Light-induced oxidative stress in choroidal endothelial cells in mice.
OBJECTIVE
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.
Wu
Tinghuai
T
Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. tingwu@jhmi.edu
Handa
James T
JT
Gottsch
John D
JD
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002829:Choroid / Q000098:blood supply*; D004247:DNA / Q000378:metabolism; D003623:Dark Adaptation; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism / Q000528:radiation effects*; D019084:Fluorescent Antibody Technique, Indirect; D020672:I-kappa B Proteins / Q000378:metabolism; D007124:Immunoenzyme Techniques; D008027:Light; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D018384:Oxidative Stress / Q000528:radiation effects*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D000072000:NF-KappaB Inhibitor alpha; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D003849:Deoxyguanosine