Purification and Characterization of VEGFNPF Secreted by Human Retinal Pigment Epithelial Cells

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Abstract
A growth factor with an apparent target cell specificity for human umbilical vein endothelial cells (HUVEC) has been identified in serum-free medium conditioned by human retinal pigment epithelial (HRPE) cells. This factor, designated vascular endothelial growth factor-like mitogen (VEGF-like mitogen), was purified 4,200-fold by a three-step chromatography consisting of Mono Q ion-exchange chromatography, heparin-Sepharose affinity chromatography and hydrophobic chromatography on a C4 reverse phase HPLC column. The VEGF-like mitogen has a molecular mass of 43,000 and is composed of two apparently identical subunits as assessed by silver-stained SDS/PAGE with or without prior reduction. The purified factor has a maximal mitogenic effect on HUVEC at a concentration of approximately 10 ng/ml. In contrast to bFGF, aFGF and PD-ECGF, the VEGF-like mitogen is a secreted dimeric glycoprotein. It is not mitogenic for human skin fibroblasts (HFIB), human RPE cells and human dermal keratinocytes (HDK). Our results show (that the VEGF-like mitogen shares biochemical, structural, antigenic and biological properties with the mitogens of the VEGFNPF family. Immunoblotting using an anti-VEGF antiserum identifies the purified mitogen as VEGF/VPF.