Darapladib Ameliorates Radiation-Induced Skin Injury and Fibrosis by Lipoprotein-Associated Phospholipase A2 Inhibition

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  Figure 1. Lp-PLA2 inhibition by darapladib ameliorates radiation-induced endothelial cell damage. For all experiments, HDMVECs were pretreated with darapladib before irradiation. (A) Chemical structure of darapladib, an Lp-PLA2 inhibitor. (B) HDMVECs were treated with 0, 250, or 500 nM darapladib 1 h before 5 Gy irradiation. After 24 h, Lp-PLA2 mRNA levels were measured by real-time PCR. (C) Cells were treated with 500 nM darapladib 1 h before 5 Gy irradiation and cultured for 2 days. Ox LDL levels were quantified by ELISA. (D) Cells were treated with 0, 250, or 500 nM darapladib 1 h before 5 Gy irradiation. After 1 h, cells were immunostained with an anti-γ-H2AX antibody (green) and DAPI (blue). γ-H2AX foci were quantified using a confocal fluorescence microscope (40×). Scale bar = 25 μm. (E) Tube formation assays were performed by treating HDMVECs with darapladib 1 h before 5 Gy irradiation. Tube morphology was evaluated 24 h after IR using an In Cell analyzer. Red boxes indicate the enlarged areas. Scale bar = 200 μm. (F) HDMVECs were stained with JC-1 dye for 30 min and examined using a confocal fluorescence microscope (10×). Green and red fluorescence indicate low and high…

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  Figure 2. Lp-PLA2 regulates radiation-induced EndoMT in HDMVECs. (A) HDMVECs were treated with 0, 250, or 500 nM darapladib 1 h before 5 Gy irradiation. After 24 h, Vimentin, Snail and α-SMA levels were analyzed by Western blotting. In parallel, cells were transfected with 200 pmole of Lp-PLA2 siRNA for 2 days and then exposed to 5 Gy IR for 24 h. Proteins were extracted and immunoblotted with the same antibodies. β-actin served as a loading control. (B) Cells were transfected with 200 pmole of Lp-PLA2 siRNA for 2 days, and Lp-PLA2 mRNA level was quantified by real-time PCR. (C) Lp-PLA2 siRNA-transfected cells were treated with IR for 48 h, and LPC generation was analyzed using ELISA. ** p < 0.01, *** p < 0.001 versus the IR group. (D) Cells were stained with anti-CD31 or anti-a-SMA antibodies and examined by confocal microscope. Blue represents DAPI staining, and red represents CD31 or α-SMA staining. Scale bar = 25 μm.

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  Figure 3. Radiation-induced LPC promotes fibroblast-to-myofibroblast transition. (A) HDFs were irradiated with 5 Gy and harvested at 48 h after irradiation. Cell lysates were analyzed for LPC species by UPLC-ESI-QTOF MS. (B) HDFs treated with 0, 5, or 10 μM LPC (18:0) for 48 h. Vimentin, α-SMA and β-actin levels were assessed by Western blotting. (C) mRNA was extracted from HDFs treated as in (B) and relative mRNA expressions of Vimentin, α-SMA and Col1A1 were analyzed using real-time PCR. Data represent target gene expression normalized to a GAPDH gene. (D) HDFs were treated with 0, 5, or 10 μM LPC (18:0) for 24 h and then stained for F-actin (green) and α-SMA or Col1A1 (red). Nuclei were counterstained with DAPI (blue). (E) HDFs were treated with 0, 250, or 500 nM darapladib 1 h before 5 Gy irradiation. After 24 h, the cells were stained for α-SMA (green) and Col1A1 (red). Scale bar = 25 μm. * p < 0.01, *** p < 0.001 versus control.

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  Figure 4. Darapladib inhibits radiation-induced skin injury and promotes restoration of barrier integrity. (A) Schematic representation of the mouse model of RISI and fibrosis. Mice received subcutaneous injections of 20 or 40 mg/kg darapladib 1 day before IR. A total of two injections were administered within 1 week. (B) IHC staining of Lp-PLA2 in radiation-induced scarred skin was performed 3 weeks after irradiation. Lp-PLA2-positive areas were quantified as the mean value from five fields per section. Scale bar = 50 μm. (C) Representative images of mouse skin showing radiation-induced scar lesions and their attenuation by darapladib treatment (n = 5). Skin hydration (D), TEWL (E), and surface pH (F) were measured in scar lesional and adjacent normal skin. Measurements for each animal were repeated until stable and consistent values were obtained (n = 5). Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. n = 3–4 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the IR group.

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  Figure 5. Darapladib attenuates radiation-induced skin fibrosis. (A) H&E and MT-stained images in skin tissues were generated 3 weeks after irradiation, with or without darapladib treatment. Red boxes indicate the enlarged areas. Scale bar = 100 µm. (B,C) The quantification of adipocyte number and area was measured per field, averaged over five fields in H&E images. (D) The quantification of collagen deposition area per field was averaged from five fields in MT images. (E) Immunofluorescence staining of PLIN-1 (green) was analyzed in skin tissues from non-irradiated and irradiated mice, with or without darapladib treatment. Scale bar = 25 µm. Data are presented as mean ± SEM (n = 3–4 independent experiments). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 versus the IR group.

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  Figure 6. Darapladib suppresses TGF-β signaling in radiation-induced skin fibrosis in vivo. (A) Proteins were extracted from mouse skin 4 weeks after irradiation. TGF-βR2, Vimentin and α-SMA levels were analyzed by Western blotting, using ponceau S staining as a control. (B) TGF-β levels were measured using ELISA. Immunohistochemical staining of α-SMA (C) and vimentin (D) in skin tissues was analyzed 4 weeks after irradiation. Scale bars = 50 µm (C) and 100 µm (D). Data are presented as mean ± SEM (n = 3–4 independent experiments). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the IR group.

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  Figure 7. Darapladib reduces radiation-induced late-stage inflammatory cytokine expression in mouse skin. mRNA was extracted from irradiated mouse skin 3 weeks after IR. Relative mRNA expressions of IL-6 (A), IL-1β (B) and TNF-α (C) were analyzed using real-time PCR. Data represent target gene expression normalized to a GAPDH gene. (D) IHC staining of iNOS expression in skin tissues was examined 3 weeks after irradiation. Scale bar = 100 μm. Data are presented as mean ± SEM (n = 3–4 independent experiments). Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the IR group.

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  Figure 8. Darapladib reduces radiation-induced macrophage infiltration and inflammatory polarization in mouse skin. (A,B) Mice were locally irradiated (40 Gy) to the skin and treated with darapladib (20 or 40 mg/kg). Skin tissues were collected and analyzed for Ly6Chigh inflammatory macrophages by flow cytometry. In the mouse skin tissues, the number of CD80+ M1 macrophages (C) and CD206+ M2 macrophages (D) were analyzed by flow cytometry. (E) Bone marrow-derived macrophages (BMDMs) were polarized with 20ng/mL IFN-γ (F) or 20 ng/mL TGF-β treatment for 2 days (G). Differentiated BMDMs were then stimulated with 20 μg/mL lysophosphatidylcholine (LPC) and treated with darapladib for 24 h. The expression of CD80 and CD206 was analyzed by flow cytometry. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001. UMI; Unique molecular identifier.