Oxidative low-density lipoprotein and shear induced calcification within a calcific aortic valve disease-on-a-chip platform

Figures

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  Figure 1. Experimental methods of static and dynamic aortic valve fibrosa model.

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  Figure 2. Early disease progression markers after 2 days in culture: static and dynamic collagen hydrogels with PAVIC and PAVIC/PAVEC co- culture. (A–F). Alkaline phosphatase activity normalized to protein content via Bradford assay (G–L). Sulfated glycosaminoglycan production and release into culture medium detected with Alcian Blue and normalized to protein content via Bradford assay. Data shown as mean ± SEM, where static (n ≥ 5 samples) and dynamic (n = 3 samples), and statistical significance shown according to two-sided Mann–Whitney test, *p < 0.05. (PAVIC, porcine aortic valve interstitial cells, PAVEC, porcine aortic valve endothelial cells, oxLDL, oxidative low-density lipoproteins).

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  Figure 3. Hydroxyapatite formation qualification and quantification in both static and microfluidic cultures after 2 days, PAVIC-only and when co-cultured with PAVEC. (A). Scanning electron microscopy (SEM) qualitative images with arrows indicating the presence of smaller nodules (scale = 500 nm). Energy dispersive x-ray spectroscopy (EDX) calcium and phosphorus elemental atomic percentages (At%) for (B). PAVIC-only and (C). PAVIC + PAVIC co-cultures, where Mean ± SEM, n ≥ 18 measurements per sample, and statistical significance shown in Supplementary Figure S3. Calcium phosphate mineralization based on EDX At% (D). PAVIC-only and (E). PAVIC + PAVEC co- cultures, where monocalcium phosphate calcium to phosphate ratio (Ca/P) = 0.5, dicalcium phosphate Ca/P = 1.0, octacalcium phosphate Ca/P = 1.33, and hydroxyapatite Ca/P = 1.67, Mean ± SEM, n ≥ 18 calculations per sample, and statistical significance shown according to Kruskal–Wallis with Dunn's multiple comparisons post-hoc test, *p < 0.05. (PAVIC, Porcine aortic valve interstitial cells; PAVEC, Porcine aortic valve endothelial cells; oxLDL25, 25 µg/ml oxidative low-density lipoproteins; oxLDL50, 50 µg/ml oxidative low-density…

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  Figure 4. Static and dynamic oxLDL-cell interactions after 2 days in culture with PAVIC and PAVIC + PAVEC co-culture. Key: Plasma membrane (CellMask = Red), DNA (Hoechst = Blue), oxLDL (Dil-oxLDL = Yellow). (scale = 50 µm, highlighted box then zoomed into show individual cells orthogonally). (COL-I, Collagen-I; PAVIC, porcine aortic valve interstitial cells; PAVEC, porcine aortic valve endothelial cells; oxLDL, oxidative low-density lipoproteins).

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  Figure 5. Endothelial dysfunction in static and dynamic PAVIC + PAVEC co-culture with oxLDL after 2 days. (A). Scanning electron microscopy (SEM) qualitative images with arrows indicating the presence of smaller nodules (scale = 1 µm). Quantification of endothelial dysfunction as shown by (B). Frequency of endothelial pores, (C). Average percent area of pores compared to total cell area, and (C). Area (µm2) of individual pores measured (n ≥ 400 measurements), where Mean ± SEM, n ≥ 3 images of cells analyzed, and statistical significance shown according to Kruskal–Wallis with Dunn's multiple comparisons post-hoc test, *p < 0.05. (PAVIC, Porcine aortic valve interstitial cells; PAVEC, Porcine aortic valve endothelial cells; oxLDL25, 25 µg/ml oxidative low-density lipoproteins; oxLDL50, 50 µg/ml oxidative low-density lipoproteins).