The role of hepatic cholesterol transporter ABCA1 for HDL metabolism in vivo

Abstract

The role of the hepatic ABCA1 cholesterol transporter for HDL metabolism was studied in this dissertation using adenoviral-mediated RNA interference to target ABCA1. The influence of hepatic siRNA-mediated ABCA1 down-regulation on HDL plasma concentration in vivo was analysed. Five different plasmid-based siRNA vectors directed against sequences within the murine ABCA1 gene were generated. The efficiency of siRNA-mediated down-regulation of a co-transfected ABCA1 construct in HEK 293 cells was judged by RT-PCR, immunofluorescence and Western blot analysis. The recombinant adenovirus was constructed using the most effective plasmid, enabling down-regulation of ABCA1 expression exclusively in the liver. This anti-ABCA1 virus was administrated into the tail vein of C57Bl/6 male mice in the dose of 2.5*10^9 ifu. One week after injection, in comparison to the control group (administrated with EGFP expressing adenovirus), Western blot analysis of liver membrane preparations indicated a significant (~50%) down-regulation of endogenous ABCA1 protein in all Ad-anti-ABCA1 treated mice. Moreover, FPLC analysis of pooled plasma samples revealed that ABCA1 protein reduction was associated with an approximately 60% reduction of HDL cholesterol levels while other lipoprotein classes were not influenced. As a secondary effect a 1.5 times elevation of apoE expression was observed in the liver as a response to ABCA1 down regulation, while other proteins involved in lipoprotein metabolism were not influenced. In conclusion, the current study demonstrates that hepatic ABCA1 silencing mediated by adenoviral delivery of siRNA markedly reduced HDL cholesterol plasma levels in mice.