Vericiguat Attenuates Vascular Calcification by Cyclic Guanosine Monophosphate‐Protein Kinase G Pathway Mediated Suppression of Matrix Metalloproteinase 10

Figures

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  Figure 1. Vericiguat attenuates vascular smooth muscle cell calcification in vitro. A, Experimental design. B, Representative Alizarin Red staining images showing calcium deposition in rat VSMCs. Scale bars: 500 μm. C, Quantification of Alizarin Red staining intensity in VSMCs. D, Calcium content in VSMCs measured by a calcium assay kit. E, Alkaline phosphatase activity in VSMCs. F, Relative mRNA expression of runt‐related transcription factor 2, bone morphogenetic protein‐2, and alpha smooth muscle Actin in VSMCs, assessed by quantitative real‐time polymerase chain reaction. G, Representative Western blot images and densitometric analysis of RUNX2, BMP2, and α‐SMA protein expression in VSMCs. Data are presented as mean±SD, n=4 per group. α‐SMA indicates alpha smooth muscle actin; ALP, alkaline phosphatase; BMP2, bone morphogenetic protein‐2; CM, calcifying medium; GM, growth medium; OD, optical density; RUNX2, runt‐related transcription factor 2; and VSMC, vascular smooth muscle cell.

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  Figure 2. Vericiguat attenuates aortic calcification in rat models. A, Experimental design for rats. B, Representative micro‐computed tomography images showing aortic calcification in rats (scale bar=1 cm). C, Whole‐mount Alizarin Red staining of aortas from rats, indicating mineral deposition (scale bar=1 cm). D, Representative Alizarin Red staining of aortic cross‐sections from rats. Scale bars: 500 μm (Upper) and 250 μm (lower). E, Quantification of Alizarin Red‐positive area in aortas from rats. F, Calcium content in aortic tissues from rats, measured by a calcium assay kit. G, ALP activity in aortic tissues from rats. H, Representative Western blot images and densitometric analysis of OPN, BMP2, and α‐SMA in aortic tissues from rats, normalized to GAPDH. Data are presented as mean±SD, n = 6 per group. α‐SMA indicates alpha smooth muscle actin; ALP, alkaline phosphatase; BMP2, bone morphogenetic protein 2; and OPN, osteopontin.

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  Figure 3. Vericiguat suppresses matrix metalloproteinase 10 expression in vitro and in vivo. A, Venn diagram illustrating the numbers of DEGs among VSMCs cultured in GM, CM, and CM supplemented with vericiguat. B, Heatmap of 23 DEGs expression profiles in VSMCs from GM, CM, and vericiguat groups. Each row represents a gene and each column represents a treatment group. Relative expression levels are shown by the color scale: red indicates higher expression, and green indicates lower expression. C, GO biological process enrichment analyses of DEGs between the vericiguat and CM groups. D, KEGG pathway enrichment analysis of DEGs between the vericiguat and CM groups. E and F, Representative Western blot images and densitometric analysis of MMP‐10 protein expression in (E) VSMCs and (F) rat aortic tissues, normalized to GAPDH. Data are presented as mean ± SD, n=4–6 per group. cGMP indicates cyclic guanosine monophosphate; CM calcifying medium; DEG, differentially expressed gene; GM, growth medium; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; IL‐17, interleukin‐17; JAK/STAT, Janus kinase/signal transducer and activator of transcription; MMP‐10, matrix metalloproteinase 10;…

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  Figure 4. Overexpression of matrix metalloproteinase 10 promotes vascular smooth muscle cell calcification in vitro. A, Representative Alizarin Red staining images showing calcium deposition in rat VSMCs. Scale bars: 500 μm. B, Quantification of Alizarin Red staining intensity in VSMCs. C, Calcium content in VSMCs measured by a calcium assay kit. D, ALP activity in VSMCs. E, Representative Western blot images and densitometric analysis of OPN and BMP2 in VSMCs, normalized to GAPDH. Data are presented as mean±SD, n=4 per group. ALP indicates alkaline phosphatase; BMP2, bone morphogenetic protein‐2; CM, calcifying medium; GM, growth medium; LV, lentiviral vector; MMP‐10, matrix metalloproteinase 10; OD, optical density; OPN, osteopontin; and VSMC, vascular smooth muscle cell.

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  Figure 5. Overexpression of matrix metalloproteinase 10 promotes mouse aortic calcification in vivo. A, Representative whole‐mount Alizarin Red staining images of aortas from mice treated with vericiguat, with or without MMP‐10 overexpression (scale bar=1 cm). B, Representative Alizarin Red‐stained images of aortic cross‐sections, showing mineral deposition. Scale bars: 250 μm (upper), 125 μm (lower). C, Quantification of Alizarin Red‐positive area in aortic tissues. D, Calcium content in aortic tissues measured by a calcium assay kit. E, Alkaline phosphatase activity in aortic tissues. F, Representative Western blot images and densitometric analysis of OPN and BMP2 protein expression in aortic tissues, normalized to GAPDH. Data are presented as mean±SD, n=6 per group. AAV indicates adeno‐associated virus; BMP2, bone morphogenetic protein‐2; MMP‐10, matrix metalloproteinase 10; and OPN, osteopontin.

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  Figure 6. Recombinant rat matrix metalloproteinase 10 mitigates vericiguat‐mediated suppression of calcification in vitro. A, Representative Alizarin Red staining images of rat VSMCs, showing mineral deposition. Scale bars: 500 μm. B, Quantification of Alizarin Red staining intensity using a microplate reader. C, Calcium content in VSMCs measured by a calcium assay kit. D, ALP activity in VSMCs. E, Representative Western blot images and densitometric analysis of OPN and BMP2 protein expression in VSMCs, normalized to GAPDH. Data are presented as mean±SD, n=4 per group. ALP indicates alkaline phosphatase; BMP2, bone morphogenetic protein‐2; CM, calcifying medium; GM, growth medium; OPN, osteopontin; r‐MMP‐10, recombinant rat MMP‐10; and VSMC, vascular smooth muscle cell.

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  Figure 7. Vericiguat activates the cGMP‐PKG signaling pathway and inhibits c‐Jun signaling in vitro and in vivo. A and C, ELISA quantification of cGMP levels in (A) rat VSMCs and (C) rat aortic tissues, showing increased cGMP in vericiguat‐treated groups vs controls. B and D, Representative Western blot images showing expression of PKG, VASP, p‐VASP, c‐Jun, and p‐c‐Jun in (B) VSMCs and (D) rat aortas, normalized to GAPDH. Data are presented as mean ± SD, n = 4–6 per group. cGMP indicates cyclic guanosine monophosphate; c‐Jun, transcription factor Jun; CM, calcifying medium; GM, growth medium; PKG, protein kinase G; p‐c‐Jun, phosphorylated c‐Jun; p‐VASP, phosphorylated VASP; VASP, vasodilator‐stimulated phosphoprotein; and VSMC, vascular smooth muscle cell.

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  Figure 8. A schematic diagram illustrating the anti‐calcification mechanism of vericiguat. Vericiguat stimulates sGC, enhancing cGMP production and activating PKG. PKG activation promotes phosphorylation of vasodilator‐stimulated phosphoprotein and suppresses osteogenic differentiation of VSMCs. In parallel, vericiguat inhibits c‐Jun/AP‐1 signaling, leading to downregulation of MMP‐10, which limits extracellular matrix degradation. These actions collectively stabilize the VSMC contractile phenotype and reduce vascular calcification. This figure was created with BioRender.com. AP‐1 indicates activator protein‐1; cGMP, cyclic guanosine monophosphate; c‐Jun, transcription factor Jun; GTP, guanosine‐5’‐triphosphate; MMP‐10, matrix metalloproteinase 10; NO, nitric oxide; PKG, protein kinase G; sGC, soluble guanylate cyclase; and VSMC, vascular smooth muscle cell.