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Reversible and irreversible differentiation of cardiac fibroblasts.

Driesen RB, Nagaraju CK, Abi-Char J, Coenen T, Lijnen PJ, Fagard RH, Sipido KR, Petrov VV - Cardiovasc. Res. (2013)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Diseases, Division of Experimental Cardiology, University of Leuven, KU Leuven, Campus Gasthuisberg O/N1 Box 704, Herestraat 49, Leuven B-3000, Belgium.

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.

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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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CVT338F6: 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) .

Mentions: We examined whether loss of mechanical stress induced dedifferentiation of p-MyoFb and non-p-MyoFb by studying stress fibre and α-SMA integrity in unrestrained 3-DCMs, cultured without serum for 2 days. After 2 days of culture, a fraction of spread p-MyoFb (41%) shows G-actin aggregates (Figure 6A,a and g). This de-polymerization of stress fibres is associated with a release of bound α-SMA and its aggregation in the cytoplasm alongside G-actin (Figure 6A,c). Co-localization of G-actin aggregates and stress fibres is often observed suggesting stress fibre de-polymerization, which in some cells is very pronounced (Figure 6A,c). In a number of cells, stress fibres are completely de-polymerized showing only G-actin aggregates (Figure 6A,b). These observations point to the activation of a dedifferentiation process in p-MyoFb.Figure 6


Reversible and irreversible differentiation of cardiac fibroblasts.

Driesen RB, Nagaraju CK, Abi-Char J, Coenen T, Lijnen PJ, Fagard RH, Sipido KR, Petrov VV - Cardiovasc. Res. (2013)

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) .
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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CVT338F6: 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) .
Mentions: We examined whether loss of mechanical stress induced dedifferentiation of p-MyoFb and non-p-MyoFb by studying stress fibre and α-SMA integrity in unrestrained 3-DCMs, cultured without serum for 2 days. After 2 days of culture, a fraction of spread p-MyoFb (41%) shows G-actin aggregates (Figure 6A,a and g). This de-polymerization of stress fibres is associated with a release of bound α-SMA and its aggregation in the cytoplasm alongside G-actin (Figure 6A,c). Co-localization of G-actin aggregates and stress fibres is often observed suggesting stress fibre de-polymerization, which in some cells is very pronounced (Figure 6A,c). In a number of cells, stress fibres are completely de-polymerized showing only G-actin aggregates (Figure 6A,b). These observations point to the activation of a dedifferentiation process in p-MyoFb.Figure 6

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiovascular Diseases, Division of Experimental Cardiology, University of Leuven, KU Leuven, Campus Gasthuisberg O/N1 Box 704, Herestraat 49, Leuven B-3000, Belgium.

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.

Show MeSH
Related in: MedlinePlus