VSMCs were obtained from Keygen Biotech. The cells were cultured in DMEM containing 10% foetal bovine serum and maintained at 37°C in 5% CO2 environment with medium changed every 2 days. For HMGB‐1 induction experiment, the cells were exposed to AGEs (100 μg/mL) for 0, 6, 12 and 24 hours. Then, 100 μg/mL AGEs for 24 hours were used in the following experiments. For the in vitro calcification experiments, VSMCs were incubated with β‐glycerophosphate (β‐GP, 10 mmol/L) in medium containing HMGB‐1 (100, 200, 400 μg/mL) or TM (1, 2, 4 mg/L) for two weeks. Then, 200 μg/mL HMGB‐1 was used in the following experiments. For the inhibitory studies, 10 mmol 4‐PBA or 100 μmol/L Gly was pre‐treated for 2 hours before coincubation with AGEs or HMGB‐1.

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