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L.C. Shaw, A. Afzal, P.E. Spoerri, H. Pan, M.B. Grant; IGF–1 and VEGF Receptor Signaling and Interaction in Human Retinal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4206.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The complex interplay between vascular endothelial growth factor (VEGF) and insulin like growth factor–1 (IGF–1) is key in maintenance and development of healthy vessels and is involved in both early and late histological abnormalities associated with diabetic retinopathy (DR). Using ribozymes specifically targeted against IGF–1 receptor (IGF–1R), VEGF receptor–1 (VEGFR–1) and VEGF receptor–2 (VEGFR–2), we investigated the relative contribution of IGF–1 and VEGF in assays relevant to neovascularization. Methods: The in vivo effect of these ribozymes on retinal neovascularization was examined using the oxygen–induced retinopathy (OIR) mouse model. Human retinal endothelial cells (HRECs) were transfected with plasmids expressing ribozymes. The levels of receptor mRNA was examined using reverse transcription and real time–PCR. Receptor protein levels were determined using western analysis and flow cytometry. Functional studies, including chemotaxis, proliferation, tube formation, were performed to examine the interaction of these growth factors and receptors. Results: Each ribozyme reduced the levels of abnormal retinal neovascularization in the OIR mouse model (IGF–1R by 73 ± 3%, VEGFR–1 by 47 ± 5%, VEGFR–2 by 75 ±5%). Cell proliferation, as measured by BrdU incorporation, was differentially inhibited by ribozyme expression. Chemotaxis experiments confirmed the specificity of the ribozymes as HRECs expressing the VEGFR–1 ribozyme did not respond to placental growth factor (PlGF, VEGFR–1 specific), but migrated to VEGF–E (VEGFR–2 specific). Conversely, HRECs expressing the VEGFR–2 ribozyme did not respond to VEGF–E, but migrated to PlGF. Endothelial tube elongation was inhibited by expression of the VEGFR–2 ribozyme in HRECs, while endothelial tube sprouting was inhibited by the VEGFR–1 ribozyme suggesting receptor specific functions. Conclusions: Hammerhead ribozymes can be used to examine receptor specific responses and receptor interactions. We have shown that IGF–1R, VEGFR–1 and VEGFR–2 have unique contributions to neovascularization and endothelial function.
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