Organ Culture Model of Aortic Valve Calcification

Figures

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  Figure 1. Schematic diagram of the experimental protocol. Following incubation of the pinned-out valve leaflets, they are fixed while still in the wells with 10% formal saline. Each leaflet is cut into 3 portions radially, processed and embedded in wax blocks and cut longitudinal sections cut on a microtome. Alizarin Red staining of a slide from each of the three leaflet portions is viewed under polarized light and then quantified using Image J, to ultimately calculate the percentage of the area of the leaflet that stains for Alizarin Red.

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  Figure 2. Optimisation of the response of valve leaflets to LPS and phosphate. (A) The effect of LPS concentration on calcium accumulation in porcine aortic valve leaflets in culture for 14 days in the presence of 3 mM phosphate (*p = 0.071, **p = 0.003, ANOVA; n = 3) and representative images of sections stained with Alizarin Red, imaged under polarized light for each concentration of LPS. (B) The effect of phosphate concentration on calcium accumulation in porcine aortic valve leaflets in culture for 14 days in the presence of 100 ng/mL LPS (*P = 0.015, **P = 0.001, ANOVA; n = 4) and representative images of sections stained with Alizarin Red, imaged under polarized light for each concentration of phosphate. (C) Time course of the effect of culture media (black bars) and 100 ng/mL LPS and 3 mM phosphate (cross-hatched bars) on calcium accumulation in porcine aortic valve leaflets in culture for 3 to 14 days (*P = 0.001, 2 way ANOVA, n = 2) and representative images of sections stained with Alizarin Red, imaged under polarized light for each time point.

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  Figure 3. Leaflet-specific and regional effects of LPS and phosphate. (A) Leaflet specific effects of 100 ng/mL LPS and 3 mM phosphate on calcium accumulation in the left-, right- and non-coronary leaflets of porcine aortic valves. Values are the mean of the measurement from the three regions of each leaflet (p > 0.05, ANOVA; n = 6). (B) Comparison of the effect of 100 ng/mL LPS and 3 mM in the 3 regions of the left- right- and non-coronary leaflet assessed for calcium accumulation (p > 0.05, ANOVA; n = 6).

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  Figure 4. Electron microscopy and EDAX analysis of calcified valve leaflets. Scanning electron micrographs of porcine valve leaflets stimulated with media alone (A) or 100 ng/mL LPS and 3 mM phosphate for 14-days (B), with presence of calcified areas (white arrows) (x100 magnification, scale bar = 100 μM). High power view and point analysis (yellow cross) of aortic valve leaflets from control and LPS/PO4 treated valves (x1,000 magnification, scale bar =10 μM) (C,D), and the spectrum showing no calcium or phosphorus peaks in the control (E), but their presence in the LPS/PO4 treated tissue (F).

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  Figure 5. Elemental mapping of LPS and phosphate treated valve leaflets. Scanning electron micrographs showing the elemental distribution of carbon, oxygen, phosphorus, calcium and silicon. Regions of greater electron density corresponding to the distribution of calcium and phosphorus.

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  Figure 6. Expression of NF-kB in calcified valve leaflets. Low and high-powered images of immunohistochemical staining for NF-kB in untreated (A,B) and 100 ng/mL LPS & 3 mM phosphate treated (C,D) valve leaflets. Nuclear expression of NF-kB can be seen in (D) (black arrows). (A,C) scale bar, 200 μM; (B,D) scale bar, 500 μM.

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  Figure 7. Colocalisation of Alizarin Red staining with osteogenic, inflammatory and apoptotic markers. Sequential sections of a valve leaflet stimulated media alone (left column) or 100 ng/mL LPS and 3 mM phosphate (right column) for 14-days. Histochemical staining for Alizarin Red (A,B) co-localizes with regions of immunohistochemical staining for osteocalcin (C,D), RUNX2 (E,F), NF-kB (G,H) and caspase 3 (I,J). Scale bar, 500 μM.

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  Figure 8. Quantification of osteogenic, inflammatory and apoptotic markers in Alizarin Red positive and negative areas of valve leaflets. Percentage area staining of 1.1 mm2 regions from non-calcified and calcified regions for (A) osteocalcin (*P = 0.012, T-Test; n = 4), (B) RUNX2 (**P < 0.001, T-Test; n = 4), (C) NF-kB (**P < 0.001, T-Test; n = 4) and (D) caspase 3 (**P < 0.001, T-Test; n = 4).

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  Figure 9. Effect of adenosine on the expression of osteogenic markers. Immunohistochemical staining in media alone (left column), 100 ng/mL LPS 3 μM phosphate treated (center column) and 100 ng/mL LPS 3 mM phosphate and 10−5M adenosine treated (right column) valve leaflets. Section were stained with RUNX2 (A–C), osteopontin (D–F) and osteocalcin (G–I). Scale bar, 100 μM.

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  Figure 10. Inhibitory effect of adenosine on calcification and expression of osteogenic markers. Effect of increasing concentrations of adenosine (10−8-10−5M) on the expression of Alizarin Red in sections of valve leaflet response to 100 ng/mL and 3 mM phosphate (*P = 0.008, T-Test; n = 8 and **P = 0.011, T-Test; n = 6) and representative images of sections stained with Alizarin Red imaged under polarized light for each treatment group (A). Quantification of the area of positive staining in control (media alone), 100 ng/mL and 3 mM phosphate and 100 ng/mL & 3 mM phosphate with 10−5M adenosine for (B) osteocalcin (**P = 0.001, *P = 0.029, ANOVA; n = 11–15 areas from 3 valves), (C) osteopontin (**P < 0.001, *P = 0.029, ANOVA; n = 13–15 areas from 3 valves) and (D) RUNX2 (*P = 0.003, **P = 0.001, ANOVA; n = 9–14 areas from 3 valves). (E) The mean area per measurement was similar in all 3 groups.