Expression of nitrotyrosine and oxidative consequences in the trabecular meshwork of patients with primary open-angle glaucoma.
OBJECTIVE
To evaluate the possible correlation between the visual field defects in patients with primary open-angle glaucoma (POAG) and the expression and enzymatic activity of nitric oxide synthase (NOS) isoenzymes and nitrotyrosine in trabecular meshwork (TM) samples.


METHODS
TM specimens were collected from 146 patients with POAG by using standard filtration surgery. Visual field defects were evaluated by perimetry. Expression of endothelial (e)NOS and inducible (i)NOS were evaluated by quantitative RT-PCR. Constitutive (Ca2+-dependent) and iNOS (Ca2+-independent) activities were measured by the conversion of L-[14C]-arginine to L-[14C]-citrulline. In four TM specimens from POAG-affected eyes and in three human donor control eyes, 3-nitrotyrosine was localized by immunohistochemistry. The marker of lipid peroxidation malondialdehyde (MDA) was measured by the thiobarbituric acid test in samples of aqueous humor (AH) from 48 patients with either POAG or cataracts.


RESULTS
The results showed an upregulation of iNOS and a downregulation of calcium-dependent NOS correlated with visual field defects. Expression and activity of iNOS increased in parallel with visual field defects. However, constitutive activity decreased as the visual field defect increased. Nitrotyrosine was observed only in the cells of the TM specimens from eyes with severe POAG.


CONCLUSIONS
The increased expression and activity of iNOS in the TM of patients with POAG are proportional to the visual field defect and could lead to the increased of nitrotyrosine levels which may serve as marker of oxidative stress in the progression of cell death of the TM in POAG.
Fernández-Durango
Raquel
R
Research Unit, Department Internal Medicine III, Hospital Clínico San Carlos, Madrid, Spain. fernandez.durango@gmail.com
Fernández-Martínez
Amalia
A
García-Feijoo
Julián
J
Castillo
Alfredo
A
de la Casa
José Martínez
JM
García-Bueno
Borja
B
Pérez-Nievas
Beatriz G
BG
Fernández-Cruz
Arturo
A
Leza
Juan Carlos
JC
D000368:Aged; D000369:Aged, 80 and over; D002118:Calcium / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005902:Glaucoma, Open-Angle / Q000378:metabolism*; D006801:Humans; D007429:Intraocular Pressure; D015227:Lipid Peroxidation; D008315:Malondialdehyde / Q000378:metabolism; D008875:Middle Aged; D052247:Nitric Oxide Synthase Type II / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics*; D018384:Oxidative Stress / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014129:Trabecular Meshwork / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D015854:Up-Regulation; D014786:Vision Disorders / Q000378:metabolism; D058609:Visual Field Tests; D014794:Visual Fields
D012333:RNA, Messenger; C002744:3-nitrotyrosine; D014443:Tyrosine; D008315:Malondialdehyde; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D002118:Calcium