Structural elements of kallistatin required for inhibition of angiogenesis
Structural elements of kallistatin required for inhibition of angiogenesis
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DOI：
10.1152/ajpcell.00524.2002
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发表时间：
2003-06-01
影响因子：
5.5
通讯作者：
Chao, J
中科院分区：
文献类型：
--
作者：
Miao, RQ;Chen, V;Chao, J
卡利斯汀是一种丝氨酸，最初被确定为组织激肽释放酶的特异性抑制物。我们最近的研究表明，激肽释放酶促进血管生成，而激肽释放酶抑制血管生成和肿瘤生长。本研究的目的是确定Kallistatin抗血管生成功能所必需的结构元件。通过定点突变，获得了KListatin铰链区(A377T)和主要肝素结合区(K312A/K313A)突变株。重组激肽释放酶突变体A377T不结合或抑制组织激肽释放酶活性。野生型Klistatin和Klistatin突变体A377T，而不是Klistatin突变体K312A/K313A缺乏肝素结合活性，可抑制VEGF诱导的人微血管内皮细胞的增殖、生长和迁移。同样，野生型Kallistatin和Klistatin突变体A337T，但不能抑制Klistatin突变体K312A/K313A，显著抑制血管内皮生长因子诱导的小鼠Matrigel内皮细胞毛细血管形成和Matrigel植入体内毛细血管形成。为了阐明肝素结合域在调节血管生成中的作用，我们发现野生型kallistatin阻断了I-125标记的血管内皮细胞与血管内皮细胞的结合，而kallistatin突变体K312A/K313A不干扰VEGF的结合。因此，野生型kallistatin而不是kallistatin突变体K312A/K313A抑制了VEGF诱导的Akt的磷酸化。综上所述，这些结果表明，抑制血管生成的关键是肝素结合区域，而不是kallistatin的反应部位环。
Kallistatin is a serpin first identified as a specific inhibitor of tissue kallikrein. Our recent studies showed that kallikrein promoted angiogenesis, whereas kallistatin inhibited angiogenesis and tumor growth. This study is aimed to identify the structural elements of kallistatin essential for its antiangiogenic function. Kallistatin mutants at the hinge region (A377T) and a major heparin-binding domain (K312A/K313A) were created by site-directed mutagenesis. Recombinant kallistatin mutant A377T did not bind or inhibit tissue kallikrein activity. Wild-type kallistatin and kallistatin mutant A377T, but not kallistatin mutant K312A/K313A lacking heparin-binding activity, inhibited VEGF-induced proliferation, growth, and migration of human microvascular endothelial cells. Similarly, wild-type kallistatin and kallistatin mutant A337T, but not kallistatin mutant K312A/K313A, significantly inhibited VEGF-induced capillary tube formation of cultured endothelial cells in Matrigel and capillary formation in Matrigel implants in mice. To elucidate the role of the heparin-binding domain in modulating angiogenesis, we showed that wild-type kallistatin interrupted the binding of I-125-labeled VEGF to endothelial cells, whereas kallistatin mutant K312A/K313A did not interfere with VEGF binding. Consequently, wild-type kallistatin, but not kallistatin mutant K312A/K313A, suppressed VEGF-induced phosphorylation of Akt. Taken together, these results indicate that the heparin-binding domain, but not the reactive site loop of kallistatin, is essential for inhibiting VEGF-induced angiogenesis.