This article was downloaded by: [New York University] On: 02 September 2015, At: 02:10 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place, London, SW1P 1WG
Experimental Lung Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ielu20
Inhibition of Notch3 prevents monocrotaline-induced pulmonary arterial hypertension a
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Yonghong Zhang , Xinming Xie , Yanting Zhu , Lu Liu , Wei Feng , Yilin Pan , Cui Zhai , Rui a
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Ke , Shaojun Li , Yang Song , Yuncun Fan , Fenling Fan , Xiaochuang Wang , Fengjuan Li & Manxiang Li
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Department of Respiratory Medicine, the First Affiliated Hospital of Medical College at Xi'an JiaoTong University, Xi'an, Shanxi, China Published online: 28 Aug 2015.
Click for updates To cite this article: Yonghong Zhang, Xinming Xie, Yanting Zhu, Lu Liu, Wei Feng, Yilin Pan, Cui Zhai, Rui Ke, Shaojun Li, Yang Song, Yuncun Fan, Fenling Fan, Xiaochuang Wang, Fengjuan Li & Manxiang Li (2015): Inhibition of Notch3 prevents monocrotaline-induced pulmonary arterial hypertension, Experimental Lung Research To link to this article: http://dx.doi.org/10.3109/01902148.2015.1060545
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Experimental Lung Research, Early Online, 1–9, 2015 Copyright © 2015 Taylor & Francis Group, LLC ISSN: 0190-2148 print / 1521-0499 online DOI: 10.3109/01902148.2015.1060545
ORIGINAL ARTICLE
Inhibition of Notch3 prevents monocrotaline-induced pulmonary arterial hypertension Yonghong Zhang, Xinming Xie, Yanting Zhu, Lu Liu, Wei Feng, Yilin Pan, Cui Zhai, Rui Ke, Shaojun Li, Yang Song, Yuncun Fan, Fenling Fan, Xiaochuang Wang, Fengjuan Li, and Manxiang Li
Downloaded by [New York University] at 02:10 02 September 2015
Department of Respiratory Medicine, the First Affiliated Hospital of Medical College at Xi’an JiaoTong University, Xi’an, Shanxi, China A B STRA CT It has been shown that activation of Notch3 signaling is involved in the development of pulmonary arterial hypertension (PAH) by stimulating pulmonary arteries remodeling, while the molecular mechanisms underlying this are still largely unknown. The aims of this study are to address these issues. Monocrotaline dramatically increased right ventricle systolic pressure to 39.0 ± 2.6 mmHg and right ventricle hypertrophy index to 53.4 ± 5.3% (P < 0.05 versus control) in rats, these were accompanied with significantly increased proliferation and reduced apoptosis of pulmonary vascular cells as well as pulmonary arteries remodeling. Treatment of PAH model with specific Notch inhibitor DAPT significantly reduced right ventricle systolic pressure to 26.6 ± 1.3 mmHg and right ventricle hypertrophy index to 33.5 ± 2.6% (P < 0.05 versus PAH), suppressed proliferation and enhanced apoptosis of pulmonary vascular cells as well as inhibited pulmonary arteries remodeling. Our results further indicated that level of Notch3 protein and NICD3 were increased in MCT-induced model of PAH, this was accompanied with elevation of Skp2 and Hes1 protein level and reduction of P27Kip1. Administration of rats with DAPT-prevented MCT induced these changes. Our results suggest that Notch3 signaling activation stimulated pulmonary vascular cells proliferation by Skp2-and Hes1-mediated P27Kip1 reduction, and Notch3 might be a new target to treat PAH. KEYWORDS Hes1, Notch3, P27Kip1, pulmonary arterial hypertension, Skp2, vascular remodeling
dothelial dysfunction [3]. The most prominent feature of pulmonary vascular remodeling is the excessive pulmonary vascular cells in pulmonary arterial wall, especially PASMCs, and the transition of PASMCs from the quiescent phenotype to the proliferative phenotype, which is commonly existed in all types of PAH[4]. Therefore, elucidating the molecular mechanisms of PASMCs proliferation and pursuing appropriate targets are critical in the treatment of PAH. The Notch signaling is an evolutionarily conserved regulatory cascade playing a critical role in cell fate decisions including proliferation, differentiation, and apoptosis [5]. Four types of Notch receptors (Notch 1, 2, 3, 4) and five Notch ligands (Jagged 1, 2, Deltalike 1, 3, 4) have been identified in mammals. Upon the binding of ligands to Notch receptors, Notch receptors undergo several proteolytic events that lead to the release of the intracellular domains (ICDs)
INTRODUCTION Pulmonary arterial hypertension (PAH) is a progressive disease of various origins which is associated with increased pulmonary vascular resistance and subsequent right ventricular failure [1]. The key pathological feature of PAH is adverse remodeling of distal pulmonary arterioles leading to narrowness and obstruction of small pulmonary arteries [2]. Pathological changes confer to vascular remodeling include increased migration/proliferation of pulmonary arterial smooth muscle cells (PASMCs) and fibroblasts proliferation and production of collagen as well as enReceived 7 December 2014; accepted 6 June 2015. Address correspondence to Manxiang Li, Department of Respiratory Medicine, the First Affiliated Hospital of Medical College at Xi’an JiaoTong University, No. 277, West Yanta Road, Xi’an, Shaanxi, China 710061. E-mail: [email protected]
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Y. Zhang et al.
of these receptors. ICDs translocate into the nucleus and activate transcription factors CBF1/RBPJk thus regulate the expression of particular target genes, such as hairy-and-enhancer of split (Hes) and Hes-related transcriptional factors (HesR or Hrt) [6, 7]. Hes and Hrt are basic helix-loop-helix (bHLH) type transcriptional repressors and act as Notch effectors by negatively regulating expression of downstream target genes such as Hash1 [8], Gata2 [9], Gata4/6 [10], as well as cell-cycle regulatory proteins p57 kip2 [11], p27Kip1 [12] and p21waf1/cip1 [13]. Skp2 is recently founded another immediate downstream target of ICDs associated with the cell proliferation [14, 15]. Recent studies have shown that Notch3 pathway is implicated in the development of pulmonary artery hypertension in both clinic patients and animal model by promoting pulmonary artery remodeling [16, 17]. Yet, detailed molecular mechanisms underlying Notch3 stimulating pulmonary artery remodeling, and which downstream targets are regulated by active Notch3 receptors are still unclear. The aim of this study is to address these issues in rat animal model of PAH induced by monocrotaline.
Center. All protocols used in this study were approved by the Laboratory Animal Care Committee of Xi’an Jiaotong University. Total 32 rats were randomly divided into three groups: control group (Con, n = 10), MCT treatment group (MCT, n = 12), MCT and DAPT treated group (MCT + DAPT, n = 10). All animals were kept in the same room and subjected to the same light/dark cycle.
MATERIALS AND METHODS
Measurement of the RVSP and the RVH
Materials N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) with a purity >99.0%, an γ -secretase inhibitor, was obtained from Santa Cruz Biotechnology; Monocrotaline (MCT) and RIPA lysis buffer were purchased from Sigma-Aldrich. The following primary antibodies were used: rabbit antibodies to Notch3 (Abcam, 1:500 dilution), Hes1 (Cell Signaling Technology, 1:1000 dilution), Skp2 (Cell Signaling Technology, 1:1000 dilution), and p27Kip1 (Cell Signaling Technology, 1:1000 dilution) as well as GAPDH (Sigma-Aldrich, 1:2,000 dilution). Horseradish peroxidase-conjugated goat anti-rabbit IgG was used as secondary antibody (Sigma-Aldrich, 1:5000 dilution). Enhanced chemiluminescence (ECL) reagents were from Millipore Corp. All other reagents were from common commercial sources.
Animals Male Sprague–Dawley (SD) rats weighing from180 to190 g were provided by the Animal Experimental Center of Xi’an Jiaotong University. All animal care and experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Xi’an Jiaotong University Animal Experiment
Generation of PAH Models and Drug Treatment MCT was dissolved in 0.1 mol/L HCl, the solution was then titrated to pH 7.4 with 0.1 mol/L NaOH with the final concentration of 20 mg/mL. The PAH model was induced by intraperitoneally injection of MCT (60 mg/kg of body weight) on day 1. DAPT was dissolved in DMSO with the final concentration of 20 mg/mL, DAPT (10 mg/kg) was administrated to rats by intraperitoneally injection every day throughout the experiment period. Three of twelve rats died in MCT treated rats. Twenty-eight days after MCT injection, rats were used for further studies as followings.
Twenty-eight days after MCT administration, rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate (30 mg/kg). Tracheal cannulation was performed after tracheostomy, animal was endotracheally intubated with positive-pressure ventilation (respiratory rate: 30 breaths per minute; tidal volume: 10 mL/kg, positive end-expiratory pressure: 2–3 cmH2 O) with room air using a small animal ventilator. Heart was exposed by left thoracotomy. A sterile 20-gauge, soft plastic-coated needle was inserted into the right ventricle (RV) to measure the right ventricle systolic pressure (RVSP). The pressure signals were recorded using a Grass polygraph (Power Lab, Australia). The data were analyzed with the Labchart software (AD Instrument). After hemodynamic measurements, hearts and lungs were harvested. For the assessment of right ventricular hypertrophy, the right ventricle was dissected from the left ventricle and interventricular septum, and was weighed separately. The index of right ventricular hypertrophy (RVH) was calculated as the ratio of weight of the right ventricle to the left ventricle plus the interventricular septum, RV/ (LV + S).
Histological Analysis Marginal right lower pulmonary lobes were harvested and fixed with 10% buffered formalin for 4 hours, and Experimental Lung Research
Downloaded by [New York University] at 02:10 02 September 2015
Involvement of Notch3 in Pulmonary Arterial Hypertension
were then embedded in paraffin wax. Tissue blocks were sectioned to 5 μm in thickness and stained with HE (hematoxylin and eosin). Pulmonary vascular remodeling was evaluated by measuring the medial wall thickness of vessels (diameter 50–250 μm) using an ocular micrometer by an observer who was blinded to the treatments of the rats. Distorted arteries were not used for the measurement. The percentage of medial wall thickness was calculated as follows: percent wall thickness = (external diameter-internal diameter) ×100/external diameter. Pulmonary arterial muscularization was assessed by immunohistochemical staining with anti-α-smooth muscle actin antibody (Sigma-Aldrich, dilution 1:500) for lung sections. In each experimental group, 80 to 100 pulmonary arteries (20 to 100 μm diameter) were counted at ×400 magnification, counted arteries were categorized as muscular, partially muscular, or nonmuscular. Arterial muscularization was defined according to the degree of muscularization: muscular (with a complete medial coat of muscle), partially muscular (with only a crescent of muscle), or nonmuscular (no apparent muscle). Morphometric analyses were performed in a blinded fashion.
Pulmonary Vascular Cells Proliferation and Apoptosis Assay To examine the proliferation and apoptosis of cells, the paraffin-embedded pulmonary tissues sections (5 μm thick) were prepared. Cell proliferation was assessed by detection of PCNA (Proliferation cell nuclear antigen) using PCNA staining kit (Zymed Laboratories Inc, San Francisco, CA, USA) according to the manufacturer’s instructions. Cell apoptosis was evaluated by the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay (Roche Diagnostics) according to the manufacture instruction. 9–10 slides were selected from each experimental group, then, 8 to 11 arteries (30 to 100 μm diameter) in each slide were randomly selected for detection of proliferation or apoptosis. Ratios of proliferation or apoptosis to total vascular cells in individual arterioles were calculated, with average of the ratio as the representative value for each experimental group.
Western Blot Analysis Lungs were removed and homogenized in ice cold RIPA lysis buffer with the phosphatase inhibitor and protease inhibitor. Equivalent amounts of protein (30 μg) from each sample were separated on SDSpolyacrylamide gels, then transferred onto nitrocellulose membranes and blocked with wash buffer containing 5% nonfat dried milk for 2 hours at C
2015 Taylor & Francis Group, LLC
3
room temperature with gentle agitation. Nitrocellulose membranes were then incubated overnight at 4◦ C in wash buffer containing primary antibody. Blots were next washed and incubated with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody in wash buffer for 1 hour at room temperature. Blots were developed using ECL reagent kit (Millipore). The signal intensity of the appropriate bands on the autoradiogram was calculated by using Scion Image software (Scion).
Statistical Analysis Data are expressed as means ± SD. In all the experiments, data were analyzed by one-way ANOVA followed by Tukey post hoc test. Frequency data were analyzed by χ 2 test. P
Experimental Lung Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ielu20
Inhibition of Notch3 prevents monocrotaline-induced pulmonary arterial hypertension a
a
a
a
a
a
a
Yonghong Zhang , Xinming Xie , Yanting Zhu , Lu Liu , Wei Feng , Yilin Pan , Cui Zhai , Rui a
a
a
a
a
a
a
Ke , Shaojun Li , Yang Song , Yuncun Fan , Fenling Fan , Xiaochuang Wang , Fengjuan Li & Manxiang Li
a
a
Department of Respiratory Medicine, the First Affiliated Hospital of Medical College at Xi'an JiaoTong University, Xi'an, Shanxi, China Published online: 28 Aug 2015.
Click for updates To cite this article: Yonghong Zhang, Xinming Xie, Yanting Zhu, Lu Liu, Wei Feng, Yilin Pan, Cui Zhai, Rui Ke, Shaojun Li, Yang Song, Yuncun Fan, Fenling Fan, Xiaochuang Wang, Fengjuan Li & Manxiang Li (2015): Inhibition of Notch3 prevents monocrotaline-induced pulmonary arterial hypertension, Experimental Lung Research To link to this article: http://dx.doi.org/10.3109/01902148.2015.1060545
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Experimental Lung Research, Early Online, 1–9, 2015 Copyright © 2015 Taylor & Francis Group, LLC ISSN: 0190-2148 print / 1521-0499 online DOI: 10.3109/01902148.2015.1060545
ORIGINAL ARTICLE
Inhibition of Notch3 prevents monocrotaline-induced pulmonary arterial hypertension Yonghong Zhang, Xinming Xie, Yanting Zhu, Lu Liu, Wei Feng, Yilin Pan, Cui Zhai, Rui Ke, Shaojun Li, Yang Song, Yuncun Fan, Fenling Fan, Xiaochuang Wang, Fengjuan Li, and Manxiang Li
Downloaded by [New York University] at 02:10 02 September 2015
Department of Respiratory Medicine, the First Affiliated Hospital of Medical College at Xi’an JiaoTong University, Xi’an, Shanxi, China A B STRA CT It has been shown that activation of Notch3 signaling is involved in the development of pulmonary arterial hypertension (PAH) by stimulating pulmonary arteries remodeling, while the molecular mechanisms underlying this are still largely unknown. The aims of this study are to address these issues. Monocrotaline dramatically increased right ventricle systolic pressure to 39.0 ± 2.6 mmHg and right ventricle hypertrophy index to 53.4 ± 5.3% (P < 0.05 versus control) in rats, these were accompanied with significantly increased proliferation and reduced apoptosis of pulmonary vascular cells as well as pulmonary arteries remodeling. Treatment of PAH model with specific Notch inhibitor DAPT significantly reduced right ventricle systolic pressure to 26.6 ± 1.3 mmHg and right ventricle hypertrophy index to 33.5 ± 2.6% (P < 0.05 versus PAH), suppressed proliferation and enhanced apoptosis of pulmonary vascular cells as well as inhibited pulmonary arteries remodeling. Our results further indicated that level of Notch3 protein and NICD3 were increased in MCT-induced model of PAH, this was accompanied with elevation of Skp2 and Hes1 protein level and reduction of P27Kip1. Administration of rats with DAPT-prevented MCT induced these changes. Our results suggest that Notch3 signaling activation stimulated pulmonary vascular cells proliferation by Skp2-and Hes1-mediated P27Kip1 reduction, and Notch3 might be a new target to treat PAH. KEYWORDS Hes1, Notch3, P27Kip1, pulmonary arterial hypertension, Skp2, vascular remodeling
dothelial dysfunction [3]. The most prominent feature of pulmonary vascular remodeling is the excessive pulmonary vascular cells in pulmonary arterial wall, especially PASMCs, and the transition of PASMCs from the quiescent phenotype to the proliferative phenotype, which is commonly existed in all types of PAH[4]. Therefore, elucidating the molecular mechanisms of PASMCs proliferation and pursuing appropriate targets are critical in the treatment of PAH. The Notch signaling is an evolutionarily conserved regulatory cascade playing a critical role in cell fate decisions including proliferation, differentiation, and apoptosis [5]. Four types of Notch receptors (Notch 1, 2, 3, 4) and five Notch ligands (Jagged 1, 2, Deltalike 1, 3, 4) have been identified in mammals. Upon the binding of ligands to Notch receptors, Notch receptors undergo several proteolytic events that lead to the release of the intracellular domains (ICDs)
INTRODUCTION Pulmonary arterial hypertension (PAH) is a progressive disease of various origins which is associated with increased pulmonary vascular resistance and subsequent right ventricular failure [1]. The key pathological feature of PAH is adverse remodeling of distal pulmonary arterioles leading to narrowness and obstruction of small pulmonary arteries [2]. Pathological changes confer to vascular remodeling include increased migration/proliferation of pulmonary arterial smooth muscle cells (PASMCs) and fibroblasts proliferation and production of collagen as well as enReceived 7 December 2014; accepted 6 June 2015. Address correspondence to Manxiang Li, Department of Respiratory Medicine, the First Affiliated Hospital of Medical College at Xi’an JiaoTong University, No. 277, West Yanta Road, Xi’an, Shaanxi, China 710061. E-mail: [email protected]
1
Downloaded by [New York University] at 02:10 02 September 2015
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Y. Zhang et al.
of these receptors. ICDs translocate into the nucleus and activate transcription factors CBF1/RBPJk thus regulate the expression of particular target genes, such as hairy-and-enhancer of split (Hes) and Hes-related transcriptional factors (HesR or Hrt) [6, 7]. Hes and Hrt are basic helix-loop-helix (bHLH) type transcriptional repressors and act as Notch effectors by negatively regulating expression of downstream target genes such as Hash1 [8], Gata2 [9], Gata4/6 [10], as well as cell-cycle regulatory proteins p57 kip2 [11], p27Kip1 [12] and p21waf1/cip1 [13]. Skp2 is recently founded another immediate downstream target of ICDs associated with the cell proliferation [14, 15]. Recent studies have shown that Notch3 pathway is implicated in the development of pulmonary artery hypertension in both clinic patients and animal model by promoting pulmonary artery remodeling [16, 17]. Yet, detailed molecular mechanisms underlying Notch3 stimulating pulmonary artery remodeling, and which downstream targets are regulated by active Notch3 receptors are still unclear. The aim of this study is to address these issues in rat animal model of PAH induced by monocrotaline.
Center. All protocols used in this study were approved by the Laboratory Animal Care Committee of Xi’an Jiaotong University. Total 32 rats were randomly divided into three groups: control group (Con, n = 10), MCT treatment group (MCT, n = 12), MCT and DAPT treated group (MCT + DAPT, n = 10). All animals were kept in the same room and subjected to the same light/dark cycle.
MATERIALS AND METHODS
Measurement of the RVSP and the RVH
Materials N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) with a purity >99.0%, an γ -secretase inhibitor, was obtained from Santa Cruz Biotechnology; Monocrotaline (MCT) and RIPA lysis buffer were purchased from Sigma-Aldrich. The following primary antibodies were used: rabbit antibodies to Notch3 (Abcam, 1:500 dilution), Hes1 (Cell Signaling Technology, 1:1000 dilution), Skp2 (Cell Signaling Technology, 1:1000 dilution), and p27Kip1 (Cell Signaling Technology, 1:1000 dilution) as well as GAPDH (Sigma-Aldrich, 1:2,000 dilution). Horseradish peroxidase-conjugated goat anti-rabbit IgG was used as secondary antibody (Sigma-Aldrich, 1:5000 dilution). Enhanced chemiluminescence (ECL) reagents were from Millipore Corp. All other reagents were from common commercial sources.
Animals Male Sprague–Dawley (SD) rats weighing from180 to190 g were provided by the Animal Experimental Center of Xi’an Jiaotong University. All animal care and experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Xi’an Jiaotong University Animal Experiment
Generation of PAH Models and Drug Treatment MCT was dissolved in 0.1 mol/L HCl, the solution was then titrated to pH 7.4 with 0.1 mol/L NaOH with the final concentration of 20 mg/mL. The PAH model was induced by intraperitoneally injection of MCT (60 mg/kg of body weight) on day 1. DAPT was dissolved in DMSO with the final concentration of 20 mg/mL, DAPT (10 mg/kg) was administrated to rats by intraperitoneally injection every day throughout the experiment period. Three of twelve rats died in MCT treated rats. Twenty-eight days after MCT injection, rats were used for further studies as followings.
Twenty-eight days after MCT administration, rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate (30 mg/kg). Tracheal cannulation was performed after tracheostomy, animal was endotracheally intubated with positive-pressure ventilation (respiratory rate: 30 breaths per minute; tidal volume: 10 mL/kg, positive end-expiratory pressure: 2–3 cmH2 O) with room air using a small animal ventilator. Heart was exposed by left thoracotomy. A sterile 20-gauge, soft plastic-coated needle was inserted into the right ventricle (RV) to measure the right ventricle systolic pressure (RVSP). The pressure signals were recorded using a Grass polygraph (Power Lab, Australia). The data were analyzed with the Labchart software (AD Instrument). After hemodynamic measurements, hearts and lungs were harvested. For the assessment of right ventricular hypertrophy, the right ventricle was dissected from the left ventricle and interventricular septum, and was weighed separately. The index of right ventricular hypertrophy (RVH) was calculated as the ratio of weight of the right ventricle to the left ventricle plus the interventricular septum, RV/ (LV + S).
Histological Analysis Marginal right lower pulmonary lobes were harvested and fixed with 10% buffered formalin for 4 hours, and Experimental Lung Research
Downloaded by [New York University] at 02:10 02 September 2015
Involvement of Notch3 in Pulmonary Arterial Hypertension
were then embedded in paraffin wax. Tissue blocks were sectioned to 5 μm in thickness and stained with HE (hematoxylin and eosin). Pulmonary vascular remodeling was evaluated by measuring the medial wall thickness of vessels (diameter 50–250 μm) using an ocular micrometer by an observer who was blinded to the treatments of the rats. Distorted arteries were not used for the measurement. The percentage of medial wall thickness was calculated as follows: percent wall thickness = (external diameter-internal diameter) ×100/external diameter. Pulmonary arterial muscularization was assessed by immunohistochemical staining with anti-α-smooth muscle actin antibody (Sigma-Aldrich, dilution 1:500) for lung sections. In each experimental group, 80 to 100 pulmonary arteries (20 to 100 μm diameter) were counted at ×400 magnification, counted arteries were categorized as muscular, partially muscular, or nonmuscular. Arterial muscularization was defined according to the degree of muscularization: muscular (with a complete medial coat of muscle), partially muscular (with only a crescent of muscle), or nonmuscular (no apparent muscle). Morphometric analyses were performed in a blinded fashion.
Pulmonary Vascular Cells Proliferation and Apoptosis Assay To examine the proliferation and apoptosis of cells, the paraffin-embedded pulmonary tissues sections (5 μm thick) were prepared. Cell proliferation was assessed by detection of PCNA (Proliferation cell nuclear antigen) using PCNA staining kit (Zymed Laboratories Inc, San Francisco, CA, USA) according to the manufacturer’s instructions. Cell apoptosis was evaluated by the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay (Roche Diagnostics) according to the manufacture instruction. 9–10 slides were selected from each experimental group, then, 8 to 11 arteries (30 to 100 μm diameter) in each slide were randomly selected for detection of proliferation or apoptosis. Ratios of proliferation or apoptosis to total vascular cells in individual arterioles were calculated, with average of the ratio as the representative value for each experimental group.
Western Blot Analysis Lungs were removed and homogenized in ice cold RIPA lysis buffer with the phosphatase inhibitor and protease inhibitor. Equivalent amounts of protein (30 μg) from each sample were separated on SDSpolyacrylamide gels, then transferred onto nitrocellulose membranes and blocked with wash buffer containing 5% nonfat dried milk for 2 hours at C
2015 Taylor & Francis Group, LLC
3
room temperature with gentle agitation. Nitrocellulose membranes were then incubated overnight at 4◦ C in wash buffer containing primary antibody. Blots were next washed and incubated with an appropriate horseradish peroxidase (HRP)-conjugated secondary antibody in wash buffer for 1 hour at room temperature. Blots were developed using ECL reagent kit (Millipore). The signal intensity of the appropriate bands on the autoradiogram was calculated by using Scion Image software (Scion).
Statistical Analysis Data are expressed as means ± SD. In all the experiments, data were analyzed by one-way ANOVA followed by Tukey post hoc test. Frequency data were analyzed by χ 2 test. P
