Abstract
This study aimed to investigate the effects of advanced glycation end products (AGEs) on the calcification of human arterial smooth muscle cells (HASMCs) and to explore whether AGEs can promote the calcification of HASMCs by activating the phosphoinositide 3-kinase (PI3K)/AKT-glycogen synthase kinase 3 beta (GSK3-β) axis. Cultured HASMCs were divided into five groups: blank control group, dimethyl sulfoxide (vehicle) group, AGEs group, LY294002 (AKT inhibitor) group, and TWS119 (GSK3-β inhibitor) group. Cells were pretreated with either vehicle, LY294002, or TWS119 for 2 hours followed by incubation with AGEs (25 μg/mL) for 5 days, and the expression levels of proteins in each group were analyzed by western blotting. AGE treatment promoted HASMC calcification, which coincided with increased expression of p-AKT and p-GSK3-β (serine 9). Also, AGEs upregulated the expression of osteoprotegerin and bone morphogenetic protein, and these effects were suppressed by LY294002 but enhanced by TWS119. In conclusion, AGEs promote calcification of HASMCs, and this effect is ameliorated by inhibition of AKT activity but potentiated by inhibition of GSK3-β activity. Hence, AGEs trigger HASMC calcification by regulating PI3K/AKT-GSK3-β signaling.
Highlights
Vascular calcification is a progressive disease [1,2,3] that is accompanied by phenotypic changes in the vascular smooth muscle cells (VSMCs) that manifest mainly as calcification in the intimal or medial layers of the involved arteries [4]
We examined the effects of advanced glycation end products (AGEs) on human aortic SMCs (HASMCs) calcification via Alizarin Red staining (Fig. 1A) and Von Kossa staining (Fig. 1B)
The expression levels of OPG, bone morphogenetic protein 2 (BMP-2), and βcatenin in the glycogen synthase kinase 3 beta (GSK3-β) knockdown group were significantly up-regulated compared with those in the control and AdGFP groups (Fig. 5C). These results indicated that AGEs promoted HASMC calcification, and this process was enhanced by inhibiting GSK3-β
Summary
Vascular calcification is a progressive disease [1,2,3] that is accompanied by phenotypic changes in the vascular smooth muscle cells (VSMCs) that manifest mainly as calcification in the intimal or medial layers of the involved arteries [4]. A growing body of evidence suggests that advanced glycation end products (AGEs) and their receptors (receptors for AGE, RAGE) play an important role in the initiation and progression of vascular calcification [5, 6]. AGEs bind to RAGE present on the membrane of VSMCs [7], which in turn promotes activation of the Wnt/β-catenin signaling pathway to trigger vascular calcification [8, 9]. Whether other signaling pathways are involved in AGE-induced vascular calcification remains largely unknown. In the field of vascular calcification, one mechanism by which AKT activation and GSK3-β inhibition promote vascular calcification is the potentiation of Runx activity [13, 14], a transcriptional factor for osteogenesis [15]
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