Abstract
Purpose:
We have found that the only significant metabolism of 7-Ketocholesterol (7KCh) in ARPE-19 cells is via fatty acid esterification catalyzed by Sterol O-acyltransferase (SOAT1/ACAT-1). The object of this study was to investigate whether adenovirus-mediated overexpression of SOAT1 can attenuate 7KCh-induced cytotoxicity and inflammation in ARPE-19 cells.
Methods:
ARPE-19 cells were infected with adenovirus encoding SOAT1 prior to 7KCh treatment. Adenovirus-GFP was used as a negative control. Overexpression of SOAT1 protein was confirmed by immunoblot using a rabbit polyclonal affinity purified anti-SOAT1 antibody. Cells were collected and analyzed for 7KCh, Cholesterol (Ch) and 7KCh-fatty acid esters (7KFAEs) 24 h after 7KCh treatment. Cell viability was determined by celluar dehydrogenase activity. Identification and quantification of 7KCh, Ch and 7KFAEs were performed by HPLC-UV and LCMS. IL-6, IL-8 and VEGF protein levels in conditioned media were measured by Milliplex Cytokine Kit using the Luminex MAGPIX instrument.
Results:
Adenovirus SOAT1 infection resulted in a high level of SOAT1 overexpression in ARPE-19 cells. In normal ARPE-19 cells (without SOAT1 overexpression) 4 main 7KFAEs are formed which account for 20% of the internalized 7KCh. SOAT1 overexpression increased 7KFAEs synthesis by 1.5-fold and reduced intracellular 7KCh levels by 40%. SOAT1 overexpression significantly decreased IL-6 and IL-8 expression in conditioned media but had no effect on VEGF levels. SOAT1 overexpression protected the cells from 7KCh-induced cytotoxicity.
Conclusions:
Overexpression of SOAT1 in ARPE-19 cells attenuates 7KCh-induced cell toxicity and inflammation. The protective effect seems to be caused by the formation of non-toxic 7KFAEs. This seems to be the only metabolic pathway available to extra-hepatic tissues.
Keywords: 583 lipids •
592 metabolism •
688 retina