The PAH rat model was established using Sugen 5416 (VEGF receptor-1,-2 inhibitor) and chronic hypoxic exposure as previously described [14 (link), 21 (link)] Briefly, adult male Sprague Dawley rats (150–180 g) were injected with Sugen 5416 (20 mg/kg; Cayman Chemical Co, MI) subcutaneously and exposed to hypobaric hypoxia (360 mmHg, 10% O2) for 3 weeks. After returning to normoxic conditions (760 mmHg, 21% O2), the rats were analyzed immediately [SuHx(3W) group] or were chronically treated with either vehicle (0.5% hydroxyethylcellulose) [SuHx(5W) group] or nintedanib [50 mg/kg/day, SuHx(5W) + Nin group] by oral gavage for 2 weeks. We determined the dose of nintedanib based on previously published experiments [15 (link)]. Control rats received a single vehicle injection and were exposed to normoxic conditions for 5 weeks, with vehicle or nintedanib (50 mg/kg/day) gavage from 3 to 5 weeks [Nx(5W)] and [Nx(5W) + Nin] groups, respectively. Hemodynamic measurements were performed at 3 or 5 weeks after vehicle or Sugen 5416 injection.
Sugen 5416
Manufactured by Cayman Chemical
Sugen 5416 is a small molecule kinase inhibitor. It functions by inhibiting the activity of the vascular endothelial growth factor receptor (VEGFR).
Automatically generated - may contain errors
Lab products found in correlation
Male balb c mice, by Jackson ImmunoResearch (1 mentions)
Su5416, by Cayman Chemical (1 mentions)
C57bl 6 mice, by Jackson ImmunoResearch (1 mentions)
Dimethyl sulfoxide (dmso), by Cayman Chemical (1 mentions)
Spr 839, by Millar (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Sds page reagents, by Bio-Rad (1 mentions)
Fibronectin, by Roche (1 mentions)
Phospho akt s473, by Cell Signaling Technology (1 mentions)
Mouse antihuman β actin monoclonal antibody, by Abcam (1 mentions)
Pd98059, by Cell Signaling Technology (1 mentions)
Tissue culture reagents, by Lonza (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Phospho p38 mapk thr180 tyr182, by Cell Signaling Technology (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Sb203580, by Cell Signaling Technology (1 mentions)
Gelatin, by Merck Group (1 mentions)
7 protocols using sugen 5416
1
PAH Rat Model: Sugen 5416 and Hypoxia
2
Investigating Lung Tissue Remodeling via CRISPR-Mediated Vegfr2 Knockdown
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Sugen 5416 (Cayman Chemicals) (20 mg/kg), dissolved in PBS containing 20% DMSO, was injected intraperitoneally into wild-type mice (4 weeks of age) three times a week for 21 days. Separate cohorts of mice were injected retro-orbitally with mixture of CRISPRCDH5 plasmid expressing Vegfr2 gRNA and PP/PEI nanoparticles at days 1 and 8. The lung tissues were collected and fixed at day 28. 5 μm paraffin sections were stained with hematoxylin and eosin. The images were taken using light microscopy. The mean linear intercept was quantified using ImageJ with MLI plugin (Crowley et al., 2019 (link)). The mean lumen area and alveoli number were determined using ImageJ with angiogenesis analyzer plugin (Carpentier et al., 2020 (link)).
3
Standardized Mouse Models for Pulmonary Hypertension
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All animal experiments were approved by the Institutional Animal Care and Use Committee at Acceleron Pharma Inc., a subsidiary of Merck & Co., Inc., Rahway, NJ, USA and performed in accordance with the guidelines from the NIH Guide for the Care and Use of Laboratory Animals. Male C57BL/6 mice (10 weeks old, Jackson Laboratory) were used for TAC and MI models as described (18 (link), 22 (link)), and male Balb/c mice (10 weeks old, Jackson Laboratory) were used for the prolonged TAC model to establish PH. Male obese ZSF1 rats (8 and 23 weeks old) and their lean littermates (Charles River, Wilmington, MA, USA) were used for the PH-HFpEF study. PH was established by a single subcutaneous injection of a vascular endothelial growth factor receptor antagonist, Sugen 5416 (SU5416, 100 mg/kg; Cayman), suspended in CMC buffer (0.5% sodium carboxymethyl cellulose, 0.4% polysorbate 80, 0.9% sodium chloride, and 0.9% benzyl alcohol) (23 (link), 24 (link)). Animals were euthanized in all experiments by heart and lung removal en bloc according to AVMA guidelines.
4
Measuring Right Ventricular Pressures in Mice
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Wildtype and mutant mice were treated weekly, three times with Sugen5416 (20 mg/kg in DMSO, s.c., Cayman Chemical) and normobaric, 10% O2. Mice were weighed at the end of the exposure period (Figure S1). Right ventricle pressures were measured as previously described (Breen et al., 2017 (link)). The right ventricular chamber was accessed by way of Sastry et al. (2006 (link)) with a small pressure‐conductance transducer. Briefly, mice were anesthetized and an incision was made on the midline of the neck to allow the right jugular vein to be exposed by blunt dissection. A distal tie (6–0 silk suture) on the vein was made to serve as a retractor while a loose silk knot was placed at the proximal end that was used to occlude the vein temporally. A small incision was made between the two sutures and a 1.4F pressure‐conductance transducer (Millar SPR‐839) was inserted and advanced into the vessel and to the RV cavity. The catheter was secured by the proximal suture once the RV pressure waveform was identified. Right ventricular pressures (RVPs) were recorded, converted digitally via an analog‐digital converter (emka Technologies, IOX 1.8), and stored on a computer for analyses. Upon completion of data collection, the mouse was euthanized by an overdose of isoflurane (5%).
5
VEGF-Induced Signaling Pathway Analysis
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Tissue culture reagents, unless otherwise specified, were obtained from Biowhittaker (Walkersville, MD). Human recombinant VEGF, bFGF and epidermal growth factor (EGF) were purchased from PrepoTech (Rocky Hill, NJ). Sugen 5416 was obtained from Cayman Chemical Company (Ann Arbor, MI) and human VEGF neutralizing antibody was purchased from R&D Systems Europe (Abingdon, UK). Inhibitors LY294002, PD98059, and SB203580 were purchased from Cell Signaling Technology, Inc. (Bioke, Leiden, NL). Antibodies against Akt, Phospho-Akt (S473), p44/42 MAPK (ERK1/2), Phospho-p44/42 MAPK (ERK1/2), p38 MAPK, and Phospho- p38 MAPK (Thr180/Tyr182) were from Cell Signaling and mouse anti-human β actin monoclonal antibody was from Abcam (Cambridge, UK). Gelatin was obtained from Sigma-Aldrich and Fibronectin from Roche Diagnostics. SDS-PAGE reagents were obtained from BioRad (Hercules, CA). TaqMan Gene Expression assays were purchased from Applied Biosystems (Carlsbad, CA), TRIzol reagent and First Strand cDNA synthesis kits were purchased from Invitrogen (Carlsbad, CA). All other reagents were from Sigma-Aldrich Chemie B.V. (Zwijndrecht, NL), unless stated otherwise.
6
Sugen-Induced Pulmonary Hypertension in Rats
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All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of South Alabama (Protocol # 829408, PI Natalie Bauer; Association for Assessment and Accreditation of Laboratory Animal Care International approved since 1999; Compliance with Public Health Service Policy on Humane Care and Use of Laboratory Animals Assurance Number: A3288-01). Adult male Sprague-Dawley rats (Harlan Laboratories, Inc.) weighing 200–225 g were randomly assigned to one of two treatment groups. Control rats were housed in normoxia (21 % O2) for the duration of the experiment. Sugen/Hypoxia/Normoxia rats, which will be referred to as PAH rats, were administered subcutaneous injections of Sugen5416 (20 mg/kg, Cayman) then placed in hypoxia (10 % O2) for 3 weeks. The rats were removed from hypoxia and placed in normoxia at the end of the 3 week period. Rats in each group (3–5 per group) were euthanized at one of two experimental time points – 3 or 8 weeks. Heparinized blood was collected from the right ventricle.
7
Investigating Lung Tissue Remodeling via CRISPR-Mediated Vegfr2 Knockdown
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