Aberrant neovascularization of the retina is a major cause of adult blindness associated with diabetic retinopathy. The 16 kDa fragment of human prolactin (16K hPRL) is a potent and specific antiangiogenic factor that inhibits neovascularization of the retina in the oxygen-induced retinopathy (OIR) model. In a variety of endothelial cell models, 16K hPRL inhibits VEGF- and bFGF-induced endothelial cell proliferation and activates apoptosis. We will determine the signaling pathways responsible for the antiangiogenic action of the 16K hPRL in the retina in vitro in bovine adrenal capillary endothelial cells (BAEC) and human retinal endothelial cells (HREC), and in vivo in the mouse OIR model. The inhibition of VEGF-induced endothelial cell proliferation by 16K hPRL is mediated via inhibition of Ras activation. We hypothesize that 16K hPRL inhibits Ras activation by stimulating the association of Ras with two Ras inhibitory proteins, Ras-GAP and Sprouty2 (Spry). In vitro we will study the protein-protein interactions between Ras-GFP and Ras-GAP-RFP and Spry2-RFP in living BAEC and HREC by measuring fluorescence resonance energy transfer (FRET) intensity in cellular compartments. Cells will be treated with VEGF and VEGF + 16K hPRL and signaling interactions measured in real-time. We hypothesize that the organization of signaling molecules occurs within calveolae. The calveolae will be identified by caveolin-1 (Cav-1-CFP) and the co-localization and FRET of signaling molecules measured in calveolae with the GFP and RFP donor/acceptor pair. Immunoblotting of proteins purified from cell compartments will be used to confirm results. We hypothesize that the 16K hPRL-induced inhibition of retinal neovascularization in the OIR model is mediated by the induction of apoptosis and inhibition of MAPK signaling. We will determine if intravitreal injection of an adenovirus (Ad) expressing 16K hPRL (16K-Ad) or a NulI-Ad into one eye activates apoptosis in areas of neovascularization measured by the TUNEL assay and a caspase-3 assay. We will utilize quantitative fluorescence microscopy to measure MAPK activation in areas of neovascularization in retinal whole mounts from the OIR model. MAPK activation will be assessed with a recently established assay that measures co-localization and FRET intensity of Ras-GFP and Raf-I-RFP. These studies will provide the basis for developing potent antiangiogenic factors for the treatment of abnormal neovascualization of the retina.
