A TRIALTO REGULATE ANGIOGENESIS BY MEANS OF GENE

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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