title: VEGF receptor binding peptide-linked high mobility box group-1 box A as a targeting gene carrier for hypoxic endothelial cells.
abstract: High mobility group box-1 (HMGB-1) is a nuclear protein that can bind to and condense plasmid DNA. In this study, we developed a recombinant VEGF receptor binding peptide (VRBP) linked to HMGB-1 box A (VRBP-HMGB1A) as a targeting gene carrier to hypoxic endothelial cells. Hypoxic endothelial cells in ischemic tissues of solid tumors are important targets for gene therapy. A recombinant VRBP-HMGB1A expression vector, pET21a-VRBP-HMGB1A was constructed. VRBP-HMGB1A was over-expressed in BL21 strain and purified by nickel-chelate affinity chromatography. Complex formation between VRBP-HMGB1A and pCMV-Luc was confirmed by gel retardation assay. pCMV-Luc was retarded completely at a 2/1 weight ratio (peptide/plasmid). For transfection assays, calf pulmonary artery endothelial (CPAE) cells were incubated under hypoxia for 24 h, prior to transfection to induce the VEGF receptors on the cells. VRBP-HMGB1A/pCMV-Luc complexes were transfected to hypoxic CPAE cells. The highest transfection efficiency was at a 30/1 weight ratio (peptide/plasmid). In addition, VRBP-HMGB1A had higher efficiency than poly-L-lysine (PLL) specifically in hypoxic CPAE cells, However, VRBP-HMGB1A had lower efficiency than PLL in 293, H9C2, and normoxic CPAE cells. In MTT assay, VRBP-HMGB1A was less toxic than PLL to cells. In conclusion, VRBP-HMGB1A is a potential gene carrier for targeting hypoxic endothelial cells and thus, may be useful for cancer gene therapy.
authors.affiliation: Department of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791, South Korea.
authors.forename: Jee Seung
authors.initials: JS
authors.lastname: Han
authors.forename: Hyun Ah
authors.initials: HA
authors.lastname: Kim
authors.forename: Sanghyun
authors.initials: S
authors.lastname: Lee
authors.forename: Minhyung
authors.initials: M
authors.lastname: Lee
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015687:Cell Hypoxia; D002460:Cell Line; D002470:Cell Survival; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D018014:Gene Transfer Techniques; D024243:HMGB1 Protein / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D040262:Receptors, Vascular Endothelial Growth Factor / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D024243:HMGB1 Protein; D010455:Peptides; D040262:Receptors, Vascular Endothelial Growth Factor