April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
The involvement of transforming growth factor beta in endoplasmic reticulum stress of corneal endothelial cells in Fuchs’ endothelial corneal dystrophy
Author Affiliations & Notes
  • Naoki Okumura
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Ryuki Minamiyama
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • EunDuck P Kay
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Satoshi Kawasaki
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Robert D Young
    School of Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom
  • Andrew J Quantock
    School of Optometry and Vision Sciences, Cardiff University, Cardiff, United Kingdom
  • Ursula Schlotzer-Schrehardt
    Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Friedrich E Kruse
    Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
  • Shigeru Kinoshita
    Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Noriko Koizumi
    Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Footnotes
    Commercial Relationships Naoki Okumura, Doshisha University (P), JCR Pharmaceuticals (P), Senju Pharmaceutical Co (P); Ryuki Minamiyama, None; EunDuck Kay, None; Satoshi Kawasaki, None; Robert Young, None; Andrew Quantock, None; Ursula Schlotzer-Schrehardt, None; Friedrich Kruse, None; Shigeru Kinoshita, Otsuka Pharmaceutical Co (C), Santen Pharmaceutical Co (P), Senju Pharmaceutical Co (P); Noriko Koizumi, Doshisha University (P), JCR Pharmaceuticals Co (P), Senju Pharmaceutical Co (P)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 2067. doi:https://doi.org/
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      Naoki Okumura, Ryuki Minamiyama, EunDuck P Kay, Satoshi Kawasaki, Robert D Young, Andrew J Quantock, Ursula Schlotzer-Schrehardt, Friedrich E Kruse, Shigeru Kinoshita, Noriko Koizumi; The involvement of transforming growth factor beta in endoplasmic reticulum stress of corneal endothelial cells in Fuchs’ endothelial corneal dystrophy. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2067. doi: https://doi.org/.

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

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Abstract

Purpose: The pathogenesis of Fuchs’ endothelial corneal dystrophy (FECD) has yet to be elucidated. Here we demonstrate the involvement of transforming growth factor beta (TGF-β) signaling to induce endoplasmic reticulum (ER) stress in FECD corneal endothelial cells (CECs).

Methods: FECD-patient human CECs (HCECs) and normal donor-cornea HCECs were cultured and immortalized to produce iFECD and iHCEC cell lines, respectively. To determine extracellular matrix (ECM) protein production and ER colocalization, iFECD and iHCEC cells were immunostained by antibodies for collagen type-I and IV, fibronectin, and ER-marker protein disulfide isomerase. iFECD and iHCEC were assessed by transmission electron microscopy (TEM). To evaluate ER stress, molecular chaperone (GRP78) and ER stress-sensor (IRE1, PERK, and ATF6) expression was analyzed by western blotting of iFECD and iHCEC stimulated with or without TGF-β. To elucidate TGF-β signaling pathway involvement in apoptosis, cells were treated with TGF-β and inhibitors SB431542, A-83-01, and ALK5 inhibitor, and Annexin V-positive apoptotic cells were then evaluated by flow cytometry.

Results: Immunocytochemistry showed that collagen type-I and IV and fibronectin were expressed higher in iFCED than in iHCEC, and that they localized in ER. TEM demonstrated that ER and mitochondria were dilated in iFECD, yet were in normal morphology in iHCEC. Expression of GRP78 was higher in iFECD than in iHCEC, and was markedly increased by TGF-β in iFECD in comparison to iHCEC. Likewise, TGF-β markedly increased the expression of IRE1, PERK, and ATF6 in iFECD in comparison to iHCEC. In iHCEC, the percentage of Annexin V-positive apoptotic cells was not increased in comparison to before TGF-β stimulation (12.3±0.5% and 11.1±0.6%, respectively). In contrast, TGF-β significantly increased Annexin V-positive cells in comparison to the control iFECD (29.9±1.5% and 19.4±1.4%, respectively). In addition, SB431542, A-83-01, and ALK5 inhibitor significantly suppressed the TGF-β induced apoptosis of iFECD.

Conclusions: Our findings suggest that ER stress caused by excessive production of ECM proteins is involved in cell loss in FECD, and that FECD cells have higher sensitivity towards TGF-β signaling. The TGF-β signaling pathway may be a potent therapeutic target for suppressing FECD CEC damage.

Keywords: 481 cornea: endothelium • 426 apoptosis/cell death  
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