title: Laser-mediated activation of human retinal pigment epithelial cells and concomitant release of matrix metalloproteinases.
abstract: OBJECTIVE
  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.
authors.affiliation: Division of Molecular Therapy, UCL Institute of Ophthalmology, London, United Kingdom. jj.zhangopth@gmail.com
authors.forename: Jin Jun
authors.initials: JJ
authors.lastname: Zhang
authors.forename: Yue
authors.initials: Y
authors.lastname: Sun
authors.forename: Ali A
authors.initials: AA
authors.lastname: Hussain
authors.forename: John
authors.initials: J
authors.lastname: Marshall
mesh_terms: D000328:Adult; D000368:Aged; D018929:Cell Culture Techniques / Q000295:instrumentation; D002465:Cell Movement; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D053844:Lasers, Solid-State; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D018613:Microscopy, Confocal; D008875:Middle Aged; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000201:enzymology; D055815:Young Adult
chemical_list: D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9