Su5416

Animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Colorado Denver. Ten week-old male Sprague Dawley rats (Charles River Laboratories) were used for all studies. A single dose of SU5416 (Tocris Bioscience; 20 mg/kg) or vehicle control (50% DMSO and 50% of a solution containing 0.5% carboxymethylcellulose sodium, 0.9% sodium chloride, 0.4% Tween-80, 0.9% benzyl alcohol in deionized water) was administered at day zero. Rats receiving SU5416 and were housed in a hypobaric chamber to simulate an altitude of 18,000 feet above sea level and create a hypoxic environment (10% 0₂). Normoxic control rats were maintained in chambers simulating sea level (21% 0₂). After three weeks, all animals were transferred to Denver altitude and treated daily for four weeks by oral gavage with vehicle (0.5% hydroxypropyl-methyl cellulose), tadalafil (10 mg/kg), ambrisentan (10 mg/kg), or a combination of tadalafil and ambrisentan (10 mg/kg of each compound). Tadalafil and ambrisentan were obtained from Sequoia Research Products.

Animals were anaesthetized with 1.5–2% isoflurane. Intravitreal injection of NMDA (100 mM in PBS, 2 μl per eye; Cat# M3262, Sigma-Aldrich, United States) was performed using a sharp 32-guage needle (micro-syringe equipped of Hamilton Storage, United States). Two days post NMDA challenge, intravitreal injection of recombinant SCF (50 ng/μl in PBS, 2 μl per eye; Cat# 455-MC, Novus Biologicals, United States) and SU 5416 (c-kit inhibitor, ic-kit, 50 ng/μl in PBS, 2 μl per eye; Cat# 3037, Tocris Bioscience, United Kingdom) were performed. Mice injected with an equal volume of PBS (2 μl per eye) were served as control.

The VEGF pathway was assessed using the VEGF-R inhibitor SU 5416 (Tocris, Bristol, UK); 10 mg/kg was intraperitoneally injected (i.p.) twice daily from E21 to P7. To investigate the role of NO synthases, we inhibited all isoforms with N(G)-nitro-L-arginine methyl ester (L-NAME; 15 mg/kg was administered twice daily via i.p. injection from E21 to P7). To investigate the role of thrombospondin-1 (TSP-1), we used a pharmacological agonist of CD36, ABT510, which was developed by Abbott (Abbott Park, US-IL) and generously provided under transfer agreement N°45068. A dose of 100 mg/kg was injected i.p. every 12 h from E21 to P7.

The Su/Hx‐PH protocol consisted of 3 weekly subcutaneous injections of the Sugen vascular endothelial growth factor receptor‐2 inhibitor SU5416 (Tocris) at 20 mg/kg in 100 μl dimethyl sulfoxide or vehicle alone. During the 3‐week SU5416 treatment, mice were exposed to hypoxia (8.5% O₂) or normoxia (Nx) for 3 weeks. For the MCT‐induced PH mouse model, cohorts of mice received weekly subcutaneous injections of MCT (60 mg/kg; Sigma) resuspended in 100 μl of saline or 100 μl of saline only (vehicle) for 4 weeks. Development of PH was determined by measurement of the right ventricular systolic pressure (RVSP) and right ventricular (RV) hypertrophy (i.e., Fulton's index). To measure RVSP, mice were anesthetized via intraperitoneal injection with ketamine (100 mg/kg) and xylazine (10 mg/kg). RVSP was measured through a trans‐thoracic route with a Millar catheter transducer PVR‐1030 (ADInstruments Inc., Colorado Springs, CO) and data were collected and analyzed using the LabChart software v8.1.3 (ADInstruments). To access RV hypertrophy, whole hearts were weighted after the atria and great vessels were trimmed. The right ventricles were then dissected away from the heart and left ventricle plus septum (LV + S) were weighted. Fulton's index (RV/LV + S) was then calculated.