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Kristine Porter, Ping Xu, W Daniel Stamer, Paloma Liton; DIMINISHED AUTOPHAGIC CAPACITY OF GLAUCOMATOUS TRABECULAR MESHWORK CELLS. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3548.
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To evaluate the autophagic capacity in non-glaucomatous and glaucomatous trabecular meshwork (TM) cells.
Experiments were performed in primary cultures and transformed cell lines of non-glaucomatous and glaucomatous human TM cells grown to confluence under normal culture conditions. mRNA and/or protein expression levels of components of the autophagic lysosomal pathway (Atg3, Atg4, Atg5, Atg12, Beclin, p62, cathepsin D, cathepsin B, LAMP1, and TFEB) were quantified by qPCR and WB analyses, respectively. Autophagy dynamics were evaluated by monitoring the LC3-I to LC3-II conversion. Intralysosomal oxidized material, lysosomal content (LTR), and senescence-associated-β-galactosidase (SA-β-gal, FDG) were monitored by flow cytometry.
Primary cultures of glaucomatous TM cells demonstrated decreased lysosomal content, but increased intralysosomal oxidized material and SA-β-gal activity compared to non-glaucomatous TM cells (LTR: 82±38.9 RFU versus 197.01±59.688; AF: 27.73±0.87 RFU versus 22.92±1.308; FDG: 89.38±18.68 versus 17.32±2.73; p<0.05). We also found a significant down-regulation of mRNA expression of LC3 (0.77 ± 0.06 fold), ATG4 (0.02 ± 0.001 fold), ATG7 (0.51 ± 0.07) and LAMP1 (0.33 ± 0.04) in glaucomatous compared to non-glaucomatous TM cells. However, no changes in the mRNA levels of beclin, LAMP2, and Atg5 were observed. At the protein level, glaucomatous TM cells showed a significant decrease (>50 %) in the content of the lysosomal marker LAMP1, the lysosomal enzymes cathepsin B and cathepsin D, as well as the autophagosome marker LC3-II compared to control cells. A decrease in LC3-II and LAMP1 was also confirmed in the transformed glaucomatous cell line compared to the control cell line.
Our results indicate that (1) TM cells isolated from the glaucomatous outflow pathway retain in vitro the senescence phenotype observed in glaucomatous outflow tissue in vivo; and (2) glaucomatous TM cells display diminished autophagic capacity, characterized by lower levels of key protein participants of the autophagic lysosomal pathway, including LC3-II. Autophagy has been shown by our laboratory to be activated in response to oxidative and mechanical stress; therefore, we hypothesize that such diminished autophagic capacity in glaucomatous TM cells jeopardizes their ability to respond to stress, possibly contributing to the loss in cellularity observed in the outflow pathway in glaucoma.
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