Pro-Calcifying Role of Enzymatically Modified LDL (eLDL) in Aortic Valve Sclerosis via Induction of IL-6 and IL-33

Figures

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  Figure 1. Viability of VICs/myofibroblasts after eLDL treatment (n = 3). The VICs/myofibroblasts were seeded on 96-well plates. The cells were treated with DMEM Ø FCS or 20 µg/mL eLDL and incubated for the indicated time periods. After each incubation period, an MTT assay was performed and the absorbance at 550 nm was measured to determine the viability of the cells.

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  Figure 2. eLDL enhances phosphate-induced calcification in cultured human VICs/myofibroblasts. (A) Representative example of confluent monolayers of human VICs/myofibroblasts treated with 1 mM phosphate containing pro-calcifying medium (PM) with (wells 11 & 33 containing 2.5 µg/mL eLDL, wells 12 & 34 containing 5 µg/mL eLDL) or without eLDL (wells 10 & 32) as indicated. Cells cultured in PM containing 1 mM inorganic phosphate (Pi) served as controls. Cells were fixed and calcium phosphate deposits were stained with alizarin red pH 4.4. (B) For quantification of the alizarin red staining, the alizarin–Ca2+ complexes were extracted by addition of CPC. The amount of released dye was measured by spectrophotometry at 570 nm. (C) qPCR analysis of osteogenic gene mRNA in cultured VICs/myofibroblasts incubated for 7 days in PM containing 1 mM inorganic phosphate (Pi) in the presence (5 µg/mL) or absence of eLDL (control). The mRNA expression levels were normalized to GAPDH according to the 2−ddCT method. Results from 12 independent experiments are shown. Bar values are means ± SD. (D) eLDL induced ANGPTL4 gene expression in VICs/myofibroblasts. ANGPTL4 gene expression was determined for cells…

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  Figure 3. p38 MAPK and eLDL in human aortic valves. (A) Detection of rapidly phosphorylated p38 MAPK upon treatment of VICs/myofibroblasts with eLDL. Cells were exposed to 10 or 20 µg/mL eLDL for 30 min. Cells without treatment served as control. Phosphorylated p38 MAPK and total p38 MAPK in whole-cell lysates were sequentially detected by Western blot analysis on the same membrane. Blots were re-probed with β-actin antibody to confirm equal loading. (B) WB quantification of the phosphorylation of p38 MAPK. (C) Expression of p38 MAPK isoforms in VICs/myofibroblasts. Relative mRNA levels for p38 subtypes quantified in VICs/myofibroblasts from 10 different patients using real-time PCR. Expression levels are given as RQ of target genes (p38α/MAPK14, p38β/MAPK11, p38γ/MAPK12, p38δ/MAPK13) normalized to the reference gene GAPDH. (D) Representative immunohistochemical staining of aortic valves sections stained with (a), anti-p38; (b), anti-phospho-p38; (c), anti-Hsp27; (d), anti-phospho-Hsp27. The fibrosa with the aortic side of the valve is on top.

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  Figure 4. eLDL increases mRNA expression of (A) IL-6 and (B) IL-33 in cultured human VICs/myofibroblasts. Confluent monolayers of VICs/myofibroblasts were exposed to 20 µg/mL eLDL for the indicated time periods. Cells cultured in DMEM alone were used as controls. Total RNA was extracted, and levels of IL-6 and IL-33 mRNA were measured via qPCR analysis. The expression levels of IL-6 and IL-33 mRNA were normalized to the housekeeping gene (GAPDH). Data are represented as mean ± SD, ** p < 0.01, and * p < 0.05.

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  Figure 5. Colocalization of eLDL with IL-6 and IL-33. Representative sequential sections of Grade 4 aortic valve calcification stained with (A) Masson–Goldner for (B) eLDL, (C) IL-6, and (D) IL-33. Note the close intermingling and overlap of the different antigens within and around calcified areas (colored red by Masson–Goldner). In all panels, the fibrosa with the aortic side of the valve is to the upper right-hand corner.

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  Figure 6. Activation of the p38 MAPK pathway in human VICs/myofibroblasts stimulated with IL-6 and IL-33. Cells from AS patients were treated with (A) 100 ng/mL IL-6 and (B) 100 ng/mL IL-33 for 24 h and 48 h. Cells without treatment served as controls. Phosphorylated p38 MAPK and total p38 MAPK protein expression were detected by WB analysis. The expression of vinculin was used for loading control. The bar graphs show the relative quantification of phospho-p38 protein compared to the untreated cells.

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  Figure 7. Effect of skepinone-L and SB203580 on the eLDL-induced expression of IL-6 and IL-33 in human VICs/myofibroblasts. Cells were incubated with skepinone-L (0.1 μM) or SB203580 (20 μM) for 2 h and further exposed to 40 μg/mL eLDL for 24 h. mRNA expression levels of (A) IL-6 and (B) IL-33 were measured via qPCR analysis. The expression levels of IL-6 and IL-33 mRNA were normalized to the housekeeping gene (GAPDH). The eLDL-stimulated cells were assigned an arbitrary value of 100, and the other gene expression values are shown in proportion to this as a percentage. Data are represented as mean ± SD, *** p < 0.001.

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  Figure 8. IL-6, IL-33, and calcification. (A) IL-33 induces IL-6 mRNA expression in human VICs/myofibroblasts. qPCR analysis of IL-6 mRNA in cultured VICs/myofibroblasts incubated for 6 days in PM containing 1.0 mM inorganic phosphate (Pi) in the presence (100 ng/mL) or absence of IL-33 (control). The mRNA expression level of IL-6 was normalized to GAPDH according to the 2−ddCT method. Results from 7 independent experiments are shown. The bar values are mean values ± SD, * p < 0.05. (B) Effects of IL-6 on phosphate-induced calcification in VICs/myofibroblasts. Confluent monolayers of human VICS/myofibroblasts cultured in 1 mM phosphate containing PM were treated with (well 3 containing 100 ng/mL IL-6) and without IL-6 (well 2) for 6 days. Calcium phosphate deposits were visualized by alizarin red staining (pH 4.4). (C) To quantify calcium deposition, alizarin–Ca2+ complexes were extracted by adding CPC. The optical density of the extract was measured at 570 nm and normalized to cellular protein content.