SU 5416

Male Sprague‐Dawley rats (≈250 g) were subjected to chronic hypoxia in a chamber (30′′ wide×20′′ deep×20′′ high) regulated by an OxyCycler Oxygen Profile Controller (Model A84XOV; BioSpherix) that was set to maintain 10% O₂ with an influx of nitrogen gas, located in the animal care facility at Georgetown University Medical Center.^{21 (link)} Ventilation to the outside of the chamber was adjusted to remove CO₂, such that its level did not exceed 5000 ppm. Control animals were subjected to ambient 21% O₂ (normoxia) in another chamber. Animals were fed normal rat chow during the treatment.
For the chronic hypoxia model of pulmonary hypertension, rats were subjected to hypoxia (10% O₂) for 2 weeks.^{21 (link)} For the SUGEN/hypoxia model,^{19 (link)–20 (link)} rats were subcutaneously injected with SU‐5416 (20 mg/kg body weight), maintained in hypoxia for 3 weeks, then in normoxia for 5 weeks.
After pulmonary hypertension and pulmonary vascular remodeling are developed, rats were then injected intraperitoneally with antitumor drugs including DNR (Sigma‐Aldrich; 2 or 5 mg/kg body weight), bortezomib (Selleck Chemicals; 1 mg/kg body weight), and MG‐132 (Selleck; 10 mg/kg body weight). Vehicles used were equal amounts of saline for DNR, and equal amounts of DMSO in saline for MG‐132 and bortezomib. Animals were placed back in hypoxia or normoxia for an additional 1 to 3 days before lungs and hearts were harvested.
At the end of the experiments, some rats were anesthetized with intraperitoneal injections of xylazine (10 mg/kg) and ketamine (100 mg/kg). They were then intubated and mechanically ventilated with a volume‐controlled Inspira Advanced Safety Ventilator (Harvard Apparatus). Rats were maintained on a heat pad and the temperature was kept at 37°C using a TR‐200 Temperature Controller connected to a rectal probe (Fine Scientific Tools). After a thoracotomy through the third left intercostal space, a Millar catheter (1.4 F) was inserted to the RV. RV pressure signals were recorded using PowerLab with Chart 5 software (ADInstruments).
The Georgetown University Animal Care and Use Committee approved all animal experiments, and the investigation conforms to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

All procedures were approved by the Johns Hopkins University School of Medicine Animal Care and Use Committee. Procedures were conducted in accordance with the NIH Guide for Care and Use of Laboratory Animals as well as ARRIVE guidelines. SU5416 (Tocris, Bio‐Techne) was prepared in a diluent composed of dimethyl sulfoxide and carboxymethylcellulose as previously described.² Male Wistar rats (250–300 g) were injected subcutaneously with 20 mg/kg of SU5416 before exposure to sustained hypoxia (FiO₂ 0.1) for 3 weeks followed by return to normoxia (room air, FiO₂ 21%) for 2 weeks. Normoxic controls were treated with vehicle and were maintained in racks adjacent to the hypoxia chamber, at room air, for 5 weeks.

ΔdblGATA mice were purchased from Jackson Laboratory (033551). Male hemizygote mice (ΔdblGATA/Y) and female heterozygote mice (ΔdblGATA/+) were maintained in a C57BL/6 genetic background to generate wild-type (WT) (+/Y) and knockout (KO) (ΔdblGATA/Y) littermates. C57BL/6 mice and Sprague Dawley rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were randomly assigned to various treatment groups for each experiment. Mice or rats were maintained on a 12-h light–dark cycle with a regular unrestricted diet. To establish a SuHx-induced PH mouse model, 8–10-week-old male or female mice received weekly subcutaneous injections of SU5416 (20 mg·kg⁻¹; S8442, Sigma Aldrich) for 3 weeks and were housed in an airtight plexiglass chamber (China Innovation Instrument) with 10% O₂ [27 (link)]. To establish a SuHx-induced PH rat model, 200 g male rats received a single SU5416 injection (20 mg·kg⁻¹) and were then housed in 10% O₂ for 3 weeks. For the EOS depletion mouse model, the anti-IL5-neutralising antibody TRFK5 (554391, BD Pharmingen) was administrated at a dose of 2 μg per mouse each week, with a total of three doses for each mouse, through intravenous injection [28 (link)]. Sex- and age-matched littermates injected with an equal dose of isotype control antibody (IgG1κ; 559072, BD Pharmingen) were used as controls. For the EOS-depletion rat model, the anti-IL5-neutralising antibody TRFK5 (14-7052-85, Invitrogen) was administrated at a dose of 17.5 μg per rat each week, three doses for each rat, and isotype control antibody was injected at an equal volume. For N-formyl peptide receptor 2 (FPR2) agonist Ac2-26 (HY-P1098A, MCE) treatment, mice were intraperitoneally injected at a dose of 50 μg per mouse every 3 days. The control mice were injected with an equal volume of vehicle. All animal experiments were conducted under the approval of the Animal Research Committee of the Institute of Laboratory Animals, Chinese Academy of Medical Sciences, and Peking Union Medical College (ACUC-A01-2020-017).
Additional materials and methods are available in the supplementary material.