Transcriptomic Changes in TGF-β1 Differentiated and PGE2 Dedifferentiated Myofibroblasts
ABSTRACT: These data show that the genes that distinguish myofibroblasts from fibroblasts are myriad, and that some genes not traditionally associated with myofibroblast differentiation may serve as novel therapeutic targets for fibrosing disorders. Gene expression levels were assessed from total RNA on the Affymetrix U219 microarray. Here, we use transforming growth factor-β1 (TGF-β1) and prostaglandin E2 (PGE2), which has recently been shown to reverse myofibroblast differentiation, to investigate the transcriptomic changes that occur during TGF-β1-induced differentiation and PGE2-induced de-differentiation of myofibroblasts.
Project description:Fibrotic diseases have significant health impact and have been associated with differentiation of the resident fibroblasts into myofibroblasts. In particular, stiffened extracellular matrix and TGF-β1 in fibrotic lesions have been shown to promote pathogenic myofibroblast activation and progression of fibrosis in various tissues. To better understand the roles of mechanical and chemical cues on myofibroblast differentiation and how they may crosstalk, we cultured primary valvular interstitial cells (VICs) isolated from porcine aortic valves and studied how traditional TCPS culture, which presents a non-physiologically stiff environment, and TGF-β1 affect native VIC phenotypes. We carried out gene expression profiling using porcine genome microarrays from Affymetrix and found that traditional TCPS culture induces major changes in gene expression of native VICs, rendering these cells more activated and similar to cells treated with TGF-β1. We also monitored time-dependent effects induced by TGF-β1 by examining gene expression changes induced by TGF-β1 at 8 hours and 24 hours. Porcine aortic VICs were isolated and cultured with or without TGF-β1 treatment for RNA extraction and hybridization on Affymetrix microarrays. We included 3 biological replicates for each condition. P0 VICs were freshly isolated cells which had not been cultured. P2 VICs were cells that had been passaged 2 times and cultured on plastic plates in low serum media. Some of the P2 VICs were treated with TGF-β1 at 5ng/ml for 8 hours or 24 hours. All the control and TGF-β1-treated conditions were collected at the same time on day 3 of culture.
Project description:Aim: Differentiation of cardiac fibroblasts (Fb) into myofibroblasts (MyoFb) is responsible for connective tissue buildup in myocardial remodeling. We examined reversibility of MyoFb differentiation. Methods and Results: Adult rat cardiac Fb were cultured on a plastic substratum providing mechanical stress, with conditions to obtain different Fb phenotypes. Fb spontaneously differentiated to proliferating MyoFb (p-MyoFb) with stress fiber formation decorated with alpha-smooth muscle actin (α-SMA). Transforming growth factor-β1 (TGF-β1) promoted terminal differentiation into α-SMA positive MyoFb showing near absence of proliferation i.e. non-p-MyoFb (2-fold increase in cell number after 12 days vs 11-fold for p-MyoFb). SD-208, a TGF-β-receptor-I kinase blocker, inhibited p-MyoFb differentiation as shown by stress fiber absence, low levels of α-SMA protein expression, and high levels of proliferation (32-fold increase after 12 days). Fb seeded in collagen matrices induced no contraction, whereas p-MyoFb and non-p-MyoFb induced 2.5- and 4-fold contraction. Fb produced low levels of collagen and secreted high levels of IL-10. Non-p-MyoFb showed high collagen production and high MCP-1 and TIMP-1 secretion. Transcriptome analysis indicated differential gene expression between all phenotypes. Dedifferentiation of p-MyoFb, but not of non-p-MyoFb, was induced by SD-208 despite maintained stress, shown by stress fiber de-polymerization in 30% of p-MyoFb vs in 8% of non-p-MyoFb. Stress fiber de-polymerization could be induced by mechanical strain release in p-MyoFb and non-p-MyoFb (2 day culture in unrestrained 3-D collagen matrices). Only p-MyoFb showed true dedifferentiation after long-term 3-D culture. Conclusions: Both reduction in mechanical strain and TGF-β-receptor-I kinase inhibition can reverse p-MyoFb differentiation but not in non-p-MyoFb. Fibroblasts isolated from each rat heart (n= 4) were cultured in specific conditions to obtain different fibroblast phenotypes: 1) spontaneously differentiation into proliferating myofibroblasts (code RC), 2) terminal differentiation into non-proliferating myofibroblasts with TGF-β1 (code RT) and 3) inhibition of myofibroblast differentiation with SD-208, a TGF-β-receptor-I kinase blocker (code RS). RNA concentration and purity from a total of 12 samples were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Using the Ambion WT Expression Kit, per sample, an amount of 100 ng of total RNA spiked with bacterial poly-A RNA positive controls (Affymetrix) was converted to double stranded cDNA in a reverse transcription reaction. Next the sample was converted and amplified to antisense cRNA in an in vitro transcription reaction which was subsequently converted to single stranded sense cDNA. Finally, samples were fragmented and labeled with biotin in a terminal labeling reaction according to the Affymetrix WT Terminal Labeling Kit. A mixture of fragmented biotinylated cDNA and hybridisation controls (Affymetrix) was hybridised on Affymetrix GeneChip® Rat Gene 2.0 ST array followed by staining and washing in a GeneChip® fluidics station 450 (Affymetrix) according to the manufacturer’s procedures. To assess the raw probe signal intensities, chips were scanned using a GeneChip® scanner 3000 (Affymetrix).
Project description: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 by a SMAD-3–dependent mechanism. 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. Experiment Overall Design: mRNA expression of genes in human fetal lung mesenchymal cells (IMR-90) treated with or without TGF-β1, as analyzed by Affymetrix (U133A) microarrays. Control (C0, C2, C3) = cells without TGF-β1 treatment (n=3). Experimental (T0, T5, T7) = cells treated with TGF-β1 (2ng/ml) (n=3). mRNA was collected for all 6 samples for 48 hours post treatment.
Project description:Endothelial to mesenchymal transition is a possible source of myofibroblasts, which play a crucial role in the pathogenesis of fibrosis. EndMT participate in tissue fibrotic processes in various organs. TGF-β family growth factors are involved in the initiation of EndMT. This process plays a crucial role n the pathogenesis of various fibrotic diseases.
Project description:The human hepatic stellate cell line LX2 was treated with 8 Gy X-ray irradiation and/or 2ng/ml recombinant human TGF-β1. The iTRAQ-based high throughput quantitative proteomic approach was used to obtain a comprehensive view of the protein ensembles affected by irradiation and/or TGF-β1 treatment on LX2. This study provides clues for further investigation of the mechanisms behind radiation-induced liver fibrosis.
Project description:CD4+ T cells that selectively produce interleukin (IL)-17, are critical for host defense and autoimmunity1-4. Crucial for T helper17 (Th17) cells in vivo5,6, IL-23 has been thought to be incapable of driving initial differentiation. Rather, IL-6 and transforming growth factor (TGF)-β1 have been argued to be the factors responsible for initiating specification7-10. Herein, we show that Th17 differentiation occurs in the absence of TGF-β signaling. Neither IL-6 nor IL-23 alone efficiently generated Th17 cells; however, these cytokines in combination with IL-1β effectively induced IL-17 production in naïve precursors, independently of TGF-β. Epigenetic modification of the Il17a/Il17f and Rorc promoters proceeded without TGF-β1, allowing the generation of cells that co-expressed Rorγt and T-bet. T-bet+Rorγt+ Th17 cells are generated in vivo during experimental allergic encephalomyelitis (EAE), and adoptively transferred Th17 cells generated with IL-23 in the absence of TGF-β1 were more pathogenic in this experimental disease. These data suggest a new model for Th17 differentiation. Consistent with genetic data linking the IL23R with autoimmunity, our findings re-emphasize the role of IL-23 and therefore have important implications for the development of new therapies. Mouse T helper 17 cell differentiation with or without TGFB
Project description:Inflammation features in diverse central nervous system disorders such as stroke, trauma, neurodegeneration, infection and autoimmunity. To better understand how inflammatory mediators may alter astrocyte functions, we examined the effects of transforming growth factor-β1 (TGF-β1), lipopolysaccharide (LPS) and interferon-gamma (IFNγ) on purified, murine, primary cortical astrocyte cultures. We used microarrays to conduct whole genome expression profiling, and measured calcium signaling, which is implicated in mediating dynamic astrocyte functions. Combinatorial exposure to TGF-β1, LPS and IFNγ significantly modulated astrocyte expression of over 6,800 genes and resulted in both additive and synergistic changes compared with individual stimuli alone. Bioinformatic analysis revealed that combinatorial treatment significantly and markedly up regulated molecular networks and pathways associated with immune signaling and with regulation of cell compromise, death, growth and proliferation. These findings provide databases of astrocyte transcriptome changes elicited by the inflammatory stimuli, TGF-β1, LPS and IFNγ alone and in combination, and show that these stimuli up regulate astrocyte molecular networks associated with immune- and injury-related functions and significantly alter astrocyte calcium signaling evoked by multiple GPCR. We used microarrays to examine the effects of transforming growth factor-β1 (TGF-β1), lipopolysaccharide (LPS) and interferon-gamma (IFNγ) on purified, murine, primary cortical astrocyte cultures.
Project description:This study aimed to establish an epithelial-mesenchymal transition (EMT) model with an immortalized human bronchial epithelial cell line, M-BE, and to identify an EMT signature gene set. The TGF-β1-induced M-BE cells got spindle-shaped fibroblast-like morphology and lost the cell-cell contact, with down-regulated expression of epithelial marker E-cadherin and up-regulated expression of mesenchymal markers N-cadherin and Vimentin. Examined by microarray, there were 2628 genes identified as significant EMT-related, including 1490 up-regulated genes (FC > 2, fdr < 0.01) and 1138 down-regulated genes (FC < 0.5, fdr < 0.01) in TGF-β1-induced M-BE cells. M-BE cells were treated with human recombinant TGF-β1 (5ng/ml) for six days. M-BE cells cultured without TGF-β1 were considered the controls. Three replicates of each were carried out for this investigation. Agilent 4x44K Human Whole Genome expression microarray (G4112F) analysis was applied to the RNA samples of the TGF-β1-treated M-BE cells and the controls.
Project description:Langerhans dendritic cells represent abundantly occuring and evolutionary highly conserved DCs specifically located in the stratified epithelial tissues. LCs are unique among DC family members in that they express epithelial-type adhesion molecules, allowing them to form a tight three-dimensional network in basal and suprabasal epidermal keratinocyte layers and developmentally dependent on the cytokine TGF-β1. In the present study, we identified BMP-7 as another key factor inducing LC differnetiation. Here we have performed comparative analysis of highly purified CD207+/CD1a+ in vitro generated Langerhans cells in the presence of BMP-7 and TGF-β1. We have identified that both BMP-7-LCs and TGF-β1-LCs are closely related to each other. CD34+ hematopoietic stem cells were isolated from freshly obtained cord blood and cultured in the presence of GM-CSF, SCF, Flt3L, TNFa and either TGF-β1 or BMP-7 for 7 days to get immature LCs. RNA was isolated from these in vitro genetared LCs.