A TRIALTO REGULATE ANGIOGENESIS BY MEANS OF GENE

血管生成细胞生长因子如FGF-2、VEGF、HGF与硫酸乙酰肝素（HS）相互作用。我们假设这些特性不仅对这些细胞生长因子的活性很重要，而且对每个细胞生长因子的特异性调节也很重要。已经进行了几个实验来证明这些证据。1)HS上每个血管生成细胞生长因子的结合结构的表征揭示了结构彼此不同，这取决于细胞生长因子。糖醛酸残基C2和葡萄糖胺残基C6的O-硫酸化似乎是这种结构的决定性反应。2)我们首次纯化了负责这些O-硫酸化的酶，并克隆了它们的cDNA，这表明涉及四种不同酶基因的存在。包括随后发现的剪接变体形式，五种酶负责这些硫酸化。3)底物特异性分析 ...更多信息 重组酶的研究表明，不同的酶具有不同的特异性，它们都参与合成与FGF-2、VEGF和HGF结合的HS结构。4)通过转染它们的cDNA过量表达酶引起HS结构的预期变化，这对于建立通过基因操作干扰HS结构的方法是重要的。对它们表达下调的一些尝试使我们认识到它们与其他组分的组装以及它们在高尔基体中的定位的重要作用。5)对HGF检查了HS结构紊乱引起的细胞生长因子信号传导（ERK磷酸化）的可能变化。此外，我们发现，在果蝇中，FGF-2信号和气管形成（对应于哺乳动物血管生成）的中断6-O-磺基转移酶的同时中断。6）在体外血管生成的模型培养系统中，我们观察到通过添加具有结合活性的HS oligo对细胞生长的显著抑制，这证实了我们提出的工作假设是相关的。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