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