June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
AGE levels are higher in capsules of human cataractous lenses than non-cataractous lenses: Relevance to TGFβ2 induced EMT response in human lens epithelial cells
Author Affiliations & Notes
  • Cibin T Raghavan
    Ophthalmology, Case Western Reserve University, Aurora, CO
    Ophthalmology, University of Colorado, Aurora, CO
  • Mareen Smuda
    Institute of Chemistry, Martin-Luther-University, Halle, Germany
  • Andrew J O Smith
    University of East Anglia, Norwich, United Kingdom
  • Annapurna Singh
    University Hospitals of Cleveland, Cleveland, OH
  • Pankaj Gupta
    University Hospitals of Cleveland, Cleveland, OH
  • Marcus Glomb
    Institute of Chemistry, Martin-Luther-University, Halle, Germany
  • Michael Wormstone
    University of East Anglia, Norwich, United Kingdom
  • Ram H Nagaraj
    Ophthalmology, Case Western Reserve University, Aurora, CO
    Ophthalmology, University of Colorado, Aurora, CO
  • Footnotes
    Commercial Relationships Cibin Raghavan, None; Mareen Smuda, None; Andrew Smith, None; Annapurna Singh, None; Pankaj Gupta, None; Marcus Glomb, None; Michael Wormstone, None; Ram Nagaraj, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 1348. doi:
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      Cibin T Raghavan, Mareen Smuda, Andrew J O Smith, Annapurna Singh, Pankaj Gupta, Marcus Glomb, Michael Wormstone, Ram H Nagaraj; AGE levels are higher in capsules of human cataractous lenses than non-cataractous lenses: Relevance to TGFβ2 induced EMT response in human lens epithelial cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1348.

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

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Abstract

Purpose: Proteins in basement membrane are long-lived and accumulate chemical modifications during aging. The reaction of carbonyl compounds that results in the formation of advanced glycation end products (AGEs) is a major chemical modification in basement membrane proteins. In this study we have investigated the influence of AGE levels in human lens capsule on TGFβ2 treated human lens epithelial cells (HLE). The overall objective was to determine the effects of capsule AGEs on posterior capsule opacification (PCO).

Methods: AGE measurements in non-cataractous and cataractous human lens capsules were done by LC-MS/MS. Tissue culture plates were coated with basement membrane extract (BME; 50 μg/ml) overnight and AGE-modified with glycating mixture (2 mM ascorbate, 25 mM glucose and 250 μM methylglyoxal) for 7 days at 37°C. HLE isolated from a 48-year donor lens was cultured on AGE-modified or unmodified BME and treated with 10 ng/ml of TGFβ2 for 24 h at 37°C. RNA was isolated, cDNA generated and real time PCR analysis was carried for EMT-associated proteins.

Results: The total as well as individual AGEs in cataractous human lens capsules were significantly higher; Total AGEs (p<0.0001), CML (p<0.0001), pyrraline (p<0.0008), formyllysine (p<0.0009), CEA (p<0.0001), glucosepane (p<0.08) and MODIC (p<0.0001) than age-matched non-cataractous lens capsules. Real time PCR analyses revealed that TGFβ2-induced expression of EMT markers (αSMA, MMP2, CTGF, integrin αV, integrin α5, integrin β1, miR 1469 and miR 4279) were significantly increased in HLE cells cultured on AGE-modified BME than on unmodified BME. The mRNA levels of TGFβ2-downregulated proteins (miR 184, miR 204, BMP4 and TGFββR3) were further reduced in cells cultured on AGE-modified BME.

Conclusions: The higher AGE content in the capsules of cataractous lenses suggest that AGEs might promote the TGFβ-induced EMT response in lens epithelial cells during PCO.

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