast activation. We show that fibrosis induced by TGFα is highly dependent on AURKB expression and treating TGFα mice with barasertib, an AURKB inhibitor, reverses fibroblast activation, and pulmonary fibrosis. Barasertib similarly attenuated fibrosis in the bleomycin model of pulmonary fibrosis. Together, our preclinical studies provide important proof‐of‐concept that demonstrate barasertib as a possible intervention therapy for IPF.

A near-infrared fluorescent probe for evaluating glutamyl transpeptidase fluctuation in idiopathic pulmonary fibrosis cell and mice models

Idiopathic pulmonary fibrosis (IPF), whose early diagnosis and effective treatment still remain the focus of clinical studies, is a chronic, irreversible and finally fatal pulmonary disease. Glutamyl transpeptidase (GGT) has a potential relationship with the occurrence and development of IPF. Therefore, explore whether GGT can be applied as a biological indicator for the clinical identification and diagnosis of IPF, sensitive and accurate detection of GGT under physiological conditions is necessary. In this research, a new fluorescent probe Cy-GGT was exhibited for detecting GGT concentrations in pulmonary fibrosis cell and mice models. Cy-GGT was capable of rapidly and selectively detecting GGT in vitro. The probe was successfully applied for visualizing GGT in oxidative stress cell models, pulmonary fibrosis cells and mice models. The results revealed that intracellular GGT increased in the cells and mice models of pulmonary fibrosis. GGT plays a significant part in pulmonary fibrosis, and the GGT level abnormally expressed in the lung tissue may be employed as a potential biological indicator to diagnose IPF lesions. Furthermore, the discovery of the close relationship between IPF and GGT will provide a new idea for effective therapy of IPF in the future.

Delayed contrast dynamics as marker of regional impairment in pulmonary fibrosis using 5D MRI - a pilot study

To analyse delayed contrast dynamics of fibrotic lesions in interstitial lung disease (ILD) using five dimensional (5D) MRI and to correlate contrast dynamics with disease severity. 20 patients (mean age: 71 years; M:F, 13:7), with chronic fibrosing ILD: n = 12 idiopathic pulmonary fibrosis (IPF) and n = 8 non-IPF, underwent thin-section multislice CT as part of the standard diagnostic workup and additionally MRI of the lung. 2 min after contrast injection, a radial gradient echo sequence with golden-angle spacing was acquired during 5 min of free-breathing, followed by 5D image reconstruction. Disease was categorized as severe or non-severe according to CT morphological regional severity. For each patient, 10 lesions were analysed. IPF lesions showed later peak enhancement compared to non-IPF (severe: p = 0.01, non-severe: p = 0.003). Severe lesions showed later peak enhancement compared to non-severe lesions, in non-IPF (p = 0.04), but not in IPF (p = 0.35). There was a tendency towards higher accumulation and washout rates in IPF compared to non-IPF in non-severe disease. Severe lesions had lower washout rate than non-severe ones in both IPF (p = 0.003) and non-IPF (p = 0.005). Continuous contrast agent accumulation, without washout, was found only in IPF lesions. Contrast agent dynamics are influenced by type and severity of pulmonary fibrosis, which might enable a more thorough characterisation of disease burden. The regional impairment is of particular interest in the context of antifibrotic treatments and was characterised using a non-invasive, non-irradiating, free-breathing method. Delayed contrast enhancement patterns allow the assessment of regional lung impairment which could represent different disease stages or phenotypes in ILD.

Registration of the extracellular matrix components constituting the fibroblastic focus in idiopathic pulmonary fibrosis.

The extracellular matrix (ECM) in idiopathic pulmonary fibrosis (IPF) drives fibrosis progression; however, the ECM composition of the fibroblastic focus (the hallmark lesion in IPF) and adjacent regions remains incompletely defined. Herein, we serially sectioned IPF lung specimens constructed into tissue microarrays and immunostained for ECM components reported to be deregulated in IPF. Immunostained sections were imaged, anatomically aligned, and 3D reconstructed. The myofibroblast core of the fibroblastic focus (defined by collagen I, α-smooth muscle actin, and procollagen I immunoreactivity) was associated with collagens III, IV, V, and VI; fibronectin; hyaluronan; and versican immunoreactivity. Hyaluronan immunoreactivity was also present at the fibroblastic focus perimeter and at sites where early lesions appear to be forming. Fibrinogen immunoreactivity was often observed at regions of damaged epithelium lining the airspace and the perimeter of the myofibroblast core but was absent from the myofibroblast core itself. The ECM components of the fibroblastic focus were distributed in a characteristic and reproducible manner in multiple patients. This information can inform the development of high-fidelity model systems to dissect mechanisms by which the IPF ECM drives fibrosis progression.

Pathognomonic remodeling of blood and lymphatic capillaries in idiopathic pulmonary fibrosis.

Numerous studies have been published investigating the pathologic alterations in various interstitial pneumonias, particularly in idiopathic pulmonary fibrosis (IPF). However, the few existing studies on capillary remodeling, which does not seem to have priority for pathologic diagnosis, are contradictory, with some reporting increased and others reduced vascularization. We hypothesized that these discrepancies were due to the temporal heterogeneity of the lesions in IPF. We subsequently developed original techniques for evaluating vascular density within the alveolar septa and discovered, for the first time, a heterogeneous increase in alveolar capillaries in the lungs of IPF patients. Notably, we consistently found that the fibrotic lesions in IPF lungs, which are composed mainly of dense collagen with myofibroblasts, featured a reduction in capillaries. This finding provides a plausible explanation for the intractability of IPF, as this reduced vascularization would result in poor delivery of anti-fibrotic agents to these lesions. We also reported the disappearance of subpleural and interlobular lymphatics in IPF lungs, which likely results in poor alveolar clearance in the diseased lungs. Finally, we assessed the autopsied lungs of patients with IPF who died because of acute exacerbation and observed increased and dilated alveolar capillaries. These capillaries are likely to be "leaky" owing to exposure to VEGF produced by regenerated alveolar type II epithelial cells. Furthermore, poor alveolar clearance may prolong the high mobility group box 1 (HMGB1)-induced lung injury in acute exacerbation of IPF. Our data obtained from the assessment of blood and lymphatic capillary alterations in IPF provide novel pathogenetic insights and may provide the basis for new therapeutic strategies targeting IPF.

Macrophage bone morphogenic protein receptor 2 depletion in idiopathic pulmonary fibrosis and Group III pulmonary hypertension.

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of unknown etiology. The development of pulmonary hypertension (PH) is considered the single most significant predictor of mortality in patients with chronic lung diseases. The processes that govern the progression and development of fibroproliferative and vascular lesions in IPF are not fully understood. Using human lung explant samples from patients with IPF with or without a diagnosis of PH as well as normal control tissue, we report reduced BMPR2 expression in patients with IPF or IPF+PH. These changes were consistent with dampened P-SMAD 1/5/8 and elevated P-SMAD 2/3, demonstrating reduced BMPR2 signaling and elevated TGF-β activity in IPF. In the bleomycin (BLM) model of lung fibrosis and PH, we also report decreased BMPR2 expression compared with control animals that correlated with vascular remodeling and PH. We show that genetic abrogation or pharmacological inhibition of interleukin-6 leads to diminished markers of fibrosis and PH consistent with elevated levels of BMPR2 and reduced levels of a collection of microRNAs (miRs) that are able to degrade BMPR2. We also demonstrate that isolated bone marrow-derived macrophages from BLM-exposed mice show reduced BMPR2 levels upon exposure with IL6 or the IL6+IL6R complex that are consistent with immunohistochemistry showing reduced BMPR2 in CD206 expressing macrophages from lung sections from IPF and IPF+PH patients. In conclusion, our data suggest that depletion of BMPR2 mediated by a collection of miRs induced by IL6 and subsequent STAT3 phosphorylation as a novel mechanism participating to fibroproliferative and vascular injuries in IPF.

Pleuroparenchymal Fibroelastosis: Its Clinical Characteristics.

Pleuroparenchymal fibroelastosis (PPFE) is a rare pulmonary fibrosis that is clinically characterized by upper-lobe predominant fibrosis. PPFE is a slowly progressive disorder and its first symptom is dyspnea or dry cough. Chest pain because of pneumothorax may be the first symptom in some patients. Patients with PPFE are slender with a flat rib cage or abnormally narrowed anterior–posterior thoracic dimension. Decreases in forced vital capacity, total lung capacity, and diffusing capacity are respiratory-function characteristics of PPFE, similar to those seen in idiopathic pulmonary fibrosis (IPF). The most remarkable difference in clinical features between PPFE and IPF is imaging findings, with upper-lobe-predominant lesions in PPFE and lower-lobe-predominant lesions in IPF.

Heterogeneous distribution of mechanical stress in human lung: A mathematical approach to evaluate abnormal remodeling in IPF

Idiopathic pulmonary fibrosis (IPF) is the most common and severe form of idiopathic interstitial pneumonia. A recently proposed pathogenic model suggests that the concurrent action of cell senescence, exposure to cigarette smoke and mechanical stress due to respiratory lung movements lead to a localized exhaustion of tissue renewal capacity with eventual alveolar loss and abnormal lung remodeling. In this study we have compared the distribution of IPF lesions, as shown by TC radiological images, with the hypothetical distribution of maximal mechanical stress obtained by a simplified mathematical model. The geometry and distribution of stress as determined by our simulation are closely similar to those demonstrated in vivo in the lungs of patients with idiopathic pulmonary fibrosis using high resolution CT scan radiological imaging. These data argue in favor of the recently proposed contribution of mechanical stress to progressive damage and remodeling of lung parenchyma in IPF. The parameters of the model can be tuned on the age of the patients.

Increased survivin expression contributes to apoptosis-resistance in IPF fibroblasts

Fibroblasts perform critical functions during the normal host response to tissue injury, but the inappropriate accumulation and persistent activation of these cells results in the development of tissue fibrosis. The mechanisms accounting for the aberrant accumulation of fibroblasts during fibrotic repair are poorly understood, although evidence supports a role for fibroblast resistance to apoptosis as a contributing factor. We have shown that TGF-β1 and endothelin-1 (ET-1), soluble mediators implicated in fibrogenesis, promote fibroblast resistance to apoptosis. Moreover, we recently found that ET-1 induced apoptosis resistance in normal lung fibroblasts through the upregulation of survivin, a member of the Inhibitor of Apoptosis (IAP) protein family. In the current study, we sought to determine the role of survivin in the apoptosis resistance of primary fibroblasts isolated from the lungs of patients with Idiopathic Pulmonary Fibrosis (IPF), a fibrotic lung disease of unclear etiology for which there is no definitive therapy. First, we examined survivin expression in lung tissue from patients with IPF and found that there is robust expression in the fibroblasts residing within fibroblastic foci (the "active" lesions in IPF which correlate with mortality). Next, we show that survivin expression is increased in fibroblasts isolated from IPF lung tissue compared to cells from normal lung tissue. Consistent with a role in fibrogenesis, we demonstrate that TGF-β1 increases survivin expression in normal lung fibroblasts. Finally, we show that inhibition of survivin enhances susceptibility of a subset of IPF fibroblasts to apoptosis. Collectively, these findings suggest that increased survivin expression represents one mechanism contributing an apoptosis-resistant phenotype in IPF fibroblasts.

Increased expression of YKL-40, a chitinase-like protein, in serum and lung of patients with idiopathic pulmonary fibrosis

YKL-40, a mammalian member of chitinase-like proteins, has been shown to play a role in pathological conditions leading to tissue remodeling and fibrosis. Recently, YKL-40 was found to be increased in severe asthma, suggesting that YKL-40 contributes to airway remodeling; however, no data are available about YKL-40 expression in idiopathic pulmonary fibrosis (IPF). The present study was conducted to investigate YKL-40 expression in the serum and lung of IPF patients, and to determine its clinical significance. Using an enzyme-linked immunosorbent assay, we measured YKL-40 levels in the serum of 63 IPF patients and in bronchoalveolar lavage fluid (BALF) of 18 IPF patients. YKL-40 levels were also assessed in the serum and BALF of healthy subjects. We further investigated the relationship between serum YKL-40 levels and clinical parameters. Additionally, immunohistochemical staining for YKL-40 was performed in lung specimens of IPF patients and control subjects. Serum and BALF YKL-40 levels were significantly higher in IPF than in controls (serum: 245.8+/-180.2ng/ml vs. 116.0+/-58.3ng/ml; BALF: 17.8+/-19.1ng/ml vs. 0.3+/-0.9ng/ml, respectively). Serum YKL-40 levels significantly correlated positively with serum KL-6 levels and AaDO(2), and negatively with DLco and PaO(2). Immunohistochemical study revealed enhanced YKL-40 expression in alveolar macrophages and bronchiolar epithelia adjacent to fibrotic lesions in IPF, but not in controls. These data suggest that YKL-40 is increased in the circulation and lungs of IPF patients, suggesting that this glycoprotein is associated with the pathophysiology of IPF.

The Quest for the Initial Lesion in Idiopathic Pulmonary Fibrosis

The Quest for the Initial Lesion in Idiopathic Pulmonary Fibrosis

Endothelial–Mesenchymal Transition in Bleomycin-Induced Pulmonary Fibrosis

The pathological hallmark lesions in idiopathic pulmonary fibrosis are the fibroblastic foci, in which fibroblasts are thought to be involved in the tissue remodeling, matrix deposition, and cross-talk with alveolar epithelium. Recent evidence indicates that some fibroblasts in fibrosis may be derived from bone marrow progenitors as well as from epithelial cells through epithelial-mesenchymal transition. To evaluate whether endothelial cells could represent an additional source for fibroblasts, bleomycin-induced lung fibrosis was established in Tie2-Cre/CAG-CAT-LacZ double-transgenic mice, in which LacZ was stably expressed in pan-endothelial cells. Combined X-gal staining and immunocytochemical staining for type I collagen and alpha-smooth muscle actin revealed the presence of X-gal-positive cells in lung fibroblast cultures from bleomycin-treated mice. To explore the underlying mechanisms, by which loss of endothelial-specific markers and gain of mesenchymal phenotypes could be involved in microvascular endothelial cells, the effects of activated Ras and TGF-beta on the microvascular endothelial cell line MS1 were analyzed. Combined treatment with activated Ras and TGF-beta caused a significant loss of endothelial-specific markers, while inducing de novo mesenchymal phenotypes. The altered expression of these markers in MS1 cells with activated Ras persisted after withdrawal of TGF-beta in vitro and in vivo. These findings are the first to show that lung capillary endothelial cells could give rise to significant numbers of fibroblasts through an endothelial-mesenchymal transition in bleomycin-induced lung fibrosis model.

Th1/Th2 Immune Response in Lung Fibroblasts in Interstitial Lung Disease

Inflammatory response in pulmonary fibrosis closely resembles a T-helper (Th) 2 immune response. For recruitment to an inflammatory lesion, the majority of Th1 cells express CXC chemokine receptor 3, recognizing monokine induced by interferon-gamma (Mig), interferon gamma-inducible protein of 10 kD (IP-10), and interferon-inducible T-cell alpha chemoattractant (I-TAC). Th2 cells express CC chemokine receptor 4, recognizing thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC). We investigated the Th1/Th2 chemokine production patterns by lung fibroblasts and their evaluation in bronchoalveolar lavage (BAL) fluid of interstitial lung disease. The production pattern of Th1/Th2 chemokines by lung fibroblasts was examined in ELISA and quantitative reverse transcriptase polymerase chain reactions. Th1/Th2 chemokine levels in BAL fluid of idiopathic pulmonary fibrosis (IPF) and nonspecific interstitial pneumonia (NSIP) were examined to evaluate the clinical relevance of Th1/Th2 chemokines. The lung fibroblasts were polarized to produce Th1-type chemokines by the pro-inflammatory cytokine, tumor necrosis factor (TNF)-alpha and the anti-fibrotic cytokine, interferon (IFN)-gamma. However, the induction patterns of chemokines by these two cytokines were different, i.e., involving predominant induction of IP-10 and I-TAC by TNF-alpha and induction of Mig by IFN-gamma. Although Mig, IP-10, and I-TAC were produced within the BAL fluid of patients, TARC and MDC were at significantly low levels. Our results suggest that lung fibroblasts tend to induce a Th1-type immune response under normal conditions, and that a Th2-type immune response does not play a significant role in smoldering inflammation around the established lesions in IPF and NSIP.

Guanine Nitration in Idiopathic Pulmonary Fibrosis and Its Implication for Carcinogenesis

Nitric oxide (NO)-induced nitrative stress of nucleic acids, as evidenced by guanine nitration, appears to be involved in inflammation-induced carcinogenesis. A high incidence of lung cancer in idiopathic pulmonary fibrosis (IPF) is the major reason for poor prognosis in patients with IPF. We immunohistochemically analyzed the formation and localization of 8-nitroguanine in lung tissues from control subjects, patients with IPF, and patients with lung cancer. Immunohistochemical analysis of control smoker and nonsmoker lungs showed weak immunoreactivity for 8-nitroguanine, mainly in cytoplasm of bronchial epithelial cells. In addition to the bronchial epithelial cells, metaplastic regenerated epithelial cells overlying dense fibrotic lesions in IPF showed strong 8-nitroguanine staining in the cytoplasm. The staining in these metaplastic cells colocalized with staining of inducible and endothelial NO synthases and 8-oxodeoxyguanosine, as evidenced by double-immunostaining analysis. Confocal and immunoelectron microscopy revealed localization of 8-nitroguanine in metaplastic epithelial cytoplasm, mostly in mitochondria. Appreciable 8-nitroguanine immunostaining was also observed in both nuclei and cytoplasm of malignant epithelial cells in squamous cell carcinoma. No significant difference was found in the epithelial 8-nitroguanine formation between control smokers and nonsmokers, but much higher guanine nitration was observed in patients with IPF than in control subjects and patients with lung cancer, via a quantitative immunofluorescence image analysis. The present study indicates that not only oxidative stress but also nitrative stress induced by NO may participate in the pathogenesis of epithelial cell damage and aberrant regeneration occurring in IPF. Thus, guanine nitration may be a major risk factor for lung cancer development in IPF.