Abstract
Purpose: :
To date experimental identification of angiogenesis inhibitors has been an empirical time-consuming process. Selected proteins were proteolytically processed and the cleaved fragments were tested for their antiangiogenic potency. In this study we introduce several antiangiogenic peptides for ocular angiogenesis that have been predicted using a bioinformatic approach.
Methods: :
: A custom built bioinformatics algorithm was used to test for similarities of known peptide inhibitors of angiogenesis derived from proteins within the whole human proteome. The predicted peptides were synthesized using standard solid phase synthesis techniques. Human retinal endothelial cells were grown in the presence of varying concentrations of peptide in complete medium with serum. After 3 days cell proliferation was quantified. The effect of peptides on migration of endothelial cells was evaluated in a modified Boyden chamber assay. Specificity of peptide effect was determined by receptor neutralization studies performed after preincubating endothelial cells with increasing concentrations of neutralizing monoclonal antibody with the peptide.
Results: :
Significant in vitro activity was associated with peptides in each of the three protein classes tested thereby validating the bioinformatics approach. Inhibition of migration and proliferation was quantitatively compared for peptides derived from the type IV collagen, CXC chemokine, and TSP1 repeat containing protein families. The tested peptides derived from the TSP1 repeat containing proteins were the most potent in suppressing both endothelial cell proliferation (49% decrease, p<0.001, n=8) and migration (84% decrease, p<0.001, n=8). The peptides derived from type IV collagen fibrils were most active in suppressing proliferation, 51% (p<0.001, n=8) while the peptides derived from CXC chemokines were potent suppressors of endothelial cell migration, 78% (p<0.001, n=8). Receptor neutralization studies have identified receptors for each peptide group.
Conclusions: :
Using a systematic approach we have identified a variety of novel peptides that are negative regulators of angiogenesis. Their in vitro activity has been quantified and is compared using endothelial cell proliferation and migration assays. Receptor neutralization studies confirm receptor mediated activity and specificity. We will present the comparative data set for the peptides tested. These studies provide mechanistic insights into angiogenesis and have clear potential therapeutic implications.
Keywords: neovascularization • retinal neovascularization • vascular endothelial growth factor