PPAR-gamma agonists inhibit profibrotic phenotypes in human lung fibroblasts and bleomycin-induced pulmonary fibrosis

Abstract

Pulmonary fibrosis is characterized by alterations in fibroblast phenotypes resulting in excessive extracellular matrix accumulation and anatomic remodeling. Current therapies for this condition are largely ineffective. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear hormone receptor superfamily, the activation of which produces a number of biological effects, including alterations in metabolic and inflammatory responses. The role of PPAR-gamma as a potential therapeutic target for fibrotic lung diseases remains undefined. In the present study, we show expression of PPAR-gamma in fibroblasts obtained from normal human lungs and lungs of patients with idiopathic interstitial pneumonias. Treatment of lung fibroblasts and myofibroblasts with PPAR-gamma agonists results in inhibition of proliferative responses and induces cell cycle arrest. In addition, PPAR-gamma agonists, including a constitutively active PPAR-gamma construct (VP16-PPAR-gamma), inhibit the ability of transforming growth factor-beta1 to induce myofibroblast differentiation and collagen secretion. PPAR-gamma agonists also inhibit fibrosis in a murine model, even when administration is delayed until after the initial inflammation has largely resolved. These observations indicate that PPAR-gamma is an important regulator of fibroblast/myofibroblast activation and suggest a role for PPAR-gamma ligands as novel therapeutic agents for fibrotic lung diseases.

Fig. 1

Human lung fibroblasts express peroxisome proliferator-activated receptor-γ (PPAR-γ). A: Western immunoblot for PPAR-γ on cells from patients with idiopathic interstitial pneumonia [usual interstitial pneumonia (UIP), respiratory bronchiolitis-associated interstitial lung disease (RBILD), and nonspecific interstitial pneumonia (NSIP)] and normal human fibroblasts. Each lane represents a different patient sample. Equal loading was confirmed by stripping and probing with antibody against β-actin. Blot is representative of a larger group of patient samples (n = 16). B: densitometric analysis of Western immunoblots in A (n = 4/group). C: immunofluorescence staining for PPAR-γ in IMR-90 fibroblasts.

Fig. 2

Activation of PPAR-γ in human lung fibroblasts. IMR-90 cells were simultaneously transfected with a PPAR-dependent luciferase reporter (pFATP-luc) and pRL-SV40 and then stimulated with vehicle (DMSO) or 1–20 µM troglitazone (Tro) or ciglitazone (Cig) for 24 h. Relative luciferase activity was calculated by normalization of firefly luciferase activity to Renilla luciferase activity. Data are averages from 3 independent experiments. *P < 0.05 vs. DMSO.

Fig. 3

PPAR-γ ligands inhibit proliferative responses of human lung fibroblasts. A: IMR-90 cells were plated in 5% FBS and then treated with 0 µM (■), 1 µM (●), 5 µM (▲), 10 µM (▼), or 20 µM (◆) Tro or Cig in DMSO. At 24-h intervals, cells were trypsinized and counted using a Coulter counter. Data are averages from 3 independent experiments. B: cultured fibroblasts from normal lungs or patients with different forms of idiopathic interstitial pneumonia (NSIP, RBILD, and UIP) were plated in 5% FBS. At 24-h intervals, cells were trypsinized and counted using a Coulter counter. Data from normal and IIP fibroblasts are from 4 individual patients in 2 separate experiments with Tro treatment. *P < 0.05; **P < 0.01 vs. 0 µM at 72 h.

Fig. 4

PPAR-γ ligands inhibit proliferative responses of human lung fibroblasts to mitogenic growth factors. A: IMR-90 cells were treated with Tro or Cig in the presence of 5% FBS followed by stimulation with the mitogen PDGF (5 ng/ml) for 24, 48, and 72 h. In both panels, concentration of drugs in DMSO increases from top line to bottom line: 0 µM ( ), 1 µM ( ), 5 µM ( ), 10 µM ( ), and 20 γM ( ). At 24-h intervals, cells were trypsinized and counted using a Coulter counter. Data are averages from 3 independent experiments. *P < 0.05; **P < 0.01 vs. 0 µM at 72 h. B: IMR-90 cells were seeded on dishes and allowed to grow to 80% confluence in medium containing 10% FBS (Ctrl); then their growth was arrested by change to serum-free medium for 48 h. Cells were treated with transforming growth factor (TGF)-β1 (2 ng/ml) for 24 h (SS) before treatment with 1–20 µM Tro or vehicle (DMSO) and then stimulated with fibroblast growth factor (FGF, 10 ng/ml) for 48 h. Cells were trypsinized and cell counts were assessed as previously described. Data are averages from 3 independent experiments. **P < 0.01 vs. 0 µM + TGF-β1 + FGF.

Fig. 5

PPAR-γ ligands result in cell cycle arrest and inhibition of cyclin D expression in human lung fibroblasts. A: number of propidium iodide-stained IMR-90 cells in G₀/G₁, S, and G₂/M phases at 72 h determined by flow cytometry. Proportion of cells in G₀/G₁, S, and G₂/M phases was determined relative to cells in the absence of Tro. Actual values used to calculate relative percentage were obtained from mean of 3 independent experiments. *P < 0.05 vs. 0 µM. B: Western blot analysis of cyclin D expression in IMR-90 cells after exposure to 20 µM Tro. Equal loading was confirmed by stripping and probing with antibody against β-actin. Blot is representative of 2 separate experiments.

Fig. 6

PPAR-γ activation inhibits fibroblast-to-myofibroblast differentiation. A: cells from IPF patients or normal human controls were grown to 80% confluence, and growth was arrested for 48 h. Cells were treated for 1–2 h with 1 or 5 µM Tro or Cig and then for 24 h with TGF-β1 (TGF; 2 ng/ml), and α-smooth muscle actin (α-SMA) was assessed by Western immunoblotting. Equal loading was confirmed by stripping and probing with antibody against β-actin. B: densitometric analysis of Western immunoblots in A (n = 4/group). *P < 0.05 vs. TGF. C: IMR-90 cells were treated as described in A or, before TGF-γ1 stimulation, transiently transfected with pcDNA3-VP16-PPAR-γ construct or empty vector (Ctrl). Cells were then assessed for α-SMA levels as described in A. D: Tro, but not Cig, decreases α-SMA transcriptional activity in rat fibroblasts. Rat fibroblasts were simultaneously transfected with an α-SMA reporter plasmid (pGAL3-α-SMAp-luc) and pRL-SV40 and then treated with 10 or 20 µM Tro or Cig and TGF-β1 (2 ng/ml) for 24 h. Relative luciferase activity was calculated by normalization of firefly luciferase activity to Renilla luciferase activity. Data are averages from 3 independent experiments. *P < 0.05; **P < 0.01 vs. DMSO.

Fig. 7

PPAR-γ activation inhibits TGF-β1-induced collagen secretion. A: Fibroblasts constitutively produce collagen, so baseline collagen secretion was set to be 100%. Cells from normal human controls (shaded bars) or patients with idiopathic pulmonary fibrosis (IPF, solid bars) were grown to 80% confluence in standard medium and then placed in serum-free medium for 48 h. Fresh serum-free medium was added, and cells were pretreated for 1–2 h in triplicate with 10 or 20 µM Tro or vehicle and then with TGF-β1 (2 ng/ml). After 24 h, cell culture supernatants were assayed for total soluble collagen on the basis of specific binding of Sirius red dye with the [Gly-X-Y]n helical structure of collagen. *P < 0.05; **P < 0.01 vs. 0 µM. B: IMR-90 cells were grown and treated as described in A. Other IMR-90 cells (solid bars) were transiently transfected with the pcDNA3-VP16-PPAR-γ (VP16-PPAR-γ) construct or empty vector (E. Vector). After 24 h, cell culture supernatants were assayed for total soluble collagen as described in A. *P < 0.05; **P < 0.01 vs. 0 µM. #P < 0.01 vs. E. Vector.

Fig. 8

Tro inhibits lung hydroxyproline and collagen accumulation in response to bleomycin (Bleo) treatment in vivo. All mice (n = 10/group) received 0.025 U of bleomycin intratracheally as a single dose. A and B: mice received Tro by oral gavage at 0, 200, or 400 mg·kg body wt⁻¹·day⁻¹ beginning 3 days before bleomycin instillation. C and D: mice received Tro at 400 mg·kg body wt⁻¹ ·day⁻¹ beginning on day 11 following bleomycin instillation. On day 21 following bleomycin instillation, mice were killed and amounts of hydroxyproline (A and C) and collagen (B and D) in their lungs were measured. *P < 0.05; **P < 0.01 vs. vehicle + bleomycin-treated mice.

Fig. 9

Tro inhibits collagen deposition in lungs of bleomycin-treated mice. Saline (A) or bleomycin (0.025 U, B–D) was administered intratracheally as a single dose. Bleomycin-treated mice were also treated with vehicle (B), Tro by oral gavage at 400 mg·kg body wt⁻¹ ·day⁻¹ beginning 3 days before bleomycin instillation (C), or Tro at 400 mg·kg body wt⁻¹·day⁻¹ beginning on day 11 following bleomycin instillation (D). On day 21 following bleomycin or saline instillation, mice were killed and their lungs were fixed, sectioned, and stained for collagen with Masson’s trichrome. Original magnification ×20.

Fig. 10

Tro decreases TGF-β1 levels in lungs of bleomycin-treated mice. Mice (n = 4/group) were treated with 0.025 U of bleomycin intratracheally as a single dose. Tro was administered by oral gavage at 0 or 400 mg·kg body wt⁻¹·day⁻¹ beginning 3 days before bleomycin instillation. On day 14 following bleomycin instillation, mice were killed and TGF-β1 in lung homogenates was measured by ELISA.