Kim Kwang Sei, Hong Yong-Kil, Lee Yoon, Shin Joo-Young, Chang Soo-Ik, Chung Soo Il, Joe Young Ae
Cancer Research Institute, Catholic Research Institute of Medicial Sciences, The Catholic University of Korea, Seoul 137-701, Korea.
Exp Mol Med. 2003 Dec 31;35(6):578-85. doi: 10.1038/emm.2003.76.
The serine protease urokinase-type plasminogen activator (uPA) is implicated in pericellular proteolysis in a variety of physiological and pathological processes including angiogenesis and tumor metastasis. The kringle domain of uPA (UK1) has proven to be an anti-angiogenic molecule with unknown mechanism and amino terminal fragment of uPA (u-ATF) with additional growth factor-like domain can be used for blocking interaction of uPA and uPA receptor. Here, we compared anti-angiogenic activities of these two molecules in vitro and in vivo. The recombinant u-ATF from E. coli and refolded in vitro was found to bind to uPAR with high affinity, whereas E. coli-derived UK1 showed no binding by Biacore analysis. In contrast to UK1 having potent inhibitory effect, u-ATF exhibited low inhibitory effect on bovine capillary endothelial cell growth (ED(50)>320 nM). Furthermore, u-ATF inhibition of VEGF-induced migration of human umbilical vein endothelial cell was far less sensitive (IC(50) = 600 nM) than those observed with UK1, and angiogenesis inhibition was marginal in chorioallantoic membrane. These results suggest that kringle domain alone is sufficient for potent anti- angiogenic activity and additional growth factor-like domain diverts this molecule in undergoing different mechanism such as inhibition of uPA/uPAR interaction rather than undergoing distinct anti- angiogenic mechanism driven by kringle domain.
丝氨酸蛋白酶尿激酶型纤溶酶原激活剂（uPA）在包括血管生成和肿瘤转移在内的多种生理和病理过程中的细胞周围蛋白水解中发挥作用。uPA的kringle结构域（UK1）已被证明是一种抗血管生成分子，其作用机制尚不清楚，而具有额外生长因子样结构域的uPA氨基末端片段（u-ATF）可用于阻断uPA与uPA受体的相互作用。在此，我们比较了这两种分子在体外和体内的抗血管生成活性。发现从大肠杆菌中重组并在体外复性的u-ATF能以高亲和力与uPAR结合，而通过Biacore分析发现大肠杆菌来源的UK1无结合能力。与具有强效抑制作用的UK1相反，u-ATF对牛毛细血管内皮细胞生长的抑制作用较低（ED(50)>320 nM）。此外，u-ATF对VEGF诱导的人脐静脉内皮细胞迁移的抑制作用远不如UK1敏感（IC(50)=600 nM），并且在尿囊绒膜上的血管生成抑制作用微弱。这些结果表明，单独的kringle结构域足以产生强效抗血管生成活性，而额外的生长因子样结构域使该分子通过不同机制发挥作用，如抑制uPA/uPAR相互作用，而不是通过kringle结构域驱动的独特抗血管生成机制。
