Cell culture and RNA sequencing sample preparation

Culturing of HUVECs and RNA preparation were broadly followed in the previous study 24. HUVECs were obtained from China Center for Type Culture Collection (CCTCC, Wuhan, China) and were maintained in modified Eagle's medium (MEM; HyClone/Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Gibco, Gaithersburg, MD, USA), 100 units·mL of penicillin, and 100 µg·mL⁻¹ of streptomycin (Gibco), and grown in a humidified atmosphere with 5% CO₂ at 37 °C. High glucose‐induced HUVECs were cultured in medium containing 25 mm glucose for 6 days, and control HUVECs were cultured with 5 mm glucose and 20 mm mannitol for the same duration.

Total RNA was isolated and purified using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The quantity and quality of the RNA samples were determined using NanoDrop 2000 (Thermo Fisher Scientific) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).