Recombinant Mojastin Mutant Disintegrins, r-Moj-MP and r-Moj-DM, Inhibit Angiogenesis

Integrins, transmembrane proteins that act as cell-matrix adhesion receptors, play an important role in the induction of angiogenesis. VEGF (vascular endothelial growth factor) activates the angiogenic pathway on endothelial cells resulting in the formation of new blood vessels and branching of those vessels.  Disintegrins are small peptides containing an RGD binding motif that have a high binding affinity to integrins. The recombinant version of the disintegrin mojastin (r-Moj-WN) was mutated to contain the amino acids MP, or DM carboxyl to the RGD motif. Using an in vitro angiogenesis assay with cultured Human Umbilical Vein Endothelial Cells, HUVEC, we tested vessel tube formation and branching in the presence of r-Moj-MP (2.5µM) or r-Moj-DM (2.5µM). We hypothesized that r-Moj-DM and r-Moj-MP will reduce vessel tube formation and branching. Angiogenesis assays were performed in triplicates.  The untreated controls formed an average of 68.3 tubes, while r-Moj-DM-treated cells formed 15.3 tubes, and r-Moj-MP-treated cells formed 18.3 tubes.  The number of branches was also drastically reduced between the untreated control (13.4 branches) and the r-Moj-DM (1.3) and r-Moj-MP-treated cells (2).  Our hypothesis was supported as both recombinant disintegrins reduced the number of vessel tubes and branches significantly (p<0.05) as compared to untreated HUVECs. Therefore, we concluded that the recombinant disintegrins r-Moj-MP and r-Moj-DM inhibited angiogenesis.