Vascular calcification is associated with cardiovascular disease as a complication of hypertension, hyperlipidemia, diabetes mellitus, and chronic kidney disease. Vitamin K2 (VK2) delays vascular calcification by an unclear mechanism. Moreover, apoptosis modulates vascular smooth muscle cell (VSMC) calcification. This paper aimed to study VK2-modified VSMC calcification and survival cell signaling mediated by growth arrest-specific gene 6 (Gas6) and its tyrosine kinase receptor Axl. Primary-cultured VSMCs were dose-dependently treated with VK2 in the presence of calcification medium for 8 days, or pre-treated for 1 h with/without the Axl inhibitor R428 (2 μmol/L) or the caspase inhibitor Z-VAD-fmk (20 μmol/L) followed by treatment with VK2 (10 μmol/L) or rmGas6 (200 nmol/L) in calcification medium for 8 days. Calcium deposition was determined by the o-cresolphthalein complexone assay and Alizarin Red S staining. Apoptosis was determined by TUNEL and flow cytometry using Annexin V-FITC and propidium iodide staining. Western blotting detected the expressions of Axl, Gas6, p-Akt, Akt, and Bcl2. VK2 significantly inhibited CaCl2- and β-sodium glycerophosphate (β-GP)-induced VSMC calcification and apoptosis, which was dependent on restored Gas6 expression and activated downstream signaling by Axl, p-Akt, and Bcl2. Z-VAD-fmk significantly inhibited CaCl2- and β-GP-induced VSMC calcification and apoptosis. Augmented recombinant mouse Gas6 protein (rmGas6) expression significantly reduced VSMC calcification and apoptosis. Furthermore, the Gas6/Axl interaction was inhibited by R428, which abolished the preventive effect of VK2 on CaCl2- and β-GP-induced apoptosis and calcification. These results suggest that Gas6 is critical in VK2-mediated functions that attenuate CaCl2- and β-GP-induced VSMC calcification by blocking apoptosis.
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This work was supported by the National Science Foundation of China (Nos. 81170256 and 81070698) and the Development Project of Scientific and Technological Research of Shanxi Province (2009K13-01).
This work was supported by the National Science Foundation of China (Nos. 81170256 and 81070698) and the Development Project of Scientific and Technological Research of Shanxi Province (No. 2009K13-01).
Conflicts of interest
The authors declare that they have no conflict of interest.
The experiments were performed in accordance with the National Institutes of Health Guidelines for the Use of Laboratory Animals and were approved by the Institutional Review Board (IRB) of the Fourth Military Medical University.
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Qiu, C., Zheng, H., Tao, H. et al. Vitamin K2 inhibits rat vascular smooth muscle cell calcification by restoring the Gas6/Axl/Akt anti-apoptotic pathway. Mol Cell Biochem 433, 149–159 (2017). https://doi.org/10.1007/s11010-017-3023-z
- Vitamin K2
- Growth arrest-specific gene 6 (Gas-6)
- Vascular calcification
- Vascular smooth muscle cells