Abstract
Purpose: :
In age–related macular degeneration (AMD), choroidal neovascularization (CNV) is often preceded by the migration of choroidal endothelial cells (CECs) across the retinal pigment epithelium (RPE). Previous studies have shown that hypoxia increases RPE expression of the angiogenic factor VEGF. The aim of this study was to investigate the role of VEGF isoforms in CEC transmigration when contact is made with RPE.
Methods: :
For all studies, ARPE passages 15–20 were plated in 6–well dishes in DMEM/F–12 with 10% FBS and grown at 37°C in 5% CO2 to assure continuity across experimental and control conditions . For hypoxia studies, ARPE were grown for three days and then exposed to 24 hours of 1% O2. Controls remained in 21% O2. mRNA concentration was determined by real time RT–PCR using Taqman primers for VEGF189, 165, and 121. Samples were normalized to endogenous 18S and protein determined by Western blot. mRNA and protein were expressed as fold increase over values measured in ARPE grown in 21% O2. To determine the change in VEGF189 protein expression when CECs made contact with ARPE, ARPE were plated on the undersides of transwell inserts with 0.4 µm pores (contacting coculture) or in the wells of a 6–well dish (noncontacting coculture). CECs were plated in the inserts in both coculture conditions and the media was changed to serum–free EBM–2. For the transmigration study, ARPE were incubated in CellTracker Red and plated onto the underside of Transwell inserts with 8 µm pores at a density of 1.8x105 cells/cm2. After 72 hours, primary human CECs incubated in CellTracker Green were plated into each insert at 1 x 105 cells/cm2. Then, after 24, 48, or 72 hours, inserts were transferred to a new well and trypsinized. Green cells taken from the well were counted on a hemocytometer to determine the number of transmigrated CECs.
Results: :
Exposure to 1% O2 conditions induced increased expression of VEGF mRNA for all three isoforms compared to 21% O2 (5.60, 8.68 and 10.01 fold increase for VEGF121, 165, 189, respectively). VEGF189 protein expression was higher in ARPE grown in contact with CECs than in ARPE grown in noncontacting coculture (0.479 vs 0.328 pixel density). Transmigration of CECs was increased in contacting culture vs. solo CEC culture (2091 cells/0.33 cm2 vs 270 cells/0.33 cm2 p = 0.016).
Conclusions: :
This study provides evidence that expression of VEGF189 is increased when ARPE are grown in hypoxia or make contact with CECs and may play an important role in the transmigration of CECs, a step involved in CNV.
Keywords: choroid: neovascularization • age-related macular degeneration • retinal pigment epithelium