Mediators of Neovascularization
We are trying to characterize the events necessary to induce neovascularization as an approach to understanding the process in the adult organism. The rabbit cornea assay has been utilized in vivo to show angiogenic capacity of tissues or chemically defined compounds. The material is implanted in the rabbit cornea, which is avascular, transparent, and an immunoprivileged site. Neovascularization of the cornea can be seen directly with a split-lamp stereomicroscope. In a first set of experiments we found that prostaglandin E1 is a most convenient angiogenesis inducer when incorporated in a slow-release pellet of a vinyl acetate copolymer. Corneas ready to be invaded by capillaries under PGE1 influence increased their concentration of copper ions and sialic acid. The increment of copper ions appears to be essential for angiogenesis to occur since (1) ceruloplasmin, the copper carrier in plasma, is angiogenic but apoceruloplasmin is not, (2) nonangiogenic compounds such as heparin or the tripeptide glycyl-L-histidyl-L-lysine became angiogenic when bound to copper, and (3) corneas of rabbits deficient in copper were unable to mount an angiogenic response to an appropriate effector, but the situation was rapidly reversed as soon as the plasma copper returned to physiological levels. The increment of sialic acid content of corneas ready to be colonized by capillaries appears to be related to the mobilization of capillary endothelium. We studied this event because it is the first morphological manifestation of angiogenesis. Bovine capillary endothelium was cultivated in vitro and cloned, and mobilization was studied with Boyden chambers and the gelatin-agarose procedures (Alessandri et al, 1983). Migration of capillary endothelium was enhanced by fragments of cornea ready to be colonized by capillaries and also by hydrolysates of the extracellular substance deposited by the capillary endothelium when cultivated for 8-10 days. Among the constituents of this material, fibronectin was found to be a most efficient mobilizer of capillary endothelium, particularly when heparin was also present. Fragments of both molecules were as effective as the complete molecules, and an antifibronectin serum could block mobilization. Motility of capillary endothelium was also substantially enhanced after incubation with a mixture of gangliosides. The cells incorporated the gangliosides and bound about twice as much fibronectin as before incubation. GT1 was particularly effective in enhancing motility. “Blockage” of binding sites by cholera toxin or γ interferon substantially reduced motility. In conclusion, copper, fibronectin, heparin, and gangliosides appear to act as mediators of angiogenesis. The sequence of events controlled or influenced by them is still unknown.
KeywordsSialic Acid Cholera Toxin Boyden Chamber Angiogenic Response Capillary Endothelium
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