Tumor Necrosis Factor-Related Apoptosis-inducing Ligand Regulate The Accumulation Of Extracelluar Matrix In Pulmonary Artery By Activating The Phoaphorylation Of Smad2/3

Background Pulmonary arterial hypertension(PAH)is a progressive,refractory and extremely malignant vascular disease,which is characterized by small pulmonary arterial stenosis and obstruction induced by vascular remodeling and vascular cells proliferation.As we know,this disease ultimately leads to right heart failure and finally death.It was reported that the incidence increases by years.Current drugs have shown short-term effects in reducing pulmonary pressure,increasing exercise tolerance and improving quality of life,but failed to improve long-term outcomes and survival rates.There are numerous evidences support that vascular remodeling plays a central pathological role in the progress and prognosis of pulmonary hypertension,which was observed as proliferation and hypertrophy of vascular wall cells like endothelial cell(EC),smooth muscle cell(SMC),adventitial fibroblast(VAF)and accumulation of extracellular matrix(ECM).It has been confirmed that the m RNA and protein of TRAIL,a tumor necrosis factor-related apoptosis inducing ligand,are significantly expressed in normal pulmonary vascular endothelial cells and smooth muscle cells.It was previously demonstrated that human and rodents serum TRAIL concentrations are positively related to the severity of PAH.Abdul G has previously demonstrated that TRAIL-KO and administration of TRAIL inhibitors can obstruct the development of PAH in mammals by reducing the expression and deposition of ECM.However,up till now there is no report on which signal pathway TRAIL participates in the expression and deposition of ECM in PAH.What is rather more clear is that the activation of transforming growth factor-?(TGF-?)and BMP signaling pathways play major roles in the expression of ECM.Activated TGF-? and BMP signaling pathways act on their phosphorylated receptors respectively located in the cell membrane and exert phosphorylation on the downstream Smad2/3 and Smad1/5/8,affecting the transcription of target gene and the expression of target protein.Objective To investigate TRAIL regulates the expression of ECM by affecting the downstream phosphorylated receptors p-Smad2/3 and p-Smad1/5/8 of TGF-? and BMP in pulmonary artery smooth muscle cells(PASMCs)under hypoxia.Methods(1)in vitro,8-week-old C57BL6 WT male mice were stratified into four groups: the first group was exposed to hypoxia(10% oxygen,90% nitrogen)for 3weeks(hypoxia-induced PAH group),the second one was treated with TRAIL-Ab for 4weeks after 3 weeks hypoxia using osmotic mini-pumps(TRAIL-Ab group),the third one was treated with Ig G antibody for 4 weeks after 3 weeks hypoxia using osmotic mini-pumps(Ig G group),and the fourth one was exposed to normoxia for 3 weeks(control group).Quantitative comparison the expression of extracellular matrix and vascular stenosis of four groups with HE staining and immunofluorescence techniques.(2)in vivo,pulmonary artery smooth muscle cells were divided into three groups: the first group was cultured with F12 K medium under hypoxia(1% oxygen,5% carbon dioxide,94% nitrogen)for 24 hours(hypoxia-induced group),the second one was treated with anti TRAIL-Ab during cultured with F12 K medium under hypoxiafor 24hours(TRAIL-Ab group),and the third one was cultured with F12 K medium under normoxia(95% oxygen,5% carbon dioxide)for 24 hours(control group).Cell lysate and Trizol were added to extract cell protein.Quantitative comparison the expression of extracellular matrix and p-Smad2/3 and p-Smad1/5/8 proteins of the three groups with Western blotting techniques.Results In vitro,Hypoxia-induced PAH mice had significant increases in right ventricle systolic pressure(RVSP),right ventricular hypertrophy(RVH),vascular stenosis,the expression of extracellular matrix compared with controls.Mice with anti-TRAIL antibody had lower levels in RVSP,RVH,vascular stenosis and the expression of extracellular matrix than PAH mice.In vivo,hypoxia-induced group had prominent increase in the expression of Smad2/3 compared with controls.PASMCs with anti-TRAIL antibody had lower levels in the expression of Smad2/3 than hypoxia-induced group.Conclusion Anti-TRAIL antibody alleviate pulmonary vascular remodeling by reducing the expression and deposition of extracellular matrix.TRAIL regulates the accumulation of ECM by activating p-Smad2/3 in pulmonary artery smooth muscle cells(PASMC)under hypoxia.These findings plays an essential theoretical roles in the treatment of pulmonary hypertension in molecular signaling pathways and provide experimental evidence for subsequent research and clinical transformation.