Inhaled Rho Kinase Inhibitors Are Potent and Selective Vasodilators in Rat Pulmonary Hypertension

Pulmonary hypertension (PH) is caused by a variety of diseases, but is characterized by common features (e.g., abnormal sustained vasoconstriction and progressive structural remodeling of pulmonary arteries) (1-3). In the past few years, there has been advancement in the treatment of PH, including continuous prostacyclin infusion and oral endothelin-1 receptor antagonists (4-7). However, current treatment is not optimal, and a range of different drug options is needed for better clinical management of PH. Although several vasodilator agents have been used to treat PH, their effects are generally limited because of a lack of pulmonary vascular selectivity.

Recent studies show that RhoA/Rho kinase signaling plays an important role in mediating pulmonary vasoconstriction induced by G protein-coupled receptor agonists (8, 9) and acute hypoxia (10, 11). Our group has recently found that RhoA/Rho kinase signaling is involved in both vasoconstriction and vascular remodeling in the mouse model of hypoxic PH (12), and that Rho kinase-mediated vasoconstriction substantially contributes to the sustained elevation of pulmonary vascular resistance in the rat model of hypoxic PH (13). In the latter study, the acute effect of intravenous Y-27632, a selective Rho-kinase inhibitor, in chronically hypoxic rats was striking (i.e., it nearly normalized the elevated pulmonary artery pressure). However, intravenous Y-27632 had no pulmonary vascular selectivity and also caused systemic vasodilation. Uehata and colleagues (14) have reported that oral administration of Y-27632 has potent hypotensive effects in rat models of systemic hypertension, but has a smaller transient effect in normotensive rats. Thus we tested in this study if oral, instead of intravenous, Y-27632 would lower the high pulmonary arterial pressure without affecting the normal systemic arterial pressure in chronically hypoxic rats. However, we found that although oral administration of Y-27632 caused a marked and sustained decrease in pulmonary artery pressure, it did not have pulmonary selectivity. We then tested whether inhaled Y-27632 would cause selective pulmonary vasodilation and exhibit similar potency to its intravenous or oral routes of administration. We also examined if inhaled Rho kinase inhibitor would cause effective and selective pulmonary vasodilation in monocrotaline (15) and fawn-hooded (16, 17) rat models of PH. Some of the results of this study have been previously reported in abstract form (18).

METHODS

Animals

All experimental procedures were approved by the Animal Care and Use Committee of the University of Colorado Health Sciences Center (Denver, CO). Adult male Sprague-Dawley rats (SDR, 240-400 g) were either exposed to hypobaric hypoxia (barometric pressure [asymptotically =] 410 mm Hg) for 3 to 4 weeks, or given a single subcutaneous injection of monocrotaline (MCT, 60 mg/kg) and allowed 3 weeks to develop PH. Fawn-hooded rats (FHR), which are genetically predisposed to spontaneously develop PH when raised from birth under the mildly hypoxic condition of Denver's altitude (5,280 feet, barometric pressure [asymptotically =] 630 mm Hg), were studied at 10 to 12 weeks of age (160-230 g) when they had severe PH (16, 17).

Catheterized Rats

All rats were anesthetized with ketamine (100 mg/kg) and xylazine (15 mg/kg) for placement of catheters in the right jugular vein and pulmonary and right carotid arteries (19). Chronically hypoxic SDR were allowed to recover for 48 hours in room air at Denver's altitude, and subsequent hemodynamic measurements were made while the animals were conscious. FHR and MCT-treated SDR were examined immediately after catheterization under anesthesia, because these rats do not recover well from the anesthesia and catheterizalion. For hemodynamic measurements, all rats were placed in a small ventilated Plexiglas chamber, and mean pulmonary arterial pressures (MPAP), systemic arterial pressures (MSAP), and heart rate were measured with pressure transducers while breathing room air at Denver's altitude. Cardiac index was determined by dividing the cardiac output (measured by dye-dilution) (19) by the animal's body weight.

Experimental Protocols

Oral administration of Y-27632 in chronically hypoxic SDR. After baseline hemodynamic measurements, Y-27632 (30 mg/kg) or vehicle (saline) was given by gavage to chronically hypoxic SDR, and measurements were repeated 1, 2, 3, 6, and 24 hours later. We chose the dose of 30 mg/kg because oral doses of 3 mg/kg and 10 mg/kg had no or only minor effects on MPAP in chronically hypoxic SDR in preliminary experiments.

Inhalation of Y-27632 and nitric oxide in chronically hypoxic SDR. After baseline hemodynamic measurements, chronically hypoxic SDR were exposed to either aerosolized Y-27632 (3, 10, 30, and 100 mM), vehicle (saline), or nitric oxide (NO) gas. Aerosol exposure was done as previously described (20). Briefly, each catheterized animal was placed in the small Plexiglas chamber and exposed for 5 minutes to aerosolized Y-27632 or vehicle. Aerosols were generated by a jet nebulizer (PARI LC PLUS, PARI Respiratory Equipment Inc., Monterey, CA) set at an airflow rate of 6 L/minute. Hemodynamic measurements were repeated 15 minutes after each exposure to Y-27632 or vehicle, and the successive doses of Y-27632 were administrated at 20-minute intervals. For the NO exposure experiment, the chamber was flushed for 5 minutes with room air containing 80 parts per million (ppm) NO and hemodynamic measurements were performed during the fifth minute of exposure. To examine the time course of inhaled Y-27632-induced pulmonary vasodilation, a separate group of chronically hypoxic SDR was exposed to Y-27632 (100 mM) or vehicle for 5 minutes, and hemodynamic changes were monitored for up to 5 hours.

Effects of inhaled fasudil in chronically hypoxic SDR, FHR, and MCT-treated SDR. We next tested if inhaled fasudil, another Rho kinase inhibitor (14), also caused a pulmonary selective vasodilation in chronically hypoxic SDR, FHR, and MCT-treated SDR. We used fasudil because it is helpful to test multiple different inhibitors to ensure the specificity of the effects and because it has already been used safely in clinical trial of cerebral and coronary vasospasm (21). The pharmacologic properties and cardiovascular effects of the two Rho kinase inhibitors have been discussed by Hu and Lee (22). After baseline measurements, chronically hypoxic SDR, FHR, and MCT-treated SDR were exposed to 5 minutes of fasudil (100 mM in each case); hemodynamic measurements were made 15 minutes later. We found inhaled fasudil effectively reduced MPAP in chronically hypoxic SDR and FHR but not in MCT-treated SDR. Because the anesthetized MCT-treated SDR were breathing very shallowly and rapidly, and because subsequent intravenous injection of fasudil did reduce MPAP in these rats, we suspected that a sufficient amount of aerosolized drug did not reach distal airways and pulmonary arteries under spontaneous breathing conditions. Therefore, we exposed a separate group of MCT-treated SDR to aerosolized fasudil under mechanically ventilated conditions. Anesthetized MCT-treated SDR were ventilated through the tracheal cannula with a constant volume ventilator (tidal volume at 10 ml/kg, frequency at 80 breaths/minute). Then, fasudil was aerosolized by an ultrasonic nebulizer (Model 40-270-000; Mabis, Lake Forest, IL) and administrated through the tracheal cannula at 100 mM for 5 minutes. Hemodynamic measurements were performed before and 15 minutes after the 5-minute exposure.

Statistical Analysis

Values are means

RESULTS

Baseline MPAP

Baseline MPAPs of chronically hypoxic SDR (re-exposed to room air at Denver's altitude for 48 hours), Denver-raised FHR, and MCT-treated SDR were 39 ± 2, (n = 20), 70 ± 5 (n = 5), and 44 ± 3 (n = 5) mmHg, respectively. Baseline MPAPs in normoxic, untreated SDR, and sea level-raised FHR were 21 ± 1 mm Hg (n = 5) (13) and 26 ± 1 mm Hg (n = 8) (unpublished data), respectively.

Effects of Acute Oral Y-27632 in Chronically Hypoxic SDR

Acute oral administration of Y-27632 (30 mg/kg) to the chronic hypoxia-exposed rats caused a marked and sustained decrease in MPAP, which peaked at approximately 2-3 hours after treatment (Figure 1). However, it also reduced MSAP. The peak decrease in MPAP averaged 35 ± 7%, whereas that in MSAP was 27 ± 4%. Oral Y-27632 had no effect on cardiac index (198 ± 7 at baseline versus 182 ± 24 ml/minute/kg at 3 hours) or heart rate (445 ± 10 at baseline versus 435 ± 21 beats/minute at 3 hours). It was apparent the hypotensive effect of oral Y-27632 lasted longer on MPAP than on MSAP (Figure 1).

Concentration Response Effects of Inhaled Y-27632 in Chronically Hypoxic SDR

Inhaled Y-27632 (5 minutes) concentration-dependently reduced MPAP with no significant effect on MSAP (121 ± 3 before versus 115 ± 4 mm Hg after 100 mM) (Figure 2). Absolute MPAP values are shown in Table 1. The baseline pulmonary artery pressures between the two groups in Table 1 were not statistically different (p = 0.23). Inhaled Y-27632 had no effect on cardiac index (218 ± 19 before versus 217 ± 30 ml/minute/kg after 100 mM) or heart rate (421 ± 10 before versus 415 ± 25 beats/minute after 100 mM).

Effects of Inhaled NO in Chronically Hypoxic SDR

Baseline pulmonary artery pressure of inhaled NO treated group did not differ from that of Y-27632 treated group (38 ± 2 versus 40 ± 3 mm Hg, NO versus Y-27632, p = 0.50). Exposure of chronically hypoxic SDR to NO gas (80 ppm) caused a selective decrease in MPAP (from 38 ± 3 to 32 ± 2 mm Hg, a 17 ± 1% reduction), which was less than that induced by inhaled Y-27632 (100 mM) (Figure 3).

Time-Course Effects of Inhaled Y-27632 on MPAP in Chronically Hypoxic SDR

The pulmonary hypotensive effect of inhaled Y-27632 (100 mM) peaked within 15 minutes, but a residual decrease in MPAP was observed for at least 5 hours after the 5-minute inhalation (Figure 4). As noted previously, inhaled Y-27632 had no significant effects on MSAP, cardiac index, or heart rate (data not shown).

Effect of Inhaled Fasudil in Chronically Hypoxic SDR, FHR, and MCT-treated SDR

Inhaled fasudil was as effective as inhaled Y-27632 in chronically hypoxic SDR (100 mM fasudil caused a 23 ± 4% reduction in MPAP versus a 26 ± 4% reduction by 100 mM Y-27632) (Figure 5), with no significant change in MSAP (from 112 ± 4 to 103 ± 7 mm Hg, p = 0.28). In FHR, inhaled fasudil also effectively reduced MPAP (from 70 ± 5 to 55 ± 9 mm Hg, a 23 ± 9% reduction by 100 mM) (Figure 5), with no significant reduction in MSAP (from 86 ± 6 to 72 ± 6 mm Hg, p = 0.17). Whereas our initial experiment with inhaled fasudil in spontaneously breathing MCT-treated SDR showed no significant reduction in MPAP (41 ± 2 before versus 40 ± 3 mm Hg after 100 mM), subsequent intravenous fasudil (10 mg/kg) in these rats nonselectively reduced MPAP from 40 ± 2 to 22 ± 2 mm Hg (a 44 ± 5% reduction). Furthermore, in mechanically ventilated MCT-treated SDR, inhaled fasudil (100 mM) was effective in selectively (change in MSAP; from 70 ± 3 to 65 ± 2 mm Hg, p = 0.22) reducing MPAP (from 44 ± 3 to 35 ± 4 mm Hg, a 21 ± 5% reduction) (Figure 5). In chronically hypoxic SDR, FHR, and MCT-treated SDR, inhaled fasudil had no effect on cardiac index or heart rate (Table 2).

DISCUSSION

One of the major problems in the treatment of PH with vasodilators is the lack of pulmonary selectivity. Although inhaled NO has been established as a selective pulmonary vasodilator, its clinical use, especially in long-term applications, is limited because of its complicated and expensive delivery system (23). Based on a report that acute oral Y-27632 (up to 30 mg/kg) had marked systemic hypotensive effects in hypertensive rat models but a smaller transient effect in normotensive Wistar rats (14), we initially tested if a Rho kinase inhibitor reduced the high MPAP without affecting the normal MSAP when given systemically to hypoxia-induced pulmonary hypertensive rats. However, in the previous (13) and present studies, we found that although both intravenous and oral administration of Y-27632 caused marked and sustained reductions in MPAP, there was no pulmonary vascular selectivity for either route of administration. It is apparent from our and other studies that constitutive Rho kinase activity contributes to normal systemic vascular tone and this contribution is increased in systemic hypertension (14, 24, 25). Although not selective, the pulmonary hypotensive effect of Y-27632 (both intravenous and oral) was marked and more potent than other vasodilators we had previously tested in chronically hypoxic SDR in our laboratory. For example, Y-27632 caused 33 ± 4% (intravenous) and 35 ± 7% (oral) reductions in MPAP, whereas intravenous NIP-121, a potassium channel opener (26), and E4021, a selective phosphodiesterase type 5 inhibitor (27), reduced MPAP by 22 ± 5% and 13 ± 4%, respectively, and nifedipine, a voltage-gated calcium channel blocker (26), at doses that decreased MSAP, caused no pulmonary vasodilation.

Airway application of aerosolized vasodilators has several advantages over systemic routes of delivery, including decreased systemic hypotensive effects, and selective delivery to the well-ventilated regions of the lung. Studies show that inhaled aerosolized vasodilators, such as iloprost (28, 29) and adrenomedullin (30, 31), effectively and selectively reduce pulmonary artery pressure in experimental animal models of PH and in patients with PH. However, these particular aerosolized agents still have limitations (i.e., relatively short-lived effects). For instance, the vasodilatory effect of iloprost lasts less than approximately 1-2 hours, and frequent inhalations are required to manage PH patients (32, 33). In this study, we found in chronically hypoxic SDR that inhalation of aerosolized Y-27632 concentration-dependently and selectively reduced MPAP (26 ± 4% at 100 mM) with a greater potency than inhaled NO (17 ± 1% at 80 ppm). Furthermore, the selective pulmonary hypotensive effect of inhaled Y-27632 lasted at least 5 hours after the 5-minute inhalation. These results suggest that inhaled Y-27632 is a potent and relatively long-lasting pulmonary selective vasodilator in chronically hypoxic SDR. The pulmonary vasodilatory potency of inhaled Y-27632 appears to be less than that of its intravenous or oral application but more than that of inhaled NO. NO-induced, cyclic guanosine monophosphate-mediated pulmonary vasodilation in chronically hypoxic rats is also attributable to inhibition of RhoA/Rho kinase signaling (34), and the relatively less potent effect of inhaled NO versus inhaled Y-27632 might be related to increased activity of pulmonary artery phosphodiesterase type-5 and increased degradation of the NO second messenger cyclic guanosine monophosphate in this model (35, 36). Although the possibility was not addressed in this study, another advantage of inhaled Y-27632 in the treatment of PH may be that relaxing both airway (37) and pulmonary vascular smooth muscle improves both ventilation and perfusion of even underventilated regions of the lung, thereby leading to better arterial oxygenation. Pulmonary vasodilation induced by inhaled Y-27632 is believed to be achieved by direct drug diffusion from airway to the peripheral pulmonary artery smooth muscle cells. However, our study does not rule out the possibility that the vasodilation may involve effects of a secondary mediator released from adjacent airway (38).

We also asked if inhaled Rho kinase inhibitors would be effective and selective pulmonary vasodilators in other experimental rat models of PH. We found that inhaled fasudil, another Rho kinase inhibitor that has been used in clinical trials (21), was an effective and selective pulmonary vasodilator in chronically hypoxic SDR. It also markedly reduced MPAP in FHR without significant effects on MSAP. We initially observed that inhaled fasudil did not reduce MPAP in anesthetized, spontaneously breathing MCT-treated SDR, whereas when given intravenously, it effectively lowered MPAP. We suspected this discrepancy was because the anesthetized MCT-treated SDR were breathing very shallowly and rapidly and an insufficient amount of aerosolized drug was delivered to distal airways and pulmonary arteries. Therefore, we examined the effects of aerosolized fasudil in mechanically ventilated MCT-treated SDR and found that it markedly decreased MPAP with no systemic effects, indicating that inhaled fasudil causes pulmonary selective vasodilation in MCT-treated SDR when adequately delivered to peripheral airways.

Our finding that inhaled fasudil acutely and effectively reversed the elevated MPAP in all three rat models of PH suggested that Rho kinase-mediated sustained vasoconstriction was an important component of the increased vascular resistance in these PH models, at least at the time points we studied them (3 weeks of hypoxic exposure, 10- to 12-week-old Denver-raised FHR, and 3 weeks after MCT injection). Multiple mediators, such as endothelin-1 and serotonin, as both vasoconstrictors and comitogens, are implicated in the complicated pathogenesis of most forms of experimental and human PH (39, 40). Recent evidence indicates that RhoA/Rho kinase signaling plays a central role in the sustained phase of vasoconstriction induced by many G protein-coupled receptor agonists, including endothclin-1 and serotonin, mainly by inhibiting myosin light chain phosphatase activity and increasing smooth muscle cell Ca^sup 2+^ sensitivity (41, 42). In addition, there is evidence that, in some arteries, Rho kinase activation is also involved in agonist-induced Ca^sup 2+^ entry (43, 44) (Figure 6). Thus we speculate that RhoA/Rho kinase signaling serves as a common downstream pathway of multiple important vasoconstrictors in various types of PH, and Rho kinase inhibitors are therefore effective in reversing the increased pulmonary vascular tone.

In summary, this study has shown that inhaled Rho kinase inhibitor, Y-27632, is a more potent and longer acting pulmonary selective vasodilator than is inhaled NO in hypoxic PH. Furthermore, the results clearly indicate that Rho kinase-mediated vasoconstriction also contributes significantly to the elevated pulmonary artery pressures in Denver-raised FHR and MCT-treated SDR. Although it will be necessary to test their effectiveness in more long-standing PH (months to years versus weeks), we speculate that inhaled Rho kinase inhibitors may be relatively long-acting, effective, and selective pulmonary vasodilators in various forms of PH when adequately delivered to peripheral airways. In addition to its role in sustained vasoconstriction, RhoA/Rho kinase signaling is involved in cell proliferation, migration, and apoptosis (21, 45-47), and a recent study shows that this pathway is involved in the serotonin-induced proliferation of bovine pulmonary artery smooth muscle cells (48). Abe and colleagues (15) have shown that long-term inhibition of Rho kinase by fasudil prevents and reverses MCT-induced pulmonary artery remodeling, and this is accompanied by enhanced apoptosis and decreased proliferation of pulmonary artery smooth muscle cells. Similarly, Fagan and colleagues (12) have observed that treatment with Y-27632 reduces the severity of PH and the neomuscularization of the distal pulmonary vasculature in hypoxic mice. Thus we propose that inhaled Rho kinase inhibitors, possessing abilities of both selective pulmonary vasodilation (potent and long-acting) and antivascular remodeling, might provide a new therapy for various forms of PH.

Conflict of Interest Statement: T.N. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; K.A.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.A.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; K.G.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; T.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; H.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; I.F.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.O. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Acknowledgment: The authors thank Welfide Corporation (Osaka, Japan) and Asahi Kasei Corporation (Tokyo, Japan) for providing Y-27632 and fasudil, respectively.