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.