Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Mar;56(3):653-664.
doi: 10.1194/jlr.M056754. Epub 2015 Jan 5.

HDL from apoA1 transgenic mice expressing the 4WF isoform is resistant to oxidative loss of function

Affiliations

HDL from apoA1 transgenic mice expressing the 4WF isoform is resistant to oxidative loss of function

Stela Z Berisha et al. J Lipid Res. 2015 Mar.

Abstract

HDL functions are impaired by myeloperoxidase (MPO), which selectively targets and oxidizes human apoA1. We previously found that the 4WF isoform of human apoA1, in which the four tryptophan residues are substituted with phenylalanine, is resistant to MPO-mediated loss of function. The purpose of this study was to generate 4WF apoA1 transgenic mice and compare functional properties of the 4WF and wild-type human apoA1 isoforms in vivo. Male mice had significantly higher plasma apoA1 levels than females for both isoforms of human apoA1, attributed to different production rates. With matched plasma apoA1 levels, 4WF transgenics had a trend for slightly less HDL-cholesterol versus human apoA1 transgenics. While 4WF transgenics had 31% less reverse cholesterol transport (RCT) to the plasma compartment, equivalent RCT to the liver and feces was observed. Plasma from both strains had similar ability to accept cholesterol and facilitate ex vivo cholesterol efflux from macrophages. Furthermore, we observed that 4WF transgenic HDL was partially (∼50%) protected from MPO-mediated loss of function while human apoA1 transgenic HDL lost all ABCA1-dependent cholesterol acceptor activity. In conclusion, the structure and function of HDL from 4WF transgenic mice was not different than HDL derived from human apoA1 transgenic mice.

Keywords: ATP binding cassette transporter A1; apolipoprotein A1; cholesterol efflux; dysfunctional high density lipoprotein; myeloperoxidase; reverse cholesterol transport.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
Characterization of plasma apoA1 and HDL-C in human apoA1 heterozygous and 4WF homozygous transgenic mice. Plasma apoA1 (A) and HDL-C (B) levels were quantified in apoA1 heterozygous and 4WF homozygous female and male transgenic mice (n = 5 per group). Data are presented as mean ± SD. Different numbers above bars show P value <0.0001 (A) by ANOVA with Bonferroni posttest. Cholesterol profiles of human apoA1 and 4WF male (C) and female (D) transgenic mice were generated by FPLC analysis of pooled plasma samples. Human apoA1 protein levels in males (E) were quantified in even-numbered FPLC fractions by an immunoturbidimetric assay.
Fig. 2.
Fig. 2.
Quantification of mouse and human apoA1 in plasma from C57BL/6, human apoA1, and 4WF transgenic mice. Mouse apoA1 (gray bars) and human apoA1 (open bars) levels were quantified in plasma samples isolated from apoA1 and 4WF female transgenic mice with matched human apoA1 levels. Data are presented as mean ± SD (n = 3 per group). For mouse apoA1 levels, different numbers above bars show P value <0.01 by ANOVA with Newman-Keuls posttest.
Fig. 3.
Fig. 3.
HDL profile and protein composition. Nondenaturing gradient gel electrophoresis (A; male samples) and SDS-PAGE (B; female samples) were performed on human apoA1 and 4WF transgenic HDL isolated by sequential gradient ultracentrifugation. HDL samples were run at 10 µg of total HDL protein per lane, stained with Coomassie blue, and scanned by densitometry.
Fig. 4.
Fig. 4.
RCT in human apoA1 and 4WF transgenic mice with matched levels of human plasma apoA1. RCT to plasma (A), liver (B), and cumulative feces (C) 72 h post [3H]labeled foam cell injection into human apoA1 and 4WF male transgenic mice hosts. Data are presented as mean ± SD (n = 5 per group). Values represent % of the injected [3H]cholesterol counts. P value shown for significant differences by unpaired t-test.
Fig. 5.
Fig. 5.
Ex vivo evaluation of apoA1 and 4WF plasma cholesterol acceptor activity. A: Plasma cholesterol acceptor activity from wild-type mouse BMDMs. B: Plasma cholesterol acceptor activity from RAW264.7 cells in the absence or presence of 8Br-cAMP treatment to distinguish ABCA1-independent, total, and ABCA1-dependent (by subtraction of independent from total) cholesterol efflux. For both panels, no plasma control is shown in black bars; 0.4% (v/v) plasma from human apoA1 (blue bars) or 4WF (green bars) male transgenic mice with matched human apoA1 levels was used as cholesterol acceptor. Data are presented as mean ± SD (n = 5 per group; each sample run in triplicate). Different numbers above bars show P values <0.001 (A) or P values of <0.05 (B) by ANOVA with Newman-Keuls posttest.
Fig. 6.
Fig. 6.
Plasma HDL-C acceptor activity in the absence or presence of ex vivo MPO-mediated oxidation. ABCA1-independent (A), ABCA1-dependent (B), and total (C) [3H]cholesterol effluxed from RAW 264.7 cells in the presence of either MPO buffer control, native (filled bars) or MPO-oxidized (open bars) HDL isolated from apoA1 (blue) or 4WF (green) transgenic mice. HDL containing human apoA1 or the 4WF isoform, isolated form male mice, was added at a final concentration of 50 µg of apoA1/ml of media. Data are presented as mean ± SD (each condition run in triplicate). Different numbers above bars show P values <0.001 (A) or <0.05 (B, C), by ANOVA with Newman-Keuls posttest.
Fig. 7.
Fig. 7.
HDL uptake and binding by primary mouse hepatocytes. Binding at 4°C (A) and uptake at 37°C (B) of DiI-labeled HDL by primary mouse hepatocytes normalized to cellular protein. Native (filled bars) and oxidized (open bars) DiI-labeled HDL from apoA1 (blue) or 4WF (green) transgenic mice was added at a final concentration of 25 µg of apoA1/ml of media. Data are presented as mean ± SD (each condition run in quadruplicates). Different numbers above bars show P values <0.05 by ANOVA with Newman-Keuls posttest.

Similar articles

Cited by

References

    1. Gordon D. J., Probstfield J. L., Garrison R. J., Neaton J. D., Castelli W. P., Knoke J. D., Jacobs D. R., Jr, Bangdiwala S., Tyroler H. A. 1989. High-density lipoprotein cholesterol and cardiovascular disease. Four prospective American studies. Circulation. 79: 8–15. - PubMed
    1. Barter P. J., Caulfield M., Eriksson M., Grundy S. M., Kastelein J. J., Komajda M., Lopez-Sendon J., Mosca L., Tardif J. C., Waters D. D., et al. 2007. Effects of torcetrapib in patients at high risk for coronary events. N. Engl. J. Med. 357: 2109–2122. - PubMed
    1. Schwartz G. G., Olsson A. G., Abt M., et al. dal-OUTCOMES Investigators. 2012. Effects of dalcetrapib in patients with a recent acute coronary syndrome. N. Engl. J. Med. 367: 2089–2099. - PubMed
    1. Michos E. D., Sibley C. T., Baer J. T., Blaha M. J., Blumenthal R. S. 2012. Niacin and statin combination therapy for atherosclerosis regression and prevention of cardiovascular disease events: reconciling the AIM-HIGH (Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Tri­glycerides: Impact on Global Health Outcomes) trial with previous surrogate endpoint trials. J. Am. Coll. Cardiol. 59: 2058–2064. - PubMed
    1. HPS2-THRIVE Collaborative Group. 2013. HPS2-THRIVE randomized placebo-controlled trial in 25 673 high-risk patients of ER niacin/laropiprant: trial design, pre-specified muscle and liver outcomes, and reasons for stopping study treatment. Eur. Heart J. 34: 1279–1291. - PMC - PubMed

Publication types