Activation of Soluble Guanylate Cyclase Reverses ExperimentalPulmonary Hypertension and Vascular Remodeling

Rio Dumitrascu, MD; Norbert Weissmann, PhD; Hossein Ardeschir Ghofrani, MD;Eva Dony; Knut Beuerlein, PhD; Harald Schmidt, MD; Johannes-Peter Stasch, PhD; Mark Jean Gnoth, PhD;

Werner Seeger, MD; Friedrich Grimminger, MD, PhD; Ralph Theo Schermuly, PhD

Background—Severe pulmonary hypertension is a disabling disease with high mortality, characterized by pulmonaryvascular 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 orthe 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 acutehypoxia 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, andpulmonary 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 significantlyreduced pulmonary hypertension, right ventricular hypertrophy, and structural remodeling of the lung vasculature. Incontrast, only minor efficacy of chronic sGC activator therapies was noted in NOS3�/� mice. In monocrotaline-injectedrats with established severe pulmonary hypertension, both compounds significantly reversed hemodynamic andstructural changes.

Conclusions—Activation of sGC reverses hemodynamic and structural changes associated with monocrotaline- andchronic hypoxia-induced experimental pulmonary hypertension. This effect is partially dependent on endogenous nitricoxide generated by NOS3. (Circulation. 2006;113:286-295.)

Key Words: cardiovascular diseases � hypertension, pulmonary � muscle, smooth � nitric oxide � pharmacology

Pulmonary arterial hypertension is characterized by lungvascular remodeling, high pulmonary blood pressure, and

right ventricular hypertrophy. Hypoxia is considered a majorfactor in the pathogenesis of pulmonary hypertension, eg, inpulmonary obstructive and restrictive diseases and at highaltitude. Acute hypoxia causes a selective pulmonary arterio-lar vasoconstriction and increases pulmonary blood pressure,whereas exposure to chronic hypoxia induces structural andfunctional changes in the pulmonary arterial bed.1,2 Thesechanges include proliferation and migration of smooth mus-cle cells as well as an increased accumulation of extracellularmatrix. Imbalances in vasodilatory and vasoconstrictiveforces have been implicated in both the predominance ofincreased vasomotor tone and the chronic remodeling ofresistance vessels. Nitric oxide (NO) synthesized by endothe-lial NO synthase (eNOS, NOS3) is a potent vasodilator and isconsidered to play an important role in regulating pulmonary

Clinical Perspective p 295

vascular tone. The downstream effector of NO is solubleguanylate cyclase (sGC), which synthesizes the second mes-senger cyclic guanosine monophosphate (cGMP).

Although impairment of the endothelium-dependent regu-lation of pulmonary vascular tone is reported consistently, theanalysis of the role of sGC in chronic hypoxia-inducedpulmonary arterial hypertension has yielded conflicting data,with both increase and decrease of sGC protein expressiondescribed.3–5 Potential therapeutic potential has been reportedfor YC-1, which acts as a “NO sensitizer,” greatly enhancingthe sensitivity of sGC toward this soluble agent.6,7 YC-1increases cGMP in smooth muscle cells and induces adose-dependent vasodilation of endothelium-denuded rat aor-tic rings.8–10 Furthermore, YC-1 has been shown to inhibit theadhesion and aggregation of platelets.11–13

Received August 6, 2005; revision received October 17, 2005; accepted October 31, 2005.From 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.); and Department ofPharmacology, Monash University, Victoria, Australia (H.S.).

Correspondence to Ralph Theo Schermuly, PhD, Zentrum für Innere Medizin, Justus-Liebig Universität Giessen, Klinikstrasse 36, 35392 Giessen,Germany. E-mail ralph.schermuly@innere.med.uni-giessen.de

© 2006 American Heart Association, Inc.

Circulation is available at http://www.circulationaha.org DOI: 10.1161/CIRCULATIONAHA.105.581405

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Vascular Medicine

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Recently, the compound Bay41-2272, which stimulatessGC directly and enhances the sensitivity of sGC to NO, wasshown to be a systemic and pulmonary vasodilator.14,15

Furthermore, it augments the vasodilative response to inhaledNO in acute pulmonary hypertension in lambs.16 WhereasBay41-2272 activates sGC in its native form, another com-pound, Bay58-2667, has recently been shown to activate sGCeven in its oxidized or heme-free form and independently ofNO.17

The aim of this study was to test the hypothesis that bothcompounds reverse pulmonary vascular remodeling inchronic experimental pulmonary hypertension in mice andrats. Chronic hypoxia was applied to induce pulmonaryhypertension in mice, and the injection of the plant alkaloidmonocrotaline was used in rats to induce a more aggressiveform of pulmonary hypertension. To investigate the role ofendogenous NO in this putative antiremodeling pathway, wetested this hypothesis in both wild-type and eNOS (NOS3)knockout mice with hypoxia-induced pulmonaryhypertension.

MethodsAnimalsAdult male Sprague-Dawley rats (350 to 400 g body wt) andC57Bl/6J and NOS3�/� (Nos3tm1Unc, Jackson Laboratories) mice wereobtained from Charles River Laboratories. Animals were housedunder controlled temperature (�22°C) and lighting (12/12-hourlight/dark cycle), with free access to food and water. All experimentswere performed according to the institutional guidelines that complywith national and international regulations.

HemodynamicsThe animals were anesthetized with ketamine/xylazine (intraperito-neally) and placed on a heating pad to maintain the body temperaturein the physiological range. They were tracheostomized and artifi-cially 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. Thesystemic arterial pressure (SAP) was monitored by cannulating theleft carotid artery with a polyethylene cannula connected to afluid-filled force transducer (Braun). The right jugular vein was usedfor catheterization of the right ventricle with a custom-made siliconecatheter. The transducers were calibrated before every measurement.

Isolated Perfused Mouse LungThe effects of Bay41-2272 and Bay58-2667 on acute hypoxicpulmonary vasoconstriction were examined in isolated ventilatedperfused mice lungs.18 Briefly, C57Bl/6J mice weighing 22 to 22 gwere anesthetized as described above. Tracheostomy was performed,and the animals were ventilated with room air with the use of aMinivent 845 (Hugo Sachs Electronics, Harvard Apparatus GmbH)respirator. After midsternal thoracotomy, catheters were placed intothe pulmonary artery and the left ventricle. The technique ofsuccessive hypoxic maneuvers in buffer-perfused lungs has beendescribed previously.19 Sequential hypoxic maneuvers of 10-minuteduration interrupted by 15-minute periods of normoxia were per-formed. The effects of the various pharmacological agents onpressure responses provoked by alveolar hypoxia (1% O2) weredetermined within such a sequence of repetitive hypoxic maneuvers.Each agent was added to the buffer fluid 5 minutes before a hypoxicchallenge, with the addition starting after the second hypoxicmaneuver was accomplished. Cumulative dose-effect curves wereestablished by addition of either Bay41-2272 or Bay58-2667 in thereservoir (dose range, 0.001 to 10 �mol/L).

RadiotelemetryFor a continuous measurement of SAP and heart rate, radiotelemetricsensors were implanted into anesthetized mice (Dataquest A.R.T.2.1; Data Science Inc). The system comprises a fluid-filled sensingcatheter (5 cm long, external diameter 0.7 mm, internal diameter0.25 mm; model TA11PA) connected to a transmitter that signals toa remote receiver (model RPC-1) and a data exchange matrixconnected to a computer. After surgery, mice were allowed torecover for 3 days. The SAP stabilized in the first 24 hours. None ofthe animals manifested signs of inflammation or infection.

Hypoxia and Treatment With Bay41-2272and Bay58-2667Pulmonary hypertension was induced by exposure to hypoxia (10%inspired O2 fraction) in a normobaric chamber as described previ-ously.18 Mice were exposed to hypoxia for 21 or 35 days in a hypoxicnormobaric chamber (n�10 each). Control animals were placed in anormoxic chamber with a normal oxygen environment (21% inspiredO2 fraction). Eight groups of chronic hypoxic C57Bl/6J mice (21days of 10% O2; n�4) were investigated for acute hemodynamiceffects of Bay41-2272 and Bay58-2667. After a stabilization period

Figure 1. Dose-response curve of Bay41-2272 and Bay58-2667on acute hypoxic pulmonary vasoconstriction in isolated mouselungs. In a sequence of repetitive hypoxic challenges (1% O2,10 minutes) alternating with normoxic ventilation periods (21%O2, 15 minutes), cumulative doses of Bay41-2272 or Bay58-2667 were applied during the normoxic periods. PAP indicatespulmonary artery pressure.

Figure 2. Hemodynamic effects of Bay41-2272 and Bay58-2667in chronic hypoxic mice. Immediate vasodilatory effects of incre-mental doses of Bay41-2272 or Bay58-2667 in anesthetizedmice that developed pulmonary hypertension in response to 5weeks of hypoxia are shown. Decreases in RVSP and SAP inresponse to different doses (1, 3, and 10 mg/kg body wt) of theagents are shown.

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of 15 minutes, each group of mice received a different dose ofBay41-2272 and Bay58-2667 (0, 1, 3, or 10 mg/kg) by gavage, andhemodynamics were recorded for 180 minutes.

In a separate set of experiments, telemetric sensors were implantedin wild-type mice, and the effect of a single oral dose of Bay41-2272or Bay58-2667 (10 mg/kg body wt each) on SAP and heart rate wasmonitored over a time range of 30 hours.

For assessment of long-term effects of sGC activation, 3 sub-groups 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 �L/g body wt; n�10) from day21 to 35. Hemodynamics were measured as described above.

Plasma Level of Bay41-2272 and Bay58-2667Samples were subjected to high-performance liquid chromatographyperformed on a 2300 HTLC system (Coesive Technologies) asdescribed.16,20 Briefly, the mobile phase consisted of 10 mmol/Lammonium acetate (pH 3.0) and acetonitrile. A linear gradient from20% 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 the2300HTLC system through a Turbospray interface. The lower limitfor quantification of Bay41-2272 and Bay58-2667 was 0.5 �g/L.

Monocrotaline and Chronic TreatmentAs described previously, hemodynamic and histological changeswere examined in rats at 4 (n�10) and 6 (n�15) weeks after a singleinjection of monocrotaline (60 mg/kg SC).21,22 Animals that wereinjected with monocrotaline for 6 weeks received placebo (methyl-cellulose 3%) from week 4 to 6. Two other groups of monocrotaline-injected rats were treated with Bay41-2272 (10 mg/kg body wt) orBay58-2667 (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 durationof 2 weeks. Hemodynamics were measured as described above.

Tissue ProcessingAfter SAP and right ventricular pressure were recorded, the animalswere exsanguinated, and the lungs and heart were isolated. The rightventricle was dissected from the left ventricle�septum (LV�S), andthese dissected samples were dried and weighed to obtain the right toleft ventricle plus septum ratio (RV/LV�S).

Figure 3. Telemetric measurement of SAP (A) and heart rate (B)in normoxic conscious mice receiving Bay41-2272 or Bay58-2667 and plasma concentrations of Bay41-2272 and Bay58-2667 (C) in chronic hypoxic mice. Continuous 30-hour profile ofthe deviation of mean blood pressure (A) and heart rate (B) innormoxic mice receiving a single oral application of Bay41-2272(10 mg/kg body wt) or Bay58-2667 (10 mg/kg body wt) is given.For measurement of plasma levels of both compounds, animalswere exposed to hypoxia for 35 days. Bay41-2272 or Bay58-2667 was applied daily by gavage from day 21 to day 35 inhypoxia-exposed animals (n�10) each at a dose of 10 mg/kgbody wt. Plasma levels of Bay41-2272 and Bay58-2667 aregiven (C). The samples were collected 6 hours after the lastapplication of the compounds.

Figure 4. Effect of Bay41-2272 and Bay58-2667 on RSVP (A)and right heart hypertrophy (B) in wild-type mice. Animals wereexposed to hypoxia for 21 or 35 days or remained in normoxiathroughout. The sGC activators Bay41-2272 or Bay58-2667were applied daily by gavage from day 21 to 35 in hypoxia-exposed animals (n�10) each at a dose of 10 mg/kg body wt.Control animals received placebo (10 �L/g body wt in 3% meth-ylcellulose). RVSP (in mm Hg) (A) and right to left ventricularratio (RV/LV�S) (B) are given. *P�0.05 vs control; †P�0.05 vshypoxia 21 days; ‡P�0.05 vs hypoxia 35 days.

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HistologyAfter the lungs were flushed with saline solution, they were perfusedthrough the pulmonary artery and through the tracheae with amixture of formaldehyde (2%) and picric acid (15%) in 0.1 mol/Lphosphate buffer with a constant pressure of 22 and 11 cm H2O,respectively. The lung and the heart were removed en block. Thelung lobes were embedded in paraffin blocks, and sections of 3 �mwere cut. The degree of muscularization of small peripheral pulmo-nary arteries was assessed by double staining the 3-�m sections withan anti–�-smooth muscle actin antibody (dilution 1:900, clone 1A4,Sigma, Saint Louis, Mo) and anti-human von Willebrand factorantibody (dilution 1:900, Dako, Hamburg, Germany), as previouslydescribed.23 Sections were counterstained with methyl green andexamined by light microscopy with the use of a computerizedmorphometric system (Qwin, Leica). At �40 magnification, 80 to100 intra-acinar vessels accompanying either alveolar ducts oralveoli were analyzed by an observer blinded to treatment in eachanimal. As described, each vessel was categorized as nonmuscular-ized, partially muscularized, or fully muscularized.24 The percentageof pulmonary vessels in each muscularization category was deter-mined by dividing the number of vessels in that category by the totalnumber counted in the same experimental group.

Western Blot AnalysisProtein concentrations were determined according to Lowry et al.25

Tissue homogenates (15 �g protein per lane) were separated bySDS-PAGE (8%), transferred to Hybond ECL nitrocellulose mem-branes (Amersham Pharmacia Biotech), and blocked with 3% nonfatdry milk in TBS (20 mmol/L Tris, 150 mmol/L NaCl, pH 7.4, 0.1%Tween-20). As described previously, immunodetection of sGC�1

and sGC�1 subunits was performed with the use of polyclonal rabbitantibodies directed and affinity-purified against synthetic peptidesequences corresponding to human sGC�1 (residues 634 to 647) andsGC�1 (residues 593 to 614), respectively.26,27 Anti-sGC�1 wasdiluted 1:3000 and anti-sGC�1 1:2000 in the aforementioned block-ing solution. Immune complexes were visualized with an ECL(enhanced chemiluminescence) immunodetection kit (AmershamPharmacia Biotech). The 80-kDa band for sGC�1 and the 70-kDa

Figure 5. Effect of Bay41-2272 and Bay58-2667 on RVSP (A)and right heart hypertrophy (B) in NOS3�/� mice. Animals wereexposed to hypoxia for 21 or 35 days or remained in normoxiathroughout. The sGC activators Bay41-2272 or Bay58-2667were applied daily by gavage from day 21 to 35 in hypoxia-exposed animals (n�10) each at a dose of 10 mg/kg body wt.Control animals received placebo (10 �L/g body wt in 3% meth-ylcellulose). RVSP (in mm Hg) (A) and right to left ventricularratio (RV/LV�S) (B) are given. *P�0.05 vs control; †P�0.05 vshypoxia 21 days.

Figure 6. Effects of Bay41-2272 and Bay58-2667 on the degreeof muscularization of pulmonary arteries in wild-type mice. Ani-mals were exposed to hypoxia for 21 or 35 days or remained innormoxia throughout. The sGC activators Bay41-2272 orBay58-2667 were applied daily by gavage from day 21 to 35 inhypoxia-exposed animals (n�10) each at a dose of 10 mg/kgbody wt. Control animals received placebo (10 �L/g body wt in3% methylcellulose). Proportions of nonmuscularized (N), par-tially muscularized (P), or fully muscularized (M) pulmonary arter-ies, as percentage of total pulmonary artery cross section (sized20 to 70 �m), are given. A total of 80 to 100 intra-acinar vesselswere analyzed in each lung. *P�0.05 vs control; †P�0.05 vshypoxia 21 days, ‡P�0.05 vs hypoxia 35 days.

Effects of 2-Week Daily Oral Administration of Bay41–2272 andBay58–2667 on SAP, Hematocrit, and Body Weight in MiceWith Hypoxia-Induced Pulmonary Hypertension

Variable/InterventionSAP,

mm HgHematocrit,

%Body Weight,

g

Wild-type normoxia 77.3�5.2 43.6�0.4 26.5�0.6

Wild-type hypoxia 3 wk 70.8�4.3 63.3�2.1 22.8�0.5

Wild-type hypoxia 5 wk 64.5�6.6 57.8�2.4 22.8�0.6

Wild-type hypoxia/Bay41–2272 62.5�2.8 54.7�1.1 23.9�0.6

Wild-type hypoxia/Bay58–2667 64.6�5.7 58.9�1.3 21.6�0.6

NOS3�/� normoxia 107.6�11.0 36.5�2.0 23.7�1.7

NOS3�/� hypoxia 3 wk 83.2�2.9 62.0�2.1 20.6�0.9

NOS3�/� hypoxia 5 wk 83.5�8.4 64.6�2.1 24.3�0.6

NOS3�/� hypoxia/Bay41–2272 80.6�2.6 65.2�2.1 23.9�0.7

NOS3�/� hypoxia/Bay58–2667 75.0�3.9 65.9�2.3 22.6�0.5

Data are mean�SEM; n�7 to 10.

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band for sGC�1 were scanned and quantified with a Kodak ImageStation IS 440F and normalized to the housekeeping gene �-actin.

Data AnalysisAll data are given as mean�SEM. Differences between groups wereassessed by ANOVA and Student-Newman-Keuls post hoc test formultiple comparisons, with a probability value �0.05 regarded to besignificant.

Results

Effects of Bay41-2272 and Bay58-2667 on AcuteHypoxic Pulmonary Vasoconstriction in IsolatedMouse LungsBoth Bay41-2272 and Bay58-2667 decreased acute hypoxicpulmonary vasoconstriction in a dose-dependent manner. Themaximum inhibitory effect on hypoxic pulmonary vasocon-striction was similar for the 2 agents, but the concentrationrequired to induce a 50% decrease in pulmonary arterypressure for Bay41-2272 was �10 times higher than forBay58-2667 (Figure 1).

Immediate Vasodilatory Effects of Bay41-2272 andBay58-2667 in Mice With Hypoxia-InducedChronic Pulmonary HypertensionBoth compounds reduced right ventricular systolic pressurein a dose-dependent manner from 1 to 10 mg/kg body wt(Figure 2). Pulmonary vasodilatation was accompanied by adecrease in SAP. Telemetric measurement showed that 1 oraladministration of either Bay41-2272 or Bay58-2667 (dose 10mg/kg) reduced SAP by �20% over a time range of 10 to 20hours (Figure 3A). Heart rate ranged from �600 bpm andincreased to �700 bpm in response to the compounds (Figure3B). This value normalized �5 hours after oral application ofBay41-2272 or Bay58-2667. Plasma samples were collected6 hours after the last application of the compounds, and thelevels of Bay41-2272 and Bay58-2667 were measured at 10and 25 nmol/L, respectively (Figure 3C).

Chronic Effects of Bay41-2272 and Bay58-2667 onHemodynamics and Right Heart Hypertrophy in MiceWith Hypoxia-Induced Pulmonary HypertensionThe hypoxic wild-type mice developed pulmonary hyperten-sion within 21 days, which was sustained until day 35.

Figure 7. sGC �1 and �1 subunit expression in hypoxia (hox)-induced pulmonary hypertension in wild-type (A) and NOS3�/� (B) mice.Western blot analysis was used to assess expression of sGC �1 and �1 subunits in lungs from controls, hypoxia-challenged animals,and animals treated with Bay41-2272 or Bay58-2667. Densitometric analysis of sGC subunit expression is given and normalized to thehousekeeping gene �-actin (C, D). Immunoblots are representative of n�3 to 5 blots for each group.

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Consequently, right ventricular systolic pressure (RVSP) wasincreased significantly compared with the control group(Figure 4A). This increase was accompanied by an increase inthe ratio of right ventricle to left ventricle plus septum weight[RV/(LV�S)] (Figure 4B). The ratio increased from0.24�0.02 (controls) to 0.38�0.02 (21 days of hypoxia) and0.42�0.03 (35 days of hypoxia), respectively (both P�0.05versus controls). Bay41-2272 and Bay58-2667, applied bygavage from day 21 to 35, significantly reduced hypoxia-induced chronic pulmonary hypertension in wild-type mice.Accordingly, Bay41-2272 and Bay58-2667 caused a decreaseof 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 treatmentgroups (Table). Likewise, NOS3�/� mice developed pulmonaryhypertension, with RVSP values increasing from 23.7�0.8(controls) to 35.5�3.0 (21 days of hypoxia) and 34.9�1.2 (35days of hypoxia) (Figure 5A) and RV/(LV�S) values increasingfrom 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 reductionof RVSP in NOS3�/� mice, whereas both compounds failed toreduce RV/(LV�S) values (Bay41-2272, 0.36�0.02; Bay58-2667, 0.39�0.06). Mean SAP did not change in any of thetreatment groups (Table).

Chronic Effects of Bay41-2272 and Bay58-2667 onDegree of Muscularization of Pulmonary Arteries inMice With Hypoxia-Induced Pulmonary HypertensionWe quantitatively assessed the degree of muscularization ofpulmonary arteries with a diameter from 20 to 70 �m. Inwild-type mice, the majority of vessels from 20 to 70 �m areusually nonmuscularized and partially muscularized (Figure6). In the hypoxia-exposed animals, both at day 21 and 35, adramatic decrease in nonmuscularized pulmonary arteriesoccurred with a concomitant increase in fully and partiallymuscularized pulmonary arteries. Treatment with Bay41-2272 and Bay58-2667 resulted in a significant increase ofnonmuscularized arteries compared with both hypoxiagroups. In addition, Bay41-2272 decreased the percentage ofpartially muscularized pulmonary arteries.

Expression of � and � Subunits of sGC in MiceWith Hypoxia-Induced Pulmonary Hypertension:Effects of Bay41-2272 and Bay58-2667The protein levels of both subunits of sGC did not changesignificantly in response to hypoxia (Figure 7). In contrast, inNOS3�/� mice, the �1 subunit of sGC was downregulated atday 35 and the �1 subunit decreased at day 35. Significantchanges in either sGC�1 or sGC�1 subunit appeared in noneof the treatment groups.

Chronic Effects of Bay41-2272 and Bay58-2667 onHemodynamics and Right Heart Hypertrophy in RatsWith Monocrotaline-Induced Pulmonary HypertensionIn rats injected with monocrotaline for 28 days, severe pulmonaryhypertension developed with marked increase in RVSP (from25.1�1.4 to 67.7�3.1 mm Hg; Figure 8A), in the ratio of rightventricular weight to left ventricle plus septum (RV/LV�S) (from0.30�0.01 to 0.63�0.01; Figure 8B), and in the percentage of

pulmonary artery muscularization (Figure 9A, 9B). In rats treatedwith vehicle, further progression of pulmonary hypertension untilday 42 was noted (RVSP�78.5�6.2 mm Hg; RV/LV�S�0.81�0.05; Figures 8 and 9). No significant changes inmean SAP were observed (control�117�9 mm Hg; monocrotalinefor 4 weeks�103�9 mm Hg; monocrotaline for 6weeks�109�10 mm Hg). Ninety percent (9/10) and 60% (9/15) ofanimals survived the 28- and 42-day monocrotaline treatment.Long-term treatment with Bay41-2272 or Bay58-2667 significantlydecreased 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). Inaddition, both compounds decreased RV/LV�S values to0.47�0.01 and 0.50�0.03, respectively (P�0.05 versus monocro-taline both at day 42 and at day 28). SAP was unchanged(Bay41-2272, 91�4 mm Hg; Bay58-2667, 102�6 mm Hg). In theanimals treated with Bay41-2272 or Bay58-2667, survival was 80%(8/10) and 70% (7/10), respectively.

Chronic Effects of Bay41-2272 and Bay58-2667on Degree of Muscularization of PulmonaryArteries in Rats With Monocrotaline-InducedPulmonary HypertensionWe quantitatively assessed the degree of muscularization ofpulmonary arteries with a diameter from 10 to 50 �m. In the

Figure 8. Influence of long-term treatment with Bay41-2272 orBay58-2667 on hemodynamics (A) and right heart hypertrophy (B)in monocrotaline (MCT)-induced pulmonary hypertension. RSVP(in mm Hg) (A) and right to left ventricular ratio (RV/LV�S) (B) aregiven. Bay41-2272 or Bay58-2667 was applied daily by gavagefrom day 28 to 42 at a dose of 10 mg/kg body wt. Control animalsreceived placebo (3% methylcellulose). *P�0.05 vs control;†P�0.05 vs MCT at day 28; ‡P�0.05 vs MCT at day 42.

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monocrotaline-injected animals, both at day 28 and 42, asignificant decrease in nonmuscularized pulmonary arteriesoccurred (Figure 9A) with a concomitant increase in fullymuscularized pulmonary arteries. Treatment with Bay41-2272 or Bay58-2667 at 10 mg/kg per day resulted in asignificant reduction of fully muscularized arteries and in-creased the percentage of nonmuscularized pulmonary arter-ies (both parameters P�0.05 versus monocrotaline both atday 42 and at day 28) (Figure 9B).

DiscussionIn this study we demonstrated that both the sGC stimulatorBay41-2272 and the sGC activator Bay58-2667 reversepulmonary hypertension in chronically hypoxic mice andmonocrotaline-injected rats. Notably, treatment with theseagents was commenced only after full establishment ofpulmonary hypertension, right heart hypertrophy, and struc-tural changes in the lung vasculature. The compound Bay41-

2272 is a novel NO-independent stimulator of sGC withcharacteristics similar to YC-1 but with higher potency of �2to 3 orders of magnitude and no phosphodiesterase-5 inhib-itory activity.17,28 Bay41-2272 also acts synergistically withNO, which was shown experimentally in NO-dependentpenile erection29 and experimental acute pulmonary hyper-tension.16 In both systems, the NO/sGC/cGMP system playsan important role in maintaining physiological function. Incontrast, the compound Bay58-2667 does not synergize withNO but stimulates the heme-oxidized or heme-depleted puri-fied enzyme14 (Figure 10). Both compounds Bay41-2272 andBay58-2667 are orally bioavailable, and both proved to havea long-lasting effect over 10 and 12 hours, respectively.14,17

Similarly, we show in our study in mice that both compoundsBay41-2272 and Bay58-2667 reduce SAP for �20 hours. Onthe basis of these findings, therapy was performed by once-daily application to achieve optimal efficacy. Detailed phar-macokinetic studies have been performed for Bay41-2272.30

Figure 9. Effects of Bay41-2272 and Bay58-2667 on the degree of muscularization of pulmonary arteries in monocrotaline (MCT)-injectedrats. Proportion of nonmuscularized (N), partially muscularized (P), or fully muscularized (M) pulmonary arteries, as percentage of total pulmo-nary artery cross section (sized 10 to 50 �m) is given (A). A total of 60 to 80 intra-acinar vessels were analyzed in each lung. B, The degreeof muscularization is demonstrated by von Willebrand (brown) and �-smooth muscle actin (purple) staining for identifying endothelium andvascular smooth muscle cells, respectively. Bay41-2272 or Bay58-2667 was applied daily by gavage from day 28 to 42 at a dose of 10mg/kg body wt. *P�0.05 vs control; †P�0.05 vs MCT at day 28; ‡P�0.05 vs MCT at day 42. Bar�20 �m.

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Hepatic metabolism quickly results in oxidation of the5-pyrimidinyl-cyclopropyl residue of Bay41-2272 to a stablemetabolite that exerts long-term persistence in plasma andthus may contribute to the sustained vascular effects seenafter oral application of the parent compound.

Chronic hypoxia induces pulmonary hypertension similarto human pulmonary hypertension secondary to disorders ofthe respiratory system, such as chronic obstructive pulmonarydisease and interstitial lung disease. It is characterized bystructural changes to the vascular system, including de novomuscularization of normally nonmuscularized small pulmo-nary arteries and an increase in medial wall thickness. Incontrast, injection of the plant alkaloid monocrotaline in ratsinduces severe progressive pulmonary hypertension that fi-nally results in death. Most impressively, both compounds donot attenuate but partially reverse the structural changesinduced by 2 independent stimuli (hypoxia and monocrotal-ine) in 2 different species (mice and rats). The pharmacolog-ical activation of sGC may thus have a broad clinicalperspective for treatment of pulmonary vascular diseases. Theregulation of the expression of sGC under pathophysiological

conditions has been addressed by several groups. Althoughthe aortic GC content was not altered in NOS3 knockoutanimals31 or NOS inhibitor–treated rats, hypertension andaging appear to result in downregulation of GC expres-sion.32–35 In experimental models of hypoxia-induced pulmo-nary hypertension, upregulation of sGC expression has beenreported in rats and mice.4,5 With the use of immunostainingand Western blotting, a �2-fold increase of sGC protein �1

subunit was noted in smooth muscle cells of the pulmonaryarteries in hypoxic rat lungs.36 The same group demonstratedin mice that both subunits of sGC, the �1 and �1 subunits,were increased under conditions of hypoxia-induced pulmo-nary hypertension.4 Interestingly, similar results were ob-served in NOS2 knockout animals but not in NOS3 knockoutanimals, suggesting NOS3 as a major regulator of sGCactivity and protein expression in the lung vasculature.4 Inthis study both the �1 and �1 subunits were not changed inwild-type mice in response to hypoxia, and the expressionwas not altered by the 2 sGC activators. In contrast, adownregulation was noted in NOS3�/� mice, which is well inagreement with the aforementioned previous report.

Against this background, sGC is an attractive target for thetreatment of hypoxia-induced pulmonary vascular diseases.In contrast to previous investigations, which investigated theinfluence of an endothelin antagonist, prostaglandin E1, orphosphodiesterase-5 inhibitors37 together with the hypoxicchallenge, we started therapeutic interventions when pulmo-nary hypertension was already fully established, from week 3to 5 in mice and from week 4 to 6 in rats. Under theseconditions, both Bay41-2272 and Bay58-2667 significantlyreversed the degree of pulmonary hypertension evolving inresponse to hypoxia and monocrotaline. This was true forsystolic pulmonary artery pressure and right heart hypertro-phy but also for structural changes including the de novomuscularization of small precapillary vessels.

The NOS3�/� mice developed pulmonary hypertensionwith hemodynamics and morphological changes similar tothose in wild-type mice, which is in contrast to 1 previousreport38 but is in agreement with 2 other publications.4,23

Interestingly, both compounds failed to reverse RVSP andright heart hypertrophy in these mice. These findings suggestthat the antiremodeling effects of Bay41-2272 and Bay58-2667 were both dependent on ongoing NOS3-dependent NOgeneration.

The antiremodeling potency of Bay58-2667 in hypoxicmice is particularly interesting because this compound acti-vates mainly the oxidized or the heme-free form of sGC,which does not occur under physiological conditions. How-ever, it has recently been shown that in lungs from mice keptunder hypoxic conditions, levels of reactive oxygen speciesmay even increase,39 which in turn might oxidize the hemegroup of sGC. Enhanced levels of oxygen radicals have alsobeen found under conditions of atherosclerosis, diabetes,hypercholesterinemia, or hypertension. However, future stud-ies must prove the incidence of heme-free or oxidized formsof sGC in vascular abnormalities such as chronic pulmonaryhypertension.

In conclusion, the compounds Bay41-2272 and Bay58-2667 caused dose-dependent pulmonary vasodilation in hyp-

Figure 10. The sGC stimulator Bay41-2272 and the activatorBay58-2667 increase cGMP production, thereby regulatingsmooth muscle function. Bay41-2272 is an NO-dependent stim-ulator acting preferentially on the physiological form of sGCcontaining the iron II heme [Fe(II) heme] (left). In contrast,Bay58-2667 is a NO-independent activator preferably address-ing the oxidized (and therefore NO-insensitive) iron III heme form[Fe(III) heme] of sGC (right). Increased levels of cGMP thenresult acutely in vasodilatation and antiaggregation and resultchronically in antiremodeling of the vascular wall as well asunloading of the right ventricle. GTP indicates guanosinetriphosphate; ox. Stress, oxidative stress; and RV, rightventricle.

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oxia-induced pulmonary hypertension in isolated mouselungs. When these agents are used for chronic treatment bydaily gavage, reversal of the hypoxia-elicited pulmonaryhypertension was demonstrated, which was true for hemody-namics, structural changes of the lung vasculature, and rightheart hypertrophy. Notably, the efficacy of both agents wasdependent on intact NOS function. We conclude that activa-tion of sGC may offer a new therapeutic option for antire-modeling therapy in severe pulmonary hypertension.

AcknowledgmentsThis work was supported by the Deutsche Forschungsgemeinschaft,SFB547, projects B5 and C6. The authors acknowledge the technicalassistance of Anke Voigt and Helmut Mueller.

DisclosuresDrs Stasch and Gnoth report that they are employed by PharmaResearch Center, Bayer HealthCare. The other authors report noconflicts.

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CLINICAL PERSPECTIVEBlood vessel remodeling in the context of chronic systemic and pulmonary disorders (eg, systemic and pulmonaryhypertension, chronic obstructive pulmonary disease, interstitial lung disease, left heart failure, diabetes, and arterioscle-rosis) shares many similarities such as medial wall thickening, neointimal formation, and endothelial dysfunction. Thenitric oxide (NO)–soluble guanylate cyclase (sGC) pathway plays a central role in maintaining physiological organfunction. Alterations of this pathway have been attributed to be centrally involved in the course of these diseases and aresubject to the development of new therapeutic agents. Among the most recent approaches, approval of thephosphodiesterase-5 inhibitor sildenafil for the treatment of pulmonary arterial hypertension represents the most intriguingtherapeutic option. In the present study we address another important molecular key player of the NO/cGMP axis byproving the therapeutic efficacy of the sGC stimulator Bay41-2272 and activator Bay58-2667 in 2 well-established modelsof chronic pulmonary hypertension (hypoxia and monocrotaline-induced pulmonary hypertension). Both compounds notonly improved pulmonary hemodynamics symptomatically (as previously shown for many other substances) but alsoreversed vascular remodeling. Notably, treatment with these agents was commenced after full establishment of pulmonaryhypertension, right heart hypertrophy, and structural changes in the lung vasculature. Targeting sGC is of considerableinterest because stimulators and activators of this enzyme represent a new class of drugs complementary to currentlyestablished therapies for chronic vascular disorders (eg, phosphodiesterase inhibitors, ACE inhibitors, endothelin receptorantagonists). Clinical trials are warranted to address the safety and efficacy of these substances.

Dumitrascu et al sGC Activation in Pulmonary Hypertension 295

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Grimminger and Ralph Theo SchermulyHarald Schmidt, Johannes-Peter Stasch, Mark Jean Gnoth, Werner Seeger, Friedrich

Rio Dumitrascu, Norbert Weissmann, Hossein Ardeschir Ghofrani, Eva Dony, Knut Beuerlein,and Vascular Remodeling

Activation of Soluble Guanylate Cyclase Reverses Experimental Pulmonary Hypertension

Print ISSN: 0009-7322. Online ISSN: 1524-4539 Copyright © 2006 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation doi: 10.1161/CIRCULATIONAHA.105.581405

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