Abstract
Purpose: :
To investigate the role of VEGF 189 isoform mRNA upregulation in retinal pigment epithelium (RPE)/choroidal endothelial cell (CEC) coculture and the effects on CEC signaling.
Methods: :
Primary human fetal RPE (hfRPE) were isolated from eyes and characterized to be well-differentiated. Primary mouse RPE (mRPE) were isolated from either wild-type (WT) or mice expressing only the vegf188 (188/188) isoform. CECs were isolated from young (< 30 years old) human adult donors. hfRPE or mRPE were plated on the underside of 1µm pore culture inserts and allowed to attach. The inserts were then uprighted and hfRPE grown in media until epithelial monolayers formed. In contacting coculture, CECs were then plated within the wells to permit cell processes to make contact with the underlying hfRPE. In non-contacting coculture, hfRPE were plated in the well of the culture plate and CECs into the insert. In both coculture conditions, analyses were performed after 3 days of coculture. Total cellular RNA was extracted from each cell type, and VEGF mRNA expression from hfRPE and VEGF receptor mRNA from CECs with or without coculture were analyzed using Real-Time PCR. GTPase Rac1 activity was determined in CECs grown in coculture with either wild type (WT) or 188/188 mRPE was analyzed by Western blot using a Rac1 pulldown assay.
Results: :
hfRPE grown in contacting coculture showed a preferential upregulation of VEGF 189 isoform (70 fold) compared to solo culture or non-contacting coculture. Only a modest increase in VEGF isoforms 121 and 165 was found in the coculture conditions. VEGF receptor 1 (VEGFR1) mRNA in CECs was upregulated in both coculture conditions, whereas VEGF receptor 2 (VEGFR2) mRNA in CECs was downregulated in coculture. Active Rac1 protein was not different in CECs grown in contacting coculture with 188/188 mRPE compared to WT mRPE.
Conclusions: :
When hfRPE are grown in contact with CECs, VEGF 189 mRNA is upregulated in hfRPE. We previously showed that active Rac1 was important in CEC transmigration across human RPE and that contact with 188/188 mRPE, rather than WT mRPE, increased CEC transmigration. However, our data suggest that the increased migration of CECs across a cell culture insert toward 188/188 mRPE rather than to WT mRPE appears independent of Rac1 signaling. Investigation of RPE/CEC interactions can be useful to develop methods to prevent a vision threatening step in neovascular age-related macular degeneration, namely, transmigration of CECs across the RPE into the neurosensory retina.
Keywords: retinal pigment epithelium • choroid: neovascularization • vascular endothelial growth factor