Crystalline ultrastructures, inflammatory elements, and neoangiogenesis are present in inconspicuous aortic valve tissue

Abstract

Morbidity from calcific aortic valve disease (CAVD) is increasing. Recent studies suggest early reversible changes involving inflammation and neoangiogenesis. We hypothesized that microcalcifications, chemokines, and growth factors are present in unaffected regions of calcific aortic valves. We studied aortic valves from 4 patients with CAVD and from 1 control, using immunohistochemistry, scanning electron microscopy, and infrared spectrography. We revealed clusters of capillary neovessels in calcified (ECC), to a lesser extent in noncalcified (ECN) areas. Endothelial cells proved constant expression of SDF-1 in ECC, ECN, and endothelial cells from valvular surface (ECS). Its receptor CXCR4 was expressed in ECC. IL-6 expression correlated with CXCR4 staining and presence of lymphocytes. VEGF was expressed by ECS, its receptor by ECC and ECN. Crystalline ultrastructures were found on the surface of histologically noncalcified areas (HNCAs), spectrography revealed calcium hydroxylapatite. Our results demonstrate that crystalline ultrastructures are present in HNCAs, undergoing neoangiogenesis in an inflammatory context. These alterations could be an early witness of disease and an opening to therapy.

Figure 1

Immunohistochemical pattern of inflammatory infiltrate and neovascular endothelial cells in microscopically calcified (ECC) and noncalcified (ECN) areas, as well as of surface endothelial cells (ECS) in aortic valves from patients with calcific aortic stenosis. (a) Hematoxylin-Eosin-Saffron staining, note multiple capillary neovessels near calcified area (arrows); (b) CD3+ lymphocytes in the range of neovessels (asterisks) near calcific lesions; (c) CD117+ cells corresponding to mast cells are sparsely present. (d) Neovessels of all subgroups are highlightened by FVIII staining (here: ECC) (e) CXCL12 (ECC); (f) CXCL12 (ECN); (g) CXCR4 (ECC); (h) CXCR4 (ECN); (i) IL-6 (ECC); (j) VEGF in surface endothelial cells (arrows on ECS); (k) VEGFR (arrows on ECC); (l) VEGFR (arrows on ECN). Images (a),(d),(f),(g),(h),(i),(j), and (k): magnification ×100; images (b),(c),(e), and (l): magnification ×200.

Figure 2

Scanning electron micrographs of crystalline ultrastructures on the surface of inconspicuous valve areas in patients suffering from CAVD. (a) Bundles of faceted microrods are inserted into the leaflet surface. Magnification ×5790. (b) Surface areas are covered with round-shaped or sphere-like, flat structures adherent to the leaflet surface. Note the additional small bundle of plate-like facetted microrods (center). Magnification ×1790.

Figure 3

EDX spectra collected for the ultrastructures presented above (blue graph) and for massively calcified lesions within the same valve (red graph) (SEM not shown). The contributions of light elements such as C (carbon), O (oxygen), and Na (sodium) are predominating within crystalline ultrastructures, while massive calcifications display higher peaks of elements such as P (phosphor) and Ca (calcium). Results are fitting into the chemical composition given by FT-IR (see beneath).