Expression of metalloproteinases from human retinal pigment epithelial cells and their effects on the hydraulic conductivity of Bruch's membrane.
OBJECTIVE
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.
Ahir
Alpa
A
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
Guo
Li
L
Hussain
Ali A
AA
Marshall
John
J
D000368:Aged; D000369:Aged, 80 and over; D001692:Biological Transport; D016570:Bruch Membrane / Q000378:metabolism*; D002455:Cell Division; D002461:Cell Line, Transformed; D017077:Culture Media, Conditioned / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism* / Q000494:pharmacology; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism* / Q000494:pharmacology; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*
D017077:Culture Media, Conditioned; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9