Oxidative Modified Proteins in Normal Macula and Exudative AMD

Abstract

Abstract: : Purpose: Proteins can be targets for oxidative modifications that frequently result in functional changes, such as impaired enzymatic activity, decreased/increased DNA binding activities of transcription factors, and decreased susceptibility to proteolytic degradation. The aim of the present study was to detect carbonyl groups introduced in protein side chains by reactive oxygen species (ROS) or by reactions with aldehydes (malondialdehyd) produced during lipid peroxidation in normal macula and choroidal neovascularisation membranes (CNV) from patients with exudative age-related macular degeneration (AMD). Method: Macula specimen of 25 donor eyes and 20 surgically excised exudative AMD lesions, with choroidal neovascularisation were examined. Carbonyl residues were detected by reaction with 2,4- dinitrophenylhydrazine (DNPH) generating dinitrophenylhydrazones. The detection was processed with an anti-dinitrophenylhydrazone antibody followed by the immunhistochemical ABC-method. Immunoreactivity was resolved with horseradish peroxidase-aminoethylcarbazole, which produces a magenta reaction product. Negative control experiments were performed using: a, control derivatization solution instead of DNPH solution, b, buffer samples in place of primary antibody. Results: Protein carbonyls were detected in endothelial cells of the choriocapillaris and only rare in the retinal pigment epithelium (RPE) in normal macula. Bruch's membrane and associated small drusen showed no positive reaction product. Within CNV membranes RPE cells showed positive staining for carbonyl residues. Associated diffuse deposits were weak reactive with stronger staining reaction at the apical side at the border to the RPE. Conclusion: The detection of oxidative modified proteins in the choriocapillary endothelium may indicate more oxidative stress due to high oxygen concentration derived from blood flow, or/and a less antioxidative defense system. The RPE in normal macula showed no accumulation of carbonyl residues, while in CNV membranes the RPE and diffuse deposits contain oxidative modified proteins. This may indicate a well adapted antioxidative defense system in the RPE of normal macula, which may break down due to additional oxidative stress associated with the development of CNV membranes.

Keywords: oxidation/oxidative or free radical damage • retinal pigment epithelium • age-related macular degeneration