Vitamin K2 inhibits rat vascular smooth muscle cell calcification by restoring the Gas6/Axl/Akt anti-apoptotic pathway

Abstract

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 CaCl- 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 CaCl- 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 CaCl- and β-GP-induced apoptosis and calcification. These results suggest that Gas6 is critical in VK2-mediated functions that attenuate CaCl- and β-GP-induced VSMC calcification by blocking apoptosis.

Signaling pathways involved in vascular smooth muscle cell calcification during hyperphosphatemia

“…In response to phosphate, a multitude of up-stream signaling cascades may lead eventually also to the activation of pro-apoptotic signaling pathways in VSMCs [ 6 , 56 , 74 , 178 , 182 , 183 ]. A key event is represented by the downregulation of growth arrest-specific gene 6 (Gas6) and its receptor tyrosine kinase Axl [ 74 , 182 , 183 ].…”

“…In response to phosphate, a multitude of up-stream signaling cascades may lead eventually also to the activation of pro-apoptotic signaling pathways in VSMCs [ 6 , 56 , 74 , 178 , 182 , 183 ]. A key event is represented by the downregulation of growth arrest-specific gene 6 (Gas6) and its receptor tyrosine kinase Axl [ 74 , 182 , 183 ]. Phosphate reduces the expression of Gas6 and Axl in VSMCs [ 184 ], leading to Bcl2 inactivation, activation of the pro-apoptotic protein Bcl2-associated death promoter (Bad), and subsequent caspase-3 activation and VSMC apoptosis [ 74 , 182 ].…”

“…A key event is represented by the downregulation of growth arrest-specific gene 6 (Gas6) and its receptor tyrosine kinase Axl [ 74 , 182 , 183 ]. Phosphate reduces the expression of Gas6 and Axl in VSMCs [ 184 ], leading to Bcl2 inactivation, activation of the pro-apoptotic protein Bcl2-associated death promoter (Bad), and subsequent caspase-3 activation and VSMC apoptosis [ 74 , 182 ]. Gas6/Axl activates Bcl2 via AKT [ 183 ], a key downstream signaling pathway of the Gas6-mediated VSMC survival [ 183 ].…”

The Inhibitory Roles of Vitamin K in Progression of Vascular Calcification

“…Interaction between Gas6 and Axl activates the PI3K/Akt pathway resulting in stimulation of cell survival pathway [64]. Therefore, apoptosis-mediated VSMC calcification can be inhibited by the Gas6/Axl-PI3K/Akt pathway activated by statin and vitamin K2 [65,66]. GRP also function as an inhibitor of vascular calcification.…”

Bavachin Protects Human Aortic Smooth Muscle Cells Against β-Glycerophosphate-Mediated Vascular Calcification and Apoptosis via Activation of mTOR-Dependent Autophagy and Suppression of β-Catenin Signaling

“…Since β-GP can induce apoptosis in HASMCs (Qiu et al, 2017), to examine the downstream apoptotic signaling during β-GP-induced apoptosis in HASMCs, we examined the cleaved form of Caspase3 and Caspase9, Bak, and Bax of which the apoptosis-related protein, and Bcl-2, an inhibitory protein for apoptosis. Results indicated that bavachin dose-dependently suppressed the β-GP-induced expression of apoptosis-related proteins Bax, Bak, Caspase3 and Caspase9, it also prevented the β-GP-mediated suppression of Bcl-2, and the ratio of Bcl-2/Bax was gradually increased ( Figure 4A, p < 0.01).…”