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Bae HK, et al. • Korean J Pediatr 2016;59(6):262­270http://dx.doi.org/10.3345/kjp.2016.59.5.262pISSN 1738-1061•eISSN 2092-7258 The effect of sildenafil on right ventricular remo­ deling in a rat model of monocrotaline­induced right ventricular failureHyun Kyung Bae, MD1, Hyeryon Lee, PhD1, Kwan Chang Kim, MD, PhD2, Young Mi Hong, MD, PhD1Departments of 1Pediatrics, 2Thoracic and Cardiovascular Surgery, Ewha Womans University School of Medicine, Seoul, Korea Purpose: Pulmonary arterial hypertension (PAH) leads to right ventricular failure (RVF) as well as an Corresponding author: Young Mi Hong, MD increase in pulmonary vascular resistance. Our purpose was to study the effect of sildenafil on right Department of Pediatrics, Ewha Womans Univer- ventricular remodeling in a rat model of monocrotaline (MCT)-induced RVF.
sity School of Medicine, 1071 Anyangcheon-ro, Yangcheon-gu, Seoul 07985, Korea Methods: The rats were distributed randomly into 3 groups. The control (C) group, the monocrotaline Tel: +82-2-2650-2841 (M) group (MCT 60 mg/kg) and the sildenafil (S) group (MCT 60 mg/kg+ sildenafil 30 mg/kg/ Fax: +82-2-2653-3718 day for 28 days). Masson Trichrome staining was used for heart tissues. Western blot analysis and immunohistochemical staining were performed. Received: 14 March, 2016 Results: The mean right ventricular pressure (RVP) was significantly lower in the S group at weeks Revised: 25 April, 2016 1, 2, and 4. The number of intra-acinar arteries and the medial wall thickness of the pulmonary Accepted: 10 May, 2016 arterioles significantly lessened in the S group at week 4. The collagen content also decreased in heart tissues in the S group at week 4. Protein expression levels of B-cell lymphoma-2 (Bcl-2)-associated X, caspase-3, Bcl-2, interleukin (IL)-6, matrix metal oproteinase (MMP)-2, endothelial nitric oxide synthase (eNOS), endothelin (ET)-1 and ET receptor A (ERA) in lung tissues greatly decreased in the S group at week 4 according to immunohistochemical staining. According to Western blotting, protein expression levels of troponin I, brain natriuretic peptide, caspase-3, Bcl-2, tumor necrosis factor-α, IL-6, MMP-2, eNOS, ET-1, and ERA in heart tissues greatly diminished in the S group at week 4. Conclusion: Sildenafil al eviated right ventricular hypertrophy and mean RVP. These data suggest that sildenafil improves right ventricular function.
Key words: Sildenafil citrate, Pulmonary hypertension, Gene expression Pulmonary arterial hypertension (PAH) affects all ages from newborns to adults. PAH leads to dysfunctions of both pulmonary vasculature and the heart. Chronic PAH results in significant peripheral and proximal arterial remodeling and right ventricular failure (RVF)1). The pathophysiology in PAH is vasoconstriction from endothelial dysfunction, and an imbalance of vasoactive mediators. Remodeling of the pulmonary artery happens from an imbalance in the cell proliferation and apoptosis in the pulmonary vessel2). Right ventricle (RV) adaptation and ventricular remodeling occurs after changes in Copyright 2016 by The Korean Pediatric Society pulmonary vasculature. Right ventricle hypertrophy (RVH) follows PAH because of compensatory mechanisms to the increased afterload. However, persistent overload results This is an open-access article distributed under the terms of the Creative Commons Attribution Non- in RV dysfunction and failure3). Commercial License (http://creativecommons.org/ Ventricular pressure-volume relationships, changes in wall thickness and geometry licenses/by-nc/4.0/) which permits unrestricted non- commercial use, distribution, and reproduction in any are included in RV remodeling. Increased myocyte number, dimension and myocardial medium, provided the original work is properly cited.
Korean J Pediatr 2016;59(6):262-270 extracellular matrix can also be noted4). RV remodeling is con- (MCT) group of subcutaneous injection of MCT (60 mg/kg), S nected with alteration in myocardial collagen content5). The (sildenafil, 30 mg/kg) group of daily gavage feeding of sildenafil larger amount of collagen is likely due to increased RV afterload for 28 days after MCT injection. We sacrificed 6 rats per each as well as the paracrine factors to provoke RVH5). The levels group at weeks 1, 2, and 4. of tumor necrosis factor (TNF)-α and collagen are increased in Protocol approval was received by the Institutional Animal RVF6). Increased gene expressions of fetal contractile proteins and Care and Use Committees of Ewha Womans University the School collagen were noted in the RV of rats7). of Medicine of (approval number: 13-0226).
RV dilatation has been correlated with higher rates of apop- tosis8). The primary crucial factor in the development of RVF is We weighed rats and monitored for general activity throughout Pharmacotherapy for PAH has been developing for the last the research period. The wet weights of RV, left ventricle (LV)+ 10 years to prevent the progress of the disease. Recently, there septum (S) and lung were gauged and RV to LV+S ratio [RV/ has been much interest in pulmonary vasodilators in therapies (LV+S)] was calculated for an index of RVH. for PAH with heart failure. Inhaled nitric oxide (NO), endothelin antagonists and phosphodiesterase (PDE)-5 inhibitors have 3. Estimation of hemodynamics usually been used as a vasodilator in PAH patients9). We put the animals in the supine position with an arterial Sildenafil is a selective agent that relaxes pulmonary vascu lar pressure line (Physiological Pressure Transducer, MLT1199; AD smooth muscle by inhibiting cyclic guanosine monophosphate Instruments, Oxfordshire, UK). We inserted catheter in the ex ternal (cGMP) specific PDE. Decrease in pulmonary vascular resistance jugular vein to measure mean RVP. After estimating RVP, we has been noted in sildenafil treatment in PAH10). Some research measured pressure in the external carotid artery. Hemo dynamic has shown that sildenafil improves endothelial function11) and parameters were measured at weeks 1, 2, and 4.
reduces the pro-inflammatory cytokines such as TNF-α and interleukin (IL)-612) and inhibits the apoptosis progress in the PAH 4. Histopathologic analysis of pulmonary arteries Neutral buffered formalin (10% formalin in 0.08M sodium However, the mechanisms of the effect of sildenafil on PAH phosphate, pH 7.4) was used for lung tissue fixation before patients associated with congestive heart failure (CHF) are not paraffin embedding. Three-μm-thick sections were made with completely understood9,14). Sildenafil as a vasodilator may lead Victoria blue staining. We captured more than 20 images of pul- to decreased vascular pressure and tone in patients with CHF monary arterioles per tissue section (diameter, 50–160 μm) at a since vasoconstriction is contributed to an increase in ventricular magnification of ×400 using a microscopic digital camera and filling pressure and pulmonary venous pressure15). Sildenafil analysis program (analySIS, Olympus Soft Imaging Solutions, decreases pulmonary arteriolar resistance and pressure in CHF Singapore). We measured the medial wall thickness between the by increasing NO availability because the defective release of NO internal and external elastic lamina from two sides of muscular may be a mechanism for the constriction of pulmonary vessels16).
arteries (M1 and M2). The wall thickness was calculated as There has been little research about the functional and struc- follows: % wall thickness=(M1+M2)/diameter×100. The number tural changes of RV in PAH. The effects of current PAH therapies of intra acinar arteries was counted. A total of randomly selected on the RV have not been thoroughly understood17).
microscopic fields per tissue section at a magnification (×200) The object of this research is to evaluate the effects of RV re- were analyzed.
modeling after sildenafil treatment in a rat model of monocrotaline The heart tissues were stained with Masson's trichrome stain- (MCT)-induced RVF.
ing. This stain was used for distinguishing collagen from mu-scle tissues. Collagen contents were quantified using Image J developed by the National Institutes of Health.
Materials and methods
5. Immunohistochemistry We sliced formalin-fixed 4-μm section from paraffin embedded Six-week-old male Sprague-Dawley rats with a weight of 200– tissue blocks and then deparaffinizing with zylene and rehydrating 290 g, were used. All rats were housed in individual cages under by serial dilutions of alcohol (70%–100%) were done. Heat anti- standard conditions. RVF was revoked by subcutaneous injection gen retrieval was achieved at 100°C for 10 minutes in micro wave of 60 mg/kg MCT (Sigma Chemicals, St. Louis, MO, USA) melted before incubation at 4°C overnight.
in 0.5 N HCl solution. Primary antibodies were used for B-cell lymphoma-2 (Bcl- The rats were divided into three groups: C (control) group, M 2)-associated X (Bax), caspase-3, Bcl-2, TNF-α, IL-6, matrix Bae HK, et al. • The effect of sildenafil on right ventricular remodeling metalloproteinase (MMP)-2, endothelial nitric oxide synthase 7. Statistical analysis (eNOS), endothelin receptor A (ERA) from Santacruz Biotechno- Results were expressed as the mean±standard deviation. A logy, Santa Cruz (Santa Cruz, CA, USA) and endothelin (ET)- Kruskal-Wallis test was used for the comparison of differences 1 from Abcam (Cambridge, UK). Slides were incubated with the in the three groups and a Mann-Whitney test was used for biotinylated secondary antibodies for 30 minutes at 4°C and then between groups comparisons with Bonferroni correction. P value with a streptavidin (Dako, Kyoto, Japan). Color development was of <0.05 was considered statistically significant. SPSS ver. 14.0 accomplished using 3-amino-9-ethylcarbazole or DAB as a chro- (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses. mogen. Densities were evaluated by using Image J and ex pressed To determine whether immunohistochemical staining were in arbitrary units. statistically significant, we randomly selected five areas in each lung region of each group and measured mean staining densities. 6. Western blot analysis The tissue was homogenized and centrifuged. The supernatant was used for sodium dodecyl sulfate polyacrylamide gel electro- phoresis. The proteins on the acrylamide gel were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, 1. Hemodynamic data MA, USA) and the membranes were incubated at 4°C overnight The mean RVP was greatly higher in the M group in compa- with the appropriated primary antibodies for troponin I, brain rison with the C group at week 1 (n=6 per each group, 17.00±0.00 natriuretic peptide (BNP), caspase-3, Bcl-2, TNF- α, IL-6, MMP-2, mmHg vs. 8.50±0.71 mmHg, P<0.05), at week 2 (n=6 per each eNOS, ERA from Santacruz Biotechnology and ET-1 from Abcam. group, 25.33±1.53 mmHg vs. 11.00±1.00 mmHg, P<0.05), and at Then, the membrane was incubated with horseradish peroxidase- week 4 (n=6 per each group, 39.67±8.50 mmHg vs. 10.00±0.00 conjugated secondary antibodies for 1 hour at room temperature. mmHg, P<0.05). RVP was significantly reduced in the S group After the washing process the membrane was measured by a in comparison with the M group at week 1 (n=6 per each group, chemilu minescent reaction using an electrochemiluminescence- 13.00±1.00 mmHg vs. 17.00±0.00 mmHg, P<0.05), at week 2 detection kit system from GE Healthcare (Amersham Bioscience, (n=6 per each group, 13.00±1.00 mmHg vs. 25.33±1.53 mmHg, Piscataway, NJ, USA). The protein content was calculated with a P<0.05) and at week 4 (n=6 per each group, 13.07±1.53 mmHg Molecular Devices ELISA reader (Sunnyvale, CA, USA). Western vs. 39.67±8.50 mmHg, P<0.05) (Table 1). There was no significant blotting band intensity values were normalized by β-actin change in aorta pressure (Data is not shown).
Table 1. The changes of right ventricular pressure at weeks 1, 2, and 4
The RV/BW ratio in the M group was greatly higher in com- parison with the C group at week 2 (n=6 per each group, 0.81±0.08 Monocrotaline (M), g vs. 0.58±0.02 g, P<0.05) and at week 4 (n=6 per each group, 1.76±0.14 g vs. 0.62±0.06 g, P<0.05). The RV weight in the S group was greatly reduced in comparison with the M group at week 4 (n=6 per each group, 1.36±0.30 g vs. 1.76±0.14 g, P< 0.05). The RV/LV+S showed significant increase in M group Values are presented as mean±standard deviation.
in comparison with C group at week 2 (n=6 per each group, *P<0.05: C vs. M at the corresponding time point. †P<0.05: M vs. S at the corresponding time point.
0.41±0.06 g vs. 0.30±0.02 g, P<0.05) at week 4 (n=6 per each Table 2. The changes of body and organ weight at weeks 1, 2, and 4 in 3 groups
Week
Values are presented as mean±standard deviation.
BW, body weight; RV, right ventricle; LV, left ventricle; S, septum; C, control; M, monocrotaline; S, sildenafil.
*P<0.05: C vs. M at the corresponding time point.

Korean J Pediatr 2016;59(6):262-270 group, 0.75±0.06 g vs. 0.32±0.03 g, P<0.05) after MCT injection. group, 2.01±0.30 vs. 1.24±0.11, P<0.05). There was a significant Although the RV/LV+S in the S group was lowered in comparison reduction of the number of intra acinar arteries in the S group with the M group, the result was not statistically significant (Table in comparison with the M group at week 2 (n=6 per each group, 1.10±0.13 vs. 1.86±0.24, P<0.05) and at week 4 (n=6 per each group, 1.13±0.41 vs. 2.01±0.30, P<0.05) (Fig. 1C).
3. Histopathological changes in the lung tissues Fully muscularized arteries were seen in the pulmonary ar- 4. Histopathological changes in the heart tissues terioles (>10μM, <100μM) in the M group and S group at weeks Since the Masson's Trichrome staining marks collagen-rich 2 and 4. Victoria blue staining identified inner and outer elastic area in blue, the collagen-rich area was well visualized at week 2 layers of arteries (Fig. 1A).
and 4 (Fig. 2A) in each group. Collagen content (%) in the heart The medial wall thickness (%) of pulmonary arterioles had tissues significantly expanded in the M group in comparison increased significantly in the M group in comparison with the with the C group at week 2 (n=6 per each group, 19%±2.55%, vs. C group at week 2 (n=6 per each group, 44.99±4.84 vs. 25.71± 8.5%±1.41%, P<0.05) and at week 4 (n=6 per each group, 30%± 7.85, P<0.05), at week 4 (n=6 per each group, 42.35±1.94 vs. 3.54% vs. 11%±2.12%, P<0.05) after MCT injection and signifi- 19.93±3.64, P<0.05). There was a significant decrease in the S cantly reduced in the S group at week 4 (n=6 per each group, group in comparison with the M group at week 2 (n=6 per each 25%±2.83 % vs. 30%±3.54%, P<0.05) (Fig. 2B).
group, 35.41±3.13 vs. 28.44±4.61, P<0.05) and at week 4 (n=6 per each group, 33.75±0.80 vs. 42.35±1.94, P<0.05) (Fig. 1B). 5. Immunohistochemical staining in lung tissues The number of intra-acinar arteries significantly increased The immunohistochemical staining assay by arbitrary unit in the M group in comparison with the C group at week 1 (n=6 revealed significantly enhanced expression of Bax, Caspase-3, per each group, 1.48±0.16 vs. 1.44±0.29, P<0.05), at week 2 Bcl-2, IL-6, MMP-2, eNOS, ET-1, ERA in the M group compared (1.86±0.24 vs. 1.23±0.08, P<0.05) and at week 4 (n=6 per each with the C group at week 4. In contrast, Bax, Caspase-3, Bcl-2, Fig. 1. Pulmonary arterioles stained with Victoria blue at the magnification of ×400 (A), medial wall
thickness (B), and the number of intra-acinar arteries (C) in the 3 groups. The medial wall thickness of pulmonary arterioles significantly decreased in the S group in comparison with the M group at weeks 2 and 4 (panels A and B). There was a significant reduction in the number of intra-acinar arteries in the S group in comparison with the M group at weeks 2 and 4 (panel C). C, control; M, monocrotaline; S, sildenafil. *P<0.05: C vs. M at the corresponding time point. †P<0.05: M vs. S at the corresponding

Bae HK, et al. • The effect of sildenafil on right ventricular remodeling Fig. 2. Masson's trichrome staining of the heart tissues (X400) (A) and collagen content (B) in the 3
groups. The collagen-rich area of heart tissue was visualized well at weeks 2 and 4 (panel A) in each group. In comparison with group M, col agen content (%) of heart tissues significantly decreased in the S group after 4 weeks (panel B). C, control; M, monocrotaline; S, sildenafil. *P<0.05: C vs. M at the correspond ing time point. †P<0.05: M vs. S at the corresponding time point.
Fig. 3. Immunohistochemical staining of lung tissues (A) and relative density (B) in 3 groups at week 4.
The immunohistochemical staining assay by arbitrary unit revealed significantly reduced expressions of Bax, Caspase-3, Bcl-2, TNF-α, IL-6, MMP-2, eNOS, ET-1, and ERA in the S group compared with the M group at week 4 (×400). Bax, Bcl-2-associated X; Bcl, B-cell lymphoma; IL, interleukin; MMP, matrix metalloproteinase; eNOS, endothelial nitric oxide synthase; ET, endothelin; ERA, endothelin receptor A; C, control; M, monocrotaline; S, sildenafil. *P<0.05: C vs. M at the corresponding time point. †P<0.05: M vs. S at the corresponding time point.
IL-6, MMP-2, eNOS, ET-1 and ERA significantly reduced in the S MMP-2 (Fig. 4G), eNOS (Fig. 4H), ET-1 (Fig. 4I) and ERA (Fig. 4J) group at week 4 (Fig. 3A, B).
in the M group were significantly increased in comparison with the C group at week 4. Protein expressions of troponin I (Fig. 4A), 6. Western blot analysis BNP (Fig. 4B), Caspase-3 (Fig. 4C), Bcl-2 (Fig. 4D), TNF-α (Fig. 4E), In western blot analyses of heart tissue, band intensity was IL-6 (Fig. 4F), MMP-2 (Fig. 4G), eNOS (Fig. 4H), ET-1 (Fig. 4I) and normalized to the expression of the β-actin protein at each ERA (Fig. 4J) in heart tissues significantly lessened in the S group week. Protein expressions of troponin I (Fig. 4A), BNP (Fig. 4B), caspase-3 (Fig. 4C), Bcl-2 (Fig. 4D), TNF-α (Fig. 4E), IL-6 (Fig. 4F), There was an unexpected increase in troponin I, BNP, TNF-α,

Korean J Pediatr 2016;59(6):262-270 Troponin-I
Caspase-3
Bcl-2 0.4
β-actin
β-ac 100
Fig. 4. (A-K) Western blot analysis in the heart tissue at weeks 1, 2, and 4. Protein expressions of troponin I (A), BNP (B), caspase-3 (C), Bcl-
2 (D), TNF-α (E), IL-6 (F), MMP-2 (G), eNOS (H), ET-1 (I), and ERA (J) in the heart tissues significantly lessened in the S group in comparison with the M group at week 4. BNP, brain natriuretic peptide; Bcl, B-cell lymphoma; TNF, tumor necrosis factor; IL, interleukin; MMP, matrix metalloproteinase; eNOS, endothelial nitric oxide synthase; ET, endothelin; ERA , endothelin receptor A; C, control; M, monocrotaline; S, sildenafil. *P<0.05: C vs. M at the corresponding time point. †P<0.05: M vs. S at the corresponding time point.
Bae HK, et al. • The effect of sildenafil on right ventricular remodeling and IL-6 protein expression levels in the C group. The small sildenafil administrations in MCT rats. The protein expressions number of animals causes the unexpected increase in protein related to endothelial cell dysfunction such as eNOS, ET-1, and expression levels because of individual differences. ERA were significantly reduced after sildenafil treatment. ET and RV dysfunction (BNP), apoptosis (caspase-3, Bcl-2), inflamma- ERA have been noted to affect tissue remodeling. MMP-2 is an tion (TNF-α, IL-6) and endothelial dysfunction (eNOS, ET-1, enzyme that degrades extracellular matrix. ET-1 plays a role as a ERA) associated genes were significantly reduced after sildenafil vasoconstrictor and enhances cell proliferation and fibrogenesis treatment at week 4.
by controlling MMP19). In our current research, the MMP-2 ex-pression was augmented in the M group and lessened in the S group. NO, produced by endothelial NO synthase, acts as a vasodilator in pulmonary vasculature. In some reports, the overproduction In this research, RVF and PAH were developed by intraperi- of eNOS in mice models prevents hypoxia-induced PAH20) and toneal injection of MCT, which is selectively toxic to the endothe- severe PAH was seen in eNOS deficient mice exposed to mild lium of the pulmonary artery and causes PAH from 1 week after hypoxia21). On the other hand, there is a report that sildenafil ele- injection. We were able to confirm this by increased RVP and vates the cGMP in target cells and then decreases NOS activity RV/LV+septum. RVF was confirmed by signs such as tachypnea, and plasma NO level22). In our results, eNOS protein expressions significant edema and ascites.
were significantly increased in the M group compared with the C RVH is the underpinning of the functional and structural group and decreased after sildenafil treatment at week 4. changes in RV remodeling. RV muscle mass is more susceptible Inflammation and apoptosis are important mechanisms in the to changes in afterload5). The functional and structural changes of molecular change of the RVF in PAH. Increased inflammatory cardiomyocytes happen because of stress on the myocardiocytes. cytokines are known to increase pulmonary circulation and dilate Myocardiocytes under stress have a long contraction time and RV of PAH patients3). In this study, the expressions of IL-6 and increased wall stress, following ventricular remodeling, con- TNF-α increased in MCT induced RVF rats and decreased after tractile dysfunction and ventricular enlargement with pressure sildenafil treatment. In addition, the failing RV may be subject to overload. Myocardiocyte changes include lengthened RV myo- cardiomyocyte apoptosis13). There was also the result that the ex- cyte diameter, expanded mean myocardial cell volume, elongated pressions of apoptosis related proteins, including caspase-3, bcl-2 myocyte length with shrunk cross-sectional areas and extended augmented in the M group and reduced in the S group. Induction extracellular space5). of apoptosis is another example of the cellular changes associated However, the critical mechanisms of change from hypertrophy with RV remodeling. Bussani et al.8) compared apop tosis in myo- to dilatation to cause RVF in PAH have not been well investi- cardium with the degree of unfavorable cardiac remodeling. gated3,18). Although incremented afterload is the first causal factor Apop tosis may be the primary critical factor to cause biventricular for RV adaptation in PAH17) neurohormonal signaling, oxidative stress, inflammation, ischemia, apoptosis and endothelial In our research, protein expressions of troponin I and BNP in dysfunction may induce the development of RV dilatation and the heart tissues significantly decreased at week 4 after sildenafil treatment. The troponin is one of the myocardial regulatory In our current research, increased inflammation (TNF-α and proteins and may be related to the pathobiology of heart failure IL-6 expressions), apoptosis abnormality (caspase-3 and Bcl- 23,24). Contractile dysfunction in heart failure is related to changes 2 expressions), endothelial dysfunction (eNOS, ET-1, and ERA in regulatory and contractile and protein expression.
expressions) and cardiac dysfunction (BNP and Troponin I Ventricular BNP level is upregulated in cardiac failure and expressions) were found in our MCT induced RVF model. locally in the area around a myocardial infarct. BNP is valuable Collagen change has also been reported as an important patho- in the diagnosis and prognosis of heart failure. It is regarded physiology in RV remodeling. Heart failure related to myocardiac to be the best biomarker in heart failure25). BNP is released by infarction in a rat model showed an increased level of collagen myocardial stretch from RV overload.
gene expressions in the RV7). The increased levels of RV collagen Hemodynamic effect of PDE-5 inhibitor on RVF with PAH has and TNF-α were seen in failing hearts of patients with end-stage been recently found26). PDE-5 inhibitors may contribute to sup- car diomyopathy6). press RVH and improve RV function as well as decrease the RV In our study, collagen content in the heart tissues significantly afterload27). PDE-5 has been reported to be prominently upre- increased at weeks 2 and 4 in MCT rats and significantly de- gulated in RVH myocardium4). creased after sildenafil treatment.
Sildenafil as a selective PDE-5 inhibitor is also known to pre- We investigated changes of RV protein expression levels after vent RV remodeling by a complex mechanism like modulating Korean J Pediatr 2016;59(6):262-270 cGMP and calcium signaling28). Sildenafil increases cardiac tute (2014) and Basic Science Research Program through the index26) and prevents progressive chamber dilation, dysfunction National Research Foundation of Korea (NRF) funded by the and fibrosis of the heart with pre-existing hypertrophy and forces Ministry of Education (2013R1A1A3004619).
the contractility of cardiomyocytes28). Sildenafil has also been reported to decrease pulmonary vascular resistance (PVR) and mean pulmonary artery pressure in patients with heart fail ure and PAH29). In our study, there was a significant decrease of RVP after sildenafil treatment. There 1. Chesler NC, Roldan A, Vanderpool RR, Naeije R. How to measure were also improvements in RVH by estimating the decrease of pulmonary vascular and right ventricular function. Conf Proc IEEE Eng Med Biol Soc 2009;2009:177-80.
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Conflict of interest
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