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Am J Physiol Lung Cell Mol Physiol 294: L24-L33, 2008. First published October 26, 2007; doi:10.1152/ajplung.00245.2007
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PDE5A inhibition attenuates bleomycin-induced pulmonary fibrosis and pulmonary hypertension through inhibition of ROS generation and RhoA/Rho kinase activation

Anna R. Hemnes, Ari Zaiman, and Hunter C. Champion

Divisions of Cardiology and Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; and Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee

Submitted 26 June 2007 ; accepted in final form 19 October 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pulmonary hypertension frequently complicates interstitial lung disease, where it is associated with a high mortality. Patients with this dual diagnosis often fare worse than those with pulmonary arterial hypertension (PAH) alone and respond poorly to standard PAH therapy, often dying of right ventricular (RV) failure. We hypothesize that nitric oxide synthase (NOS) uncoupling is important in the pathogenesis of interstitial lung disease-associated pulmonary hypertension, and this process can be abrogated by phosphodiesterase type 5 (PDE5) inhibition to improve pulmonary vascular remodeling and right ventricular function. Intratracheal bleomycin (4 U/kg) or saline control was administered to C57/BL6 mice after anesthesia. After recovery, animals were fed a diet of sildenafil (100 mg·kg–1·day–1) or vehicle for 2 wk when they underwent hemodynamic measurements, and tissues were harvested. Survival was reduced in animals treated with bleomycin compared with controls and was improved with sildenafil (100.0 vs. 73.7 vs. 84.2%, P < 0.05). RV/LV+S ratio was higher in bleomycin-alone mice with improvement in ratio when sildenafil was administered (33.00 ± 0.01% vs. 20.98 ± 0.01% P < 0.05). Histology showed less pulmonary vascular and RV fibrosis in the group cotreated with sildenafil. Bleomycin was associated with a marked increase in superoxide generation by DHE histological staining and luminol activity in both heart and lung. Treatment with sildenafil resulted in a concomitant reduction in superoxide levels in both heart and lung. These data demonstrate that PDE5 inhibition ameliorates RV hypertrophy and pulmonary fibrosis associated with intratracheal bleomycin in a manner that is associated with improved NOS coupling and a reduction in reactive oxygen species signaling.

right ventricular hypertrophy

PULMONARY HYPERTENSION (PH) is a devastating complication of a number of diverse disease processes. While much attention has been given to pulmonary arterial hypertension (PAH), including idiopathic and connective tissue disease associated, PH often complicates interstitial lung disease. The presence and severity of PH is associated with increased mortality in idiopathic pulmonary fibrosis (IPF) (17). PH also frequently complicates scleroderma-associated pulmonary fibrosis (5, 42). There are several other diseases that combine pathology of interstitial lung disease and PH, such as sarcoidosis and nonspecific interstitial pneumonitis.

There have been significant advances in the treatment of PAH in the last several years, notably the use of prostacyclines, endothelin receptor antagonists, and phosphodiesterase type 5 (PDE5) inhibition (7, 12, 31). Large-scale trials for these medications have not included patients with significant interstitial lung disease-associated PH. However, one small study of patients with scleroderma-associated pulmonary fibrosis and PH treated acutely with sildenafil, a potent and selective PDE5 inhibitor, showed improvement in pulmonary hemodynamics with maintenance of ventilation-perfusion ratios (8). In addition, others have shown improved 6-min walk time in patients with PH associated with IPF (4). Despite these studies, little progress has been made in the understanding of basic mechanisms or specific therapy of PH associated with interstitial lung disease.

Bleomycin, a peptide antibiotic produced from Streptomyces verticillus, causes oxidant-mediated DNA scission leading to fibrogenic cytokine release and has been used as an animal model of pulmonary fibrosis as initial histology resembles human IPF. Bleomycin can cause either bronchiolocentric fibrosis or subpleural disease, depending on the route of administration, intratracheal or systemic (3). The initial histology is similar to that seen in human IPF. In a previous study by our group, mice exposed to bleomycin showed increased pulmonary arterial pressure and pulmonary vascular resistance after bleomycin treatment that was attenuated by adenoviral-associated endothelial nitric oxide synthase (eNOS) administration (2).

We hypothesized that PDE5 inhibition alters pulmonary and right ventricular (RV) response to intratracheal bleomycin and that this inhibition acts through suppression of tissue free radicals and the Rho kinase pathway. To test this hypothesis, we used a mouse model of bleomycin-induced pulmonary fibrosis that results in severe PAH and RV failure.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal model. Adult male C57/BL6 mice (8–11 wk of age, Jackson Laboratory) were used in the present study. The Johns Hopkins Animal Care and Use Committee approved all animal experiments. Animals were anesthetized with 3% inhaled isoflurane and intraperitoneal etomidate (40–80 ml/kg) and placed in the supine position. Using a sterile technique, the trachea was approached via a midline neck incision and isolated via blunt dissection. Using a 27-gauge needle attached to a microliter syringe, 4 U/kg bleomycin (Sigma-Aldrich) or the equivalent volume of saline was instilled into the trachea as previously described (2). Immediately before instillation, a forced exhalation was achieved by circumferential compression of the thorax. Compression was released after endotracheal instillation of bleomycin. This resulted in a forceful inspiration facilitating bleomycin dispersion to distal air spaces. The incision was then sutured closed, and the animal was allowed to recover. Fourteen days after intratracheal instillation, terminal RV and pulmonary hemodynamics were measured, and tissues were harvested for morphological, biochemical, and histochemical analyses. Tissue was immediately processed or quick-frozen in liquid nitrogen.

PDE5 inhibition. Sildenafil citrate (Viagra/Pfizer, Sandwich, UK) was used in these studies. Animals were fed a diet of vehicle or sildenafil within 24 h after undergoing sham surgery or intratracheal bleomycin. For in vivo chronic studies, we used 100 mg·kg–1·day–1 of sildenafil, yielding a mean free plasma concentration of 10.4 ± 2.3 nM as previously described (36). This is comparable to levels obtained in humans at doses of 1 mg·kg–1·day–1 reflecting much higher murine drug metabolism.

In a separate series of experiments, animals were treated with bleomycin as described above and fed a vehicle diet for 7 days. Animals began a diet of sildenafil or continued control from days 8–21 at which time tissues were harvested as described above.

In vivo hemodynamics. Intact heart hemodynamic analysis was performed as previously described 14 days after intratracheal instillation of bleomycin or vehicle. These studies used a four-electrode pressure-volume catheter (model SPR-839, Millar Instruments) placed through the RV apex in the open chest, anesthetized mouse and positioned along the longitudinal axis to record chamber volume by impedance and pressure by micromanometry in a manner as previously described (11).

Histology. Hearts and lungs were fixed with 10% formalin overnight, then embedded in paraffin, sectioned at a thickness of 5 µm, and stained with periodic acid-silver methenamine. We determined cardiomyocyte diameter and interstitial collagen fraction using computer-assisted image analysis (Adobe Photoshop 5.0, NIH Image J) with the observer blinded as to tissue source. At least four or five different hearts, with five separate fields of cells (total 50-70 cells for each heart), were quantified for cellular analysis.

PDE5A activity and PKG activity. Total low-Km cGMP phosphodiesterase activity was assayed at 1 µM/l substrate using fluorescence polarization assay (Molecular Devices) under linear conditions as previously described. PKG-1 activity was assayed by colorimetric analysis (CycLex) from RV and lung lysates as previously described.

Reactive oxygen species and nitrotyrosine analysis. Reactive oxygen species (ROS) generation was examined by several independent methods. Superoxide production in LV tissue homogenates was determined by luminol-enhanced chemiluminescence (EMD Biosciences). Flash-frozen RV myocardium was homogenized in iced PBS buffer and centrifuged, and the precipitate was resuspended in assay buffer to a final concentration of 100 µM luminol in accordance with the manufacturer's instructions. Phorbol-12-myristate-13-acetate or other oxidase stimulators were not used in the assay. Data were normalized by sample weight. In addition, fresh frozen LV myocardium (8-µm slices) was incubated for 1 h at 37°C with dihydroethidium (DHE) (2 µM; Invitrogen) assessing O2– formation (typically nuclear localization). Imaging was performed on a Zeiss inverted epifluorescence microscope (Carl Zeiss) attached to an argon-krypton laser confocal scanning microscope (UltraVIEW; Perkin Elmer Life Sciences). The excitation/emission spectrum for DHE was 488 and 610 nm, respectively, with detection at 585 nm, and for dichlorofluorescein was 480 and 535 nm, respectively, with detection at 505 nm. Nitrotyrosine was also quantitatively assessed by ELISA assay (Oxis International) as previously described.

Determination of NOS activity. Ca2+-dependent and -independent NOS activity was determined from myocardial homogenates by [3H]-L-arginine to [3H]-L-citrulline conversion (Sigma-Aldrich) as previously described (27).

Cardiac gelatinase analysis. In vitro gelatin lysis by matrix metalloproteinase (MMP)-2 and MMP-9 was assessed by zymography and by ELISA. Briefly, modified Laemmli buffer without mercaptoethanol was added to lysed tissue samples and loaded on a 10% gelatin (Invitrogen). After electrophoresis, gels were washed twice with renaturing buffer at room temperature followed by developing buffer (Invitrogen) and then stained to visualize lytic bands (SimplyBlue; Invitrogen). For quantification of MMP-9 and MMP-2, RV and lung lysates were processed using ELISA (R&D Systems, Minneapolis, MN).

RhoA and ROCK activity. RhoA activity (Cytoskeleton, Denver, CO) and ROCK activity (Cyclex) were assayed in whole RV and lung lysates by 96-well activity assays according to manufacturer specifications and read on a Molecular Devices M-5 microplate reader.

Lung hydroxyproline content. Lung collagen content was quantitated by measuring the total hydroxyproline content of the lung. Lung hydroxyproline concentration was determined spectrophotometrically according to the method of Kivirikko and colleagues (18a). Briefly, stored left lungs were homogenized in 5% trichloroacetic acid (1:9, wt/vol) and centrifuged for 10 min at 4,000 g. The pellet was then washed twice with distilled water and hydrolyzed for 16 h at 100°C in 6 N HCl. Hydroxyproline in the hydrolysate was assessed colorimetrically at 561 nm with p-dimethylaminobenzaldehyde. Hydroxyproline content was computed as micrograms of hydroxyproline per whole left lung and indexed to body weight. Results were expressed as the percent increase compared with control (saline-treated) values.

Statistical analyses. All data are presented as means ± SE. Data were analyzed using GraphPad Prism Plus software, version 5.1, using ANOVA with Tukey's post hoc test.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PDE5A inhibition reduces RV hypertrophy and mortality in bleomycin-induced lung injury. There was significant death associated with administration of intratracheal bleomycin in mice under control conditions. In contrast, concomitant administration of sildenafil (100 mg·kg–1·day–1 orally) and intratracheal bleomycin improved survival in the 2 wk following injury (Fig. 1A). Divergence of the curves occurred within 5 days of the bleomycin dose and was persistent over the remainder of the experiment. Survival was 73.7% in the group treated with bleomycin and 84.2% in the group cotreated with bleomycin and sildenafil (P < 0.05). After 2 wk of exposure to bleomycin, RV hypertrophy, as measured by the RV/LV+S ratio, was increased significantly in bleomycin-treated animals compared with controls (20.00 ± 0.03 vs. 33.0 ± 0.01, P < 0.05). RV hypertrophy did not occur with concomitant administration of sildenafil (Fig. 1); there was no significant difference between RV/LV+S ratio in sham-treated control mice vs. those treated with bleomycin and sildenafil [20.00 ± 0.03 vs. 20.98 ± 0.01, P = not significant (ns)]. Adverse mortality associated with bleomycin administration was reversed after institution of sildenafil therapy at day 8, with mortality in bleomycin group 63.64 ± 10.50 vs. 75.00 ± 13.06 (Fig. 1C; P < 0.05).


Figure 1
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  		Fig. 1. A: Kaplan-Meier survival curve animals treated with sildenafil and bleomycin (bleo + sild) vs. those treated with bleomycin alone (bleo; n = 57 each). B: RV/LV+S ratio is increased with bleomycin (Bleo, n = 6) compared with sham-treated wild-type (WT, n = 6) (P < 0.05), and this is ameliorated with sildenafil administration (Bleo + Sild, n = 13) (P < 0.05 Bleo vs. Bleo + Sild, P = ns Bleo + Sild vs. Con). *P < 0.05 vs. control, #P < 0.05 vs. Bleo alone. C: Kaplan-Meier survival curve in animals treated with bleomycin alone (bleo) vs. bleomycin and sildenafil 1 wk after bleomycin exposure (BleoSildRev) (P < 0.05 bleo vs. control, P = ns control vs. BleoSildRev, n = 57 control, 11 BleoSildRev). ns, Not significant.

 
PDE5A inhibition attenuates bleomycin-induced pulmonary vascular and RV fibrosis. Bleomycin administration induced significant fibrosis in the RV of exposed animals, and this fibrosis was attenuated by simultaneous sildenafil administration (Fig. 2A). Additionally, when lung tissue stained for smooth muscle actin and medial hypertrophy assessed, there was a statistically significant increase in medial thickness in bleomycin-treated tissue compared with sildenafil (17.72 ± 1.42 bleomycin vs. 14.3 ± 0.77 µm bleomycin + sildenafil, P < 0.05, Fig. 2, B and C).


Figure 2
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  		Fig. 2. Histology. A: bleomycin (Bleo) induced significant RV myocardial fibrosis as seen on this representative hematoxylin and eosin stain. This was ameliorated by concomitant sildenafil administration (Bleo+Sild). B: smooth muscle actinin stain for medial thickness, bleomycin alone at left, bleomycin + sildenafil at right. C: medial thickness of fully muscularized vessels (in µm) associated with an airway. P < 0.05 bleomycin compared with bleomycin + sildenafil.

 
PDE5A inhibition improves RV function and reduces RV afterload in bleomycin-induced PH. The effects of a 14-day exposure to bleomycin and to the combination of bleomycin and sildenafil on pulmonary vascular and RV hemodynamics were measured in the open chest, mechanically ventilated, and anesthetized animal (Fig. 3). Heart rate was not altered by bleomycin or bleomycin plus sildenafil. Bleomycin resulted in PH with increased mean pulmonary artery pressure, RV peak pressures, and effective arterial elastance (P < 0.05, bleomycin vs. control), and diminished RV function with increased RV end diastolic pressure (RV EDP) and decreased contractility index, ejection fraction, and cardiac output (P < 0.05 bleomycin vs. control). Additionally, Tau (time constant of isovolumic relaxation) was prolonged consistent with worsening relaxation (P < 0.05 vs. control).


Figure 3
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  		Fig. 3. In vivo hemodynamic measurements in sham-treated controls, bleomycin (Bleo), and bleomycin + sildenafil (Bleo + Sild). N = 7 each group. *P < 0.05 compared with WT, #P < 0.05 compared with bleomycin. RV EDP, RV end diastolic pressure; Tau, time constant; SW/PVA, stroke work/pressure-volume area.

 
Administration of sildenafil to bleomycin-treated mice resulted in diminished PH, as measured by RV peak pressure, effective arterial elastance, and mean pulmonary artery pressure (data not shown). However, the pulmonary vascular resistance did not completely normalize with sildenafil treatment (P > 0.05 bleomycin + sildenafil vs. control). After concomitant administration of sildenafil, markers of RV function, including RV EDP, ejection fraction, and cardiac output were improved and indistinguishable from normal controls (P < 0.05 bleomycin + sildenafil vs. bleomycin alone, P = ns bleomycin + sildenafil compared with control).

Impaired pulmonary arterial and RV coupling as demonstrated by stroke work/pressure-volume area and arterial elastance/slope of end-systolic pressure-volume relationship (Ea/Ees) was evident after bleomycin administration (P < 0.05), but improved with bleomycin and sildenafil together (P < 0.05 stroke work/pressure-volume area bleomycin + sildenafil vs. bleomycin and bleomycin + sildenafil vs. control, P < 0.05 Ea/Ees bleomycin + sildenafil vs. bleomycin, P = ns bleomycin + sildenafil vs. control; Fig. 3).

PDE5A/PKG pathway is altered by both bleomycin and bleomycin and sildenafil cotreatment in lung and RV. After 2 wk of bleomycin exposure, RV and lung tissue were harvested and homogenized, and PDE5A activity was measured (Fig. 4). PDE5A activity was higher in both the lung and the right ventricle after bleomycin administration. When downstream measures of cGMP activity were measured, PKG activity was found to be decreased with bleomycin exposure in the lung and increased in the RV (Fig. 4, P < 0.05 bleomycin vs. control). When these assays were repeated in bleomycin and sildenafil-treated animals, PKG activity was markedly increased in both tissues compared with control and bleomycin alone (P < 0.05).


Figure 4
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  		Fig. 4. A: phosphodiesterase type 5 (PDE5) activity assay in sham-treated controls vs. bleomycin in both lung and RV (*P < 0.05). B: PKG activity assay in lung (n = 6–8) and heart (n = 7–8) in sham-treated controls; bleomycin (Bleo) and bleomycin and sildenafil-treated animals (Bleo + Sild). *P < 0.05 compared with control, **P < 0.05 compared with Bleo.

 
Mediators of tissue damage after bleomycin and bleomycin and sildenafil cotreatment. Calcium-dependent and -independent NOS activity was measured in control, control and sildenafil, bleomycin, and bleomycin and sildenafil cotreated lung tissue to evaluate constitutive NOS activity (Fig. 5). Bleomycin profoundly decreased constitutive NOS (eNOS) activity and increased Ca2+-independent NOS activity [inducible NOS (iNOS), NOS2], thus preferentially promoting ROS generation (P < 0.05 vs. control). This reduction in eNOS activity was ameliorated with sildenafil cotreatment (P < 0.05 vs. bleomycin) with subsequently less ROS generation. To specifically determine the presence of ROS generation in the lung, a luminol activity assay was performed. Bleomycin generated a marked increase in superoxide formation, and this was abrogated with sildenafil (P < 0.05 vs. control, P < 0.05 bleomycin vs. bleomycin and sildenafil).


Figure 5
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  		Fig. 5. A: lung calcium-dependent and -independent nitric oxide synthase (NOS) activity in sham-treated controls, control animals fed a diet of sildenafil (control/sild), bleomycin (Bleo)-treated animals, and bleomycin and sildenafil (Bleo/Sild). *P < 0.05 compared with control, **P < 0.05 compared with Bleo. B, left: luminol activity assay to evaluate superoxide activity in lung of control, control and sildenafil (control/sild), bleomycin (Bleo), and bleomycin and sildenafil (Bleo/Sild). Activity is expressed in relative light units (RLU). *P < 0.05 compared with control, **P < 0.05 compared with Bleo. Right: luminol activity assay in bleomycin-treated lungs with coadministration of L-NAME, apocynin, oxypurinol, and SOD. *P < 0.05 compared with control. C: nitrotyrosine level in sham-treated control (Control), bleomycin (Bleo), and bleomycin with sildenafil (Bleo + Sild) in lung and RV. *P < 0.05 compared with control, **P < 0.05 compared with Bleo.

 
Potential mechanisms by which bleomycin increases superoxide activity was studied. Uncoupled NOS production of O2– was greatest since L-NAME blocked superoxide formation in bleomycin-treated lungs by >50% (P < 0.05). NADPH oxidase inhibition by apocynin and xanthine oxidase inhibition by oxypurinol blocked superoxide formation, suggesting a role for each of these enzymes in superoxide formation due to bleomycin (P < 0.05 vs. control). As an internal control, superoxide dismutase nearly abrogated superoxide formation in this luminol assay.

Nitrotyrosine levels, as a marker of oxidative stress (particularly by OONO–), were measured in lung and heart in control, bleomycin, and bleomycin + sildenafil animals (Fig. 5). Bleomycin effected a distinct increase in nitrotyrosine in bleomycin-treated animals (P < 0.05 vs. control), and this was partially improved with sildenafil therapy (P < 0.05 vs. bleomycin and vs. control).

The time course of iNOS and eNOS activation was studied (Fig. 6). There was a marked drop in calcium-dependent NOS activity in the first 3 days after bleomycin exposure that continued through day 14. There is an initial peak in iNOS activity, as measured by calcium-dependent citrulline formation, that remains elevated throughout the 14 days. There is a trend toward a difference in sildenafil and bleomycin treatment in iNOS activity. Furthermore, eNOS ELISA showed a 25% increase in eNOS protein at day 14 (data not shown).


Figure 6
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  		Fig. 6. Time course of iNOS and eNOS activity after bleomycin exposure as measured by calcium-dependent and calcium-independent citrulline formation in myocardial homogenates.

 
MMP-2 and -9 activity were profoundly upregulated by bleomycin, and this was attenuated by cotreatment with sildenafil (P < 0.05 bleomycin vs. control, bleomycin vs. bleomycin and sildenafil; Fig. 7).


Figure 7
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  		Fig. 7. Matrix metalloproteinase (MMP)-9 and MMP-2 activity assay measured in relative units in lung and RV of sham-treated control (Control), bleomycin (Bleo), and bleomycin with sildenafil (Bleo + Sild). *P < 0.05 compared with control, **P < 0.05 compared with Bleo. A representative zymogram is shown below. Lanes are from left to right: standard, bleo lung x2, bleo + sild lung x3, bleo RV x2, bleo + sild RV x3.

 
PDE5A inhibition modulates RhoA/Rho kinase pathway. RhoA and ROCK activity was measured in RV and lung tissue after bleomycin or bleomycin and sildenafil (Fig. 8). In this series of experiments, sham-treated animals were fed a diet of vehicle or sildenafil as well as bleomycin and bleomycin and sildenafil cotreatment. Both RhoA and ROCK activity were unaltered by sildenafil alone (P < 0.05 vs. control) but markedly higher in mice treated with bleomycin (P < 0.05 vs. control). PDE5A inhibition decreased the activity of RhoA and ROCK in the face of bleomycin exposure (P < 0.05 vs. bleomycin and vs. control).


Figure 8
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  		Fig. 8. A: RhoA activity assay in sham-treated control (Control), control with sildenafil (control/sild), bleomycin (Bleo), and bleomycin with sildenafil (bleo/sild). *P < 0.05 compared with control, **P < 0.05 compared with Bleo. B: ROCK activity assay in sham-treated control (Control), control with sildenafil (control/sild), bleomycin (Bleo), and bleomycin with sildenafil (bleo/sild). *P < 0.05 compared with control, **P < 0.05 compared with Bleo.

 
Lung and RV fibrosis is induced by bleomycin and attenuated by concomitant PDE5A inhibition. Hydroxyproline content was measured in lungs after bleomycin exposure and after coadministration of sildenafil (Fig. 9). There was a marked increase in collagen content after bleomycin, and this was attenuated by sildenafil (P < 0.05 bleomycin vs. control, bleomycin vs. bleomycin + sildenafil). These findings were recapitulated in the RV tissue as well.


Figure 9
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  		Fig. 9. Fibrosis (as a percent of control) and hydroxyproline content in both RV and whole lung homogenates. *P < 0.05 vs. control, **P < 0.05 compared with bleomycin alone.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we show that PDE5 inhibition ameliorates RV hypertrophy and fibrosis as well as pulmonary vascular changes associated with intratracheal bleomycin. Furthermore, we showed that PDE5 inhibition in the bleomycin model is associated with improved NOS coupling and a reduction in ROS signaling. Moreover, this is the first study, to our knowledge, to demonstrate that bleomycin-induced fibrosis of the lung and right ventricle is associated with alteration of the RhoA/Rho kinase pathway and that this aberration can be ameliorated by PDE5 inhibition.

Recent studies have shown the antihypertrophic effect of PDE5 inhibition on the cardiac myocyte in vivo and in cultured myocytes (36); however, these investigations have focused on the left ventricle, which has been shown to be functionally and molecularly distinct from the right ventricle (23, 39). Given the high concentration of PDE5 in the mammalian lung and the importance of PDE5 in the stressed human heart, it is logical that PDE5 inhibition affects both organs concomitantly in the bleomycin model.

Bleomycin has been shown in other studies to be an important effector of PH and RV hypertrophy in multiple vertebrate animals (2, 9, 29, 40). Additionally, we have shown that transfer of the eNOS gene to the pulmonary parenchyma attenuates bleomycin-induced PH. PDE5A inhibitors such as sildenafil increase levels of cGMP, the downstream messenger of nitric oxide, thereby enhancing its effects. Administration of PDE5A inhibitor is a convenient, safe method of achieving a similar effect as eNOS gene transfer. Indeed, we were able to show improvement in pulmonary vascular remodeling associated with bleomycin and ameliorated RV hypertrophy, fibrosis, and ultimately mortality, similar to findings in the eNOS transfer model (2). Importantly, the mortality benefit was sustained in reversal as well as prevention experiments, suggesting a possible clinical application, as it is often difficult to capture early pulmonary fibrosis. While mortality in our experiments was somewhat lower than others have published, this may be accounted for by gender differences in bleomycin response or dose or dosing technique (25). These data, together, suggest that PDE5A inhibition has important pulmonary vascular and RV effects in pulmonary fibrosis-associated PH.

The importance of oxidant stress in bleomycin-induced lung injury has been well documented (13, 22), as the administration of the antioxidants superoxide dismutase or N-acetyl-L-cysteine has been shown to partially reverse lung injury (16, 37). Furthermore, increased myocardial oxidative stress by enhanced ROS generation has been shown to mediate myocyte hypertrophy stimulated by angiotensin II and TNF, and we and others have previously shown the angiotensin and TNF pathways to be important mediators in the pathology of bleomycin-induced lung pathology (24, 29). Among the potential mechanisms linking ROS to hypertrophy and remodeling are its posttranslational stimulation of MMPs that facilitate further structural remodeling and are linked to fibrosis and matrix turnover involving the activation of MMPs (35). In mice treated with the antioxidant dimethylthiourea (18) or overexpressing glutathione peroxidase (34), remodeling after myocardial infarction is reduced and accompanied by diminution of MMP abundance (34). The present data support these findings in that both MMP-9 and MMP-2 activation occurred in bleomycin-treated mice. MMP-9 has also been shown to play a key role in early stages of vascular remodeling in response to hypertensive distending pressures (20).

Although the role of ROS in hypertrophic signaling is increasingly accepted, its sources remain unclear. One candidate is NADPH oxidase, first shown to play a central role in ROS generation in vascular endothelium in response to mechanical (e.g., hypertension) and humoral (e.g., angiotensin II) stimulation (10, 11). Investigations showing that NADPH-dependent ROS generation rose in response to pressure overload and LV decompensation focused interest on these enzymes in the heart as well (21). Another alternative source is xanthine oxidase, and although xanthine oxidase inhibition blunts postinfarction chamber remodeling (6), its role in bleomycin-induced PH and RV failure remains unclear. Inducible NOS (NOS2) also can contribute to oxidant stress, and iNOS activity was increased modestly with bleomycin treatment as shown in Fig. 6, but this did not appear important for the beneficial effect of sildenafil in the current studies, because Ca2+-independent NOS activity was relatively low and unaltered by sildenafil treatment. This was also true for both the xanthine oxidase (XO) and NADPH pathways as well since an inhibitor of NADPH (apocynin) and the XO inhibitor (oxypurinol) resulted in statistically significant, but modest, reductions in luminol activity. In contrast, the NOS inhibitor, L-NAME, resulted in a ~50% reduction in luminol chemiluminescence after bleomycin treatment. Under control conditions, L-NAME blocks <20% of luminol activity in both the heart and the lung (data not shown). Additionally, our evaluation of iNOS and eNOS activation time course suggests that sildenafil treatment preserves eNOS activity after bleotreament and may have an effect on quelling iNOS activity in the acute inflammatory phase after bleomycin treatment.

The notion that NOS3 might serve as a dominant source for ROS is supported by prior work conducted in vascular tissue. NOS function is beneficial under normal conditions, but its exposure to mechanical/oxidant stress can shift it to a ROS generator contributing to vascular disease. For example, deoxycorticosterone acetate-salt-induced hypertension stimulates endothelial-dependent ROS generation that is markedly blunted by L-NAME and absent in vessels from mice lacking NOS3 (19). ROS generation in this model may be associated with BH4 oxidation, resulting in NOS uncoupling, and NADPH oxidases may provide an important initial trigger to this change. NOS uncoupling also occurs directly by oxidation of the zinc-thiolate complex, as shown by Zou et al. (43) in studies employing ONOO–.

The RhoA/Rho kinase pathway has been linked to ROS signaling (38, 41) and shown to be important in maintenance of vascular tone in PH where Rho kinase inhibitors, such as fasudil, have been used in animal models and humans with PH (14, 15, 26, 27). Furthermore, the RhoA/Rho kinase pathway has been shown to be affected by sildenafil in pulmonary vessels in the chronic hypoxia model of PH (32, 33). It is known that there are multiple levels of the RhoA/ROCK pathway that can be inhibited by the cGMP/PKG system. Since RhoA/ROCK signaling is also linked to ROS signaling, we hypothesized that the RhoA/Rho kinase pathway is activated by bleomycin in both lung and right ventricle and that these alterations would be potentially improved by sildenafil coadministration via a PKG-mediated process. Our results show that RhoA activity in lung is increased by bleomycin exposure, which concurs with what has been shown in the chronic hypoxia model and concurs with the increased vascular tone described in PH (28). Although the RhoA/Rho kinase pathway is known to be important in LV hypertrophy, there is evidence that the right and left ventricles are functionally and biochemically distinct (1, 23, 30, 39). Here we demonstrate activation in RV RhoA and ROCK activity following bleomycin administration. As was found in the lungs, this activation was ameliorated by sildenafil administration.

Interestingly, both ROS signaling and RhoA/ROCK activation occurs in both the lungs and the RV with bleomycin treatment, suggesting a common pathway in bleomycin-induced pulmonary fibrosis and RV failure from PH. Our data suggest that in the bleomycin pulmonary fibrosis model in which NOS uncoupling is associated with ROS generation resulting in both ROCK activation and increased MMP activity, sildenafil may directly affect both RhoA/ROCK and ROS, and, in an additive manner, the improvement in ROS may inactivate RhoA.

It is impossible to completely segregate the effects of sildenafil on the pulmonary vasculature from those in the right ventricle in this animal model; however, this is the only model currently available for the study of interstitial lung disease-associated PH, and the model does closely resemble clinical disease, where the right ventricle and pulmonary vasculature are integrally linked. Additionally, there is recent evidence that afterload induced heart failure, and hypertrophy can be significantly reduced even in the setting of increased afterload.

In summary, the results of the present study show that PH due to bleomycin exposure is associated with the activation of the RhoA/Rho kinase pathway in both the right ventricle and the pulmonary vasculature. We show improvement in mortality, RV hypertrophy, and in vivo hemodynamics after sildenafil administration. Moreover, the RhoA and ROCK changes seen in the lung and right ventricle with bleomycin exposure are improved with sildenafil, thereby linking the pathways in both tissues. These results have important clinical applications to all types of pulmonary fibrosis-associated PH and may point to novel targets for treatments of both disorders simultaneously. Further research is needed to determine the utility of PDE5A inhibitors and Rho kinase inhibitors alone or in conjunction in treatment of PH associated with acute and chronic interstitial lung disease.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by the Bernard A. and Rebecca S. Bernard Foundation, a scientist development grant from the American Heart Association, and the W. W. Smith Foundation. A. R. Hemnes' work is supported by National Heart, Lung, and Blood Institute Grant 7 F32 HL-82132-02.


   ACKNOWLEDGMENTS
 
H. C. Champion is a recipient of the Shin Chun-Wang Young Investigator Award and the Giles F. Filley Memorial Award from the American Physiological Society.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. C. Champion, Johns Hopkins Univ. School of Medicine, 720 Rutland Ave., Ross 850, Baltimore, MD 21205 (e-mail: hcc{at}jhmi.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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