D valine

Cell lines of PASMCs (passages 5 to 10) from normal subjects (Lonza, Walkersville, USA), IPAH patients [12 (link)], and CTEPH patients [24 (link)] were cultured in Medium 199 supplemented with 10% fetal bovine serum, 100 U/ml penicillin plus 100 μg/ml streptomycin (Invitrogen/GIBCO, Grand Island, USA), 50 μg/ml D-valine (Sigma-Aldrich, St. Louis, USA), and 20 μg/ml endothelial cell growth supplement (BD Biosciences, Franklin Lakes, USA) at 37°C.

PASMCs of patients with idiopathic PAH (IPAH-PASMCs) was established as previously reported and used in the present study.^{39 (link)} For the control experiment, PASMCs of normal subjects (Normal-PASMCs) were purchased from Lonza (Walkersville, MD). The cells were cultured in Medium 199 supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin plus 100 µg/mL streptomycin (Invitrogen/GIBCO, Grand Island), 50 µg/mL D-valine (Sigma-Aldrich) and 20 µg/mL endothelial cell growth supplement (BD Biosciences, Franklin Lakes) at 37°C. The cells were used in the experiments at passages 5 to 10. Three independent lines of IPAH- and Normal-PASMCs were used respectively.

Mouse tracheal cells were isolated from three-to-six week old female BALB/c mice by pronase digestion. The cells were seeded in a petri dish and incubated for 3–4 hours to adhere fibroblasts. Then non-adherent cells were collected and reseeded in a 10cm petri dish and cultured with DMEM/F12 medium supplemented with 10% FBS, 15 mM HEPES (Gibco), 0.03% NaHCO3 (Gibco), 0.01uM Retinoic acid (Sigma, cat R-2625), Amphotericin B (Gibco, cat A2942: final concentration 250ng/ml), EGF (BD, cat 354001: final concentration: 25ng/ml), D-valine (Sigma, V1255: final concentration: 0.1mg/ml), bovine pituitary extract (Gibco, cat 13028–014: final concentration: 30 ug/ml), Cholera Toxin (Sigma, cat C8052: final concentration: 0.1ug/ml), Insulin-Transferrin-Selium (Gibco, cat 41400045: 1:100). Medium was changed every two days. After 14 days incubation and differentiation, the epithelial cells were harvested for experimental use.

D-methionine, D-tryptophan, D-leucine, D- or L-cysteine, D- or L-aspartic acid, D- or L-glutamic acid, D-alanine, D-threonine, D-valine, D-isoleucine, D-glutamine, D-phenylalanine, D-histidine, D-proline, D-lysine, D-arginine, and glycine (Sigma-Aldrich, St. Louis, MO, USA) were all prepared at a concentration of 40 mM, and D-tyrosine was prepared at a concentration of 0.8 mM due to its low solubility. Nisin powder (2.5% purity, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in a solution at pH 2 to obtain a stock solution with a concentration of 1 g/L.

iMPMECs were generated in our laboratory [34 (link)]. The cells were used at passages 3–10, and maintained in DMEM-F12 (Gibco, USA) supplemented with 5% foetal bovine serum (FBS) (ExCell, China), 1% penicillin and streptomycin (Gibco, USA), 1% endothelial cell growth supplement (ECGS) (ScienCell, USA), 100 IU/ml heparin (Solarbio, China), and 92 mg/L D-valine (Sigma, USA) and incubated at 37 °C in 5% CO₂. The cells were grown to 70–80% confluence, washed 3 times with PBS, and treated with or without 1 µg/ml LPS (Sigma, USA) for 24 h in conditioned culture medium (CCM) containing exosome-depleted FBS. CCM was collected after 24 h. Exosome-depleted FBS was prepared by centrifuging FBS for at least 18 h overnight at 100,000 × g at 4 °C, after which the centrifuged FBS were passed through a 0.22 μm filter (Millipore, USA). MSCs derived from the bone marrow of C57BL/6 mice (Cyagen Biosciences, China) were used at passages 3–8 and cultured in DMEM-F12 supplemented with 10% FBS and 1% penicillin and streptomycin, and these cells were tested for their osteogenic, adipogenic, and chondrogenic differentiation potentials to ensure they met the characteristic of stem cells (Supplementary Fig. S1).