Fibrinogen stimulates in vitro angiogenesis by choroidal endothelial cells via autocrine VEGF
Fibrinogen stimulates in vitro angiogenesis by choroidal endothelial cells via autocrine VEGF
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DOI：
10.1007/s00417-004-0910-2
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发表时间：
2004-04
期刊：
影响因子：
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通讯作者：
S. Shiose;Y. Hata;Y. Noda;Y. Sassa;A. Takeda;H. Yoshikawa;K. Fujisawa;T. Kubota;T. Ishibashi
中科院分区：
文献类型：
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作者：
S. Shiose;Y. Hata;Y. Noda;Y. Sassa;A. Takeda;H. Yoshikawa;K. Fujisawa;T. Kubota;T. Ishibashi
本研究旨在探讨纤维蛋白原对体外培养的牛脉络膜内皮细胞(BCECs)血管生成的影响以及血管内皮生长因子(VEGF)在其中的作用。方法将牛脉络膜内皮细胞接种于含纤维蛋白原(0~1.5 mg/ml)的胶原凝胶上进行体外成管实验。培养3天后，用MacScope分析仪测量肾小管结构的总长度。收集纤维蛋白原处理后的总RNA和条件培养液，分别进行Northern和Western印迹分析。免疫印迹法检测转录因子HIF-1α的表达。用抗血管内皮细胞生长因子中和抗体或血管内皮生长因子受体2选择性抑制剂(SU5416)检测血管内皮生长因子在纤维蛋白原依赖的体外小管形成中的作用。结果含纤维蛋白原凝胶以浓度依赖的方式促进小管结构形成20~50倍。经纤维蛋白原处理后，血管内皮细胞血管内皮生长因子基因和蛋白表达明显增加。纤维蛋白原处理后，胞核中可见HIF-1α蛋白积聚。抗血管内皮细胞生长因子中和抗体可显著抑制纤维蛋白原诱导的新生血管形成(1μg/ml抑制率为52.0%，P<0.05)或SU5416(3μΜ浓度时抑制率为54.8%，P<0.05)。结论血管外源性纤维蛋白原在老年性黄斑变性脉络膜新生血管的形成中可能起重要作用，至少部分是通过血管内皮生长因子的自分泌作用。转录因子HIF-1似乎参与了纤维蛋白原诱导的血管内皮生长因子的表达。
AbstractsBackgroundThe purpose of this study is to investigate the effect of fibrinogen on angiogenesis in vitro formed by cultured bovine choroidal endothelial cells (BCECs) and the involvement of vascular endothelial growth factor (VEGF) in this mechanism.MethodsFor in vitro tube formation assay, BCECs were seeded on collagen gel containing fibrinogen (0–1.5 mg/ml). After 3 days of cultivation, the total length of the tubular structure was measured using Macscope Analyzer. Total RNA and conditioned media were collected after fibrinogen treatment and subjected to Northern and Western blot analyses, respectively. Transcription factor HIF-1α was also analyzed by Western blot analysis using cytosolic and nuclear fraction of BCECs. Involvement of VEGF in fibrinogen-dependent in vitro tube formation was evaluated using anti-VEGF neutralizing antibody or VEGF receptor 2-selective inhibitor (SU5416).ResultsFormation of the tubular structure was enhanced 20~50 times in fibrinogen-containing gel in a concentration-dependent manner. The treatment of BCECs with fibrinogen resulted in a significant increase in VEGF gene and protein expression. Accumulation of HIF-1α protein in the nuclear fraction was also detected after the treatment with fibrinogen. Finally, fibrinogen-induced tube formation was significantly inhibited in the presence of anti-VEGF-neutralizing antibody (52.0% inhibition at the concentration of 1 μg/ml,P<0.05) or SU5416 (54.8% inhibition at the concentration of 3 μΜ,P<0.05).ConclusionsExtravasated fibrinogen might play an important role in the development of choroidal neovascularization associated with age-related macular degeneration, at least in part, through the function of VEGF in an autocrine manner. Transcription factor HIF-1 appears to be involved in fibrinogen-induced VEGF expression.