IGFBP-3 reduces eNOS and PKCzeta phosphorylation, leading to lowered VEGF levels

PURPOSE In models of diabetic retinopathy, insulin-like growth factor binding protein-3 (IGFBP-3) is protective to the retina, especially retinal microvascular endothelial cells (RECs), but the underlying mechanisms are unclear. For this study, we hypothesized that IGFBP-3 may reduce vascular endothelial growth factor (VEGF) levels through reduced endothelial nitric oxide synthase (eNOS) activity, which may be protective against macular edema. METHODS To test this hypothesis, we grew primary human retinal endothelial cells in normal glucose (5 mM) or high glucose (25 mM) for three days, treated with IGFBP-3 NB plasmid (a plasmid of IGFBP-3 that cannot bind IGF-1), followed by western blotting for eNOS, protein kinase C zeta (PKCzeta), and VEGF. Additionally, we treated some cells with recombinant eNOS or PKCzeta, after IGFBP-3 NB plasmid transfection to validate that these pathways regulate VEGF levels. Immunoprecipitation experiments were done with the eNOS antibody, followed by western blotting for PKCzeta, to determine if eNOS and PKCzeta interact directly. RESULTS Our results suggest that 1) IGFBP-3 inhibits the endothelial nitric oxide synthase (eNOS) and protein kinase C zeta (PKCzeta) pathway, which in turn inhibits VEGF production, and 2) that eNOS plays a role in activating PKCzeta to increase VEGF levels in diabetic retinopathy. CONCLUSIONS In conclusion, IGFBP-3 may be a novel treatment for macular edema through the inhibition of eNOS and PKCzeta activation, leading to reduced VEGF levels.