June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Prdx6 abates the process of senescence and restores trabecular meshwork cell integrity by regulating Telomerase, p16 and p21 Expression
Author Affiliations & Notes
  • Nigar Fatma
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, NE
  • Eri Kubo
    Ophthalmology, Kanazawa Medical University, Kanazawa, Japan
  • Bhavana Chhunchha
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, NE
  • W Daniel Stamer
    Ophthalmology, Duke Eye Center, Duke University, Durham, NC
  • Dhirendra Singh
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, NE
  • Footnotes
    Commercial Relationships Nigar Fatma, None; Eri Kubo, None; Bhavana Chhunchha, None; W Daniel Stamer, Allergan (F), Alcon (F), Acucela (C), Aerie (C), Cytokinetics (C); Dhirendra Singh, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3545. doi:
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      Nigar Fatma, Eri Kubo, Bhavana Chhunchha, W Daniel Stamer, Dhirendra Singh; Prdx6 abates the process of senescence and restores trabecular meshwork cell integrity by regulating Telomerase, p16 and p21 Expression. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3545.

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

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Abstract

Purpose: Normal functioning of Trabecular Meshwork (TM) is vital in regulating intraocular pressure (IOP). Glaucomatous TM cells contain increased levels of extracellular matrix (ECM) and oxidative DNA and protein adducts, and undergo senescence, suggesting a role for oxidative stress. Peroxiredoxin 6 (Prdx6) provides cytoprotection from oxidative stress. Using human TM cells facing oxidative stress as a model in vitro, we tested the hypothesis that Prdx6 overexpression is protective in glaucomatous or aging TM cells.

Methods: pGFP-Prdx6 and/or pGRN145-hTERT (ATCC) or mutant plasmids were overexpressed in TM cells from normal and glaucomatous human subjects of different ages and different passages and were exposed to H2O2 (0-200μM). Prdx6 fused to TAT transduction domain was also used to deliver Prdx6 to assess cytoprotection. Cell viability, cell proliferation, lipid peroxidation (LPO) were measured by MTS assay, BrdU incorporation and LPO assay kit, respectively. Reactive oxygen species (ROS) were quantified using H2DCF dye. Real-time PCR and Western analysis assessed the level of Prdx6, telomerase reverse transcriptase (TERT), ECM proteins and senescence markers, p16 and p21. β-galactosidase staining was done for senescence status. 8-OHdG assay was used for oxidative DNA damage. Telomerase activity and TGFβs were assessed by Telomerase and Emax assays kits, respectively.

Results: Glaucomatous or aging cells, or cells facing oxidative stress showed reduced Prdx6 expression. Such cells had increased expression of ECM proteins; αsm-actin, fibronectin, thrombospondin1, TGase2, PAI1, tropomyocin, p16, p21; and reduced expression and activity of telomerase, hTERT. Glaucomatous cells bore increased ROS and active TGFβ1 and 2 and LPO adducts. The abnormalities were reversed in cells overexpressing Prdx6; having significantly reduced β-galactosidase staining, with increased proliferation/cell division and telomerase activity.

Conclusions: The study shows Prdx6’s ability reverse the senescence process of glaucomatous TM cells by increasing telomerase activity, and argues a role for oxidative damage in replicative senescence. Since loss of Prdx6 expression in TM cells is causally related to TM cells etiopathogenesis, development of Prdx6 expression-based therapeutics may delay or prevent TM cell dysfunction, representing a novel therapeutic strategy for glaucoma.

Keywords: 634 oxidation/oxidative or free radical damage • 424 antioxidants • 449 cell survival  
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