Differential Suppressive Effects of Rho Kinase Inhibitor Fasudil on Serotonin- and Noradrenaline-Induced Contractions of Human Internal Thoracic Arteries and Saphenous Veins

Abstract

Rho kinase inhibitor fasudil exerts therapeutic effects against vasospasms. In this study, we aimed to compare its suppressive effects on serotonin (5-HT)- and noradrenaline (NAd)-induced contractions of human endothelium-denuded internal thoracic arteries (ITAs) and saphenous veins (SVs). NAd and 5-HT induced concentration-dependent contractions in both ITAs and SVs. However, fasudil (3 µmol/L) pretreatment decreased these constrictor-induced contractions in both ITAs and SVs. Fasudil exerted similar inhibitory effects on 5-HT and NAd in ITAs. However, in SVs, fasudil exerted stronger inhibitory effects on NAd-induced contractions than on 5-HT-induced contractions. Therefore, inhibitory effects of fasudil on 5-HT-induced contractions were stronger in ITAs than in SVs. Overall, these results suggest that Rho kinases exert different effects on the two vasoconstrictors in SVs, but not in ITAs, thus explaining their different graft patencies.

INTRODUCTION

Coronary artery bypass graft (CABG) surgery is an effective therapeutic strategy for coronary artery disease.¹⁾ In CABG surgery, prevention of vasospasms during and post-operation and reduction of thrombotic occlusion and stenosis after surgery are important for prolonged patency of vessels and for improved prognosis.²⁾

Internal thoracic arteries (ITAs) and saphenous veins (SVs) are commonly used for CABG surgery. ITAs harvested from the chest wall are elastic arteries with excellent long-term patency.³⁾ SVs are the predominantly used grafts owing to their sufficient length, accessibility, and adjustable size matching those of the coronary artery.³⁾ A comprehensive understanding of the types of vessels involved, their signaling, and susceptibility to drugs is necessary for CABG surgery. Several studies have compared ITAs and SVs during relaxation and contraction.^4,5)

Rho kinases plays a crucial role in the pathogenesis of vasospasms.⁶⁾ Fasudil, a specific Rho kinase inhibitor, is used to prevent cerebral vasospasms and ischemic injury after subarachinoid hemorrhage surgery.⁷⁾ Many clinical studies have investigated the potential of fasudil to treat various cardiovascular diseases and conditions, including pulmonary hypertension, ischemic stroke, aortic stiffness, and heart failure-associated vascular resistance and contraction, kidney transplantation, essential hypertension, and coronary vasospasms, and atherosclerosis.⁸⁾ Although fasudil exerts beneficial effects against coronary artery disease,^9,10) its specific effects on the vasoconstriction of ITAs and SVs remains unclear.

In this study, we aimed to determine the effects of fasudil on the vasoconstriction of human ITAs and SVs using serotonin (5-HT) and noradrenaline (NAd), two potent vasoconstrictors associated with vasospasm.¹¹⁾ Endothelium and endothelium-derived factors, such as nitric oxide, affect ITA and SV vasoconstriction.¹²⁾ Here, we assessed vasoconstriction of the vessels without endothelium. Even with caution, handling blood vessels during surgical procedures can cause varying degrees of damage to the vascular endothelial cells. Therefore, in this study, we used endothelium-denuded ITAs and SVs to avoid the influence of endothelial cell damage on the vessels.

PATIENTS AND METHODS

Chemicals

NAd and 5-HT were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Fasudil was purchased from Asahi Kasei Pharmaceutical (Tokyo, Japan).

Patients

We examined the ITA and SV segments of 16 patients who underwent CABG surgery. This study was conducted according to the principles of the Declaration of Helsinki. All patients provided written informed consent to participate in the study. This study was approved by the Ethics Committees of the Miyazaki Prefectural Nobeoka Hospital and Kyusyu University of Medical Science (formerly, Kyusyu University of Health and Welfare; Approval Number: 09-004).

Blood Vessel Preparation and Functional Studies

Human ITAs and SVs were obtained from patients who underwent CABG surgery at the Miyazaki Prefectural Nobeoka Hospital (Miyazaki, Japan). Portions of each ITA and SV grafts were sectioned to bypass the occluded coronary arteries, and the remaining portions were used in the experiments. Small ITA and SV segments were placed in a modified Krebs buffer solution aerated with 95% O₂ and CO₂ and immediately transported to the laboratory. The modified Krebs buffer solution consisted of 118.4 NaCl, 4.7 KCl, 25.0 NaHCO₃, 1.2 MgSO₄, 1.2 KH₂PO₄, 2.5 CaCl₂, 0.026 ethylenediaminetetraacetic acid, and 11.1 glucose (pH 7.4; in mmol/L). After careful removal of the fat and connective tissues, the vessels were cut into 2-mm rings. The rings were denuded of endothelium by inserting an injection needle into the lumen and gently rolling it back and forth to eliminate the influence of the endothelium on vasoconstriction. Vasoconstriction was evaluated as previously described.^13,14) Briefly, the ITA and SV rings were stretched progressively to optimal tension (2.0 g) and allowed to equilibrate for 60 min. The ring was washed several times with fresh buffer solution. Cumulative concentration vs. response curves for 5-HT or NAd were plotted over a concentration range of 10⁻⁹ to 10⁻⁵ mol/L. Subsequently, after washing and achieving re-stabilization, to investigate the inhibitory effects of fasudil (3 µmol/L) on 5-HT- and NAd-induced vasoconstrictions, fasudil (3 µmol/L) was added to the bath for 30 min before administering 5-HT or NAd in the corresponding bath. Each contractile response of the ring was evaluated using the percentage of vasoconstriction induced by 60 mmol/L KCl as 100%.

To compare the inhibitory effects of fasudil on contractions between ligands and vessels, the inhibitory ratio of fasudil (3 µmol/L) on the contractions of ITAs and SVs induced by submaximal concentrations (3 µmol/L) of 5-HT or NAd was calculated. Using the contractile values of each concentration vs. contractile curve for 5-HT or NAd (at 3 µmol/L: submaximal concentration in the absence and presence of fasudil), the inhibitory ratio of fasudil (%) was calculated as follows: 100 − the value of contraction in the presence of fasudil divided by the value of contraction in the absence of fasudil ×100.

Statistical Analysis

Data are represented as the mean ± standard error of the mean obtained from different numbers (n) of ring preparations. GraphPad Prism software was used for the statistical analyses. Differences between values were analyzed using repeated-measures two-way ANOVA with post hoc Sidak’s test (Fig. 1) or post hoc Tukey’s test after one-way ANOVA (Fig. 2). Statistical significance was set at p < 0.05.

Fig. 1. Concentration–Response Curves for Serotonin and Noradrenaline in Endothelium-Denuded Rings of Human Internal Thoracic Arteries and Saphenous Veins

Effects of fasudil (3 µmol/L) on serotonin (5-HT)-induced contractions of internal thoracic arteries (ITAs) (a) and saphenous veins (SVs) (b). Effects of fasudil (3 µmol/L) on NAd-induced contractions of ITAs (c) and SVs (d). After conducting concentration–contractile curves for 5-HT or NAd in the absence of fasudil in both endothelium-denuded rings, fasudil was added to the bath for 30 min before administering 5-HT and NAd as second curves. Data are expressed as a percentage of the response to 60 mM KCl in each vessel and represented as the mean ± standard error of the mean (S.E.M.; n = 8 (ITA/5-HT), 10 (ITA/NAd), 26 (SV/5-HT), and 16 (SV/NAd)). * p < 0.05, control vs. fasudil (two-way repeated ANOVA followed by Sidak’s test).

Fig. 2. Inhibitory Ratio of Fasudil (3 µmol/L) on the Contractions of ITAs and SVs Induced by Submaximal Concentrations (3 µmol/L) of 5-HT or NAd

Using the contractile values of concentration–contractile curve for 5-HT or NAd (at 3 µmol/L: submaximal concentration in the absence and presence of fasudil, the inhibitory ratio of fasudil was calculated as follows: 100–the value of contraction in the presence of fasudil divided by the value of contraction in the absence of fasudil ×100 (%). Data are expressed as the mean ± S.E.M. (n = 8–26). * p < 0.05, ITA 5-HT vs. SV 5-HT. ^# p < 0.05, SV 5-HT vs. SV NAd (by one-way ANOVA followed by Tukey’s test).

RESULTS

Effects of Rho Kinase Inhibitor Fasudil on 5-HT- and NAd-Induced Contractions of Human Endothelium-Denuded ITAs and SVs

Cumulative application of 5-HT (10⁻⁹ to 10⁻⁵ mol/L) induced concentration-dependent contractions in ITAs (Fig. 1a) and SVs (Fig. 1b). However, 5-HT-induced contractions were partly suppressed by fasudil (3 µmol/L) in both vessels.

Cumulative application of NAd (10⁻⁹ to 10⁻⁵ mol/L) induced concentration-dependent contractions in ITAs (Fig. 1c) and SVs (Fig. 1d), which were partly suppressed by fasudil (3 µmol/L).

Next, the inhibitory effects of fasudil on 5-HT- and NAd-induced contractions were compared between ITAs and SVs and are shown as the ratio of the inhibitory effect of fasudil based on the value of each concentration–contractile curve at the submaximal concentration of each ligand. As shown in Fig. 2, fasudil exerted similar inhibitory effects on 5-HT- and NAd-induced contractions in the ITAs. However, the inhibitory effects of fasudil were stronger on NAd-induced contractions than on 5-HT-induced contractions in the SVs.

The suppressive effects of fasudil on 5-HT- and NAd-induced contractions were further compared between ITAs and SVs and are shown as a percentage of the maximal contraction at each concentration. The inhibitory effects of fasudil on 5-HT-induced contractions were more potent in the ITAs than in the SVs. The inhibitory effects of fasudil on NAd-induced contractions were similar in ITAs and SVs (Fig. 2).

DISCUSSION

This study showed that the specific Rho kinase inhibitor fasudil suppressed vasoconstriction induced by 5-HT and NAd in both ITAs and SVs, with different extents of inhibitory effects based on the ligand and vessel. Fasudil inhibited 5-HT- and NAd-induced contractions more potently in ITAs than in SVs. In the SVs, the inhibitory effects of fasudil on NAd-induced contractions were stronger than those on 5-HT-induced contractions summarizing as Fig. 3.

Fig. 3. Schematic Summary of the Inhibitory Effects of Fasudil on 5-HT- and NAd-Induced Contractions of ITAs and SVs

Fasudil inhibited 5-HT and NAd-induced contractions. 5-HT-mediated contractions were more potent in ITAs than in SVs. In SVs, the inhibitory effects of fasudil on NAd-induced contractions were stronger than those on 5-HT-induced contractions. The inhibitory effects of fasudil on NAd-induced contractions were similar between ITA and SV. In ITA, the inhibitory effects of fasudil were similar in contractions between 5-HT and NAd. ITA, internal thoracic artery; SV, saphenous vein; NAd, noradrenaline; 5-HT, 5-hydroxytriptamine (serotonin).

In CABG surgery, prevention of intraoperative and postoperative vasospasms is crucial to prolong vessel patency and improve the long-term prognosis.²⁾ Therefore, understanding the effects and molecular signaling induced by spasmogens in ITAs and SVs is important to improve the patient outcomes. Exogenous and endogenous sympathomimetic amines and 5-HT act as spasmogens for blood vessels.^11,15) We previously demonstrated that co-treatment with 5-HT_2A and 5-HT_1B receptor antagonists prevents 5HT-induced vasospasms in ITAs.¹⁴⁾

Gq-coupled receptor ligand-mediated vasoconstriction is mediated by various signal transduction pathways, including the protein kinase C, mitogen-activated protein kinase, and Rho/Rho kinase pathways.⁶⁾ Specifically, Rho/Rho kinases play key roles in vasospasms.⁶⁾ Rho kinase activation contributes to vasospasms⁶⁾ and abnormal hyperreactivity to vasoconstrictors, including 5-HT¹³⁾ and NAd.¹⁶⁾ In this study, we compared the inhibitory effects of fasudil on 5-HT- and NAd-induced contractions of ITAs and SVs (Fig. 2). When the inhibitory effects of fasudil were compared to those of the ligands in each vessel, similar extent of inhibition of both 5-HT- and NAd-induced contractions was observed in ITAs, whereas stronger inhibition of NAd-induced contractions than 5-HT-induced contractions was observed in SVs. Moreover, when the inhibitory effects of fasudil were compared in both vessels for each constrictor, stronger suppressive effects were observed in ITAs than in SVs for 5-HT, but not for NAd (Fig. 2). Overall, our data suggest that the suppressive effect of fasudil on vasoconstriction depends on the vessel type and ligands. However, further investigations are necessary to determine the expression and activity of Rho/Rho kinases in these vessels, with and without stimulations.

Fasudil is the only Rho kinase inhibitor approved for systemic application. It has been used in Japan and China since 1995 for the treatment of vasospasms following subarachnoid hemorrhage,^17,18) and has been considered for the treatment of angina. Several phase I studies were conducted in patients with vasospastic angina (either angiographically detectable or microvascular) who developed electrocardiographic evidence of ischemia in response to acetylcholine.^19,20) The results showed that acetylcholine-induced ischemia was prevented in ≥85% of patients by a 15-min intracoronary infusion of 4.5 mg fasudil.^19,20) Fasudil is bioavailable after oral administration.⁹⁾ In phase II dose-finding trials conducted in Japanese patients with stable effort angina, fasudil monotherapy at doses ranging from 5 mg three times daily to 40 mg three times daily increased maximum exercise time and time to the onset of ≥1 mm ST-segment depression compared with baseline. Fasudil was well tolerated, with minimal effects on blood pressure or heart rate at rest or during exercise.⁹⁾ It is marked under the name ERIL® and approved for intravenous (i.v.) treatment up to three times daily for two weeks. After i.v. administration, fasudil is rapidly converted in the liver to its active metabolite hydroxyfasudil,²¹⁾ corresponding to a serum half-life of 20 min for fasudil and 100–200 min for hydroxyfasudil,^22,23) followed by renal excretion.²⁴⁾ In addition, fasudil has shown promising preclinical results for various chronic diseases, including neurodegenerative diseases and cancer,^24,25) for which long-term i.v. administration might not be suitable. An intravenous formulation of fasudil is licensed in Japan, oral (tablet) formulations, including extended-release formulations, were previously used in clinical trials in humans,^10,26) and a good enteric bioavailability of the drug has been documented.^18,24,27) The longest published exposure to fasudil in humans was 8 and 12 weeks (angina pectoris and pulmonary arterial hypertension).^10,26) In the present study, we used fasudil at 3 µmol/L, which is an effective concentration for Rho kinase inhibition in various in vitro studies.^28,29) In CABG surgery, prevention of intra- and post-operative vasospasms and reduction of post-operative thrombotic occlusion and stenosis prolong the patency of vessels and improve the long-term prognosis.²⁾ Papaverine is widely used worldwide to prevent intra-operative vasospasm during CABG surgery.³⁰⁾ Since papaverine may impair vascular endothelial function,³¹⁾ the optimal concentration of papaverine should be determined based on the usage.³²⁾ Fasudil may be a new drug to prevent vasospasm during intraoperation in addition to papaverine. In the present study, fasudil showed different inhibitory effects on ITA and SV. These differences may be attributable to the different amounts of smooth muscle Rho kinase and/or to the different extents of Rho kinase activation upon receptor stimulation (viz. α₁, 5-HT_2A, and 5-HT_1B receptors) between ITA and SV, and to the different components of smooth muscle signaling pathways upon these stimuli between ITA and SV. Future investigations are required on these points, including determination of the optimal concentration of fasudil for intra- and post-operation, elucidation of detailed signaling pathways between vessels, and the optimal dose and route of treatment after CABG surgery.

This study has many limitations. First, this study only measured the contractile responses in endothelium-denuded vessels. As Rho kinase activation affects the endothelial function,⁶⁾ the influence of fasudil on endothelium-derived factor(s) in ITAs and SVs must be further explored. Additional studies are needed to elucidate the roles of Rho kinases in the endothelial and smooth muscle cells of ITAs and SVs. Second, we specifically focused on the contractile responses induced by two ligands, 5-HT and NAd, which are major constrictors and spasmogens.¹¹⁾ As Rho kinase possibly modulate vascular responses in other ligands related to vasospasm, such as endothelin-1 and thromboxane/prostanoid receptor agonists,³³⁾ further investigation are necessary to determine the suppressive effects of fasudil on other vasoconstrictors in ITAs and SVs. Third, we only assessed Rho kinase inhibition by fasudil in this study. As 5-HT and NAd affect various signaling pathways and molecules in addition to Rho kinases and the reactions to these vasoconstrictors possibly differ between ITAs and SVs,³⁴⁾ further investigations are essential to compare other signaling pathways in ITAs and SVs.

In conclusion, this study demonstrated that fasudil suppressed 5-HT- and NAd-induced contractions of ITAs and SVs, with different extent of inhibition based on the ligands and vessel types. These findings highlight the therapeutic potential of fasudil to treat vasospasms during CABG surgery.

Acknowledgments

This work received no grants from any funding agency in the public, commercial, or not-for-profit sectors.

Funding

The authors declare no specific funding for this work.

Author Contributions

Conceptualization: NTT. Data curation: TM, TN, RY, NTT. Formal analysis: NTT. Investigation: AY, EN, MK, NTT. Methodology: NTT. Project administration: NTT. Resources: AY, EN, MK, RY, NTT. Visualization: TM, TN, NTT. Writing-original draft: TM. Writing-review & editing: TM, TN, AY, EN, MK, RY, NTT.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

Patient data presented in this study are not publicly available and can be made available upon justified request.

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