Table 1.
Human valve specimen information.
Fig 1.
Representative images of human aortic valve leaflets stained with Movat’s Pentachrome.
Top: Images of a complete leaflet are shown for a representative (A) healthy and (B) diseased valve. F = fibrosa, S = spongiosa, V = ventricularis. Scale bar = 100 μm. Bottom: Higher magnification images of the boxed area in the top images show the three leaflet layers in greater detail. Scale bar = 50 μm.
Fig 2.
Visualization of collagen fibers via picrosirius red staining and quantification of collagen content.
Picrosirius red staining of (A) healthy and (B) diseased leaflets was visualized using brightfield microscopy and polarized light. Birefringence hue and amount were quantified as a percent of total tissue area (N = 5, n = 3) in the (C) fibrosa and (D) spongiosa. Collagen production was also quantified via (E) qRT-PCR analysis of COL1A1 gene expression (N = 4, n = 3) and (F) measurement of total collagen protein via dot blot (N = 5 healthy; N = 4 diseased, n = 3).
Fig 3.
Visualization of collagen fiber microarchitecture using SHG microscopy.
Representative images from (A) healthy and (B) diseased leaflets show microarchitectural changes in collagen in the fibrosa and spongiosa. Scale bar = 50 μm.
Table 2.
Quantification of collagen fiber characteristics in the fibrosa and spongiosa of healthy vs. diseased aortic valve leaflets.
Fig 4.
Immunohistochemical detection and quantification of lysyl oxidase (LOX) expression.
Immunohistochemical staining of (A) healthy and (B) diseased leaflets shows distribution of LOX production throughout leaflet, with some areas of localized high-intensity staining (indicated by arrows). F = fibrosa, S = spongiosa. N = 5. Scale bar = 0.50 mm. (C) Layer-specific quantification of LOX staining, indicating significantly greater amounts of LOX in the diseased fibrosa relative to the healthy condition (p = 0.001728). N = 5, n = 3. Total LOX expression across the entire leaflet was also quantified via (D) qRT-PCR for LOX (N = 5, n = 3) and (E) measurement of LOX protein via dot blot (N = 5, n = 3).