Laser-Mediated Activation of Human Retinal Pigment Epithelial Cells and Concomitant Release of Matrix Metalloproteinases

Abstract

PURPOSE: To investigate cellular dynamics and associated matrix metalloproteinase (MMP) release patterns of human retinal pigment epithelium (RPE) cells subsequent to irradiation by nanosecond pulsed laser at energy levels below visual threshold. METHODS: Following a stabilization period, human RPE-Bruch's-choroid explants were irradiated with a nanosecond laser system (Q-switched, frequency doubled YAG laser, 532 nm), using a 400 μm spot size with a discontinuous energy distribution and total irradiance of 240 mJ/cm², and returned to the incubator for a further 14 days. RPE cellular dynamics were assessed using confocal laser scanning, conventional microscopy, cell viability, and proliferation assays. MMPs were quantified by gelatine zymography and densitometry. RESULTS: Within 4 hours of laser intervention, 47% ± 8% (mean ± SEM, n = 6) of the RPE cells within the treatment zone showed clear signs of injury. By posttreatment days 10 to 14, most of the injured beds were repopulated by migrating RPE cells from regions surrounding the lesion. Release of inactive MMP-2 was little altered over the 2-week experimental period, whereas levels of inactive MMP-9 increased 1.3-fold by day 1 to reach a 2.8-fold threshold by day 7 (n = 4; P < 0.05). However, changes in activated MMP-2 and MMP-9 were much more profound with levels increasing 6.7 ± 2.6-fold (mean ± SEM, n = 6; P < 0.001) and 4.4 ± 1.1-fold (mean ± SEM, n = 5; P < 0.01), respectively, above controls at day 7 post laser. CONCLUSIONS: The nanosecond laser pulse modality provides an avenue for transiently increasing the RPE-mediated release of active MMP enzymes. The likely impact of this enzymatic release on the structural and functional aspects of aging Bruch's membrane requires further evaluation.