Influence of apolipoprotein A-I domain structure on macrophage reverse cholesterol transport in mice

Abstract

Objective: The goal of this study was to determine the influence of apolipoprotein A-I (apoA-I) tertiary structure domain properties on the antiatherogenic properties of the protein. Two chimeric hybrids with the N-terminal domains swapped (human-mouse apoA-I and mouse-human apoA-I) were expressed in apoA-I-null mice with adeno-associated virus (AAV) and used to study macrophage reverse cholesterol transport (RCT) in vivo.

Methods and results: The different apoA-I variants were expressed in apoA-I-null mice that were injected with [H(3)]cholesterol-labeled J774 mouse macrophages to measure RCT. Significantly more cholesterol was removed from the macrophages and deposited in the feces via the RCT pathway in mice expressing mouse-H apoA-I compared with all other groups. Analysis of the individual components of the RCT pathway demonstrated that mouse-H apoA-I promoted ATP-binding cassette transporter A1-mediated cholesterol efflux more efficiently than all other variants, as well as increasing the rate of cholesterol uptake into liver cells.

Conclusions: The structural domain properties of apoA-I affect the ability of the protein to mediate macrophage RCT. Replacement of the N-terminal helix bundle domain in the human apoA-I with the mouse apoA-I counterpart causes a gain of function with respect to macrophage RCT, suggesting that engineering some destabilization into the N-terminal helix bundle domain or increasing the hydrophobicity of the C-terminal domain of human apoA-I would enhance the antiatherogenic properties of the protein.

Fig 1. Particle size and apoprotein composition analysis of HDL from mice expressing the human apoA-I variants

ApoA-I-null mice were infected with AAV and bled at 2,4 and 6 weeks post infection. HDL were isolated from pooled plasma by ultracentrifugation. (A) 10 μg of total HDL protein was loaded on a 4–20% Tris-Glycine non-denaturing gradient gel and stained with Coomassie brilliant blue. (B) To determine the apoprotein composition of the HDL, 2 μg of total protein was analyzed by reducing 8–25% SDS-PAGE. Lanes: (1) WT Human apoA-I, (2) WT Mouse apoA-I, (3) Human-M apoA-I, (4) Mouse-H apoA-I, (5) LacZ.

Figure 2. Reverse Cholesterol Transport Assay

Male apoA-I-null mice (n = 6 per group) were injected with AAV (1 X 10¹² GC) expressing either WT Human apoA-I, WT Mouse apoA-I, Human-M apoA-I, Mouse-H apoA-I or LacZ 10 weeks prior to the RCT assay. The mice were then injected IP with [³H]cholesterol-labeled, acLDL-loaded J774 macrophages. Data are expressed as percentage ± SD of the [³H]cholesterol tracer relative to total cpm tracer injected. (A) Time course of [³H]cholesterol appearance in plasma. Mice were bled at 2, 6, 24, and 48 hours after injection. (B) Fecal [³H]cholesterol tracer levels (mean ± SD, n = 6). Feces were collected continuously from 0 to 48 hours post-injection. WT Human apoA-I (■), WT Mouse apoA-I (▲), Human-M apoA-I (▼), Mouse-H apoA-I (◇) and LacZ (●). * Indicates p<0.05 compared to WT mouse apoA-I.

Figure 3. Influence of apoA-I structure on ABCA1- and ABCG1-mediated cholesterol efflux from cells

(A) J774 macrophages were labeled with [³H] cholesterol as described under “Materials and Methods.” These labeled cells were then treated with cAMP overnight to up-regulate ABCA1. The lipid efflux was then initiated by the addition of the recombinant apoA-I variants (0–20 μg/mL). After 4 h incubation, the medium was removed, filtered and extracted for lipids and ³H-radioactivity was determined. The catalytic efficiency (V_max/K_m) ((%FC efflux/4h)(μg apoA-I/ml)⁻¹) was calculated from kinetic parameters generated by fitting the fractional efflux at 4 h, measured at various concentrations of apoA-I, to the Michaelis-Menten equation. The V_max/K_m values are plotted as mean ± SD. (B) BHK cells were labeled as described in “Materials and Methods” and treated with mifepristone for 18h to upregulate ABCG1. Cholesterol efflux was initiated by the addition of rHDL (20 μg/mL apoA-I) containing the recombinant apoA-I variants for 4 h. * Indicates p<0.05 compared to WT mouse apoA-I.

Figure 4. LCAT cholesterol esterification rates

(A) Cholesterol esterification rate in the plasma of mice expressing the apoA-I variants or LacZ. Whole plasma was radiolabeled with [³H]cholesterol at 4 °C overnight and then incubated at 37 °C for 30 min. LCAT activity was determined as described in “Materials and Methods” and is expressed as nanomoles of CE formed per milliliter of plasma during the 30 min incubation. (B) Percentage of cholesterol as CE in the plasma of mice expressing the apoA-I variants or LacZ. Values are expressed as mean ± SD (n = 6/group). * Indicates p<0.05 compared to WT mouse apoA-I.

Figure 5. Influx of cholesterol to hepatic cells from serum of mice expressing apoA-I variants

[³H]cholesterol- and [³H]cholesteryl ester-labeled sera from the 48 h timepoint of the experiment described in Fig. 2A was diluted to 20% with MEM and incubated with Fu5AH rat hepatoma cells for 6 h with BLT-1 (□)to block SR-BI-mediated influx or without BLT-1 (■). Influx was calculated as described in “Materials and Methods”. * Indicates p<0.05 compared to + BLT-1 group.