April 2014
Volume 55, Issue 13
ARVO Annual Meeting Abstract  |   April 2014
Ascorbic Acid Modulation of Iron Homeostasis and Lysosomal Function in Trabecular Meshwork
Author Affiliations & Notes
  • Paloma Borrajo Liton
    Ophthalmology, Duke University Eye Center, Durham, NC
  • Joshua Hirt
    Ophthalmology, Duke University Eye Center, Durham, NC
  • Kristine M Porter
    Ophthalmology, Duke University Eye Center, Durham, NC
  • Footnotes
    Commercial Relationships Paloma Liton, None; Joshua Hirt, None; Kristine Porter, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5660. doi:
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      Paloma Borrajo Liton, Joshua Hirt, Kristine M Porter; Ascorbic Acid Modulation of Iron Homeostasis and Lysosomal Function in Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5660.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: To investigate the antioxidant properties and biological functions of ascorbic acid (AA) on trabecular meshwork (TM) cells.

Methods: Primary cultures of porcine TM cells were supplemented for ten days with increasing concentrations of AA (0, 100, 250, 500 μg/mL) in the presence or absence of an iron chelator (SIH or DFO). Antioxidant properties against cytotoxic effect of H2O2 (0, 500, 1000, 1500 μM) were evaluated by monitoring cell viability. Redox-active iron was quantified using calcein-AM. Intracellular reactive oxygen species (ROS) production was quantified using H2DCFDA. Ferritin and cathepsin protein levels were analyzed by western-blot. Autophagy was evaluated by monitoring lipidation of LC3-I to LC3-II. Lysosomal proteolysis and cathepsins activities were quantified using DQ-BSA and specific fluorogenic substrates, respectively.

Results: Ascorbic acid exerts a dual effect against oxidative stress in TM cells, acting as an anti-oxidant or a pro-oxidant depending on the concentration used. The pro-oxidant effect of AA was mediated by free intracellular iron (p=0.0011, n=4) and correlated with increased protein levels of ferritin (> 2-fold) and elevated iROS (28.44 ± 1.26 % increase, p=0.025, n=4). In contrast, antioxidant properties correlated with lower ferritin (< 0.5 fold) and basal iROS content (23.2 ± 2.5 % decrease, p=0.03, n=4). Ascorbic acid supplementation also caused iron-dependent induction of autophagy, as determined by increased LC3 lipidation (ANOVA, p=0.0011); as well as increased lysosomal proteolysis, this latter resulting from higher proteolytic activation of lysosomal cathepsins in treated cultures.

Conclusions: Our results suggest that the reported decrease of AA levels in plasma and aqueous humor can compromise lysosomal degradation in the outflow pathway cells with aging and contribute to the pathogenesis of glaucoma. Restoration of physiological levels of vitamin C inside the cells might improve their ability to degrade proteins within the lysosomal compartment and recover tissue function.

Keywords: 735 trabecular meshwork • 634 oxidation/oxidative or free radical damage  

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