Suppression of Graft Spasm by the Particulate Guanylyl Cyclase Activator in Coronary Bypass Surgery Takeshi Kinoshita, MD, PhD, Masashi Tawa, PhD, Tomoaki.

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Suppression of Graft Spasm by the Particulate Guanylyl Cyclase Activator in Coronary Bypass Surgery Takeshi Kinoshita, MD, PhD, Masashi Tawa, PhD, Tomoaki Suzuki, MD, PhD, Yoshinari Aimi, MD, PhD, Tohru Asai, MD, PhD, Tomio Okamura, MD, PhD The Annals of Thoracic Surgery Volume 104, Issue 1, Pages 122-129 (July 2017) DOI: 10.1016/j.athoracsur.2016.10.003 Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Typical example of en face staining of the luminal surface using silver nitrate solution in (left panel) endothelium-intact and (right panel) endothelium-denuded internal thoracic artery strip (bar = 50 μm). Histologic endothelial integrity was clearly differentiated in the intact and rubbed strips, indicating that the endothelium remained morphologically intact even after surgical intervention and other experimental protocols. The Annals of Thoracic Surgery 2017 104, 122-129DOI: (10.1016/j.athoracsur.2016.10.003) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Concentration-response curve for carperitide (10−11 to 10−7 M) in (A) internal thoracic artery (ITA) segments and (B) gastroepiploic artery (GEA) segments contracted by 30 mM KCl (dashed line), phenylephrine (solid line) 10−7 to 10−6 M, prostaglandin F2α (broken line) 10−7 to 10−6 M), and endothelin-1 (dotted line) 10−9 M. Effect of removal of endothelium and inhibition of nitric oxide synthase on carperitide-induced relaxation in (C) internal thoracic artery segments and (D) gastroepiploic artery segments contracted with phenylephrine. Relaxation induced by 10−4 M papaverine were taken as 100%. Each point and bar represents the mean ± standard error of the mean; n indicates the number of segments obtained from different patients. *p less than 0.05 compared with other vasoconstrictors (two-way analysis of variance). (Solid line indicates control; dotted line indicates NG-nitro-L-arginine plus carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide.) The Annals of Thoracic Surgery 2017 104, 122-129DOI: (10.1016/j.athoracsur.2016.10.003) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Concentration-response curve for carperitide (solid line), nitroglycerin (broken line), and nifedipine (dotted line) in (A) internal thoracic artery segments and (B) gastroepiploic artery segments contracted with phenylephrine. Relaxation induced by 10−4 M papaverine was taken as 100%. Each point and bar represents the mean ± standard error of the mean; n indicates the number of segments obtained from different patients. *p less than 0.05 compared with nitroglycerin and nifedipine (two-way analysis of variance). The Annals of Thoracic Surgery 2017 104, 122-129DOI: (10.1016/j.athoracsur.2016.10.003) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 (A) Maximal relaxation and (B) the pD2 values for carperitide, nitroglycerin, and nifedipine in internal thoracic artery (ITA) segments (blue bars) and gastroepiploic artery (GEA) segments (orange bars) contracted with phenylephrine. Relaxation induced by 10−4 M papaverine were taken as 100%. Mean levels among the three groups were compared by one-way analysis of variance; post-hoc Bonferroni test was used to detect the individual differences; unpaired t test was used to compare between internal thoracic artery and gastroepiploic artery segments. Values are expressed as mean ± standard error of the mean. **Indicates statistical significance at p less than 0.01. *Indicates p less than 0.05. The effective concentration of vasodilators that caused 50% of maximal relaxation (Emax) was defined as EC50 and expressed as pD2 (= −log EC50). The Annals of Thoracic Surgery 2017 104, 122-129DOI: (10.1016/j.athoracsur.2016.10.003) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Concentration-contraction curves for (A) phenylephrine (7 segments obtained from different patients), (B) prostaglandin F2α (6 segments obtained from different patients), and (C) endothelin-1 (5 segments obtained from different patients) in internal thoracic artery (ITA) segments with treatment (dotted line) or without treatment (solid line) with 10−9 M carperitide. Two segments from the same patient were allocated to each group. Contraction induced by 30 mM KCl was taken as 100%. *p less than 0.05 (two-way analysis of variance). Values are expressed as mean ± standard error of the mean. The Annals of Thoracic Surgery 2017 104, 122-129DOI: (10.1016/j.athoracsur.2016.10.003) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 6 Concentration-contraction curves for (A) phenylephrine (8 segments obtained from different patients), (B) prostaglandin F2α (8 segments obtained from different patients), and (C) endothelin-1 (6 segments obtained from different patients) in gastroepiploic artery (GEA) segments with treatment (dotted line) or without treatment (solid line) with 10-9 M carperitide. Two segments from the same patient were allocated to each group. Contraction induced by 30 mM KCl was taken as 100%. *p less than 0.05 (two-way analysis of variance). Values are expressed as mean ± standard error of the mean. The Annals of Thoracic Surgery 2017 104, 122-129DOI: (10.1016/j.athoracsur.2016.10.003) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Fig 7 Effects of carperitide and nitroglycerin on cyclic guanosine monophosphate (GMP) accumulation in (A) internal thoracic artery (ITA) and (B) gastroepiploic artery (GEA) segments. Arterial segments obtained from the same patients were separated into three groups: control, nitroglycerin, and carperitide. Precontraction was obtained with KCl (red bars) or prostaglandin F2α (PGF2α) (blue bars). The cyclic GMP level in the tissue was expressed as the relative value divided by the frozen tissue weight (pmol/mg). Mean levels of cyclic GMP among the three groups were compared by using one-way analysis of variance; post-hoc Bonferroni test was used to detect the individual differences; unpaired t test was used to compare between segments precontracted with KCl and PGF2α. Values are expressed as mean ± standard error of the mean. **Indicates statistical significance at p less than 0.01. The Annals of Thoracic Surgery 2017 104, 122-129DOI: (10.1016/j.athoracsur.2016.10.003) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Supplemental Figure 1 The Annals of Thoracic Surgery 2017 104, 122-129DOI: (10.1016/j.athoracsur.2016.10.003) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions

Supplemental Figure 2 The Annals of Thoracic Surgery 2017 104, 122-129DOI: (10.1016/j.athoracsur.2016.10.003) Copyright © 2017 The Society of Thoracic Surgeons Terms and Conditions