June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
The Role of 7-Ketocholesteryl Esters on 7KCh-Mediated Cytotoxicity and Inflammation in ARPE-19 cells
Author Affiliations & Notes
  • Jung 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 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 2013, Vol.54, 6093. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Jung Lee, Jiahn-Dar Huang, Ignacio Rodriguez; The Role of 7-Ketocholesteryl Esters on 7KCh-Mediated Cytotoxicity and Inflammation in ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6093.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose: 7-Ketocholesterol (7KCh) is known to have potent pro-inflammatory and cytotoxic effects in cultured RPE cells. Our previous study has shown that the only significant metabolism of 7KCh in ARPE-19 cells is by esterification to membrane fatty acids catalyzed by acyl-coenzyme A: cholesterol acyltransferase (ACAT-1). In this study, we investigated the role of 7-ketocholesteryl ester (7KEs) on 7KCh-mediated cytotoxicity and inflammation in ARPE-19 cells.

Methods: ARPE-19 cells were incubated with 7KCh alone or pre-incubated with either ACAT-1 inhibitors or phospholipase A2 inhibitors prior to 7KCh treatment. For identification and quantification of 7KCh, Ch and 7KEs, cells were collected over time after 7KCh treatment and analyzed by HPLC-UV and LCMS. Caspase 3/7 activity was measured by Promega Apo-One® kit. Cell viability was measured by cellular dehydrogenase activity. ACAT-1 and cPLA2a were knockdown using siRNAs. VEGF, IL-6 and IL-8 mRNA levels were measured by qRT-PCR.

Results: 7KCh was measured inside the ARPE-19 cells 5 min after 7KCh treatment. At 3 h after treatment, 7KCh reached a maximum level and 7KE began to form in the cells. The 7KE levels steadily increased for 24 h before starting to plateau. A minimum concentration of 4 µM 7KCh is needed for 7KE formation. ACAT-1 inhibition completely abolished 7KE formation and suppressed 50% of caspase 3/7 activity. By contrast, cPLA2a inhibition completely ablated both 7KE formation and caspase 3/7 activity. Knockdown of ACAT-1 caused a synergistic effect with 7KCh resulting in a markedly increase VEGF, IL-6, IL-8 mRNA levels whereas knockdown of cPLA2a had no effect. Inhibition of iPLA2 caused a marked increase in caspase 3/7 activity but did not increase the cytotoxicity of 7KCh. None of the cPLA2a or ACAT-1 inhibitors or the siRNAs protected the cells from 7KCh-mediated cytotoxicity.

Conclusions: Both ACAT-1 and cPLA2a inhibition reduced caspase 3/7 activity but failed to rescue the cells from 7KCh-mediated cytotoxicity. Inhibition of iPLA2 increased caspase 3/7 activity but did not enhance 7KCh cytotoxicity. This suggests that caspase 3/7 activity is not related to the 7KCh-mediated cytotoxicity. The synergistic effect on cytokine induction caused by the knockdown of ACAT-1 in combination with 7KCh treatment suggests that free arachidonic acid released at the membrane by the 7KCh-activated cPLA2a may enhance the cytokine response.

Keywords: 583 lipids • 592 metabolism • 688 retina  
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×