| Persistent pulmonary hypertension of the newborn (PPHN) is a clinical syndrome characterized by abnormal pulmonary vascular tone, reactivity, and structure. A sustained elevation of pulmonary vascular resistance at birth leads to extrapulmonary right-to-left shunting of blood, and severe hypoxemia. PPHN patients are usually full-term or post-term infants who have had perinatal asphyxia, meconium aspiration, diaphragmatic hernia, pneumonia, or sepsis. Further research is required to fully understand the mechanism(s) behind these types of lung disorders to enable the development of effective treatments.Lung development during the saccular period occurs between embryonic day 17.5 and postnatal day 5 and the alveolar stage occurs during the postnatal weaning period in mouse and rat. During this time alveolar septation initiates and leads to a tremendous increase in the surface area of the lung. Exposure to hypoxia during this critical period can impair both alveolar and pulmonary vascular structure and function. Postnatal exposure to 10-15% oxygen during the first 3 weeks of life impairs lung development, characterized by decreased alveolarization and reduced lung vascular development. Deruelle et al. found that exposure of infant rats to hypoxia for 14 days impairs alveolarization as reflected by reduced radial alveolar counts, and decreased vascular volume density. The pathogenesis of pulmonary hypertension (PH) appears to involve an initial, active vasoconstriction of pulmonary resistance vessels that may progress to fixed luminal narrowing, elevated pulmonary vascular resistance (PVR), thrombi formation, and, ultimately, right heart failure and death. After the structural remodeling of pulmonary vessels has occurred, therapeutic interventions to restore PVR to normal levels are largely ineffective. Therefore, drug therapies targeting cellular pathways that mediate the PH and reversible rise in PVR may be advantageous.Ghrelin is a 28 amino acid peptide originally isolated from rat stomach as an endogenous ligand for the growth hormone (GH) secretagogue receptor (GHS-R). The ghrelin gene peptides include acylated ghrelin, unacylated ghrelin., and obestatin. Acylated ghrelin, exerts its central and peripheral effects through the GHS-Rla. Indeed, acylated ghrelin was demonstrated to act as an autocrine/paracrine factor, regulating cell proliferation and survival, apoptosis, inflammation, cardiovascular and gastric functions, metabolism, angiogenesis, development, and reproduction. Ghrelin is able to attenuate the development of pulmonary artery hypertension in a monocrotaline-treated adult animal model. However, this monocrotaline-induced PH model, including the response to potential therapeutic treatments, differs considerably from PH induced by chronic hypoxia (CH). A previous study has shown that ghrelin can directly stimulate the production of nitric oxide (NO) from vascular endothelial cells through PI 3-kinase-dependent signaling pathways that mimic the effects of insulin. PPHN in utero causes sustained alteration of fetal pulmonary artery endothelial cell (PAEC) phenotypes, as determined in vitro. Also, endothelial nitric oxide synthetase (eNOS) protein expression was decreased in PPHN PAECs and NO enhanced growth and tube formation was observed in PPHN PAECs. There is increasing evidence that ghrelin has a potent vasodilator effect. We hypothesize that ghrelin would prevent PPHN by altering signal transduction pathways.In this study, using a CH-induced PPHN model in rats, we first explored the molecular signaling cascades and gene expression patterns in lung tissue. We then addressed whether ghrelin protected neonatal rats from hypoxia-induced PH, including its effects on hemodynamics and pulmonary vasculature. Finally, we analyzed signaling transduction pathways regulated by ghrelin.Part I Multiplexed profiling of candidate genes for hypoxic pulmonary hypertension in neonatal rats using a PCR Array assay Aim:1. To establish PPHN animal model and assess pulmonary hemodynamics and morphometry.2. To screen multiplexed profiling of candidate genes for lung tissues of hypoxic pulmonary hypertension in neonatal rats by PCR Array assay.Methods:1. PPHN model and groupsHypoxic group:neonatal rats were exposed to 12% oxygen in a forced-air environmental chamber for 14 days.Normoxic group:rats were exposed to 21% oxygen (room air) for 14 days.2. Assessment of pulmonary hypertensionThe right ventricular systolic pressure (RVSP) was detected in rats after 14 days' hypoxic exposure. The degree of muscularization of pulmonary vessels was assessed by immunohistochemistry staining of a-SMA. The ratio of the right ventricle (RV) to the left ventricle plus septum (LV+S) was used to evaluate the hypertrophy of right ventricle. 3. PCR Array assay to screen multiplexed profiling of candidate genes for lung tissues of hypoxic pulmonary hypertension in neonatal rats.Results:1. RVSP increased significantly in neonatal rats after 14 days'hypoxic exposure than that of normoxic group (37.29±3.15 mmHg vs 20.02±1.02 mmHg, P<0.05). The number of muscular pulmonary vessels increased significantly in rats following 14 days'hypoxic exposure than in normoxic group. The ratio of RV/(LV+S) was also significantly increased after hypoxic exposure (P<0.01).2. Two signal transduction pathways were significantly upregulated by a threshold of?3.0 when hypoxic group compared with the normoxic group:Wnt pathway (lefl, 4.79; Wnt,3.87) and CREB pathway (Cypl9al,4.87; Egrl,7.79; Fos,3.86).Conclusions:1. We established PPHN model successfully.2. Wnt and CREB signaling pathway are most important signaling pathways in hypoxic pulmonary hypertension in newborns.Part?The mechanism of ghrelin ameliorates hypoxia-induced pulmonary hypertensionAim:1. To investigate the change of pulmonary vascular remodeling and right ventricle hypertrophy in neonatal rats after 14 days'hypoxic exposure after the ghrelin administration.2. Confirmatory real-time PCR assay for lung tissues of hypoxic pulmonary hypertension in neonatal rats after ghrelin administration.Methods:1. PPHN model and groups Rats were divided into four groups (n=10/each group):hypoxia and vehicle treatment (sc,0.2ml) daily for 14 days;hypoxia and ghrelin (Phoenix Biotech Co., Ltd., Beijing, China) treatment (sc,150?g/kg (Schwenke, et al.2008),0.2ml) daily for 14 days;Sham control, sham chamber was not subjected to hypoxia and was treated with the vehicle control (sc,0.2ml) daily for the same period.Ghrelin control (sc,150?g/kg,0.2ml) ghrelin treatment daily for 14 days in normoxic rats.2. The morphological changes of the small pulmonary arteries were examined by media wall thickness (a-SMAarea/LD) after ghrelin administration. The ratio of RV/(LV+S) was calculated to evaluate the hypertrophy of right ventricle. Serum active ghrelin were determined by ELISA. Wnt Pathway verification by qRT-PCR analysis and Western blot analysisResults:1. In the hypoxia group, a significant increase in mean RVSP (mRVSP), above the control value, was observed (37.29±3.15 mmHg versus 20.02±1.02 mmHg, P<0.05). Daily administration of ghrelin during hypoxia significantly attenuated the development of neonatal PH. There was also a significant increase in the RV/(LV+S) ratio in hypoxia group (0.51±0.09 versus 0.19±0.01, P<0.05), indicating RVH. Therefore, Ghrelin treatment reduced both the magnitude of PH and the RV/(LV+S) ratio. A significant decrease in the a-SMAarea/LD ratio was observed for ghrelin-treated rats (107.4±6.8; P=0.000) versus vehicle-treated hypoxia.2. The levels of active ghrelin measured in hypoxia+ghrelin animals versus hypoxia group were 43.75±4.94 and 27.38±5.29 pg/ml (P<0.05), respectively.3. We also found that the Wnt pathway (Birc5,5.05; Myc,3.09; Pparg,4.82) and the PI3K/Akt pathway (Fn1,3.60) were upregulated in hypoxia+ghrelin rats compared with hypoxia group. Wnt pathway (Birc5,-8.76; Myc,1.12; Pparg,-1.59) and the PI3K/ Akt pathway (Fn1,-2.19) when hypoxic group compared with the normoxic group.4. Notably, a significant increase in expression of p-GSK3?/GSK3?and?-catenin was detected in ghrelin treated CH rats. Surprisingly, significant differences in the ID of GHSR-1a were observed between the groups with and without ghrelin treatment in the pulmonary arteriole. The results were also verified by western blot analysis (hypoxia: 0.7±0.2 versus hypoxia+ghrelin group:1.6±0.3; P<0.05).Conclusions:1. Ghrelin ameliorates hypoxia-induced pulmonary hypertension, pulmonary vascular remodeling and right ventricular hypertrophy.2. The mechanisms of ghrelin ameliorates hypoxia-induced pulmonary hypertension involving PI3-K/Akt/GSK-3?and Wnt signaling pathway.3. GHSR-la receptor is involved as an intermediary through PI3-K/Akt/GSK-3?signaling pathway.Part III Ghrelin inhibits hypoxic pulmonary microvascular endothelial cell apoptosis and its p-GSK3?/GSK3?signaling pathwayAim:1. To observe the impact of ghrelin on apoptosis induced by hypoxia.2. To observe the changes in PI3-K/Akt/GSK-3?protein expression and distribution of?-catenin after ghrelin intervention.Methods:1. Rat pulmonary microvascular endothelial cells (RPMECs) were isolated using a modification of the technique.2. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labelling (TUNEL) assays. 3. Administration of Ghrelin Receptor Antagonist and PI3K/AKT Inhibitor, then p-Akt and p-GSK3?/7GSK3?Pathway verification by Western blot analysis.4. Luciferase activity was assayed 48 hours after transfection, using a dual-luciferase reporter assay system.Results:1. RPMECs formed a confluent monolayer of cobble stone-like cells, EC markers such as labeled anti-mouse CD31 antibody accounted for 95%.2. TUNEL showed that the hypoxia group, normoxia+ghrelin group and hypoxia+ ghrelin apoptosis rate were 2.77±0.28,0.92±0.07,1.63±0.19 respectively (P<0.05)3. Treatment of RPMECs with ghrelin rapidly activated p-Akt in a time and dose-dependent manner. GSK3?phosphorylation was increased after 30min of ghrelin treatment and lasted for 120min. The inhibition of ghrelin receptor with [D-Lys3]-GHRP-6 reduced ghrelin-induced Akt phosphorylation and Ly294002 also reduced GSK3?phosphorylation.4. We found ghrelin induced phosphorlation of GSK3?is associated with?-catenin translocation to the nucleus under hypoxia condition.Conclusions:1. Ghrelin inhibits hypoxic pulmonary microvascular endothelial cell apoptosis involving PI3-K/Akt/GSK-3?and Wnt signaling pathway.2. GHSR-la receptor is involved as an intermediary through p-GSK3p/GSK3p signaling pathway in RPMECs.3. We built a Wnt/?-catenin signaling pathway reporter gene model. Activities of?-catenin/TCF reporter gene was significantly up-regulated by ghrelin analyzed by using the reporter gene model. |
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