Statins Protect Human Aortic Smooth Muscle Cells From Inorganic Phosphate-Induced Calcification by Restoring Gas6-Axl Survival Pathway

Abstract

Abstract-Vascular calcification is clinically important in the development of cardiovascular disease. It is reported that hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) inhibited vascular calcification in several clinical trials. However, the mechanism is poorly understood. Recently, it has been suggested that apoptosis is one of the important processes regulating vascular smooth muscle cell (VSMC) calcification. In this study, we investigated the effect of statins on VSMC calcification by testing their effect on apoptosis, focusing in particular on regulation of the survival pathway mediated by growth arrest-specific gene 6 (Gas6), a member of the vitamin K-dependent protein family, and its receptor, Axl. In human aortic smooth muscle cells (HASMC), statins significantly inhibited inorganic phosphate (Pi)-induced calcification in a concentration-dependent manner (reduced by 49% at 0.1 mol/L atorvastatin). The inhibitory effect of statins was mediated by preventing apoptosis, which was increased by Pi in a concentration-dependent manner, and not by inhibiting sodium-dependent phosphate cotransporter (NPC) activity, another mechanism regulating HASMC calcification. Furthermore, the antiapoptotic effect of statins was dependent on restoration of Gas6, whose expression was downregulated by Pi. Restoration of Gas6 mRNA by statins was mediated by mRNA stabilization, and not by an increase in transcriptional activity. Suppression of Gas6 using small interfering RNA and the Axl-extracellular domain abolished the preventive effect of statins on Pi-induced apoptosis and calcification. These data demonstrate that statins protected HASMC from Pi-induced calcification by inhibiting apoptosis via restoration of the Gas6-Axl pathway. Key Words: calcification Ⅲ statins Ⅲ apoptosis Ⅲ Gas6 Ⅲ Axl V ascular calcification, such as coronary and aortic calcification, is a significant feature of vascular pathology, because this lesion is associated with cardiovascular disease. 1,2 It has been recognized that statins exhibit various protective effects against atherosclerosis, including modification of endothelial function, 3 decreased inflammation, 4 and inhibition of vascular smooth muscle cell (VSMC) proliferation and migration, 5 all of which cannot be accounted for by lipid reduction. One of the interesting pleiotropic effects of statins is the inhibition of vascular calcification. Results from clinical trials suggest an association of statin use with slowed progression of calcific aortic stenosis 6 -8 and coronary artery calcification. 9 Statins also inhibited calcification of atherosclerotic plaques in experimental hyperlipidemic animals. 10,11 On the other hand, some recent clinical trials were not able to find such an inhibitory effect. 12,13 To clarify these discrepancies, it is important to identify the detailed regulatory mechanism of vascular calcification and the target of effect of statins.Based on clinical findings, 14 inorganic phosphate (Pi) has been shown to be an important inducer of VSM...

Mechanism of Pi-induced Vascular Calcification - Regulation of Growth Arrest-Specific Gene 6 (Gas6)-Mediated Survival Pathway

“…VSMCs exposed to elevated calcium and phosphate levels that are equivalent to levels found in uremic patients, release vesicles containing preformed apatite crystals within the vesicle sap 24) . We also observed that Pi induces apoptosis in association with calcification in human aortic smooth muscle cells (HASMCs) 25) (Fig. 1).…”

“…In Pi-induced VSMC calcification, the expression of Gas6 and Axl is markedly downregulated 25) . We examined the role of Gas6-Axl interaction in the processes of apoptosis and calcification.…”

Clones of Interstitial Cells From Bovine Aortic Valve Exhibit Different Calcifying Potential When Exposed to Endotoxin and Phosphate

“…However, when Clone 1 was seeded on collagen scaffolds the treatment with Pi was sufficient to induce the calcification of the matrix, accompanied by apoptosis of clonal cells. Previous studies carried out in both VIC and SMC have demonstrated that blocking apoptosis pathways can abate the in vitro process of calcification, 16,20 suggesting that apoptosis could represent the final differentiation stage of cells acquiring a procalcific profile (as it happens at the end of chondrocyte maturation). Apoptosis cannot be considered per se a trigger for calcific degeneration of vascular tissues as suggested by Clarke et al, 21 who showed that the chronic induction of apoptosis in vascular SMC was not accompanied in vivo by calcium deposition in the arterial wall.…”

Vitamin K-dependent Proteins, Warfarin, and Vascular Calcification

“…Indeed, recent in vitro studies have shown Gas-6 prevents vascular calcification. In a model of phosphate-induced vascular calcification, Son et al have shown that Gas-6 prevents vascular smooth muscle cell apoptosis and calcification in a dosedependent manner (7). Elevated phosphate levels directly suppressed Gas-6 activity (8).…”