Effects of angiogenic growth factor combinations on retinal endothelial cells

The aim of this paper was to determine if growth factors, known to be upregulated in proliferative diabetic retinopathy, exerted combined effects on retinal endothelial cells. The authors explored the individual and collective actions of insulin-like growth factor I (IGF-I), vascular endothelial growth factor (VEGF), platelet-derived growth factor-BB (PDGF-BB), fibroblast growth factor-2 (FGF-2) and placenta growth factor (PlGF) on several parameters that reflect the angiogenic potential of endothelial cells. The effect of growth factors on cell migration and survival/proliferation was examined using primary cultures of bovine retinal endothelial cells (BREC). The authors also determined the growth factor action on capillary-like tube formation on a reconstituted basement membrane matrix and on the newly described phenomenon of secondary sprouting, in which endothelial cell colonies spontaneously survive, proliferate, migrate and invade the matrix after the original capillary-like tubes have collapsed. Sprouting cells were positive for von Willebrand factor and could aggregate into larger tubes with lumens. Incubation with VEGF+IGF-I or PlGF+FGF-2 enhanced tube stability by 40-50%, more than each growth factor alone or other combinations (5-20%). The concurrent addition of four growth factors did not improve the response seen with growth factor pairs. Surprisingly, PDGF-BB induced tube collapse. IGF-I and FGF-2 mildly enhanced BREC proliferation/survival (5-15%). However, VEGF+IGF-I or PlGF+FGF-2 increased BREC proliferation/survival by 25% under low serum conditions, whereas combinations of all four growth factors exerted a clearly synergistic effect (250% increase). PDGF-BB or FGF-2 stimulated secondary sprouting and were the only factors capable of exerting this effect alone. Even though VEGF, IGF-I or PlGF were not effective, if administered in pairs, they demonstrated increased responses. PDGF-BB was also able to enhance the effect of FGF-2+IGF-I+VEGF on BREC secondary sprouting, but not of any of them individually. No other growth factor tested was able to significantly improve the action of combinations of three other growth factors. VEGF increased cell migration in a wounded monolayer assay two-fold and PDGF-BB, 2.5 times, but other individual growth factors were ineffective. PlGF+FGF-2 enhanced cell migration more than each factor alone. VEGF+IGF-I+PlGF+FGF-2, however, increased cell migration four-fold. In summary, this study indicates that growth factors, overexpressed in diabetic retinopathy eyes, enhance the angiogenic characteristics of cultured cells (tube formation, proliferation, secondary sprouting and migration). Their effects, however, can be greatly augmented by other growth factors that alone exert little or no action. Therefore, diabetic retinal neovascularization may result from the additive or synergistic action of several growth factors.