In Vitro Retinal Secretion of MMP-2, MMP-9, TIMP-1, TIMP-2 and Their Levels in Retinal Extracts of C3H Control and rd/rd Mice

Abstract

Abstract: : Purpose: Our unpublished work showed that PN7 rd retinal explants (RE) secrete more of different proteases into retinal conditioned medium (RCM) than those of control mice. The levels of MMPs and TIMPs in the retinal extracts and RCM of the two genotypes of increasing age in vivo and in vitro were therefore determined. Methods: The mean of protein content (µg/ 50µL retinal extract), levels of MMP-2, MMP-9 (δ Absorbance ₄₅₀ /h ^2* 1000 for RCM, δ Absorbance ₄₅₀ /h ^2* 1000 /mg proteins) TIMP-1, TIMP-2 (pg/ml RCM, pg/mg proteins) in the retinal extracts of control and rd mice of PN2, PN7, PN14, PN21 and PN28; and those secreted into RCM for 2-3 d over a period of 14-21 d by PN7 RE were determined by spectrophotometry (Proteins) and ELISA Kits (MMPs- Amersham Biosciences; TIMPs- R&D Systems) . Results: The following differences in rd and control retinal extracts/RCM were observed: 1. The protein content of control retinal extracts was higher than those of rd and increased from PN2 till PN28, whereas those of rd were decreased. 2. Compared to the control, in rd retinal extracts the activities of active MMP-9 and active MMP-2 were respectively higher from PN7 and PN14 onwards. The levels of active- and total- MMP-2 and MMP-9 in PN2 rd retinal extracts were always lower. The levels of MMP-9 but not of MMP-2 generally increased with age in both the genotypes. 3. Irrespective of the age and genotype of the explant, secretion of total MMP-2 into RCM was higher than that of active MMP-2, total- and active- MMP-9. The secretion of total- MMP-2 by control RE increased with age in vitro and was generally higher than that by rd RE; secretion of total MMP-2 by rd RE was high only at PN7+div9 while secretion of active MMP-2 increased upto PN7+div5 after which it decreased. 4. The level of TIMP-1 in rd and control retinal extracts was lower than that in RCM; TIMP-1 in retinal extracts decreased with age and its level in RCM of both genotypes was higher than that present in retinal extracts. The level of TIMP-1 in rd RCM was lower upto PN7+div5 and it was higher than that in the control RCM. Level of TIMP-2 in rd and control retinal extracts was low. Conclusion: The higher level of MMP-9, TIMP-1 in PN7 to PN28 rd retinal extracts and lower secretion of TIMP-1 by rd RE upto PN7+div5 indicate an imbalance in MMP-9/TIMP-1, which coincides with initiation of rd degeneration. The lower level of TIMP-1 in retinal extracts and higher level in RCM of rd and control mice indicates secretion of TIMP-1 without retinal accumulation which may have effects other than inhibition of MMPs.

Keywords: retinal degenerations: hereditary • retina: neurochemistry • regeneration