June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Epithelial Mesenchymal Transition and Human Vitreous Samples in 2D and 3D culture of ARPE-19 Cells
Author Affiliations & Notes
  • Eri Takahashi
    Ophthalmology, Kumamoto Univ Grad School of Med Sci, Kumamoto, Japan
  • Takahiro Kawaji
    Ophthalmology, Kumamoto Univ Grad School of Med Sci, Kumamoto, Japan
  • Yasuhiro Ito
    Ophthalmology, Kumamoto Univ Grad School of Med Sci, Kumamoto, Japan
  • Hidenobu Tanihara
    Ophthalmology, Kumamoto Univ Grad School of Med Sci, Kumamoto, Japan
  • Footnotes
    Commercial Relationships Eri Takahashi, None; Takahiro Kawaji, None; Yasuhiro Ito, None; Hidenobu Tanihara, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1193. doi:
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      Eri Takahashi, Takahiro Kawaji, Yasuhiro Ito, Hidenobu Tanihara; Epithelial Mesenchymal Transition and Human Vitreous Samples in 2D and 3D culture of ARPE-19 Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1193.

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

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Abstract

Purpose: To investigate the effects of exposure to vitreous samples on human retinal pigment epithelial (RPE) cells.

Methods: Human vitreous samples were collected from eyes with rhegmatogenous retinal detachment (RRD), epiretinal membrane (ERM), or macular hole (MH). For 2D culture, human RPE cells (ARPE-19) were cultured in serum free medium with/ without vitreous samples. For mechanical stress assay, after scraping with a silicon rubber, cells were seeded on poly 2-hydroxyethyl methacrylate coated dish in serum free medium with/ without vitreous samples for 60 min at 37°C and 5% CO2. For 3D culture, cells were grown in 2% growth factor reduced Matrigel with or without vitreous humor. The 3D structures were scored based on the resemblance to images (round, branching, stellate).

Results: In 2D culture, exposure to vitreous samples induced neither morphological changes nor acceleration of wound closure. Some samples showed the increase of p-Smad2 and nuclear translocation of NF-κB. In mechanical stress assay, level of p-Smad2 was elevated independently of vitreous samples, p-NF-κB increased dependently of vitreous, and p-p38 was not changed. ARPE-19 cells appeared the round spheroid structures in Matrigel without vitreous samples. In contrast, cells in Matrigel with vitreous humor formed stellate phenotype and vitreous humor from RRD patients accelerated stellate phenotype compared to samples from MH patiens(p<0.05).

Conclusions: Our findings showed that mechanical stress and vitreous samples induced EMT-associated signals, p-Smad2 and p-NF-κB, and that vitreous samples induced invasive phenotype in 3D not in 2D culture. Taken together, exposure of vitreous humor triggered EMT in the status of loss of polarity in RPE cells.

Keywords: 701 retinal pigment epithelium • 512 EMT (epithelial mesenchymal transition) • 763 vitreous  
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