Mechanism Of Fibrin(Ogen) Promoting Vascular Endothelial Cell Proliferation And Vascule-like Formation

Objective:To detect the mechanasim and effect of cell proliferation and vascule-like formation in HUVECs induced by fibrin.Methods: Partâ… Culturing HUVECs, we detected the impact on vascule-like formation by fibrin treatment at lower(0.5mg/ml),middle(3mg/ml),higher(6mg/ml) concentration; using MTT and flow cytometry, we detected the cell proliferation by fibrin at higher concentration. Partâ…¡using real-time quantitative RT-PCR, to detect the expression of microtubule-associated protein 1 light chain 3 (MAP1 LC3) under different concentrations (0mg/ml, 0.5mg/m, 1.5mg/ml, 3mg/ml, 4.5mg/ml, 6mg/ml) and time (6 h, 12 h, 24 h) of fibrin treatment,and using immunofluorescence labeling, to detect the expression of specific marker of late autophagic vesicles (L-AVs) and LC3 in the cytoplasm with fibrin treatment; using real-time quantitative RT-PCR, to detect the expression of VEGF by fibrin treatment with concentrations and time aforementioned. Partâ…¢using real-time quantitative RT-PCR, to detect the expression of vascular endothelial-cadherin (VE-cadherin) and a novel connecting molecule IQGAP3 under different concentrations and time of fibrin treatment as aforementioned,and using immunofluorescence labeling, to detect the the change of F-actin structure.Results: Partâ… There was vascule-like structure formation in different doses of fibrin , and strongest in higher concentration(6mg/ml); cell proliferation was increased compared to the control group. In the control group, there was free of vascule-like formation. Partâ…¡The gene expression of LC3 was changed in the pocess of angiogenesis, increasing in a fibrin concentration- and time-dependent manner and the expressionof L-AVs and LC3 in the cytoplasm was also increased by immunofluorescence labeling; VEGF gene expression was increased accompanying with activatied autophagy. Partâ…¢In the process of angiogenesis, VE-cadherin expression was increased treated with fibrin in a concentration- and time-dependent manner, especially significant in the higher concentration; while F-actin, which was well-distributed around nuclei and cell under normal conditions, was switched into disordered stress fiber under fibrin treatment. The expression of IQGAP3 was also increased accompanying with VE-cadherin levels.Conclusion:Partâ… Fibrin can promote vascule-like structure formation in different doses, showing in a concentration- and time-dependent fashion.Partâ…¡There was activation of autophagy in the pocess of angiogenesis, increasing in a fibrin concentration- and time-dependent manner, demonstrating that autophagy activated by fibrin pomoted cell proliferation. In addition, it's also concerned with the expression of VEGF, VE-cadherin and IQGAP3 and the change of cytoskeletal structure.