Light-induced oxidative stress in choroidal endothelial cells in mice.

PURPOSE: Although light-induced oxidative stress in the retina has been extensively reported, little information regarding light-induced oxidative stress in choroidal endothelial cells (CECs) is available. In the current study, light-induced DNA oxidation and the activation of nuclear factor-kappaB (NF-kappaB), a major oxidative responsive transcription factor, were investigated in mouse CECs.
METHODS: Mice were exposed to green light. Light-induced DNA oxidation in CECs was detected by in situ 8-hydroxy-2-deoxyguanosine (8-oxo-dG) immunolabeling. CECs were isolated from the retinal pigment epithelium (RPE)/choroid by using immunomagnetic beads. The isolated CECs were immunochemically characterized by the expression of endothelial markers, CD31, and P1H12. The quality of total RNA from CECs was assessed by a bioanalyzer and RT-PCR. NF-kappaB activation in situ and in isolated CECs was investigated.
RESULTS: After a 3-hour exposure to light, the immunoreactivity to anti-8-oxo-dG antibody or anti-NF-kappaB p65 antibody in CECs in situ was significantly increased when compared with unexposed mice. Isolated CECs expressed CD31 and P1H12. The 28S/18S rRNA ratio of RNA isolated from CECs was 1.5:1. CD31 and von Willebrand Factor (vWF) transcripts were predominantly expressed in the RNA from isolated CECs. IkappaBalpha was more heavily phosphorylated in light-exposed than untreated CECs. IkappaBalpha expression levels were increased fivefold in isolated CECs after exposure to light compared to unexposed control subjects.
CONCLUSIONS: Exposure to light induces oxidative stress in CECs in vivo. A method for CEC isolation from the mouse RPE/choroid with preservation of RNA quality has been developed. The results of this study may facilitate the ability to identify CEC-specific genes and gene products that respond to photo-oxidative stress.