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.
ABSTRACT
PURPOSE: Calcific aortic valve disease (CAVD), has been characterized as a cascade of cellular changes leading to leaflet thickening and valvular calcification. In diseased aortic valves, glycosaminoglycans (GAGs) normally found in the valve spongiosa migrate to the collagen I-rich fibrosa layer near calcified nodules. Current treatments for CAVD are limited to valve replacement or drugs tailored to other cardiovascular diseases. METHODS: Porcine aortic valve interstitial cells and porcine aortic valve endothelial cells were seeded into collagen I hydrogels of varying initial stiffness or initial stiffness-matched collagen I hydrogels containing the glycosaminoglycans chondroitin sulfate (CS), hyaluronic acid (HA), or dermatan sulfate (DS). Assays were performed after 2 weeks in culture to determine cell gene expression, protein expression, protein secretion, and calcification. Multiple regression analyses were performed to determine the importance of initial hydrogel stiffness, GAGs, and the presence of endothelial cells on calcification, both with and without osteogenic medium. RESULTS: High initial stiffness hydrogels and osteogenic medium promoted calcification, while for DS or HA the presence of endothelial cells prevented calcification. CS was found to increase the expression of pro-calcific genes, increase activated myofibroblast protein expression, induce the secretion of collagen I by activated interstitial cells, and increase calcified nodule formation. CONCLUSION: This study demonstrates a more complete model of aortic valve disease, including endothelial cells, interstitial cells, and a stiff and disease-like ECM. In vitro models of both healthy and diseased valves can be useful for understanding the mechanisms of CAVD pathogenesis and provide a model for testing novel therapeutics.
