Grützkau A, Krüger-Krasagakes S, Baumeister H, Schwarz C, Kögel H, Welker P, Lippert U, Henz B M, Möller A
Department of Dermatology, Virchow Clinics, Humboldt Universität zu Berlin, 13353 Berlin, Germany.
Mol Biol Cell. 1998 Apr;9(4):875-84. doi: 10.1091/mbc.9.4.875.
Mast cells have been implicated in various diseases that are accompanied by neovascularization. The exact mechanisms by which mast cells might mediate an angiogenic response, however, are unclear and therefore, we have investigated the possible expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in the human mast cell line HMC-1 and in human skin mast cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that mast cells constitutively express VEGF121, VEGF165, and VEGF189. After a prolonged stimulation of cells for 24 h with phorbol 12-myristate 13-acetate (PMA) and the ionophore A23187, an additional transcript representing VEGF206 was detectable, as could be verified by sequence analysis. These results were confirmed at the protein level by Western blot analysis. When the amounts of VEGF released under unstimulated and stimulated conditions were compared, a significant increase was detectable after stimulation of cells. Human microvascular endothelial cells (HMVEC) responded to the supernatant of unstimulated HMC-1 cells with a dose-dependent mitogenic effect, neutralizable up to 90% in the presence of a VEGF-specific monoclonal antibody. Flow cytometry and postembedding immunoelectron microscopy were used to detect VEGF in its cell-associated form. VEGF was exclusively detectable in the secretory granules of isolated human skin mast cells. These results show that both normal and leukemic human mast cells constitutively express bioactive VEGF. Furthermore, this study contributes to the understanding of the physiological role of the strongly heparin-binding VEGF isoforms, since these were found for the first time to be expressed in an activation-dependent manner in HMC-1 cells.
肥大细胞与多种伴有新血管形成的疾病有关。然而，肥大细胞介导血管生成反应的确切机制尚不清楚，因此，我们研究了血管内皮生长因子/血管通透因子（VEGF/VPF）在人肥大细胞系HMC-1和人皮肤肥大细胞中的可能表达。逆转录聚合酶链反应（RT-PCR）分析显示，肥大细胞组成性表达VEGF121、VEGF165和VEGF189。在用佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）和离子载体A23187对细胞进行24小时的长时间刺激后，可检测到另一种代表VEGF206的转录本，序列分析证实了这一点。这些结果在蛋白质水平上通过蛋白质印迹分析得到了证实。当比较未刺激和刺激条件下释放的VEGF量时，刺激细胞后可检测到显著增加。人微血管内皮细胞（HMVEC）对未刺激的HMC-1细胞的上清液有剂量依赖性的促有丝分裂作用，在存在VEGF特异性单克隆抗体的情况下，这种作用可被中和高达90%。流式细胞术和包埋后免疫电子显微镜用于检测细胞相关形式的VEGF。VEGF仅在分离的人皮肤肥大细胞的分泌颗粒中可检测到。这些结果表明，正常和白血病人类肥大细胞都组成性表达生物活性VEGF。此外，这项研究有助于理解强肝素结合VEGF异构体的生理作用，因为首次发现这些异构体在HMC-1细胞中以激活依赖方式表达。
