Activation of the Interleukin-33/ST2 Pathway Exerts Deleterious Effects in Myxomatous Mitral Valve Disease

Figure 1. IL-33 and ST2 expression in myxomatous mitral valves. Representative microphotographs at low and high magnification of human myxomatous mitral valve sections stained for IL-33 (A), ST-2 (B) and double immunohistochemistry for IL-33 (brown) and ST-2 (red) (C). Correlation between IL-33 and ST-2 (r = 0.548, p < 0.0001) protein levels in human myxomatous mitral valves (D). Representative microphotographs of immunohistochemistry double staining for IL-33 (brown) and α-SMA (red) on the left and ST2 (brown) and α-SMA (red) on the right (E), IL-33 (brown) and VE-cadherin (red) (left) and ST2 (brown) and VE-cadherin (red) (right) (F) and IL-33 (brown) and CD68 (red) (left) and ST2 (brown) and CD68 (red) (right) (G). Magnification 50× (Scale bar 500 µm) and 400× (Scale bar 50 µm). Protein data in pg/mL. IL-33: Interleukin-33; α-SMA: Alpha-smooth muscle actin.

Figure 2. Correlations between IL-33 protein levels and principal features that characterize human myxomatous mitral valves. Correlation between valve tissue levels of IL-33 and IL-6 (r = 0.468, p < 0.0001) (A), IL-1β (r = 0.496, p < 0.0001) (B), RANTES (r = 0.331, p = 0.0037) (C), aggrecan (r = 0.711, p < 0.0001) (D), fibronectin (r = 0.634, p < 0.0001) (E), Gal-3 (r = 0.314, p < 0.0076) (F), MMP-1 (r = 0.618, p < 0.0001) (G), MMP-2 (r = 0.638, p < 0.0001) (H) and TIMP-2 (r = 0.606, p < 0.0001) (I). Protein data in pg/mL. IL-33: Interleukin-33; IL-6: Interleukin-6; IL-1β: Interleukin-1 beta; Gal-3: Galectin-3; MMP-1: Matrix metalloproteinase-1; MMP-2: Matrix metalloproteinase-2: TIMP-2: Tissue inhibitor of metalloproteinase-2.

Figure 3. Effects of IL-33 on human mitral VICs. (A) Quantification of VICs activation markers (vimentin and α-SMA) in VICs treated with IL-33; (B,C) Quantification of secreted inflammatory markers (IL-1β and IL-6) in VICs treated with IL-33; (D) Quantification of mRNA of IL-6 in VICs treated with IL-33; (E) Quantification of extracellular proteoglycans aggrecan, lumican and biglycan in VICs treated with IL-33; (F) Quantification of secreted proteoglycans syndecan-1 and decorin in VICs treated with IL-33; (G) Quantification of mRNA of decorin and biglycan in VICs treated with IL-33; (H) Quantification of fibronectin, elastin and Galectin-3 at protein levels in VICs treated with IL-33; (I) Quantification of secreted MMP-1 and MMP-9, (J) TIMP-1, MMP-2, TIMP-2 at protein levels in VICs treated with IL-33; (K) Quantification of mRNA of MMP-1, TIMP-1, MMP-2 and TIMP-2 in VICs treated with IL-33; (L) Representative Stain free. Box plots show the individual datapoints and the horizontal bars indicate the mean and SEM in arbitrary units versus the control group. For Western blot experiments, protein densitometry was expressed in arbitrary units (AU) once normalized to stain-free and β-actin.…

Figure 4. Effects of IL-33 on human mitral VECs. (A) Quantification of VECs markers (CD31, VE-cadherin and Von Willebrand factor) in VECs treated with IL-33 at protein levels. (B) Quantification of VICs activation markers (vimentin and α-SMA) in VECs treated with IL-33 at protein levels. (C,D) Quantification of secreted inflammatory markers IL-1β and IL-6 in VECs treated with IL-33. (E) Quantification of mRNA of IL-6 in VICs treated with IL-33; (F) Quantification of fibronectin and Galectin-3 at protein levels in VICs treated with IL-33; (G) Quantification of extracellular proteoglycans aggrecan, lumican and biglycan in VECs treated with IL-33; (H) Quantification of secreted decorin in VECs treated with IL-33; (I) Quantification of mRNA of lumican, decorin and biglycan in VECs treated with IL-33; (J) Quantification of secreted MMP-1 and MMP-9, (K) TIMP-1, MMP-2, TIMP-2 at protein levels in VECs treated with IL-33; (L) Quantification of mRNA of MMP-1, TIMP-1, MMP-2 and TIMP-2 in VECs treated with IL-33; (M) Representative Stain free. Box plots show the individual datapoints and the horizontal bars indicate the mean and SEM in arbitrary units versus the control group. For Western blot…