FIGURE 4.

Activated myofibroblasts (aVICs) exhibit a senescent‐associated secretory phenotype (SASP) with a reduced capacity for autophagy. (A) Representative confocal immunofluorescent images of ATG7, p21^CIP1 and α‐SMA expressions in canine healthy and myxomatous mitral valves, scale bar 20 μm. (B) Quantitative analysis of mean fluorescence intensity (MFI) of ATG7, p21^CIP1 and α‐SMA in canine healthy and myxomatous mitral valves. (C) Representative images of co‐localization ratio analysis of ATG7 (red) and α‐SMA (green), p21^CIP1 (red) and α‐SMA (green) fluorescence signals in canine mitral valve tissues. (D) Quantitative analysis of co‐localization parameters (Pearson's correlation and overlap coefficient) of ATG7 (red) and α‐SMA (green), p21^CIP1 (red) and α‐SMA (green) fluorescence signals. (E) Representative images of SA‐β‐gal (blue) staining and quantitative analysis of the percentage of SA‐β‐gal positive cells, scale bar 50 μm (n = 12 microscopic fields/treatment). (F) Cell cycle analysis (left panel) and BrdU incorporation assay (right panel) of canine aVICs and qVICs (n = 6). (G) Representative western blot of p16^INK4A, p21^CIP1, p53 and β‐actin protein expression and quantification of the relative protein expression in VICs (two biological replicates shown in blots, n = 6). (H) Quantitative RT‐PCR for SASP cytokine expression (left panel) and secreted TGF‐β1, IL‐6 and MMP‐9 (right panel) in collected supernatant from VIC cultures (n = 6). Results are presented as mean ± SEM. ANOVA followed by Tukey's range test. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control. ANOVA, analysis of variance; VIC, valve interstitial cell.