Hypoxia induces vascular endothelial growth factor in cultured human endothelial cells
Hypoxia induces vascular endothelial growth factor in cultured human endothelial cells
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DOI：
10.1074/jbc.270.52.31189
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发表时间：
1995-12-29
影响因子：
4.8
通讯作者：
Isner, JM
中科院分区：
文献类型：
--
作者：
Namiki, A;Brogi, E;Isner, JM
平滑肌细胞、巨噬细胞、胶质细胞、角化细胞和转化细胞已被确定为血管内皮生长因子（VEGF）的合成位点。VEGF的调节作用主要局限于内皮细胞（ECs），这是唯一一种持续表达VEGF受体的细胞类型。因此，VEGF被认为完全通过旁分泌途径起作用。我们试图确定在特定条件下，人类内皮细胞的作用是否可能超出目标范围，涉及VEGF的偶然性合成。在未受刺激的人脐静脉内皮细胞（HUVECs）和人皮肤来源的微血管内皮细胞（HUVECs）中，Northern分析均未检测到VEGF转录物。然而，phorboll -12-肉豆酸13醋酸酯（10(-7)M）处理诱导了HUVECs和HMECs中VEGF mRNA的表达，分别在3和6小时达到峰值，并在12小时后恢复到无法检测到的水平。在体外，HUVECs暴露于缺氧环境（pO(2) = 35 mm汞）12、24和48小时，HMECs暴露于6、12、24和48小时，以时间依赖性的方式诱导VEGF mRNA。缺氧24小时后，再次暴露于常氧环境（pO(2) = 150 mm汞）24小时，使huvec中VEGF mRNA转录物达到无法检测的水平。氯化钴和氯化镍处理均可诱导内皮细胞中VEGF mRNA表达。环己亚胺处理进一步增强了氯化钴、氯化镍和缺氧诱导的HUVECs中VEGF mRNA的表达。免疫组织化学方法证实了缺氧HUVECs中VEGF蛋白的产生。来自缺氧HUVECs的条件培养基导致氚化胸苷的掺入增加2倍。最后，用抗tyr (P)抗体探测的抗kdr免疫沉淀在缺氧而非常氧HUVECs中显示了受体自磷酸化的证据。因此，这些发现确立了自分泌途径的可能性，该途径可能增强和/或放大VEGF刺激血管生成的旁分泌作用。
Smooth muscle cells, macrophages, glial cells, keratinocytes, and transformed cells have been established as synthesis sites for vascular endothelial growth factor (VEGF). The modulating effects of VEGF are essentially limited to endothelial cells (ECs), the only cell type consistently shown to express VEGF receptors. VEGF has thus been considered to act exclusively via a paracrine pathway. We sought to determine whether the role of human ECs might, under selected conditions, extend beyond that of a target to involve contingency synthesis of VEGF. In both unstimulated human umbilical vein ECs (HUVECs) and human derma-derived microvascular ECs (HUVECs), Northern analysis detected no VEGF transcripts. Phorbol-12-myristate 13 acetate (10(-7) M) treatment, however, induced VEGF mRNA expression in both HUVECs and HMECs, peaking at 3 and 6 h, respectively, and returning to undetectable levels by 12 h. In vitro exposure of HUVECs to a hypoxic environment (pO(2) = 35 mm of mercury) for 12, 24, and 48 h and exposure of HMECs for 6, 12, 24, and 48 h induced VEGF mRNA in a time-dependent fashion. Re-exposure to normoxia (pO(2) = 150 mm of mercury) for 24 h after 24 h of hypoxia returned VEGF mRNA transcripts to undetectable levels in HUVECs. Cobalt chloride and nickel chloride treatment each induced VEGF mRNA in ECs. Cyclo-heximide treatment further augmented expression of VEGF mRNA induced by cobalt chloride, nickel chlo ride, and hypoxia in HUVECs. VEGF protein production in hypoxic HUVECs was demonstrated immunohistochemically. Conditioned media from hypoxic HUVECs caused a 2-fold increase in the incorporation of tritiated thymidine. Finally, immune precipitates of anti-KDR probed with anti-Tyr(P) antibodies demonstrated evidence of receptor autophosphorylation in hypoxic but not normoxic HUVECs. These findings thus establish the potential for an autocrine pathway that may augment and/or amplify the paracrine effects of VEGF in stimulating angiogenesis.