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P. Geisen, M. Zayed, L. V. Parise, M. E. Hartnett; Vegf 189 Isoform Plays an Important Role in Retinal Pigment Epithelium/Choroidal Endothelial Cell Transmigration. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2526.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the role VEGF 189 isoform plays in retinal pigment epithelium (RPE) stressed with hypoxia and in choroidal endothelial cell (CEC) transmigration across RPE.
Primary human fetal RPE (hfRPE) were isolated from donor eyes and characterized to be well-differentiated with strong cell junctions evidence by high TER (>100Ω.cm2) and occludin localization to cell boundaries. CECs were isolated from young (< 30 years old) human adult donors. Three separate experiments were performed: 1) hfRPE grown in 1% O2 overnight were analyzed for VEGF mRNA expression by real-time PCR; 2) hfRPE were grown in coculture with CECs in the insert of 1.0 µm pore inserts, and VEGF 189 protein was analyzed by Western blot; 3) hfRPE infected with lentivirus containing short hairpin RNA (shRNA) to VEGF 189 and GFP or a non-specific shRNA with GFP as control were plated to confluence on the underside of 8.0 µm culture inserts. After the hfRPE formed epithelioid monolayers, CECs (stained with CellTracker Red, Invitrogen, IL) were plated in the insert. After 3 days, transmigrated red CECs on the underside of the inserts were counted.
hfRPE grown in 1% O2 showed preferential upregulation of VEGF 189 over other isoforms and a 6.6 fold increase in VEGF 189 mRNA expression compared to room air controls (p<0.024, Student's t-Test). hfRPE that were in contact with CECs showed upregulation of VEGF 189 by western blot compared to hfRPE grown in non-contacting coculture with CECs (4.3 fold increase). Transmigration of CECs to hfRPE infected with shRNA to VEGF 189 was reduced by 29% compared to control shRNA.
When RPE become stressed or injured, VEGF is upregulated. We found an increase in VEGF189 in two conditions where hfRPE was stressed (contact with CECs and hypoxia) that provide insight into RPE/CEC interaction in vitro and possibly age-related macular degeneration in vivo.
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