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Dissection of human fibroblast populations by lung regions: genomic differences and functional pathway analysis

ABSTRACT: ABSTRACT Background: Primary human distal lung/parenchymal fibroblasts (DLF) exhibit a different phenotype from airway fibroblasts (AF), including the expression of high levels of alpha-SMA. The scope of the differences and the mechanisms driving them are unknown. We hypothesized that the distinct fibroblast characteristics based on lung region predicted a broad range of genomic differences which contribute to distinctly different functional pathway activation in AF and DLF. Methods and Findings: To investigate whether comprehensive gene expression patterns vary between AF and DLF, we compared global gene expression profiles of 3 matched pairs of primary human fibroblasts isolated from proximal and distal lung. 194 transcripts were upregulated in AF, and 290 transcripts upregulated in DLF. Quantitative real-time PCR (qRT-PCR) from both asthmatic and normal subjects confirmed the validity of microarray data (n = 8). Further analysis identified distinct pathway activation patterns, including the identification of the TGF-β receptor/SMAD3 signaling pathway as critical to the phenotypic differences in AF and DLF. The functional impact of these molecular differences on AF and DLF was then analyzed using Western blot, qRT-PCR, ELISA and gene knock-down approaches. TGF-β receptor/SMAD2-3 pathway analysis confirmed the contribution of higher TGF-β1 expression and accompanying SMAD3 and JNK activation to the increased alpha-SMA level seen in DLF. There are two limitations associated with our work. First, the microarray sample size was small. Second, for ethical reasons, a large majority of our data were from asthmatic subjects, only one pair of airway and distal lung tissues from a subject without knowing preexisting lung disease matched for age and smoking status was available. In this single normal subject, the same pattern of differences existed, supporting the concept that these genomic differences are regional, rather than disease-related. Conclusions: These findings demonstrated marked molecular and functional differences for these two lung regional fibroblast populations. These results suggest that airway and parenchymal fibroblasts differ in their responses to injury, repair, and remodeling in the lungs. There are likely profound implications of these observations for understanding mechanisms of development of fibrotic lung diseases as well as approaching therapy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE19362 | GEO | 2011/07/22

SECONDARY ACCESSION(S): PRJNA120397

REPOSITORIES: GEO

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Project description:ABSTRACT Background: Primary human distal lung/parenchymal fibroblasts (DLF) exhibit a different phenotype from airway fibroblasts (AF), including the expression of high levels of alpha-SMA. The scope of the differences and the mechanisms driving them are unknown. We hypothesized that the distinct fibroblast characteristics based on lung region predicted a broad range of genomic differences which contribute to distinctly different functional pathway activation in AF and DLF. Methods and Findings: To investigate whether comprehensive gene expression patterns vary between AF and DLF, we compared global gene expression profiles of 3 matched pairs of primary human fibroblasts isolated from proximal and distal lung. 194 transcripts were upregulated in AF, and 290 transcripts upregulated in DLF. Quantitative real-time PCR (qRT-PCR) from both asthmatic and normal subjects confirmed the validity of microarray data (n = 8). Further analysis identified distinct pathway activation patterns, including the identification of the TGF-β receptor/SMAD3 signaling pathway as critical to the phenotypic differences in AF and DLF. The functional impact of these molecular differences on AF and DLF was then analyzed using Western blot, qRT-PCR, ELISA and gene knock-down approaches. TGF-β receptor/SMAD2-3 pathway analysis confirmed the contribution of higher TGF-β1 expression and accompanying SMAD3 and JNK activation to the increased alpha-SMA level seen in DLF. There are two limitations associated with our work. First, the microarray sample size was small. Second, for ethical reasons, a large majority of our data were from asthmatic subjects, only one pair of airway and distal lung tissues from a subject without knowing preexisting lung disease matched for age and smoking status was available. In this single normal subject, the same pattern of differences existed, supporting the concept that these genomic differences are regional, rather than disease-related. Conclusions: These findings demonstrated marked molecular and functional differences for these two lung regional fibroblast populations. These results suggest that airway and parenchymal fibroblasts differ in their responses to injury, repair, and remodeling in the lungs. There are likely profound implications of these observations for understanding mechanisms of development of fibrotic lung diseases as well as approaching therapy. In order to identify the underlying molecular differences between airway- and distal lung fibroblasts, microarray analysis was performed on 3 different matched pairs of fibroblasts.

Project description:Background: Asthma is the most common chronic lung disease in children and young adults worldwide. Airway remodelling (including increased fibroblasts and myofibroblasts in airway walls due to chronic inflammation) differentiates asthmatic from non-asthmatic airways. The increase in airway fibroblasts and myofibroblasts occurs via epithelial to mesenchymal transition (EMT) where epithelial cells lose their tight junctions and are transdifferentiated to mesenchymal cells, with further increases in myofibroblasts occurring via fibroblast-myofibroblast transition (FMT). Transforming growth factor (TGF)-β is the central EMT- and FMT-inducing cytokine. In this study, we have used next generation sequencing to delineate the changes in the fibroblast transcriptome induced by TGF-β treatment in both the short term and after differentiation into myofibroblasts, to gain an understanding of the contribution of TGF-b induced transdifferentiation to the asthmatic phenotype. The data obtained from RNAseq analysis was confirmed by quantitative PCR (qPCR). Results: As expected, we found that genes coding for intermediates in the TGF-β signalling pathways (SMADs) were differentially expressed after treatment, and genes involved in cytoskeletal pathways (FN1, LAMA, ITGB1) were differentially expressed in myofibroblasts compared to fibroblasts. Importantly, genes that were previously shown to be changed in asthmatic lungs (ADAMTS1, DSP, TIMPs, MMPs) were differentially expressed in myofibroblasts, strongly suggesting that TGF-β mediated differentiation of fibroblasts to myofibroblasts may underlie important changes in the asthmatic airway. We also identified new signalling pathways (AKT, PTEN) that are changed in myofibroblasts compared to fibroblasts. Conclusion: We have found a significant number of genes that are altered after differentiation of fibroblasts into myofibroblasts by TGF-β treatment, many of which were expected or predicted. However, we also identified novel genes and pathways that were affected after treatment of fibroblasts with TGF-β, which suggests additional pathways that that are activated during the transition between fibroblasts and myofibroblasts, and may contribute to the asthma phenotype.

Project description:Background: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease of the lungs that is currently the fourth leading cause of death worldwide. Genetic factors account for only a small amount of COPD risk, but epigenetic mechanisms including DNA methylation, have the potential to mediate the interactions between an individual?s genetics and environmental exposure. DNA methylation is highly cell type specific and individual cell type studies of DNA methylation in COPD are sparse. Fibroblasts are present within the airway and parenchyma of the lung and contribute to the aberrant deposition of extracellular matrix in COPD. No assessment or comparison of genome-wide DNA methylation profiles in airway and parenchymal fibroblasts from individuals with and without COPD has been undertaken. These data provide valuable insight into the molecular mechanisms contributing to COPD and the differing pathologies of small airways disease and emphysema in COPD. Methods: Genome-wide DNA methylation was evaluated at over 485,000 CpG sites using the Illumina Infinium HumanMethylation450 BeadChip array in airway (non-COPD n=8, COPD n=7) and parenchymal fibroblasts (non-COPD n=18, COPD n=28) isolated from individuals with and without COPD. Targeted gene expression was assessed by qPCR in matched RNA samples. Results: Differentially methylated DNA regions were identified between cells isolated from individuals with and without COPD in both airway and parenchymal fibroblasts. Only in parenchymal fibroblasts was differential DNA methylation associated with differential gene expression. A second analysis of differential DNA methylation variability identified 359 individual differentially variable CpG sites in parenchymal fibroblasts. No differentially variable CpG sites were identified in airway fibroblasts. Five differentially variable methylated CpG sites, associated with three genes were subsequently assessed for gene expression differences. Two genes (OAT and GRIK2) displayed significantly increased gene expression in cells isolated from individuals with COPD. Conclusions: Differential and variable DNA methylation was associated with COPD status in parenchymal fibroblasts but not airway fibroblasts. Aberrant DNA methylation was associated with altered gene expression imparting biological function to DNA methylation changes. Changes in DNA methylation are therefore implicated in the molecular mechanisms underlying COPD pathogenesis and may represent novel therapeutic targets.

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Dissection of human fibroblast populations by lung regions: genomic differences and functional pathway analysis

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