Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that is caused by a variety of diseases and characterized by an increase in pulmonary vascular resistance, abnormal sustained pulmonary vasoconstriction, and progressive structural remodeling of pulmonary arteries, ultimately leading to right ventricular failure and death.1-3 The pathological changes of hypertensive pulmonary arteries include endothelial injury, proliferation and hypercontraction of vascular smooth muscle cells, and migration of inflammatory cells.4-8 Although several vasodilator agents, such as vasodilator prostaglandins, calcium channel blockers, and endothelin-1 receptor antagonists, have been used for the treatment of PAH, their effects are limited because of a lack of pulmonary vascular selectivity.9-11 Nitric oxide (NO) inhalation could cause a selective pulmonary vasodilatation without affecting systemic blood pressure in patients with PAH.12,13 However, since NO is unstable in air and undergoes spontaneous oxidation to nitrogen dioxide (NO2),14 continuous and on-line monitoring NO and NO2 concentrations is mandatory during the therapy with inhaled NO.15
In the 1990s, Rho-kinase/ROK/ROCK was identified as an effector of the small GTPase Rho,16-18 which plays an important role in various cellular functions, including smooth muscle contraction, actin cytoskeleton organization, cell adhesion and motility, cytokinesis, and gene expressions.19-23 We have recently demonstrated that long-term inhibition of Rho-kinase with a Rho-kinase inhibitor, fasudil, ameliorates monocrotaline (MCT)-induced pulmonary hypertension (PH) in rats23,24 and hypoxia-induced PH in mice.25 However, it remains to be examined in more detail whether fasudil has a selective vasodilator effect at a given dose. In the present study, we thus examined whether acute oral administration with different doses of fasudil exert an effective and selective pulmonary vasodilator effect in rats with MCT-induced PH under conscious conditions.
MATERIALS AND METHODS
All procedures were approved by the Institutional Animal Care and Use Committee and were conducted in conformity with the institutional guidelines of Kyushu University Graduate School of Medical Sciences. Fasudil was provided by Asahi Kasei Pharma (Tokyo, Japan).
We used a total of 75 rats, including 40 for hemodynamic study, 31 for Western blot analyses, and 4 for drug concentration measurements. Adult male Sprague-Dawley rats (Charles River, Yokohama, Japan; 200 to 250 g body weight) were given a single subcutaneous injection of MCT (60 mg/kg; Wako, Tokyo, Japan), which induces severe PH in 3 weeks with a subsequent high mortality rate.24,26 Control rats received an equal volume of saline.24
After 3 weeks of a single MCT injection, all surviving rats were anesthetized with pentobarbital sodium (45 mg/kg, intraperitoneally) and a pulmonary artery catheter was inserted via the right external jugular vein into the pulmonary artery by a closed-chest technique.27 An aortic catheter was also passed from the left carotid artery into the ascending aorta to measure arterial pressure. At 24 to 48 hours after the catheterization and under conscious conditions in which the animals were calm and free-moving, a single oral administration of fasudil (3, 10, and 30 mg/kg) or vehicle was performed, and hemodynamic variables were continuously monitored with a polygraph system (AP-601G; Nihon Kohden, Tokyo, Japan) before and after the fasudil administration at 0.5, 1, 3, 5, 7, and 24 hours.
Western Blot Analyses
To quantify Rho-kinase activity, the protein expression of the total and phosphorylated ERM (ezrin, radixin, and moesin) family, a substrate of Rho-kinase, was analyzed by Western blot analyses in the normal and MCT-induced PH rats treated with vehicle or fasudil.24 At 3 weeks after the MCT or saline injection, a single oral administration with fasudil (10 mg/kg) or vehicle was performed, and 30 minutes after the administration, bilateral pulmonary arteries and the aorta were isolated and immediately frozen by immersion in acetone containing 10% trichloroacetic acid (TCA) cooled with dry ice for 50 minutes. The frozen specimens were washed 5 times with acetone containing dithiothreitol (10 mmol/L) to remove the TCA and were then dried. The dried samples were cut and exposed to 200 μL of SDS-PAGE sample buffer for protein extraction. The extracted samples (10 μg of protein) were subjected to SDS-PAGE/immunoblot analysis by using the specific ERM antibody.28 The regions containing ERM family proteins were visualized by ECL Western blotting luminal reagent (Santa Cruz Biotechnology, Santa Cruz, CA). The extent of the ERM phosphorylation was normalized by that of total ERM.24
Plasma Concentration of Fasudil and Hydroxyfasudil
Blood samples were obtained from carotid arteries in rats with MCT-induced PH at 0.5, 1, 3, and 6 hours after a single oral administration with fasudil (10 mg/kg). Plasma concentrations of fasudil and hydroxyfasudil, which is an active oral metabolite of fasudil and acts as a more specific inhibitor of Rho-kinase,29 were serially measured by an HPLC method.30
All results are expressed as the mean ± SEM. Comparisons between groups were made with Student's t-test or analysis of variance with Fisher's post-hoc test or Tukey-Kramer test for multiple comparisons. Differences were considered to be statistically significant at P < 0.05.
Induction of Pulmonary Hypertension and Baseline Hemodynamics
Under basal conditions, mean pulmonary arterial pressure (mPAP) was significantly elevated in MCT-treated rats (49 ± 3 mm Hg; n = 17) compared with saline-treated control rats (19 ± 1 mm Hg; n = 9; P < 0.01) (Table 1). In the rats with MCT-induced PH, mean systemic arterial pressure (mSAP) tended to be decreased and heart rate was significantly increased compared with the control rats (Table 1).
Acute Effects of Fasudil on Hemodynamics
In the rats with MCT-induced PH, acute oral administration of fasudil (3, 10, and 30 mg/kg), but not that of vehicle, decreased mPAP in a dose-dependent manner, which peaked within 30 minutes and lasted for about 5 hours (Figure 1A). While a low dose (3 mg/kg) had no significant effect on mPAP, a middle (10 mg/kg) and a high dose (30 mg/kg) of fasudil significantly reduced mPAP by 20% ± 3% (n = 7) and 35% ± 3% (n = 6), respectively. By contrast, mSAP was significantly reduced to a high dose (31% ± 6% at peak; n = 3) but not to a middle dose of fasudil (Figure 1B). All doses of fasudil had no effect on heart rate (data not shown).
Acute Effects of Fasudil on Rho-kinase Activity
The extent of ERM phosphorylation, a marker of Rho-kinase activity, was significantly increased in pulmonary arteries in rats with MCT-induced PH and was markedly inhibited by fasudil (10 mg/kg; n = 7) (Figure 2A). By contrast, the extent of ERM phosphorylation was unchanged by the same dose of fasudil (10 mg/kg) in the aorta in both normal and PH rats (n = 3-4) (Figure 2B).
Plasma Concentrations of Hydroxyfasudil
The mean peak value of plasma concentration of fasudil/hydroxyfasudil was 516 ng/mL, noted 1 hour after oral administration of fasudil (10 mg/kg) in MCT-induced PH rats (n = 4) (Figure 3). This value was within the clinical concentrations of the drugs in humans.30
The novel finding of the present study is that oral single administration of fasudil, a Rho-kinase inhibitor, causes dose-dependent and selective pulmonary vasodilatation at a given dose in rats with MCT-induced PH under conscious conditions.
Acute Pulmonary Vasodilatation by Rho-kinase Inhibition
It was previously suggested that pulmonary vascular proliferation and remodeling, but not vasoconstriction, are the major mechanisms of PAH.6 However, in the present study with a rat model of MCT-induced PH, acute oral administration of fasudil significantly reduced mPAP, suggesting that Rho-kinase-mediated pulmonary vasoconstriction plays an important role in the pathogenesis of PH, at least at the present time point (3 weeks after the MCT injection). Rho/Rho-kinase pathway is substantially involved in vasoconstriction induced by various factors, including angiotensin II,31 endothelin-1,32 serotonin,28 and hypoxia,33 all of which may be involved in the pathogenesis of PH.33-36 A recent report suggested that Rho-kinase inhibition is involved in the beneficial effect of the type 5 phosphodiesterase inhibitor, sildenafil, on PH.37 Furthermore, it was also demonstrated that inhaled Rho-kinase inhibitors cause selective pulmonary vasodilatation in animal model of PH with various etiologies.38 Thus, Rho-kinase apparently is an important therapeutic target for the treatment of PH.
Therapeutic Importance of Fasudil for the Treatment of PH
One of the major problems in the current treatments of PH is that all vasodilators used lack pulmonary selectivity and reduce systemic arterial pressure.9-11 Those vasodilators could cause systemic hypotension in patients with PH associated with tachycardia, sympathetic stimulation, and right ventricular ischemia.11 NO inhalation could reduce PAP alone without affecting systemic arterial pressure.12,13 However, the mandatory delivery and monitoring system, which is complicated and expensive, is another important issue to overcome.39 Furthermore, it has been recently demonstrated that inhaled NO does not prevent pulmonary vascular remodeling in both the MCT-induced and the chronic hypoxic models of PH in rats.40,41 In the present study, baseline heart rate was significantly increased in rats with MCT-induced PH, which could be caused by the slight (but insignificant) decrease in systemic pressure. Under these conditions, oral administration of a middle dose of fasudil (10 mg/kg) significantly reduced mPAP alone without affecting mSAP or heart rate. Plasma concentrations of fasudil/hydroxyfasudil that were achieved by the same dose of fasudil (10 mg/kg) were within their clinical therapeutic range in humans.30 We also have previously demonstrated that neither acute intravenous infusion nor chronic oral administration of fasudil lowers mSAP.24,42 Taken together, it is possible that Rho-kinase inhibitors could cause selective pulmonary vasodilatation in humans.
The Activity of Rho-kinase
Another important finding of the present study is that Rho-kinase was activated in pulmonary arteries but not in the aorta of MCT-induced PH rats and that oral administration of fasudil only suppressed activated Rho-kinase in pulmonary arteries without affecting the physiological Rho-kinase activity in the aorta. We have also demonstrated that fasudil only suppresses activated Rho-kinase in atherosclerotic porcine coronary arteries without affecting the activity in normal coronary segment.43 These results suggest that fasudil exerts more effective inhibitory effects on activated Rho-kinase than on basal physiological activity, which could explain, at least partially, why fasudil exerts selective inhibitory effects on pulmonary arteries of MCT-induced PH rats.
Limitations of the Study
Several limitations should be mentioned for the present study. First, due to the technical difficulty, we were unable to measure other hemodynamic variables, such as cardiac output and pulmonary vascular resistance. Therefore, further studies on those hemodynamic variables remain to be performed. Second, we only examined the acute inhibitory effects of fasudil in MCT-induced PH model. Nagaoka et al reported that both oral and inhaled Y-27632, another Rho-kinase inhibitor, have vasodilator effects on the pulmonary circulation, whereas only the inhaled route induces selective effects in a rat model of hypoxia-induced PH.38 Therefore, it remains to be examined whether oral administration of fasudil also exerts selective vasodilator effects on pulmonary circulation at a given dose in a PH model with different etiology as well (eg, chronic hypoxia).
Recently, we and others have demonstrated acute inhibitory effects of intravenous fasudil in patients with PH.42,44 We have recently confirmed the effectiveness and safety of the oral form of fasudil in patients with stable effort angina.45 PH continues to be a serious clinical problem with high morbidity and mortality for which no satisfactory treatment is yet available. The present results suggest that Rho-kinase could be a novel therapeutic target for the treatment of PH and that oral form of a Rho-kinase inhibitor, such as fasudil, may be useful for the acute treatment of PH in humans without hypotension.
In conclusion, we were able to demonstrate that oral administration of fasudil could selectively reduce mPAP without affecting mSAP at a given dose in a rat model of MCT-induced PH. When combined with our previous findings on the chronic inhibitory effect of fasudil on PH,24,25 the present findings suggest that Rho-kinase inhibitors may be useful and safe for the treatment of PAH in humans.
We thank Asahi Kasei Pharma (Tokyo, Japan) for providing fasudil. We also thank E. Gunshima and Y. Matsuo for excellent technical assistance.
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