Pulmonary matrix-derived hydrogels from patients with idiopathic pulmonary fibrosis induce a proinflammatory state in lung fibroblasts in vitro.

Idiopathic pulmonary fibrosis (IPF), one of the most common forms of interstitial lung disease, is a poorly understood, chronic, and often fatal fibroproliferative condition with only two FDA-approved medications. Understanding the pathobiology of the fibroblast in IPF is critical to evaluating and discovering novel therapeutics. Using a decellularized lung matrix derived from patients with IPF, we generate three-dimensional hydrogels as in vitro models of lung physiology and characterize the phenotype of fibroblasts seeded into the hydrogels. When cultured in IPF extracellular matrix hydrogels, IPF fibroblasts display differential contractility compared with their normal counterparts, lose the classical myofibroblast marker α-smooth muscle actin, and increase expression of proinflammatory cytokines compared with fibroblasts seeded two-dimensionally on tissue culture dishes. We validate this proinflammatory state in fibroblast-conditioned media studies with monocytes and monocyte-derived macrophages. These findings add to a growing understanding of the lung microenvironment effect on fibroblast phenotypes, shed light on the potential role of fibroblasts as immune signaling hubs during lung fibrosis, and suggest intervention in fibroblast-immune cell cross-talk as a possible novel therapeutic avenue.

Serum levels of small HDL particles are negatively correlated with death or lung transplantation in an observational study of idiopathic pulmonary fibrosis.

BACKGROUND: Serum lipoproteins, such as high-density lipoproteins (HDL), may influence disease severity in idiopathic pulmonary fibrosis (IPF). Here, we investigated associations between serum lipids and lipoproteins and clinical end-points in IPF. METHODS: Clinical data and serum lipids were analysed from a discovery cohort (59 IPF subjects, 56 healthy volunteers) and validated using an independent, multicentre cohort (207 IPF subjects) from the Pulmonary Fibrosis Foundation registry. Associations between lipids and clinical end-points (forced vital capacity, 6-min walk distance, gender age physiology (GAP) index, death or lung transplantation) were examined using Pearson's correlation and multivariable analyses. RESULTS: Serum concentrations of small HDL particles measured using nuclear magnetic resonance spectroscopy (S-HDLPNMR) correlated negatively with the GAP index in the discovery cohort of IPF subjects. The negative correlation of S-HDLPNMR with GAP index was confirmed in the validation cohort of IPF subjects. Higher levels of S-HDLPNMR were associated with lower odds of death or its competing outcome, lung transplantation (OR 0.9 for each 1-µmol·L-1 increase in S-HDLPNMR, p<0.05), at 1, 2 and 3 years from study entry in a combined cohort of all IPF subjects. CONCLUSIONS: Higher serum levels of S-HDLPNMR are negatively correlated with the GAP index, as well as with lower observed mortality or lung transplantation in IPF subjects. These findings support the hypothesis that S-HDLPNMR may modify mortality risk in patients with IPF.

Global Gene Expression Analysis in an in vitro Fibroblast Model of Idiopathic Pulmonary Fibrosis Reveals Potential Role for CXCL14/CXCR4.

Idiopathic Pulmonary Fibrosis (IPF) is a progressive disorder that is marked by an over accumulation of activated fibroblast populations. Despite the improved understanding of many mechanisms within this disease, global gene expression analysis has few focused studies on the fibroblast, the central effector cell of progressive fibrosis. We present a unique analysis of IPF pulmonary fibroblasts as they transition through cell culture and identify in vitro altered cellular processes. Fibroblasts were isolated from diseased (n = 8) and non-diseased (n = 4) lungs. Global gene expression analysis was carried out at the initial point of isolation and after 3 weeks of culture. We identify several genes that are altered by removal of the fibroblast from the IPF environment. Comparison of this subset of genes to four previously published whole lung analyses refined our list to a small subset of key fibroblast specific genes important in IPF. Application of STRING database analysis and confirmation via in-vitro and histological assay highlights the CXCL14/CXCR4 chemokine axis with a possible role in the progression and/or activation of fibroblasts within the IPF lung. Our findings, present a possible therapeutic target for IPF and a model for the study and discovery of novel protein and processes in this terrible disease.

Sustained activation of toll-like receptor 9 induces an invasive phenotype in lung fibroblasts: possible implications in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive scarring of the lung parenchyma, resulting in a steady decline of lung function and ultimately respiratory failure. The disease course of IPF is extremely variable, with some patients exhibiting stability of symptoms for prolonged periods of time, whereas others exhibit rapid progression and loss of lung function. Viral infections have been implicated in IPF and linked to disease severity; however, whether they directly contribute to progression is unclear. We previously classified patients as rapid and slow progressors on the basis of clinical features and expression of the pathogen recognition receptor, Toll-like receptor 9 (TLR9). Activation of TLR9 in vivo exacerbated IPF in mice and induced differentiation of myofibroblasts in vitro, but the mechanism of TLR9 up-regulation and progression of fibrosis are unknown. Herein, we investigate whether transforming growth factor (TGF)-ß, a pleiotropic cytokine central to IPF pathogenesis, regulates TLR9 in lung myofibroblasts. Results showed induction of TLR9 expression by TGF-ß in lung myofibroblasts and a distinct profibrotic myofibroblast phenotype driven by stimulation with the TLR9 agonist, CpG-DNA. Chronic TLR9 stimulation resulted in stably differentiated α-smooth muscle actin(+)/platelet-derived growth factor receptor α(+)/CD44(+)/matrix metalloproteinase-14(+)/matrix metalloproteinase-2(+) myofibroblasts, which secrete inflammatory cytokines, invade Matrigel toward platelet-derived growth factor, and resist hypoxia-induced apoptosis. These results suggest a mechanism by which TGF-ß and TLR9 responses in myofibroblasts collaborate to drive rapid progression of IPF.

Lactic acid is elevated in idiopathic pulmonary fibrosis and induces myofibroblast differentiation via pH-dependent activation of transforming growth factor-ß.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a complex disease for which the pathogenesis is poorly understood. In this study, we identified lactic acid as a metabolite that is elevated in the lung tissue of patients with IPF. OBJECTIVES: This study examines the effect of lactic acid on myofibroblast differentiation and pulmonary fibrosis. METHODS: We used metabolomic analysis to examine cellular metabolism in lung tissue from patients with IPF and determined the effects of lactic acid and lactate dehydrogenase-5 (LDH5) overexpression on myofibroblast differentiation and transforming growth factor (TGF)-ß activation in vitro. MEASUREMENTS AND MAIN RESULTS: Lactic acid concentrations from healthy and IPF lung tissue were determined by nuclear magnetic resonance spectroscopy; α-smooth muscle actin, calponin, and LDH5 expression were assessed by Western blot of cell culture lysates. Lactic acid and LDH5 were significantly elevated in IPF lung tissue compared with controls. Physiologic concentrations of lactic acid induced myofibroblast differentiation via activation of TGF-ß. TGF-ß induced expression of LDH5 via hypoxia-inducible factor 1α (HIF1α). Importantly, overexpression of both HIF1α and LDH5 in human lung fibroblasts induced myofibroblast differentiation and synergized with low-dose TGF-ß to induce differentiation. Furthermore, inhibition of both HIF1α and LDH5 inhibited TGF-ß-induced myofibroblast differentiation. CONCLUSIONS: We have identified the metabolite lactic acid as an important mediator of myofibroblast differentiation via a pH-dependent activation of TGF-ß. We propose that the metabolic milieu of the lung, and potentially other tissues, is an important driving force behind myofibroblast differentiation and potentially the initiation and progression of fibrotic disorders.

Genomic phenotype of non-cultured pulmonary fibroblasts in idiopathic pulmonary fibrosis.

Activated fibroblasts are the central effector cells of the progressive fibrotic process in idiopathic pulmonary fibrosis (IPF). Characterizing the genomic phenotype of isolated fibroblasts is essential to understanding IPF pathogenesis. Comparing the genomic phenotype of non-cultured pulmonary fibroblasts from advanced IPF patients' and normal lungs revealed novel genes, biological processes and concomitant pathways previously unreported in IPF fibroblasts. We demonstrate altered expression in proteasomal constituents, ubiquitination-mediators, Wnt, apoptosis and vitamin metabolic pathways and cell cycle regulators, suggestive of loss of cellular homeostasis. Specifically, FBXO32, CXCL14, BDKRB1 and NMNAT1 were up-regulated, while RARA and CDKN2D were down-regulated. Paradoxically, pro-apoptotic inducers TNFSF10, BAX and CASP6 were also found to be increased. This comprehensive description of altered gene expression in isolated IPF fibroblasts underscores the complex biological processes characteristic of IPF and may provide a foundation for future research into this devastating disease.

Evaluation of imatinib mesylate in the treatment of pulmonary arterial hypertension.

Imatinib mesylate is a small molecule inhibitor that selectively inhibits the PDGF receptor kinase as well the cKIT and Abl kinases, among other targets. Various studies have implicated the PDGF pathway in the pathogenesis of pulmonary arterial hypertension (PAH). Inhibition with imatinib mesylate has shown efficacy in human case reports and experimental models of PAH. Results from a Phase II trial of imatinib mesylate in PAH did not meet the primary end point but showed improvement in several secondary end points and in a subgroup analysis. As suggested by this study as well as a few case reports, imatinib may be effective in a subset of patients with more severe disease. However, this remains to be further validated through a Phase III study, which is already underway. In conclusion, it appears that imatinib mesylate may hold promise as an adjunct drug in PAH therapy, especially since it is directed at a pathway not previously targeted.

Hepatic stellate cell and myofibroblast-like cell gene expression in the explanted cirrhotic livers of patients undergoing liver transplantation.

BACKGROUND: Hepatic stellate cells (HSC) are involved in hepatic fibrogenesis. Cell signaling associated with an insult to the liver affects an HSC transdifferentiation to fibrogenic myofibroblast-like cells. AIMS: To investigate the transcriptional expression distinguishing HSC and myofibroblast-like cells between livers with and without cirrhosis. METHODS: Tissue from ten cirrhotic livers (undergoing transplant) and four non-cirrhotic livers from the National Disease Research Interchange underwent cell separation to extract HSC and myofibroblast-like cell populations. Separated cell types as well as LI-90 cells were subjected to microarray analysis. Selected microarray results were verified by quantitative real-time PCR. RESULTS: Differential expression of some genes, such as IL-1beta, IL-1alpha, and IL-6, was associated with both transdifferentiation and disease. Other genes, such as fatty acid 2-hydroxylase only show differential expression in association with disease. Functional analysis supported these findings, indicating some signal transduction pathways (IL-6) are involved in disease and activation, whereas retinoid X receptor signaling in HSC from cirrhotic and non-cirrhotic livers varies in scope and quality. CONCLUSIONS: These findings indicate distinct phenotypes for HSC from cirrhotic and non-cirrhotic livers. Furthermore, coordinated differential expression between genes involved in the same signal transduction pathways provides some insight into the mechanisms that may control the balance between fibrogenesis and fibrolysis.

Potential of imatinib mesylate as a novel treatment for pulmonary fibrosis.

Pulmonary fibrosis is a disease characterized by progressive scarring of the lungs, with idiopathic pulmonary fibrosis (IPF) being the most aggressive form. The diagnosis of IPF is made after other conditions are excluded and is based on a characteristic clinical presentation, radiographic features and, sometimes, pathologic specimen. Existing IPF drug regimens, including corticosteroids and cytotoxic medications, are generally ineffective. To date, only lung transplantation has been shown to improve mortality in carefully selected patients. Multiple therapeutic agents have been investigated but none have proven to be successful. Novel drugs are constantly being sought in an attempt to find a therapy that halts or reverses this disease. Imatinib mesylate is used for chronic myelogenous leukemia and gastrointestinal stromal tumors. It also has antifibrotic properties, as demonstrated in several studies using mouse models of pulmonary fibrosis. Currently, trials are underway to investigate its efficacy in human subjects with IPF.

Application of a correlation correction factor in a microarray cross-platform reproducibility study.

BACKGROUND: Recent research examining cross-platform correlation of gene expression intensities has yielded mixed results. In this study, we demonstrate use of a correction factor for estimating cross-platform correlations. RESULTS: In this paper, three technical replicate microarrays were hybridized to each of three platforms. The three platforms were then analyzed to assess both intra- and cross-platform reproducibility. We present various methods for examining intra-platform reproducibility. We also examine cross-platform reproducibility using Pearson's correlation. Additionally, we previously developed a correction factor for Pearson's correlation which is applicable when X and Y are measured with error. Herein we demonstrate that correcting for measurement error by estimating the "disattenuated" correlation substantially improves cross-platform correlations. CONCLUSION: When estimating cross-platform correlation, it is essential to thoroughly evaluate intra-platform reproducibility as a first step. In addition, since measurement error is present in microarray gene expression data, methods to correct for attenuation are useful in decreasing the bias in cross-platform correlation estimates.

Hepatic gene expression in patients with obesity-related non-alcoholic steatohepatitis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is among the most common causes of chronic liver disease. NAFLD includes a spectrum of clinicopathologic syndromes that includes non-alcoholic steatohepatitis (NASH) that has potential for progression. The pathogenesis of NASH is poorly characterized. AIM: This study was designed to identify differences in hepatic gene expression in patients with NASH and to relate such differences to their clinical characteristics. DESIGN: Consecutive patients undergoing bariatric surgery were prospectively recruited. Extensive clinical data and two liver biopsy specimens were obtained at the time of enrollment. A single hepatopathologist reviewed and classified the liver biopsies. Patients with excessive alcohol use and other causes of liver disease were excluded. A group of 29 NASH patients, 12 with steatosis alone, seven obese controls and six non-obese controls were selected for further investigation. Customized cDNA microarrays containing 5220 relevant genes were designed specifically for this study. Microarray experiments were run in triplicate for each sample and a selected group of genes were confirmed using real-time PCR. OUTCOME MEASURE: Differential hepatic gene expressions in patients with NASH as compared with controls. RESULTS: Thirty-four genes with significant differential expression were identified in patients with NASH when compared with non-obese controls. Moreover, 19 of these genes showed no significant expression differences in obese vs. non-obese controls, suggesting a stronger association of these genes to NASH. CONCLUSIONS: Several differentially expressed genes in patients with NASH are related to lipid metabolism and extracellular matrix remodeling. Additionally, genes related to liver regeneration, apoptosis, and the detoxification process were differentially expressed. These findings may help clarify the molecular pathogenesis of NASH and identify potential targets for therapeutic intervention.

Microarrays in cancer research.

Microarray technology has presented the scientific community with a compelling approach that allows for simultaneous evaluation of all cellular processes at once. Cancer, being one of the most challenging diseases due to its polygenic nature, presents itself as a perfect candidate for evaluation by this approach. Several recent articles have provided significant insight into the strengths and limitations of microarrays. Nevertheless, there are strong indications that this approach will provide new molecular markers that could be used in diagnosis and prognosis of cancers. To achieve these goals it is essential that there is a seamless integration of clinical and molecular biological data that allows us to elucidate genes and pathways involved in various cancers. To this effect we are currently evaluating gene expression profiles in human brain, ovarian, breast and hematopoetic, lung, colorectal, head and neck and biliary tract cancers. To address the issues we have a joint team of scientists, doctors and computer scientists from two Virginia Universities and a major healthcare provider. The study has been divided into several focus groups that include; Tissue Bank Clinical & Pathology Laboratory Data, Chip Fabrication, QA/QC, Tissue Devitalization, Database Design and Data Analysis, using multiple microarray platforms. Currently over 300 consenting patients have been enrolled in the study with the largest number being that of breast cancer patients. Clinical data on each patient is being compiled into a secure and interactive relational database and integration of these data elements will be accomplished by a common programming interface. This clinical database contains several key parameters on each patient including demographic (risk factors, nutrition, co-morbidity, familial history), histopathology (non genetic predictors), tumor, treatment and follow-up information. Gene expression data derived from the tissue samples will be linked to this database, which allows us to query the data at multiple levels. The challenge of tissue acquisition and processing is of paramount importance to the success of this venture. A tissue devitalization timeline protocol was devised to ensure sample and RNA integrity. Stringent protocols are being employed to ascertain accurate tumor homogeneity, by serial dissection of each tumor sample at 10 microM frozen sections followed by histopathological evaluation. The multiple platforms being utilized in this study include Affimetrix, Oligo-Chips and custom-designed cDNA arrays. Selected RNA samples will be evaluated on each platform between the groups. Analysis steps will involve normalization and standardization of gene expression data followed by hierarchical clustering to determine co-regulation profiles. The aim of this conjoint effort is to elucidate pathways and genes involved in various cancers, resistance mechanisms, molecular markers for diagnosis and prognosis.

DNA damage assessment by comet assay of human lymphocytes exposed to jet propulsion fuels.

Exposure to jet fuel damages DNA and results in a number of physiological changes in liver, lung, immune, and neurological tissue. In this study the single-cell gel electrophoresis assay or comet assay was used to compare the DNA damage in human peripheral lymphocytes produced by three jet propulsion fuels: JP-8, JP-5, and JP-8+100. These fuels consist of complex mixtures of aliphatic, aromatic, and substituted naphthalene hydrocarbons. Two exposure times were investigated which correspond to estimated occupational exposure times and concentrations of fuels were used that were based on previous fuel toxicity studies. Analysis of samples for the extent of DNA damage as determined by tail moment and percent tail DNA was performed on exposed cells following a brief recovery time. All fuels produced significant increases in DNA damage; however, only JP-8+100 was genotoxic at the lowest exposure concentration (1:500). At the highest exposure concentration (1:75), the mean tail moments for JP-8 and JP-8+100 (32.041 +/- 2.599 and 45.774 +/- 4.743, respectively) were significantly greater than for JP-5 (1.314 +/- 0.474). These results indicate that JP-8+100 is the most potent inducer of DNA damage in human peripheral lymphocytes and that both JP-8+100 and JP-8 are capable of damaging lymphocyte DNA to a greater extent than JP-5.