Glycosaminoglycans affect endothelial to mesenchymal transformation, proliferation, and calcification in a 3D model of aortic valve disease

Abstract

Calcific nodules form in the fibrosa layer of the aortic valve in calcific aortic valve disease (CAVD). Glycosaminoglycans (GAGs), which are normally found in the valve spongiosa, are located local to calcific nodules. Previous work suggests that GAGs induce endothelial to mesenchymal transformation (EndMT), a phenomenon described by endothelial cells’ loss of the endothelial markers, gaining of migratory properties, and expression of mesenchymal markers such as alpha smooth muscle actin (α-SMA). EndMT is known to play roles in valvulogenesis and may provide a source of activated fibroblast with a potential role in CAVD progression. In this study, a 3D collagen hydrogel co-culture model of the aortic valve fibrosa was created to study the role of EndMT-derived activated valvular interstitial cell behavior in CAVD progression. Porcine aortic valve interstitial cells (PAVIC) and porcine aortic valve endothelial cells (PAVEC) were cultured within collagen I hydrogels containing the GAGs chondroitin sulfate (CS) or hyaluronic acid (HA). The model was used to study alkaline phosphatase (ALP) enzyme activity, cellular proliferation and matrix invasion, protein expression, and calcific nodule formation of the resident cell populations. CS and HA were found to alter ALP activity and increase cell proliferation. CS increased the formation of calcified nodules without the addition of osteogenic culture medium. This model has applications in the improvement of bioprosthetic valves by making replacements more micro-compositionally dynamic, as well as providing a platform for testing new pharmaceutical treatments of CAVD.