Examples are shown from mice at 12 months of age. A–C: transdifferentiation from quiescent valve interstitial cells to pro-fibrotic myofibroblasts, indicated by staining for α-smooth muscle actin (α-SMA). D–F: staining for transforming growth factor-β1 (TGF-β1), a pro-fibrotic signaling molecule. G–I: staining for p-Smad2, a mediator of pro-fibrotic TGF-β1 signaling. J–L: antibody-free negative controls for immunostaining. The image in Panel L is identical to the image in Panel K, except that Panel L was electronically brightened to show the location of valve tissue (V). % refers to the proportion of valve tissue exhibiting positive staining. Autofluorescence (AF) arises from coherently arranged fibers in valve annulus, but not in valve. Scale bar = 100 μm. *p < 0.05 vs. age-matched and treatment-matched Control.