Effects of native and myeloperoxidase-modified apolipoprotein a-I on reverse cholesterol transport and atherosclerosis in mice

Abstract

Objective: Preclinical and clinical studies have shown beneficial effects of infusions of apolipoprotein A-I (ApoA-I) on atherosclerosis. ApoA-I is also a target for myeloperoxidase-mediated oxidation, leading in vitro to a loss of its ability to promote ATP-binding cassette transporter A1-dependent macrophage cholesterol efflux. Therefore, we hypothesized that myeloperoxidase-mediated ApoA-I oxidation would impair its promotion of reverse cholesterol transport in vivo and the beneficial effects on atherosclerotic plaques.

Approach and results: ApoA-I(-/-) or apolipoprotein E-deficient mice were subcutaneously injected with native human ApoA-I, oxidized human ApoA-I (myeloperoxidase/hydrogen peroxide/chloride treated), or carrier. Although early postinjection (8 hours) levels of total ApoA-I in plasma were similar for native versus oxidized human ApoA-I, native ApoA-I primarily resided within the high-density lipoprotein fraction, whereas the majority of oxidized human ApoA-I was highly cross-linked and not high-density lipoprotein particle associated, consistent with impaired ATP-binding cassette transporter A1 interaction. In ApoA-I(-/-) mice, ApoA-I oxidation significantly impaired reverse cholesterol transport in vivo. In advanced aortic root atherosclerotic plaques of apolipoprotein E-deficient mice, native ApoA-I injections led to significant decreases in lipid content, macrophage number, and an increase in collagen content; in contrast, oxidized human ApoA-I failed to mediate these changes. The decrease in plaque macrophages with native ApoA-I was accompanied by significant induction of their chemokine receptor CCR7. Furthermore, only native ApoA-I injections led to a significant reduction of inflammatory M1 and increase in anti-inflammatory M2 macrophage markers in the plaques.

Conclusions: Myeloperoxidase-mediated oxidation renders ApoA-I dysfunctional and unable to (1) promote reverse cholesterol transport, (2) mediate beneficial changes in the composition of atherosclerotic plaques, and (3) pacify the inflammatory status of plaque macrophages.

Keywords: apolipoprotein A-I; atherosclerosis; myeloperoxidase.

Figure 1. Distribution of injected human native and oxidized ApoA-I in the plasma of ApoE^−/− mice

ApoE^−/− mice (16 weeks on Western diet) were injected s.c. with 15 mg of either native or oxidized (oxApoA-I) human ApoA-I. Blood was collected at 8 hours after the injections. A-B) Fast performance liquid chromatography (FPLC) on a Superdex 200 column was performed on pooled plasma (100 μl) from 3 mice. C-D) Western blot analysis of human ApoA-I in numbered FPLC fractions probed with anti-total human ApoA-I monoclonal antibody (mAb 10G1.5). E-G) HDL-containing lipoprotein fraction and lipoprotein deficient fraction (LPDF) were isolated from 40 μl of plasma after sequential buoyant density ultracentrifugation. Human ApoA-I levels were quantified by Western blot analysis using mAb 10G1.5. E) Percentage of injected ApoA-I within HDL versus LPDF, F) percentage of cross-linked ApoA-I within HDL versus LPDF and G) illustrative Western blot analyses probed with mAb 10G1.5 of the distribution of injected human native and oxidized ApoA-I in the plasma compartments.

Figure 2. Plasma HDL-C levels and reverse cholesterol transport (RCT) after treatment with human native or oxidized ApoA-I

A) Experimental timeline and plasma HDL-cholesterol (HDL-C) levels, B) RCT to the plasma, C) liver and D) feces over 3 days after s.c. injection of [³H]cholesterol labeled foam cells into ApoA-I^−/− mouse hosts. Significance values for (A) and (B) are given in the text. RCT to plasma, liver and feces is shown as the % of the injected radioactivity. Data are shown as mean ± SD, n = 5-6 mice for controls and n = 4 mice each for native ApoA-I and oxidized ApoA-I (oxApoA-I).

Figure 3. Plaque size, macrophages, lipid and collagen content after treatment with human native or oxidized ApoA-I

Aortic root sections: A) plaque size, B-C) macrophage (CD68+) plaque cells (magnification 4×, n = 10 in each group), D) lipid content (Oil Red O, magnification 10×, n = 10 in each group), E) collagen content (Sirius Red, magnification 5×, n = 5 in each group) after s.c. injection (every other day) of ApoE^−/− mice with native ApoA-I, oxidized ApoA-I (oxApoA-I) or carrier (control) over 1 week. Data are shown as mean ± SEM; n.s. = not significant.

Figure 4. Attenuated induction CCR7 expression and HMG-CoA reductase in plaque macrophages by oxidation of ApoA-I

A) Chemokine receptor CCR7 mRNA from laser-captured CD68+ aortic root plaque cells measured by qRT-PCR, B) representative immunohistochemistry for CCR7 in aortic root plaques (magnification 20×), and C) HMG-CoA reductase mRNA expression from laser-captured CD68+ aortic root plaque cells measured by qRT-PCR from ApoE^−/− mice (16 weeks on Western diet) after s.c. injection (every other day) of native ApoA-I, oxidized ApoA-I (oxApoA-I) or carrier (control) over 1 week; minimum of 7 mice in each group. Data are shown as mean ± SEM; L = lumen.

Figure 5. Inflammatory state of the plaque: changes in M1 and M2 macrophage markers

A) IL1-β and B) MCP-I mRNA expression of laser-captured CD68+ aortic root plaque cells measured by qRT-PCR; minimum of 8 mice in each group, and immunohistochemistry for C) MCP-I, D) mannose receptor 1 (MR) and E) arginase-I (Arg-I) in aortic root plaques from ApoE ^−/− mice (16 weeks on Western diet) after s.c. injection (every other day) of native ApoA-I, oxidized ApoA-I (oxApoA-I) or carrier (control) over 1 week; magnification 10×, arginase-I 20×; minimum of 5 mice in each group; Data are shown as mean ± SEM; L = lumen.