All cell culture reagents were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Written informed consent was obtained from patients and approval was received from the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China). The work described in the present study was conducted in accordance with The Code of Ethics of the World Medical Association (www.wma.net/). VSMCs were isolated from human femoral arteries (4 patients, 45.4±4.6 years old, 2 male and 2 female, recruited from Guangzhou, China, from June 2016 to May 2017) using the explant method described previously (16). VSMCs were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum at 37°C in 5% CO₂. Both DMEM and fetal serum were purchased from Sigma-Aldrich (Merck KGaA). Cells between passages 3 and 6 were used in this study. To induce calcification, growth medium was replaced with osteogenic DMEM, which was supplemented with 10 mM β-glycerophosphate, which was purchased from Sigma-Aldrich (Merck KGaA).

LDLs (1.019–1.063 g/ml) were separated from human plasma by sequential density gradient ultracentrifugation (145,000 × g at 10°C for 20 h). The LDL fraction was dialyzed against PBS (pH 7.4) at 4°C for 24 h. Ox-LDL was prepared by incubation of LDL with 5 mM copper sulfate (Sigma-Aldrich, Merck KGaA) at 37°C for 3 h, and was subsequently sterilized by passaging through a 0.22-µm filter. Cells were treated with Ox-LDL (10, 50 or 100 µg/ml) for 14 days (37°C, treated once every two days).

Mineral deposition in cultured VSMCs was assessed by Alizarin Red S staining as previously described (17). Calcified VSMCs (2×10⁶) were fixed in 4% formaldehyde for 10 min at room temperature and exposed to 2% Alizarin Red S (pH 4.2) for 5 min at room temperature. Subsequently, cells were washed with deionized water to remove excess dye and imaged using an inverted phase contrast microscope. For the quantification of Alizarin Red S staining, 10% formic acid was used to elute the Alizarin Red S dye, and the absorbance at 405 nm was determined with a microplate reader and normalized to the protein content. For quantification of calcium content, cells were washed with PBS and decalcified with 0.6 M hydrogen chloride for 24 h. The calcium content of the cultures was determined colorimetrically using the O-cresolphthalein complexone method and normalized to protein content as previously described (18).

Total RNA was isolated from cultured VSMCs using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and was reverse transcribed using an avian myeloblastosis virus Reverse Transcriptase (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's protocol. The thermocycling conditions were 37°C for 1 h and then 94°C for 10 min. RT-qPCR was performed using a SYBR^®-Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) in a StepOne Real Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers used for RT-qPCR were as follows: CBFA1 forward, 5′-CCGTGGCCTTCAAGGTTGT-3′ and reverse, 5′-TTCATAACAGCGGGAGGCATTTC-3′; SM22α forward, 5′-TTGGATCCGACATGGCCAACAAG-3′ and reverse, 5′-GCCCTAAGCTTTCTAACTGATGATCT-3′; and β-actin forward, 5′-CCAGCTCACCATGGATGATG-3′ and reverse, 5′-GAGCCGTTGTCGACGACG-3′. Values were normalized to those of β-actin. The results were calculated using the comparative threshold cycle (Cq) method (19).

In all assays human VSMCs were cultured in osteogenic medium at 37°C with 5% CO₂ for 14 days. Experiment 1 (Fig. 1) human VSMCs were treated with Ox-LDL (0, 10, 50 and 100 µg/ml). In experiment 2 (Fig. 2) cells were treated with 0 or 50 µg/ml Ox-LDL and different concentrations of FPS (0, 10, 100 or 1,000 µg/ml). Experiment 3 (Fig. 3) was performed with 0 or 50 µg/ml Ox-LDL and 1,000 µg/ml FPS. In experiment 4 (Fig. 4) human VSMCs with 0 or 50 µg/ml Ox-LDL, 1,000 µg/ml FPS and 5 mM 3MA.

Cells (1×10⁷) were plated in 100-mm diameter Petri dishes and were treated as indicated above following 70–80% confluency. Following treatment, cells were harvested and resuspended in ice-cold cell lysis buffer (Sigma-Aldrich, Merck KGaA), and the homogenate was centrifuged at 10,000 × g for 15 min at 4°C. The total supernatant protein concentration was measured using a bicinchoninic acid protein assay kit. Total protein (50 µg) from each sample was separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% fat-free dry milk in TBS with Tween-20 for 1.5 h at room temperature, followed by incubation with primary antibodies against microtubule-associated protein 1A/1B light chain 3 (LC3B), nucleoporin P62 and GAPDH (cat. nos. 2775, 5114 and 5014; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight with gentle agitation at 4°C. The next day, the membranes were washed with PBS and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (cat. no. 7075; 1:1,000; Cell Signaling Technology, Danvers, MA, USA) for 1.5 h at room temperature. Following three washes with TBS with Tween-20, membranes were developed using an enhanced chemiluminescence kit (Applygen Technologies, Inc., Beijing, China) and exposed to X-ray films. ImageJ software (1.48u version; National Institutes of Health, Bethesda, MD, USA) was used to quantify the protein expression levels.

Cells were transfected with pEGFP-LC3 plasmids (cat. no. 24920; Addgene, Cambridge, MA, USA) using Lipofectamine™ LTX (cat. no. 11668019; Thermo Fisher Scientific, Inc.) reagent as previously described (20); a minimum of 40% transfection efficiency was achieved and GFP-LC3-transfected cells were visualized using a fluorescence microscope.

All assays were performed ≥4 times and the values were expressed as the mean ± standard error. Statistical analysis of between-group differences was performed by one-way analysis of variance with a Bonferroni post hoc test with using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Human VSMCs were treated with 10, 50 or 100 µg/ml Ox-LDL for 14 days to determine the effect of Ox-LDL on vascular calcification. Alizarin Red S staining was used to assess mineralization, which is positively associated with VSMC mineralization. As illustrated in Fig. 1A and B, Ox-LDL increased human VSMC calcification in a concentration-dependent manner, particularly in the 50 and 100 µg/ml Ox-LDL treatment groups. Additionally, RT-qPCR revealed that Ox-LDL increased CBFA1 mRNA expression in a concentration-dependent manner, whereas it decreased SM22α mRNA expression (Fig. 1C), further confirming that Ox-LDL increased human VSMC calcification. For the subsequent experiments, 50 µg/ml Ox-LDL treatment for 14 days was selected as this induced VSMC calcification and increased Alizarin Red S staining along with increasing CBFA1 mRNA expression and decreasing SM22α mRNA expression.

Various concentrations of FPS were added to the cells to determine its protective effect against vascular calcification induced by Ox-LDL. As demonstrated in Fig. 2A, FPS treatment attenuated the Ox-LDL-induced increase in Alizarin Red S staining in human VSMCs, particularly in the 1,000 µg/ml FPS-treatment group (Fig. 2B). Furthermore, the CBFA1 and SM22α mRNA changes induced by Ox-LDL were significantly attenuated by 1,000 µg/ml FPS (Fig. 2C), suggesting that FPS treatment protected against Ox-LDL-induced calcification in human VSMCs.

It was also investigated whether autophagy was upregulated or downregulated following 14 days of Ox-LDL treatment, and whether FPS treatment affected autophagic activity. The protein expression levels of LC3-I, LC3-II, P62 and GAPDH were measured by western blotting. As presented in Fig. 3A and B, following 50 µg/ml Ox-LDL-treatment for 14 days, the LC3-II/LC3-I ratio was decreased and P62 protein expression was increased, indicating that autophagy was inhibited in the vascular calcification progress. Notably, treatment with 1,000 µg/ml FPS attenuated the aforementioned changes in LC3-II/LC3-I ratio and P62 protein intensity induced by Ox-LDL, suggesting that FPS treatment protected against the Ox-LDL-induced downregulation of autophagic activity. Additionally, no change in autophagic activity was detected in native FPS-treated cells compared with the control group of untreated cells. As demonstrated in Fig. 3C, GFP-LC3 puncta, as indicators of autophagosomes, were also used to monitor autophagic activity. A greater number of GFP-LC3 dots in each VSMC represents increased autophagy. Compared with the control group, fewer GFP-LC3 dots were detected in the Ox-LDL treatment group. When 1,000 µg/ml FPS was added, the Ox-LDL-induced reduction in the number of GFP-LC3 dots was attenuated. This further confirmed that FPS treatment alleviated the Ox-LDL-induced downregulation of autophagy.

Whether increased autophagy activity contributed to FPS-mediated protection against vascular calcification was also investigated. Since 3MA is commonly used as a pharmacological agent for the inhibition of autophagy in vitro, VSMCs were treated with 5 mM 3MA (Sigma-Aldrich, Merck KGaA) to inhibit autophagy, and the effects on FPS-mediated protection against vascular calcification were assessed. As presented in Fig. 4A and B, when 3MA was added, the protective effect of FPS against VSMC calcification was also inhibited. Additionally, 3MA treatment decreased the LC3-II/LC3-I ratio and increased the P62 protein level, indicating that autophagic activity was inhibited (Fig. 4C and D). Furthermore, a decrease in the number of GFP-LC3 dots also demonstrated that autophagy was inhibited (Fig. 4E). Therefore, it may be concluded that inhibition of autophagy by 3MA decreased the protective effect of FPS against VSMC calcification.