Role of Rho Kinase in Regulating Arterial Stiffness in Anesthetized Rabbits

Abstract

The effects of Rho kinase inhibitors fasudil and ripasudil on arterial stiffness were assessed using anesthetized rabbits, where the aortic β and femoral β were measured as a stiffness parameter at each arterial region. Intravenous administration of fasudil and ripasudil dose-dependently decreased blood pressure and femoral vascular resistance and increased femoral arterial blood flow, which appeared according to their in vitro potencies for Rho kinase inhibition. Both drugs increased the aortic β but decreased the femoral β at hypotensive doses. These results suggest that the inhibition of Rho kinase contributes to reducing elastic recoil in the aorta and an increase in compliance in the femoral artery. In addition, the Rho kinase-associated Ca²⁺-independent mechanism of arterial vascular smooth muscle contraction may be essential in the regulation of femoral arterial stiffness.

INTRODUCTION

Arterial stiffness secondarily and functionally regulates systemic circulation via the Windkessel effect.¹⁾ To quantitatively measure arterial stiffness from the origin of the aorta to the tibial artery, the cardio-ankle vascular index (CAVI) was clinically introduced based on a combination of the theory of stiffness parameter β and the Bramwell–Hill equation.^2–4) Arterial stiffness could be modified by acute pharmacological interventions in humans and rabbits,^5–9) which may explain the in vivo association between the function of vascular smooth muscle cells and the physiological control of arterial stiffness. Recently, a measurement system was established to simultaneously and separately assess the arterial stiffness of the aortic and femoral regions in anesthetized rabbits, where the effects of Ca²⁺ channel blockers on arterial stiffness were analyzed.¹⁰⁾ To better understand the mechanism underlying the regulation of arterial stiffness, this study analyzed the role of Rho kinase-associated Ca²⁺-independent vascular smooth muscle contraction in the regulation of arterial stiffness using two Rho kinase inhibitors, fasudil and ripasudil, in anesthetized rabbits.

MATERIALS AND METHODS

All experiments were approved by the Ethics Committee of Toho University (22-51-509). All experiments were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, authorized by The Japanese Pharmacological Society. Male New Zealand White rabbits were purchased from Japan SLC (Hamamatsu, Japan).

Animal Preparations

Approximately 17-week old rabbits were initially anesthetized with an intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg). After intubation, the rabbits were ventilated with 1.5% isoflurane vaporized with pure oxygen. The core temperature was maintained at 37 °C using a heated operating table. A surface lead II electrocardiogram was obtained using the limb electrodes. To measure the blood pressure in the right brachial artery, aortic bifurcation, and right tibial artery, heparinized catheters were inserted into each artery and connected to a pressure transducer (DX-100; Nihon Kohden, Tokyo, Japan), which was amplified with a strain pressure amplifier (AP-610J; Nihon Kohden). The femoral arterial blood flow was measured using an ultrasonic blood flow meter (TS420; Transonic Systems Inc., Ithaca, NY, U.S.A.). The femoral vascular resistance was calculated using the following basic equation: mean blood pressure/femoral arterial blood flow. Phonocardiograms were recorded using a microphone placed on the breastbone. The right and left ear veins were used for drug administration. All signals were recorded using the PowerLab^® system (ADInstruments, Sydney, Australia) and VaSera^® (VS-1500, Fukuda Denshi Co., Ltd., Tokyo, Japan) for evaluating arterial stiffness.

Evaluation of Changes in Arterial Stiffness of Aortic and Femoral Arterial Segments

Arterial stiffness was measured in the three segments: between the origin of the aorta and tibial artery (denoted as ha β), the origin of the aorta and aortic bifurcation (denoted as aortic β), and the aortic bifurcation and femoral artery (denoted as femoral β), as shown in Supplementary Figure. β was calculated by modifying the formula for the CAVI (denoted as ha β) used in humans²⁾ as follows:

  

where ρ is blood density, ln is logarithm natural, Ps is systolic blood pressure, Pd is diastolic blood pressure, and ∆P is Ps–Pd. Blood pressure at the brachial artery and aortic bifurcation was utilized for aortic β and femoral β, respectively. PWV is the pulse wave velocity calculated as follows:

  

where L₁ is the length from the aortic valve to the aortic bifurcation and T₁ is the time taken for the pulse wave to propagate from the aortic valve to the aortic bifurcation. L₂ is the distance from the aortic bifurcation to the ankle, and T₂ is the time taken for the pulse wave to propagate from the aortic bifurcation to the tibial artery. T₁ was measured using VaSera^® and T₂ was measured using LabVIEW^® (National Instruments Co., Austin, TX, U.S.A.) with the same algorism as that used by VaSera^®.

Experimental Protocols

The experiments were conducted after assessing the basal conditions. Fasudil (0.3 mg/kg) was intravenously administered over 10 min, and each parameter was sequentially measured until 30 min after the start of drug infusion. The effects of fasudil at doses of 1, 3, and 10 mg/kg were assessed using the same procedure. The effects of ripasudil at doses of 0.03, 0.1, 0.3, and 1 mg/kg were also assessed in another group of animals.

Drugs

Fasudil hydrochloride was purchased from neo CritiCare Pharma Co., Ltd. (Atsugi, Japan) as an injectable form, and ripasudil hydrochloride was obtained from Kowa Company Ltd. (Nagoya, Japan) as an ophthalmic solution (GLANATEC^®). Drugs were diluted in saline and administered intravenously. Ketamine (Ketalar^® for intramuscular injection, Daiichi Sankyo, Tokyo, Japan), xylazine (Selactar^®, Bayer, Osaka, Japan), isoflurane (Isoflurane Inhalation Solution, Pfizer Japan Inc., Tokyo, Japan) and heparin sodium (AY Pharma, Tokyo, Japan) were used in commercial formulations.

Statistical Analysis

Data are presented as the mean ± standard error of the mean. The statistical significance of within-group comparisons for each parameter measured over time was assessed using one-way repeated-measures ANOVA, followed by Dunnett’s test to compare the mean values. Statistical analyses were conducted using Prism^® 8 software (GraphPad Software, Inc., San Diego, CA, U.S.A.). Statistical significance was set at p < 0.05.

RESULTS

The time courses of changes in the hemodynamic parameters, aortic β, femoral β, and ha β after fasudil administration are summarized in Fig. 1. Intravenous administration of fasudil at 0.3 mg/kg did not affect any cardiovascular parameters. Fasudil at doses of 1, 3, and 10 mg/kg decreased blood pressure and femoral vascular resistance in a dose-dependent manner, with an increase in femoral arterial blood flow. A significant increase in heart rate was detected at a dose of 3 mg/kg. Significant increases in aortic β were detected at 3 and 10 mg/kg doses. Femoral β decreased at 3 mg/kg (p = 0.057, 10 min after the start of drug administration), and a significant decrease in femoral β was detected at a dose of 10 mg/kg. Meanwhile, no significant change was observed in ha β.

Fig. 1. Time Courses of the Effects of Fasudil on the Heart Rate, Systolic (Inverted Triangles) and Diastolic (Triangles) Brachial Arterial Blood Pressures, Aortic β, Femoral β, ha β, Femoral Arterial Blood Flow, and Femoral Vascular Resistance

The pre-drug control values are 197 ± 16 beats/min, 63.0 ± 1.9, 41.0 ± 0.7 mmHg, 3.0 ± 0.1, 7.4 ± 2.0, 3.5 ± 0.3, 3.8 ± 0.5 mL/min, and 13.8 ± 1.6 mmHg/(mL/min), respectively. Fasudil at 1, 3, and 10 mg/kg decreased the mean blood pressure by up to 7.6 ± 2.3, 14.9 ± 2.1, and 14.1 ± 2.3 mmHg, respectively. Data are presented as the mean ± standard error of the mean (S.E.M.) (n = 6). The closed symbols represent significant differences from the corresponding control value of each parameter at p < 0.05. HR; heart rate, BP; blood pressure, FBF; femoral arterial blood flow, FVR; femoral vascular resistance.

The time courses of changes in the hemodynamic parameters, aortic β, femoral β, and ha β after ripasudil administration are summarized in Fig. 2. Ripasudil at doses of 0.1, 0.3, and 1 mg/kg decreased blood pressure and femoral vascular resistance in a dose-dependent manner, with an increase in femoral arterial blood flow. Significant increases in aortic β and decreases in femoral β were observed after administration of fasudil at 0.3 and 1 mg/kg doses. Meanwhile, no significant change was observed in ha β.

Fig. 2. Time Courses of the Effects of Ripasudil on the Heart Rate, Systolic (Inverted Triangles) and Diastolic (Triangles) Brachial Arterial Blood Pressures, Aortic β, Femoral β, ha β, Femoral Arterial Blood Flow, and Femoral Vascular Resistance

The pre-drug control values are 201 ± 11 beats/min, 69.3 ± 4.2, 44.8 ± 3.6 mmHg, 3.4 ± 0.2, 7.7 ± 1.3, 4.1 ± 0.1, 5.6 ± 0.5 mL/min, and 9.6 ± 0.50 mmHg/(mL/min), respectively. Ripasudil at 0.1, 0.3, and 1 mg/kg decreased the mean blood pressure by up to 6.9 ± 0.7, 15.1 ± 2.7, and 24.2 ± 3.6 mmHg, respectively. Data are presented as the mean ± S.E.M. (n = 6). The closed symbols represent significant differences from the corresponding control value of each parameter at p < 0.05. HR; heart rate, BP; blood pressure, FBF; femoral arterial blood flow, FVR; femoral vascular resistance.

DISCUSSION

This study assessed the effects of Rho kinase inhibitors fasudil and ripasudil on aortic and femoral arterial stiffness (aortic β and femoral β) in anesthetized rabbits. Fasudil and ripasudil at hypotensive doses increased aortic β but decreased femoral β, suggesting that the increase in aortic β and decrease in femoral β are common effects of Rho kinase inhibitors on conduit arteries.

Effects of Rho Kinase Inhibitors on Hemodynamics

Fasudil and ripasudil dose-dependently decreased blood pressure and femoral vascular resistance, in addition to increasing femoral arterial blood flow, indicating that the drugs adequately dilated the arterial smooth muscle of the peripheral arteries, including femoral arterial trees. The in vivo potencies of fasudil and ripasudil for the hemodynamic actions were almost comparable to those of previous in vitro studies, where IC₅₀ values of fasudil and ripasudil for Rho-associated Coiled-coil-containing Protein Kinase-1 (ROCK-1) were 0.29 and 0.051 µM, respectively.¹¹⁾ Thus, obvious Rho kinase inhibition in anesthetized rabbits appeared at ≥1 mg/kg of fasudil and ≥0.1 mg/kg of ripasudil. Since a higher concentration of a representative ROCK inhibitor Y-27632 depolymerizes actin filament,¹²⁾ higher doses of fasudil or ripasudil might partly include the cytoskeletal action.

Effects of Rho Kinase Inhibitors on Arterial Stiffness

Both fasudil and ripasudil increased the aortic β but decreased the femoral β. The rabbit aorta contains a high proportion of elastin fibers that allow systolic distension, whereas the femoral artery has less elastin and rich vascular smooth muscle cells,¹⁰⁾ which advances the blood forward in the aorta owing to elastic recoil and less compliance of the distal artery, respectively. These results suggest that Rho kinase inhibition may contribute to reduced elastic recoil in the aorta and increased compliance in the femoral artery of rabbits. The previous study using the isolated rabbit aorta has shown that fasudil effectively suppressed KCl or α₁-adrenoceptor agonist phenylephrine-induced contraction.¹³⁾ However, the aortic β increased after the administration of higher fasudil and ripasudil doses, which resembles our previous results of hypotensive doses of the L-type Ca²⁺ channel blocker nifedipine and the L/N-type Ca²⁺ channel blocker cilnidipine.¹⁰⁾ We speculate that mechanisms other than the relaxation of smooth muscle cells mainly contribute to the control of arterial stiffness in the aorta.

In contrast to the regulation of aortic stiffness, the femoral β decreased after the administration of hypotensive doses of fasudil as well as ripasudil. We have confirmed that the femoral β is sympathetically regulated by α₁-adrenoceptor activation in rabbits, leading to an increment of femoral arterial stiffness.¹⁰⁾ On the other hand, our recent study has demonstrated that the L-type Ca²⁺ channel blocker nifedipine hardly affected the femoral β in the same experimental condition of vasodilator-induced hypotension at approximately −15 mmHg.¹⁰⁾ These observations at the condition of a potential increase in sympathetic tone suggest that the Rho kinase-associated Ca²⁺-independent mechanism in the femoral arterial cells during the activation of α₁-adrenoceptors is critical in the regulation of the femoral β, in contrast to the L-type Ca²⁺ channel-mediated mechanism in anesthetized rabbits. The interrelation between Ca²⁺-dependent and Ca²⁺-sensitization processes in the regulation of arterial stiffness may be essentially similar to those in the previous study using isolated rabbit aorta, where fasudil inhibited contractile responses to phenylephrine whereas the L-type Ca²⁺ channel blocker nicardipine was much less effective in blocking the contractions induced by phenylephrine.¹³⁾

Clinical Relevance

Fasudil is clinically used for the suppression of cerebral vasospasm after subarachnoid hemorrhage surgery for a ruptured cerebral aneurysm. Since subarachnoid hemorrhage often complicates hypertension via sympathetic nerve activation, pharmacological inhibition of Rho kinase in such patients will increase compliance in the muscular arteries including the femoral artery, which may contribute to maintaining peripheral circulation.

CONCLUSION

Rho kinase inhibition may contribute to reduced elastic recoil in the aorta and increased compliance in the femoral artery. Furthermore, the Rho kinase-associated Ca²⁺-independent mechanism of arterial vascular smooth muscle contraction may be essential in the regulation of femoral arterial stiffness.

Acknowledgments

This study was supported by JSPS KAKENHI (Grant Number: JP23K11836).

Conflict of Interest

This study was funded by Fukuda Denshi Co., Ltd.

Supplementary Materials

This article contains supplementary materials.

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