Short Peptide Mutant RLj-RGD4 And Its Prototype RLj-RGD3 Comparative Study Of Inhibition Of Angiogenesis

Objective:To reduce the potential risk of immunogenicity in the biopharmaceutical application of rLj-RGD3,the rLj-RGD4 peptide with 4 Arg-Gly-Asp（RGD）motifs designed using the Lj-RGD3 gene as a prototype,and the recombinant protein was expressed and purified.In this study,we investigated the inhibitory effect of the truncated peptide rLj-RGD4on angiogenesis in human umbilical vein endothelial cells（HUVECs）.As a positive control,rLj-RGD3 was also examined.Methods and Results:An MTT assay revealed that rLj-RGD4 inhibited basic fibroblast growth factor（bFGF）-induced proliferation of HUVECs in a dose-dependent manner with an IC₅₀0 value of 19.95?Μ,whereas the IC₅₀value of rLj-RGD3 was 23.99μM.Moreover,migration and invasion assay,the results show that rLj-RGD4 at different concentrations（20?and 30μM）inhibited the bFGF-induced migration and invasion of HUVEC cells,and this inhibitory effect was dose dependent.In addition,chick chorioallantoic membrane models and tube formation assays showed that rLj-RGD4 reduced bFGF-induced vessel formation.HUVEC apoptosis was detected by Annexin-V fluorescein isothiocyanate（FITC）/propidium iodide（PI）double staining、Hoechst 33258 staining、FITC-phalloidin staining,and the expression levels of Bcl-2 and caspase-3 were detected by Western blotting.The results showed that rLj-RGD4 significantly induced apoptosis and notably degraded the cytoskeleton of HUVECs at different concentrations（20?and 30μM）.Conclusion:In summary,the above results demonstrate that rLj-RGD4 has anti-angiogenesis functions similar to rLj-RGD3.Due to its low molecular weight,rLj-RGD4shows promise for utilization as an anti-angiogenesis drug.