A Secreted Phospholipase A2 Induces Formation of Smooth Muscle Foam Cells Which Transdifferentiate to Macrophage-Like State

Figures

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  Figure 1. MT-III induces LD formation in VSMCs. (A) Effect of MT-III on LD formation in VSMCs stimulated with selected concentrations of MT-III or with DMEM (control) for 1 h. (B) Time-course of MT-III-induced LD formation. VSMCs (8 × 103 cells/coverslip) were incubated with MT-III (0.4 µM) or DMEM (control) for 1, 3, 6, or 12 h. LDs were quantified using light microscopy after OsO4 staining. (C) Osmium-stained LDs observed in control or MT-III (0.4 µM) stimulated cells for 1 or 12 h. Each bar represents the mean ± SEM of the number of LDs/cell in 50 cells. Values represent mean ± SEM for six animals (n = 6) (ANOVA).Note: * p < 0.05 compared with vehicle treated cells.

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  Figure 2. MT-III induces PLIN2 and PLIN3 recruitment to LDs in VSMCs. VSMCs (8 × 103 cells/coverslip) were incubated with DMEM (control) or MT-III (0.4 µM) for 12 h and labelled for LDs (with Nile red) and for PLIN2 (A) or PLIN3 (B) (with fluorescein isothiocyanate- FITC-conjugated secondary antibody). The merged image shows co-localization of PLIN2 and PLIN3 to LDs. The pictures are representative of three independent experiments (n = 9).

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  Figure 3. MT-III does not affect PLIN2 or PLIN3 protein expression in VSMCs. VSMCs (5 × 104 cells/well) were incubated with DMEM (control) or MT-III (0.4 µM) for 1, 3, 6, and 12 h. Western blot analysis of PLIN2 (A) and PLIN3 (B) and β-actin (loading control) of the VSMC extracts and the densitometric analysis (in arbitrary units - A.U.) of PLIN2 and PLIN3 expression normalized to β-actin. The results are expressed as the mean ± SEM for three independent experiments (n = 9) (ANOVA).

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  Figure 4. Effect of DGAT and ACAT inhibitors on LD formation induced by MT-III. VSMCs (5 × 103 cells/well) were incubated with A922500 (100 nM) or TMP-153 (100 nM) or vehicle (< 1% DMSO or RPMI) for 1 h and then with MT-III (0.4 μM) for 12 h. LDs were quantified using light microscopy after osmium staining. Each bar represents the mean ± SEM LDs/cell in 50 counted cells. Values represent means ± SEM from three independent experiments (n = 9) (ANOVA). Note: * p < 0.05 compared with vehicle treated cells; # p < 0.05 compared with vehicle treated cells stimulated with MT-III.

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  Figure 5. ABCA1 and ABCG1 are upregulated in VSMCs incubated with MT-III. VSMCs (5 × 104 cells/well) were incubated with MT-III (0.4 μM) or DMEM (control) for 1, 3, 6, and 12 h. (A) Western blotting and densitometric analysis of ABCA1 (A) and ABCG1 (B) and β-actin (loading control) in VSMC extracts. The densities were normalized with those of β-actin. The results are expressed as the mean ± SEM from three experiments (n = 9) (ANOVA). Note: * p < 0.05 compared with control cells.

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  Figure 6. MT-III induces activation and upregulation of PPAR-γ and PPAR-δ/β expression in VSMCs. VSMCs (5 × 104 cells/well) were stimulated with MT-III (0.4μM) or DMEM (control) for 1, 3, 6, and 12 h. (A,B) Western blotting and densitometric analysis of PPAR-γ or PPAR-δ/β and β-actin (loading control) in VSMC extracts. The densities were normalized with those of β-actin. (C) VSMCs (8 × 103 cells/coverslip) incubated with DMEM (control) or MT-III (0.4 μM) for 1 or 3 h were labeled with anti-PPAR-γ or PPAR-δ/β and visualized with a secondary antibody conjugated to Alexa488 (green). The nucleus was stained with iodide propidium. The pictures prove an activation and translocation of the PPARs to the nucleus. Shown pictures are representative of three independent experiments. (D) VSMCs (8 × 103 cells/coverslip) were incubated with GW9662 or GSK0660 (10 μM) PPAR inhibitors for 1 h and then stimulated with MT-III (0.4 μM) for 12 h. LDs were quantified using light microscopy subsequent to osmium staining. Each bar represents the mean ± SEM LDs/cell in 50 counted cells from three independent experiments (n = 9) (ANOVA). Note: * p < 0.05 compared with vehicle treated cells; # p < 0.05 compared with…

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  Figure 7. Effect of MT-III on protein expression of SR-A1, LOX-1, and CD36 by VSMCs and the effect of scavenger receptor inhibitors on LD formation induced by MT-III. VSMCs (5 × 104 cells/well) were incubated with MT-III (0.4 μM) or DMEM (control) for 1, 3, 6, and 12 h. Western blotting and densitometric analysis of CD36 (A), SR-A1 (B), LOX-1 (C), and β-actin (loading control) in VSMC extracts. The densities were normalized with those of β-actin. (D) Treatment of VSMCs (8 × 103 cells/coverslip) with SSO (250 μM) or fucoidin, dextran sulphate sodium salt (250 μM), or kappa-carrageenan (250 μM) for 1 h before stimulation with MT-III (0.4 µM) for 12 h. LDs were counted using light microscopy after osmium staining. The results are expressed as the mean ± SEM from three independent experiments (n = 9) (ANOVA). Note: * p < 0.05 compared with control cells.

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  Figure 8. Effect of MT-III on acLDL uptake by VSMCs. (A) The first column illustrates the result image from the high content analysis made with the CellProfiler (scale bar 250 µM). The second column illustrates the single channel of the incorporated AlexFluor488 labeled acLDL. The third column shows a magnified view of the result image (Scale bar 25 µM). (B) Quantification of the number of Alexa Fluor 488-acLDL particles captured by VSMCs upon stimulation by the sPLA2 MT-III. The results are expressed as the mean ± SEM from two independent experiments (n = 6) (ANOVA). Note: * p < 0.05 compared with control cells.

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  Figure 9. Effect of inhibitors of macropinocytosis and receptor-mediated endocytosis in MT-III-induced LD formation in VSMCs. VSMCs (8 × 103 cells/coverslip) were incubated with one of the following inhibitors before stimulation with MT-III (0.4 µM) for 12 h: (A) dynasore (100 µM) for 1 h or (B) LY294002 (100 µM) or wortmaninn (2 µM) for 1 h. LDs were counted using light microscopy after osmium staining. Each bar represents the mean ± SEM of the number of LDs/cell in 50 cells. Values represent means ± SEM for three independent experiments (n = 9) (ANOVA). Note: * p < 0.05 compared with vehicle treated cells; # p < 0.05 compared with vehicle treated cells incubated with MT-III.

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  Figure 10. Phagocytotic activity and upregulation of macrophage markers in VSMCs induced by MT-III. VSMCs (2.5 × 103 cells/well) were treated with MT-III (0.4 and 0.8 μM) or DMEM (Control) for 12, 24, and 48 h, followed by incubation with 0.5 μm fluorescent beads (red) for 6 h. Cells were then washed extensively, fixed with paraformaldehyde (4%), counterstained with Hoechst 33342 (blue-nuclei), and analyzed with an automated Axiovert 200 m followed by a computerized automatic quantification in the CellProfiler analysis software. (A) The number of latex beads per cell was counted to obtain numerical data for the total phagocytotic activity. (B) A representative field of view. (C) The effect of MT-III on VSMC and macrophage-related gene expression in rat VSMCs. Subconfluent rat VSMCs (8 × 105 cells/well) were treated with MT-III (0.4 and 0.8 μM) or with DMEM medium (control) for 48 h. Total RNA was extracted and subjected to RT-PCR analysis. Values represent the mean ± SEM from six animals (n = 6) (ANOVA). Note: * p < 0.05 compared with control cells.