3.6 The effect of Twist1 inhibitor on tube formation by primary
HUVECs under normal or hyperoxia conditions
Tube formation is a rapid and quantifiable measure of angiogenesisin vitro and an indicator of endothelial cell function. Tube
formation ability was represented by the total tube length and the
number of tube branches in each group of HUVEC cells. Figure 5A, 5B, and
5C show that the total tube length (9058.75±575.78μm) and the number of
tube branches (38.5±5.97) in the normoxia+harmine group were
significantly reduced compared with the total tube length
(12392.75±924.78μm) and the number of tube branches (68±11.11) in the
control group. The total tube length (7717.75±825.55μm) and the number
of tube branches (36±8.04) in the hyperoxia group were also
significantly lower than those in the control group. Compared with the
hyperoxia group, the total tube length (11135.50±307.45μm) and the
number of tube branches (53.25±5.44) was higher in the hyperoxia+harmine
group. These results suggest that under normal conditions, the knockdown
of Twist1 reduces the tube formation ability of endothelial cells.
Knocking down Twist1 in hyperoxia increased endothelial tube formation.
. Discussion
With the continuous development of neonatal medical technology, an
increasing number of very low birth weight infants or ultra-low birth
weight infants have been successfully treated27. The
”old” form of BPD, which is characterized by pulmonary inflammation and
pulmonary fibrosis, is becoming increasingly rare. However, the
incidence of ”new” BPD, characterized by pulmonary vascular dysplasia,
is increasing year by year1,28. Therefore, research
focus on the key pathogenesis of BPD has shifted from alveolar
epithelial cells to endothelial cells. Vascular endothelial cells in
different organs can form specific microenvironments and vascular
endothelial cells provide vascular secretory factors to adjacent cells
through this microenvironment to regulate the development, homeostasis,
and regeneration of corresponding organs29. Vascular
endothelial cells can also form a specific microenvironment in lung
tissue and interact with airway epithelial cells, fibroblasts, and
macrophages to promote the formation of the alveolar septum and the
remodeling of pulmonary microvessels in the process of alveolization;
they also regulate the normal development of
alveoli30. This also seems to indicate that
endothelial cells play a greater role than other cells in lung tissue.
In this study, we used the primary HUVEC in vitro model and
intervention with a Twist1 inhibitor (harmine) to study the effects of
the Twist1-Tie2-Angs signaling pathway on endothelial cell related
functions under normal and hyperoxia conditions. The multiple functions
and stability of Twist1 depends on the dimerization of itself and E2A
protein. Harmine targets the Twist1 pathway by promoting the degradation
of the twist1-E2A protein heterodimer31. In this
study, we found that as a regulatory factor of Tie2, the reduction of
Twist1 expression would have a negative effect under normal
circumstances, thus resulting in the increased permeability of
endothelial cells and the reduced ability to form tubes. In contrast,
reducing the expression of Twist1 under hyperoxia would play a positive
role, reduce the permeability of endothelial cells, and promote tube
formation ability. However, reducing the levels of Twist1 protein has
differing outcomes, depending on whether Ang1 or Ang2 is dominant in the
environment (Figure 6).
Ang1-Tie2 signaling has been fully demonstrated to promote angiogenesis
and protect vascular stability32. The fibrin-like
domain of Ang1 binds to Tie2 and induces Tie2 oligomerization; this is a
key process that activates the Tie2 receptor and initiate the downstream
signaling pathway33. VE-cadherin is the most important
protein in the interconnection between endothelial cells. When it
phosphorylated in the cytoplasmic region, VE-cadherin will be lost from
the junction between endothelial cells, thus resulting in an increase in
vascular permeability. Ang1-Tie2 signaling mainly protects vascular
permeability via three key mechanisms. First, Ang1-Tie2 signaling
mediates anti-inflammatory signals which attenuate the tumor necrosis
factor α-mediated JNK pathway to dephosphorylate VE-cadherin, thereby
protecting the connections between endothelial
cells34. Second, vascular endothelial growth factor
(VEGF) phosphorylates VE-cadherin through the Src-RAC-PAK pathway.
Ang1-Tie2 signals can counteract the effect of VEGF and induce the
retention of tyrosine kinase Src in cells, thus stabilizing the
expression of VE-cadherin on the surface of endothelial
cells35. Third, Ang1 can promote the Tie2/Akt
signaling pathway to play an anti-apoptotic role so as to promote tube
formation in endothelial cells36. According to the
results of our western blot experiments, Ang1 was expressed normally
under normoxia while Ang 2 is expressed at lower levels; thus Ang1 is
dominant under normoxia. When Twist1 was inhibited in HUVECs under
physiological conditions, we found that the expression of the downstream
Tie2 receptor was also reduced. At this time, even if there were
sufficient Ang1 ligands, there would not be enough Tie2 receptor to bind
to Ang1; therefore, this could not protect the stability of the
endothelial cells. Therefore, after harmine intervention under normoxic
conditions, the integrity of the connection between endothelial cells
would be destroyed; the permeability of the endothelial cells would
increase, and the ability of angiogenesis in vitro would be
reduced.
Previous studies of diseases characterized by vascular leakage and
tissue injury have proven that there is an association between high
expression levels of Ang2 and adverse outcomes37,38.
For example, when the imbalance between Ang2 and Ang1 tends to be
advantageous to Ang2, the permeability of the endothelial cells will
increase following coronary artery bypass grafting36.
The transient increase of trans-endothelial permeability in patients
with Dengue fever is also known to be due to an increase of the
Ang2/Ang1 ratio39. An increase in the Ang2/Ang1 ratio
was also detected in an animal model of sepsis-induced BPD40. In this experiment, we used the in vitromodel of hyperoxia-induced BPD and analyzed the protein expression of
Ang1 and Ang2 under hyperoxia. We observed a reduction of Ang1 protein
expression and an increase in Ang2 protein expression. Therefore, under
hyperoxia, the permeability of endothelial cells increased, and the tube
formation ability of endothelial cells in vitro decreased. Ang2
mainly leads to the destruction of endothelial barrier function through
two mechanisms. First, Ang2 is a vascular inflammatory medium that can
destroy vascular endothelial cells. Secondly, when Ang2 competes with
Ang1 for the Tie2 receptor, it inhibits the barrier protection function
of endothelial cells mediated by Ang1. Because when Ang1 binds to the
Tie2 receptor, it activates small guanosine triphosphatases Rac1 (GTPase
Rac1). The activation of this enzyme not only promotes the accumulation
of VE-cadherin among endothelial cells, but also activates protein
p190Rho-GAP, thus resulting in the inactivation of RhoA. The
inactivation of RhoA can inhibit the formation of actin stress fibers
and stabilize the function of the endothelial
barrier41. To prevent the adverse consequences of
elevated Ang2 under hyperoxia, it is important to reduce the expression
of its receptor Tie2. This is because after the significant reduction of
the Tie2 receptor, even if the level of Ang2 increases, there will not
be enough receptors to bind Ang2, thus preventing the increase of
endothelial cell permeability caused by Ang2. It has been reported that
the reduction of Tie2 expression caused by the knockdown of Twist1
expression can inhibit the increase in pulmonary vascular permeability
in a mouse model of lung injury induced by
endotoxin19. Mammoto et al. previously used a neonatal
mouse model of hyperoxia-induced acute lung injury to show that the
reduction in Tie2 expression caused by the knockdown of low-density
lipoprotein receptor related protein 5 (LRP5) expression can also reduce
the permeability of pulmonary vessels14. In the
present study, we showed that under hyperoxia stimulation, the further
reduction of Twist1 expression following the treatment of endothelial
cells with harmine would lead to a further reduction of Tie2 expression.
This would reduce the permeability of endothelial cells, promote the
connection between endothelial cells, and promote tube formation
ability; this was consistent with the results derived from the twoin vivo experiments mentioned above.
In conclusion, the regulatory effects of the Twist1-Tie2 signaling
pathway on vascular endothelial cells under physiological and
pathological (hyperoxia) conditions can result in opposing outcomes and
depends on the balance between Ang1 and Ang2. Severe BPD in childhood,
and even adulthood, will have respiratory sequelae such as low pulmonary
function, pulmonary airflow obstruction, and even nervous system injury.
This will seriously affect their survival rate and long-term quality of
life, and bring a heavy economic burden to the family and
society42. In this experiment, we demonstrated that
the Twist1-Tie2-Angs signaling pathway may play an important role in the
increased endothelial cell permeability induced by hyperoxia. Reducing
the expression of Twist1 may become a potential target to treat BPD in
the future. However, this experiment also has some limitations. Onlyin vitro experiments were carried out; our hypothesis was not
verified in an in vivo animal model. In our future research, we
aim to knockout the Twist1 in an animal model to further explore the
protective mechanisms of the Twist1 under hyperoxia.