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 premature death.1-3 The pathological changes of hypertensive pulmonary arteries include endothelial injury, proliferation and hypercontraction of vascular smooth muscle cells (VSMC), and migration of inflammatory cells.4-6 In the clinical setting, prostacyclin is most frequently used for the treatment of PAH, with moderate success.7-9
In the 1990s, Rho-kinase/ROK/ROCK was identified as an effector of the small GTPase Rho,10-12 which plays an important role in various cellular functions, including smooth muscle contraction, actin cytoskeleton organization, cell adhesion and motility, cytokinesis, and gene expressions.13-17 We and others have previously demonstrated that Rho-kinase is involved in the pathogenesis of pulmonary hypertension (PH) in animal models18-20 and patients.21,22 We also have reported that prostacyclin and its oral analogue, beraprost sodium (BPS), lack inhibitory effect on serotonin-induced VSMC contraction and Rho-kinase activation in vitro.23 Because the vasodilator and antiproliferative mechanisms of prostacyclin and BPS are mediated by cyclic adenosine mono-phosphate (AMP),5,24-26 these results suggest that combination therapy with a Rho-kinase inhibitor and prostacyclin could be superior to each monotherapy in the treatment of PH. In the present study, we thus examined whether the combination of fasudil and BPS is more effective than each monotherapy for ameliorating monocrotaline (MCT)-induced PH in rats.
All procedures were performed according to the protocols approved by the Institutional Committee for Use and Care of Laboratory Animals of Tohoku University. Fasudil and BPS were provided by Asahi Kasei Pharma Co. (Tokyo, Japan).
We used a total of 75 rats for the present study, including hemodynamic measurements, morphometric analysis, and drug concentration measurement. Adult male Sprague-Dawley rats (7 weeks of age, 220-280 g body weight; Charles River, Yokohama, Japan) were randomly assigned into experimental groups and anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg). The osmotic pumps (model 2ML4; Alzet, Cupertino, CA) filled by fasudil (125 mg/mL of saline) or saline were implanted intraperitoneally in rats. These pumps delivered a volume of 2.5 μL/hour, which was equivalent to 30 mg/kg/day of fasudil for a 250-g rat. On the next day, animals were given a single subcutaneous injection of MCT (60 mg/kg, Wako, Tokyo, Japan) or saline and started to receive oral administration of BPS (200 μg/kg/day in drinking water at a concentration of 3 μg/mL).27 Thus, the present study included five experimental groups: normal rats (control group), untreated MCT rats (PH group), and MCT rats treated with either fasudil alone (fasudil group), BPS alone (BPS group), or both of them (combination group).
Three weeks after the single MCT injection, all surviving rats were anesthetized by intraperitoneal injection of pentobarbital (45 mg/kg) and placed on a heating pad to maintain body temperature in the physiological range throughout the study. Polyethylene catheters were inserted into the right ventricle (RV) through the right jugular vein and the left carotid artery for hemodynamic measurements. RV systolic pressure (RVSP), mean arterial pressure (MAP) and heart rate were measured with a polygraph system (AP-601G, Nihon Kohden Co., Tokyo, Japan).18
Right Ventricular Hypertrophy
The RV was dissected from the left ventricle (LV) and the septum (S). These samples were weighed to determine the extent of RV hypertrophy (RVH) as follows; RV/(LV + S), RV/body weight (BW).18,28
Morphometric Analysis of Pulmonary Arteries
After the hemodynamic measurements, the lung was flushed with physiologic salt solution and fixed with 10% formalin for morphometric analysis.18,28 Paraffin sections were obtained from the left lung and stained with elastica Masson for examination by light microscopy (BX51, Olympus Optical Co., Tokyo, Japan). Pulmonary arteries of 50 to 200 μm in external diameter were divided into two groups (50-100 and 100-200 μm in diameter) and evaluated for the measurement of medial thickness. Medial thickness was expressed as follows; percent wall thickness = [(medial thickness × 2)/external diameter] × 100.18 For each animal, at least 40 pulmonary arteries were scanned at a magnification of × 400 and measured using Adobe Photoshop 7.0 software in a blind manner.18
Plasma Concentration of Fasudil and Hydroxyfasudil
After the hemodynamic measurements, blood samples were obtained from the abdominal aorta and transferred into chilled tubes. Plasma samples were isolated by centrifugation at 4°C. Plasma concentrations of fasudil and hydroxyfasudil, which is an active metabolite of fasudil and acts as a more specific inhibitor of Rho-kinase,29 were serially measured by high-performance liquid chromatography.30
All results are expressed as means ± SEM. Comparisons between groups were made with Student's t-test or analysis of variance with Tukey test for multiple comparisons. Differences were considered to be statistically significant at P < 0.05.
Effects of the Combination Therapy on Hemodynamic Variables
Body weight was significantly decreased by MCT injection and did not differ significantly between the drug treatment groups and the PH group (Table 1). MAP was slightly but significantly decreased in the combination group compared with the control group (Table 1). There were no significant differences in heart rate among the experimental groups.
Effects of the Combination Therapy on PH and RVH
Hemodynamic measurements demonstrated that RVSP (a marker of pulmonary artery systolic pressure) was elevated in the PH groups compared with the control groups (54 ± 3 and 32 ± 1 mm Hg; P < 0.001; n = 15 each; Fig. 1). Fasudil and BPS did not significantly decrease RVSP because the decrease was relatively small (47 ± 2 and 48 ± 2 mm Hg; n = 15 each) and the combination therapy caused a further and significant decrease in RVSP by 35% (35 ± 1 mm Hg; P < 0.001 compared with the PH group, n = 15). RVH also was developed in the PH group compared with the control group (Fig. 2A, B). Fasudil monotherapy tended to inhibit and BPS monotherapy significantly inhibited the development of RVH, and the combination therapy with both of them further inhibited the development of RVH (Fig. 2).
Morphometric Analysis of Pulmonary Arteries
Representative photomicrographs showed that medial thickening of the pulmonary artery was markedly inhibited in the combination group (Fig. 3). Quantitative analysis of different-sized pulmonary arteries (the ranges of 50-100 μm and 100-200 μm in diameter) demonstrated that MCT caused a significant increase in medial thickness of the pulmonary artery and that fasudil or BPS monotherapy tended to inhibit and the combination therapy significantly inhibited the development of the MCT-induced medial thickening in both-sized pulmonary arteries (Fig. 4).
Plasma Concentrations of Fasudil and Hydroxyfasudil
There was no significant difference in the plasma concentrations of fasudil and hydroxyfasudil between the fasudil and the combination groups (187 ± 21 and 168 ± 18 ng/mL, respectively; n = 6 each). Furthermore, these values were within the clinical concentrations of the drugs in humans.30
The major finding of the present study is that the combination therapy with fasudil, a Rho-kinase inhibitor, and BPS, an oral prostacyclin analogue, is more effective than each monotherapy for ameliorating MCT-induced PH in rats.
Prostacyclin activates adenylate cyclase and increases cAMP, which decreases intracellular Ca2+.24 Recent report also suggested that cAMP inhibits Rho activation and resultant myosin phosphatase suppression in a manner independent of the Ca2+-lowering effect of cAMP in vitro.31 By contrast, Rho-kinase inhibitors decrease Ca2+ sensitization through dephosphorylation of myosin phosphatase.32 Thus, it is possible that the combination of fasudil and BPS suppresses pulmonary VSMC hypercontraction through both Ca2+-dependent and -independent mechanisms. Furthermore, Rho-kinase inhibitors exert cGMP-mediated vasodilatation through the increase in endothelial nitric oxide synthase expression and activation.33,34 Thus, these drugs that produce similar effects through different mechanisms may have additive or synergistic effects when combined. Indeed, in the present study, the combination of fasudil and BPS markedly attenuated the increases in RVSP and RVH as compared with each monotherapy. These findings suggest that the combination of fasudil and BPS is superior to each monotherapy for inhibiting the development of MCT-induced PH. Previous reports also suggested that the combination of BPS and an endothelin-A receptor antagonist or a phosphodiesterase type-5 inhibitor is superior to each agent alone on MCT-induced PH rats.28,35 However, there is no evidence for superiority among these combination therapy for the treatment of PH.
In the present study, the combination therapy slightly but significantly decreased MAP when compared to the control group. However, this effect is lower than pulmonary artery vasodilatation (10% reduction in MAP vs. 35% reduction in RVSP), suggesting that the combination therapy with fasudil and BPS causes relatively selective vasodilatation of pulmonary arteries.
MCT is known to cause endothelial injury of pulmonary arteries with subsequent proliferation of pulmonary VSMC, infiltration of inflammatory cells, and platelet activation, resulting in PH and pulmonary vascular remodeling.18,36,37 Prostacyclin inhibits platelet aggregation and VSMC proliferation.5 Rho-kinase is substantially involved in the pathogenesis of arteriosclerosis, and Rho-kinase inhibitors have an inhibitory effect on proliferation and migration of VSMC and infiltration of inflammatory cells induced by many proinflammatory agonists.17 In the present study, morphometric analysis demonstrated that the combination therapy with fasudil and BPS effectively inhibited the MCT-induced medial wall thickening of the pulmonary artery as compared with each monotherapy. These findings suggest that the combination therapy is more effective than each monotherapy for inhibiting the development of pulmonary vascular remodeling in PH.
Fasudil and hydroxyfasudil, an active metabolite of fasudil, inhibit Rho-kinase activity to the same extent.38 In the present study, the plasma concentrations of fasudil and hydroxyfasudil were within the clinical therapeutic range in humans.30 In addition, there was no significant difference in the plasma concentrations between the fasudil and the combination groups, suggesting that the combination of fasudil and BPS does not affect the plasma concentrations of fasudil and hydroxyfasudil in vivo. Recently, we have developed a sustained-release form of fasudil, with which we are going to start clinical trials in patients with PAH in Japan. We have confirmed that this long-acting form of fasudil can maintain the drug concentration in the blood for a longer time and the pattern of drug concentration is similar to that observed in the present study with an intraperitoneal infusion (unpublished data).
We have previously demonstrated that long-term inhibition of Rho-kinase with fasudil markedly suppressed the development of MCT-induced PH in rats.18 In the present study, fasudil alone did not show statistically significant inhibitory effect on PH, RVH, or medial thickening of the pulmonary artery, although the dose of fasudil (30 mg/kg/day) was comparable between the previous and the present study. This discrepancy may be explained, at least in part, by the difference in the route of administration and subsequent pharmacokinetics between the previous (oral administration in tap water) and the present study (intraperitoneal infusion with a minipump).
Several limitations should be mentioned for the present study. First, we only examined the preventive effects of the combination therapy against MCT-induced PH. From clinical point of view, it may be more important to examine the therapeutic effects of the combination therapy after the establishment of MCT-induced PH. This point remains to be examined in a future study. Second, due to the technical difficulty, we were unable to measure plasma concentration of BPS in the BPS or the combination group. However, because it was reported that beraprost (100 μg/kg by gavage administration) increases plasma cAMP levels in rats,28 we consider that it also is the case in the present study, although this point remains to be examined in a future study. Finally, we only examined the effects of the combination therapy in a single-animal model (MCT-induced PH model in rats). This point remains to be examined in animal models of PH with different etiology and then carefully examined in clinical trials in the future.
PH continues to be a serious clinical problem with high morbidity and mortality, for which no satisfactory treatment is yet available. Recently, we and others have demonstrated acute effectiveness and safety of fasudil in patients with PH.21,22 We also have confirmed the effectiveness and safety of the oral form of fasudil in patients with stable effort angina.39 Rho-kinase inhibitors may have beneficial effects on PH through different mechanisms from prostacyclin.18,19 Thus, the present results suggest that the combination therapy with a Rho-kinase inhibitor and prostacyclin may be a novel therapeutic strategy for the treatment of PAH in humans.
The combination of fasudil and BPS ameliorates MCT-induced PH in rats. The present in vivo study, when combined with our previous in vitro study, suggests that combination therapy with a Rho-kinase inhibitor and prostacyclin could exert further beneficial effects on PAH.
We thank H. Hase, N. Yamaki, and H. Tatebayashi for excellent technical assistance and Asahi Kasei Pharma Co. (Tokyo, Japan) for providing fasudil and BPS.
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