Alpa Ahir1, Li Guo, Ali A Hussain, John Marshall. 1. Department of Ophthalmology, the Guy's, King's and St. Thomas' Hospitals' Medical and Dental School, King's College London, St. Thomas' Campus, Lambeth Palace Road, London, United Kingdom. alpaahir@hotmail.com
Abstract
PURPOSE: To evaluate the expression pattern of matrix metalloproteinases (MMPs) from retinal pigment epithelial (RPE) cells in culture, and to determine their ability to alter the transport properties of human Bruch's membrane. METHODS: Human RPE cells from either primary cultures or a cell line were maintained under culture conditions. At different time intervals after subculturing of cells the presence of MMPs in the bathing medium was determined by zymography. Cellular MMP activity was determined in a similar series of experiments where serum was omitted from the culture medium. Cultured cells were introduced onto Bruch's membrane, mounted in a modified Ussing chamber, to assess entry of MMPs into the membrane. Fluid flow across Bruch's membrane was determined by hydraulic conductivity for different ages of donor tissue, before and after 24 hours' incubation with active MMPs from the RPE-conditioned medium or after incubation with purified activated MMPs. Latent (inactive) MMPs from medium containing serum were used in control experiments. RESULTS: Cultured RPE cells expressed both MMP-2 and -9, with active MMP-2 becoming detectable from 4 days after subculture through to confluence and activated MMP-9 becoming abundant up to 24 hours after subculture. Both active MMPs significantly increased hydraulic conductivity of Bruch's membrane, with the increase after MMP-9 incubation being far greater than that for MMP-2. Both enzymes showed a trend in hydraulic conductivity change with age such that, MMP-2 produced a relatively constant change, whereas MMP-9 showed a greater increase in older eyes. CONCLUSIONS: Activation of both MMP-2 and -9 by cultured RPE cells appeared to show a temporal relationship with the growth cycle of the cells. The activated enzymes increased fluid flow of Bruch's membrane, and the marked effect observed with MMP-9 in older eyes suggests a mechanism that may allow debris removal.
PURPOSE: To evaluate the expression pattern of matrix metalloproteinases (MMPs) from retinal pigment epithelial (RPE) cells in culture, and to determine their ability to alter the transport properties of human Bruch's membrane. METHODS:Human RPE cells from either primary cultures or a cell line were maintained under culture conditions. At different time intervals after subculturing of cells the presence of MMPs in the bathing medium was determined by zymography. Cellular MMP activity was determined in a similar series of experiments where serum was omitted from the culture medium. Cultured cells were introduced onto Bruch's membrane, mounted in a modified Ussing chamber, to assess entry of MMPs into the membrane. Fluid flow across Bruch's membrane was determined by hydraulic conductivity for different ages of donor tissue, before and after 24 hours' incubation with active MMPs from the RPE-conditioned medium or after incubation with purified activated MMPs. Latent (inactive) MMPs from medium containing serum were used in control experiments. RESULTS: Cultured RPE cells expressed both MMP-2 and -9, with active MMP-2 becoming detectable from 4 days after subculture through to confluence and activated MMP-9 becoming abundant up to 24 hours after subculture. Both active MMPs significantly increased hydraulic conductivity of Bruch's membrane, with the increase after MMP-9 incubation being far greater than that for MMP-2. Both enzymes showed a trend in hydraulic conductivity change with age such that, MMP-2 produced a relatively constant change, whereas MMP-9 showed a greater increase in older eyes. CONCLUSIONS: Activation of both MMP-2 and -9 by cultured RPE cells appeared to show a temporal relationship with the growth cycle of the cells. The activated enzymes increased fluid flow of Bruch's membrane, and the marked effect observed with MMP-9 in older eyes suggests a mechanism that may allow debris removal.
Authors: Sharon Elliot; Paola Catanuto; William Stetler-Stevenson; Scott W Cousins Journal: Invest Ophthalmol Vis Sci Date: 2006-04 Impact factor: 4.799
Authors: Gary E Striker; Francoiçe Praddaude; Oscar Alcazar; Scott W Cousins; Maria E Marin-Castaño Journal: Am J Physiol Cell Physiol Date: 2008-10-15 Impact factor: 4.249
Authors: Michael J Koss; Paulo Falabella; Francisco R Stefanini; Marcel Pfister; Biju B Thomas; Amir H Kashani; Rodrigo Brant; Danhong Zhu; Dennis O Clegg; David R Hinton; Mark S Humayun Journal: Graefes Arch Clin Exp Ophthalmol Date: 2016-06-22 Impact factor: 3.117
Authors: A M Joussen; P Walter; C P Jonescu-Cuypers; K Koizumi; V Poulaki; K U Bartz-Schmidt; G K Krieglstein; B Kirchhof Journal: Br J Ophthalmol Date: 2003-09 Impact factor: 4.638