Cultured Porcine Trabecular Meshwork Cells Display Altered Lysosomal Function when Subjected to Chronic Oxidative Stress

¹ Ophthalmology, Duke University, AERI 4004, Durham, North Carolina, 27710, United States
² Ophthalmology, Duke University, Durham, North Carolina, United States

^* To whom correspondence should be addressed. E-mail: paloma.liton{at}duke.edu.

	Abstract

Purpose: 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 two weeks at physiological (5% O₂) or hyperoxic conditions (40% O₂) in the presence or absence of the protease inhibitor leupeptin (10 µM). The following parameters were quantified using the fluorogenic probes indicated within parentheses: autofluorescence, intracellular ROS (H₂DCFDA), mitochondrial membrane potential (JC-1), mitochondrial content (mitotracker red), lysosomal content (acridine orange and lysotracker red), autophagic vacuoles content (MDC), SA--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: Chronic 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 vacuoles content was confirmed using 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: Our 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.

Key Words: trabecular meshwork, lysosomes, aging, oxidative stress, SA-beta-gal