Measurement of TIMP-3 Expression and Bruch's Membrane Thickness in Human Macula

Abstract

An increase or accumulation in tissue inhibitor of matrix metalloproteinases-3 (TIMP-3) protein in Bruch's membrane with ageing in normal eyes, and in age related macular degeneration (AMD) has been previously demonstrated. The purpose of this study was to determine whether the expression of TIMP-3 mRNA increases with age, and to define any relationship between altered expression and Bruch's membrane thickness. Normal eyes were obtained from 30 donors (age range 15–90 years). Full-thickness 8mm macular punches centred on the fovea were taken to allow removal of the chorioretinal complex, for subsequent nucleic acid extraction. Samples were normalized for RNA degradation using beta-actin reverse transcriptase–polymerase chain reaction (RT–PCR). A competitive RT–PCR was then used to allow measurement of TIMP-3 gene expression in each sample. The tissue adjacent to that used for nucleic acid extraction was processed histologically to allow determination of Bruch's membrane thickness. Bruch's membrane thickness was found to increase with age (P<0.01), but TIMP-3 expression, as measured by competitive RT–PCR, was not significantly increased with age (P=0.19). An inverse correlation was noted between TIMP-3 expression and Bruch's membrane thickness after controlling for age (P=0.032). The results of this study suggest that TIMP-3 expression does not alter significantly with age. Therefore, accumulation of the TIMP-3 protein must occur by a mechanism other than increased expression. TIMP-3 protein levels may still prove to contribute to events associated with ageing in the macula, such as matrix remodelling in Bruch's membrane. Further studies are required to elucidate the precise interactions and turnover of the TIMP-3 protein, and resulting changes in the control of matrix metalloproteinase activity in the ageing macula.