J. Pineal Res. 2014; 57:442–450
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Doi:10.1111/jpi.12184
Journal of Pineal Research
Melatonin attenuates hypoxic pulmonary hypertension by inhibiting the inflammation and the proliferation of pulmonary arterial smooth muscle cells Abstract: Hypoxia-induced inflammation and excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) play important roles in the pathological process of hypoxic pulmonary hypertension (HPH). Melatonin possesses anti-inflammatory and antiproliferative properties. However, the effect of melatonin on HPH remains unclear. In this study, adult Sprague– Dawley rats were exposed to intermittent chronic hypoxia for 4 wk to mimic a severe HPH condition. Hemodynamic and pulmonary pathomorphology data showed that chronic hypoxia significantly increased right ventricular systolic pressures (RVSP), weight of the right ventricle/left ventricle plus septum (RV/ LV+S) ratio, and median width of pulmonary arterioles. Melatonin attenuated the elevation of RVSP, RV/LV+S, and mitigated the pulmonary vascular structure remodeling. Melatonin also suppressed the hypoxia-induced high expression of proliferating cell nuclear antigen (PCNA), hypoxia-inducible factor-1a (HIF-1a), and nuclear factor-jB (NF-jB). In vitro, melatonin concentration-dependently inhibited the proliferation of PASMCs and the levels of phosphorylation of Akt and extracellular signal-regulated kinases1/2 (ERK1/2) caused by hypoxia. These results suggested that melatonin might potentially prevent HPH via anti-inflammatory and antiproliferative mechanisms.
Introduction Hypoxic pulmonary hypertension (HPH) is a serious disease with poor prognosis. It is characterized by hypoxiainduced pulmonary vasoconstriction, pulmonary vascular remodeling, and elevated pulmonary artery pressure [1–3]. According to the updated classification of pulmonary hypertension (PH) in the Fourth World Symposium on PH, HPH belongs to Group 3: pulmonary hypertension owing to lung diseases and/or hypoxia [4, 5]. In this form of PH, patients are commonly encountered with chronic obstructive pulmonary disease, interstitial lung disease, respiratory disorders with a mixed restrictive, and obstructive pattern [5]. Although HPH is very common, there is no specific treatment for this serious disease [6]. Pulmonary vasodilators such as phosphodiesterase-type 5 inhibitors, endothelin receptor antagonists, and prostanoids often nonselectively vasodilate the pulmonary vessels and ultimately lead to worsened gas exchange and hypoxemia [5, 7]. Therefore, hunting for novel effective pharmacologic treatments for HPH is urgent. Hypoxia-induced inflammation, endothelium dysfunction, production of reactive oxygen species (ROS), and proliferation of pulmonary artery smooth muscle cells (PASMCs) are all involved in the pathological process of HPH. Among them, inflammation participates in the initiation and progression of HPH by actively contributing 442
Haifeng Jin1,2, Yueyue Wang1, Lei Zhou1, Lu Liu1, Peng Zhang1, Wuguo Deng1,3 and Yuhui Yuan1 1
Institute of Cancer Stem Cell, The First Affiliated Hospital, Dalian Medical University Cancer Center, Dalian, China; 2Department of Anatomy, Qiqihar Medical University, Qiqihar, China; 3Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China Key words: hypoxia, inflammation, melatonin, pulmonary artery smooth muscle cells, pulmonary hypertension Address reprint requests to Yuhui Yuan, Institute of Cancer Stem Cell, First Affiliated Hospital, Dalian Medical University Cancer Center, 9 West Section of Lvshun South Road, Dalian 116044, China. E-mails: [email protected]; [email protected] Received August 18, 2014; Accepted September 21, 2014.
to chronic vasoconstriction and remodeling of the pulmonary vessel wall [8, 9]. Exposure of animals to even moderate hypoxia results in increased expression of inflammatory cytokines, chemokines, adhesion molecules, and accumulation of leukocytes in the lungs [1]. These factors disrupt endothelial membrane integrity and facilitate deregulation of vascular cell proliferation in PH [8]. In addition, the hypoxia-induced excessive proliferation of PASMCs is also a key factor, which leads to media thickening and vascular resistance, in the development of HPH [10, 11]. Melatonin, an indolamine, is a small lipophilic molecule and ubiquitous physiological mediator that is synthesized in the pineal gland [12]. In addition, using specific melatonin antibodies, melatonin had been found in multiple extrapineal tissues including the liver, spleen, stomach, intestine, thymus, cerebral cortex, striatum, lens, skin, etc., and most of these tissues express melatonin-synthesizing enzymes [13–15]. Numerous studies have shown that melatonin plays crucial roles in several vital physiological and pathological processes, such as regulation of circadian rhythms, inhibition of tumor growth and metastasis, inhibition of inflammation and cell proliferation [16–20]. In the cardiovascular system, melatonin has protective effects through the free radical scavenger activity and antioxidant properties [21–24]. More recently, it has been confirmed that melatonin’s metabolic derivatives,
Melatonin attenuates HPH development N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), and N1-acetyl-5-methoxykynuramine (AMK), also possess free radical scavenger activity and antioxidant properties, which makes melatonin more effective and exerts continuous protection in oxidative damage [25, 26]. For the relative free radical scavenging activity of melatonin and its metabolic derivatives, melatonin is a better ˙OOH and ˙OOCCl3 radicals scavenger than AMK, AMK is better than melatonin for scavenging ˙OH radical, and AFMK is a poorer scavenger than AMK and melatonin [25]. Meanwhile, studies also reported that melatonin could promote the generation of ROS at pharmacological concentrations in several vitro cultured cells [26]. Melatonin’s anti-inflammatory effects are related to the limitation of nitric oxide, prostanoids, leukotrienes, inflammatory cytokines, chemokines, adhesion molecules production, and the modulation of a number of transcription factors such as nuclear factor-jB (NF-jB), hypoxia-inducible factor (HIF), nuclear factor erythroid 2-related factor 2, etc. [19]. Moreover, its antiproliferative activity has been demonstrated in numerous cell systems, such as breast cancer cells, prostate cancer cells, human umbilical vein endothelial cells (HUVECs) [20, 27, 28]. Although melatonin has been found to reduce inflammation and inhibit cell proliferation, whether melatonin has effects on HPH has never been reported. The aim of this study was to investigate the possible effect and the underlying mechanism of melatonin on HPH.
Materials and methods Reagents and antibodies Melatonin was purchased from Sigma (St. Louis, MO, USA). High-glucose Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and Trizol reagent were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against phospho-Akt, phospho-ERK1/2, total-Akt, total-ERK1/2, HIF-1a, NF-jB p65, PCNA, GAPDH, b-actin, horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit were purchased from Cell Signaling (Beverly, MA, USA). Anti-a-SMA was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Animal experiments Twenty-eight male Sprague–Dawley rats (200–250 g) were obtained from the animal center of Dalian Medical University. The rats were randomly divided into four groups (seven rats per group): (1) normoxia group; (2) normoxia group treated with melatonin; (3) hypoxia group; and (4) hypoxia group treated with melatonin. For group (2) and (4), each rat received melatonin (15 mg/kg/day) via intraperitoneal injection every morning prior to hypoxia exposure for 1 wk before and for the entire 4 wk of normoxia or hypoxia exposure. The rats designated for exposure to chronic hypoxia were housed intermittently in a hypobaric hypoxia chamber for 10 hr/day lasting 4 wk. The hypoxic chamber was flushed with room air and 100% N2 to maintain 10% O2 concentration. The normoxic rats were housed at room air.
Hemodynamic experiments After 4 wk hypoxia exposure, the rats were anesthetized with 4.8% tribromoethanol (7.5 mL/kg via intraperitoneal injection). A polyethylene catheter linked to a transducer was inserted through the right jugular vein into the right ventricle. Then the right ventricle systolic pressure (RVSP) was recorded using Power Lab Software (ADI Instruments). After that, the thorax was opened, and lungs were perfused free of blood via the right ventricle. Then lungs together with heart were removed to the culture plate with cold PBS. The right ventricle (RV) and left ventricle plus septum (LV+S) were collected, and the weight ratio of (RV/LV+S) was calculated as an index of RV hypertrophy. The lungs were dissected into 4-mm-thick slices and placed in 4% paraformaldehyde solution for 72 hr. The left lungs were stored at 80°C for subsequent experiments. Morphological investigation The lung slices were embedded in paraffin and cut into 4-lm-thick sections and stained with hematoxylin and eosin as we previously reported [29]. Morphologic changes in the small pulmonary artery (50–200 lm) were detected using a Zeiss microscope digital camera (Carl Zeiss, Jena, Germany). Five vessels with approximate round shape were obtained from each rat, total 35 arteries were got from every group. The outside diameter, inside diameter, medial wall area, and total vessel area of pulmonary arterioles were measured. The percent medial wall thickness (WT%) and percent medial wall area (WA%) were calculated to present pulmonary vascular structure remodeling. WT% = (outside diameterinside diameter)/(outside diameter) 9 100; WA% = (medial wall area)/(total vessel area) 9 100. Immunohistochemical staining Sections were deparaffinized, rehydrated, retrieved the antigens, and then incubated with 1% H2O2 in methanol for 15 min at room temperature to block endogenous peroxidase. After blocked with 5% bovine serum albumin, sections were incubated overnight with anti-a-SMA mouse monoclonal antibody (1:500 dilution), anti-PCNA mouse monoclonal antibody (1:2000 dilution), anti-HIF-1a mouse monoclonal antibody (1:100 dilution), or anti-NFjB p65 rabbit monoclonal antibody (1:800 dilution) at 4°C. Then, a biotinylated anti-mouse or rabbit IgG antibody and an avidin-biotinylated peroxidase complex were applied with 3,3-diaminobenzidine as a peroxidase substrate. Immunoreactivity was visualized using diaminobenzidine. After that a light hematoxylin counterstain was applied. For the a-smooth muscle actin (a-SMA), a marker for smooth muscle cells (SMCs) to demonstrate the expression of SMCs, quantitative immunohistochemical assessments were performed as previously reported [30]. The integrated optical density, which relates to immunohistochemical staining intensity and area, was calculated in the vessel wall of pulmonary arterioles. In addition, to 443
Jin et al. determine cell proliferation in pulmonary arterial media walls, the proliferating cell nuclear antigen (PCNA) was originally identified as an antigen that is expressed in the nuclei of cells during the DNA synthesis phase of the cell cycle [31]. The PCNA-positive cells were brown in nuclei. The numbers of positive cells and all the cells in the pulmonary arterial media walls were counted. The percentage of positive cell number was calculated as positive cells/all cells. Primary cells culture and in vitro hypoxia Primary PASMCs were cultured from explants. The pulmonary arteries were isolated from adult male Sprague– Dawley rats, and then, the outer and inner membranes were removed under anatomy microscope. Minced arteries were placed in a culture flask. The culture flask was overturned placed and DMEM supplemented with 100 U/mL penicillin, and 10% FBS was added in. After 2–3 hr, the culture flask was carefully turned over, and the medium immersed the tissue pieces. The PASMCs grew out in 5–7 days, and cell passage was performed when the cells grew to 70% confluence. PASMCs were identified by immunocytochemical staining for a-SMA at each passage. Cells were used for experiments between passages 3 and 6. For all experiments, cells were exposed to 21% or 1% O2 for 48 hr. MTT assay The cell viability of PASMCs under normoxia and hypoxia was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) assay. Cells were seeded at a density of 1 9 104 cells per well in a 96-well culture plate, and 4 dosages of melatonin (1 nM, 100 nM, 10 lM, 1 mM) was added in. After cultured for 48 hr, 10 lL MTT (5 mg/mL) was added to each well of 96-well and the cells were incubated for another 4 hr; then, dimethyl sulfoxide was added in. The optical density values were detected at 490 nm wavelength using a spectrophotometer. Western blotting analysis PASMCs and lung homogenates were lysed in a protein extraction buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM NaF, 5 mM EDTA (pH 8.0), 1 mM sodium orthovanadate. The protease inhibitor of phenylmethylsulfonyl fluoride (PMSF, 1 mM) was added to the buffer in advance. The samples were separated on 10% SDS-polyacrylamide gels, and then transferred to a nitrocellulose membrane. The primary antibodies were phosphorylation of ERK1/2 (p-ERK1/2) antibody (1:1000), phosphorylation of Akt (p-Akt) antibody (1:1000), and NF-jB p65 antibody (1:1000). The signals were detected by ECL kit (Amersham Biosciences, Little Chalfont, UK). Quantitative real-time RT-PCR analysis Total RNAs of lung tissues were extracted using Trizol agent. Total RNAs were reverse-transcribed with oligo-dT 444
primers (TaKaRa Bio, Shiga, Japan). Quantitative realtime RT-PCR (qRT-PCR) was performed with the use of the Applied Lightcycler 2.0 detection system (Roche Applied Science, Mannheim, Germany) and the SYBR Green I reagent following the manufacturers’ instructions. PCR program was as follows: 94°C for 30 s, 40 cycles of 94°C for 5 s, 56°C for 30 s, 72°C for 20 s. The relative amount of the mRNA expression for hypoxia-inducible factor-1a (HIF-1a) was represented using the 2DDCt value. The primer pairs for HIF-1a PCR (138 bp) were (forward) 50 -TGAGCTCACATCTTGATAAAGCTTCT-30 and (reverse) 50 -GGGCTTTCAGATAAAAACAGTCCAT-30 , and for the housekeeping gene GAPDH (359 bp) were (forward) 50 -TGAAGGTCGGTGTGAACGGATTTG-30 and (reverse) 50 -GGCGGAGATGATGACCCTTTTG-30 , respectively. Statistical analyses Results were expressed as means S.E.M. Statistical analysis was performed by SPSS (version 11.5; SPSS Inc., Chicago, IL, USA). Statistical comparisons between groups were performed using ANOVA with a Holms– Sidak post hoc test. Significant difference was accepted at P < 0.05.
Results After exposure to hypoxia for 4 wk, the average RVSP of hypoxia group was increased significantly compared with the normoxia group. However, the average RVSP of hypoxia treated with melatonin group was much lower than that of hypoxia alone group (Fig. 1A). In accordance with the RVSP, hypoxia-induced elevation of the ratio of RV/LV+S was inhibited by the application of melatonin (Fig. 1B). To investigate the effect of melatonin on hypoxia-induced pulmonary artery remodeling, WT% and WA% of pulmonary arterioles which stained by hematoxylin and eosin were evaluated. As shown in Fig. 2, hypoxia markedly elevated WT% and WA%. However, WT% and WA% in hypoxia group treated with melatonin were much lower than in the hypoxia group. Next, to determine the cellular basis for the increased thickness and area of pulmonary arterioles, the hyperplastic smooth muscularization in lung sections was evaluated using an antibody against a-SMA (Fig. 3). Integrated optical density value of a-SMA in hypoxia group was significantly higher than in normoxia group. Thus, increased thickness and area of pulmonary arterioles are associated with enhanced proliferation of SMCs. However, with the treatment of melatonin, the integrated optical density value of a-SMA was reduced significantly. In addition, the cell proliferation in pulmonary vessels was determined. Increased PCNA-positive cells were detected primarily in the medial wall of pulmonary arterioles (Fig. 4). The percentage of PCNA-positive cells in hypoxia group was significantly higher than in normoxia group. However, increased cell proliferation in hypoxia group was inhibited markedly by treatment of melatonin. With the hope of learning whether melatonin could reverse the hypoxia-induced upregulation of HIF-1a, expression of HIF-1a in the medial wall of pulmonary
Melatonin attenuates HPH development (A)
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Fig. 1. Right ventricular systolic pressure (RVSP) in rats exposed to normoxia or hypoxia (A). Weight ratio of the right ventricular wall (RV) and the leftventricular wall (LV) and septum (S) [RV/(LV+S)] in rats exposed to normoxia or hypoxia (B). MLT means melatonin. Values are means S.E.M., *P < 0.01 compared with normoxia group. # P < 0.01 compared with hypoxia group (n = 7).
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Fig. 2. Hematoxylin and eosin staining of pulmonary arterioles (original magnification 920) (A). Percentage of medial wall thickness (WT%) of pulmonary arterioles (B). Percentage of medial wall area (WA%) of pulmonary arterioles (C). MLT means melatonin. Values are means S.E.M., *P < 0.01 compared with normoxia group. #P < 0.01 compared with hypoxia group (n = 7).
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(B) Fig. 3. Immunohistochemical staining of a-SMA of pulmonary arterioles (original magnification 920) (A). Bar graph shows the quantitative analysis of optical density (OD) value of a-SMA immunoreactivity in pulmonary arterioles (B). MLT means melatonin. Values are means S.E.M., *P < 0.01 compared with normoxia group. #P < 0.01 compared with hypoxia group (n = 7).
arterioles and the mRNA levels of HIF-1a in the four experimental groups were compared. Immunohistochemical staining showed that HIF-1a expression in the medial wall of pulmonary arterioles was overexpressed under hypoxic conditions, and mainly expressed in the nucleus, whereas melatonin could reduce HIF-1a expression significantly (Fig. 5A). In the whole lungs analysis, HIF-1a mRNA levels were significantly elevated on hypoxic
exposure compared with normoxic controls; however, HIF-1a mRNA levels were obviously downregulated by the treatment of melatonin (Fig. 5B). NF-jB is a protein transcription factor that is required for maximal transcription of many proinflammatory molecules which are important in the generation of inflammation [32]. In this study, immunohistochemical staining showed that hypoxia significantly increased NF-jB p65 445
Jin et al. (A)
(B) Fig. 4. Immunohistochemical staining of PCNA of pulmonary arterioles (original magnification 920) (A). Bar graph shows that the number of PCNA-positive cells relative to the total smooth muscle cells in the medial wall of pulmonary arterioles (%) (B). MLT means melatonin. Values are means S.E.M., *P < 0.01 compared with normoxia group. #P < 0.01 compared with hypoxia group (n = 7).
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Fig. 5. Immunohistochemical staining of HIF-1a of pulmonary arterioles (original magnification 920). HIF-1a was expressed under hypoxic conditions mainly in the nucleus, whereas melatonin decreased HIF-1a expression significantly (A). Bar graph shows the relative expression of HIF-1a mRNA levels in rat lungs (B). MLT means melatonin. Values are means S.E.M., *P < 0.01 compared with normoxia group. # P < 0.01 compared with hypoxia group (n = 3).
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expression in rat lungs. Especially in bronchiolar epithelial cells and pulmonary artery endothelial cells, the positive cells for NF-jB p65 staining were much higher in the hypoxia group compared with the normoxia group. With the treatment of melatonin, the staining of NF-jB p65 was reduced notably in rat lungs (Fig. 6A). Western blotting results also demonstrated that NF-jB p65 expression was upregulated in the hypoxia group (Fig. 6B). However, melatonin suppressed NF-jB p65 expression in rat lungs markedly. Fig. 7 showed the results of MTT assay. Optical density (OD) which represents the proliferation of PASMCs was significantly elevated in hypoxia compared with the cells under normoxic conditions. However, 100 nM melatonin significantly inhibited cell proliferation, and this inhibitory effect on PASMCs proliferation was enhanced with increasing melatonin concentrations. Melatonin of 1 mM had the greatest antiproliferative effect on PASMCs in both normoxic and hypoxic conditions. To further investigate the cellular and molecular mechanisms underlying melatonin-induced PASMCs growth inhibition, we evaluated the effect of melatonin on the 446
PI3K/AKT and ERK1/2 pathways. Western blotting results showed that hypoxia notably increased the p-AKT and p-ERK1/2 protein levels in PASMCs. In both normoxic and hypoxic conditions, only 1 mM melatonin repressed the protein expression of p-AKT. However, melatonin in 100 nM repressed the protein expression of p-ERK1/2, and in 1 mM had the greatest inhibition effect on the protein expression of p-ERK1/2 (Fig. 8).
Discussion We successfully established an animal model of hypoxiainduced pulmonary hypertension by exposing rats to normobaric hypoxia for 4 wk. Consistent with previous reports [33], this rat model exhibited significant structural remodeling of small pulmonary vessels and elevated pulmonary artery pressure. Melatonin supplementation markedly inhibited the elevation of RVSP and [RV/(LV+S)], as well as the increased WT% and WA% in hypoxic rats. Furthermore, the increased cellular proliferation in pulmonary arteries as evident by increased PCNA-positive cells, and enhanced a-SMA, the marker for SMCs,
Melatonin attenuates HPH development (A)
Fig. 6. Immunohistochemical staining of NF-jB p65 of rat lungs (original magnification 920). The positive cells for NF-jB p65 staining in the hypoxia group was significantly increased, whereas melatonin decreased the positive cells markedly (A). Western blotting analysis of NF-jB p65 protein levels in rat lungs (B). Bar graph shows the statistic results of NF-jB p65 protein levels (C). MLT means melatonin. Values are means S.E.M., *P < 0.01 compared with normoxia group. #P < 0.01 compared with hypoxia group (n = 3).
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immunoreactivity in pulmonary arteries were also inhibited by melatonin. These results indicated that melatonin could prevent pulmonary artery pressure elevation, right ventricular hypertrophy, and vascular remodeling in a chronic hypoxia rat model. Substantial evidence is accumulating to suggest that inflammation plays an important role in the pathogenesis of PH. Moreover, inflammatory processes are increasingly recognized as major pathogenic components of pulmonary vascular remodeling [34]. Hypoxia stimulates specific inflammatory responses, including the pronounced perivascular inflammatory cell infiltration, and a marked increase in the expression of different cytokines, chemokines, and adhesion molecules [9, 35]. NF-jB, as a protein transcription factor, is required for maximal transcription
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of numerous proinflammatory molecules, such as adhesion molecules, enzymes, and cytokines [32]. Previous studies reported that NF-jB played a pivotal role in the pathogenesis of pulmonary hypertension and inhibition of NFjB in the lungs could prevent pulmonary hypertension [36, 37]. Furthermore, it has been confirmed that melatonin modulates the NF-jB signaling pathway during inflammation [38]. And we have previously demonstrated that melatonin could suppress proinflammatory mediators and enhance the antitumor effect of drug by targeting multiple signaling, including inhibition of NF-jB activation [39, 40]. In this study, immunohistochemical staining and Western blotting results showed that the increased expression of NF-jB in rat lungs caused by hypoxia was significantly downregulated by melatonin. 447
Jin et al. The pulmonary vascular medial hyperplasia mainly due to excessive PASMCs proliferation is also a key factor in the pathogenesis of PH [1]. Increasing amounts of evidence have shown that hypoxia-induced abnormal proliferation of PASMCs is one of the major causes for hypoxic pulmonary arterial remodeling [11, 41]. The ERK1/2 and PI3K/Akt signaling pathways have been recognized to mediate a wide range of functions, including proliferation, growth, and survival [42–44]. Besides, it has been demonstrated that they were crucial in mediating vascular smooth muscle cell proliferation in response to hypoxia exposure [45, 46]. Consistent with this notion, our study showed that hypoxia significantly increased the protein
Fig. 7. Proliferation of PASMCs was measured by the MTT method in different concentrations of melatonin under normoxic and hypoxic conditions. Values are means S.E.M., *P < 0.01 compared with normoxia control. #P < 0.05 compared with normoxia control. MP < 0.05 compared with hypoxia control (n = 3).
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expression of p-ERK1/2 and p-Akt in PASMCs. A Previous report has been demonstrated that melatonin reduced the proliferation of HUVECs through markedly inhibiting the activation of ERK1/2 and PI3K/Akt [47]. So far, no report is available about the effect of melatonin on the SMCs proliferation. Here, we discovered that melatonin significantly inhibited the hypoxia-induced proliferation of PASMCs and the levels of p-ERK1/2 and p-Akt in dosage-dependent manner. HIF-1a as a critical oxygen-sensitive transcriptional factor participates in many physiological and pathological processes including PH [48]. Under normoxic conditions, HIF-1a protein is ubiquitinated and subjected to proteasomal degradation. However, exposure of PASMCs to hypoxia causes increased HIF-1a protein levels and HIF1a DNA-binding activity [49]. Moreover, HIF-1a can promote hypoxia-induced proliferation of vascular smooth muscle cells and plays an important role in hypoxiainduced pulmonary vascular remodeling [50, 51]. About the effect of melatonin on HIF-1a, melatonin was reported to suppress tumor progression through the inhibition of HIF-1a and vascular endothelial growth factor (VEGF) mediated angiogenesis in the tumor [12, 52]. Herein, our data showed that melatonin obviously reversed the hypoxia-induced expression of HIF-1a in the medial wall of pulmonary arterioles and level of HIF-1a mRNA in lung tissues. In this study, we showed that hypoxia-induced high expression of HIF-1a, p-ERK1/2, p-Akt might contribute to the proliferation of PASMCs. For the relation between HIF-1a and ERK1/2, Akt signaling, it is well known that overexpression of HIF-1a can lead to the activation of various signaling molecules, including ERK1/2 and Akt [53]. Additionally, ERK1/2 and Akt signaling also can facilitate the activation of HIF-1a protein [54]. Therefore, both streams from ERK1/2, Akt to HIF-1a, and from HIF-1a to ERK1/2, Akt may participate in the PASMCs
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Fig. 8. Representative Western blotting analysis of p-AKT and p-ERK1/2 protein levels in PASMCs in different concentrations of melatonin under normoxic and hypoxic conditions (A, B). Bar graph shows the statistic results of p-AKT and p-ERK1/2 protein levels, respectively (C, D). ‘Nor’ means normoxia; ‘Hyp’ means hypoxia; MLT means melatonin. Values are means S.E.M., *P < 0.01 compared with normoxia control. #P < 0.01 compared with normoxia control. MP < 0.01 compared with hypoxia control (n = 3).
Melatonin attenuates HPH development proliferation caused by hypoxia. The exactly mechanism of inhibition of PASMCs proliferation by melatonin and the association between HIF-1a and ERK1/2, Akt signaling need to be further investigated. Currently, the effect of melatonin on the prevention and treatment of cardiovascular diseases has been fully confirmed. Melatonin has beneficial effects on hypertension, cardiac reperfusion injury, cardiac hypertrophy, drug-mediated damage to the heart, and atherosclerosis [21–24]. Although endogenous melatonin production decreases with age and in cardiovascular disease, when exogenously administered, melatonin is quickly distributed throughout the organism and has beneficial effects on the cardiovascular system [15, 21, 55]. As a nontoxic, inexpensive, widely available, and safe drug, melatonin makes possible its long-term use [21]. Furthermore, these protective actions of melatonin may have potential clinical applicability for individuals with cardiovascular disease. Although melatonin has multiple beneficial effects on the cardiovascular system, to our knowledge, this is the first report that melatonin directly attenuates HPH development in vivo. In conclusion, melatonin supplementation prevents the changes in hemodynamics and pulmonary vascular remodeling that occur in rats exposed to chronic hypoxia. Moreover, our data also show that melatonin could attenuate the inflammation and the proliferation of PASMCs caused by hypoxia. These findings provide clues that melatonin might be used to prevent HPH and open new perspectives in the field of prevention of HPH.
Acknowledgements This work was supported by the funds from the Liaoning Provence Natural Science Foundation of China (2013023043). Authors who received the funding: Yuhui Yuan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conflict of interest The authors have stated no conflict of interest.
Author contributions HJ, WD, and YY designed the research study; HJ, YW, LZ, and PZ performed the research; HJ, YW, LZ, and WD analyzed the data; HJ, LL, and YY wrote the paper.
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© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Doi:10.1111/jpi.12184
Journal of Pineal Research
Melatonin attenuates hypoxic pulmonary hypertension by inhibiting the inflammation and the proliferation of pulmonary arterial smooth muscle cells Abstract: Hypoxia-induced inflammation and excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) play important roles in the pathological process of hypoxic pulmonary hypertension (HPH). Melatonin possesses anti-inflammatory and antiproliferative properties. However, the effect of melatonin on HPH remains unclear. In this study, adult Sprague– Dawley rats were exposed to intermittent chronic hypoxia for 4 wk to mimic a severe HPH condition. Hemodynamic and pulmonary pathomorphology data showed that chronic hypoxia significantly increased right ventricular systolic pressures (RVSP), weight of the right ventricle/left ventricle plus septum (RV/ LV+S) ratio, and median width of pulmonary arterioles. Melatonin attenuated the elevation of RVSP, RV/LV+S, and mitigated the pulmonary vascular structure remodeling. Melatonin also suppressed the hypoxia-induced high expression of proliferating cell nuclear antigen (PCNA), hypoxia-inducible factor-1a (HIF-1a), and nuclear factor-jB (NF-jB). In vitro, melatonin concentration-dependently inhibited the proliferation of PASMCs and the levels of phosphorylation of Akt and extracellular signal-regulated kinases1/2 (ERK1/2) caused by hypoxia. These results suggested that melatonin might potentially prevent HPH via anti-inflammatory and antiproliferative mechanisms.
Introduction Hypoxic pulmonary hypertension (HPH) is a serious disease with poor prognosis. It is characterized by hypoxiainduced pulmonary vasoconstriction, pulmonary vascular remodeling, and elevated pulmonary artery pressure [1–3]. According to the updated classification of pulmonary hypertension (PH) in the Fourth World Symposium on PH, HPH belongs to Group 3: pulmonary hypertension owing to lung diseases and/or hypoxia [4, 5]. In this form of PH, patients are commonly encountered with chronic obstructive pulmonary disease, interstitial lung disease, respiratory disorders with a mixed restrictive, and obstructive pattern [5]. Although HPH is very common, there is no specific treatment for this serious disease [6]. Pulmonary vasodilators such as phosphodiesterase-type 5 inhibitors, endothelin receptor antagonists, and prostanoids often nonselectively vasodilate the pulmonary vessels and ultimately lead to worsened gas exchange and hypoxemia [5, 7]. Therefore, hunting for novel effective pharmacologic treatments for HPH is urgent. Hypoxia-induced inflammation, endothelium dysfunction, production of reactive oxygen species (ROS), and proliferation of pulmonary artery smooth muscle cells (PASMCs) are all involved in the pathological process of HPH. Among them, inflammation participates in the initiation and progression of HPH by actively contributing 442
Haifeng Jin1,2, Yueyue Wang1, Lei Zhou1, Lu Liu1, Peng Zhang1, Wuguo Deng1,3 and Yuhui Yuan1 1
Institute of Cancer Stem Cell, The First Affiliated Hospital, Dalian Medical University Cancer Center, Dalian, China; 2Department of Anatomy, Qiqihar Medical University, Qiqihar, China; 3Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China Key words: hypoxia, inflammation, melatonin, pulmonary artery smooth muscle cells, pulmonary hypertension Address reprint requests to Yuhui Yuan, Institute of Cancer Stem Cell, First Affiliated Hospital, Dalian Medical University Cancer Center, 9 West Section of Lvshun South Road, Dalian 116044, China. E-mails: [email protected]; [email protected] Received August 18, 2014; Accepted September 21, 2014.
to chronic vasoconstriction and remodeling of the pulmonary vessel wall [8, 9]. Exposure of animals to even moderate hypoxia results in increased expression of inflammatory cytokines, chemokines, adhesion molecules, and accumulation of leukocytes in the lungs [1]. These factors disrupt endothelial membrane integrity and facilitate deregulation of vascular cell proliferation in PH [8]. In addition, the hypoxia-induced excessive proliferation of PASMCs is also a key factor, which leads to media thickening and vascular resistance, in the development of HPH [10, 11]. Melatonin, an indolamine, is a small lipophilic molecule and ubiquitous physiological mediator that is synthesized in the pineal gland [12]. In addition, using specific melatonin antibodies, melatonin had been found in multiple extrapineal tissues including the liver, spleen, stomach, intestine, thymus, cerebral cortex, striatum, lens, skin, etc., and most of these tissues express melatonin-synthesizing enzymes [13–15]. Numerous studies have shown that melatonin plays crucial roles in several vital physiological and pathological processes, such as regulation of circadian rhythms, inhibition of tumor growth and metastasis, inhibition of inflammation and cell proliferation [16–20]. In the cardiovascular system, melatonin has protective effects through the free radical scavenger activity and antioxidant properties [21–24]. More recently, it has been confirmed that melatonin’s metabolic derivatives,
Melatonin attenuates HPH development N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), and N1-acetyl-5-methoxykynuramine (AMK), also possess free radical scavenger activity and antioxidant properties, which makes melatonin more effective and exerts continuous protection in oxidative damage [25, 26]. For the relative free radical scavenging activity of melatonin and its metabolic derivatives, melatonin is a better ˙OOH and ˙OOCCl3 radicals scavenger than AMK, AMK is better than melatonin for scavenging ˙OH radical, and AFMK is a poorer scavenger than AMK and melatonin [25]. Meanwhile, studies also reported that melatonin could promote the generation of ROS at pharmacological concentrations in several vitro cultured cells [26]. Melatonin’s anti-inflammatory effects are related to the limitation of nitric oxide, prostanoids, leukotrienes, inflammatory cytokines, chemokines, adhesion molecules production, and the modulation of a number of transcription factors such as nuclear factor-jB (NF-jB), hypoxia-inducible factor (HIF), nuclear factor erythroid 2-related factor 2, etc. [19]. Moreover, its antiproliferative activity has been demonstrated in numerous cell systems, such as breast cancer cells, prostate cancer cells, human umbilical vein endothelial cells (HUVECs) [20, 27, 28]. Although melatonin has been found to reduce inflammation and inhibit cell proliferation, whether melatonin has effects on HPH has never been reported. The aim of this study was to investigate the possible effect and the underlying mechanism of melatonin on HPH.
Materials and methods Reagents and antibodies Melatonin was purchased from Sigma (St. Louis, MO, USA). High-glucose Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and Trizol reagent were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against phospho-Akt, phospho-ERK1/2, total-Akt, total-ERK1/2, HIF-1a, NF-jB p65, PCNA, GAPDH, b-actin, horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit were purchased from Cell Signaling (Beverly, MA, USA). Anti-a-SMA was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Animal experiments Twenty-eight male Sprague–Dawley rats (200–250 g) were obtained from the animal center of Dalian Medical University. The rats were randomly divided into four groups (seven rats per group): (1) normoxia group; (2) normoxia group treated with melatonin; (3) hypoxia group; and (4) hypoxia group treated with melatonin. For group (2) and (4), each rat received melatonin (15 mg/kg/day) via intraperitoneal injection every morning prior to hypoxia exposure for 1 wk before and for the entire 4 wk of normoxia or hypoxia exposure. The rats designated for exposure to chronic hypoxia were housed intermittently in a hypobaric hypoxia chamber for 10 hr/day lasting 4 wk. The hypoxic chamber was flushed with room air and 100% N2 to maintain 10% O2 concentration. The normoxic rats were housed at room air.
Hemodynamic experiments After 4 wk hypoxia exposure, the rats were anesthetized with 4.8% tribromoethanol (7.5 mL/kg via intraperitoneal injection). A polyethylene catheter linked to a transducer was inserted through the right jugular vein into the right ventricle. Then the right ventricle systolic pressure (RVSP) was recorded using Power Lab Software (ADI Instruments). After that, the thorax was opened, and lungs were perfused free of blood via the right ventricle. Then lungs together with heart were removed to the culture plate with cold PBS. The right ventricle (RV) and left ventricle plus septum (LV+S) were collected, and the weight ratio of (RV/LV+S) was calculated as an index of RV hypertrophy. The lungs were dissected into 4-mm-thick slices and placed in 4% paraformaldehyde solution for 72 hr. The left lungs were stored at 80°C for subsequent experiments. Morphological investigation The lung slices were embedded in paraffin and cut into 4-lm-thick sections and stained with hematoxylin and eosin as we previously reported [29]. Morphologic changes in the small pulmonary artery (50–200 lm) were detected using a Zeiss microscope digital camera (Carl Zeiss, Jena, Germany). Five vessels with approximate round shape were obtained from each rat, total 35 arteries were got from every group. The outside diameter, inside diameter, medial wall area, and total vessel area of pulmonary arterioles were measured. The percent medial wall thickness (WT%) and percent medial wall area (WA%) were calculated to present pulmonary vascular structure remodeling. WT% = (outside diameterinside diameter)/(outside diameter) 9 100; WA% = (medial wall area)/(total vessel area) 9 100. Immunohistochemical staining Sections were deparaffinized, rehydrated, retrieved the antigens, and then incubated with 1% H2O2 in methanol for 15 min at room temperature to block endogenous peroxidase. After blocked with 5% bovine serum albumin, sections were incubated overnight with anti-a-SMA mouse monoclonal antibody (1:500 dilution), anti-PCNA mouse monoclonal antibody (1:2000 dilution), anti-HIF-1a mouse monoclonal antibody (1:100 dilution), or anti-NFjB p65 rabbit monoclonal antibody (1:800 dilution) at 4°C. Then, a biotinylated anti-mouse or rabbit IgG antibody and an avidin-biotinylated peroxidase complex were applied with 3,3-diaminobenzidine as a peroxidase substrate. Immunoreactivity was visualized using diaminobenzidine. After that a light hematoxylin counterstain was applied. For the a-smooth muscle actin (a-SMA), a marker for smooth muscle cells (SMCs) to demonstrate the expression of SMCs, quantitative immunohistochemical assessments were performed as previously reported [30]. The integrated optical density, which relates to immunohistochemical staining intensity and area, was calculated in the vessel wall of pulmonary arterioles. In addition, to 443
Jin et al. determine cell proliferation in pulmonary arterial media walls, the proliferating cell nuclear antigen (PCNA) was originally identified as an antigen that is expressed in the nuclei of cells during the DNA synthesis phase of the cell cycle [31]. The PCNA-positive cells were brown in nuclei. The numbers of positive cells and all the cells in the pulmonary arterial media walls were counted. The percentage of positive cell number was calculated as positive cells/all cells. Primary cells culture and in vitro hypoxia Primary PASMCs were cultured from explants. The pulmonary arteries were isolated from adult male Sprague– Dawley rats, and then, the outer and inner membranes were removed under anatomy microscope. Minced arteries were placed in a culture flask. The culture flask was overturned placed and DMEM supplemented with 100 U/mL penicillin, and 10% FBS was added in. After 2–3 hr, the culture flask was carefully turned over, and the medium immersed the tissue pieces. The PASMCs grew out in 5–7 days, and cell passage was performed when the cells grew to 70% confluence. PASMCs were identified by immunocytochemical staining for a-SMA at each passage. Cells were used for experiments between passages 3 and 6. For all experiments, cells were exposed to 21% or 1% O2 for 48 hr. MTT assay The cell viability of PASMCs under normoxia and hypoxia was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) assay. Cells were seeded at a density of 1 9 104 cells per well in a 96-well culture plate, and 4 dosages of melatonin (1 nM, 100 nM, 10 lM, 1 mM) was added in. After cultured for 48 hr, 10 lL MTT (5 mg/mL) was added to each well of 96-well and the cells were incubated for another 4 hr; then, dimethyl sulfoxide was added in. The optical density values were detected at 490 nm wavelength using a spectrophotometer. Western blotting analysis PASMCs and lung homogenates were lysed in a protein extraction buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM NaF, 5 mM EDTA (pH 8.0), 1 mM sodium orthovanadate. The protease inhibitor of phenylmethylsulfonyl fluoride (PMSF, 1 mM) was added to the buffer in advance. The samples were separated on 10% SDS-polyacrylamide gels, and then transferred to a nitrocellulose membrane. The primary antibodies were phosphorylation of ERK1/2 (p-ERK1/2) antibody (1:1000), phosphorylation of Akt (p-Akt) antibody (1:1000), and NF-jB p65 antibody (1:1000). The signals were detected by ECL kit (Amersham Biosciences, Little Chalfont, UK). Quantitative real-time RT-PCR analysis Total RNAs of lung tissues were extracted using Trizol agent. Total RNAs were reverse-transcribed with oligo-dT 444
primers (TaKaRa Bio, Shiga, Japan). Quantitative realtime RT-PCR (qRT-PCR) was performed with the use of the Applied Lightcycler 2.0 detection system (Roche Applied Science, Mannheim, Germany) and the SYBR Green I reagent following the manufacturers’ instructions. PCR program was as follows: 94°C for 30 s, 40 cycles of 94°C for 5 s, 56°C for 30 s, 72°C for 20 s. The relative amount of the mRNA expression for hypoxia-inducible factor-1a (HIF-1a) was represented using the 2DDCt value. The primer pairs for HIF-1a PCR (138 bp) were (forward) 50 -TGAGCTCACATCTTGATAAAGCTTCT-30 and (reverse) 50 -GGGCTTTCAGATAAAAACAGTCCAT-30 , and for the housekeeping gene GAPDH (359 bp) were (forward) 50 -TGAAGGTCGGTGTGAACGGATTTG-30 and (reverse) 50 -GGCGGAGATGATGACCCTTTTG-30 , respectively. Statistical analyses Results were expressed as means S.E.M. Statistical analysis was performed by SPSS (version 11.5; SPSS Inc., Chicago, IL, USA). Statistical comparisons between groups were performed using ANOVA with a Holms– Sidak post hoc test. Significant difference was accepted at P < 0.05.
Results After exposure to hypoxia for 4 wk, the average RVSP of hypoxia group was increased significantly compared with the normoxia group. However, the average RVSP of hypoxia treated with melatonin group was much lower than that of hypoxia alone group (Fig. 1A). In accordance with the RVSP, hypoxia-induced elevation of the ratio of RV/LV+S was inhibited by the application of melatonin (Fig. 1B). To investigate the effect of melatonin on hypoxia-induced pulmonary artery remodeling, WT% and WA% of pulmonary arterioles which stained by hematoxylin and eosin were evaluated. As shown in Fig. 2, hypoxia markedly elevated WT% and WA%. However, WT% and WA% in hypoxia group treated with melatonin were much lower than in the hypoxia group. Next, to determine the cellular basis for the increased thickness and area of pulmonary arterioles, the hyperplastic smooth muscularization in lung sections was evaluated using an antibody against a-SMA (Fig. 3). Integrated optical density value of a-SMA in hypoxia group was significantly higher than in normoxia group. Thus, increased thickness and area of pulmonary arterioles are associated with enhanced proliferation of SMCs. However, with the treatment of melatonin, the integrated optical density value of a-SMA was reduced significantly. In addition, the cell proliferation in pulmonary vessels was determined. Increased PCNA-positive cells were detected primarily in the medial wall of pulmonary arterioles (Fig. 4). The percentage of PCNA-positive cells in hypoxia group was significantly higher than in normoxia group. However, increased cell proliferation in hypoxia group was inhibited markedly by treatment of melatonin. With the hope of learning whether melatonin could reverse the hypoxia-induced upregulation of HIF-1a, expression of HIF-1a in the medial wall of pulmonary
Melatonin attenuates HPH development (A)
(B)
Fig. 1. Right ventricular systolic pressure (RVSP) in rats exposed to normoxia or hypoxia (A). Weight ratio of the right ventricular wall (RV) and the leftventricular wall (LV) and septum (S) [RV/(LV+S)] in rats exposed to normoxia or hypoxia (B). MLT means melatonin. Values are means S.E.M., *P < 0.01 compared with normoxia group. # P < 0.01 compared with hypoxia group (n = 7).
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Fig. 2. Hematoxylin and eosin staining of pulmonary arterioles (original magnification 920) (A). Percentage of medial wall thickness (WT%) of pulmonary arterioles (B). Percentage of medial wall area (WA%) of pulmonary arterioles (C). MLT means melatonin. Values are means S.E.M., *P < 0.01 compared with normoxia group. #P < 0.01 compared with hypoxia group (n = 7).
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(B) Fig. 3. Immunohistochemical staining of a-SMA of pulmonary arterioles (original magnification 920) (A). Bar graph shows the quantitative analysis of optical density (OD) value of a-SMA immunoreactivity in pulmonary arterioles (B). MLT means melatonin. Values are means S.E.M., *P < 0.01 compared with normoxia group. #P < 0.01 compared with hypoxia group (n = 7).
arterioles and the mRNA levels of HIF-1a in the four experimental groups were compared. Immunohistochemical staining showed that HIF-1a expression in the medial wall of pulmonary arterioles was overexpressed under hypoxic conditions, and mainly expressed in the nucleus, whereas melatonin could reduce HIF-1a expression significantly (Fig. 5A). In the whole lungs analysis, HIF-1a mRNA levels were significantly elevated on hypoxic
exposure compared with normoxic controls; however, HIF-1a mRNA levels were obviously downregulated by the treatment of melatonin (Fig. 5B). NF-jB is a protein transcription factor that is required for maximal transcription of many proinflammatory molecules which are important in the generation of inflammation [32]. In this study, immunohistochemical staining showed that hypoxia significantly increased NF-jB p65 445
Jin et al. (A)
(B) Fig. 4. Immunohistochemical staining of PCNA of pulmonary arterioles (original magnification 920) (A). Bar graph shows that the number of PCNA-positive cells relative to the total smooth muscle cells in the medial wall of pulmonary arterioles (%) (B). MLT means melatonin. Values are means S.E.M., *P < 0.01 compared with normoxia group. #P < 0.01 compared with hypoxia group (n = 7).
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Fig. 5. Immunohistochemical staining of HIF-1a of pulmonary arterioles (original magnification 920). HIF-1a was expressed under hypoxic conditions mainly in the nucleus, whereas melatonin decreased HIF-1a expression significantly (A). Bar graph shows the relative expression of HIF-1a mRNA levels in rat lungs (B). MLT means melatonin. Values are means S.E.M., *P < 0.01 compared with normoxia group. # P < 0.01 compared with hypoxia group (n = 3).
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expression in rat lungs. Especially in bronchiolar epithelial cells and pulmonary artery endothelial cells, the positive cells for NF-jB p65 staining were much higher in the hypoxia group compared with the normoxia group. With the treatment of melatonin, the staining of NF-jB p65 was reduced notably in rat lungs (Fig. 6A). Western blotting results also demonstrated that NF-jB p65 expression was upregulated in the hypoxia group (Fig. 6B). However, melatonin suppressed NF-jB p65 expression in rat lungs markedly. Fig. 7 showed the results of MTT assay. Optical density (OD) which represents the proliferation of PASMCs was significantly elevated in hypoxia compared with the cells under normoxic conditions. However, 100 nM melatonin significantly inhibited cell proliferation, and this inhibitory effect on PASMCs proliferation was enhanced with increasing melatonin concentrations. Melatonin of 1 mM had the greatest antiproliferative effect on PASMCs in both normoxic and hypoxic conditions. To further investigate the cellular and molecular mechanisms underlying melatonin-induced PASMCs growth inhibition, we evaluated the effect of melatonin on the 446
PI3K/AKT and ERK1/2 pathways. Western blotting results showed that hypoxia notably increased the p-AKT and p-ERK1/2 protein levels in PASMCs. In both normoxic and hypoxic conditions, only 1 mM melatonin repressed the protein expression of p-AKT. However, melatonin in 100 nM repressed the protein expression of p-ERK1/2, and in 1 mM had the greatest inhibition effect on the protein expression of p-ERK1/2 (Fig. 8).
Discussion We successfully established an animal model of hypoxiainduced pulmonary hypertension by exposing rats to normobaric hypoxia for 4 wk. Consistent with previous reports [33], this rat model exhibited significant structural remodeling of small pulmonary vessels and elevated pulmonary artery pressure. Melatonin supplementation markedly inhibited the elevation of RVSP and [RV/(LV+S)], as well as the increased WT% and WA% in hypoxic rats. Furthermore, the increased cellular proliferation in pulmonary arteries as evident by increased PCNA-positive cells, and enhanced a-SMA, the marker for SMCs,
Melatonin attenuates HPH development (A)
Fig. 6. Immunohistochemical staining of NF-jB p65 of rat lungs (original magnification 920). The positive cells for NF-jB p65 staining in the hypoxia group was significantly increased, whereas melatonin decreased the positive cells markedly (A). Western blotting analysis of NF-jB p65 protein levels in rat lungs (B). Bar graph shows the statistic results of NF-jB p65 protein levels (C). MLT means melatonin. Values are means S.E.M., *P < 0.01 compared with normoxia group. #P < 0.01 compared with hypoxia group (n = 3).
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immunoreactivity in pulmonary arteries were also inhibited by melatonin. These results indicated that melatonin could prevent pulmonary artery pressure elevation, right ventricular hypertrophy, and vascular remodeling in a chronic hypoxia rat model. Substantial evidence is accumulating to suggest that inflammation plays an important role in the pathogenesis of PH. Moreover, inflammatory processes are increasingly recognized as major pathogenic components of pulmonary vascular remodeling [34]. Hypoxia stimulates specific inflammatory responses, including the pronounced perivascular inflammatory cell infiltration, and a marked increase in the expression of different cytokines, chemokines, and adhesion molecules [9, 35]. NF-jB, as a protein transcription factor, is required for maximal transcription
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of numerous proinflammatory molecules, such as adhesion molecules, enzymes, and cytokines [32]. Previous studies reported that NF-jB played a pivotal role in the pathogenesis of pulmonary hypertension and inhibition of NFjB in the lungs could prevent pulmonary hypertension [36, 37]. Furthermore, it has been confirmed that melatonin modulates the NF-jB signaling pathway during inflammation [38]. And we have previously demonstrated that melatonin could suppress proinflammatory mediators and enhance the antitumor effect of drug by targeting multiple signaling, including inhibition of NF-jB activation [39, 40]. In this study, immunohistochemical staining and Western blotting results showed that the increased expression of NF-jB in rat lungs caused by hypoxia was significantly downregulated by melatonin. 447
Jin et al. The pulmonary vascular medial hyperplasia mainly due to excessive PASMCs proliferation is also a key factor in the pathogenesis of PH [1]. Increasing amounts of evidence have shown that hypoxia-induced abnormal proliferation of PASMCs is one of the major causes for hypoxic pulmonary arterial remodeling [11, 41]. The ERK1/2 and PI3K/Akt signaling pathways have been recognized to mediate a wide range of functions, including proliferation, growth, and survival [42–44]. Besides, it has been demonstrated that they were crucial in mediating vascular smooth muscle cell proliferation in response to hypoxia exposure [45, 46]. Consistent with this notion, our study showed that hypoxia significantly increased the protein
Fig. 7. Proliferation of PASMCs was measured by the MTT method in different concentrations of melatonin under normoxic and hypoxic conditions. Values are means S.E.M., *P < 0.01 compared with normoxia control. #P < 0.05 compared with normoxia control. MP < 0.05 compared with hypoxia control (n = 3).
(A)
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expression of p-ERK1/2 and p-Akt in PASMCs. A Previous report has been demonstrated that melatonin reduced the proliferation of HUVECs through markedly inhibiting the activation of ERK1/2 and PI3K/Akt [47]. So far, no report is available about the effect of melatonin on the SMCs proliferation. Here, we discovered that melatonin significantly inhibited the hypoxia-induced proliferation of PASMCs and the levels of p-ERK1/2 and p-Akt in dosage-dependent manner. HIF-1a as a critical oxygen-sensitive transcriptional factor participates in many physiological and pathological processes including PH [48]. Under normoxic conditions, HIF-1a protein is ubiquitinated and subjected to proteasomal degradation. However, exposure of PASMCs to hypoxia causes increased HIF-1a protein levels and HIF1a DNA-binding activity [49]. Moreover, HIF-1a can promote hypoxia-induced proliferation of vascular smooth muscle cells and plays an important role in hypoxiainduced pulmonary vascular remodeling [50, 51]. About the effect of melatonin on HIF-1a, melatonin was reported to suppress tumor progression through the inhibition of HIF-1a and vascular endothelial growth factor (VEGF) mediated angiogenesis in the tumor [12, 52]. Herein, our data showed that melatonin obviously reversed the hypoxia-induced expression of HIF-1a in the medial wall of pulmonary arterioles and level of HIF-1a mRNA in lung tissues. In this study, we showed that hypoxia-induced high expression of HIF-1a, p-ERK1/2, p-Akt might contribute to the proliferation of PASMCs. For the relation between HIF-1a and ERK1/2, Akt signaling, it is well known that overexpression of HIF-1a can lead to the activation of various signaling molecules, including ERK1/2 and Akt [53]. Additionally, ERK1/2 and Akt signaling also can facilitate the activation of HIF-1a protein [54]. Therefore, both streams from ERK1/2, Akt to HIF-1a, and from HIF-1a to ERK1/2, Akt may participate in the PASMCs
(B)
(D)
Fig. 8. Representative Western blotting analysis of p-AKT and p-ERK1/2 protein levels in PASMCs in different concentrations of melatonin under normoxic and hypoxic conditions (A, B). Bar graph shows the statistic results of p-AKT and p-ERK1/2 protein levels, respectively (C, D). ‘Nor’ means normoxia; ‘Hyp’ means hypoxia; MLT means melatonin. Values are means S.E.M., *P < 0.01 compared with normoxia control. #P < 0.01 compared with normoxia control. MP < 0.01 compared with hypoxia control (n = 3).
Melatonin attenuates HPH development proliferation caused by hypoxia. The exactly mechanism of inhibition of PASMCs proliferation by melatonin and the association between HIF-1a and ERK1/2, Akt signaling need to be further investigated. Currently, the effect of melatonin on the prevention and treatment of cardiovascular diseases has been fully confirmed. Melatonin has beneficial effects on hypertension, cardiac reperfusion injury, cardiac hypertrophy, drug-mediated damage to the heart, and atherosclerosis [21–24]. Although endogenous melatonin production decreases with age and in cardiovascular disease, when exogenously administered, melatonin is quickly distributed throughout the organism and has beneficial effects on the cardiovascular system [15, 21, 55]. As a nontoxic, inexpensive, widely available, and safe drug, melatonin makes possible its long-term use [21]. Furthermore, these protective actions of melatonin may have potential clinical applicability for individuals with cardiovascular disease. Although melatonin has multiple beneficial effects on the cardiovascular system, to our knowledge, this is the first report that melatonin directly attenuates HPH development in vivo. In conclusion, melatonin supplementation prevents the changes in hemodynamics and pulmonary vascular remodeling that occur in rats exposed to chronic hypoxia. Moreover, our data also show that melatonin could attenuate the inflammation and the proliferation of PASMCs caused by hypoxia. These findings provide clues that melatonin might be used to prevent HPH and open new perspectives in the field of prevention of HPH.
Acknowledgements This work was supported by the funds from the Liaoning Provence Natural Science Foundation of China (2013023043). Authors who received the funding: Yuhui Yuan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conflict of interest The authors have stated no conflict of interest.
Author contributions HJ, WD, and YY designed the research study; HJ, YW, LZ, and PZ performed the research; HJ, YW, LZ, and WD analyzed the data; HJ, LL, and YY wrote the paper.
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