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

Abstract

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.

Keywords: antioxidant; apoptosis; calcific aortic heart valve disease; oxidative stress; valvular interstitial cell.

Figure 1

(A) Phase (10×) image of control day 2 porcine aortic VIC spheroids formed in non-adherent agarose mold. Each mold is seeded with one million VICs. The red circle indicates the outline of a single well in the mold, with the darker circles being formed spheroids. Scale bar = 100 μm. (B) Calcified Spheroid: Phase (20×) image of Von Kossa stain showing calcification on a day 4 porcine aortic VIC spheroid. The red arrow points to a calcific nodule, which became black once stained. Scale bar = 100 μm. (C) Change in spheroid size over the course of seven days in culture.

Figure 2

Control spheroids (top) and ascorbic acid-supplemented spheroids (bottom) after four days in culture. Fluorescent (right) with live (green)/Dead (red) stain and 10× phase (left). Passage 7 porcine aortic VICs were used. Supplementation with 250 μm ascorbic acid almost completely inhibited cell death and prevented calcification. Scale bar = 100 μm.

Figure 3

Z-VAD supplementation reduces apoptosis: Day 2 Control (top), Day 4 (middle), and Day 7 (bottom) spheroids 10× phase (left) and with caspase stain (right). Passage 9, day 4 porcine aortic VIC spheroids treated with 20µM caspase inhibitor Z-VAD for five days prior to creating the spheroids and for the duration of the experiment. Scale bar = 100 μm.

Figure 4

Z-VAD supplementation inhibits calcification. Images (20×, transmitted light) of passage 9, day 4 (top), and day 7 (bottom) of porcine aortic VIC spheroids stained with Von Kossa. Control spheroids (left) show pronounced calcification as indicated by dark nodules. In contrast, spheroids treated with Z-VAD show only slight calcification (indicated with a red arrow). Scale bars = 100 μm.

Figure 5

Calcium stain on antioxidant-supplemented spheroids on day 4: Images (20×) of passage 4 porcine aortic VIC spheroids on day 4 with Von Kossa staining for various antioxidant treatments. Scale bar = 100 μm.

Figure 6

Calcium stain on antioxidant-supplemented spheroids on day 7: Phase image (20×) of passage 4 porcine aortic VIC spheroids on day 7 with Von Kossa staining for various antioxidant treatments. Scale bar = 100 μm.

Figure 7

Quantification of results from antioxidant treatment: Percent of calcification per total area of spheroids treated with different antioxidants (AA 250 μm, Trolox 500 μm, Methionine 200 μm) on days 4 and 7. Asterixs (*) represent a significant difference from the same-day control group (no antioxidants), and double asterixs (**) represent a significant difference from the same-day AA-treated group (p < 0.05; one-way ANOVA on ranks with Dunn’s test post hoc pairwise comparisons).