US EPA

3238765 

Journal Article 

Elements of the nitric oxide pathway can degrade TIMP-1 and increase gelatinase activity 

Brown, DJ; Lin, B; Chwa, M; Atilano, SR; Kim, DW; Kenney, MC 

2004 

1 

Molecular Vision
ISSN: 1090-0535 

10 

36 

281-288 

WOS:000220992700001 

Purpose: 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.