Figure 3.

PCSK9 Is Highly Abundant in Calcified Leaflets

(A) Representative explanted aortic valve leaflet with normal structure (control valve) and (B) representative calcified aortic valve leaflet (stenotic valve), both stained with Von Kossa to visualize calcium deposits (upper panels) and with anti-PCSK9 antibody (lower panels). PCSK9 immunohistochemical (IHC) staining is presented as 3,3′-diaminobenzidine (DAB) and hematoxylin (H) counter staining (DAB + H) and as deconvoluted image visualizing only the DAB staining. Panoramic images were taken with a 10× magnification. Black boxes indicate the higher magnification areas. (C and D) High magnification areas of aortic valve leaflets (20× left and 40× right). Arrows indicate representative PCSK9 expression in close proximity to cell nuclei. (E) Box and whisker plots represent DAB quantification by ImageJ with IHC Tool box plugin on normal (control; n = 6) and calcified (stenotic; n = 6) leaflets. (F) Box and whisker plots represent PCSK9 levels measured by enzyme-linked immunosorbent assay on control (n = 6) and stenotic (n = 6) whole tissue extracts.