Abstract
Abstract: :
Purpose: To determine whether peroxynitrite and/or nitric oxide donors (SIN-1 and SNAP, respectively) altered the tissue inhibitor of metalloproteinase-1 (TIMP-1) and/or matrix metalloproteinase-2 (MMP-2) produced by normal and keratoconus stromal cells in vitro. Methods: A total of 22 normal and 24 keratoconus corneas were analyzed in this study. The RNA levels of TIMPs and MMPs from intact keratoconus and age-matched normal corneas were evaluated by semi-quantitative RT-PCR and Southern blotting. Normal and keratoconus stromal cells were incubated in vitro with 1 mM of SIN-1 or SNAP. RNA levels for TIMP-1, MMP-2, and beta-actin were measured by Northern blot analyses. Peroxynitrite and TIMP-1 levels were measured by Western blots and dot blot analyses. Culture media were analyzed by gelatin zymography and a collagenolytic fibril assay. Results: Keratoconus corneas had decreased TIMP-1 mRNA (p<0.005) and protein levels compared to normal intact corneas. Within 4 hours in SIN-1 treated cultures, there was an accumulation of peroxynitrite (NT positive proteins). After 18 hours, SIN-1 treated cultures showed degradation of the TIMP-1 protein and increased TIMP-1 RNA levels. The MMP-2 RNA levels increased with SIN-1 but remained the same with SNAP treatment. Media from the SIN-1 and SNAP treated samples had increased gelatinase activities. Conclusion: Peroxynitrite was associated with altered RNA levels for MMP-2/TIMP-1 and fragmentation of TIMP-1 protein. In light of the multiple cellular functions of TIMP-1 (i.e., inhibition of MMP activity, suppression of apoptosis) this relationship may play an important role in the pathogenesis of keratoconus.
Keywords: 450 keratoconus • 374 cornea: stroma and keratocytes