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. 2014 Feb;73(2):446-54.
doi: 10.1136/annrheumdis-2012-202716. Epub 2013 Mar 20.

A synthetic PPAR-γ agonist triterpenoid ameliorates experimental fibrosis: PPAR-γ-independent suppression of fibrotic responses

Jun Wei  1 , Hongyan Zhu, Kazuhiro Komura, Gabriel Lord, Michal Tomcik, Wenxia Wang, Sruthi Doniparthi, Zenshiro Tamaki, Monique Hinchcliff, Joerg H W Distler, John Varga
Affiliations

A synthetic PPAR-γ agonist triterpenoid ameliorates experimental fibrosis: PPAR-γ-independent suppression of fibrotic responses

Jun Wei et al. Ann Rheum Dis. 2014 Feb.

Abstract

Background: Persistent fibroblast activation initiated by transforming growth factor β (TGF-β) is a fundamental event in the pathogenesis of systemic sclerosis, and its pharmacological inhibition represents a potential therapeutic strategy. The nuclear receptor, peroxisome proliferator-activated receptor γ (PPAR-γ), exerts potent fibrotic activity. The synthetic oleanane triterpenoid, 2-cyano-3,12-dioxo-olean-1,9-dien-28-oic acid (CDDO), is a PPAR-γ agonist with potential effects on TGF-β signalling and dermal fibrosis.

Objective: To examine the modulation of fibrogenesis by CDDO in explanted fibroblasts, skin organ cultures and murine models of scleroderma.

Material and methods: The effects of CDDO on experimental fibrosis induced by bleomycin injection or by overexpression of constitutively active type I TGF-β receptor (TgfbR1ca) were evaluated. Modulation of fibrotic gene expression was examined in human skin organ cultures. To delineate the mechanisms underlying the antifibrotic effects of CDDO, explanted skin fibroblasts cultured in two-dimensional monolayers or in three-dimensional full-thickness human skin equivalents were studied.

Results: CDDO significantly ameliorated dermal fibrosis in two complementary mouse models of scleroderma, as well as in human skin organ cultures and in three-dimensional human skin equivalents. In two-dimensional monolayer cultures of explanted normal fibroblasts, CDDO abrogated fibrogenic responses induced by TGF-β. These CDDO effects occurred via disruption of Smad-dependent transcription and were associated with inhibition of Akt activation. In scleroderma fibroblasts, CDDO attenuated the elevated synthesis of collagen. Remarkably, the in vitro antifibrotic effects of CDDO were independent of PPAR-γ.

Conclusions: The PPAR-γ agonist triterpenoid CDDO attenuates fibrogenesis by antagonistically targeting canonical TGF-β/Smad and Akt signalling in a PPAR-γ-independent manner. These findings identify this synthetic triterpenoid as a potential new therapy for the control of fibrosis.

Keywords: CDDO; PPAR-γ; TGF-β; fibroblast; fibrosis; murine scleroderma; triterpenoid.

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Conflict of interest statement

Competing interests None.

Figures

Figure 1
Figure 1. CDDO induces PPAR-γ activation and promotes adipogenesis
A, B. Confluent 3T3L1 preadipocytes were incubated with CDDO (5 μM or indicated concentration) or rosiglitazone (10 μM) for 7 days before harvesting. A. Cultures were immunostained with antibody to perilipin-1. Upper panels, phase contrast microscopy; lower panels, immunofluorescence. Representative images. Original magnification x 200. B. RNA was examined by real-time qPCR. The results, normalized with 36B4, represent the means ±SD of triplicate determinations from a representative experiment. C. Human skin fibroblasts transiently transfected with PPRE-luc were incubated in media with or without CDDO for 24 h. Cell lysates were assayed for their luciferase activities. The results, normalized with renilla luciferase, represent the means ± SD of three transfection assays in triplicate. * p<0.05.
Figure 2
Figure 2. CDDO ameliorates dermal fibrosis in mouse models of scleroderma
Mice were given s.c. injections of bleomycin daily for 14 days (A,B, F,G), or Ad-TβRIca or control adenovirus twice separated by two weeks (C–E) along with daily i.p. injections of CDDO from days 1–27 (A–E) or from days 15–27 (F,G). At the end of the experiments, lesional skin was harvested for analysis. A, F. Left panels, Trichrome (original magnification 200 x). Arrows delineate the dermis. Right panel, quantitation of dermal thickness. The results, representing the means ± SD of 5 determinations/hpf from 5 mice/group, are shown as -fold change compared to control (PBS-treated) mice. B, G. RNA was isolated and examined by real-time qPCR. The results, normalized with 36b4 or β-actin, represent the means ± SD of triplicate determinations from 3–5 mice/group. * p<0.05. C. Left panels, hematoxylin and eosin (original magnification 200 x). Right panel, quantitation of dermal thickness. The results, representing the means ± SEM of 6–8 mice/group, are shown as fold change compared to control (Ad-lacZ). D. Hydroxyproline assays. The results, representing the means ± SEM of triplicate determinations from 6–8 mice/group, are shown as -fold change compared to control (Ad-lacZ). E. Immunohistochemistry. α-SMA positive cells were quantified as described under Materials and Methods. Results are the means ± SEM. * p<0.05.
Figure 3
Figure 3. CDDO abrogates stimulation of collagen synthesis and myofibroblast differentiation
A–D. Confluent skin fibroblasts were incubated with CDDO (2.5 μM unless otherwise indicated) and TGF-β (10 ng/ml) following (A–C) or preceding (D) CDDO. A. Whole cell lysates were examined by Western analysis. Type I cgn, Type I collagen. Representative images. B, D. RNA was examined by real-time qPCR. The results, normalized with GAPDH, are the means ± SD of triplicate determinations from a representative experiment. * p<0.05. C. Immunofluorescence. Red, Type I collagen. Blue, DAPI. Representative images. Original magnification, 400 X. E, F. 3-dimensional human skin equivalents populated with skin fibroblasts were incubated with CDDO (5 μM) for 24 h, followed by TGF-β (5 ng/ml) for 5 days, and rafts were harvested. E. Total RNA was isolated and examined by real-time qPCR. The results, normalized with GAPDH, are the means ± SD of triplicate determinations from a representative experiment. * p<0.05. F. 4-μM thick paraffin-embedded sections were stained with Picrosirius Red. Mature collagen fibers in the dermal compartment appear red when visualized under polarized light, whereas less mature collagen fibers containing fewer cross-links appear yellow/green. Representative images (original magnification, x 40). Dashed lines, dermal-epidermal junction. G, H. Human skin organ cultures were incubated in media with TGF-β (10 ng/ml) for six days. CDDO (5 μM) was added to the cultures 15 min before or 48 h following addition of TGF-β. G. Four-μM thick paraffin-embedded sections were stained with Masson’s trichrome. Left panels, representative images (original magnification, x 400); right panel, relative -fold change in staining intensity. H. Skin tissues were harvested, and total RNA was isolated and examined by real-time qPCR. The results, normalized with GAPDH, are the means ± SD of triplicate determinations from a representative experiment. * p<0.05.
Figure 4
Figure 4. CDDO blocks Smad-dependent transcription and Akt activation
Confluent skin fibroblasts were transiently transfected with [SBE]4-luc (A), or left untransfected (B, C). Cultures were preincubated with CDDO (2.5 μM or indicated concentrations), followed by further incubation with TGF-β (10 ng/ml) for up to 24 h (A) or indicated time periods (B). A. Whole cell lysates were assayed for their luciferase activities. The results, normalized with renilla luciferase, represent the means ± SD of triplicate experiments. * p<0.05. B. Whole cell lysates (left panel) or cytosolic and nuclear fractions (right panel) were examined by Western analysis. Representative images. C. Whole cell lysates were examined by Western analysis. Bands were quantitated by densitometry. Relative phospho-Akt levels normalized with total Akt are shown below the images. Representative images.
Figure 5
Figure 5. CDDO normalizes collagen overproduction in scleroderma fibroblasts
Skin fibroblasts explanted from SSc patients (n=5) were incubated with CDDO (5 μM) for 24 h. A. Whole cell lysates and culture supernatants were analyzed by Western analysis. Bands were quantitated by densitometry. Relative levels normalized with GAPDH are shown below the images. Representative images. B. RNA was examined by real-time qPCR. Results are expressed as dot plots of mRNA levels relative to untreated cultures.
Figure 6
Figure 6. Inhibition of profibrotic responses is PPAR-γ independent
Confluent skin fibroblasts untransfected (A), or transiently transfected with [SBE]4-luc (B), were preincubated GW9662, followed by CDDO (2.5 μM) and TGF-β (10 ng/ml) for 24 h. A. RNA was examined by real-time qPCR. The results, normalized with GAPDH, represent the means ± SD of triplicate determinations from a representative experiment. * p<0.05. B. Luciferase activities, normalized with renilla luciferase in each sample, are means ± SD of triplicate experiments. * p<0.05.
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References

    1. Bhattacharyya S, Wei J, Varga J. Understanding fibrosis in systemic sclerosis: shifting paradigms, emerging opportunities. Nat Rev Rheumatol. 2012;8(1):42–54. - PMC - PubMed
    1. Rosenbloom J, Castro SV, Jimenez SA. Narrative review: fibrotic diseases: cellular and molecular mechanisms and novel therapies. Ann Intern Med. 2010;152(3):159–66. - PubMed
    1. Varga J, Pasche B. Transforming growth factor beta as a therapeutic target in systemic sclerosis. Nat Rev Rheumatol. 2009;5(4):200–6. - PMC - PubMed
    1. Sargent JL, Milano A, Bhattacharyya S, et al. A TGFbeta-responsive gene signature is associated with a subset of diffuse scleroderma with increased disease severity. J Invest Dermatol. 2010;130(3):694–705. - PMC - PubMed
    1. Milano A, Pendergrass SA, Sargent JL, et al. Molecular subsets in the gene expression signatures of scleroderma skin. PLoS One. 2008;3(7):e2696. - PMC - PubMed

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