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. 2014 Mar 1;101(3):411-22.
doi: 10.1093/cvr/cvt338. Epub 2013 Dec 23.

Reversible and Irreversible Differentiation of Cardiac Fibroblasts

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Free PMC article

Reversible and Irreversible Differentiation of Cardiac Fibroblasts

Ronald B Driesen et al. Cardiovasc Res. .
Free PMC article

Abstract

Aims: Differentiation of cardiac fibroblasts (Fbs) into myofibroblasts (MyoFbs) is responsible for connective tissue build-up in myocardial remodelling. We examined MyoFb differentiation and reversibility.

Methods and results: Adult rat cardiac Fbs were cultured on a plastic substratum providing mechanical stress, with conditions to obtain different levels of Fb differentiation. Fb spontaneously differentiated to proliferating MyoFb (p-MyoFb) with stress fibre formation decorated with alpha-smooth muscle actin (α-SMA). Transforming growth factor-β1 (TGF-β1) promoted differentiation into α-SMA-positive MyoFb showing near the absence of proliferation, i.e. non-p-MyoFb. SD-208, a TGF-β-receptor-I (TGF-β-RI) kinase blocker, inhibited p-MyoFb differentiation as shown by stress fibre absence, low α-SMA expression, and high proliferation levels. Fb seeded in collagen matrices induced no contraction, whereas p-MyoFb and non-p-MyoFb induced 2.5- and four-fold contraction. Fb produced little collagen but high levels of interleukin-10. Non-p-MyoFb had high collagen production and high monocyte chemoattractant protein-1 and tissue inhibitor of metalloproteinases-1 levels. Transcriptome analysis indicated differential activation of gene networks related to differentiation of MyoFb (e.g. paxilin and PAK) and reduced proliferation of non-p-MyoFb (e.g. cyclins and cell cycle regulation). Dedifferentiation of p-MyoFb with stress fibre de-polymerization, but not of non-p-MyoFb, was induced by SD-208 despite maintained stress. Stress fibre de-polymerization could also be induced by mechanical strain release in p-MyoFb and non-p-MyoFb (2-day cultures in unrestrained 3-D collagen matrices). Only p-MyoFb showed true dedifferentiation after long-term 3-D cultures.

Conclusions: Fb, p-MyoFb, and non-p-MyoFb have a distinct gene expression, ultrastructural, and functional profile. Both reduction in mechanical strain and TGF-β-RI kinase inhibition can reverse p-MyoFb differentiation but not non-p-MyoFb.

Keywords: Cardiac fibroblast; Dedifferentiation; Myofibroblast.

Figures

Figure 1
Figure 1
Differentiation of Fb cells in 2-D cultures and MRTF-A/B expression. (AD) Different phenotypes, cells are stained for F-actin (a), α-SMA (b), and vinculin (c); scale bars represent 40 µm. (A) Spontaneously differentiation to p-MyoFb during 4 days in standard culture medium. (B) Treatment with SD-208 (3 µmol/L), an inhibitor for TGF-β-RI kinase, for 4 days maintains the Fb phenotype. (C) Treatment with TGF-β1 (400 pmol/L) for 6 days induces non-p-MyoFb. (D) Fb cultures are treated simultaneous with SD-208 (3 µmol/L) and TGF-β1 (400 pmol/L) for 4 days. (E) Proliferative capacity of cells after 12 days in culture. (F) Quantification of immunostained α-SMA-positive cells in different Fb cultures. Western blotting (G) of α-SMA in p-MyoFb, SD-208-treated Fb, and Y-27632-treated Fb (4-day-old cultures) and in TGF-β1-treated MyoFb (6-day-old cultures). (H) mRNA expression of MRTF-A/B in different Fb phenotypes. *P < 0.05 vs. p-MyoFb (control) or non-p-MyoFb (N = 5).
Figure 2
Figure 2
Structural adaptations of Fb cells in unrestrained 3-DCMs. (A) Fb phenotypes; Rhodamin-Phalloidin (red) marks stress fibres; nuclei stained with DAPI (blue); scale bars represent 10 µm. (a) Round phenotype, (b) dendritic phenotype without stress fibres, (c) dendritic phenotype with stress fibres, and (d)elongated phenotype with stress fibres. (B) Quantification of the cell fractions with specific phenotypes in the absence of serum. Fbs in 3-D cultures without mechanical strain and serum acquire a dendritic phenotype without stress fibres. p-MyoFb and non-p-MyoFb acquire an elongated phenotype with stress fibres. (C) Contraction of unrestrained 3-DCM by Fb cells. Volume of unrestrained 3-DCM after 3-day cultures. 3-DCMs were populated with: p-MyoFb; Fb pre-treated in 2-D cultures with SD-208 (3 µmol/L); non-p-MyoFb pre-treated with TGF-β1 (400 pmol/L); Fb pre-treated with SD-208 (3 µmol) and TGF-β1(400 pmol/L); Fb pre-treated with Y-27632 (10 µmol/L). No cells represent control 3-DCM without cells. *P < 0.05 vs. 3-DCM without cells (N = 3). Scale bars represent 5 (Aa) and 20 (Ab, c, and d) µm.
Figure 3
Figure 3
Collagen production and cytokine secretion in Fb cells. (A) Intracellular collagen production in Fb, p-MyoFb, and non-p-MyoFb (N = 5). (B) Release of IL-10 (a) MCP-1 (b), TIMP-1, and (c) in conditioned media of different Fb phenotypes (N = 6). *P < 0.05 vs. p-MyoFb or non-p-MyoFb.
Figure 4
Figure 4
Differential gene expression in different Fb phenotypes. (A) Heat-maps showing distribution of the top 100 of differentially expressed genes (P < 0.05) between Fb, p-MyoFb, and non-p-MyoFb. (B) List of differentially regulated canonical pathways derived from the comparison between Fb, p-MyoFb, and non-p-MyoFb (N = 4). Bar graphs indicate the percentage of down-regulated (green) and up-regulated genes (red). The number of differentially expressed genes for each pathway is shown adjacent to the bar graph. Values higher than –log (P-value) of >1.3 indicate significant changed pathways (orange).
Figure 5
Figure 5
Effect of SD-208 on dedifferentiation of p-MyoFb and non-p-MyoFb in 2-D cultures. Treatments of p-MyoFb (Aa–d) and non-p-MyoFb (Ba–d) for 4 days with SD-208 (3 µmol/L). Cells are stained for stress fibres (Aa and Ba) and α-SMA (Ab and Bb); scale bars represent 20 µm. Insert in Ab shows α-SMA cytosolic aggregates. (Ac and Bc) Quantification of cells with α-SMA-positive stress fibres. Western blotting of α-SMA in p-MyoFb (Ad) and non-p-MyoFb (Bd) treated with SD-208 during 4 days. *P < 0.05 vs. p-MyoFb or non-p-MyoFb (N = 5).
Figure 6
Figure 6
Dedifferentiation of p-MyoFb and non-p-MyoFb during cultures in 3-DCM. (A) Two-day cultures in unrestrained 3-DCM without serum; p-MyoFb (Aa–c) and non-p-MyoFb (Ad–f) double-labelled with Rhodamin-Phalloidin (red, Aa–f) and α-SMA antibodies (green, Ac and Af). Stress fibre de-polymerization in p-MyoFb into aggregates is marked in Aa and Ab by white arrows; α-SMA aggregates are observed in spread MyoFb (Ac; insert). (d–f) Non-p-MyoFbs also show stress fibre de-polymerization; magnification (Ae) of the stress fibre area (Ad; white rectangle) shows actin aggregates between stress fibres; Af shows α-SMA aggregates. (Ag) Quantification of p-MyoFb and non-p-MyoFb fractions with actin aggregates, mean ± SEM of three independent cultures. (B) Two-week cultures in restrained 3-DCM, p-MyoFb (Ba) and non-p-MyoFb (Bb) labelled for α-SMA (brown). Note the absence of α-SMA staining in p-MyoFb indicating dedifferentiation (edges are α-SMA positive, arrowheads). (Bc) Quantification of α-SMA-positive cells in restrained 3-DCM compared with 2-D cultures, mean ± SEM of five independent cultures. Scale bars represent 5 µm (Ac and Ae), 10 µm (Aa, Af); 20 µm (Ab, Ac insert, Ad), 100 µm (Ba), and 125 µm (Bb) .

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