The Role of Apoptosis and Oxidative Stress in a Cell Spheroid Model of Calcific Aortic Valve Disease

Calcific aortic valve disease (CAVD) is the most common heart valve disease among aging populations. There are two reported pathways of CAVD: osteogenic and dystrophic, the latter being more prevalent. Current two-dimensional (2D) in vitro CAVD models have shed light on the disease but lack three-dimensional (3D) cell-ECM interactions, and current 3D models require osteogenic media to induce calcification. The goal of this work is to develop a 3D dystrophic calcification model. We hypothesize that, as with 2D cell-based CAVD models, programmed cell death (apoptosis) is integral to calcification. We model the cell aggregation observed in CAVD by creating porcine valvular interstitial cell spheroids in agarose microwells. Upon culture in complete growth media (DMEM with serum), calcium nodules form in the spheroids within a few days. Inhibiting apoptosis with Z-VAD significantly reduced calcification, indicating that the calcification observed in this model is dystrophic rather than osteogenic. To determine the relative roles of oxidative stress and extracellular matrix (ECM) production in the induction of apoptosis and subsequent calcification, the media was supplemented with antioxidants with differing effects on ECM formation (ascorbic acid (AA), Trolox, or Methionine). All three antioxidants significantly reduced calcification as measured by Von Kossa staining, with the percentages of calcification per area of AA, Trolox, Methionine, and the non-antioxidant-treated control on day 7 equaling 0.17%, 2.5%, 6.0%, and 7.7%, respectively. As ZVAD and AA almost entirely inhibit calcification, apoptosis does not appear to be caused by a lack of diffusion of oxygen and metabolites within the small spheroids. Further, the observation that AA treatment reduces calcification significantly more than the other antioxidants indicates that the ECM stimulatory effect of AA plays a role inhibiting apoptosis and calcification in the spheroids. We conclude that, in this 3D in vitro model, both oxidative stress and ECM production play crucial roles in dystrophic calcification and may be viable therapeutic targets for preventing CAVD.