Complete ecm medium

ECs from fresh lung tissue of mice were isolated as described previously (61 (link)). Briefly, lung lobes were carefully dissected out and washed in ice-cold DMEM containing 20% FBS (Corning). Lung tissues were minced and incubated in 15 mL prewarmed HBSS (Thermo Fisher Scientific) containing 0.2 mg/mL type I collagenase (Worthington Biochemical Corporation) for 45 minutes at 37°C with shaking. After centrifugation (at 280g for 5 minutes at room temperature), cells were resuspended in DMEM and incubated with anti-CD31–coated (553369, BD Biosciences) Dynal magnetic beads (Thermo Fisher Scientific) on an end-over-end rotator for 10 minutes at room temperature. The beads/cell pellets were washed 5 times using the magnetic separator (Thermo Fisher Scientific) and transferred to a 0.1% gelatin-coated (MilliporeSigma) 100 mm cell culture dish (Corning) with complete ECM medium (ScienCell) supplemented with 20% FBS, 1% ECGS, and 1% P/S. To improve MLEC purity, the primary cells that reached around 80% confluence were sorted a second time using anti-CD102–coated (553326, BD Biosciences) magnetic beads.

TAECs were isolated as described before (16 (link)). TAECs were grown in complete ECM medium (Sciencell) and used at passages 1–6.

Primary lung microvascular endothelial cells (LMEVCs) were extracted from male neonatal C57BL/6N mice (3 days old) using a tissue block attachment method (Additional file 1: Supplementary content). Complete ECM medium (ScienCell, USA) containing 5% fetal bovine serum, 1% triple antibiotics, and endothelial cell growth factors was used. Cells were identified by immunocytochemistry with the endothelial marker CD31. Third-generation LMVECs were inoculated into a 6-well Bioflex plate (Flexcell, USA). Cells were subjected to cyclic mechanical stretch (MS) in the Flexcell FX-5000 system using the following parameters: 0.5 Hz (30 times/minute); 20% max elongation; 4-h duration. After modeling, the cells were treated with rFGF21 or phosphate-buffered saline (PBS), transferred to a conventional incubator (37 °C, 5% CO₂), and cultured for 24 h before subsequent experiments.

The umbilical cords were obtained with informed donor consent following healthy births. The umbilical veins were perfused with HBSS (Gibco) that was supplemented with 200 U/mL penicillin and 200 mg/mL streptomycin. One end of each umbilical vein was closed with a hemostat, and 0.1% type I collagenase in HBSS was introduced to the lumen. The open end of the umbilical vein was clamped shut with another hemostat and incubated at 37 °C for 30 min. After a gentle massage of the umbilical cord, the endothelial cell-collagenase suspension was poured off and diluted 1:1 with HBSS. The vessel lumen was washed twice with HBSS, and the combined cell suspension was centrifuged at 1000 rpm for 10 min. The cell pellet was washed once with HBSS, suspended in 10 mL complete ECM medium (Sciencell), seeded in a 10 cm petri dish, and incubated at 37 °C, with 5% CO₂. The HUVECs were not used for experiments beyond passage seven. The HUVECs were identified by immunofluorescent staining for an endothelial cell marker, vWF.

TAECs were obtained from surgical HCC specimens immediately after removal from patients. Specimens were incubated and digested for 1 h at 37 °C in RPMI 1640 medium (Hyclone, Logan, UT, USA) containing 0.1% collagenase IV (Sigma‐Aldrich, St. Louis, MO, USA). The cell suspension was filtrated through a graded series of meshes to separate the cell components from the stroma. The cell suspension was then aggregated after washing in PBS. TAECs were isolated from the cell suspension using magnetic beads coupled with anti‐CD31 monoclonal antibody (Miltenyi Biotech, Bergisch Gladbach, Germany), and magnetic cells were stored with the MACS system (Miltenyi Biotech, Bergisch Gladbach, Germany). To ensure the purity of isolated TAECs, the cell pellets underwent second isolation with anti‐CD31 monoclonal antibodies. Cells were maintained in complete ECM medium (Sciencell, Carlsbad, CA, USA) with 5% FBS, 1% ECGS, 100 U mL⁻¹ penicillin, and 100 µg mL⁻¹ streptomycin at 37 °C in 5% CO₂.

Human umbilical vein endothelial cells (HUVECs) were purchased from ScienCell and cultured in complete ECM medium (ScienCell, CA, USA) that contained 5% FBS, 1% endothelial cell growth supplement, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Beyotime Biotechnology, Shanghai, China) at 37°C under 5% CO₂. The experimental groups were designed as follows: (a) Control group: normal cells without Hcy (1 mmol/L) and Gas (200‐800 μg/mL) treatment for 24 hours; (b) Model group: cells treated with Hcy (1 mmol/L) for 24 hours; (c) Protective group: cells treated with Hcy (1 mmol/L) and Gas (200‐800 μg/mL) for 24 hours. Gastrodin (IUPAC: (2R,3S,4S,5R,6S)‐2‐(hydroxymethyl)‐6‐[4‐(hydroxymethyl)phenoxy]oxane‐3,4,5‐triol) was purchased from Chengdu Must Bio‐Technology Co., Ltd. (≥98%, A0138, Chengdu, China) and dissolved in PBS. Hcy was purchased from Sigma (≥98%, H4628, Missouri, USA) and dissolved in PBS. For the Akt inhibition experiment, the Akt inhibitor MK‐2206 was purchased from Selleckchem (S1078, Houston, USA) and added to cell culture medium with Gas and Hcy for 24 hours.