Activation of Soluble Guanylate Cyclase Reverses Experimental Pulmonar
http://www.100md.com
《循环学杂志》
Medical Clinic II/V, University Hospital, Giessen, Germany (R.D., N.W., H.A.G., E.D., W.S., F.G., R.T.S.)
Pharma Research Center, Bayer HealthCare, Wuppertal, Germany (J.S., M.J.G.)
Rudolf Buchheim Institute for Pharmacology, Giessen, Germany (K.B., H.S.)
Department of Pharmacology, Monash University, Victoria, Australia (H.S.).
Abstract
Background— Severe pulmonary hypertension is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. Using wild-type and homozygous endothelial nitric oxide synthase (NOS3–/–) knockout mice with pulmonary hypertension induced by chronic hypoxia and rats with monocrotaline-induced pulmonary hypertension, we examined whether the soluble guanylate cyclase (sGC) stimulator Bay41-2272 or the sGC activator Bay58-2667 could reverse pulmonary vascular remodeling.
Methods and Results— Both Bay41-2272 and Bay58-2667 dose-dependently inhibited the pressor response of acute hypoxia in the isolated perfused lung system. When wild-type (NOS3+/+) or NOS3–/– mice were housed under 10% oxygen conditions for 21 or 35 days, both strains developed pulmonary hypertension, right heart hypertrophy, and pulmonary vascular remodeling, demonstrated by an increase in fully muscularized peripheral pulmonary arteries. Treatment of wild-type mice with the activator of sGC, Bay58-2667 (10 mg/kg per day), or the stimulator of sGC, Bay41-2272 (10 mg/kg per day), after full establishment of pulmonary hypertension from day 21 to day 35 significantly reduced pulmonary hypertension, right ventricular hypertrophy, and structural remodeling of the lung vasculature. In contrast, only minor efficacy of chronic sGC activator therapies was noted in NOS3–/– mice. In monocrotaline-injected rats with established severe pulmonary hypertension, both compounds significantly reversed hemodynamic and structural changes.
Conclusions— Activation of sGC reverses hemodynamic and structural changes associated with monocrotaline- and chronic hypoxia-induced experimental pulmonary hypertension. This effect is partially dependent on endogenous nitric oxide generated by NOS3.
Key Words: cardiovascular diseases hypertension, pulmonary muscle, smooth nitric oxide pharmacology
Introduction
Pulmonary arterial hypertension is characterized by lung vascular remodeling, high pulmonary blood pressure, and right ventricular hypertrophy. Hypoxia is considered a major factor in the pathogenesis of pulmonary hypertension, eg, in pulmonary obstructive and restrictive diseases and at high altitude. Acute hypoxia causes a selective pulmonary arteriolar vasoconstriction and increases pulmonary blood pressure, whereas exposure to chronic hypoxia induces structural and functional changes in the pulmonary arterial bed.1,2 These changes include proliferation and migration of smooth muscle cells as well as an increased accumulation of extracellular matrix. Imbalances in vasodilatory and vasoconstrictive forces have been implicated in both the predominance of increased vasomotor tone and the chronic remodeling of resistance vessels. Nitric oxide (NO) synthesized by endothelial NO synthase (eNOS, NOS3) is a potent vasodilator and is considered to play an important role in regulating pulmonary vascular tone. The downstream effector of NO is soluble guanylate cyclase (sGC), which synthesizes the second messenger cyclic guanosine monophosphate (cGMP).
Clinical Perspective p 295
Although impairment of the endothelium-dependent regulation of pulmonary vascular tone is reported consistently, the analysis of the role of sGC in chronic hypoxia-induced pulmonary arterial hypertension has yielded conflicting data, with both increase and decrease of sGC protein expression described.3–5 Potential therapeutic potential has been reported for YC-1, which acts as a "NO sensitizer," greatly enhancing the sensitivity of sGC toward this soluble agent.6,7 YC-1 increases cGMP in smooth muscle cells and induces a dose-dependent vasodilation of endothelium-denuded rat aortic rings.8–10 Furthermore, YC-1 has been shown to inhibit the adhesion and aggregation of platelets.11–13
Recently, the compound Bay41-2272, which stimulates sGC directly and enhances the sensitivity of sGC to NO, was shown to be a systemic and pulmonary vasodilator.14,15 Furthermore, it augments the vasodilative response to inhaled NO in acute pulmonary hypertension in lambs.16 Whereas Bay41-2272 activates sGC in its native form, another compound, Bay58-2667, has recently been shown to activate sGC even in its oxidized or heme-free form and independently of NO.17
The aim of this study was to test the hypothesis that both compounds reverse pulmonary vascular remodeling in chronic experimental pulmonary hypertension in mice and rats. Chronic hypoxia was applied to induce pulmonary hypertension in mice, and the injection of the plant alkaloid monocrotaline was used in rats to induce a more aggressive form of pulmonary hypertension. To investigate the role of endogenous NO in this putative antiremodeling pathway, we tested this hypothesis in both wild-type and eNOS (NOS3) knockout mice with hypoxia-induced pulmonary hypertension.
Methods
Animals
Adult male Sprague-Dawley rats (350 to 400 g body wt) and C57Bl/6J and NOS3–/– (Nos3tm1Unc, Jackson Laboratories) mice were obtained from Charles River Laboratories. Animals were housed under controlled temperature (&22°C) and lighting (12/12-hour light/dark cycle), with free access to food and water. All experiments were performed according to the institutional guidelines that comply with national and international regulations.
Hemodynamics
The animals were anesthetized with ketamine/xylazine (intraperitoneally) and placed on a heating pad to maintain the body temperature in the physiological range. They were tracheostomized and artificially ventilated with 10 mL/kg body wt (SAR830A/P, IITC). Inspiratory oxygen (FIO2) was set at 0.5, and a positive end-expiratory pressure of 1.0 cm H2O was used throughout. The systemic arterial pressure (SAP) was monitored by cannulating the left carotid artery with a polyethylene cannula connected to a fluid-filled force transducer (Braun). The right jugular vein was used for catheterization of the right ventricle with a custom-made silicone catheter. The transducers were calibrated before every measurement.
Isolated Perfused Mouse Lung
The effects of Bay41-2272 and Bay58-2667 on acute hypoxic pulmonary vasoconstriction were examined in isolated ventilated perfused mice lungs.18 Briefly, C57Bl/6J mice weighing 22 to 22 g were anesthetized as described above. Tracheostomy was performed, and the animals were ventilated with room air with the use of a Minivent 845 (Hugo Sachs Electronics, Harvard Apparatus GmbH) respirator. After midsternal thoracotomy, catheters were placed into the pulmonary artery and the left ventricle. The technique of successive hypoxic maneuvers in buffer-perfused lungs has been described previously.19 Sequential hypoxic maneuvers of 10-minute duration interrupted by 15-minute periods of normoxia were performed. The effects of the various pharmacological agents on pressure responses provoked by alveolar hypoxia (1% O2) were determined within such a sequence of repetitive hypoxic maneuvers. Each agent was added to the buffer fluid 5 minutes before a hypoxic challenge, with the addition starting after the second hypoxic maneuver was accomplished. Cumulative dose-effect curves were established by addition of either Bay41-2272 or Bay58-2667 in the reservoir (dose range, 0.001 to 10 μmol/L).
Radiotelemetry
For a continuous measurement of SAP and heart rate, radiotelemetric sensors were implanted into anesthetized mice (Dataquest A.R.T. 2.1; Data Science Inc). The system comprises a fluid-filled sensing catheter (5 cm long, external diameter 0.7 mm, internal diameter 0.25 mm; model TA11PA) connected to a transmitter that signals to a remote receiver (model RPC-1) and a data exchange matrix connected to a computer. After surgery, mice were allowed to recover for 3 days. The SAP stabilized in the first 24 hours. None of the animals manifested signs of inflammation or infection.
Hypoxia and Treatment With Bay41-2272 and Bay58-2667
Pulmonary hypertension was induced by exposure to hypoxia (10% inspired O2 fraction) in a normobaric chamber as described previously.18 Mice were exposed to hypoxia for 21 or 35 days in a hypoxic normobaric chamber (n=10 each). Control animals were placed in a normoxic chamber with a normal oxygen environment (21% inspired O2 fraction). Eight groups of chronic hypoxic C57Bl/6J mice (21 days of 10% O2; n=4) were investigated for acute hemodynamic effects of Bay41-2272 and Bay58-2667. After a stabilization period of 15 minutes, each group of mice received a different dose of Bay41-2272 and Bay58-2667 (0, 1, 3, or 10 mg/kg) by gavage, and hemodynamics were recorded for 180 minutes.
In a separate set of experiments, telemetric sensors were implanted in wild-type mice, and the effect of a single oral dose of Bay41-2272 or Bay58-2667 (10 mg/kg body wt each) on SAP and heart rate was monitored over a time range of 30 hours.
For assessment of long-term effects of sGC activation, 3 subgroups of animals were treated once per day with either Bay41-2272 (10 mg/kg body wt; n=10), Bay58-2667 (10 mg/kg body wt; n=10), or vehicle (methylcellulose 3% at 10 μL/g body wt; n=10) from day 21 to 35. Hemodynamics were measured as described above.
Plasma Level of Bay41-2272 and Bay58-2667
Samples were subjected to high-performance liquid chromatography performed on a 2300 HTLC system (Coesive Technologies) as described.16,20 Briefly, the mobile phase consisted of 10 mmol/L ammonium acetate (pH 3.0) and acetonitrile. A linear gradient from 20% to 85% acetonitrile (vol/vol) within 1 minute was applied. Tandem mass spectrometry was performed on an API 3000 triple-quadruple mass spectrometer (PE Sciex) connected to the 2300HTLC system through a Turbospray interface. The lower limit for quantification of Bay41-2272 and Bay58-2667 was 0.5 μg/L.
Monocrotaline and Chronic Treatment
As described previously, hemodynamic and histological changes were examined in rats at 4 (n=10) and 6 (n=15) weeks after a single injection of monocrotaline (60 mg/kg SC).21,22 Animals that were injected with monocrotaline for 6 weeks received placebo (methylcellulose 3%) from week 4 to 6. Two other groups of monocrotaline-injected rats were treated with Bay41-2272 (10 mg/kg body wt) or Bay58-2667 (10 mg/kg body wt) by once-daily gavage (n=10 each). Treatment was started 4 weeks after injection of monocrotaline, when pulmonary hypertension was fully established, for the duration of 2 weeks. Hemodynamics were measured as described above.
Tissue Processing
After SAP and right ventricular pressure were recorded, the animals were exsanguinated, and the lungs and heart were isolated. The right ventricle was dissected from the left ventricle+septum (LV+S), and these dissected samples were dried and weighed to obtain the right to left ventricle plus septum ratio (RV/LV+S).
Histology
After the lungs were flushed with saline solution, they were perfused through the pulmonary artery and through the tracheae with a mixture of formaldehyde (2%) and picric acid (15%) in 0.1 mol/L phosphate buffer with a constant pressure of 22 and 11 cm H2O, respectively. The lung and the heart were removed en block. The lung lobes were embedded in paraffin blocks, and sections of 3 μm were cut. The degree of muscularization of small peripheral pulmonary arteries was assessed by double staining the 3-μm sections with an anti--smooth muscle actin antibody (dilution 1:900, clone 1A4, Sigma, Saint Louis, Mo) and anti-human von Willebrand factor antibody (dilution 1:900, Dako, Hamburg, Germany), as previously described.23 Sections were counterstained with methyl green and examined by light microscopy with the use of a computerized morphometric system (Qwin, Leica). At x40 magnification, 80 to 100 intra-acinar vessels accompanying either alveolar ducts or alveoli were analyzed by an observer blinded to treatment in each animal. As described, each vessel was categorized as nonmuscularized, partially muscularized, or fully muscularized.24 The percentage of pulmonary vessels in each muscularization category was determined by dividing the number of vessels in that category by the total number counted in the same experimental group.
Western Blot Analysis
Protein concentrations were determined according to Lowry et al.25 Tissue homogenates (15 μg protein per lane) were separated by SDS-PAGE (8%), transferred to Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech), and blocked with 3% nonfat dry milk in TBS (20 mmol/L Tris, 150 mmol/L NaCl, pH 7.4, 0.1% Tween-20). As described previously, immunodetection of sGC1 and sGC1 subunits was performed with the use of polyclonal rabbit antibodies directed and affinity-purified against synthetic peptide sequences corresponding to human sGC1 (residues 634 to 647) and sGC1 (residues 593 to 614), respectively.26,27 Anti-sGC1 was diluted 1:3000 and anti-sGC1 1:2000 in the aforementioned blocking solution. Immune complexes were visualized with an ECL (enhanced chemiluminescence) immunodetection kit (Amersham Pharmacia Biotech). The 80-kDa band for sGC1 and the 70-kDa band for sGC1 were scanned and quantified with a Kodak Image Station IS 440F and normalized to the housekeeping gene -actin.
Data Analysis
All data are given as mean±SEM. Differences between groups were assessed by ANOVA and Student-Newman-Keuls post hoc test for multiple comparisons, with a probability value <0.05 regarded to be significant.
Results
Effects of Bay41-2272 and Bay58-2667 on Acute Hypoxic Pulmonary Vasoconstriction in Isolated Mouse Lungs
Both Bay41-2272 and Bay58-2667 decreased acute hypoxic pulmonary vasoconstriction in a dose-dependent manner. The maximum inhibitory effect on hypoxic pulmonary vasoconstriction was similar for the 2 agents, but the concentration required to induce a 50% decrease in pulmonary artery pressure for Bay41-2272 was &10 times higher than for Bay58-2667 (Figure 1).
Immediate Vasodilatory Effects of Bay41-2272 and Bay58-2667 in Mice With Hypoxia-Induced Chronic Pulmonary Hypertension
Both compounds reduced right ventricular systolic pressure in a dose-dependent manner from 1 to 10 mg/kg body wt (Figure 2). Pulmonary vasodilatation was accompanied by a decrease in SAP. Telemetric measurement showed that 1 oral administration of either Bay41-2272 or Bay58-2667 (dose 10 mg/kg) reduced SAP by &20% over a time range of 10 to 20 hours (Figure 3A). Heart rate ranged from &600 bpm and increased to &700 bpm in response to the compounds (Figure 3B). This value normalized &5 hours after oral application of Bay41-2272 or Bay58-2667. Plasma samples were collected 6 hours after the last application of the compounds, and the levels of Bay41-2272 and Bay58-2667 were measured at 10 and 25 nmol/L, respectively (Figure 3C).
Chronic Effects of Bay41-2272 and Bay58-2667 on Hemodynamics and Right Heart Hypertrophy in Mice With Hypoxia-Induced Pulmonary Hypertension
The hypoxic wild-type mice developed pulmonary hypertension within 21 days, which was sustained until day 35. Consequently, right ventricular systolic pressure (RVSP) was increased significantly compared with the control group (Figure 4A). This increase was accompanied by an increase in the ratio of right ventricle to left ventricle plus septum weight [RV/(LV+S)] (Figure 4B). The ratio increased from 0.24±0.02 (controls) to 0.38±0.02 (21 days of hypoxia) and 0.42±0.03 (35 days of hypoxia), respectively (both P<0.05 versus controls). Bay41-2272 and Bay58-2667, applied by gavage from day 21 to 35, significantly reduced hypoxia-induced chronic pulmonary hypertension in wild-type mice. Accordingly, Bay41-2272 and Bay58-2667 caused a decrease of the RV/(LV+S) ratio to 0.32±0.02 and 0.31±0.02, respectively. Mean SAP did not change in any of the treatment groups (Table). Likewise, NOS3–/– mice developed pulmonary hypertension, with RVSP values increasing from 23.7±0.8 (controls) to 35.5±3.0 (21 days of hypoxia) and 34.9±1.2 (35 days of hypoxia) (Figure 5A) and RV/(LV+S) values increasing from 0.24±0.01 (controls) to 0.34±0.02 (21 days of hypoxia) and 0.41±0.08 (35 days of hypoxia) (Figure 5B). Bay58-2667, but not Bay41-2272, caused a moderate but significant reduction of RVSP in NOS3–/– mice, whereas both compounds failed to reduce RV/(LV+S) values (Bay41-2272, 0.36±0.02; Bay58-2667, 0.39±0.06). Mean SAP did not change in any of the treatment groups (Table).
Effects of 2-Week Daily Oral Administration of Bay41–2272 and Bay58–2667 on SAP, Hematocrit, and Body Weight in Mice With Hypoxia-Induced Pulmonary Hypertension
Chronic Effects of Bay41-2272 and Bay58-2667 on Degree of Muscularization of Pulmonary Arteries in Mice With Hypoxia-Induced Pulmonary Hypertension
We quantitatively assessed the degree of muscularization of pulmonary arteries with a diameter from 20 to 70 μm. In wild-type mice, the majority of vessels from 20 to 70 μm are usually nonmuscularized and partially muscularized (Figure 6). In the hypoxia-exposed animals, both at day 21 and 35, a dramatic decrease in nonmuscularized pulmonary arteries occurred with a concomitant increase in fully and partially muscularized pulmonary arteries. Treatment with Bay41-2272 and Bay58-2667 resulted in a significant increase of nonmuscularized arteries compared with both hypoxia groups. In addition, Bay41-2272 decreased the percentage of partially muscularized pulmonary arteries.
Expression of and Subunits of sGC in Mice With Hypoxia-Induced Pulmonary Hypertension: Effects of Bay41-2272 and Bay58-2667
The protein levels of both subunits of sGC did not change significantly in response to hypoxia (Figure 7). In contrast, in NOS3–/– mice, the 1 subunit of sGC was downregulated at day 35 and the 1 subunit decreased at day 35. Significant changes in either sGC1 or sGC1 subunit appeared in none of the treatment groups.
Chronic Effects of Bay41-2272 and Bay58-2667 on Hemodynamics and Right Heart Hypertrophy in Rats With Monocrotaline-Induced Pulmonary Hypertension
In rats injected with monocrotaline for 28 days, severe pulmonary hypertension developed with marked increase in RVSP (from 25.1±1.4 to 67.7±3.1 mm Hg; Figure 8A), in the ratio of right ventricular weight to left ventricle plus septum (RV/LV+S) (from 0.30±0.01 to 0.63±0.01; Figure 8B), and in the percentage of pulmonary artery muscularization (Figure 9A, 9B). In rats treated with vehicle, further progression of pulmonary hypertension until day 42 was noted (RVSP=78.5±6.2 mm Hg; RV/LV+S=0.81±0.05; Figures 8 and 9). No significant changes in mean SAP were observed (control=117±9 mm Hg; monocrotaline for 4 weeks=103±9 mm Hg; monocrotaline for 6 weeks=109±10 mm Hg). Ninety percent (9/10) and 60% (9/15) of animals survived the 28- and 42-day monocrotaline treatment. Long-term treatment with Bay41-2272 or Bay58-2667 significantly decreased RVSP to 55.5±1.7 and 53.9±2.9 mm Hg, respectively (P<0.05 versus monocrotaline both at day 42 and at day 28). In addition, both compounds decreased RV/LV+S values to 0.47±0.01 and 0.50±0.03, respectively (P<0.05 versus monocrotaline both at day 42 and at day 28). SAP was unchanged (Bay41-2272, 91±4 mm Hg; Bay58-2667, 102±6 mm Hg). In the animals treated with Bay41-2272 or Bay58-2667, survival was 80% (8/10) and 70% (7/10), respectively.
Chronic Effects of Bay41-2272 and Bay58-2667 on Degree of Muscularization of Pulmonary Arteries in Rats With Monocrotaline-Induced Pulmonary Hypertension
We quantitatively assessed the degree of muscularization of pulmonary arteries with a diameter from 10 to 50 μm. In the monocrotaline-injected animals, both at day 28 and 42, a significant decrease in nonmuscularized pulmonary arteries occurred (Figure 9A) with a concomitant increase in fully muscularized pulmonary arteries. Treatment with Bay41-2272 or Bay58-2667 at 10 mg/kg per day resulted in a significant reduction of fully muscularized arteries and increased the percentage of nonmuscularized pulmonary arteries (both parameters P<0.05 versus monocrotaline both at day 42 and at day 28) (Figure 9B).
Discussion
In this study we demonstrated that both the sGC stimulator Bay41-2272 and the sGC activator Bay58-2667 reverse pulmonary hypertension in chronically hypoxic mice and monocrotaline-injected rats. Notably, treatment with these agents was commenced only after full establishment of pulmonary hypertension, right heart hypertrophy, and structural changes in the lung vasculature. The compound Bay41-2272 is a novel NO-independent stimulator of sGC with characteristics similar to YC-1 but with higher potency of &2 to 3 orders of magnitude and no phosphodiesterase-5 inhibitory activity.17,28 Bay41-2272 also acts synergistically with NO, which was shown experimentally in NO-dependent penile erection29 and experimental acute pulmonary hypertension.16 In both systems, the NO/sGC/cGMP system plays an important role in maintaining physiological function. In contrast, the compound Bay58-2667 does not synergize with NO but stimulates the heme-oxidized or heme-depleted purified enzyme14 (Figure 10). Both compounds Bay41-2272 and Bay58-2667 are orally bioavailable, and both proved to have a long-lasting effect over 10 and 12 hours, respectively.14,17 Similarly, we show in our study in mice that both compounds Bay41-2272 and Bay58-2667 reduce SAP for &20 hours. On the basis of these findings, therapy was performed by once-daily application to achieve optimal efficacy. Detailed pharmacokinetic studies have been performed for Bay41-2272.30 Hepatic metabolism quickly results in oxidation of the 5-pyrimidinyl-cyclopropyl residue of Bay41-2272 to a stable metabolite that exerts long-term persistence in plasma and thus may contribute to the sustained vascular effects seen after oral application of the parent compound.
Chronic hypoxia induces pulmonary hypertension similar to human pulmonary hypertension secondary to disorders of the respiratory system, such as chronic obstructive pulmonary disease and interstitial lung disease. It is characterized by structural changes to the vascular system, including de novo muscularization of normally nonmuscularized small pulmonary arteries and an increase in medial wall thickness. In contrast, injection of the plant alkaloid monocrotaline in rats induces severe progressive pulmonary hypertension that finally results in death. Most impressively, both compounds do not attenuate but partially reverse the structural changes induced by 2 independent stimuli (hypoxia and monocrotaline) in 2 different species (mice and rats). The pharmacological activation of sGC may thus have a broad clinical perspective for treatment of pulmonary vascular diseases. The regulation of the expression of sGC under pathophysiological conditions has been addressed by several groups. Although the aortic GC content was not altered in NOS3 knockout animals31 or NOS inhibitor-treated rats, hypertension and aging appear to result in downregulation of GC expression.32–35 In experimental models of hypoxia-induced pulmonary hypertension, upregulation of sGC expression has been reported in rats and mice.4,5 With the use of immunostaining and Western blotting, a >2-fold increase of sGC protein 1 subunit was noted in smooth muscle cells of the pulmonary arteries in hypoxic rat lungs.36 The same group demonstrated in mice that both subunits of sGC, the 1 and 1 subunits, were increased under conditions of hypoxia-induced pulmonary hypertension.4 Interestingly, similar results were observed in NOS2 knockout animals but not in NOS3 knockout animals, suggesting NOS3 as a major regulator of sGC activity and protein expression in the lung vasculature.4 In this study both the 1 and 1 subunits were not changed in wild-type mice in response to hypoxia, and the expression was not altered by the 2 sGC activators. In contrast, a downregulation was noted in NOS3–/– mice, which is well in agreement with the aforementioned previous report.
Against this background, sGC is an attractive target for the treatment of hypoxia-induced pulmonary vascular diseases. In contrast to previous investigations, which investigated the influence of an endothelin antagonist, prostaglandin E1, or phosphodiesterase-5 inhibitors37 together with the hypoxic challenge, we started therapeutic interventions when pulmonary hypertension was already fully established, from week 3 to 5 in mice and from week 4 to 6 in rats. Under these conditions, both Bay41-2272 and Bay58-2667 significantly reversed the degree of pulmonary hypertension evolving in response to hypoxia and monocrotaline. This was true for systolic pulmonary artery pressure and right heart hypertrophy but also for structural changes including the de novo muscularization of small precapillary vessels.
The NOS3–/– mice developed pulmonary hypertension with hemodynamics and morphological changes similar to those in wild-type mice, which is in contrast to 1 previous report38 but is in agreement with 2 other publications.4,23 Interestingly, both compounds failed to reverse RVSP and right heart hypertrophy in these mice. These findings suggest that the antiremodeling effects of Bay41-2272 and Bay58-2667 were both dependent on ongoing NOS3-dependent NO generation.
The antiremodeling potency of Bay58-2667 in hypoxic mice is particularly interesting because this compound activates mainly the oxidized or the heme-free form of sGC, which does not occur under physiological conditions. However, it has recently been shown that in lungs from mice kept under hypoxic conditions, levels of reactive oxygen species may even increase,39 which in turn might oxidize the heme group of sGC. Enhanced levels of oxygen radicals have also been found under conditions of atherosclerosis, diabetes, hypercholesterinemia, or hypertension. However, future studies must prove the incidence of heme-free or oxidized forms of sGC in vascular abnormalities such as chronic pulmonary hypertension.
In conclusion, the compounds Bay41-2272 and Bay58-2667 caused dose-dependent pulmonary vasodilation in hypoxia-induced pulmonary hypertension in isolated mouse lungs. When these agents are used for chronic treatment by daily gavage, reversal of the hypoxia-elicited pulmonary hypertension was demonstrated, which was true for hemodynamics, structural changes of the lung vasculature, and right heart hypertrophy. Notably, the efficacy of both agents was dependent on intact NOS function. We conclude that activation of sGC may offer a new therapeutic option for antiremodeling therapy in severe pulmonary hypertension.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft, SFB547, projects B5 and C6. The authors acknowledge the technical assistance of Anke Voigt and Helmut Mueller.
Disclosures
Drs Stasch and Gnoth report that they are employed by Pharma Research Center, Bayer HealthCare. The other authors report no conflicts.
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Matsui H, Shimosawa T, Itakura K, Guanqun X, Ando K, Fujita T. Adrenomedullin can protect against pulmonary vascular remodeling induced by hypoxia. Circulation. 2004; 109: 2246–2251.(Rio Dumitrascu, MD; Norbert Weissmann, P)
Pharma Research Center, Bayer HealthCare, Wuppertal, Germany (J.S., M.J.G.)
Rudolf Buchheim Institute for Pharmacology, Giessen, Germany (K.B., H.S.)
Department of Pharmacology, Monash University, Victoria, Australia (H.S.).
Abstract
Background— Severe pulmonary hypertension is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. Using wild-type and homozygous endothelial nitric oxide synthase (NOS3–/–) knockout mice with pulmonary hypertension induced by chronic hypoxia and rats with monocrotaline-induced pulmonary hypertension, we examined whether the soluble guanylate cyclase (sGC) stimulator Bay41-2272 or the sGC activator Bay58-2667 could reverse pulmonary vascular remodeling.
Methods and Results— Both Bay41-2272 and Bay58-2667 dose-dependently inhibited the pressor response of acute hypoxia in the isolated perfused lung system. When wild-type (NOS3+/+) or NOS3–/– mice were housed under 10% oxygen conditions for 21 or 35 days, both strains developed pulmonary hypertension, right heart hypertrophy, and pulmonary vascular remodeling, demonstrated by an increase in fully muscularized peripheral pulmonary arteries. Treatment of wild-type mice with the activator of sGC, Bay58-2667 (10 mg/kg per day), or the stimulator of sGC, Bay41-2272 (10 mg/kg per day), after full establishment of pulmonary hypertension from day 21 to day 35 significantly reduced pulmonary hypertension, right ventricular hypertrophy, and structural remodeling of the lung vasculature. In contrast, only minor efficacy of chronic sGC activator therapies was noted in NOS3–/– mice. In monocrotaline-injected rats with established severe pulmonary hypertension, both compounds significantly reversed hemodynamic and structural changes.
Conclusions— Activation of sGC reverses hemodynamic and structural changes associated with monocrotaline- and chronic hypoxia-induced experimental pulmonary hypertension. This effect is partially dependent on endogenous nitric oxide generated by NOS3.
Key Words: cardiovascular diseases hypertension, pulmonary muscle, smooth nitric oxide pharmacology
Introduction
Pulmonary arterial hypertension is characterized by lung vascular remodeling, high pulmonary blood pressure, and right ventricular hypertrophy. Hypoxia is considered a major factor in the pathogenesis of pulmonary hypertension, eg, in pulmonary obstructive and restrictive diseases and at high altitude. Acute hypoxia causes a selective pulmonary arteriolar vasoconstriction and increases pulmonary blood pressure, whereas exposure to chronic hypoxia induces structural and functional changes in the pulmonary arterial bed.1,2 These changes include proliferation and migration of smooth muscle cells as well as an increased accumulation of extracellular matrix. Imbalances in vasodilatory and vasoconstrictive forces have been implicated in both the predominance of increased vasomotor tone and the chronic remodeling of resistance vessels. Nitric oxide (NO) synthesized by endothelial NO synthase (eNOS, NOS3) is a potent vasodilator and is considered to play an important role in regulating pulmonary vascular tone. The downstream effector of NO is soluble guanylate cyclase (sGC), which synthesizes the second messenger cyclic guanosine monophosphate (cGMP).
Clinical Perspective p 295
Although impairment of the endothelium-dependent regulation of pulmonary vascular tone is reported consistently, the analysis of the role of sGC in chronic hypoxia-induced pulmonary arterial hypertension has yielded conflicting data, with both increase and decrease of sGC protein expression described.3–5 Potential therapeutic potential has been reported for YC-1, which acts as a "NO sensitizer," greatly enhancing the sensitivity of sGC toward this soluble agent.6,7 YC-1 increases cGMP in smooth muscle cells and induces a dose-dependent vasodilation of endothelium-denuded rat aortic rings.8–10 Furthermore, YC-1 has been shown to inhibit the adhesion and aggregation of platelets.11–13
Recently, the compound Bay41-2272, which stimulates sGC directly and enhances the sensitivity of sGC to NO, was shown to be a systemic and pulmonary vasodilator.14,15 Furthermore, it augments the vasodilative response to inhaled NO in acute pulmonary hypertension in lambs.16 Whereas Bay41-2272 activates sGC in its native form, another compound, Bay58-2667, has recently been shown to activate sGC even in its oxidized or heme-free form and independently of NO.17
The aim of this study was to test the hypothesis that both compounds reverse pulmonary vascular remodeling in chronic experimental pulmonary hypertension in mice and rats. Chronic hypoxia was applied to induce pulmonary hypertension in mice, and the injection of the plant alkaloid monocrotaline was used in rats to induce a more aggressive form of pulmonary hypertension. To investigate the role of endogenous NO in this putative antiremodeling pathway, we tested this hypothesis in both wild-type and eNOS (NOS3) knockout mice with hypoxia-induced pulmonary hypertension.
Methods
Animals
Adult male Sprague-Dawley rats (350 to 400 g body wt) and C57Bl/6J and NOS3–/– (Nos3tm1Unc, Jackson Laboratories) mice were obtained from Charles River Laboratories. Animals were housed under controlled temperature (&22°C) and lighting (12/12-hour light/dark cycle), with free access to food and water. All experiments were performed according to the institutional guidelines that comply with national and international regulations.
Hemodynamics
The animals were anesthetized with ketamine/xylazine (intraperitoneally) and placed on a heating pad to maintain the body temperature in the physiological range. They were tracheostomized and artificially ventilated with 10 mL/kg body wt (SAR830A/P, IITC). Inspiratory oxygen (FIO2) was set at 0.5, and a positive end-expiratory pressure of 1.0 cm H2O was used throughout. The systemic arterial pressure (SAP) was monitored by cannulating the left carotid artery with a polyethylene cannula connected to a fluid-filled force transducer (Braun). The right jugular vein was used for catheterization of the right ventricle with a custom-made silicone catheter. The transducers were calibrated before every measurement.
Isolated Perfused Mouse Lung
The effects of Bay41-2272 and Bay58-2667 on acute hypoxic pulmonary vasoconstriction were examined in isolated ventilated perfused mice lungs.18 Briefly, C57Bl/6J mice weighing 22 to 22 g were anesthetized as described above. Tracheostomy was performed, and the animals were ventilated with room air with the use of a Minivent 845 (Hugo Sachs Electronics, Harvard Apparatus GmbH) respirator. After midsternal thoracotomy, catheters were placed into the pulmonary artery and the left ventricle. The technique of successive hypoxic maneuvers in buffer-perfused lungs has been described previously.19 Sequential hypoxic maneuvers of 10-minute duration interrupted by 15-minute periods of normoxia were performed. The effects of the various pharmacological agents on pressure responses provoked by alveolar hypoxia (1% O2) were determined within such a sequence of repetitive hypoxic maneuvers. Each agent was added to the buffer fluid 5 minutes before a hypoxic challenge, with the addition starting after the second hypoxic maneuver was accomplished. Cumulative dose-effect curves were established by addition of either Bay41-2272 or Bay58-2667 in the reservoir (dose range, 0.001 to 10 μmol/L).
Radiotelemetry
For a continuous measurement of SAP and heart rate, radiotelemetric sensors were implanted into anesthetized mice (Dataquest A.R.T. 2.1; Data Science Inc). The system comprises a fluid-filled sensing catheter (5 cm long, external diameter 0.7 mm, internal diameter 0.25 mm; model TA11PA) connected to a transmitter that signals to a remote receiver (model RPC-1) and a data exchange matrix connected to a computer. After surgery, mice were allowed to recover for 3 days. The SAP stabilized in the first 24 hours. None of the animals manifested signs of inflammation or infection.
Hypoxia and Treatment With Bay41-2272 and Bay58-2667
Pulmonary hypertension was induced by exposure to hypoxia (10% inspired O2 fraction) in a normobaric chamber as described previously.18 Mice were exposed to hypoxia for 21 or 35 days in a hypoxic normobaric chamber (n=10 each). Control animals were placed in a normoxic chamber with a normal oxygen environment (21% inspired O2 fraction). Eight groups of chronic hypoxic C57Bl/6J mice (21 days of 10% O2; n=4) were investigated for acute hemodynamic effects of Bay41-2272 and Bay58-2667. After a stabilization period of 15 minutes, each group of mice received a different dose of Bay41-2272 and Bay58-2667 (0, 1, 3, or 10 mg/kg) by gavage, and hemodynamics were recorded for 180 minutes.
In a separate set of experiments, telemetric sensors were implanted in wild-type mice, and the effect of a single oral dose of Bay41-2272 or Bay58-2667 (10 mg/kg body wt each) on SAP and heart rate was monitored over a time range of 30 hours.
For assessment of long-term effects of sGC activation, 3 subgroups of animals were treated once per day with either Bay41-2272 (10 mg/kg body wt; n=10), Bay58-2667 (10 mg/kg body wt; n=10), or vehicle (methylcellulose 3% at 10 μL/g body wt; n=10) from day 21 to 35. Hemodynamics were measured as described above.
Plasma Level of Bay41-2272 and Bay58-2667
Samples were subjected to high-performance liquid chromatography performed on a 2300 HTLC system (Coesive Technologies) as described.16,20 Briefly, the mobile phase consisted of 10 mmol/L ammonium acetate (pH 3.0) and acetonitrile. A linear gradient from 20% to 85% acetonitrile (vol/vol) within 1 minute was applied. Tandem mass spectrometry was performed on an API 3000 triple-quadruple mass spectrometer (PE Sciex) connected to the 2300HTLC system through a Turbospray interface. The lower limit for quantification of Bay41-2272 and Bay58-2667 was 0.5 μg/L.
Monocrotaline and Chronic Treatment
As described previously, hemodynamic and histological changes were examined in rats at 4 (n=10) and 6 (n=15) weeks after a single injection of monocrotaline (60 mg/kg SC).21,22 Animals that were injected with monocrotaline for 6 weeks received placebo (methylcellulose 3%) from week 4 to 6. Two other groups of monocrotaline-injected rats were treated with Bay41-2272 (10 mg/kg body wt) or Bay58-2667 (10 mg/kg body wt) by once-daily gavage (n=10 each). Treatment was started 4 weeks after injection of monocrotaline, when pulmonary hypertension was fully established, for the duration of 2 weeks. Hemodynamics were measured as described above.
Tissue Processing
After SAP and right ventricular pressure were recorded, the animals were exsanguinated, and the lungs and heart were isolated. The right ventricle was dissected from the left ventricle+septum (LV+S), and these dissected samples were dried and weighed to obtain the right to left ventricle plus septum ratio (RV/LV+S).
Histology
After the lungs were flushed with saline solution, they were perfused through the pulmonary artery and through the tracheae with a mixture of formaldehyde (2%) and picric acid (15%) in 0.1 mol/L phosphate buffer with a constant pressure of 22 and 11 cm H2O, respectively. The lung and the heart were removed en block. The lung lobes were embedded in paraffin blocks, and sections of 3 μm were cut. The degree of muscularization of small peripheral pulmonary arteries was assessed by double staining the 3-μm sections with an anti--smooth muscle actin antibody (dilution 1:900, clone 1A4, Sigma, Saint Louis, Mo) and anti-human von Willebrand factor antibody (dilution 1:900, Dako, Hamburg, Germany), as previously described.23 Sections were counterstained with methyl green and examined by light microscopy with the use of a computerized morphometric system (Qwin, Leica). At x40 magnification, 80 to 100 intra-acinar vessels accompanying either alveolar ducts or alveoli were analyzed by an observer blinded to treatment in each animal. As described, each vessel was categorized as nonmuscularized, partially muscularized, or fully muscularized.24 The percentage of pulmonary vessels in each muscularization category was determined by dividing the number of vessels in that category by the total number counted in the same experimental group.
Western Blot Analysis
Protein concentrations were determined according to Lowry et al.25 Tissue homogenates (15 μg protein per lane) were separated by SDS-PAGE (8%), transferred to Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech), and blocked with 3% nonfat dry milk in TBS (20 mmol/L Tris, 150 mmol/L NaCl, pH 7.4, 0.1% Tween-20). As described previously, immunodetection of sGC1 and sGC1 subunits was performed with the use of polyclonal rabbit antibodies directed and affinity-purified against synthetic peptide sequences corresponding to human sGC1 (residues 634 to 647) and sGC1 (residues 593 to 614), respectively.26,27 Anti-sGC1 was diluted 1:3000 and anti-sGC1 1:2000 in the aforementioned blocking solution. Immune complexes were visualized with an ECL (enhanced chemiluminescence) immunodetection kit (Amersham Pharmacia Biotech). The 80-kDa band for sGC1 and the 70-kDa band for sGC1 were scanned and quantified with a Kodak Image Station IS 440F and normalized to the housekeeping gene -actin.
Data Analysis
All data are given as mean±SEM. Differences between groups were assessed by ANOVA and Student-Newman-Keuls post hoc test for multiple comparisons, with a probability value <0.05 regarded to be significant.
Results
Effects of Bay41-2272 and Bay58-2667 on Acute Hypoxic Pulmonary Vasoconstriction in Isolated Mouse Lungs
Both Bay41-2272 and Bay58-2667 decreased acute hypoxic pulmonary vasoconstriction in a dose-dependent manner. The maximum inhibitory effect on hypoxic pulmonary vasoconstriction was similar for the 2 agents, but the concentration required to induce a 50% decrease in pulmonary artery pressure for Bay41-2272 was &10 times higher than for Bay58-2667 (Figure 1).
Immediate Vasodilatory Effects of Bay41-2272 and Bay58-2667 in Mice With Hypoxia-Induced Chronic Pulmonary Hypertension
Both compounds reduced right ventricular systolic pressure in a dose-dependent manner from 1 to 10 mg/kg body wt (Figure 2). Pulmonary vasodilatation was accompanied by a decrease in SAP. Telemetric measurement showed that 1 oral administration of either Bay41-2272 or Bay58-2667 (dose 10 mg/kg) reduced SAP by &20% over a time range of 10 to 20 hours (Figure 3A). Heart rate ranged from &600 bpm and increased to &700 bpm in response to the compounds (Figure 3B). This value normalized &5 hours after oral application of Bay41-2272 or Bay58-2667. Plasma samples were collected 6 hours after the last application of the compounds, and the levels of Bay41-2272 and Bay58-2667 were measured at 10 and 25 nmol/L, respectively (Figure 3C).
Chronic Effects of Bay41-2272 and Bay58-2667 on Hemodynamics and Right Heart Hypertrophy in Mice With Hypoxia-Induced Pulmonary Hypertension
The hypoxic wild-type mice developed pulmonary hypertension within 21 days, which was sustained until day 35. Consequently, right ventricular systolic pressure (RVSP) was increased significantly compared with the control group (Figure 4A). This increase was accompanied by an increase in the ratio of right ventricle to left ventricle plus septum weight [RV/(LV+S)] (Figure 4B). The ratio increased from 0.24±0.02 (controls) to 0.38±0.02 (21 days of hypoxia) and 0.42±0.03 (35 days of hypoxia), respectively (both P<0.05 versus controls). Bay41-2272 and Bay58-2667, applied by gavage from day 21 to 35, significantly reduced hypoxia-induced chronic pulmonary hypertension in wild-type mice. Accordingly, Bay41-2272 and Bay58-2667 caused a decrease of the RV/(LV+S) ratio to 0.32±0.02 and 0.31±0.02, respectively. Mean SAP did not change in any of the treatment groups (Table). Likewise, NOS3–/– mice developed pulmonary hypertension, with RVSP values increasing from 23.7±0.8 (controls) to 35.5±3.0 (21 days of hypoxia) and 34.9±1.2 (35 days of hypoxia) (Figure 5A) and RV/(LV+S) values increasing from 0.24±0.01 (controls) to 0.34±0.02 (21 days of hypoxia) and 0.41±0.08 (35 days of hypoxia) (Figure 5B). Bay58-2667, but not Bay41-2272, caused a moderate but significant reduction of RVSP in NOS3–/– mice, whereas both compounds failed to reduce RV/(LV+S) values (Bay41-2272, 0.36±0.02; Bay58-2667, 0.39±0.06). Mean SAP did not change in any of the treatment groups (Table).
Effects of 2-Week Daily Oral Administration of Bay41–2272 and Bay58–2667 on SAP, Hematocrit, and Body Weight in Mice With Hypoxia-Induced Pulmonary Hypertension
Chronic Effects of Bay41-2272 and Bay58-2667 on Degree of Muscularization of Pulmonary Arteries in Mice With Hypoxia-Induced Pulmonary Hypertension
We quantitatively assessed the degree of muscularization of pulmonary arteries with a diameter from 20 to 70 μm. In wild-type mice, the majority of vessels from 20 to 70 μm are usually nonmuscularized and partially muscularized (Figure 6). In the hypoxia-exposed animals, both at day 21 and 35, a dramatic decrease in nonmuscularized pulmonary arteries occurred with a concomitant increase in fully and partially muscularized pulmonary arteries. Treatment with Bay41-2272 and Bay58-2667 resulted in a significant increase of nonmuscularized arteries compared with both hypoxia groups. In addition, Bay41-2272 decreased the percentage of partially muscularized pulmonary arteries.
Expression of and Subunits of sGC in Mice With Hypoxia-Induced Pulmonary Hypertension: Effects of Bay41-2272 and Bay58-2667
The protein levels of both subunits of sGC did not change significantly in response to hypoxia (Figure 7). In contrast, in NOS3–/– mice, the 1 subunit of sGC was downregulated at day 35 and the 1 subunit decreased at day 35. Significant changes in either sGC1 or sGC1 subunit appeared in none of the treatment groups.
Chronic Effects of Bay41-2272 and Bay58-2667 on Hemodynamics and Right Heart Hypertrophy in Rats With Monocrotaline-Induced Pulmonary Hypertension
In rats injected with monocrotaline for 28 days, severe pulmonary hypertension developed with marked increase in RVSP (from 25.1±1.4 to 67.7±3.1 mm Hg; Figure 8A), in the ratio of right ventricular weight to left ventricle plus septum (RV/LV+S) (from 0.30±0.01 to 0.63±0.01; Figure 8B), and in the percentage of pulmonary artery muscularization (Figure 9A, 9B). In rats treated with vehicle, further progression of pulmonary hypertension until day 42 was noted (RVSP=78.5±6.2 mm Hg; RV/LV+S=0.81±0.05; Figures 8 and 9). No significant changes in mean SAP were observed (control=117±9 mm Hg; monocrotaline for 4 weeks=103±9 mm Hg; monocrotaline for 6 weeks=109±10 mm Hg). Ninety percent (9/10) and 60% (9/15) of animals survived the 28- and 42-day monocrotaline treatment. Long-term treatment with Bay41-2272 or Bay58-2667 significantly decreased RVSP to 55.5±1.7 and 53.9±2.9 mm Hg, respectively (P<0.05 versus monocrotaline both at day 42 and at day 28). In addition, both compounds decreased RV/LV+S values to 0.47±0.01 and 0.50±0.03, respectively (P<0.05 versus monocrotaline both at day 42 and at day 28). SAP was unchanged (Bay41-2272, 91±4 mm Hg; Bay58-2667, 102±6 mm Hg). In the animals treated with Bay41-2272 or Bay58-2667, survival was 80% (8/10) and 70% (7/10), respectively.
Chronic Effects of Bay41-2272 and Bay58-2667 on Degree of Muscularization of Pulmonary Arteries in Rats With Monocrotaline-Induced Pulmonary Hypertension
We quantitatively assessed the degree of muscularization of pulmonary arteries with a diameter from 10 to 50 μm. In the monocrotaline-injected animals, both at day 28 and 42, a significant decrease in nonmuscularized pulmonary arteries occurred (Figure 9A) with a concomitant increase in fully muscularized pulmonary arteries. Treatment with Bay41-2272 or Bay58-2667 at 10 mg/kg per day resulted in a significant reduction of fully muscularized arteries and increased the percentage of nonmuscularized pulmonary arteries (both parameters P<0.05 versus monocrotaline both at day 42 and at day 28) (Figure 9B).
Discussion
In this study we demonstrated that both the sGC stimulator Bay41-2272 and the sGC activator Bay58-2667 reverse pulmonary hypertension in chronically hypoxic mice and monocrotaline-injected rats. Notably, treatment with these agents was commenced only after full establishment of pulmonary hypertension, right heart hypertrophy, and structural changes in the lung vasculature. The compound Bay41-2272 is a novel NO-independent stimulator of sGC with characteristics similar to YC-1 but with higher potency of &2 to 3 orders of magnitude and no phosphodiesterase-5 inhibitory activity.17,28 Bay41-2272 also acts synergistically with NO, which was shown experimentally in NO-dependent penile erection29 and experimental acute pulmonary hypertension.16 In both systems, the NO/sGC/cGMP system plays an important role in maintaining physiological function. In contrast, the compound Bay58-2667 does not synergize with NO but stimulates the heme-oxidized or heme-depleted purified enzyme14 (Figure 10). Both compounds Bay41-2272 and Bay58-2667 are orally bioavailable, and both proved to have a long-lasting effect over 10 and 12 hours, respectively.14,17 Similarly, we show in our study in mice that both compounds Bay41-2272 and Bay58-2667 reduce SAP for &20 hours. On the basis of these findings, therapy was performed by once-daily application to achieve optimal efficacy. Detailed pharmacokinetic studies have been performed for Bay41-2272.30 Hepatic metabolism quickly results in oxidation of the 5-pyrimidinyl-cyclopropyl residue of Bay41-2272 to a stable metabolite that exerts long-term persistence in plasma and thus may contribute to the sustained vascular effects seen after oral application of the parent compound.
Chronic hypoxia induces pulmonary hypertension similar to human pulmonary hypertension secondary to disorders of the respiratory system, such as chronic obstructive pulmonary disease and interstitial lung disease. It is characterized by structural changes to the vascular system, including de novo muscularization of normally nonmuscularized small pulmonary arteries and an increase in medial wall thickness. In contrast, injection of the plant alkaloid monocrotaline in rats induces severe progressive pulmonary hypertension that finally results in death. Most impressively, both compounds do not attenuate but partially reverse the structural changes induced by 2 independent stimuli (hypoxia and monocrotaline) in 2 different species (mice and rats). The pharmacological activation of sGC may thus have a broad clinical perspective for treatment of pulmonary vascular diseases. The regulation of the expression of sGC under pathophysiological conditions has been addressed by several groups. Although the aortic GC content was not altered in NOS3 knockout animals31 or NOS inhibitor-treated rats, hypertension and aging appear to result in downregulation of GC expression.32–35 In experimental models of hypoxia-induced pulmonary hypertension, upregulation of sGC expression has been reported in rats and mice.4,5 With the use of immunostaining and Western blotting, a >2-fold increase of sGC protein 1 subunit was noted in smooth muscle cells of the pulmonary arteries in hypoxic rat lungs.36 The same group demonstrated in mice that both subunits of sGC, the 1 and 1 subunits, were increased under conditions of hypoxia-induced pulmonary hypertension.4 Interestingly, similar results were observed in NOS2 knockout animals but not in NOS3 knockout animals, suggesting NOS3 as a major regulator of sGC activity and protein expression in the lung vasculature.4 In this study both the 1 and 1 subunits were not changed in wild-type mice in response to hypoxia, and the expression was not altered by the 2 sGC activators. In contrast, a downregulation was noted in NOS3–/– mice, which is well in agreement with the aforementioned previous report.
Against this background, sGC is an attractive target for the treatment of hypoxia-induced pulmonary vascular diseases. In contrast to previous investigations, which investigated the influence of an endothelin antagonist, prostaglandin E1, or phosphodiesterase-5 inhibitors37 together with the hypoxic challenge, we started therapeutic interventions when pulmonary hypertension was already fully established, from week 3 to 5 in mice and from week 4 to 6 in rats. Under these conditions, both Bay41-2272 and Bay58-2667 significantly reversed the degree of pulmonary hypertension evolving in response to hypoxia and monocrotaline. This was true for systolic pulmonary artery pressure and right heart hypertrophy but also for structural changes including the de novo muscularization of small precapillary vessels.
The NOS3–/– mice developed pulmonary hypertension with hemodynamics and morphological changes similar to those in wild-type mice, which is in contrast to 1 previous report38 but is in agreement with 2 other publications.4,23 Interestingly, both compounds failed to reverse RVSP and right heart hypertrophy in these mice. These findings suggest that the antiremodeling effects of Bay41-2272 and Bay58-2667 were both dependent on ongoing NOS3-dependent NO generation.
The antiremodeling potency of Bay58-2667 in hypoxic mice is particularly interesting because this compound activates mainly the oxidized or the heme-free form of sGC, which does not occur under physiological conditions. However, it has recently been shown that in lungs from mice kept under hypoxic conditions, levels of reactive oxygen species may even increase,39 which in turn might oxidize the heme group of sGC. Enhanced levels of oxygen radicals have also been found under conditions of atherosclerosis, diabetes, hypercholesterinemia, or hypertension. However, future studies must prove the incidence of heme-free or oxidized forms of sGC in vascular abnormalities such as chronic pulmonary hypertension.
In conclusion, the compounds Bay41-2272 and Bay58-2667 caused dose-dependent pulmonary vasodilation in hypoxia-induced pulmonary hypertension in isolated mouse lungs. When these agents are used for chronic treatment by daily gavage, reversal of the hypoxia-elicited pulmonary hypertension was demonstrated, which was true for hemodynamics, structural changes of the lung vasculature, and right heart hypertrophy. Notably, the efficacy of both agents was dependent on intact NOS function. We conclude that activation of sGC may offer a new therapeutic option for antiremodeling therapy in severe pulmonary hypertension.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft, SFB547, projects B5 and C6. The authors acknowledge the technical assistance of Anke Voigt and Helmut Mueller.
Disclosures
Drs Stasch and Gnoth report that they are employed by Pharma Research Center, Bayer HealthCare. The other authors report no conflicts.
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