HASMCs were obtained from ScienCell American and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 U/mL) in a 37 ${}^{\circ }$C incubator with humidified air containing 5% CO${}_2$. The HASMCs were previously characterized [16]. The culture medium was replenished twice per week. First, HASMCs were divided into the following three groups: HASMCs were treated with vehicle (control group), AGEs (Biovision, Japan), $\beta$-phosphoglycerine (10 mM) (Beyotime Biotechnology, Shanghai, China), or AGEs + $\beta$-phosphoglycerine for 5 days. Also, the following five groups were designed: (1) the blank control group, (2) the dimethyl sulfoxide (DMSO, vehicle) group, (3) the AGEs group, in which cells were treated with 25 $\mu$g/mL AGEs in culture for 5 days, (4) the LY294002 group, in which cells were pretreated with LY294002 (Selleck, USA), an AKT inhibitor, at 20 $\mu$M for 2 hours followed by AGEs treatment, and (5) the TWS119 group, in which cells were pretreated with TWS119 (Selleck, USA), an inhibitor of GSK3-$\beta$, at 10 $\mu$M for 2 hours followed by AGEs treatment. HASMCs of passages 3–6 were used for this study.

Briefly, HASMCs were seeded on glass slides and cultured in an incubator (37 ${}^{\circ }$C, 5% CO${}_2$). When cells reached approximately 40–50% confluency, they were fixed with 4% paraformaldehyde at 4 ${}^{\circ }$C on a shaker for 15 min, followed by three repeated washes with distilled H${}_2$O. Next, the cells were incubated with 0.5% silver nitrate (Beyotime Biotechnology, Shanghai, China) at room temperature under sunlight for 20 min, washed twice with distilled H${}_2$O and visualized using a phase microscope. Calcification was quantified using the software Motic Images Advanced.

Briefly, whole cell lysates were extracted from cultured HASMCs with radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, Shanghai, China), and the total protein concentrations determined with the Bradford method (Beyotime Biotechnology, Shanghai, China). An equal amount of protein lysate per sample was loaded onto 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel (Sigma, USA) and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The PVDF membrane was blocked with 5% non-fat dried skim milk powder for 1 hour at room temperature. The membrane was then incubated with the primary antibody of interest, including osteoprotegerin (OPG) and bone morphogenetic protein 2 (BMP-2) (Abcam, Cambridge, UK. Dilution 1 : 500) at 4 ${}^{\circ }$C overnight, followed by another incubation with the appropriate secondary antibody (dilution 1 : 2000) for 1 hour at room temperature. The specific protein bands were visualized with an ECL Plus kit (Beyotime Biotechnology, Shanghai, China) and quantified with the Quantity One software (BioRad, USA).

The initial experiments confirmed the transfection efficiency of AKT-siRNA (Ruibo Biotec, Guangzhou, China). Briefly, HASMCs were transfected with AKT siRNA using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. After transfection, cells were cultured at 37 ${}^{\circ }$ in a 5% CO${}_2$ atmosphere for another 6 hours, and then the medium was replaced with complete medium. The efficiency of AKT knockdown was evaluated by Western blotting.

Lentiviral vectors expressing green fluorescent protein (GFP) (LV-GFP) or siRNA against GSK3-$\beta$ (LV-GSK3-$\beta$-RNAi) were provided by Shanghai Genechem Co. Ltd (China). HASMCs were infected with LV-GFP as a control or LV-GSK3-$\beta$-RNAi at a multiplicity of infection (MOI) of 100. Green fluorescence was observed at 72 hours post-infection, and screening of positive cells was performed with 4 $\mu$g/mL puromycin (Sigma, USA) for 1 week after obtaining 70–80% cell fusion. The expression level was evaluated by Western blotting.

All data were analyzed using SPSS 19.0 statistical software. The measurement data were expressed by means $\pm$ standard deviations (SDs), and single-factor analysis of variance (ANOVA) was used for data comparison among multiple groups, followed by q test for two-group comparison. Differences were considered statistically significant at a level of P 0.05.

We next examined the effects of AGEs on HASMC calcification via Alizarin Red staining (Fig. 1A) and Von Kossa staining (Fig. 1B). HASMCs were treated with vehicle (control group), AGEs, $\beta$-phosphoglycerine (10 mM), or AGEs + $\beta$-phosphoglycerine for 5 days. As expected, no calcified plaques were observed in the control group. However, calcified plaques were observed in both the AGEs and the $\beta$-phosphoglycerine groups, while the highest number of calcified plaques was observed in the AGEs + $\beta$-phosphoglycerine group (Fig. 1). Collectively, these findings indicated that either AGEs or $\beta$-phosphoglycerine promote the calcification of HASMCs and that they have synergistic effects on the calcification of HASMCs. Hence, AGEs and PI3K cooperatively trigger HASMC calcification.

Fig. 1.

Effect of AGEs on calcification of HASMCs. Cultured HASMCs were treated with vehicle (control), AGEs, $\beta$-phosphoglycerin, or AGEs + $\beta$-phosphoglycerin. Calcification of HASMCs was observed by Alizarin Red staining (A) and Von Kossa staining (B) after 14 days of treatment. The ratio of calcified cells was calculated using Motic Images Advanced 3.2. *P 0.05, versus control group; #P 0.05, versus $\beta$-phosphoglycerin group.

We next investigated the effects of AGEs on the expression of AKT and GSK3-$\beta$ by western blotting. As shown in Fig. 2A, AGEs upregulated the expression of p-AKT and p-GSK3-$\beta$ (serine 9) in a dose-dependent manner with the concentration of 25 $\mu$g/mL having the greatest effect, while AGEs showed no significant effects on the total levels of AKT and GSK3-$\beta$. Notably, GSK3-$\beta$ phosphorylation at serine 9 suppresses the ability of GSK3-$\beta$ to phosphorylate substrates [17]. Thus, we chose the concentration of 25 $\mu$g/mL AGEs to test the temporal effects of AGEs treatment. As shown in Fig. 2B, after 5 min of treatment with 25 $\mu$g/mL AGEs, the greatest effects on the expression levels of p-AKT and p-GSK3-$\beta$ were observed. Our findings suggest that AGEs activate the AKT signaling pathway and inhibit the downstream GSK3-$\beta$ signaling.

Fig. 2.

Effects of AGEs on AKT and GSK3-$\beta$ expression. (A) Effects of different concentrations of AGEs on the expression levels of p-AKT and p-GSK3-$\beta$. (B) Effects of AGEs on the expression levels of p-AKT and p-GSK3-$\beta$ at different time points as indicated.

To investigate the involvement of the AKT signaling pathway in HASMC calcification induced by AGEs, HASMCs were divided into four groups: a normal control group, DMSO (vehicle) group, AGEs group, and AGEs + LY294002 group (in which the cells were pretreated with LY294002, a specific inhibitor of AKT, followed by AGEs treatment for 5 days. Consistent with the above observations, the expression levels of p-AKT and p-GSK3-$\beta$ were significantly increased by AGEs compared with the control and DMSO treatments, and these increases were attenuated by LY294002 (Fig. 3A). The results confirmed that the inhibitor significantly reduced the activation of AKT. The expression of p-GSK3-$\beta$ in the LY294002 group was significantly reduced compared with that in the AGEs group (Fig. 3A), further indicating that AGEs activate AKT signaling and inhibit the downstream GSK3-$\beta$ signaling.

To further investigate the involvement of the AKT signaling pathway in HASMC calcification, the cells were pretreated with LY294002 for 2 hours and then incubated with 25 $\mu$g/mL AGEs for 5 days. As shown in Fig. 3B, compared with the control and DMSO groups, the AGEs group showed significantly increased expression of OPG and BMP-2, both of which are osteogenic factors. This upregulation was suppressed by LY294002 pretreatment.

AKT-siRNA treatment was used to deplete the expression of AKT. As shown in Fig. 3C, AKT expression was significantly decreased in the AKT-siRNA group compared with the expression levels in the control and NC groups. Compared with the corresponding levels in the AGEs group, the expression levels of OPG, BMP-2, and $\beta$-catenin were up-regulated in the AKT-siRNA group (Fig. 3D). Hence, AGEs promote the expression of osteogenic factors in cultured HASMCs, and this effect is alleviated by inhibition of the AKT signaling pathway.

Fig. 3.

Effect of AKT on HASMC calcification. (A) LY294002 inhibited the AGE-induced upregulation of p-AKT (top panel) and p-GSK3-$\beta$ (bottom panel) in cultured HASMCs. Quantitated data are shown in the graphs in the left panels. (B) LY294002 attenuated the AGE-induced upregulation of osteogenic factors OPG and BMP-2 in cultured HASMCs. Quantitated data are shown in the graphs in the left panels. Western blotting was performed using whole cell lysates purified from cultured HASMCs that were divided into control, DMSO (vehicle), AGEs, and AGEs + LY294002 groups. In the AGEs + LY294002 group, cells were pretreated with LY294002 for 2 hours followed by AGEs stimulation for 30 min. *P 0.05, versus control group; #P 0.05, versus AGEs group. (C) AKT- siRNA depleted the expression of AKT in cultured HASMCs. (D) AKT-siRNA attenuated the AGE-induced upregulation of osteogenic factors OPG and BMP-2 in cultured HASMCs. *P 0.05, versus control group; #P 0.05, versus AGEs group.

Because AGEs activated the AKT signaling pathway but inhibited the activity of GSK3-$\beta$, we next investigated the role of GSK3-$\beta$ in AGE-mediated HASMC calcification. HASMCs were divided into the following four groups: control, DMSO, AGEs, and AGEs + TWS119 groups. In the AGEs + TWS119 group, cells were pretreated with TWS119, which is a specific inhibitor of GSK3-$\beta$, followed by AGEs treatment for 25 min. Compared with control and DMSO groups, the AGEs group showed significantly upregulated expression of p-GSK3-$\beta$, which was further potentiated by TWS119 pretreatment (Fig. 4A). We further pretreated the cells with LY294002 or TWS119 for 2 hours followed by treatment with 25 $\mu$g/mL AGEs for 5 days. The expression levels of OPG and BMP-2 were significantly reduced in the AGEs + LY294002 group (P 0.05) compared with the AGEs group (Fig. 4B), but significantly up-regulated in the AGEs + TWS119 group (P 0.05).

Fig. 4.

Effect of GSK3-$\beta$ on HASMC calcification. (A) TWS199 increased the expression of p-GSK3-$\beta$. (B) TWS199 promoted the AGE-induced expression of osteogenic factors, BMP-2 and OPG, in HASMCs. Western blotting was performed using whole cell lysates purified from cultured HASMCs that were divided into control, DMSO (vehicle), AGEs, AGEs + LY294002, and AGEs + TWS199 groups. In the AGEs + LY294002 and AGEs + TWS199 groups, cells were pretreated with LY294002 or TWS199 for 12 hours followed by AGE stimulation for 30 min. *P 0.05, versus control group; #P 0.05, versus AGEs group.

To further investigate the effects of GSK3-$\beta$ on HASMC calcification, GSK3-$\beta$ was knocked down by adenovirus-mediated siRNA expression, and Ad-GFP was used as a control. The adenoviral infection efficiency after 72 hours of infection in either group was over 90% (Fig. 5A). As expected, GSK3-$\beta$ expression was significantly decreased in the Ad-GSK3-$\beta$-RNAi group compared with normal control (Ad-GFP) group (Fig. 5B). Cells of the blank control group, Ad-GFP group, and Ad-GSK3-$\beta$-RNAi group were co-cultured with 25 $\mu$g/mL AGEs for 5 days. The expression levels of OPG, BMP-2, and $\beta$-catenin in the GSK3-$\beta$ knockdown group were significantly up-regulated compared with those in the control and Ad-GFP groups (Fig. 5C). These results indicated that AGEs promoted HASMC calcification, and this process was enhanced by inhibiting GSK3-$\beta$. Thus, GSK3-$\beta$ may play a key role in AGE-induced HASMC calcification.

Fig. 5.

GSK3-$\beta$ knockdown promotes HASMC calcification. (A) Images showing HASMCs infected with Ad-GFP and Ad-GSK3-$\beta$-RNAi. Left, brightfield; right, fluorescence imaging of GFP expression. Magnification: 20$\times$. (B) Western blot showing efficient knockdown of GSK3-$\beta$ by Ad-GSK3-$\beta$-RNAi. NC, Ad-GFP group. *P 0.05, versus NC group. (C) Western blot showing that GSK3-$\beta$ knockdown promoted the expression of osteogenic factors, $\beta$-catenin, BMP-2 and OPG, in HASMCs. *P 0.05, versus NC group.

To further validate the role of the PI3K/AKT-GSK3-$\beta$ signaling pathway in AGE-triggered HASMC calcification, the cells were divided into the following four groups: control, AGEs, AGEs + LY29002, and AGEs + TWS199. Except for cells in the control group, those in the other three groups were treated with 25 $\mu$g/mL AGEs for 14 days. Von Kossa staining was used to detect calcification in each group. As shown in Fig. 6, the amount of calcified plaques was significantly lower in the AGEs + LY294002 group but higher in the AGEs + TWS119 group compared with the AGEs group (Fig. 6).

Fig. 6.

Effects of suppression of AKT and GSK3-$\beta$ on HASMC calcification. Cultured HASMCs were divided into control, AGEs, AGEs + LY294002, and AGEs + TWS199 groups. In the AGEs + LY294002 and AGEs + TWS199 groups, cells were pretreated with LY294002 or TWS199 for 2 hours followed by AGEs stimulation for 14 days. Calcification was detected by Von Kossa staining. *P 0.05, versus control group; #P 0.05, versus $\beta$-phosphoglycerin group.