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TGF-beta dependent regulation of oxygen radicals during transdifferentiation of activated hepatic stellate cells to myofibroblastoid cells.

Proell V, Carmona-Cuenca I, Murillo MM, Huber H, Fabregat I, Mikulits W - Comp Hepatol (2007)

Bottom Line: We found reactive oxygen species (ROS) significantly upregulated in M1-4HSCs within 72 hours of TGF-beta administration.The data provide evidence that TGF-beta induces NADPH oxidase activity which causes radical production upon the transdifferentiation of activated HSCs to M-HTs.Myofibroblastoid cells are equipped with high levels of superoxide dismutase activity as well as glutathione to counterbalance NADPH oxidase dependent oxidative stress and to avoid cellular damage.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine I, Division Institute of Cancer Research, Medical University of Vienna, Borschke-Gasse 8a, A-1090 Vienna, Austria. verena.proell@meduniwien.ac.at

ABSTRACT

Background: The activation of hepatic stellate cells (HSCs) plays a pivotal role during liver injury because the resulting myofibroblasts (MFBs) are mainly responsible for connective tissue re-assembly. MFBs represent therefore cellular targets for anti-fibrotic therapy. In this study, we employed activated HSCs, termed M1-4HSCs, whose transdifferentiation to myofibroblastoid cells (named M-HTs) depends on transforming growth factor (TGF)-beta. We analyzed the oxidative stress induced by TGF-beta and examined cellular defense mechanisms upon transdifferentiation of HSCs to M-HTs.

Results: We found reactive oxygen species (ROS) significantly upregulated in M1-4HSCs within 72 hours of TGF-beta administration. In contrast, M-HTs harbored lower intracellular ROS content than M1-4HSCs, despite of elevated NADPH oxidase activity. These observations indicated an upregulation of cellular defense mechanisms in order to protect cells from harmful consequences caused by oxidative stress. In line with this hypothesis, superoxide dismutase activation provided the resistance to augmented radical production in M-HTs, and glutathione rather than catalase was responsible for intracellular hydrogen peroxide removal. Finally, the TGF-beta/NADPH oxidase mediated ROS production correlated with the upregulation of AP-1 as well as platelet-derived growth factor receptor subunits, which points to important contributions in establishing antioxidant defense.

Conclusion: The data provide evidence that TGF-beta induces NADPH oxidase activity which causes radical production upon the transdifferentiation of activated HSCs to M-HTs. Myofibroblastoid cells are equipped with high levels of superoxide dismutase activity as well as glutathione to counterbalance NADPH oxidase dependent oxidative stress and to avoid cellular damage.

No MeSH data available.


Related in: MedlinePlus

Cellular model of hepatic fibrosis. (A) Morphological changes of M1-4HSCs treated with TGF-β1 either for 72 hours or for long-term (myofibroblastoid M-HT) as analyzed by phase contrast microscopy. (B) Nuclear translocation of Smad2/3 as visualized by confocal immunofluorescence analysis. (C) Confocal immunofluorescence images after staining of cells with anti-desmin antibody.
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Figure 1: Cellular model of hepatic fibrosis. (A) Morphological changes of M1-4HSCs treated with TGF-β1 either for 72 hours or for long-term (myofibroblastoid M-HT) as analyzed by phase contrast microscopy. (B) Nuclear translocation of Smad2/3 as visualized by confocal immunofluorescence analysis. (C) Confocal immunofluorescence images after staining of cells with anti-desmin antibody.

Mentions: The cell line M1-4HSC represents activated HSCs, which undergo further activation to myofibroblast-like cells in response to TGF-β [25]. The induction phase refers to 72 hours of TGF-β treatment, which is characterized by the change to a myofibroblastoid morphology (Fig. 1A). After 20 days of TGF-β administration, the cells represent activated MFBs with a stable phenotype, termed M-HTs. Transdifferentiation of M1-4HSCs to M-HTs shows increased nuclear accumulation of Smad2/3 (Fig. 1B), indicating a further activation of TGF-β signaling. In addition, M-HTs exhibit decreased expression of desmin (Fig. 1C), as reported recently [25]. This cellular model provides the unique ability to monitor late stage events during fibrogenesis, since spontaneous activation has already occurred. In order to examine whether ROS are implicated in this transdifferentiation from activated HSCs to MFBs, we analyzed intracellular hydrogen peroxide during the induction phase compared to untreated M1-4HSCs and M-HTs. Hydrogen peroxide was used as a general marker of oxidative stress since all forms of oxygen radicals that occur intracellularly are finally converted into H2O2. We observed a significant increase in ROS levels after 48 and 72 hours of TGF-β treatment (Fig. 2A), whereas no elevation of hydrogen peroxide levels was determined after 24 hours. Since basal levels of ROS are already induced in M1-4HSCs compared to quiescent HSCs, as reported by several investigators [20,28-30], TGF-β is obviously able to provide accumulation of hydrogen peroxide in M1-4HSCs. In contrast, M-HTs showed about 40% reduced intracellular hydrogen peroxide content compared to untreated M1-4HSCs (Fig. 2B). Hence, these data raised the question whether the lowered ROS levels in M-HTs were caused by reduced ROS production or by the upregulation of cellular antioxidant defense mechanisms. To properly tackle this issue we asked for the major source of ROS in M1-4HSCs caused by TGF-β administration.


TGF-beta dependent regulation of oxygen radicals during transdifferentiation of activated hepatic stellate cells to myofibroblastoid cells.

Proell V, Carmona-Cuenca I, Murillo MM, Huber H, Fabregat I, Mikulits W - Comp Hepatol (2007)

Cellular model of hepatic fibrosis. (A) Morphological changes of M1-4HSCs treated with TGF-β1 either for 72 hours or for long-term (myofibroblastoid M-HT) as analyzed by phase contrast microscopy. (B) Nuclear translocation of Smad2/3 as visualized by confocal immunofluorescence analysis. (C) Confocal immunofluorescence images after staining of cells with anti-desmin antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1804283&req=5

Figure 1: Cellular model of hepatic fibrosis. (A) Morphological changes of M1-4HSCs treated with TGF-β1 either for 72 hours or for long-term (myofibroblastoid M-HT) as analyzed by phase contrast microscopy. (B) Nuclear translocation of Smad2/3 as visualized by confocal immunofluorescence analysis. (C) Confocal immunofluorescence images after staining of cells with anti-desmin antibody.
Mentions: The cell line M1-4HSC represents activated HSCs, which undergo further activation to myofibroblast-like cells in response to TGF-β [25]. The induction phase refers to 72 hours of TGF-β treatment, which is characterized by the change to a myofibroblastoid morphology (Fig. 1A). After 20 days of TGF-β administration, the cells represent activated MFBs with a stable phenotype, termed M-HTs. Transdifferentiation of M1-4HSCs to M-HTs shows increased nuclear accumulation of Smad2/3 (Fig. 1B), indicating a further activation of TGF-β signaling. In addition, M-HTs exhibit decreased expression of desmin (Fig. 1C), as reported recently [25]. This cellular model provides the unique ability to monitor late stage events during fibrogenesis, since spontaneous activation has already occurred. In order to examine whether ROS are implicated in this transdifferentiation from activated HSCs to MFBs, we analyzed intracellular hydrogen peroxide during the induction phase compared to untreated M1-4HSCs and M-HTs. Hydrogen peroxide was used as a general marker of oxidative stress since all forms of oxygen radicals that occur intracellularly are finally converted into H2O2. We observed a significant increase in ROS levels after 48 and 72 hours of TGF-β treatment (Fig. 2A), whereas no elevation of hydrogen peroxide levels was determined after 24 hours. Since basal levels of ROS are already induced in M1-4HSCs compared to quiescent HSCs, as reported by several investigators [20,28-30], TGF-β is obviously able to provide accumulation of hydrogen peroxide in M1-4HSCs. In contrast, M-HTs showed about 40% reduced intracellular hydrogen peroxide content compared to untreated M1-4HSCs (Fig. 2B). Hence, these data raised the question whether the lowered ROS levels in M-HTs were caused by reduced ROS production or by the upregulation of cellular antioxidant defense mechanisms. To properly tackle this issue we asked for the major source of ROS in M1-4HSCs caused by TGF-β administration.

Bottom Line: We found reactive oxygen species (ROS) significantly upregulated in M1-4HSCs within 72 hours of TGF-beta administration.The data provide evidence that TGF-beta induces NADPH oxidase activity which causes radical production upon the transdifferentiation of activated HSCs to M-HTs.Myofibroblastoid cells are equipped with high levels of superoxide dismutase activity as well as glutathione to counterbalance NADPH oxidase dependent oxidative stress and to avoid cellular damage.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine I, Division Institute of Cancer Research, Medical University of Vienna, Borschke-Gasse 8a, A-1090 Vienna, Austria. verena.proell@meduniwien.ac.at

ABSTRACT

Background: The activation of hepatic stellate cells (HSCs) plays a pivotal role during liver injury because the resulting myofibroblasts (MFBs) are mainly responsible for connective tissue re-assembly. MFBs represent therefore cellular targets for anti-fibrotic therapy. In this study, we employed activated HSCs, termed M1-4HSCs, whose transdifferentiation to myofibroblastoid cells (named M-HTs) depends on transforming growth factor (TGF)-beta. We analyzed the oxidative stress induced by TGF-beta and examined cellular defense mechanisms upon transdifferentiation of HSCs to M-HTs.

Results: We found reactive oxygen species (ROS) significantly upregulated in M1-4HSCs within 72 hours of TGF-beta administration. In contrast, M-HTs harbored lower intracellular ROS content than M1-4HSCs, despite of elevated NADPH oxidase activity. These observations indicated an upregulation of cellular defense mechanisms in order to protect cells from harmful consequences caused by oxidative stress. In line with this hypothesis, superoxide dismutase activation provided the resistance to augmented radical production in M-HTs, and glutathione rather than catalase was responsible for intracellular hydrogen peroxide removal. Finally, the TGF-beta/NADPH oxidase mediated ROS production correlated with the upregulation of AP-1 as well as platelet-derived growth factor receptor subunits, which points to important contributions in establishing antioxidant defense.

Conclusion: The data provide evidence that TGF-beta induces NADPH oxidase activity which causes radical production upon the transdifferentiation of activated HSCs to M-HTs. Myofibroblastoid cells are equipped with high levels of superoxide dismutase activity as well as glutathione to counterbalance NADPH oxidase dependent oxidative stress and to avoid cellular damage.

No MeSH data available.


Related in: MedlinePlus