June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Metabolism of 7-ketocholesterol in ARPE-19 cells occurs by esterification to fatty acids via cPLA2α/SOAT1 followed by selective efflux to HDL
Author Affiliations & Notes
  • Jung W Lee
    Mechanisms of Retinal Diseases Section, LRCMB, National Eye Institute/NIH, Bethesda, MD
  • Jiahn-Dar Huang
    Mechanisms of Retinal Diseases Section, LRCMB, National Eye Institute/NIH, Bethesda, MD
  • Ignacio R Rodriguez
    Mechanisms of Retinal Diseases Section, LRCMB, National Eye Institute/NIH, Bethesda, MD
  • Footnotes
    Commercial Relationships Jung Lee, None; Jiahn-Dar Huang, None; Ignacio Rodriguez, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 1532. doi:
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      Jung W Lee, Jiahn-Dar Huang, Ignacio R Rodriguez; Metabolism of 7-ketocholesterol in ARPE-19 cells occurs by esterification to fatty acids via cPLA2α/SOAT1 followed by selective efflux to HDL. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1532.

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

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Abstract

Purpose: We have previously reported that overexpression of sterol O-acyltransferase (SOAT1/ACAT-1) in ARPE-19 cells reduced the 7-Ketocholesterol (7KCh)-induced inflammatory response and provided some protection from cell death. The protective effect seems to be due to the increased conversion of 7KCh to 7KCh-fatty acid esters (7KFAEs). The object of this study was to determine if HDL aids in the efflux of 7KFAEs from the cells.

Methods: ARPE-19 cells were infected with adenovirus encoding SOAT1 prior to 7KCh treatment. Adenovirus-GFP was used as a negative control. ARPE-19 cells were treated with a lethal dose of 7KCh (12 µM) in combination with different amounts of HDL (1-10 µg) in the media with and without SOAT1 overexpression. Cell viability was determined by celluar dehydrogenase activity 24 h after 7KCh treatment. Identification and quantification of 7KCh, cholesterol and 7KFAEs was performed by HPLC-UV.

Results: SOAT1 overexpression protected the cells from 7KCh-induced cytotoxicity but this protection is enhanced by the presence of HDL at 5 μg/ml and higher. The presence of HDL in the conditioned media (CM) significantly increased the intracellular levels of 7KFAEs as well as the levels in the CM. The intracellular levels of 7KCh were decreased in a dose-dependent manner in both cells with and without SOAT1 overexpression.

Conclusions: Addition of HDL selectively increased the efflux of 7KFAEs and enhanced the effect of SOAT1 overexpression. Our data suggests that HDL may also function to aid extra-hepatic tissues in returning 7KFAEs to the liver, which is the only tissue that can fully metabolize and detoxify 7KCh.

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