Abstract
Purpose:
Choroidal endothelial cell (CEC) activation and migration precede the development of choroidal neovascularization in neovascular AMD. Thy-1 is a cell surface protein expressed on different cells, including neurons and endothelial cells. As a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, Thy-1 is located in lipid raft microdomains within the cell membrane, which brings Thy-1 into proximity of signaling molecules including cytoplasmic tyrosine kinases that can modulate adhesive and migratory events. In the retina, Thy-1 is well known as a retinal ganglion cell marker. Given the possibility that Thy-1 might be expressed in CECs, we addressed the hypothesis that upregulated Thy-1 in CECs by age-related stresses contributes to CEC migration.
Methods:
Western blots of Thy-1 were determined in retinal pigment epithelial cells (RPE) and CECs. By real time quantitative PCR, Thy-1 mRNA was measured in CECs treated with vascular endothelial growth factor (VEGF) (20 ng/ml), CCL11 (100 ng/ml) or PBS for 24 hours, or in RPE/choroids from young (<40 yrs) and old (>60 yrs) donor eyes. Immunohistochemistry of Thy-1 was performed in posterior globe sections of human retina/RPE/choroids in the maculas of young and old donor eyes with or without AMD. Colabeling with VE-cadherin was used to identify CECs. CECs transfected with Thy-1 siRNA or control siRNA were stimulated with VEGF, and CEC migration and phosphorylation of VEGF receptor 2 (VEGFR2) were measured. Statistics were performed using ANOVA.
Results:
Thy-1was highly expressed in CECs but not in RPE. Thy-1 staining was not only detected in the retinal ganglion cell layer, but also in the choroid, and colocalized to a greater extent with VE-cadherin labeled CECs in sections from donors with AMD compared to age-matched controls without AMD. Thy-1 mRNA was significantly increased in CECs treated with VEGF or CCL11 (p<0.05 vs. PBS) and greater in RPE/choroids from aged donor eyes (p<0.001 vs. young). Knockdown of Thy-1 in CECs by siRNA transfection significantly inhibited VEGF-induced CEC migration (p<0.001) and VEGFR2 activation.
Conclusions:
Thy-1is expressed in CECs and its expression is upregulated by stresses associated with neovascular AMD, including elderly age and increased VEGF. Upregulated Thy-1 in CECs contributes to VEGF-induced VEGFR2 activation and CEC migration. Future studies into the potential role of Thy-1 in neovascular AMD are being considered.
Keywords: 412 age-related macular degeneration •
453 choroid: neovascularization •
748 vascular endothelial growth factor