Inhibiting TNAP Attenuates the Aortic Valve Calcification and Reduces Oxidative Stress

Figures

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  Figure 1. TNAP is overexpressed in CAVD-induced mouse model. (A) Schematic representation of vitamin D3-induced CAVD mouse model. (B) H&E and von Kossa (VK) staining of aortic valve tissue sections from normal and vitamin D3-induced CAVD mice at the indicated doses (6.5 × 105, 7.5 × 105, 8.5 × 105 IU/kg) (scale bar = 100 μm). (Calcified areas are stained in black) (C) TNAP expression in normal and CAVD mouse valve tissues, assessed by immunohistochemical staining and quantitative analysis (scale bar = 50 μm). (Brown indicates positive staining; blue indicates cell nuclei counterstained with hematoxylin). Data are shown as mean ± SD. * p-value < 0.05.

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  Figure 2. Proteomics analysis of calcified human aortic valve tissues. (A) Volcano plot between normal and calcified aortic valve tissues. (B) Heatmap of 277 proteins. (C) Gene Ontology enrichment analysis of the 34 up-regulated proteins. (D) Protein–protein interaction network of the 34 up-regulated proteins generated using the STRING database.

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  Figure 3. Effects of TNAP inhibitors in patients-derived VICs. (A) Alizarin Red-S staining in SaOS-2 cells. (B) Quantitative analysis of calcification in SaOS-2. (C) Alizarin Red-S staining in VICs from 3 individual patients. (D) Quantitative analysis of calcification in VICs. (OM, osteogenic medium). * p-value < 0.05.

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  Figure 4. Effects of TNAP inhibitors on calcification in a CAVD mouse model. (A) H&E and von Kossa staining of aortic valve tissue from normal, CAVD, DS-1211 treated, and RNV-1002 treated mice (DS-1211 at 10 mg/kg; RNV-1002 at 5, 10, and 20 mg/kg) (Scale bar = 100 μm). (Calcified areas are stained in black) (B) Quantitative analysis of annulus thickness, immune cell infiltration, and calcified regions, respectively. (C) Immunohistochemical staining of aortic valves for TNAP and ENPP2, respectively (Scale bar = 100 μm). (Brown indicates positive staining; blue indicates cell nuclei counterstained with hematoxylin) (D) Quantification of TNAP and ENPP2-positive cells per mm2 of the aortic valve region across the indicated groups. (E) Relative mRNA expression levels of pro-osteogenic markers (RANKL, OPG, and BMP-2) and an anti-calcific marker (MGP) in heart tissue. Data are shown as mean ± SD. * p-value < 0.05.

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  Figure 5. Effects of TNAP inhibitors on oxidative stress, lipid peroxidation, and RNS signaling. (A) mRNA level of ROS response markers (Nrf2 and NF-κB). (B) Activity levels of antioxidant markers (SOD, GSH and CAT) in heart tissue. (C) Levels of lipid peroxidation markers (MDA, 4-HNE, and MPO) in heart tissue. (D) RNS signaling markers (iNOS and NT) in heart tissue. * p-value < 0.05.

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  Figure 6. Effects of TNAP inhibitors on inflammation, apoptosis and fibrosis in a CAVD mouse model. (A,B) Immunohistochemical staining and quantitative analysis of IL-1β, TNF-α, iNOS, and COX-2 in aortic valve tissues (Scale bar = 100 μm). (C,D) Immunohistochemical staining and quantitative analysis of Cleaved Cas-3 and Cleaved PARP in aortic valve tissues (Scale bar = 100 μm). (E,F) Immunohistochemical staining and quantitative analysis of fibrosis markers TGF-β1 and α-SMA in aortic valve tissues. Data are shown as mean ± SD. * p-value < 0.05.

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  Figure 7. Effects of RNV-1002 on in vitro osteoclastogenesis. (A) TRAP staining and quantitative analysis of osteoclast-like cell differentiation in RANKL-induced osteoclasts treated with RNV-1002. The lower row of images represents magnified views of the areas highlighted by the red circles in the upper row. (B) Relative fluorescence intensity of RANKL-induced osteoclasts treated with RNV-1002. (C) Relative mRNA expression levels of osteoclast differentiation markers (CsK, NFATc1, RANK and MMP-9) in RANKL-induced osteoclasts treated with RNV-1002. (D) Cell viability of RAW264.7 cells treated with RNV-1002. (E) Cell viability of RANKL-induced osteoclasts treated with RNV-1002. Data are shown as mean ± SD. * p-value < 0.05, ns: not significant.