Transcriptomics

Dataset Information

2

Reversible and irreversible differentiation of cardiac fibroblasts


ABSTRACT: 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).

ORGANISM(S): Rattus norvegicus  

SUBMITTER: Joke Allemeersch   Paul J Lijnen  Victor V Petrov  Joelle Abi-Char  Chandan K Nagaraju  Robert H Fagard  Ronald B Driesen  Tamara Coenen  Karin R Sipido 

PROVIDER: E-GEOD-51824 | ArrayExpress | 2014-01-07

SECONDARY ACCESSION(S): GSE51824PRJNA225508

REPOSITORIES: GEO, ArrayExpress

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