The relevant informations of the first author are as follows:
Name: Shasha He
Mail address: Affiliated Hospital of Southwest Medical University, Jiangyang District, Luzhou City, Sichuan Province, China.
Telephone number:+86 18883939583
E-mail:763879233@qq.com
Keywords: bronchopulmonary dysplasia, hyperoxia, endothelial cell permeability, tube forming ability, Twist1, Tie2, Ang1, Ang2
Abstract
Background:Bronchopulmonary dysplasia (BPD) is a chronic lung disease of premature infants that involves pulmonary vascular development disorder as the main pathological feature; hyperoxia is its main etiology. Twist1 strictly controls the development of blood vessels via the Tie2-Angs signaling axis. However, previous research on Twist1 mainly focuses on various tumors; its effect on BPD has yet to be reported. The present study represents the first investigation of the role and related mechanisms of the Twist1-Tie2-Angs signaling pathway in hyperoxia-induced endothelial cell injury.
Methods: Primary human umbilical vein endothelial cells were used as anin vitro model. A Twist1 inhibitor (harmine) was applied to normal and hyperoxia-exposed endothelial cells. Then, we observed the permeability and tube formation ability of endothelial cells after reducing Twist1 protein.
Results: Hyperoxia increased the permeability of endothelial cells and decreased tube formation ability. Under physiological conditions dominated by angiogenin 1 (Ang1), reducing the expression of Twist1 increases the permeability of endothelial cells and reduces tube formation ability. In contrast, under hyperoxia conditions dominated by angiogenin 2 (Ang2), reducing the expression of Twist1 reduced the permeability of endothelial cells and increased tube formation ability.
Conclusion: Twist1 depends on the balance of Ang1 and Ang2 to control the permeability and tube formation of endothelial cells. Reducing the levels of Twist1 may be a protective mechanism for BPD.
Introduction
The use of prenatal steroids and postpartum surfactants has increased the survival rate of preterm infants over recent years, thus resulting in a tendency for bronchopulmonary dysplasia (BPD) to occur in preterm infants with an earlier gestational age (gestational age < 29 weeks); this condition is referred to as ”new” BPD1. The pathological feature of ”new” BPD is the obstruction of pulmonary vascular development; this leads to aberrant alveolar formation and the simplification of alveolar structure2. The lungs of premature infants usually develop in a relatively low oxygen intrauterine environment. When exposed to high oxygen concentration after birth, the lungs are the first organ to be affected. All types of cells in lung tissues are affected by high oxygen levels; endothelial cells are known to be more sensitive than epithelial cells3. Therefore, the normal function and structure of endothelial cells are an important basis for pulmonary angiogenesis and alveolarization in neonates4. Coincident with the dissemination of a hypothesis for BPD-related vascular development disorder, research relating to the pathogenesis of BPD has gradually shifted from alveolar epithelial cells to endothelial cells; however, the molecular mechanisms underlying BPD remain unclear.
Twist1 is a basic transcription factor containing helix-loop-helix domains and is a key regulator of embryonic development and organogenesis. Many experimental studies have investigated the role of Twist1 in various tumor diseases; however, no research group has studied the specific role of Twist1 in BPD. This protein not only mediates the pathological process underlying epithelial mesenchymal transformation in a variety of fibrotic diseases5; it also participates in the formation of physiological and pathological blood vessels6 and pulmonary vascular remodeling by regulating endothelial mesenchymal transformation (EndoMT)7. Twist1 contains a b-HLH sequence that regulates its own transcription by identifying a common E-box motif in the promoter region of the target gene8. There is an E-box motif in the promoter region of the Tie2 receptor; therefore, Twist1 acts as an upstream regulator of Tie2 and regulates the expression of Tie29. Angiopoietins (Angs) are ligands of Tie2 receptors and includes Ang1 and Ang2. Angs and Tie2 are widely expressed in the lungs10,11 and the Angs-Tie2 signaling pathway is known to regulate postnatal angiogenesis, vascular remodeling, vascular permeability, and inflammation, by regulating endothelial cell remodeling, thus regulating the dynamic balance of blood vessels12.
Ang1 is secreted by cells surrounding blood vessels and acts as an agonist on Tie2 receptors in a paracrine manner, thereby promoting vascular maturation and stability13. The protective effect of Ang1-Tie2 on vascular endothelial cells depends on a variety of upstream regulatory factors, including miR-34a, Twist1, YAP1, and LRP514-16. Ang2 is secreted by endothelial cells and acts on the Tie2 receptor in an autocrine manner. The excitatory/inhibitory effect of Ang2 on the Tie2 receptor is determined by vascular endothelial protein tyrosine phosphatase (VEPTP)17. Lymphatic endothelial cells do not express VEPTP which reduces the threshold for Tie2 activation. Therefore, Ang2 has an exciting effect on the Tie2 receptor on lymphatic endothelial cells. In contrast, vascular endothelial cells express VEPTP which increases the threshold of Tie2 activation. Therefore, Ang2 has an inhibitory effect on the Tie2 receptor of vascular endothelial cells, thus leading to vascular instability and increased vascular permeability18.
Animal studies have shown that when normal mice (Twist1flox|flox ) and Twist1 gene knockout mice (Tie2-Twist1ko ) were fed under normal conditions, there was no difference in the levels of Ang1 in the lung tissues of the two types of mice; however, the pulmonary vascular permeability of the Tie2-Twist1ko mice was found to increase. The two types of mice were simultaneously exposed to lipopolysaccharide (LPS) to generate an acute respiratory distress syndrome (ARDS) model. Following LPS treatment, the protein levels of Ang2 in the lung tissue of both types of mice increased, but the pulmonary vascular permeability ofTie2-Twist1ko mice was lower than that inTwist1flox|floxmice19. This animal experiment demonstrated that downregulated Twist1-Tie2 signaling could prevent the endotoxin-induced increase in pulmonary vascular permeability by inhibiting damage in the ligand of Ang2 thus preserving the integrity of endothelial cell connectivity. Our research group previously used a neonatal rat model of hyperoxia acute lung injury to show that although Twist1-Tie2 signaling was down regulated, this did not inhibit the damage caused by its ligand Ang2 with regards to the integrity of endothelial intercellular connection; furthermore, there was an increase in pulmonary vascular permeability 20.
In conclusion, we speculate that this different result may be because we used newborn rats without Twist1 knockdown as the animal model for our study. Therefore, it is possible that without the knockdown of Twist1, the reduced extent of Twist1 induced by hyperoxia might not be sufficient to reduce Tie2 to reverse the increase of hyperoxia vascular permeability induced by Ang2-Tie2. Human umbilical vein endothelial cells (HUVECs) are considered as a reliable in vitro model to study the physiological and pathological functions of vascular endothelial cells21,22. Therefore, in this study, we used the Twist1 inhibitor (harmine) to further reduce the expression of the Twist1 in vitro in hyperoxia. Because Ang2-Tie2 destroys the integrity of endothelial cell connections, we hypothesize that the further down-regulation of Twist1-Tie2 signaling may be sufficient to reverse the damage incurred by Ang2 in hyperoxia. We speculate that reducing the expression of Twist1 may become a new method with which to prevent BPD.
Materials and methods