Comparison of Different Calcification Media on Gene Expression Using a Porcine Aortic Valve Ex Vivo Assay

Background: Calcification of aortic valves is associated with gene expression changes at molecular level. Less is known about the impact of different calcification media on these changes. Hence, this study aimed to compare the impact of different calcification media on gene expression of a subset of calcification-related genes.

Method: Aortic valves of 18 swine were calcified by two commonly used calcification media Osteo (StemXvivo Osteogenetic) and PI (2.6 mM NaH2PO4) for 2 weeks. The basic cell culture DMEM medium containing 10% FKS and 2% penicillin/streptomycin was defined as noncalcification control medium (Basis). Calcification deposit was analyzed by histological examination, scanning electron microscopic, calcium-titration (µg calcium per cm² valve area) and evaluation of tissue thickness. Gene expression of 11 calcification-related genes were investigated and quantified using qPCR analysis: ACTA3 (human smooth muscle actin gene), MMP2 and MMP9 (matrix metalloproteinase-2 and 9), RUNX2 (runt-related transcription factor 2), SSP1 (small secreted protein 1), TNFRSF1 (tumor necrosing factor receptor superfamily 1), COL1A1 (collagen type I α 1), COL3A1 (collagen type III α 1), COL5A1 (collagen type V α 1), VIM (vimentin), and TGF-β1 (transforming growth factor β 1).

Results: Histological examination (Alizarine red S staining) and calcium absorption revealed very low calcification in valves treated with Basis control medium (23.0 ± 4.3 µg/cm²), moderate and isolated calcification noduli after treatment with Osteo (40.7 ± 30 µg/cm²), and distinct calcification pattern in valves after treatment with PI (142.9 ± 37.5 µg/cm²). At molecular level, most of the genes were found to be regulated as response to calcification as compared with Basis medium. RUNX2, ACTA3, and VIM were found significantly upregulated (p < 0.036). SSP1 was found to be down-regulated (Basis 1.225 ± 0.666 vs. Osteo 0.469 ± 0.328; p = 0.019). The highest upregulation was noticed for RUNX2, known as master regulator of calcification with 4.19-fold change in Osteo medium (Basis 1.074 ± 0.461 vs. Osteo 4.192 ± 2.086; p = 0.002).

Conclusion: Our data demonstrated the upregulation of RUNX2 with calcification. RUNX2 may be used as reliable marker together with histological analysis to quantify the extent of calcification in aortic valves at molecular level. In addition, such findings can be used to investigate specific gene questions involved in valve calcification pathomechanism.

Publication History

Article published online:
28 January 2023