Elements of the nitric oxide pathway can degrade TIMP-1 and increase gelatinase activity.
OBJECTIVE
Keratoconus is a non-inflammatory thinning disorder of the corneal stroma. Recently, we showed that these corneas contain inducible nitric oxide synthase and an accumulation of nitrotyrosine, representing oxidative damage from peroxynitrite. Previously, we suggested that keratoconus corneas and their cell cultures have alterations in a gelatinase system with increased matrix metalloproteinase-2 (MMP-2) activity and decreased tissue inhibitor of metalloproteinase-1 (TIMP-1). This study examines whether a peroxynitrite donor (3-morpholinosydomine N-ethylcarbamide, SIN-1) or nitric oxide donor (S-nitroso-N-acetylpenicillamine, SNAP) could alter TIMP-1 and/or MMP-2 in vitro.


METHODS
Normal stromal fibroblasts were cultured in the presence or absence of either SIN-1 or SNAP for varying times. These cultures were analyzed by western and northern blot analyses, gelatin zymography, and a quantitative gelatinase/MMP assay.


RESULTS
In vitro, SIN-1 treatment led to protein nitration, increased RNA levels of TIMP-1 and MMP-2, and loss of TIMP-1 immunostaining, but did not diminish gelatinase activity. SNAP treatment led to activation of MMP-2 and significantly increased gelatinase/MMP activity, without a change in TIMP-1 levels.


CONCLUSIONS
Our data show that peroxynitrite or nitric oxide can decrease TIMP-1 and increase gelatinase activity, respectively. This demonstrates a relationship between elements of oxidative stress and tissue degradation in human corneal fibroblasts. This effect may play a significant role in the stromal thinning that occurs in keratoconus.
Brown
Donald J
DJ
Department of Ophthalmology, College of Medicine, University of California, Irvine, Orange, CA, USA. dbrown@uci.edu
Lin
Brian
B
Chwa
Marilyn
M
Atilano
Shari R
SR
Kim
Dae W
DW
Kenney
M Cristina
MC
D000328:Adult; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects; D018093:Gelatinases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008875:Middle Aged; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
D020030:Nitric Oxide Donors; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D009569:Nitric Oxide; C002385:linsidomine; D026423:S-Nitroso-N-Acetylpenicillamine; D008981:Molsidomine; D018093:Gelatinases