Cultured porcine trabecular meshwork cells display altered lysosomal function when subjected to chronic oxidative stress.
OBJECTIVE
To investigate the effects of chronic oxidative stress on lysosomal function in trabecular meshwork (TM) cells.


METHODS
Confluent cultures of porcine TM cells were grown for 2 weeks in physiological (5% O(2)) or hyperoxic conditions (40% O(2)) in the presence or absence of the protease inhibitor leupeptin (10 microM). The following parameters were quantified using the fluorogenic probes indicated within parentheses: autofluorescence, intracellular reactive oxygen species (ROS; H(2)DCFDA), mitochondrial membrane potential (JC-1), mitochondrial content (Mitotracker Red; Invitrogen-Molecular Probes, Eugene, OR), lysosomal content (acridine orange and Lysotracker Red [Invitrogen-Molecular Probes]), autophagic vacuole content (MDC), SA-beta-galactosidase (FDG), and cathepsin activities (z-FR-AMC). Cathepsin levels were quantified by qPCR and Western blot analysis. Ultrastructural analysis was performed by transmission electron microscopy.


RESULTS
Prolonged exposure of porcine TM cells to a hyperoxic environment led to an increase in ROS production and oxidized material. Electron micrographs revealed the cytoplasmic accumulation of lipofuscin-loaded lysosomes. Augmented lysosomal and autophagic vacuole content was confirmed with specific fluorophores. The mRNA and protein levels of several cathepsins were upregulated with oxidative stress. This upregulated expression did not correlate with increased lysosomal activity.


CONCLUSIONS
The results indicate that chronic exposure of TM cells to oxidative stress causes the accumulation of nondegradable material within the lysosomal compartment, leading to diminished lysosomal activity. Since the lysosomal system is responsible for the continuous turnover of cellular organelles, impaired lysosomal activity may lead to progressive failure of cellular TM function with age.
Liton
Paloma B
PB
Department of Ophthalmology, Duke University Eye Center, Durham, North Carolina, USA.
Lin
Yizhi
Y
Luna
Coralia
C
Li
Guorong
G
Gonzalez
Pedro
P
Epstein
David L
DL
D000375:Aging / Q000502:physiology; D000818:Animals; D001343:Autophagy / Q000502:physiology; D002403:Cathepsins / Q000235:genetics; D048429:Cell Size; D002478:Cells, Cultured; D018496:Hyperoxia; D008247:Lysosomes / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D051336:Mitochondrial Membranes / Q000502:physiology; D018384:Oxidative Stress; D010100:Oxygen / Q000502:physiology; D016133:Polymerase Chain Reaction; D013552:Swine; D014129:Trabecular Meshwork / Q000166:cytology* / Q000254:growth & development / Q000502:physiology*; D014617:Vacuoles / Q000502:physiology
D002403:Cathepsins; D010100:Oxygen