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doi:10.1510/icvts.2006.147033

Interactive CardioVascular and Thoracic Surgery 6 (2007) 608–613

� 2007 Published by European Association for Cardio-Thoracic Surgery

Institutional report - Experimental

Oral sildenafil prevents and reverses the development of pulmonaryhypertension in monocrotaline-treated rats

Hao Liu, Zhi-yong Liu*, Qin Guan

Department of Cardiothoracic Surgery, Zhongda Hospital, Southeast University, Nanjing 210009, China

Received 20 October 2006; received in revised form 20 June 2007; accepted 22 June 2007

Abstract

The endothelin system plays an importment role in the development of pulmonary hypertension. Several studies have suggested thatinterfering with the function of the endothelin system will be helpful in pulmonary hypertension treatment. In the present study, weinvestigated the preventive and therapeutic effects of sildenafil on pulmonary hypertension in monocrotaline-treated rats. In the preventivestudy, the level of mean pulmonary arterial pressure, right ventricular divide, left ventricular and septum, small pulmonary arterialmorphologic and elastic fiber changes were highly improved in the treated group (P-0.05). The expressions of endothelin-1 A typereceptors on small pulmonary arterial hypertension were significantly reduced in the sildenafil-treated group (P-0.05). The ET-1 level inplasma was increased in the sildenafil-treated group, but did not reach significance. Emphysema, interstitial pneumonia were significantlyimproved in the sildenafil-treated group. The same findings were also observed in the therapeutic study. The present results suggest thatsildenafil can prevent and reverse the development of pulmonary hypertension in monocrotaline-treated rats by improving the function ofendothelin system in pulmonary arteries.� 2007 Published by European Association for Cardio-Thoracic Surgery. All rights reserved.

Keywords: Endothelium; Experimental surgery; Myocardial remodeling; Pulmonary arteries; Vascular tone and reactivity

1. Introduction

Pulmonary arterial hypertension (PH) is a severe diseasecharacterized by vasoconstriction, intimal lesions, medialhypertrophy, and adventitial thickening of the precapillarypulmonary arteries. The progressive pulmonary hyperten-sion leads to increased afterload, right ventricular hyper-trophy, right heart failure and death. In primary pulmonaryarterial hypertension the median survival is considered tobe 2.8 years from the time of diagnosis. Therefore, a noveltherapeutic strategy for pulmonary hypertension is desir-able w1x.

Treatment for pulmonary arterial hypertension was revo-lutionized in 1996 with the widespread introduction ofprostacyclin, but some patients still progress to lung trans-plant or death despite state-of-the-art medical manage-ment. This monograph highlights some of the recentpre-clinical work that holds exciting potential for PH treat-ment w2x.

Although the precise mechanism of pulmonary arterialhypertension is unclear, release by endothelial cells of anendogenous substance that acts on a target receptor hasgained acceptance. Endothelin, a product of endothelialcells, has been reported to contribute to pulmonary arterialhypertension. Endothelial cells of the pulmonary microvas-culature are frequently exposed to extraneous substancessuch as drugs, toxins or abnormal metabolic products, and

*Corresponding author. Tel.: q86-25-83272003; fax: q86-25-83272011.E-mail address: zyliu3@jsmail.com.cn (Z-y. Liu).

injury to these cells can result in pathological changes inthe alveoli w3x. Monocrotaline injures the endothelial cellsof the pulmonary blood vessels, causing pulmonary hyper-tension and interstitial pulmonary fibrosis w4x. This sub-stance also induces the proliferation of muscular intimalcells in arterioles and fibroblasts in alveolar walls at thecapillary level. Various growth factors are considered to beinvolved in cell proliferation following injury to endothelialcells. Among them, the potent vasoconstrictor peptideendothelin-1, comprising 21 amino acids, induces the pro-liferation and migration of smooth muscle cells and fibro-blasts w5x and its association with pulmonary hypertensionhas been indicated. Endothelin-1 was discovered in a cul-ture medium of endothelial cells from porcine aorta w6x. Itis known to act through two types of receptors, ET -R andA

endothelin B type receptors w7x. Plasma endothelin-1 levelsare reportedly high in pulmonary hypertension patients w8x.These data suggest that endogenous endothelin-1 isinvolved in the progression of pulmonary hypertension, andreduction of endothelin-1 or downregulation of endothelin-1 A type receptors are expected to prove effective in thetreatment of this condition.

In the present study, we investigated the effects ofendothelin-1 on pulmonary arterial hypertension and thepossible protective role of sildenafil. The study was firstlyundertaken to determine the ability of pulmonary arterialhypertension on pulmonary and heart remolding. Secondly,the study was designed to determine the preventive andtherapeutic effects of sildenafil by measuring not only

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pulmonary arterial pressure and right ventricular hypertro-phy but also histopathological changes and endothelin-1level in plasma.

2. Methods

2.1. Animals and materials

Male Sprague Dawley rats (eight weeks old) were obtainedfrom the Experimental Animal Center Medical College ofSoutheast University (Nanjing, China). Sildenafil was pur-chased from Pfizer Inc. (Pfizer, USA). Sildenafil was dis-solved or suspended in 1% sodium chloride. Monocrotalinewas purchased from Sigma (Sigma, USA). An ET RIA Kit waspurchased from Beijing Huaying Biotechnology Institute. Allother chemicals were of analytical grade. Monocrotalinewas dissolved in alcohol and distilled water (1:5). Pulmo-nary hypertensive rats were prepared by injecting themonocrotaline at a subcutaneous dose of 60 mgØkg intoy1

the dorsum of the neck.

2.2. Experimental design

2.2.1. Protocol 1: Drug efficacy on pulmonaryhypertension: preventive study

In the preventive study, 20 monocrotaline-treated ratswere used. One day after the injection of monocrotaline,the animals were randomly divided into two sub-groups(ns10 each), and given a daily administration of eithersodium chloride as the control group, or sildenafil(1.7 mgØkg Ød ) as the treated group for three weeks.y y1 1

The other ten rats were not injected monocrotaline andwere given a daily administration of sodium chloride forthree weeks as sham group. Twenty-four hours after thefinal administration, hemodynamic variables were record-ed. All of the animals were anesthetized with sodiumpentobarbital (50 mgØkg , i.p.). A catheter (PE-50) wasy1

inserted into the left carotid artery to measure meanarterial blood pressure and heart rate. The second catheterwas inserted into the pulmonary artery to measure pulmo-nary pressure, and the shape of the pressure tracing wasdisplayed on a polygraph system (RM-6000, Nihon Konden,Tokyo, Japan). All hemodynamic variables were measuredand recorded with a polygraph system. After measurement,the rats were exsanguinated, and cardiopulmonary tissueswere excised and the wet weights of the right ventricle(RV), left ventricle including septum (LVqS) and lungswere measured. The RV- to LVqS ratios wRVy(LVqS)x wereused as indices of right ventricular hypertrophy. Two milli-liters of blood were conserved (–70 8C) to detect plasmaET-1 levels.

2.2.2. Protocol 2: Drug efficacy on pulmonaryhypertension: therapeutic study

In the preliminary test, a significant increase in rightventricular systolic pressure was also observed three weeksafter monocrotaline injection, suggesting that pulmonaryhypertension was already established even at this stage.This model was, therefore, used to study the therapeuticeffects of sildenafil. The variables measured in the thera-peutic study were the same as those in the preventive

study. Three weeks after the injection of monocrotaline,20 rats were randomly divided into two sub-groups, andorally given sodium chloride as the vehicle (ns10) orsildenafil (1.7 mgØkg , ns10) once daily for three weeks.y1

The other ten rats were not injected monocrotaline andwere given a daily administration of sodium chloride forthree weeks as sham group. One day after the final drugadministration, hemodynamic variables were measured inthe same way as in the preventive study. Histopathologicalexaminations were done in all of the rats which had beenpre-treated in the same manner as in the preventive study.Two milliliters of blood were conserved (–70 8C) to detectplasma ET-1 levels.

2.3. Histopathological examination

Histopathological examination of tissue slides was per-formed at the Pathology Department of Zhongda Hospital(Nanjing, China). From the left lobe of lung, five regions,including the hilus and the two anterior and two posteriorregions thereto, were cut in cross-section and stained withhematoxylin and eosin in the usual manner. Small pulmo-nary arterioles were found (diameter -100 mm) by anobserver blinded to the experimental group. The percentwall thickness of muscular arteries was calculated for eachcategory using the following formula and the mean valuewas obtained for each animal. Percent wall thickness(%)sw(cross-sectional area–luminal area)ycross-sectionalareax=100.

2.4. Immunohistochemistry examination

Immunohistochemical staining by streptavidin biotin com-plex method was performed for the sections of the leftlung. We used the following commercially available anti-bodies at dilutions of 1:50 rabbit polyclonal anti-rat endo-thelin-1 A type receptors antibody (endothelin-1 A typereceptors, lot AN-05; Peptide Peptide (C) NHNT ERSSHKDSMN, Alomone Labs, Israel). Control slides were treatedwith normal diluted rabbit serum. The slides were reviewedby a pathologist and were semiquantitatively gradedaccording to the degrees of positivity of the immunoreac-tivity as 0 for absence of staining and 1q for weak, 2qfor moderate, and 3q for strong staining. The slides werecompared with the respective control slides to excludenon-specific staining. The degrees of positivity wereassessed for endothelia of the same peripheral pulmonaryarteries in which the percent medial thickness wasdetermined.

2.5. Plasma ET-1 level

We took blood samples from inferior vena cava immedi-ately after measuring pulmonary artery pressure. The sam-ples were put into Eppendorf tubes containing a finalconcentration of 300 kIUyml of aprotinin and 2 mgyml ofEDTA.Na2. The samples were centrifuged for 15 min at3000 rpm at 4 8C. The plasma was stored at –80 8C.Radioimmunology was performed for the determination ofplasma ET-1 levels.

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Fig. 1. The results of monocrotaline-treated rats administered sidenafil inthe preventive study ( "s). Following subcutaneous administration ofxmonocrotaline, sildenafil was administered once daily for three weeks. Theeffects on right ventricle hypertrophy (a), wall thickness (b), ET -R (c) wereA

shown. We used right ventricle to left ventricle including septum wRVy(LVqS)x for right ventricular hypertrophy, percent wall thickness for pulmonaryarterial remolding, and endothelin-1 A type receptors. Each column repre-sents the mean"S.E.M. (ns10). The doses of sildenafil were1.7 mgØkg Ød , respectively. *P-0.01 compared with the sham group byy y1 1

one-way analysis of variance. **P-0.05 compared with the control group byANOVA and Scheffe F-test.

Table 1The results of monocrotaline-treated rats administered sidenafil in the preventive study ( "s)x

RV (mg) LVqS (mg) Mean arterial blood pressure (mmHg) ET-1 (pgØml )y1

Sham 140"4 519"11 6.17"2.24 52.55"4.35Control group 198"7* 484"10* 15.03"2.20* 71.56"1.90*Treated group 162"10** 492"10** 8.18"1.61** 83.87"5.99***

Following subcutaneous administration of monocrotaline, sildenafil was administered once daily for three weeks. Values represent the mean"S.E.M. (ns10).RVsright ventricle; LVqSsleft ventricle with septum. The doses of sildenafil were 1.7 mgØkg Ød . *P-0.01 compared with the sham group. **P-0.05 com-y y1 1

pared with the control group. ***P-0.01 compared with the sham group.

2.6. Data analysis

Data are expressed as the means"S.D. Data were ana-lyzed by the one-way analysis of variance (ANOVA) andScheffe F-test. A P-value -0.05 was considered to bestatistically significant.

3. Results

3.1. Protocol 1: Preventive effects of sildenafil inmonocrotaline-treated rats: effect on hemodynamicvariables, right ventricular hypertrophy, pulmonaryvascular remodeling and histopathological changesin the lung

Compared to the sham group, the rats in the control groupshowed a marked increase in mean pulmonary arterialpressure at three weeks after monocrotaline injection,demonstrating the presence of pulmonary hypertension.These rats also showed increases in RV and RVy(LVqS)values, indicating marked right ventricular hypertrophy.Three-week administration of sildenafil (1.7 mgØkg Ød )y y1 1

significantly suppressed the increases in mean pulmonaryarterial pressure, RV and RVy(LVqS) (Table 1, Fig. 1).

The percent wall thickness (WT%) of small arteries in thecontrol group rats receiving vehicle was greater than thatin the sham group in all diameter categories, demonstratingthe progression of pulmonary vascular remodeling. Admin-istration of sildenafil significantly prevented these mono-crotaline-induced increases in arterial medial thickness(Fig. 1).

On histopathological examination, emphysema wasobserved in six out of ten of the control group, whereas nosuch findings were noted in the sham or treated group(Table 2). Interstitial pneumonia, characterized by thick-ening of the alveolar septum and inflammatory cell infiltra-tion into the thickened area, was observed more obviouslyin the control group than in the sham group (Table 2). Theincidence of interstitial pneumonia in the treated groupwas significantly lower than those in the control group (Fig.2).

3.1.1. Preventive effects of sildenafil inmonocrotaline-treated rats: endothelin-1 A typereceptors and plasma ET-1 levels

On immunohistochemical examination, the expression ofendothelin-1 A type receptors on pulmonary small artery(diameter-100 mm) smooth muscular cell was upregulatedin the control group. Three weeks after the administrationof sildenafil, the expression was significantly downregulat-ed. In the treated group, the plasma ET-1 levels were

higher than those in the control group, but did not reachsignificance (Table 1, Fig. 1).

3.2. Protocol 2: Therapeutic effects of sildenafil inpulmonary hypertension rats: effect on hemodynamicvariables, right ventricular hypertrophy, pulmonaryvascular remodeling and histopathological changesin the lung

Mean pulmonary arterial pressure was also observed inthe control group three weeks after injection. In compari-

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Table 2Histopathological findings in the lung in monocrotaline-treated rats in the preventive study

Findings Group grade Sham (ns10) Control group (ns10) Treated group (ns10)

Emphysema – 10 4* 10*1q 0 3 02q 0 3 03q 0 0 0

Interstitial pneumonia – 10 2* 7**1q 0 4 32q 0 3 03q 0 1 0

(–) Negative, (1q) mild, (2q) moderate, (3q) severe. *P-0.01 compared with the sham group. **P-0.05 compared with the control group by x test.2

Fig. 2. Effects of sildenafil on lung vascular morphology in monocrotaline-treated rats in preventive study. Three weeks after subcutaneous adminis-tration of monocrotaline, sildenafil was administered once daily for threeweeks. Lung and pulmonary arterioles were stained with hematoxylin andeosin (HE=50). (a) Sham rats, (b) control rats, (c) treated rats.

Table 3The results monocrotaline-treated rats administered sildenafil in the therapeutic study ( "s)x

RV (mg) LVqS (mg) Mean arterial blood pressure (mmHg) ET-1 (pgØml )y1

Sham 140"4 519"11 6.17"2.24 52.55"4.35Control group 242"6* 457"13* 24.01"1.96* 74.88"2.17*Treated group 207"8** 493"10** 8.03"2.03** 85.62"4.35***

Three weeks after subcutaneous administration of monocrotaline, drugs were administered once daily for four weeks. Values represent the mean"S.E.M.(ns9–10). RVsright ventricle; LVqSsleft ventricle with septum; the doses of sildenafil were 1.7 mgØkg Ød . *P-0.01 compared with the sham group.y y1 1

**P-0.05 compared with the control group. ***P-0.01 compared with the sham group.

son with the sham group, the developments of pulmonaryhypertension were confirmed three weeks after the injec-tion of monocrotaline. The increase in mean pulmonaryarterial pressure was still present at six weeks after mono-crotaline injection (24.01"1.96 mmHg), indicating persist-ent pulmonary hypertension. Increases in RV and RVy(LVqS)were observed at six weeks after monocrotaline injection.These changes were closely similar to those observed threeweeks after monocrotaline injection in the preventivestudy. Three weeks after administration of sildenafil, theincreases in mean pulmonary arterial pressure, RV, RVy(LVqS) were suppressed, without affecting heart rate andmean arterial blood pressure (Table 3, Fig. 3).

In the control group, the percent wall thickness of pul-monary arterioles was significantly greater than that in thesham group, indicating the progression of pulmonary vas-cular remodeling. Administration of sildenafil significantlyreduced the arterial medial thickening (Fig. 3). Emphysemawas observed in five of ten rats in the control group onhistopathological examination, whereas no such findingswere noted in the sham or treated group (Table 4). Inter-stitial pneumonia was present in ten out of eight of thecontrol group. These changes were not observed in thesham group (Table 4).

3.2.1. Therapeutic effects of sildenafil in pulmonaryhypertension rats: endothelin-1 A type receptors andplasma ET-1 levels

On immunohistochemical examination, the expression ofendothelin-1 A type receptors on pulmonary small artery(diameter-100 mm) smooth muscular cell was upregulatedin the control group. Three weeks after administration ofsildenafil, the expression of endothelin-1 A type receptorswas significantly downregulated. In the treated group, theplasma ET-1 levels were higher than those in the controlgroup, but did not reach significance (Table 3, Fig. 3).

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Fig. 3. The results of monocrotaline-treated rats administered sildenafil inthe therapeutic study ( "s). Following subcutaneous administration ofxmonocrotaline, sildenafil was administered once daily for three weeks. Theeffects on right ventricle hypertrophy (a), wall thickness (b), ET -R (c) wereA

shown. We used right ventricle to left ventricle including septum wRVy(LVqS)x for right ventricular hypertrophy, percent wall thickness for pulmonaryarterial remolding, and endothelin-1 A type receptors. Each column re-presents the mean"S.E.M. (ns10). The doses of sildenafil were1.7 mgØkg Ød , respectively. *P-0.01 compared with the sham group byy y1 1

one-way analysis of variance. **P-0.05 compared with the control group byANOVA and Scheffe F-test.

Table 4Histopathological findings in the lung in monocrotaline-treated rats in the therapeutic study

Findings Group grade Sham (ns10) Control group (ns8) Treated group (ns10)

Emphysema – 10 3* 10*1q 0 2 02q 0 3 03q 0 0 0

Interstitial pneumonia – 10 5 81q 0 2 12q 0 1 13q 0 0 0

(–) Negative, (1q) mild, (2q) moderate, (3q) severe. *P-0.01 compared with the sham group.

4. Comment

The main finding of this study was that oral administrationof sildenafil significantly reduced monocrotaline-inducedpulmonary hypertension without evident effects on thesystemic circulation. In addition, sildenafil administrationpartially prevented or reversed pulmonary arterial remold-ing, interstitial pneumonia and emphysema that accompa-nied monocrotaline.

In the present study, we investigated in detail the preven-tive and therapeutic effects of sildenafil on the develop-ment of pulmonary hypertension. A marked increase inpulmonary arterial pressure and right ventricular hypertro-phy were observed in rats three weeks after administrationof monocrotaline. These conditions closely resemble thecorruption changes of pulmonary hypertension patients.Histopathological examination indicated the existence ofarterial medial thickening in the small pulmonary arteryand alveolar disorders such as emphysema or interstitialpneumonia, suggesting that, in addition to disorders inpulmonary circulation, respiratory function was also aggra-vated in this model.

In the preventive study, the arterial medial thickening inthe pulmonary microvasculature and alveolar disorderswere significantly suppressed in the sildenafil-treated ratsas well as the increases in pulmonary arterial pressure andright ventricular hypertrophy. In addition to these preven-tive effects, the same improvements were also seen in thetherapeutic study. These findings indicate that sildenafilprevented and deferred the development of pulmonaryhypertension in monocrotaline-induced pulmonary hyper-tensive rats, and strongly suggest that sildenafil mayameliorate the condition of patients with pulmonary hyper-tension and cor pulmonale in clinical use.

In the preventive study, the plasma ET-1 levels in thecontrol group were significantly higher than those in thesham group. At the same time, they were higher in thetreated group than those in the control group, but did notreach significance (P)0.05). Despite higher ET-1 in theserats, mPAP remained lower after sildenafil administration.It is likely that sildenafil’s effect on vasodilatation isenough to overpower the vasoconstrictive signal elicited byhigh levels of ET-1. The same findings were also found inthe therapeutic study. This finding suggests that sildenafilcontributes to the increase in ET-1. Jefferson et al. w9xfound that sildenafil has the effect to elevate intracellularcyclic guanosine monophosphate. The mechanism behindthe increase in ET-1 is that sildenafil may upregulateexpression of both inducible and endothelial isoforms ofNO synthase. Salloum et al. w10x also described that silden-afil may upregulate expression of both inducible and endo-thelial isoforms of NO synthase. Sildenafil’s mechanism ofvasodilatation attributes to the NO pathway (Pearl et al.w11x). Wiley and Davenport w12x reported that NO has beenshown to compete with ET-1 for the endothelin receptor,which may cause signal termination and increase ET-1 levels

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by displacing off its receptor. NO has also been suggestedto terminate the vasoconstrictive signal of ET-1 by inter-ruption of downstream signaling processes not yet defined(Goligorsky et al. w13x). So, we can easily draw the conclu-sion that sildenafil may upregulate expression NO, theninduces the effects of vasodilatation.

In the preventive and therapeutic study, the expressionof endothelin-1 A type receptors on pulmonary small artery(diameter-100 mm) smooth muscular cell was upregulatedin the control group by immunohistochemical examination.Three weeks after administration of sildenafil, the expres-sion of endothelin-1 A type receptors was significantly downregulated. It indicates that sildenafil has the effect todownregulate expression of endothelin-1 A type receptorson pulmonary small artery smooth muscular cell. Besidesupregulating expression of NO, another mechanism behindthe vasodilatation is that sildenafil may downregulateexpression of endothelin-1 A type receptors.

Gillespie et al. w14x described pulmonary function inmonocrotaline-treated rats. They demonstrated decreasesin tidal volume, lung compliance and respiratory frequency.They also demonstrated decreased diffusion capacity ofthe lung. These changes indicate the impairment of gasexchange in the lung and suggest the existence of intersti-tial and parenchymatous injury in the lung. Chen et al.w15x also described the impairment of endothelial cell andmuscularization in pulmonary artery in monocrotaline-treated rats. In the present study, we demonstrated theameliorative effects of sildenafil on lung interstitial andparenchyma by effecting the endothelin system. As far aswe are aware, this is the first report to demonstrate theeffectiveness of sildenafil on lung interstitial and parenchy-ma in monocrotaline-treated rats. The mechanism of pre-ventive and therapeutic effect is that sildenafil inhibits theendothelin system and downregulates expression of endoth-elin-1 A type receptors.

5. Conclusion and perspectives

In conclusion, we have demonstrated that sildenafil effec-tively prevented and reversed the development of pulmo-nary hypertension, and reduced the pulmonary vascularremodeling and parenchymal injury in monocrotaline-treat-ed rats. This effect was associated with a mild improvementin pulmonary arterial remolding. These data strongly sug-

gest that sildenafil may be clinically useful in patients withboth primary and secondary pulmonary hypertension.

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