Changes Of TGF-β-ALK-Smad Signaling Pathway In Hyperoxia-induced Human Lung Microvascular Endothelial Cell Injury

Objective:To investigate the alteration of TGF-β-ALK-Smad signaling pathway in hyperoxia-induced pulmonary vascular endothelial cells,and to provide experimental basis for the new understanding of the mechanism of impaired pulmonary vascular development in bronchopulmonary dysplasia.Methods:Human pulmonary microvascular endothelial cells（HPMECs）were randomly divided into two groups,the hyperoxia and control group.The hyperoxia group was cultured in a multi-gas incubator with regulated delivery of 5%CO₂+60%O₂+35%N₂mixed gas.The control group was grown in a humidified incubator containing 5%CO ₂at 37℃as recommended by the manufacturer.At the beginning of the experiment,3h,6h,9h,12h,24h and 48h time points were set to observe the morphology of vascular formation between the hyperoxia group and the control group and compare the total tube length between the two groups.The protein levels of TGF-β1,ALK1,AKL5,P-Smad1/5 and P-Smad2/3 were detected by western blot at the beginning of the experiment,6h,12h and 24h.Results:1.In the control group,the PMVECs began to form tubular structures when they grew on the matrigel for 3h,and the peak of tube formation occurred between 12h and 24h.At 24h of control group,typical rope-like structures were formed with obvious lumen and complete structure,send out multiple branches and the branches clear and closed into tubes.While began to deteriorate at 48h.The ability of tube formation was inhibited in the hyperoxia group with prolonged oxygen exposure.The total length of tube formation in hyperoxia group was significantly decreased compared with control group at 24h after cell inoculation（P<0.05）.2.Compared with control group,the expression levels of TGF-β1 in hyperoxia group increased gradually at 24h（P<0.01）.Compared with control group,the expression levels of ALK1 and P-Smad1/5 in the hyperoxia group were decreased in each time point,but there was no significant difference between two groups（P>0.05）,while the expression levels of ALK5 and P-Smad2/3 in hyperoxia group were significantly increased at 24h（P<0.01）.Conclusions:1.Hyperoxia environment can significantly inhibit the angiogenesis of pulmonary vascular endothelial cells.2.Hyperoxia environment altered the balance between TGF-β-ALK1-Smad1/5 and TGF-β-ALK5-Smad2/3 signaling pathways in pulmonary vascular endothelial cells and affects angiogenesis.3.It is speculated that hyperoxia inhibits the angiogenesis of pulmonary vascular endothelial cells primarily through promoting TGF-β-ALK5-Smad2/3 signaling pathway.