Roles Of Autophagy And ER Stress In Tube Formation In HUVEC

Aims:Recovery of blood flow mediated by angiogenesis in ischemic area is the most important measuring for relieving the heart impairment and dysfunction in ischemic heart diseases. Autophagy and endoplasmic reticulum (ER) stress play cytoprotection and contribute to cell differentiation. Increasing evidence reported that autophagy is involved in hypoxia-induced angiogenesis. However, it is not well known whether vascular endothelial growth factor (VEGF), the most important angiogenesis inducer, can induce tube formation via ER stress and autophagy. So, we planed to explore the roles of ER stress and autophagy in VEGF-induced angiogenesis in vitro, providing a clues for developing the angiogenic medicines against the targets of ER stress and autophagy.Method:A cellular model of angiogenesis was prepared in human umbilical vein endothelial cells (HUVECs) treaied with VEGF. Generation of reactive oxygen species (ROS) was assessed by using DHR123staining. Genes expression in mRNA and protein levels were observed by RT-PCR and immunoblotting respectively. HUVECs were planted on the Matrigel for analysis of tube formation in vitro. The migration ability of HUVEC was detected by Wound-Healing method. Meanwhile, blockage of ER stress and autophagy were mediated by responsive inhibitor or gene knockdown. Myocardial ischemia model in mice was achieved by permanently occluding the left anterior descending coronary artery (LAD), and confirmed by visual observation (e.g. cyanosis) and continuous ECG monitoring. Ischemic heart proteins were prepared for detecting the expression of angiogenesis-related gene VE-cadherin and autophagy-associated gene LC3-II.Results:It was found that100ng/ml VEGF initiated generation of ROS in HUVECs, and unregulated the expression of ER-stress related genes (Bip, TRE-1) and autophagy-related genes (beclin-1, ATG4and ATG5) both in mRNA and protein level. Immunoblotting showed LC3-Ⅱ is increased after exposure to VEGF. Moreover, pretreatment with3-methyladenine and siRNA of Beclin-1, inhibited significantly VEGF-mediated tube formation and cell migration. Both of them did not block the ER-stress as indicated by the expression of responsive markers (IRE-1and Bip). However, treatment with a specific ER-stress inhibitor tauroursodeoxycholate (TUDC) prior to VEGF exposure, the expressions of autophagy makers (Beclinl and LC3-Ⅱ) were decreased and the capacity of tube formation and migration of HUVECs also were blocked partially. Angiogenesis-related gene VE-cadherin and LC3-Ⅱ is increased in myocardial tissue at15days after occlusion of LAD.Conclusions:Our results suggested that1) Ischemia mediated angiogenesis in mice heart was accompanied by increase of autophagy;2) VEGF-induced tube formation is partially due to autophagy which is potentially mediated by ROS and ER stress.