Genistein, a Phytoestrogen, Attenuates Monocrotaline-Induced Pulmonary HypertensionHomma N. · Morio Y. · Takahashi H. · Yamamoto A. · Suzuki T. · Sato K. · Muramatsu M. · Fukuchi Y.
Department of Respiratory Medicine, Juntendo University School of Medicine, Tokyo, Japan Corresponding Author
Background: Pulmonary hypertension is characterized by high pulmonary blood pressure, vascular remodeling, and right ventricular hypertrophy. Although recent studies suggest that an imbalance between endothelial mediators on pulmonary vasculature may contribute to the development of pulmonary hypertension, the pathogenesis is not fully understood and the treatment of pulmonary hypertension is still unresolved. Objective: The purpose of this study was to investigate whether genistein, a phytoestrogen derived from soybean, would prevent the development of monocrotaline (MCT)-induced pulmonary hypertension in rats. Hemodynamic parameters of catheterized rats and morphological feature of lungs were evaluated among MCT-treated rats receiving or not receiving genistein. Furthermore, examination of expression in endothelial nitric oxide synthase and endothelin-1 peptide level was performed. Methods: Daily supplementation with either genistein (0.2 mg/kg) or vehicle was started 2 days prior to a single-dose injection of MCT (60 mg/kg). On day 28, rats underwent catheterization, and right ventricular hypertrophy and morphological features were assessed. Furthermore, endothelial nitric oxide synthase and endothelin-1 were examined by Western blot analysis and radioimmunoassay, respectively, in homogenated lungs. Results: In rats that received daily supplementation of genistein, mean pulmonary arterial pressure was significantly reduced, whereas mean systemic arterial pressure and heart rate were unaltered compared with MCT control rats on day 28 after MCT injection. Right ventricular hypertrophy, medial wall thickness of pulmonary arteries corresponding to the terminal bronchioles, and the degree of neomuscularization of more distal arteries were less severe in genistein-treated rats. Genistein supplementation improved MCT-induced downregulation of expression of endothelial nitric oxide synthase in the lungs. However, endothelin-1 peptide levels did not differ among all groups of lungs. Conclusions: We conclude that daily supplementation of genistein potently attenuates MCT-induced pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular remodeling in rats. The underlying mechanism responsible for this effect may be partly related to the restoration of a decreased expression of endothelial nitric oxide synthase.
Copyright © 2006 S. Karger AG, Basel
Pulmonary hypertension (PH) is characterized by hemodynamic abnormalities such as high pulmonary arterial pressure (PAP), vascular remodeling, and right ventricular hypertrophy. The disease, including primary PH and chronic obstructive pulmonary disease, has various causes and leads to right ventricular failure and death. Although the pathogenesis of PH differs among these disease states and is not fully understood, it is likely that abnormal pulmonary vasoconstriction precedes the vascular process in all forms of PH. Although several vasodilator agents have been used in the treatment of PH, there is a limitation in the treatment with agents because of their lack of pulmonary selectivity.
A recent study has shown a decrease in nitric oxide (NO) production after intravenous injection of L-arginine, a precursor for NO synthesis, in patients with primary PH, suggesting a possibility of impaired NO-mediated signaling . Since impairment of NO-mediated signaling is considered as one of the causes of PH during the imbalance between endothelial mediators with opposing actions on pulmonary vasculature, inhaled NO has been used in the treatment of patients with PH. However, the utility of this therapy has been limited due to its toxicity and the difficulties with the delivery system [2, 3]. Recently, studies of PH in the animal model have demonstrated that prevention or reversal of development of PH is associated with restoration of endothelial NO synthase activity in various treatments [4,5,6,7,8,9]. This evidence suggests the possibility of an alternative strategy instead of inhaled NO in the treatment of PH through NO-mediated signaling.
Genistein, a phytoestrogen derived from soybean, has estrogen-like cardiovascular effects and inhibits tyrosine kinase activity via binding to estrogen receptors [10, 11]. Recent reports have shown that genistein supplementation suppresses vascular remodeling in a hypertensive animal model  and that it enhances endothelial NO synthase activity and NO-mediated vasorelaxation in aortic rings from ovariectomized rats . Furthermore, studies in rat pulmonary arteries have demonstrated that genistein causes NO-mediated vasorelaxation and improves impairment of the vasorelaxation after 2 weeks of exposure to hypoxia [14, 15]. Collectively, this evidence suggests the possibility that genistein may be able to suppress PH through enhancement of NO-mediated signaling.
Thus the purpose of this study was to investigate whether daily supplementation of genistein would prevent the development of PH. Therefore, we tested the effects of genistein on hemodynamic parameters and vascular remodeling in monocrotaline (MCT)-induced PH of rat lungs. Since MCT-induced PH exhibits various alterations in the expression of endothelial NO synthase [4,6,7,8,9, 16, 17] and endothelin-1 (ET-1) [18,19,20,21,22,23,24,25], we examined whether genistein would affect the change in the expression of endothelial NO synthase and endothelin-1 (ET-1) peptide in the MCT-induced hypertensive rat lungs.
All protocols and surgical procedures were approved by the Institutional Animal Use Committee of the Juntendo University School of Medicine (Tokyo, Japan) in accordance with the National Institutes of Health and American Physiological Society guidelines.
Experimentswere performed with adult male Sprague-Dawley rats (250–350 g). Four groups of rats were formed and studied: control (sham) vehicle, control genistein, MCT vehicle, and MCT-genistein. MCT (Sigma) was dissolved in 1 N HCl, neutralized to pH 7.4 with 0.5 N NaOH, and diluted with saline. Rats were given a single subcutaneous injection of MCT (60 mg/kg) or an equivalent volume of saline (2 ml/kg; control). Genistein (Sigma) was dissolved in a mixture of dimethyl sulfoxide (DMSO; Sigma) and polyethylene glycol (PEG; Sigma). Supplementation with genistein (0.2 mg/kg in 100 μl of a mixture of vehicle) was started 2 days prior to MCT or sham injection. Rats were given a subcutaneous injection of genistein or vehicle (100 μl of the mixture of 1.25% DMSO and 98.75% PEG) daily throughout the experiments. This protocol was based on the previous report by Squadrito et al. . Studies were performed 28 days after MCT or sham injection.
Twenty-eight days after MCT or sham injection, the rats of the four groups were anesthetized with intramuscular ketamine (90 mg/kg) and pentobarbital sodium (15 mg/kg), and implanted in the pulmonary and right carotid arteries and right jugular vein as previously described . We used polyethylene tubes (internal diameter: 0.58 mm, external diameter: 0.96 mm; Intramedic Clay Adams) for catheters. The intravascular location of the catheter tips was determined by blood pressure tracings, and the catheters were secured, filled with heparinized saline, sealed, and tunneled subcutaneously to the back of the neck where they were exteriorized and enclosed in a small plastic container. The cutdown procedures were repaired, and the rats were allowed to recover for 48 h. After recovery, the conscious rat was placed in a ventilated plastic box for hemodynamic measurements. PAP and systemic arterial pressure (SAP) were measured with pressure transducers (MPU-0.5 and TP-200T, respectively; Nihon Kohden, Tokyo, Japan) and displayed continuously on an oscilloscope. The heart rate was assessed from the arterial pressure pulse (AT-600G cardiotachometer; Nihon Kohden). If the heart rate fell below 300 beats/min, it was suspected that the animal’s cardiovascular function had been compromised by the catheterization procedure, and the measurements were excluded from the analysis. At the end of each hemodynamic study, the rat was sacrificed with an overdose of pentobarbital sodium, and the heart was removed to assess the severity of PH. An index of right ventricular hypertrophy was calculated as the ratio of wet weight of right ventricular wall to wet weight of left ventricular wall plus septum (RV/LV + S).
Because daily genistein supplementation was without any effects on the hemodynamic parameters of control rats, histological analysis was performed on the rats of three groups: control, MCT, and MCT-genistein. The histological changes were quantified by morphometry as previously described . The rats were sacrificed with an overdose of pentobarbital sodium, and the heart and lungs were removed en bloc. The pulmonary arteries were cannulated and perfused with phosphate-buffered saline (37°C, 20 cm H2O pressure) to wash out the residual blood and then injected with a barium-gelatin mixture (60°C) at 74 mm Hg pressure for 3 min. The trachea was intubated, and the lung was inflated with 10% formalin at 36 cm H2O pressure and fixed in the inflated state for 3 days. A block of tissue (1.0 × 0.7 × 0.2 cm) was taken from the midportion of the left lung parallel to the hilum. Sections were stained with elastic van Gieson stain and assessed microscopically for the degree of arterial muscularization and wall thickness. In each tissue section, at least 50 consecutive barium-filled arteries (>15 μm external diameter) were analyzed at ×400 magnification. Each artery was classified by the structure of the accompanying airway such as terminal bronchiole, alveolar duct, or alveolar wall. For each completely muscular artery corresponding to a terminal bronchiole, the medial wall thickness was measured at two locations of each artery. The medial wall thickness is expressed as the summation of the two points of (medial thickness/external diameter) × 100 (in percent).
The effect of genistein on the abnormal extension of smooth muscle into usually nonmuscular alveolar duct and wall vessels was assessed and classified as completely muscular, partially muscular, or nonmuscular in each barium-containing vessel at ×400 magnification.
Western blot analysis of endothelial NO synthase expression was performed in rat lungs of the three groups: control, MCT, and MCT-genistein. After anesthetization of the rats with 30 mg of intraperitoneal pentobarbital sodium, the heart and lungs were removed. A lateral peripheral sample of lung tissue was perfused with phosphate-buffered saline (37°C, 20 cm H2O pressure) to wash out the residual blood through pulmonary arterial cannulation. The sample was immediately frozen in liquid nitrogen after separation from the heart. Frozen lung tissues were homogenized in radioimmunoprecipitation assay buffer [150 mM NaCl, 1.5 mM MgCl2, 10 mM NaF, 10% glycerol, 4 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 50 mM HEPES, pH 7.4, 1% deoxycholate with 1 mM protease inhibitor cocktail; Calbiochem-Novabiochem, La Jolla,Calif., USA], and centrifuged for 15 min at 12,000 g to remove cellular debris. The protein concentration was estimated using a Micro BCA Protein Assay Reagent Kit (Pierce Biotechnology, Rockford, Ill., USA) with BSA as a standard. Twenty micrograms of protein from each sample were diluted in 2× reducing sample buffer (0.5 M Tris-Cl, 2% mercaptoethanol, 87% glycerol, 10% SDS, and 0.1% bromphenol blue) and boiled for 5 min. The protein suspensions were electrophoretically separated on 6% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Micropore). The membranes were blocked for nonspecific binding in a 5% skimmed milk solution at room temperature for 1 h, then incubated with 1:1,000 diluted monoclonal anti-eNOS antibody (Transduction Laboratory) overnight at 4°C. Thereafter, the membranes were washed 3 × 10 min with 0.1% Tween 20/TBS, incubated for 1 h with horseradish peroxidase-conjugated anti-mouse IgG at 1:10,000 dilution in 1% BSA/0.1% Tween/TBS, reacted with ECL substrate (Amersham Pharmacia Biotech), and finally exposed to radiographic films (Amersham). The films were scanned and relative density of the obtained products was measured using the QuantityOne software (BioRad, Hercules, Calif., USA).
Rat lung tissues were separated from the three groups as described in Western blot analysis of endothelial NO synthase expression, immediately frozen in liquid nitrogen, and stored at –80°C. ET-1 peptide was measured in lung homogenate using a radioimmunoassay kit as described previously by Naruse et al. .
Data are expressed as mean ± SE. Statistical analysis was done by unpaired t test or ANOVA followed by Dunnett-type multiple comparisons. The Mann-Whitney U test was used for comparison of muscularization of resistance vessels corresponding to alveolar duct and alveolar wall. Differences were considered significant at p < 0.05.
Twenty-eight days after injection of MCT, the body weight was smaller in the MCT-treated group (357.2 ± 4.1 g for control vehicle, 362.6 ± 6.0 g for control genistein, 334.5 ± 4.0 g for MCT vehicle, 339.8 ± 6.9 g for MCT-genistein, n = 8 animals/group, p < 0.05). Supplementation of genistein did not prevent the loss in body weight of MCT-treated rats. MCT-treated rats exhibited increases in mean PAP (8.7 ± 2.0 mm Hg for control vehicle, 9.3 ± 1.7 mm Hg for control genistein, 26.9 ± 3.1 mm Hg for MCT vehicle, 16.9 ± 2.3 mm Hg for MCT-genistein, n = 8 animals/group, p < 0.05) and RV/LV + S (0.338 ± 0.01 for control vehicle, 0.345 ± 0.02 for control genistein, 0.581 ± 0.03 for MCT vehicle, 0.378 ± 0.02 for MCT-genistein, n = 8 animals/group, p < 0.05) without any changes in mean SAP (96 ± 12 mm Hg for control vehicle, 98 ± 20 mm Hg for control genistein, 107 ± 9 mm Hg for MCT vehicle, 108 ± 4 mm Hg for MCT-genistein, n = 8 animals/group, p > 0.05). Daily genistein supplementation reduced mean PAP and RV/LV + S but not mean SAP in MCT-treated rats. However, genistein supplementation altered neither mean PAP, SAP, nor RV/LV + S in control rats. Heart rate did not differ among all groups of rats (369 ± 67 beats/min for control vehicle, 315 ± 15 beats/min for control genistein, 356 ± 24 beats/min for MCT vehicle, 340 ± 35 beats/min for MCT-genistein, n = 8 animals/group, p < 0.05) (fig. 1, 2).
|Fig. 1. Daily supplementation with genistein reduced MCT-induced increases in mean pulmonary arterial pressure (MPAP) (a) and right ventricular hypertrophy (RV/LV + S) (b). Genistein supplementation altered neither MPAP nor RV/LV + S in control rats. Values are means ± SE; n = 8 animals/group. C = Control vehicle; CG = control genistein; M = MCT vehicle; MG = MCT-genistein; S = septum. * p < 0.05 vs. the other group values by ANOVA.|
|Fig. 2. Daily supplementation with genistein did not affect mean systemic arterial pressure (MSAP) (a) and heart rate (HR) (b). Values are means ± SE; n = 8 animals/group. C = Control vehicle; CG = control genistein; M = MCT vehicle; MG = MCT-genistein.|
MCT-treated rats had significantly increased medial wall thickness of muscular pulmonary arteries corresponding to terminal bronchioles (vessel diameter of 50–150 μm) when compared with control rats. Genistein supplementation reduced the MCT-induced increase in medial wall thickness (6.0 ± 0.5% for control, 13.5 ± 1.0% for MCT, 6.1 ± 0.4% for MCT-genistein, vessel diameter of 50–100 μm, n = 8 animals/group, p < 0.05; 5.3 ± 0.9% for control, 14.1 ± 1.9% for MCT, 6.6 ± 0.6% for MCT-genistein, vessel diameter of 101–150 μm, n = 8 animals/group, p < 0.05) (fig. 3). Furthermore, MCT-treated rats caused neomuscularization of previously nonmuscular small pulmonary arteries. Genistein supplementation also reduced the degree of muscularization of the smaller resistance vessels corresponding to the alveolar duct and alveolar wall in MCT-treated rat lungs (table 1).
|Table 1. Daily supplementation with genistein reduces the degree of muscularization of the smaller resistance vessels corresponding to alveolar duct and alveolar wall in MCT-treated rat lungs|
|Fig. 3. Daily supplementation with genistein reduced MCT-induced increase in medial wall thickness of muscular pulmonary arteries corresponding to terminal bronchioles. Values are means ± SE; n = 8 animals/group. □ = Vessel diameter of 50–100 μm; ■ = vessel diameter of 101–150 μm; C = control; M = MCT; MG = MCT-genistein. * p < 0.05 vs. the other respective group values by ANOVA.|
Immunoblotting analysis showed that a 140-kDa band for endothelial NO synthase protein was found in lung tissue from control rats. Endothelial NO synthase expression was markedly downregulated in MCT-treated rats, and this was restored toward the level of control rats by daily supplementation of genistein (fig. 4).
|Fig. 4. Daily supplementation with genistein restored MCT-induced downregulation of endothelial NO synthase (eNOS) expression. Representative Western blot of whole lung homogenate protein in each group and its densitometric quantification are shown at the top and bottom, respectively. Values are means ± SE; n = 8 animals/group. C = Control; M = MCT; MG = MCT-genistein. * p < 0.05 vs. the other group values by ANOVA.|
Administration of MCT did not affect ET-1 peptide levels in lung homogenates. Neither did daily supplementation with genistein alter the peptide levels (7,271 ± 1,682 pg/g for control, 8,810 ± 1,480 pg/g for MCT, 7,808 ± 1,759 pg/g for MCT-genistein, n = 8 animals/group, p > 0.05) (fig. 5).
|Fig. 5. Neither MCT administration nor genistein supplementation affected ET-1 peptide levels in the lungs. Values are means ± SE; n = 8 animals/group. C = Control; M = MCT; MG = MCT-genistein.|
Genistein has multidirectional action in live cells, including beneficial estrogen-like effects on cardiovascular disease, with low toxicity [10, 11, 28]. Contrary to genistein, an increase in the risk of breast cancer by estrogen treatment has been considered despite its protective effect on cardiovascular disease . In addition, genistein is known to inhibit tyrosine kinase and has been reported to suppress proliferation of vascular smooth muscle cell(s) in an experimental model of hypertension [30,31,32]. Recently, genistein supplementation revealed inhibition of vascular remodeling in the hypertensive animal model , and it also improved endothelial dysfunction in ovariectomized rats  and postmenopausal women . Furthermore, studies in rat pulmonary arteries have shown that genistein causes NO-mediated vasorelaxation and improves hypoxic impairment of the vasorelaxation [14, 15]. Thus, the protective effects of genistein on cardiopulmonary disease may be a therapeutic potential, while the precise mechanism of the effects is not fully understood.
The major new finding of the present study is that daily supplementation with genistein attenuates MCT-induced PH, right ventricular hypertrophy, and vascular remodeling. Although this observation is similar to those made in a recent study on systemic circulation, where daily administration of genistein restores morphological changes of the basilar artery during chronic hypertension , this is the first case to show the beneficial effect of daily treatment with genistein, the supplementation of which is based on the previous report by Squadrito et al. , in an experimental model of PH. It should be necessary to avoid the possibility that genistein inhibits metabolization of MCT. Further studies are needed to examine the effect of genistein supplementation several days after MCT injection during serial experiments in the model of PH. Then, what mechanisms are involved in the protective effect of genistein in attenuating PH?
Impairment of endothelial vasodilator signaling has been considered as one of the various pathogeneses of PH. In fact, MCT-induced PH exhibits a reduction of endothelial NO synthase expression [4, 6, 8, 9] and NO production [7, 34]. These observations are consistent with our present data. However, Nakazawa et al. [16 ] demonstrated that endothelial NO synthase expression was augmented during the establishment of the PH. Furthermore, Resta et al. [17 ] demonstrated potentiation of both endothelial NO synthase expression and NO-mediated vasodilation in MCT-induced PH. Although the reason for this discrepancy is unclear, the augmentation may be due to compensation for the defect of NO-mediated signaling or in response to the elevation of PAP in PH. Because recent studies have shown that prevention or reversal of the PH is related to restoration of impaired endothelial NO synthase activity in various treatments [4,6,7,8,9], we examined whether genistein treatment would enhance decreased endothelial NO synthase expression. Our results showed that genistein supplementation augmented NO synthase expression similar to the study in ovariectomized rats . This evidence suggests that the attenuation of MCT-induced PH by genistein may be at least partly related to the restoration of decreased endothelial NO synthase expression.
Genistein has been reported to inhibit proliferation of vascular smooth muscle cells in stroke-prone spontaneously hypertensive rats  or transmural pressurization , and its inhibitory effect may result from the arrest of cell cycle progression by inactivation of mitogen-activated protein kinases . Furthermore, genistein attenuated angiotensin II-induced proliferation of vascular smooth muscle cells through suppression of oxidant stress in rats . Genistein may cause an antioxidant effect in our present preparation, since MCT-induced PH revealed an augmentation of oxidant stress . Thus, there may be the possibility that genistein supplementation directly or indirectly suppresses MCT-induced neomuscularization of pulmonary arteries.
ET-1, a potent vasoconstrictor and promitogenic peptide, importantly contributes to the pathophysiology of PH as its increased expression seems to be related to disease severity and survival . Peptide levels of ET-1 have been reported to be increased in pulmonary artery of chronic obstructive pulmonary disease complicated by PH but not chronic obstructive pulmonary disease alone . MCT-induced PH increased plasma ET-1 levels and exhibited various alterations of ET-1 expression in lungs [18,19,20,21,22,23,24,25]. Since long-term treatment with genistein has been reported to decrease plasma ET-1 levels in postmenopausal women who exhibit endothelial dysfunction , we investigated whether genistein supplementation would affect ET-1 levels in MCT-treated lungs. In the present study, lung ET-1 peptide levels did not differ among the groups. The reason why MCT-induced PH revealed no effect on ET-1 peptide levels in the lungs is unclear. One explanation of our result is that although we did not examine plasma ET-1 levels, the source of ET-1 production may be the kidneys but not the lungs and the circulating ET-1 may contribute to the pathogenesis of PH in MCT-treated rats . This result indicated the possibility that genistein supplementation attenuated MCT-induced PH independently through stimulation of the ET system in the lungs. Alternatively, some in vivo studies with bosentan, a dual ET receptor blocker, in MCT-induced PH suggested that other important mediators could be likely to be involved in the development of PH because a higher dose of bosentan was required in the study .
In summary, our results in this study showed that daily supplementation with genistein, a phytoestrogen derived from soybean, potently attenuated the development of MCT-induced PH, right ventricular hypertrophy, and vascular remodeling. Genistein supplementation also restored MCT-induced downregulation of endothelial NO synthase expression in the lungs. However, lung ET-1 peptide levels were unaltered in all groups. Protective effect of genistein on MCT-induced PH may be partly associated with restoration of endothelial NO synthase expression in the lungs. Further studies are required to delineate the exact relationship between the reduction of the severity of PH and the restoration of endothelial NO synthase expression. Although our present study was undertaken in an experimental model, which could not be predictive of a response to therapy in humans, genistein may prove to be a new potential therapeutic agent for the treatment of PH.
Department of Respiratory Medicine, Juntendo University School of Medicine
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Received: February 15, 2005
Accepted after revision: June 1, 2005
Published online: October 31, 2005
Number of Print Pages : 8
Number of Figures : 5, Number of Tables : 1, Number of References : 39
Respiration (International Journal of Thoracic Medicine)
Vol. 73, No. 1, Year 2006 (Cover Date: February 2006)
Journal Editor: Bolliger, C.T. (Cape Town)
ISSN: 0025–7931 (print), 1423–0356 (Online)
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