PPARδ Agonist GW501516 Suppresses the TGF-β-Induced Profibrotic Response of Human Bronchial Fibroblasts from Asthmatic Patients

Figures

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  Figure 1. The PPARδ agonist GW501516 and antagonist GSK0660 administered alone or in combination to HBF cultures had a negligible effect on cell viability and proliferation. HBF populations (n = 7, all in duplicate) were exposed to increasing concentrations (0–25 µM) of GW501516 and GSK0660 administered alone or in combination (0–10 µM) for four days. (A) The viability of HBFs was determined using an FDA+/EtBr- assay. (B) The proliferation rates were determined using a crystal violet assay, and the absorbances were measured spectrophotometrically (λ = 540 nm). The results are expressed as a percentage of the control and presented as the mean ± SEM. Statistical significance was tested using the nonparametric Kruskal–Wallis test with Dunn’s multiple comparisons post hoc test; * p < 0.05, ** p < 0.01, *** p < 0.001. The red box indicates the concentration used for further analyses (10 µM).

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  Figure 2. The profibrotic response of TGF-β1-stimulated HBFs was significantly attenuated by GW501516 when administered alone or in combination with GSK0660. HBFs derived from asthmatic patients were cultured in a serum-free medium containing GW501516 and GSK0660 alone or in combination, and in the absence or presence of TGF-β1 (5 ng/mL) for four days. (A) The FMT potential of HBFs was determined via immunostaining for α-SMA (green) with DNA (blue) visualization. Representative images are presented. Scale bar = 50 µm. (B) The percentage of myofibroblasts in HBF cultures (n = 10) are presented in the graph. The α-SMA content in the HBFs was determined via (C) in-cell ELISA (n = 8; each condition in triplicate) and (D,E) Western blotting (n = 3). RCU–relative colorimetric units. The relative expressions of selected FMT-related genes: (F) ACTA2 (α-smooth muscle actin), (G) COL1A1, (H) COL1A2 (collagens 1a1 and 1a2), and (I) TNC (tenascin C), in HBFs (n = 5) cultured for 24 h under the conditions described above were determined via real-time PCR. The results are presented as 2−ΔΔCt mean values in relation to the control gene (GAPDH). The results are presented as the mean ± SEM. Statistical…

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  Figure 3. The actin cytoskeleton architecture in TGF-β-treated HBFs was modulated by GW501516 alone or in combination with GSK0660. HBFs derived from asthmatic patients (n = 3) were cultured in a serum-free medium containing GW501516 and GSK0660 alone or in combination, and in the absence or presence of TGF-β1 (5 ng/mL) for 24 h. (A) Representative images of HBFs immunostained for vinculin (green) and F-actin (red) with visualization of DNA (blue) are presented; scale bar = 50 µm. Vinculin-rich focal adhesion sites are shown enlarged in boxes; scale bar = 25 µm. (B) F-actin fluorimetry was quantified in relation to DNA fluorescence and is presented in the graph (n = 3, 260 cells/condition). (C) The lengths of vinculin-rich focal adhesion sites were measured and are presented in the graph. (D,E) The contents of focal-adhesion-related proteins vinculin and talin, were determined via in-cell ELISA (n = 8; each condition in triplicate). (F) The relative expression of TLN (talin) in HBFs (n = 3) cultured for 24 h under the conditions described above was determined using real-time PCR. The results are presented as 2−ΔΔCt mean values in relation to the control gene (GAPDH). RCU–relative…

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  Figure 4. GW501516 and GSK0660, when administered alone or in combination, suppressed the TGF-β1-induced upregulation of Cx43 in HBF populations. HBFs derived from asthmatic patients were cultured in a serum-free medium containing GW501516 and GSK0660 alone or in combination, and in the absence or presence of TGF-β1 (5 ng/mL) for four days. (A) Representative images of HBFs immunostained for Cx43 (red) with DNA visualization (blue) are presented. Scale bar = 50 µm. Cx43 levels in HBFs were determined via (B) the quantification of the fluorescence signal in the collected images (n = 5) or via (C) Western blot analyses (n = 1, densitometry is presented above the bands). (D) The relative expression of GJA1 (the Cx43-encoding gene) in HBFs (n = 3) cultured for 24 h under the conditions described above was determined using real-time PCR. The results are presented as 2−ΔΔCt mean values in relation to the control gene (GAPDH). All results are presented as the mean ± SEM. Statistical significance was tested using the nonparametric Kruskal–Wallis test with Dunn’s multiple comparisons post hoc test; * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 5. Canonical TGF-β1/Smad2-dependent signaling was modulated in HBFs in response to the administration of GW501516 and GSK0660 alone or in combination. HBFs derived from asthmatic patients were cultured in a serum-free medium containing GW501516 and GSK0660 alone or in combination, and in the absence or presence of TGF-β1 (5 ng/mL) for 1 h. (A) Representative images of HBFs immunostained for pSmad2 (red) with DNA visualization (blue) are presented. Scale bar = 100 µm. (B) The number of cells expressing pSmad2 in the nuclei area was quantified. (C) The levels of pSmad2 in the nuclei area of HBFs were determined via the quantification of the fluorescence signal in the collected images (n = 4; 420 cells/condition). The relative expression of genes encoding (D) PPARδ (PPARD), (E) the p300 cofactor (P300), and (F) the Sox9 transcription factor (SOX9) were measured using real-time PCR. The results are presented as 2−ΔΔCt mean values in relation to the control gene (GAPDH). The results are presented as the mean ± SEM. Statistical significance was tested using the nonparametric Kruskal–Wallis test with Dunn’s multiple comparisons post hoc test; * p < 0.05, ** p < 0.01, *** p < 0.001.