Overexpression of apolipoprotein F reduces HDL cholesterol levels in vivo

Abstract

Objective: Apolipoprotein F (ApoF) is a protein component of several lipoprotein classes including HDL. It is also known as lipid transfer inhibitor protein (LTIP) based on its ability to inhibit lipid transfer between lipoproteins ex vivo. We sought to investigate the role of ApoF in HDL metabolism.

Methods and results: Adeno-associated viruses (AAV) based on serotype 8, were used to overexpress either murine or human ApoF in mice. Overexpression of murine ApoF significantly reduced total cholesterol levels by 28% (P<0.001), HDL by 27% (P<0.001), and phospholipid levels by 19% (P<0.001). Overexpression of human ApoF had similar effects. Human ApoF was nearly exclusively HDL-associated in mice. In agreement with this finding, greater than 90% of the ApoF in human plasma was found on HDL(3), with only a small amount on LDL. Overexpression of mouse ApoF accelerated the plasma clearance of [(3)H]-cholesteryl ether labeled HDL. Plasma from mice overexpressing ApoF showed improved macrophage cholesterol efflux on a per HDL-C basis.

Conclusions: ApoF overexpression reduces HDL cholesterol levels in mice by increasing clearance of HDL-CE. ApoF may be an important determinant of HDL metabolism and reverse cholesterol transport.

Figure 1

N-linked glycosylation and cleavage of Apo F. A. Expression constructs for human and mouse Apo F. B. N-linked glycosylation and propeptide cleavage mutants in HEK293 cells (Amino acid substitutions given above each lane). Arrows indicate the mature peptide (Mature), proprotein (Pro), and maturely glycosylated proprotein (Pro *).

Figure 2

AAV overexpression of human ApoF reduces HDL cholesterol. Total cholesterol was measured in FPLC fractions of pooled plasma from mice overexpressing either Beta-Galactosidase (LacZ) or human ApoF (hApoF). Apo A-I, Apo E, and human Apo F were detected by Western blotting.

Figure 3

AAV overexpression of mouse Apo F reduces HDL cholesterol levels. Total plasma cholesterol, HDL cholesterol, phospholipids, triglycerides in mice overexpressing LacZ or mApoF (*** p< 0.001).

Figure 4

The effects of Apo F on RCT. A. Lipoprotein profile 48 hours after macrophage injection- total cholesterol (left), [³H]-cholesterol (right). B. [³H]-cholesterol in plasma following macrophage injection. C. Final ³H counts in bile. D. Final ³H counts in liver. E. Total fecal ³H counts (* p < 0.05).

Figure 5

Plasma from mice overexpressing Apo F has improved efflux capacity per HDL-C. A. Free cholesterol efflux to plasma from mice overexpressing Lac Z or mApoF B. Efflux capacity to plasma normalized to HDL-C concentrations. Significant differences indicated with * p < 0.05 and *** p < 0.001.