Esomeprazole

Human chorionic trophoblast cells (HTR-8/Svneo) seeded at a density of 2 * 10⁶ cells into 10-cm dishes were kept at 37℃ in 5% CO₂ and 20% O₂ and cultured in 1640 media (Gibco) with a 10% Australian fetal bovine serum.
Human umbilical vein endothelial cells (HUVECs) seeded at a density of 2 × 10⁶ cells into 10-cm dishes were kept at 37 ℃ in 5% CO₂ and 20% O₂ and cultured in ECM media (Sciencell) with a 5% Australian fetal bovine serum and EGF.
When performing hypoxia, L-NAME (MCE, HY-18729), and esomeprazole (Sigma-Aldrich, PHR-1585) treatments, HTR-8/Svneo and HUVEC cells were treated with and without either L-NAME (70 mM) or esomeprazole (50 mM) and hypoxia for 48 h.
All cell experiments were repeated three times.

Animal experiments were performed in accordance with the guidelines set by the Animal Care and Use Committee of Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (no. XHEC-F-2020-004). All male and female wild-type (C57BL/6J) mice were purchased from the Mode I Animal Research Center of Xin Hua Hospital (SYXK 2018-0038). All mice were housed in ventilated cages with up to five mice and a diurnal light cycle providing 12 h of light. Virgin female mice (8–10 weeks old) were placed with a male overnight, and detection of a plug was set as embryonic day (E) 0.5. Pregnant mice were given Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME; MedChemExpress) 50 mg/kg per day in drinking water from E8.5 to E18.5. The plugged females were randomly divided into three groups: control group (n = 10), l-NAME group (n = 10), and pregnant l-NAME–treated mice with proton pump inhibitor (PPI) (esomeprazole, n = 10; Sigma-Aldrich). Pregnant l-NAME–treated mice were given 150 μg of esomeprazole by intraperitoneal injection from E8.5 to E18.5 daily (22 (link)). The tail artery systolic pressure was measured with the non-invasive indirect tail-cuff method at E8.5 and E18.5. Pregnant mice were sacrificed at E18.5, and vessels and kidney were collected for further Western blot and morphologic analysis.

Esomeprazole, Pargyline, Leflunomide, Clofibrate, Ciprofloxacin, Ketorolac, Paroxetine, Dobutamine, Tranilast, Loperamide, Xylazine, Capsaicin, Papaverine, Oxymetazoline, Dexamethasone, Docetaxel, Topotecan, Paclitaxel were purchased from Sigma. Almotriptan Malate and Sirolimus were purchased from Selleckchem, Gefitinib from Invivogen, Cefprozil Monohydrate from Abcam and Imexon from MicroSource Discovery Systems Inc.; ATP, Apyrase, Suramin and Mefloquine were purchased from Sigma; Latrunculin B was purchased from Abcam.

Esomeprazole, Lansoprazole, Amiloride and Cariporide were obtained from Sigma (Sigma Aldrich, Milano, Italy). The inhibitors came as a powder and were then dissolved in DMSO to prepare a mother stock solution that was diluted at the moment of the experiment at several concentrations. A final volume of 100 µL was prepared for each well of the 96-well plate, where cells were seeded the day before at a density of 3 × 10⁴. Cell viability tests were conducted after 24 h and 48 h of drug treatments performed in normoxia and in hypoxia, whereas for pH measurements they were conducted only in normoxia after 24 h exposure. The medium of control cells was added with DMSO to match the same concentration in drug-treated cells. Cells were incubated in hypoxia with a hypoxic incubator (New Brunswick™ Galaxy^® 48 R, Eppendorf S.r.l., Milan, Italy) set to 1% O₂, 5% CO₂, and 95% humidity during the whole experiment.