Construction And Characterization Of Fc Fusion Proteins As Extracellular Matrices

The large network of blood vessels in vivo provides the efficient oxygen andnutrients for cells growth, which plays a key role on cell metabolism and function.Vascular endothelial cells, the important part of vascular, regulate angiogenesis andvascular permeability and the expression of cell activity molecules. Therefore, theconstruction of biomimic extracellular matrix (ECM) with the functions of vascularendothelial cell adhesion, proliferation and survival regulation is an importantsolution for artificial functional tissue or organ lacking of adequate vascular system.In this study, owing to the development of recombinant DNA technology, theplasmid of pcDNA3.1-VEGF-Fc containing the vascular endothelial growth factor(VEGF) and immunoglobulin IgG Fc fragment coding sequence and the plasmid ofpcDNA3.1-VEGF-SMP-Fc containing VEGF, MMP-2specific substrate and the Fcfragment coding sequence were constructed, respectively. Then, the fusion proteinVEGF-Fc and VEGF-SMP-Fc were expressed by the eukaryocyte expression systemand purified by protein A affinity chromatography column with the protein purityover85%. Furthuremore, the purified fusion proteins were physical immobilized onthe surface of hydrophobic polystyrene (PS) surface for forming a growthfactor-extracellular matrix and a protease sensitive-extracellular matrix.As the results shown, the ECM constructed by fusion protein VEGF-Fcsignificantly enhanced the ability of human umbilical vein endothelial cells(HUVECs) adhered to the surface of hydrophobic materials, and markedly promotedthe proliferation of HUVECs longer than72h. As the result of cytoskeleton staining,endothelial cells culturing on fusion protein VEGF-Fc matrix induced thereorganization of actin filaments into larger stress fibers. The fusion protein VEGFcould also effectively regulate the expression of functional molecules in endothelialcells to induce the endothelium formation, thus contributing to regulate blood vesselformation and permeability.In addition, compared with the half-life of commercial VEGF (12h), the VEGFexisted in fusion protein had the higher half-life nearly to96h. Due to thehydrophobic interaction between Fc domain and the surface of PS, MMP-2sensitivefusion protein VEGF-SMP-Fc was stabily immobilized to form the proteasesensitive-ECM. The control release of VEGF was realized with the changes of MMP-2content.