INTRODUCTION

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PULMONARY HYPERTENSION (PH) is a hemodynamic abnormality caused by a variety of disease states, including primary PH and chronic obstructive pulmonary disease. PH is characterized by increased pulmonary vascular resistance that frequently results in an inability of the right ventricle to sustain its output and leads to right ventricular failure and death. Although the pathogenesis of PH differs among those disease states, it is likely that abnormal vasoconstriction contributes significantly to the vascular process of all forms of PH. Although a wide variety of vasodilator agents have been used in the treatment of PH, their effects are generally limited because of the lack of pulmonary selectivity.

Inhaled nitric oxide (NO) has been established as a selective pulmonary vasodilator (10, 24). However, although this therapy has been reported to be very effective in some types of PH, such as persistent PH of the newborn and heart and/or lung perioperative PH in which the PH is reversible in a relatively short term, there are problems associated with long-term use of NO inhalation, including its potential toxicity and difficulty in ambulatory inhalation (33, 36). Although the exact mechanism of its efficacy is not fully elucidated, promising results have been accumulated regarding long-term treatment of primary PH patients with continuous intravenous prostacyclin (3, 19). However, this modality also has limitations because of its high cost and complexity of delivery system. To date, unfortunately, no ideal agent for the therapy of PH has yet been identified.

Recently, cGMP-specific phosphodiesterase (PDE) 5 inhibitors have been demonstrated to be potent, acute, selective pulmonary vasodilators (5, 6, 35). Braner et al. (5) have shown that M & B 22948 (zaprinast) produces selective dose-dependent pulmonary vasodilation during PH induced by U-46619 or by alveolar hypoxia in intact newborn lambs. Cohen et al. (6) have reported that E-4021 is a pulmonary vasodilator as selective and effective as inhaled NO in conscious pulmonary hypertensive rats. The mechanism responsible for the pulmonary selectivity of these PDE5 inhibitors is not fully understood, but it is likely due to abundant distribution of PDE5 in the lung tissue (6, 21, 32, 35).

Thus we hypothesized that long-term treatment with a selective PDE5 inhibitor would increase cGMP levels in lung tissue and prevent the development of chronic hypoxia-induced PH without systemic hypotension. To test this hypothesis, we used in this study a newly synthesized compound, E-4010 [4-(3-chloro-4-metoxybenzyl)amino-1-(4-hydroxypiperidino)-6-phthalazinecarbonitrile monohydrochloride], as a selective PDE5 inhibitor because this orally effective agent has been shown to be highly selective for PDE5 compared with other PDE isoenzymes (PDE1-4) (16). We first examined the acute hemodynamic effects of oral administration of E-4010 in conscious chronic hypoxia-induced pulmonary hypertensive rats. We then investigated the effects of long-term oral administration of E-4010 on the development of chronic hypoxia-induced PH in rats and measured cGMP levels in lung and aortic tissues and plasma of these rats.

	MATERIALS AND METHODS

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Acute Hemodynamic Effects of E-4010 on Chronically Hypoxic Rats

Conscious catheterized rats. After a 3 wk-exposure to hypoxia or normoxia, the rats were anesthetized with intramuscular ketamine (90 mg/kg) and pentobarbital sodium (15 mg/kg) for catheterization of the pulmonary and right carotid arteries and right jugular vein as previously described (23). The intravascular location of the catheter tips was determined by the blood pressure tracings, and the catheters were secured, filled with heparinized saline, sealed, and tunneled subcutaneously to the back of the neck where they were exteriorized and enclosed in a small plastic container. The cutdowns were repaired, and the rats were allowed to recover for 48 h in room air. After recovery, the conscious rat was placed in a ventilated (room air) plastic box for hemodynamic measurements under the protocol described in Experimental protocol. Pulmonary (PAP) and systemic (SAP) arterial pressures were measured with pressure transducers (MPU-0.5 and TP-200T, respectively, Nihon Koden, Tokyo, Japan) and displayed continuously on an oscilloscope. The heart rate (HR) was determined from the arterial pressure pulse (AT-600G cardiotachometer, Nihon Koden). Cardiac output (CO) was measured by the cardiogreen dye-dilution method as previously described (4, 23). Total pulmonary (TPR) and systemic (TSR) resistances were calculated by dividing the respective mean pressure by the CO.

Experimental protocol. The hemodynamic measurements were performed under normoxic conditions (the rats were breathing 21% O₂). After blood samples (0.1 ml) were taken from the carotid artery for measurement of hematocrit, baseline PAP, SAP, CO, and HR were measured. Then, either 0.1, 0.3, or 1.0 mg/kg of E-4010 or its vehicle (0.5% methylcellulose) was given by gavage to conscious catheterized CH rats. PAP, SAP, and HR were monitored for 480 min, and CO was measured at 15 and 480 min after oral administration of E-4010. At the end of each hemodynamic study, the rat was killed by an overdose of pentobarbital sodium, and the heart was removed for measurement of right ventricle (RV) and left ventricle plus septum (LV+S) weights and for calculation of RV-to-(LV+S) weight ratio [RV/(LV+S)] as an index of right ventricular hypertrophy.

Prevention of Chronic Hypoxic PH

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Measurements. Hemodynamic measurements were made 48 h after catheterization under normoxic conditions. After blood samples (0.1 ml) were taken for measurement of hematocrit, the conscious rat was placed in a ventilated (room air) plastic box, and PAP, SAP, CO, and HR were measured. Blood samples (6 ml) were again collected for measurement of plasma cyclic nucleotide (cAMP and cGMP) levels. They were immediately centrifuged at 3,000 rpm for 10 min, and the supernatants were kept at 20°C until cyclic nucleotide levels were determined as described below. At the end of each study, the rat was killed by an overdose of pentobarbital sodium, and the heart [for calculation of RV/(LV+S)] and the lungs and thoracic aorta (for measurement of tissue cyclic nucleotide levels) were removed. The lung and aortic tissues were rapidly frozen in liquid N₂ and stored at 80°C. The concentration of cyclic nucleotides was measured with a radioimmunoassay kit (Amersham, Little Chalfont, UK). For measurement of cyclic nucleotide levels in plasma, 400 µl of ice-cold ethanol were added to 100 µl of plasma and centrifuged at 4°C at 11,000 rpm for 20 min. Then the supernatant was evaporated under a stream of N₂ gas and used for cyclic nucleotide measurement. For the measurement of intracellular cyclic nucleotide levels in the lung and aorta, each preparation was homogenized in 1 ml of 0.1 N HCl. The homogenate was centrifuged at 4°C at 3,000 rpm for 10 min, and the supernatant was evaporated under a stream of N₂ gas. The residue was subjected to radioimmunoassay. The protein content of the pellet was determined with bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). Tissue cyclic nucleotide levels were normalized by the amount of the protein.

Histological analysis. In subsets of N, CH_Con, and CH_E0.1 groups of rats, histological changes were quantified by morphometry as previously described (4, 14). The rats were killed with an overdose of pentobarbital sodium, and the heart and lungs were removed en bloc. The pulmonary artery was cannulated and perfused with phosphate-buffered saline (37°C, 20 cmH₂O pressure) to remove residual blood and then injected with a barium-gelatin mixture (60°C) at 74 mmHg pressure for 3 min. The trachea was cannulated, and the lung was distended with 10% Formalin at 36 cmH₂O pressure and fixed in an inflated state for 3 days. A block of tissue (1.0 × 0.7 × 0.2 cm) was taken from the midportion of the left lung parallel to the hilum. Sections were stained with elastic Van Gieson stain and assessed microscopically for the degree of arterial muscularization and wall thickness. In each tissue section, at least 50 consecutive barium-filled arteries (>15-µm external diameter) were analyzed at ×400 magnification. Each artery was classified by the structure of the accompanying airway as terminal bronchiole, respiratory bronchiole, alveolar duct, or alveolar wall. For each completely muscular artery corresponding to a respiratory or terminal bronchiole, we measured the medial thickness at two places of each artery. Medial wall thickness is expressed as the summation of the two points of (medial thickness/external diameter) × 100 (in percent).

The effect of the drug on the abnormal extension of smooth muscle into usually nonmuscular alveolar duct and wall vessels, which occurs during chronic hypoxia (14), was assessed as follows: each barium-containing vessel identified was viewed at ×400 magnification, and the structure of each vessel was then noted as either completely muscular, partially muscular, or nonmuscular.

Statistical Analysis

	RESULTS

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Acute Hemodynamic Effects of E-4010

The time course of the acute effects of oral administration of E-4010 on mean PAP and SAP is shown in Fig. 1. E-4010 at the highest dose tested in this study (1.0 mg/kg) significantly reduced mean PAP, although its lower doses did not change mean PAP. The PAP-lowering effect of E-4010 was rapid and reached its peak within 60 min [mean PAP decreased from 23.8 ± 0.9 to 17.3 ± 0.6 mmHg (27.1 ± 1.2%) 60 min after administration of E-4010] and lasted over 360 min. In contrast, E-4010 did not significantly affect mean SAP even at the highest dose tested. The hypotensive effect of E-4010 on PAP was accompanied by no change in either HR or CO (Table 1).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Time course of acute effects of E-4010 on mean pulmonary (MPAP; A) and systemic (MSAP; B) arterial pressures in chronically hypoxic pulmonary hypertensive rats. E-4010 was given orally by gavage. , 0.5% methylcellulose (vehicle control); , 0.1 mg/kg of E-4010; , 0.3 mg/kg of E-4010; , 1.0 mg/kg of E-4010. Values are means ± SE of 6-8 animals/group. * Significant difference from corresponding vehicle control values, P < 0.05 (by repeated-measures ANOVA).

Prevention of Chronic Hypoxic PH

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Hemodynamic effects of long-term oral treatment with E-4010 during exposure to chronic hypoxia on MPAP (A), MSAP (B), cardiac output (CO; C), heart rate (HR; D), and total pulmonary (TPR; E) and systemic (TSR; F) resistances in conscious rats. N, normoxic control rats; CHCon, CHE0.01, and CHE0.1, chronically hypoxic rats receiving 0, 0.01, and 0.1%, respectively, E-4010-containing food. Values are means ± SE; n, no. of animals/group (nos. in parentheses). Significant difference (P < 0.05) from: * N;  CHCon;  CHE0.01 (all by ANOVA).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effects of long-term oral treatment with E-4010 during exposure to chronic hypoxia on ratio of right ventricle to (left ventricle plus septum) weights [RV/(LV+S)]. Values are means ± SE; n, no. of animals/group (nos. in parentheses). Significant difference (P < 0.05) from: * N;  CHCon;  CHE0.01 (all by ANOVA).

As shown in Table 3, although cGMP levels in plasma and tissue (both lung and aorta) were not affected by chronic hypoxic exposure, E-4010 increased cGMP levels significantly in plasma and lung tissue but not in aortic tissue after chronic exposure to hypoxia. cAMP levels were decreased in lung tissue and unchanged in aortic tissue but tended to increase in plasma after chronic exposure to hypoxia. These levels were unaffected by E-4010.

Tables 4 and 5 show the effect of E-4010 treatment on the development of hypoxia-induced pulmonary vascular remodeling. In the muscular pulmonary arteries corresponding to the respiratory and terminal bronchioles (vessel diameter ranging from 50 to 150 µm), there was a significant increase in medial wall thickness in CH_Con rats compared with that in N rats. Treatment with E-4010 resulted in a significant reduction in the chronic hypoxia-induced increased medial wall thickness (Table 4, Fig. 4). In addition to a reduction in medial wall thickness in E-4010-treated rats, there was also a reduction in the degree of muscularization in the smaller resistance vessels corresponding to the alveolar duct and alveolar wall (Table 5).

View larger version (64K): [in this window] [in a new window]   Fig. 4.   Representative light micrographs of pulmonary arteries accompanying terminal bronchioles (100-150 µm in diameter) from normoxic control (A), chronically hypoxic, 0.1% E-4010-treated (B), and chronically hypoxic control (C) rats. Magnification, ×400.

	DISCUSSION

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This study has demonstrated that long-term oral administration of a selective PDE5 inhibitor, E-4010, markedly reduced the development of chronic hypoxia-induced PH in rats. Long-term treatment with E-4010 significantly attenuated the chronic hypoxia-induced increase in mean PAP and TPR. In addition, compared with chronically hypoxic control rats, the hypoxia-induced right ventricular hypertrophy and pulmonary vascular remodeling were less severe in E-4010-treated rats. These effects of E-4010 were associated with the increase in cGMP (but not cAMP) levels in lung but not in aortic tissue.

It is well known that cGMP plays an essential role in the regulation of vascular smooth muscle tone, including the pulmonary vascular bed. Several endogenous vasodilators such as NO and atrial natriuretic peptide cause vascular smooth muscle cell relaxation by increasing the intracellular concentration of cGMP ([cGMP]_i) via activation of soluble or particulate guanylate cyclase (GC) and consequent activation of protein kinase G (2, 7). In addition to GC activity, the [cGMP]_i is determined by PDE activity that rapidly inactivates cGMP to GMP (1, 25). In other words, the [cGMP]_i and thus smooth muscle cell relaxation is governed by the balance between GC and PDE activities.

The PDE isoenzymes have been subdivided into at least seven distinct families (PDE1-7) based on their genetic and functional characteristics and appear to show tissue-specific distribution (21, 32). Because of the presence of different complements of these isoenzymes in tissues, it is reasonable to expect that selective PDE inhibitors could increase cyclic nucleotide levels in specific target tissues and organs. The PDE5 family is termed cGMP specific because this family of isoenzymes specifically hydrolyzes cGMP and has been reported to be distributed abundantly in lung tissue (21, 32). In fact, Yamaguchi et al. (35) have previously shown that PDE5 activity is much higher in lung than in aortic tissue of chronically hypoxic pulmonary hypertensive rats. Thus inhibition of this enzyme by selective PDE5 inhibitors is likely to increase lung tissue cGMP levels and cause selective pulmonary vasodilation. Indeed, previous studies (5, 6, 35) have demonstrated that the selective PDE5 inhibitors zaprinast and E-4021 produce selective acute pulmonary vasodilation.

In this study, we used a novel selective PDE5 inhibitor, E-4010, which Ishihara et al. (16) recently synthesized. This agent completely inhibited PDE5 purified from porcine and human platelets (inhibition constants 0.092 and 0.104 nM, respectively). The IC₅₀ value for PDE5 from porcine platelets is 0.000560 µM, which is less than of that of zaprinast (1.11 µM) and almost of that of E-4021 (0.0039 µM) (29; Adachi, unpublished observations). The IC₅₀ value of E-4010 for the next most sensitive PDE isoenzyme (PDE2) is 0.984 µM (Adachi, unpublished observations). Thus this PDE5 inhibitor can be considered to be highly selective and potent.

The acute experiment of the present study showed that oral administration of E-4010 (1 mg/kg) in chronic hypoxia-induced PH rats decreased PAP, with no significant effects on SAP, HR, and CO, confirming that this selective PDE5 inhibitor is an acute selective pulmonary vasodilator similar to other PDE5 inhibitors (5, 6, 35). In addition, we studied the time course of its hemodynamic effects and found that the selective pulmonary hypotensive effect was rapid, reached its peak within 60 min, and lasted a relatively long time (>6 h).

Long-term treatment with E-4010 during chronic exposure of rats to hypoxia dose dependently reduced PAP without affecting SAP, suggesting that this PDE5 inhibitor had a sustained selective PAP-lowering effect. Similarly, chronic hypoxia-induced right ventricular hypertrophy, which is thought to be the result of increased pulmonary vascular tone, was attenuated by E-4010. Furthermore, E-4010 (higher concentration) increased the impaired CO after exposure to chronic hypoxia, which is also probably due to increased PAP. These results indicate that this selective PDE5 inhibitor provides a beneficial response (28) in the treatment of PH hemodynamically.

E-4010 also reduced the increased medial wall thickness in pulmonary arteries corresponding to the respiratory and terminal bronchioles and the degree of muscularization of more distal arteries induced by exposure to chronic hypoxia. Although the mechanism(s) responsible for the development of chronic hypoxia-induced vascular remodeling has not been fully elucidated, chronic vasoconstriction with resultant increases in pressure and shear stress is believed to play a major role in this process (8, 26). Therefore, the pulmonary vasodilating effect of E-4010 is considered to contribute, at least in part, to the prevention of vascular remodeling. Furthermore, because it has been reported that cGMP mediates the antimyogenic effects of NO (11, 30) apart from its vasodilating effect, E-4010 may have directly inhibited pulmonary smooth muscle cell proliferation via increasing cGMP levels in the smooth muscle.

In addition to reversible vasoconstriction and structural remodeling of pulmonary arteries, one more factor that is thought to contribute to the development and maintenance of chronic hypoxia-induced PH is hyperviscosity resulting from polycythemia (13, 27). It has been shown that chronic hypoxia-induced polycythemia is attributable to increased erythropoietin (EPO) activity that is directly stimulated by hypoxia (9, 12). Theoretically, therefore, even if the degree of PH is attenuated by some therapeutic agents, chronic hypoxia-induced polycythemia might not be affected. Indeed, previous studies (20, 34) have shown that although a variety of agents reduced the development of PH induced by chronic hypoxia, most of them had little effect on hematocrit. In this study, however, long-term treatment with E-4010 during chronic exposure to hypoxia of rats significantly decreased hematocrit. Although it is not clear why E-4010 suppressed the erythropoiesis induced by chronic hypoxia, it has been suggested that cGMP plays a modulatory role in the regulation of EPO activity (18). Thus a possible explanation may be that increased cGMP levels in the EPO-secreting cells of rats treated with E-4010 during exposure to chronic hypoxia may have influenced the EPO activity. This issue needs to be studied further. Although this effect of E-4010 on hematocrit is favorable for prevention of the development of PH, it may decrease systemic oxygen transport to organs of patients with chronic obstructive pulmonary disease. However, this problem can be overcome practically by long-term oxygen therapy concomitant with E-4010 treatment. Furthermore, long-term administration of E-4010 had no effects on hematocrit in rats during normoxic conditions (Oka, unpublished observations).

We measured cyclic nucleotide levels in this study and found that long-term treatment with E-4010 increased cGMP levels in lung but not in aortic tissue and did not change cAMP levels in these tissues. These results suggest that E-4010 is more effective in lung than in aortic tissue and is specific to cGMP, supporting our hypothesis that chronic treatment with a selective PDE5 inhibitor would preferentially decrease cGMP degradation rate and increase [cGMP]_i in lung tissue due to the predominant distribution of this isoenzyme in the lung. This may be the reason why selective PDE5 inhibitors have pulmonary selectivity.

Although we investigated in the present study the chronic effects of E-4010 on the development of hypoxia-induced PH only, one previous study (31) has demonstrated the protective effects of another selective PDE5 inhibitor, E-4021, on the development of right ventricular overload and medial thickening of pulmonary arteries in a different rat model of PH, i.e., monocrotaline-induced PH. In addition, several laboratory and clinical studies (15, 17, 22) have shown that PDE5 inhibitors potentiate the vasodilator effects of inhaled NO. Taken together, these reports suggest the possibility that selective PDE5 inhibitors may be useful in the treatment of PH.

In summary, data of this study have shown that an orally active selective PDE5 inhibitor, E-4010, caused selective pulmonary vasodilation and attenuated the increase in PAP, right ventricular hypertrophy, and pulmonary arterial remodeling induced by chronic hypoxia. These hemodynamic effects of E-4010 were associated with an increase in cGMP levels in lung but not in aortic tissue. These results suggest that E-4010 prevented the development of chronic hypoxia-induced PH, probably through increasing cGMP levels in the pulmonary vascular smooth muscle. We conclude that selective PDE5 inhibitors, including E-4010, may provide a new strategy for the treatment of PH.