Early events in angiogenesis: cloning an alpha-prolyl 4-hydroxylase-like gene.
To gain further insight into angiogenesis we sought to clone genes which are actively expressed during this complex process. Using the Matrigel-induced in vitro model we were able to show that although several cell-types form reticular arrays of cells on the gels (align), only endothelial cells were able to go on and form the capillary-like structures reminiscent of patent vessels. Although this alignment process did not require gene activation we show that tube formation was ultimately dependent upon gene expression occuring during the first few hours that cells are seeded onto Matrigel. We generated a cDNA library enriched for the expression of those genes and have sequenced an alpha-prolyl 4-hydroxylase-like clone (angio 0.9). This clone shares 66% overall homology to the carboxy-terminal 106 amino-acids of the published human sequence. 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 cells which have aligned on Matrigel. The dependence of collagen, and collagen hydroxylation in angiogenesis is well documented. Thus, our results are demonstrable proof that the principle of this approach has the potential to generate novel discoveries.
Cockerill
G W
GW
Cardiovascular Medicine Unit, National Heart and Lung Institute, Imperial College School of Medicine, Hammersmith Hospital, London W12 ONN, UK.
Varcoe
L
L
Meyer
G T
GT
Vadas
M A
MA
Gamble
J R
JR
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003094:Collagen / Q000096:biosynthesis; D018076:DNA, Complementary / Q000235:genetics; D004338:Drug Combinations; D004730:Endothelium, Vascular / Q000378:metabolism; D006801:Humans; D007797:Laminin; D008969:Molecular Sequence Data; D018919:Neovascularization, Physiologic / Q000502:physiology*; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics*; D011509:Proteoglycans; D012333:RNA, Messenger / Q000378:metabolism; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D015533:Transcriptional Activation; D014471:Umbilical Veins / Q000378:metabolism; D015854:Up-Regulation
D018076:DNA, Complementary; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; D012333:RNA, Messenger; C059485:matrigel; D003094:Collagen; D011393:Procollagen-Proline Dioxygenase