Hypoxic Adaptability Of Yak Pulmonary Artery Smooth Muscle Cells Based On TGF-β/BMP Signal Pathway

Animals living at low altitude will suffer from severe mountain sickness after being transferred to the hypoxia environment at high altitude,which is characterized by a series of pathophysiological changes such as hypoxic pulmonary hypertension(Hypoxic pulmonary hypertension,HPH).This change is mainly due to the decrease of oxygen content in the air.in order to give the body enough oxygen,the function of pulmonary circulation is enhanced,and the blood pressure of pulmonary circulation is increased to increase the blood volume of pulmonary circulation.In order to resist the increase of pulmonary artery blood pressure and prevent vascular swelling and even rupture,pulmonary artery thickens and enhances vascular remodeling through functional phenotypic changes,proliferation and fibroblast activation of vascular smooth muscle cells(Pulmonary artery smooth muscle cells,PASMCs).Yak(Bos Grunniens)is a unique breed in the pastoral area of Qinghai-Tibet Plateau.It has anatomical and physiological characteristics that can adapt to extreme hypoxia on the plateau.It is one of the ideal models for the study of hypoxia adaptability at high altitude.Yaks can live in extremely hypoxic environment for a long time and avoid pulmonary hypertension caused by hypoxia.However,little is known about the molecular regulatory mechanism of how yaks avoid HPH.The crosstalk of the two TGF-β/BMP signaling pathways in the transforming growth factors-β(TGF-β)superfamily plays a key role in maintaining the balance of pulmonary vascular smooth muscle cells.Therefore,in this experiment,we first detected the preliminary transduction site of TGF-β signal and the activation of TGF-β signal pathway in yak pulmonary vessels of different ages,and then further studied the mechanism of TGF-β/BMP signal crosstalk expression in yak pulmonary artery smooth muscle cells induced by hypoxia and its effect on cell proliferation.The specific results are shown as follows:1.Immunohistochemical method was used to detect the TGF-β/Smad signal transduction site in yak lungs of different ages.The results showed that the signal transduction site was mainly located in pulmonary artery smooth muscle cells and vascular endothelial cells.Through fluorescence quantitative PCR and Western blotting,it was found that TGF-β/Smad signaling pathway related factors were expressed in the lung tissues of yaks of different ages,and the expression levels were high in adult and new groups,and significantly higher than those in young and old groups.2.Through the culture of yak PASMCs in vitro,it was found that transient hypoxia(12 h and 24 h)did not up-regulate the expression of PASMCs TGF-β1 and the phosphorylation level of Smad2/3 in yak.With the increase of hypoxia exposure time,the expression level of TGF-β1 and the phosphorylation level of Smad2/3 were significantly up-regulated.However,transient hypoxia(12 h and 24 h)induced transient activation of BMP signal pathway,up-regulated BMPRII expression and Smad1/5 phosphorylation,and then this trend was gradually reversed.3.In order to evaluate the effect of inhibition of TGF-β signal pathway activation on adaptation to hypoxic stress after short-term hypoxia treatment of yak PASMCs,exogenous TGF-β1 of different concentrations was added under hypoxia condition.The results showed that exogenous TGF-β1 significantly activated TGF-β signal pathway and up-regulated the phosphorylation levels of downstream Smad2 and Smad3.The levels of CCK8,cell scratches and cell proliferative marker proteins all showed that exogenous TGF-β1 significantly promoted the proliferation and migration of yak PASMCs,suggesting that inhibition of TGF-β signal pathway activation after transient hypoxia stress may play a role in balancing hypoxia-induced PASMCs proliferation.In addition,TGF-β1 also activated the PASMCs BMP signal pathway of yak cross stimulation,but the role of BMP signal of TGF-β1 cross stimulation is still uncertain.4.In order to further verify the positive effect of transient activation and transduction of PASMCs BMP signal pathway in yak on hypoxia adaptation after shortterm hypoxia induction,noggin,a classic inhibitor of BMP signal pathway,was used to inhibit BMP/Smad1/5 signal transduction.CCK8,cell scratch test and cell proliferation marker protein level test showed that inhibition of BMP/Smad1/5 signal pathway could significantly promote the proliferation and migration of yak PASMCs.It is suggested that the activation of BMP signal pathway induced by hypoxia inhibits the proliferation of yak PASMCs,which may be achieved by inhibiting TGF-β signal pathway.In conclusion,it was found that TGF-β signal transduction in yak pulmonary vessels was mainly located in pulmonary artery smooth muscle cells,and the activation degree of TGF-β signal pathway was different in lung tissues of yaks of different ages.The level of TGF-β signal transduction in lung tissues of young and old yaks was lower than that of adult and newborn groups.The hypoxia model was established by culturing PASMCs in vitro.It was found that the activation of TGF-β signal pathway was inhibited and the level of BMP signal transduction was up-regulated after short-term hypoxia induction.Exogenous TGF-β1 and noggin were used to further explore the effects of dynamic regulation of TGF-β/BMP signaling pathways on cell proliferation balance in yak pulmonary artery smooth muscle cells induced by hypoxia.These results suggest that the dynamic regulation of TGF-β/BMP signal in yak PASMCs plays a positive role in maintaining the homeostasis of pulmonary artery smooth muscle cells and adapting to hypoxia.