Calcific aortic valve disease (CAVD) is one of the most common heart valve diseases 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 and extracellular matrix (ECM) interactions, and current 3D models induce calcification using osteogenic media. The goal of this work is to develop a 3D dystrophic calcification model. We hypothesize that, as with 2D models, mechanical stress-induced apoptosis is integral to calcification and that oxidative stress plays a minor role. We model the cell aggregation observed in CAVD by creating porcine valvular interstitial cell spheroids in agarose microwells. Upon culture in standard growth media, calcium nodules form within the spheroids. In contrast, supplementation with ascorbic acid (AA) virtually eliminates calcium buildup. As this effect has previously been attributed to the antioxidant properties of AA rather than its stimulation of ECM production, other antioxidants (Trolox and Methionine) were also used as supplements as these reduce oxidative stress while having differing effects on ECM formation. All three antioxidants significantly reduced calcification as measured by Von Kossa staining, with percent calcification per area of AA, Trolox, Methionine, and the control on day 7 equaling 0.17%, 2.46%, 5.99%, and 7.71%, respectively. As AA inhibited calcification significantly more than the other antioxidants, other effects of AA such as ECM stimulation may also play a role. Inhibiting apoptosis with Z-VAD also significantly reduced calcification demonstrating that the calcification observed in this model is dystrophic rather than osteogenic. We conclude that while not the sole factor, oxidative stress plays a crucial role in calcification in this 3D in vitro model and may be a viable therapeutic target for CAVD.
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