Reversible and irreversible differentiation of cardiac fibroblasts.

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-ß1 (TGF-ß1) promoted differentiation into α-SMA-positive MyoFb showing near the absence of proliferation, i.e. non-p-MyoFb. SD-208, a TGF-ß-receptor-I (TGF-ß-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-ß-RI kinase inhibition can reverse p-MyoFb differentiation but not non-p-MyoFb.

Oxidative stress and transforming growth factor-ß1-induced cardiac fibrosis.

A chronic increase in reactive oxygen species (ROS) plays a critical role in the development and progression of cardiac remodeling associated with heart failure. Oxidative stress is indeed increased in heart failure, hypertension, cardiac fibrosis and hypertrophy. In vitro exposure of cardiac fibroblasts to superoxide anion stimulates their proliferation by increasing the production of transforming growth factor-ß1 (TGF-ß1), a potent fibrogenic cytokine. TGF-ß1 plays an important role in cardiac development, cardiac hypertrophy, ventricular remodeling and the early response to myocardial infarction. In this review the role of TGF-ß1 and ROS in the production and deposition of collagens by cardiac fibroblasts and in the induction of gene expression in relation to the development of myocardial fibrosis and to myocardial tissue repair will be discussed.

Angiotensin II-induced mitochondrial reactive oxygen species and peroxiredoxin-3 expression in cardiac fibroblasts.

OBJECTIVE: The aim of this study was to determine whether angiotensin II (ANG II) affects the protein and mRNA expression of the mitochondrial antioxidant peroxiredoxin-3 (Prx-3) in cardiac fibroblasts, thereby contributing to the oxidative stress in the myocardium. METHOD: Cardiac fibroblasts (passage 2) from normal male adult rats were cultured to confluency and incubated in Dulbecco's modified Eagle's medium for 24  h. The cells were then preincubated with(out) the tested inhibitors for 1  h and further incubated with/without ANG II (1 µmol/l) for 24  h. RESULTS: ANG II increased (P < 0.001) the mitochondrial production of reactive oxygen species in cardiac fibroblasts from 187.8 ±â€Š38.6 to 313.8 ±â€Š30.6 a.u./mg mitochondrial protein (n = 15). ANG II decreased (P < 0.01) the mRNA and protein expression of Prx-3 by 36.9 ±â€Š3.0% and 29.7 ±â€Š2.7% (n = 4), respectively. The ANG II-induced decrease in mRNA expression of Prx-3 was prevented by the angiotensin type 1 receptor blocker, losartan but not by the angiotensin type 2 receptor blocker, PD 123 319. CONCLUSION: Our data indicate that ANG II-stimulated mitochondrial reactive oxygen species production in rat cardiac fibroblasts is accompanied by a reduction in the expression of the mitochondrial antioxidant Prx-3, and thereby potentially contributing to oxidative stress in the myocard.