title: Ascorbic acid modulation of iron homeostasis and lysosomal function in trabecular meshwork cells.
abstract: OBJECTIVE
  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 10 days with increasing concentrations of AA. Antioxidant properties against cytotoxic effect of H2O2 were evaluated by monitoring cell viability. Redox-active iron was quantified using calcein-AM. Intracellular reactive oxygen species (iROS) 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 specific fluorogenic substrates.
  
  
  RESULTS
  AA 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 and correlated with increased protein levels of ferritin and elevated iROS. In contrast, antioxidant properties correlated with lower ferritin and basal iROS content. Ascorbic acid supplementation also caused induction of autophagy, as well as increased lysosomal proteolysis, with the 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.
authors.affiliation: Department of Ophthalmology, Duke University , Durham, North Carolina.
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
authors.forename: Yizhi
authors.initials: Y
authors.lastname: Lin
authors.forename: Kristine
authors.initials: K
authors.lastname: Porter
authors.forename: Paloma B
authors.initials: PB
authors.lastname: Liton
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000008:administration & dosage / Q000378:metabolism*; D001082:Aqueous Humor / Q000378:metabolism; D001205:Ascorbic Acid / Q000008:administration & dosage / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D005452:Fluoresceins / Q000737:chemistry; D006706:Homeostasis / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000633:toxicity; D007501:Iron / Q000378:metabolism*; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000008:administration & dosage / Q000378:metabolism; D013552:Swine; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects*
chemical_list: D000975:Antioxidants; D005452:Fluoresceins; D017382:Reactive Oxygen Species; C085925:calcein AM; D006861:Hydrogen Peroxide; D007501:Iron; D001205:Ascorbic Acid