Pan PPAR agonist IVA337 is effective in prevention and treatment of experimental skin fibrosis

Abstract

Background: The pathogenesis of systemic sclerosis (SSc) involves a distinctive triad of autoimmune, vascular and inflammatory alterations resulting in fibrosis. Evidence suggests that peroxisome proliferator-activated receptors (PPARs) play an important role in SSc-related fibrosis and their agonists may become effective therapeutic targets.

Objective: To determine the expression of PPARs in human fibrotic skin and investigate the effects of IVA337, a pan PPAR agonist, in in vitro and in vivo models of fibrosis.

Methods: The antifibrotic effects of IVA337 were studied using a bleomycin-induced mouse model of dermal fibrosis. The in vivo effect of IVA337 on wound closure and inflammation were studied using an excisional model of wound healing.

Results: Low levels of PPARα and PPARγ were detected in the skin of patients with SSc compared with controls. In mice, IVA337 was associated with decreased extracellular matrix (ECM) deposition and reduced expression of phosphorylated SMAD2/3-intracellular effector of transforming growth factor (TGF)-β1. Although the antifibrotic effect of pan PPAR was similar to that of PPARγ agonist alone, a significant downregulation of several markers of inflammation was associated with IVA337. Despite its anti-inflammatory and antifibrotic properties, IVA337 did not interfere with wound closure. In vitro effects of IVA337 included attenuation of transcription of ECM genes and alteration of canonical and non-canonical TGF-β signalling pathways.

Conclusions: These findings indicate that simultaneous activation of all three PPAR isoforms exerts a dampening effect on inflammation and fibrosis, making IVA337 a potentially effective therapeutic candidate in the treatment of fibrotic diseases including SSc.

Keywords: Fibroblasts; Inflammation; Systemic Sclerosis.

Conflict of interest statement

YA consulted and received research funding from Actelion, Bayer, Biogen Idec, BMS, Genentech/Roche, Inventiva, Medac, Pfizer, Sanofi/Genzyme, Servier and UCB. YA is a member of the advisory board for the upcoming clinical study of IVA337 in systemic sclerosis. J-ML, PB and J-LJ are employed by Inventiva.

Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Figures

Figure 1

Increased expression of peroxisome proliferator-activated receptor (PPAR)α and PPARγ in human systemic sclerosis (SSc) skin. (A) Representative images of formalin-fixed and paraffin-embedded samples of human healthy control, limited SSc (lSSc) and diffused (dSSc) skin stained with anti-PPARα (red), anti-CD90 (green) and α-smooth muscle actin (SMA) (green) antibodies. (B) Human healthy control, lSSc and dSSc skin stained with anti-PPARγ (red), anti-CD90 (green) and α-SMA (green) antibodies. In all images 4′,6-diamidino-2-phenylindole (DAPI) (blue) was used for nuclear counterstaining. Images were captured using 100× objective lens. Scale bar is 20 µm. In normal skin, PPARα and PPARγ are expressed by CD90+ dermal fibroblasts. In SSc skin, PPARα and PPARγ are expressed by endothelial and CD90+ perivascular cells. In SSc skin, PPARα and PPARγ appear to be expressed by endothelial cells and α-SMA+ myofibroblasts, but not by α-SMA+ cells localised around blood vessels and postcapillary venules of the superficial plexus. (C) Graphical representation of the expression of PPARα+ and PPARγ+ cells in dermis of normal, lSSc and dSSc skin. All values represent mean±SEM; n=5 (lSSc), n=6 (dSSc) and n=6 (normal control). *p<0.05; **p<0.01.

Figure 2

IVA337 attenuates dermal thickness, collagen content and myofibroblast accumulation in preventative model of fibrosis. (A) Representative images of H&E-stained sections of mouse skin treated with subcutaneous NaCl or bleomycin injections. Scale bar is 100 μm. (B) Graphical representation of dermal thickness of mouse skin harvested after 3 weeks of NaCl or bleomycin treatment. (N) denotes NaCl; (B) denotes bleomycin; (30 mg) denotes IVA337 at 30 mg/kg; (100 mg) denotes IVA337 at 100 mg/kg; (rosi) denotes rosiglitazone at 5 mg/kg. Four high-power field images were captured and two measurements per image were made. Results represent the relative fold change compared with NaCl-treated control mice. (C) Representative images of Masson's trichrome-stained sections of mouse skin harvested after 3 weeks of treatment with NaCl or bleomycin. Scale bar is 100 μm. (D) Graphical representation of hydroxyproline assay. Results are represented as mean±SEM of triplicate measurements obtained from n≥6 mice (two biopsies per mouse) and shown as relative fold change compared with NaCl-treated control samples. (E) Representative images of α-smooth muscle actin (SMA) immunohistochemistry. Negative controls included replacing primary antibodies with normal species-specific IgG. Scale bar is 50 μm. (F) Graphical representation of relative number of α-SMA-positive cells in dermis of NaCl or bleomycin-treated mice. Results represent the relative fold change compared with NaCl-treated control mice. (G) Body weight changes of mice treated with NaCl or bleomycin for 3 weeks. All values represent mean±SEM; n=6 each group. *p

Figure 3

Infiltration of immune effector cells into mouse skin treated with bleomycin and IVA337. (A) Representative immunohistochemistry images of mouse skin incubated with anti-CD107/b (macrophage marker), anti-F4/80 (macrophage marker) and anti-CD45 (leucocyte marker) antibodies. Black arrows indicate immune-positive cells. (B + vehicle) denotes bleomycin/vehicle; (B + IVA337) denotes bleomycin/IVA337 at 30 mg/kg; (B + rosi) denotes bleomycin/rosiglitazone at 5 mg/kg. Scale bar is 20 µm. (B) Representative immunohistochemistry images of mouse skin staining for CD8 and CD3 (lymphocytes). (C) Graphical representation of number of CD107/b, F4/40 and CD45+ cells and (D) CD8+ and CD3+ cells in mouse skin treated with either B + vehicle, B + IVA337 at 30 mg/kg or B + rosi at 5 mg/kg for 21 days. All values represent mean±SEM; n=6 each group. *p

Figure 4

Microscopic and histological analysis of wounds treated with vehicle, IVA337 (30 and 100 mg/kg) and rosiglitazone (5 mg/kg). (A) Representative images of H&E-stained sections of wounds at 7 and 21 days after wounding. Arrows indicate the wound margins. Scale bar is 100 μm. (B) Representative images of CD107b-stained (macrophage), F4/80-stained (macrophage) and Ly6G-stained (neutrophil) sections of mouse wounds treated with vehicle, IVA337 (30 and 100 mg/kg) and rosiglitazone (5 mg/kg) at 7 days after surgery. Scale bar is 20 µm. (C) Graphical representation of rate of wound re-epithelialisation at 7 and 14 days after wounding in mice treated with vehicle, IVA337 (30 and 100 mg/kg) and rosiglitazone at 5 mg/kg for 7 and 14 days. (D) Graphical representation of number of CD107b (macrophage), F4/80 (macrophage) and Ly6G (neutrophil) immuno-positive cells in mouse wounds treated with vehicle, IVA337 (30 and 100 mg/kg) and rosiglitazone (5 mg/kg) at 7 days after surgery. Scale bar is 20 µm. All values represent mean±SEM; n=6 each group. *p

Figure 5

Effect of IVA337 on formation of stress fibres and transforming growth factor (TGF)-β signalling in primary human fibroblasts. (A) Systemic sclerosis (SSc) fibroblasts were treated with TGF-β1 (10 ng/mL) and IVA337 (10 µM) for 24 h, after which they were fixed with 4% paraformaldehyde, immunostained for phalloidin (red) and α-SMA (green). Scale bar is 25 μm. (B–D) RNA was isolated from scratch wounded confluent monolayers of SSc fibroblasts. Real-time quantitative (RTq) PCR analysis of (B) Col1A1, (C) Col1A2 and (D) α-SMA in response to treatment with IVA337 (10 µM) for 24 h. Differences were calculated using the Ct and comparative Ct methods for relative quantification. Results were expressed in arbitrary units, where Col1A1, Col1A2 and α-SMA genes were normalised with respect to HPRT1gene. All values represent mean±SEM; n=10 each group. (E) Representative western blots show the effect of IVA337 (10 µM) and the influence of TGF-β1 inhibitor SB431542 (10 µM) (as a positive control) on TGF-β-induced expression of collagen 1 and α-SMA proteins. (F and G) Bar graph based on western blot band densitometry showing the decrease in collagen and α-SMA protein expression after incubation with IVA337 (10 µM) for 24 h (n=6). *p

Figure 6

Effect of IVA337 on transforming growth factor (TGF)-β1-induced phosphorylation of downstream signalling molecules. (A) Primary systemic sclerosis (SSc) fibroblasts were isolated from fibrotic lesions of four individuals (n=4). Cells were used at passages 2–4 and incubated in the presence or absence of IVA337 (10 µM) for 24 h, fixed with methanol and immunostained for phosphorylated SMAD (pSMAD) 2/3 (red). Nucleus was counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (green). TGF-β1 (10 ng/mL) caused an increase in pSMAD2/3 (red) immunofluorescence signal, which was found to be present in the nucleus and cytoplasm. Reduction in pSMAD2/3 signal was evident upon incubation with IVA337. Scale bar is 10 µm. (B) Representative images of mouse skin treated with bleo + vehicle or bleo + IVA337 and stained for pSMAD2/3. (C) Graphical representation of pSMAD2/3 immunohistochemistry of mouse skin treated with bleo + IVA337 or bleo + vehicle for 42 days. All values represent mean±SEM; n≥6 each group. **p