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P. Geisen, J.R. McColm, M.E. Hartnett; Soluble Vascular Endothelial Growth Factor Promotes Endothelial Cell Transmigration Across the Retinal Pigment Epithelium . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5369.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In AMD, the transformation from occult choroidal neovascularization (CNV) under the retinal pigment epithelium (RPE) to CNV in the neurosensory retina is a major cause of legal blindness. Therefore, we were interested in mechanisms causing EC transmigration across the RPE, in particular, the effects of soluble VEGF. Methods: Transmigration assays were performed in 8µm pore Transwells that permit cell migration. ARPE–19 cells were grown 72 hours on the underside of Transwell inserts. Primary human CECs stained with Cell Tracker Green were then placed in the upright insert. ARPE–19 were plated in some wells to provide growth factors. 72 hrs after CEC were plated, green cells were counted within the well as transmigrated CECs. The ARPE–19 on the underside were then trypsinized and any green cells counted as CECs that had migrated, but not transmigrated. VEGF protein in the media was determined by ELISA. Neutralizing antibody to soluble VEGF165,121 was added to the wells of the Transwells 72 hours before termination of the assay. The effect of conditioned media from solo ARPE–19 on the proliferation of CECs was also studied. All experiments were done in triplicate. Results: CEC migration and transmigration gradually increased over 72 hours. However, ARPE–19 plated in the well significantly increased CEC migration and transmigration. The concentration of VEGF in the media was also significantly increased (998 ± 105 vs 175 ± 28 pg/ml, ARPE–19 in the well vs no ARPE–19 in the well, p<0.004, students T–Test). Neutralizing antibody to soluble isoforms of VEGF (121 and 165) significantly reduced both migrating and transmigrating CECs in a dose dependent fashion (200ng/ml antibody: 1745 ± 223 vs 1264 ± 75, p<0.03, students T–Test). CEC proliferation was significantly increased when cultured in ARPE–19 conditioned media for 3 days compared to control media (21,820 ± 2,580 vs 11,770 ± 1,915 cells/cm2, p<0.009). Conclusions: Soluble VEGF provides a chemotactic gradient for human CEC to migrate to and transmigrate across a monolayer of ARPE–19. Neutralization of soluble VEGF in the media partially reduces the effect.
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