Does MMP-2 expression and secretion change with increasing serial passage of keratocytes in culture?

The effects of ageing on matrix metalloprotease degradation of the: extrace llular matrix during corneal wound healing are largely unknown. The followi ng study used an in vitro model of ageing to assess changes in MMP-2 RNA ex pression and protein secretion. Early passage (EP) EK1.BR keratocyte cultur es from 14 to 18 cumulative population doublings (cpds) and late passage (L P) cultures from 40 to 47 cpds were used to isolate protein and mRNA sample s. Total protein from EP and LP cultures was measured using the Bradford pr otein assay. Zymographic analysis of EP and LP samples was carried out to c ompare MMP-2 activity. Northern blot analysis was used to assess changes in MMP-2 mRNA expression by EP and LP cultures, using a digoxigenin (DIG) bas ed chemiluminescent detection system. LP cultures secreted more total prote in per cell. MMP-2 but not MMP-9 activity was detected in keratocyte cultur es. Densitometric analysis of zymograms and calculation of MMP-2 activity i ndicated a significant increase in MMP-2 activity per cell (P < 0.05, n = 1 1). No difference was observed in the levels of MMP-2 mRNA expressed by EP and LP cultures. An increase in MMP-2 activity per cell by LP cultures sugg ests that senescent keratocytes increase their degradative capacity. Simila r changes in the keratocyte phenotype within the ageing cornea may alter th e balanced response necessary for adequate wound healing and may have impli cations for the therapeutic use of MMP inhibitors in the eye. (C) 2001 Else vier Science Ireland Ltd. All rights reserved.