Abstract
Purpose: :
Increased oxidative damage has been hypothesized to be involved in the pathogenesis of primary open angle glaucoma. Here we investigate the role of the autophagic lysosomal degradative pathway as a defense mechanism against oxidative stress in trabecular meshwork (TM) cells.
Methods: :
Porcine TM cells were subjected to chronic oxidative stress using the hyperoxic model. Autophagic activity was monitored by analyzing autophagosome formation and maturation by electron microscopy, immunoblotting (LC3-I to LC3-II conversion), and confocal microscopy using a recombinant adenovirus containing the tandem fluorescence tagged LC3 (tfLC3) reporter gene. The following parameters were quantified by flow cytometry using the fluorogenic probes indicated within parentheses: intralysosomal oxidized material, lysosomal content (lysotracker red), and SA-β-galactosidase (FDG). Cathepsin activities were assayed using fluorogenic substrates. Rapamycin, bafilomycin A1, chloroquine, and 3-MA were used to pharmacologically modulate different steps within the autophagic pathway.
Results: :
PTM cells treated with rapamycin (1 µM) showed a significant increase in LC3-II levels (> 2-fold), as well as tfLC3 punctuate staining by confocal microscopy starting 30 min post-treatment, followed by a decrease after 120 min. PTM cells exposed to chronic oxidative stress showed higher presence of autophagolysosomes and LC3-II levels (7.2±1.3 fold, p=0.001). Inhibition of lysosomal degradation using chloroquine (0.3 µM) or bafilomycin (0.1 µM) indicated that such increased levels of LC3-II were due to autophagy induction rather than decrease autophagy flux. These results were further confirmed using the autophagy inhibitor 3-MA. Incubation of TM cells with 3-MA (100 nM) resulted in decreased levels of LC3-II, as well as lipofuscin content (25.83±2.13, p=0.002), lysosomal mass (53.47±6.64, p=0.006) and SA-β-Gal staining (51.61±5.11, p=0.003) in the stressed cultures.
Conclusions: :
Our results validate the use of LC3 as a marker to monitor autophagy dynamics in TM cells and demonstrate that TM cells respond to an oxidative challenge by inducing the autophagic lysosomal pathway. Pharmacological modulation of autophagy might represent a novel therapeutic approach to promote the degradation of oxidized material and organelles, and thus restore TM function in aging and in disease.
Keywords: trabecular meshwork • stress response • oxidation/oxidative or free radical damage