Vasoreactivity - Contraction and Relaxation Using Aortic Rings
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Do you have test articles that cause changes in blood pressure? Would you like to screen your lead compounds or backups? Check out these slides describing our vasoreactivity assays. These are a perfect compliment to our cardiovascular toxicology and safety pharmacology assays. Uses only milligrams of compound!
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Vasoreactivity - Contraction and Relaxation Using Aortic Rings
- 2. AERS - Adverse Event Reporting System Laverty HG et a, Br J Pharmacol. 2011 Changes is Blood Pressure Lead Optimization Lead Generation Target Selection In Vivo: Anesthetized Animals/Telemetry Early Investigation of Cardiovascular Safety Liabilities In Vitro: Vasoreactivity Assay Blood pressure effect Contraction and relaxation in isolated rings Drug Discovery Process
- 3. Data Acquisition Test Article Adjustment of Passive Tension Vascular Ring 37°C Water Circulation Physiological Solution Isometric Force Transducer Pipette in out 95% O2 5% CO2 Vascular Ring in a Vertical Tissue Bath Adapted from Yildiz O. et. al. Dental Polymers: Vascular Tone Effects, 2015 Tissue Bath System: Pharmacological tool for evaluating concentration-response relationships in contractile tissues. Vascular Rings: Thoracic Aorta, Mesenteric Arteries and Veins, Coronary Arteries, Pulmonary Arteries, Mammary Arteries
- 4. 15 min Vasodilator Curve Pre-constrictor: 0.4 µM Phe TA Conc-response curve (4-5 conc.) 10 µM CCh: Positive control 15 min Vasoconstrictor Curve TA Conc-response curve (4-5 conc.) 0.4 µM Phe: Positive Control 15 min Vasodilator Control Pre-constrictor – 0.4 µM Phe 0.3 µM SNP: Vasodilator Control Washout 15 min Viability Test 15 min Repeat Viability Test Intact muscle layers: Constriction with Phe Intact endothelial cells: Relax with CCh Confirm ring viability Washout Washout Washout 40 to 60 min Equilibration (Washouts every 15min) Phe: Phenylephrine CCh: Carbamylcholine Contraction Discard Rings Viability Test (15 min) Relaxation No Contraction No Relaxation 2 µM Phe 10 µM CCh TA Curve Intact Smooth Muscle Intact Endothelium Concentration-response curves are obtained under controlled conditions without extrinsic neural factors, circulating hormones interactions, blood flow or shear stress. Rings can be tested with or without endothelial layer. Method can reliably predict in vivo vasoactive responses
- 5. Thoracic Aortic Rings Obtained from Sprague Dawley rats (300-400 g) Preservation solution: None Ring size: 3-4 mm Perfusion medium will consist of modified oxygenated Krebs- Henseleit buffer (35 ± 2°C at the bath) Passive tension = 2.0 g Viability criteria: Develop 1.0 g of tension when stimulated with 2 µM Phe and relax by 50-100% of the tension developed following exposure to 10 µM CCh RAT THORACIC AORTIC RINGS Phe: Phenylephrine CCh: Carbamylcholine 3-4 mm rings Thoracic Aorta 5-6 rings/rat
- 6. Mean±SD Relaxation of Rat Thoracic Aorta Precontracted by 400 nM of Phenylephrine Phe: Phenylephrine CCh: Carbamylcholine SNP: Sodium nitroprusside SNP
- 7. Superior Mesenteric Artery Rings Obtained from Sprague Dawley rats (300-400 g) Preservation solution: None Ring size: 3-4 mm Perfusion medium will consist of modified oxygenated Krebs- Henseleit buffer (35 ± 2°C at the bath) Passive tension = 1.0 g Viability criteria: Develop 0.25 g of tension when stimulated with 2 µM Phe and relax by 50-100% of the tension developed following exposure to 10 µM CCh RAT MESENTERIC ARTERY RINGS Phe: Phenylephrine CCh: Carbamylcholine Pourageaud F. et. al. Am J Physiol. 1997
- 8. Phe SNP Mean±SD Phe: Phenylephrine CCh: Carbamylcholine SNP: Sodium nitroprusside Constriction of Rat Mesenteric Artery Relaxation of Rat Mesenteric Artery Precontracted by 400 nM of Phenylephrine
- 9. Thoracic Aortic Rings Preservation solution: Oxygenated Tyrode’s solution. Aorta can be used for 3-4 days. Ring size: 2 mm Perfusion medium will consist of oxygenated Tyrode’s solution buffer (35 ± 2°C at the bath) Passive tension: 10 g Viability criteria: Develop 3.0 g of tension when stimulated with 2 µM Phe and relax by 50-100% of the tension developed following exposure to 10 µM CCh DOG THORACIC AORTIC RINGS Phe: Phenylephrine CCh: Carbamylcholine
- 10. SNP Mean±SD Relaxation of Dog Thoracic Aorta Precontracted by 400 nM of Phenylephrine Phe: Phenylephrine CCh: Carbamylcholine SNP: Sodium nitroprusside Phe Constriction of Dog Thoracic Aorta
- 11. Mesenteric Artery Rings Preservation solution: None Ring size: 3-4 mm Perfusion medium will consist of oxygenated Tyrode’s solution buffer (35 ± 2°C at the bath) Passive tension: 1.0 g Viability criteria: Develop 1.0 g of tension when stimulated with 2 µM Phe and relax by 50-100% of the tension developed following exposure to 10 µM CCh DOG MESENTERIC ARTERY RINGS Phe: Phenylephrine CCh: Carbamylcholine
- 12. Phe SNP Mean±SD Constriction of Dog Mesenteric Artery Phe: Phenylephrine CCh: Carbamylcholine SNP: Sodium nitroprusside Relaxation of Dog Mesenteric Artery Precontracted by 400 nM of Phenylephrine
- 13. Circumflex and Left Anterior Descending Coronary Artery Rings Preservation solution: None Ring size: 4-5 mm Perfusion medium will consist of oxygenated Tyrode’s solution buffer (35 ± 2°C at the bath) Passive tension: 4.0 g Viability criteria: Develop 3.0 g of tension when stimulated with 27 mM KCl and relax by 50-100% of the tension developed following exposure to 10 µM CCh DOG CORONARY ARTERY RINGS KCl: Potassium Chloride CCh: Carbamylcholine
- 14. U46619 SNP Constriction of Dog Coronary Artery Relaxation of Dog Coronary Artery Precontracted by 0.3 µM of U46619 CCh: Carbamylcholine SNP: Sodium nitroprusside U46: U46619
- 15. Mammary Artery Rings Obtained from patients undergoing coronary artery bypass graft surgery Preservation solution: TiProtec® (4-8°C) Preservation time: Approximately 48 hours Dissection after gently warmed to room temperature in Krebs- Henseleit buffer Perfusion medium will consist of modified oxygenated Krebs- Henseleit buffer (35 ± 2°C at the bath) Passive tension = 2.0 g Viability criteria: Develop 1.0 g of tension when stimulated with 2 µM Phe, relaxation with CCh will not be a criterion for exclusion HUMAN INTERNAL MAMMARY ARTERY RINGS
- 16. Characteristics No. of donors Male 5 Female 0 Caucasian/White 4 African American/Black 1 Coronary Artery Vascular Disease 5 Arterial Hypertension 5 Hypercholesterolemia 5 Cardiac Disease 5 Angiotensin Converting Enzyme Inhibitor 1 Statins 4 β-Blockers 3 Calcium Chanel Blocker 3 Acetylsalicylic Acid 3 Clopidogrel 2 Sex Diagnosis Risk Factor Medication Ethnic Background Donor Demographics (n=5)Five donor tissues were procured - IRB- reviewed and approved. Written consent for research use was received prior to surgery. The median age was 58 years (range: 55−76 years). The warm ischemic time (length of time from tissue removal to placement in preservation solution) ranged from 5−8 minutes. The cold storage time (length of time from placement in preservation solution to beginning of tissue isolation) in TiProtec® preservation solution ranged from 46−47 hours.
- 17. CCh Mean±SD Relaxation on Human Mammary Artery Precontracted by 400 nM of Phenylephrine Phe: Phenylephrine CCh: Carbamylcholine SNP: Sodium nitroprusside
- 18. SNP Mean±SD Phe: Phenylephrine CCh: Carbamylcholine SNP: Sodium nitroprusside Relaxation on Human Mammary Artery Precontracted by 400 nM of Phenylephrine