A TRIALTO REGULATE ANGIOGENESIS BY MEANS OF GENE

血管生成细胞生长因子如成纤维细胞生长因子2、血管内皮生长因子、肝细胞生长因子与硫酸肝素(HS)相互作用。我们假设，这些特性不仅对那些细胞生长因子的活性重要，而且对每种细胞生长因子的特定调节也是重要的。已经进行了几次实验来证明这些证据。1)每种血管生成细胞生长因子在HS上的结合结构的表征表明，根据细胞生长因子的不同，其结构是不同的。糖醛酸残基的C2位和氨基葡萄糖残基的C6位的0-硫化反应似乎是此类结构的决定性反应。2)首次分离纯化了参与O-硫代反应的酶，并克隆了它们的cDNAs，推测可能存在四种不同的酶基因。包括后来发现的剪接变体，有五种酶负责这些硫酸盐化。3)…底物专一性分析此外，重组酶还表明，不同的酶具有不同的特异性，都参与了结合成纤维细胞生长因子-2、血管内皮生长因子和肝细胞生长因子的HS结构的合成。4)通过转染酶的cDNA过表达导致了HS结构的预期变化，这对于建立一种通过基因操作干扰HS结构的方法是重要的。一些下调它们表达的尝试使我们认识到它们与其他成分的组装以及它们在高尔基体中的定位所起的重要作用。5)研究了HS结构紊乱对HGF细胞生长因子信号转导途径(ERK的磷酸化)的影响。此外，我们还发现在果蝇中，6-O-磺基转移酶同时阻断了成纤维细胞生长因子-2信号转导和气管形成(相当于哺乳动物的血管生成)。6)在体外血管生成模型培养体系中，我们观察到加入具有结合活性的HS寡核苷酸对细胞生长有明显的抑制作用，这证实了我们提出的工作假说是相关的。较少

Angiogenetic cell growth factors, such as FGF-2, VEGF, HGF interact with heparan sulfate (HS). We hypothesized that these properties should be important not only for the activities of those cell growth factors but also for the specific regulation of each cell growth factor. Several experiments have been performed to show the evidences. 1) Characterization of the binding structure on HS for each angiogenetic cell growth factor has revealed that the structures are different from each other, depending on the cell growth factors. And 0-sulfations at C2 of uronic acid residues and at C6 of glucosamine residues seem to be determinative reactions for such structures. 2) We for the first time purified the enzymes responsible for those O-sulfations and cloned their cDNAs, which suggested the occurrence of four different enzyme genes involved. Including a spliced variant form that was subsequently found, five enzymes are responsible for those sulfations. 3) Analysis for substrate specificities o … More f the recombinant enzymes has revealed that different enzymes have different specificities and all are involved in the syntheses of HS structures for the bindings of FGF-2, VEGF and HGF. 4) Over expression of the enzymes by transfections of their cDNAs caused expected changes in the HS structures, which is important to establish a way to disturb the HS structures by the gene manipulation. Some trial toward down-regulation of their expressions have made us realize essential roles of their assembly with other components and their localizations in the Golgi apparatus. 5) A possible change of cell growth factor signaling (phosphorylation of ERK) by disturbance in HS structure was examined for HGF. In addition, we found the simultaneous interruption of FGF-2 signaling and tracheal formation (corresponding to mammalian angiogenesis) by the interruption of the 6-O-sulfotransferase in Drosophila. 6)We observe significant inhibition of cell growth by the addition of HS oligo with the binding activity in a model culture system for in vitro angiogenesis, which has confirmed us that the proposed working hypothesis is relevant. Less