EARLY EVENTS IN ANGIOGENESIS - CLONING AN ALPHA-PROLYL 4-HYDROXYLASE-LIKE GENE

To gain further insight into angiogenesis we sought to clone genes whi ch are actively expressed during this complex process. Using the Matri gel-induced in vitro model we were able to show that although several cell-types form reticular arrays of cells on the gels (align), only en dothelial cells were able to go on and form the capillary-like structu res reminiscent of patent vessels. Although this alignment process did not require gene activation we show that tube formation was ultimatel y dependent upon gene expression occuring during the first few hours t hat cells are seeded onto Matrigel. We generated a cDNA library enrich ed for the expression of those genes and have sequenced an a-prolyl 4- hydroxylase-like clone (angio 0.9). This clone shares 66% overall homo logy to the carboxy-terminal 106 amino-acids of the published human se quence. In the region corresponding to the co-factor binding domains, His 1 and His 2, angio 0.9 has >90% homology to the published sequence . Using an RNAse protection assay we show that the level of expression of the message of this clone is five fold elevated in endothelial cel ls which have aligned on Matrigel. The dependence of collagen, and col lagen hydroxylation in angiogenesis is well documented. Thus, our resu lts are demonstrable proof that the principle of this approach has the potential to generate novel discoveries.