HBD-3 inhibits angiogenesis in a tissue culture model

Human b defensin-3 (hBD-3) is an antimicrobial and immunoregulatory peptide expressed primarily in basal epithelial cells of the oral mucosae. We previously reported that hBD-3 internalizes the chemokine receptor CXCR4 on T cells and competes off its natural ligand SDF-1 (Feng et al, J immunol, 177: 782, 2006). CXCR4 is important in a number of vital biological functions, including angiogenesis. Objective: To test if hBD-3 has anti-angiogenic activity in an in vitro angiogenesis model. Methods: Recombinant hBD-3 was generated as described previously (Feng et al, J. Dent. Res., 84: 445, 2005). CM-DiI loaded HUVEC cells were added to the top of confluent primary human dermal fibroblasts in 24 well plates, as previously described (Sorrell et al, Cells Tissues Organs, 186: 157, 2007). HBD-3 was added to the co-culture simultaneously with HUVEC cells. Spent medium was replaced every three to four days by fresh medium containing HBD-3. To assess the contribution of SDF-1, fibroblasts were transfected with SDF-1 siRNA or a negative control by transfection reagents. Co-cultures were maintained for 10 days, followed by fixing and staining with anti-human PECAM-1 and FITC labeled secondary antibody. Plates were observed by fluorescence microscopy. Results: Endothelial cell tube formation was significantly reduced in the presence of hBD-3, and the peptide appeared to induce aggregation of HUVEC cells. Results were not due to hBD-3 induced cytotoxicity to either fibroblasts or HUVEC, as there was no evidence of cell killing by hBD-3. Moreover, SDF-1 silencing caused similar HUVEC aggregation. Conclusion: HBD-3 inhibited HUVEC cell migration and tube formation, probably through interaction with CXCR4 on HUVEC cells. This discovery is consistent with our previous findings and justifies further studies on the possible application of hBD-3 or its derivatives in cancer therapy. This study was supported by NIH/NIDCR R01DE015510 and the L. David and Virginia Baldwin Foundation.