Retina-derived growth factor (RDGF) stimulates the proliferation and migration of capillary and aortic endothelial cells (EC) in vitro and is angiogenic in vivo. Biochemical characterization indicates that RDGF is a heat-stable anionic polypeptide (pI = 5) with a molecular weight between 16,000 and 18,000. A high level of RDGF purification, about 60,000-fold, can be accomplished by a combination of anion exchange and heparin-affinity chromatography. RDGF purified by this method contains only a few polypeptide contaminants and is mitogenic at about 5 ng/ml. The tight binding of RDGF, an anionic protein to heparin, an anionic glycosaminoglycan is unexpected and suggests an affinity between 2 macromolecules. The binding of RDGF to heparin greatly facilitates purification and makes large scale production of pure RDGF feasible. The affinity of RDGF for heparin also suggests that heparin-like molecules mediate RDGF stimulation of EC and capillary growth. Such a mechanism is suggested by observations that heparin-like molecules are the major glycosaminoglycans of the capillary basement membrane. Heparin, at 10 ug/ml, potentiates the activity of RDGF, implicating a three-way interaction among RDGF, heparin-like molecules and EC. Anionic heparin binding growth factors biochemically similar to RDGF are also found in hypothalamus and optic nerve, suggesting that RDGF may be transported between the tissues. The availability of large amounts of pure RDGF will allow us to conduct structural, immunological, mechanistic and physiological studies. Structural analysis of purified RDGF will permit us to isolate its heparin-binding domain and to investigate its structural relationship to other heparin-binding growth factors. Antibodies to RDGF will be used to localize RDGF in situ in neural and other tissues. The role of heparin in EC growth will be ascertained. The physiological role of pure RDGF as an angiogenesis factor will be ascertained. The long-range goal is to understand the role of RDGF in vascular disease, in particular in diabetic retinopathy. A complete knowledge of the structure and function of RDGF may lead to the development of angiogenesis inhibitors that could be used to prevent the vision loss that results from proliferative retinopathy.
The specific aims of the proposed research are: 1) to purify and characterize RDGF; 2) to investigate the role of heparin in the action of RDGF; 3) to study the angiogenic activity of RDGF and to investigate inhibitors of RDGF-stimulated angiogenesis and 4) to produce antibodies to RDGF.
| Wagner, J A; D'Amore, P A (1986) Neurite outgrowth induced by an endothelial cell mitogen isolated from retina. J Cell Biol 103:1363-7 |
