Superoxide (red fluorescence) in a normal (A) and stenotic (B) aortic valve detected with dihydroethidine (DHE) fluorescence. Superoxide levels were markedly elevated near the calcified (calc) region of the valve and were markedly reduced by the addition of polyethylene glycol superoxide dismutase. Magnified images of noncalcified (non-calc) and calcified regions of a stenotic valve with DHE staining are shown in C and D. (E) Superoxide levels measured with lucigenin-enhanced chemiluminescence in corresponding regions of normal and stenotic valves (n = 14 control valves, n = 20 stenotic valves; *p < 0.05 vs. noncalcified stenotic tissue; #p < 0.05 vs. base and tip of normal valves).

Hydrogen peroxide (H₂O₂) in a normal (A) and stenotic (C) aortic valve detected with dichlorofluorescein (DCF) fluorescence. Levels of H₂O₂ were markedly elevated near the calcified regions of the valve, and most of the DCF fluorescence was eliminated by pre-incubation of the slide with polyethylene glycol (PEG)-catalase (CAT) (B and D). (E) The PEG-CAT–inhibitable fraction of DCF fluorescence in normal (base and tip regions) and stenotic (calcified and noncalcified regions) aortic valves (n = 4 normal valves, n = 7 stenotic valves; *p < 0.05 vs. noncalcified stenotic tissue, #p < 0.05 vs. base region of normal valves). Abbreviations as in Figure 1.

(A) Expression of the 3 superoxide dismutase (SOD) isoforms in normal and noncalcified and calcified regions of stenotic aortic valves (n = 16 normal valves, n = 15 stenotic valves). (B) Regional total SOD activity in stenotic aortic valves (n = 10 normal valves, n = 11 stenotic valves; *p < 0.05 vs. normal valves; #p < 0.05 vs. noncalcified stenotic tissue). mRNA = messenger ribonucleic acid; other abbreviations as in Figure 1.

n = 10 normal valves, n = 11 stenotic valves; *p < 0.05 versus normal valves; #p < 0.05 versus noncalcified stenotic tissue. Abbreviations as in Figures 1 and 3.

(A) Expression of subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in normal (n = 16) and stenotic aortic valves (n = 15 valves, with 15 noncalcified and 15 calcifed regions). (B) The NADPH oxidase activity in normal (n = 8 base and tip regions) and stenotic aortic valves (n = 14 calcified and noncalcified regions). Effects of inhibitors of enzymatic sources of superoxide in noncalcified (C) and calcified (D) regions of stenotic aortic valves measured with lucigenin-enhanced chemiluminescence (n = 5 to 11/group). APO = apocynin; DPI = diphenylidonium; RLU = relative light units; other abbreviations as in Figures 1 and 3.

Antibodies against CD68 and -SMA were used to detect macrophages and activated myofibroblasts, respectively, in noncalcified and calcified regions. Representative images from n = 5. Inset images are negative control images from adjacent sections.

Immunofluorescent staining for Msx1 (A and B), Msx2 (C and D), and CBFA1 (E and F) in noncalcified and calcified regions of stenotic aortic valves (inset images are negative control images from adjacent sections; representative images from n = 5/stain). Panels G and H show messenger ribonucleic acid (mRNA) levels of osteopontin and CBFA1/Runx2 mRNA in normal valves and in noncalcified and calcified regions of stenotic aortic valves (*p < 0.05 vs. normal tissue, #p < 0.05 vs. noncalcified stenotic tissue, n = 8/group). Abbreviations as in Figure 1.

= no change; +/– = increases or decreases have been reported; NADPH = nicotinamide adenine dinucleotide phosphate; NOS = nitric oxide synthase.