Secreted Phospholipase A2-IIA Modulates Transdifferentiation of Cardiac Fibroblast through EGFR Transactivation: An Inflammation–Fibrosis Link

Figures

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  Figure 1. BALB/c mice were immunised with the MyCHα614–629 peptide. (A) Heart weight (HW)/body weight (BW) ratio of mice sacrificed 21 or 65 days after initial immunisation. (B–D) Representative photomicrographs of heart sections from control and EAM mice on day 65: (B) H&E and MTC stain, (C) DHE stain (for detection of O2−), and (D) LOX immunohistochemistry. Objective lens 2× and 20×, magnification 20× and 200×, respectively. (C) sPLA2-IIA protein levels on sera and heart tissue from control and EAM mice on day 65. Values are means ± SD. * p < 0.001 versus control mice, n = 7 in each group.

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  Figure 2. sPLA2-IIA modulates cardiac fibroblast (CFs) phenotype. Rat CFs were treated with the indicated stimuli. (A) After 24 h of incubation, cell proliferation was investigated and expressed as mean ± SD. After 24 h of incubation, αSMA (B) and paxillin (C) expression were examined by fluorescence microscopy. Representative images of resting and sPLA2-IIA-stimulated cells (objective lens 20×, magnification 200×) and quantitative analysis. (D) After 72 h of incubation, total collagen accumulation was stained with picro-Sirius red and quantified by spectrophotometric analysis. (E) Collagen I protein expression: (i) Western blot of CF stimulated at the indicated times, and (ii) flow cytometry histograms of CF upon 24 h stimulation (untreated-CFs (open black curves) and treated-CFs (open grey curves) were compared with isotype controls (solid grey curves)). (F) TGFβ expression was evaluated after 24 h of stimulation by flow cytometry analysis. The graphic represents the mean fluorescence intensity (MFI) and was shown by fold control. *p < 0.001 vs. control cells, n = 3.

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  Figure 3. sPLA2-IIA induces oxidative stress in cardiac fibroblast (CFs). CFs stained with (A) DCFHDA, or (C) DHE were untreated (solid grey curves) or 1 μg/mL of sPLA2-IIA-treated (solid black curves) for different times. Representative histogram and mean fluorescence intensity (MFI) quantification expressed by fold of control. Resting and 24 h sPLA2-IIA-stimulated cells stained with (B) DCFHDA or (D) DHE, were examined by fluorescence microscopy. Representative microphotographs and quantification. (E) CFs pretreated with the indicated inhibitor, and stimulated with 1 μg/mL of sPLA2-IIA for 24 h, were stained with DCHFDA or DHE. Representative histograms of flow cytometry analysis, n = 3. sPLA2-IIA-treated (open black curves) and inhibitor+sPLA2-IIA-treated (open grey curves) CFs were compared with untreated (solid grey curves) cells. CFs stimulated with the indicated agonist for 24 h were stained with (F) MitoSox – representative microphotographs and quantitative analysis or (G) rhodamine 123—flow cytometry analysis, expressed as mean fluorescence intensity (MFI). Bar graphs represent the mean ± SD; n = 3. Objective lens 20×, magnification 200×; bars = 100 μm. * p < 0.001, ** p < 0.01…

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  Figure 4. sPLA2-IIA-activated intracellular signaling cascades in cardiac fibroblast (CFs) requires EGFR transactivation Cells were stimulated with 1 μg/mL of sPLA2-IIA for indicated times: using phospho-specific antibodies, (A) ERK, Akt, P70S6 and S6 phosphorylation were determined by immunoblotting, while (B) Src and (C) EGFR phosphorylation was examined by flow cytometry. Untreated (solid black curves) and sPLA2-IIA-treated (solid dark grey curves) CFs were compared with isotype controls (solid light grey curves). (D–F) Cells were pretreated with AG1478 before stimulation with sPLA2-IIA. (D) After 15 min, phospho-EGFR was studied by flow cytometry. (E) After 15 min, ERK, Akt, P70S6 and S6 phosphorylation were determined by immunoblotting, while (F) Src phosphorylation was studied by flow cytometry. Untreated (open black curves), sPLA2-IIA-treated (open grey curves), AG1478+sPLA2-IIA-treated (open blue curves) CFs were compared with isotype controls (solid light grey curves). * p < 0.001, ** p < 0.01 and *** p < 0.5 vs. control cells; mean ± SD; n = 3.

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  Figure 5. sPLA2-IIA-activated intracellular signaling cascades in cardiac fibroblasts (CFs) require HB-EGF shedding. (A) Cells were stimulated with 1 μg/mL of sPLA2-IIA for the indicated times, and HB-EGF expression was determined by flow cytometry: untreated (solid black curves) and sPLA2-IIA-treated (solid dark grey curves) CFs were compared with isotype controls (solid light grey curves). * p < 0.001, ** p < 0.01, and *** p < 0.5 vs. control cells; mean ± SD CFs were pretreated with the indicated inhibitors and then incubated with 1 μg/mL of sPLA2-IIA: after 15 min, HB-EGF (B) and phospho-EGFR (C) expression were analysed by flow cytometry: untreated cells (open black curves) were compared with sPLA2-IIA-treated cells in the absence (open dark grey curves) or presence of the indicated inhibitors (open blue curves). Solid light grey curves show isotype controls. (D) After 15 min, ERK, Akt, P70S6 and S6 phosphorylation were determined by immunoblotting. n = 3.

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  Figure 6. sPLA2-IIA induces collagen production in cardiac fibroblast (CFs). CFs pretreated without/with the indicated inhibitors, were stimulated with 1 μg/mL of sPLA2-IIA. (A) After 24 h, collagen I protein expression was evaluated on cells by flow cytometry. Untreated (solid black curves) and sPLA2-IIA-treated (solid dark grey curves) CFs were compared with isotype controls (solid light grey curves). Representative histograms are shown, n = 3. (B) After 72 h, total collagen accumulation on CFs cultures was stained with picro-Sirius red and quantified by spectrophotometric analysis. Bar graphs represent the mean ± SD; n = 3. * p < 0.001 vs. control cells, # p < 0.001 vs. sPLA2-IIA-treated cells.

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  Figure 7. sPLA2-IIA induces BMP-1 expression in cardiac fibroblast (CFs) via EGFR transactivation. CFs were treated with 1 μg/mL of sPLA2-IIA or 1 μM AngII for 24 h and BMP-1 protein expression was evaluated by (A) flow cytometry analysis. The graphic represents the mean fluorescence intensity (MFI) and was shown by fold control. (B) Representative microphotographs of resting and 24 h sPLA2-IIA-stimulated cells examined by fluorescence microscopy (objective lens 20×, magnification 200×) and quantification. (C) CFs, pretreated with the EGFR inhibitor AG1476, were stimulated with 1 μg/mL of sPLA2-IIA for 24 h. BMP-1 expression was analyzed by flow cytometry: untreated cells (open black curves) were compared with sPLA2-IIA-treated cells in the absence (open dark grey curves) or presence of the inhibitor (open blue curves). Solid light grey curve shows isotype control. Representative histograms are shown, n = 3. Bar graphs represent the mean ± SD; n = 3. * p < 0.001 vs. control cells.

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  Figure 8. sPLA2-IIA induces LOX expression in cardiac fibroblast (CFs) via EGFR transactivation. CFs were treated with 1 μg/mL of sPLA2-IIA or 1 μM AngII for 24 h and LOX protein expression was evaluated by (A) flow cytometry analysis, (B) fluorescence microscopy and (C) Western blot. (A) The graphic represents the mean fluorescence intensity (MFI) and is shown by fold control. (B) Representative microphotographs (objective lens 20×, magnification 200×) and quantification. (C) Representative Western blotting in total cell lysates. (D) CFs, pretreated with the EGFR inhibitor AG1476, were stimulated with 1 μg/mL of sPLA2-IIA for 24 h. LOX expression was analysed by flow cytometry. Representative histograms are shown, n = 3. Untreated cells (open black curves) were compared with sPLA2-IIA-treated cells in the absence (open dark grey curves) or presence of inhibitor (open blue curves). Solid light grey curve represent isotype control. CFs were stimulated with the indicated agonists (E) or with 1 μg/mL of sPLA2-IIA in presence of the indicated inhibitors (F) for 24 h, and LOX activity was measured in cell culture medium. Bar graphs represent the mean ± SD; n = 3. * p < 0.001 vs. control cells,…