Inhibition of ABCA1 protein degradation promotes HDL cholesterol efflux capacity and RCT and reduces atherosclerosis in mice

Abstract

ABCA1 plays a key role in the initial lipidation of apoA-I, which generates circulating HDL cholesterol. Whereas it is known that the transcriptional upregulation of ABCA1 promotes HDL formation and reverse cholesterol transport (RCT), it is not known how the inhibition of ABCA1 protein degradation impacts HDL function. Employing the small molecule triacetyl-3-hydroxyphenyladenosine (IMM-H007), we determined how the attenuation of ABCA1 protein degradation affects HDL cholesterol efflux capacity, RCT, and atherosclerotic lesion formation. Pulse-chase analysis revealed that IMM-H007 inhibits ABCA1 degradation and facilitates its cell-surface localization in macrophages, and additional studies in macrophages showed that IMM-H007 thereby promotes cholesterol efflux. IMM-H007 treatment of Paigen diet-fed mice caused an increase in circulating HDL level, it increased the cholesterol efflux capacity of HDL, and it enhanced in vivo RCT from macrophages to the plasma, liver, and feces. Furthermore, ABCA1 degradation suppression by IMM-H007 reduced atherosclerotic plaque formation in apoE(-/-) mice. Thus, via effects on both ABCA1-expressing cells and circulating HDL function, the inhibition of ABCA1 protein degradation by IMM-H007 promotes HDL cholesterol efflux capacity and RCT and attenuates atherogenesis. IMM-H007 potentially represents a lead compound for the development of agents to augment HDL function.

Keywords: ATP binding cassette transporter A1; high density lipoprotein; macrophage; reverse cholesterol transport.

Fig. 1.

IMM-H007 increases ABCA1 protein expression in vitro and in vivo. A: Chemical structure of IMM-H007. B, C: IMM-H007 promotes the expression of ABCA1 in time- and concentration-dependent manners. THP-1 cells were treated with 10 μM IMM-H007 for the indicated times (B), or with the indicated concentrations of IMM-H007 for 1 h (C). Cell lysates were analyzed by immunoblotting. D, E: IMM-H007 does not affect ABCA1 mRNA level in THP-1 cells. Cells were treated as described in (B) and (C), total RNA was isolated, and quantitative RT-PCR was performed. F, G: IMM-H007 increases hepatic ABCA1 protein but not ABCA1 mRNA level. Hepatic ABCA1 protein (F) and mRNA (G) were evaluated in Paigen diet-fed mice administered the indicated doses of IMM-H007 for 2 weeks. Data are mean ± SEM, n = 3 for (B–E) and n = 10 for (F, G). **P < 0.01, ***P < 0.001 versus control. NS, nonsignificant.

Fig. 2.

IMM-H007 retards ABCA1 protein degradation and facilitates its surface localization. A: IMM-H007 delays ABCA1 protein degradation. THP-1 cells were incubated with CHX (20 μg/ml) for the indicated times after 1 h of vehicle or IMM-H007 treatment. Cell lysates were processed for immunoblotting. B: IMM-H007 increases cell surface ABCA1. THP-1 cells were treated with vehicle or IMM-H007 (10 μM) for the indicated times, labeled with sulfo-SS-biotin at 4°C, and biotinylated protein was selectively absorbed by streptavidin-agarose and analyzed by immunoblotting. C: IMM-H007 retards surface ABCA1 internalization. After biotinylation, surface ABCA1 was internalized in the presence or absence of IMM-H007 at 37°C for the indicated times. Surface biotinylation was cleaved and the remaining intracellular biotinylated ABCA1 was quantified to evaluate the internalized fraction. D, E: IMM-H007 facilitates the recycling of internalized ABCA1 to the plasma membrane. After internalization at 37°C for 1 h, biotinylated protein in the vehicle- or IMM-H007-treated cells was recycled at 37°C for the indicated times. Remaining intracellular biotinylated ABCA1 (ic) and total biotinylated ABCA1, which includes intracellular biotinylated ABCA1 (ic) plus surface biotinylated ABCA1 (s), after recycling were detected by immunoblotting. The difference between ic and ic+s represents recycled ABCA1. F: THP-1 cell surface ABCA1 was evaluated by flow cytometry after treatment with vehicle or 10 μM IMM-H007 for 16 h, using anti-ABCA1 primary antibody and green fluorescent Alexa Fluor 488-conjugated secondary antibody. G: Confocal microscopy was also performed on similarly-treated cells. Images shown display relative fluorescent intensity with identical exposure time for vehicle- and IMM-H007-treated cells. Data shown are mean ± SEM, for three independent assays in (A), (B), and (E), and in two studies in (C). *P < 0.05 versus vehicle. In (G), scale bar: 10 μm.

Fig. 3.

IMM-H007 inhibits ABCA1 protein degradation by suppressing calpain activity, and it promotes ABCA1 cell surface trafficking by palmitoylation. A: Calcium chelating-agents and the calpain inhibitor, calpeptin, reduce ABCA1 degradation. Cells were incubated with CHX (20 μg/ml) and vehicle (1); EGTA, 100 μM (2); BAPTA, 100 μM (3); EGTA+BAPTA, 1 μM+100 μM (4); calpeptin, 100 μM (5); or IMM-H007, 10 μM (6) for 1 h, and ABCA1 protein abundance was determined. B: IMM-H007 suppresses Ca²⁺-dependent ABCA1 protein degradation. Cells were exposed to various Ca²⁺ concentrations in the presence or absence of 10 μM IMM-H007 at 37°C for 30 min, and analyzed for ABCA1 abundance. C: Intracellular calpain activity is suppressed by IMM-H007. Cells were treated as described in (A), and calpain activity was determined. D: IMM-H007 does not affect ABCA1 stability in calpain-deficient cells. Cells were incubated in the presence of CHX for 1 h in the absence or presence of IMM-H007 (10 μM) following transfection with control siRNA or siRNA targeting calpain. ABCA1 abundance was then evaluated. In (A–C), data are mean ± SEM of three independent assays. *P < 0.05 and **P < 0.01 versus control or vehicle.

Fig. 4.

IMM-H007 increases circulating HDL cholesterol levels and facilitates cholesterol excretion via feces. Animals (n = 8–12/group) were fed a control chow or Paigen diet and treated with vehicle, probucol (200 mg/kg), or the indicated doses of IMM-H007. A: Plasma HDL levels of Paigen diet-fed apoE^−/− mice treated for 6 weeks. B: Plasma HDL levels of Paigen diet-fed WT mice treated with IMM-H007 for 2 weeks. C. Plasma HDL levels of vehicle- and IMM-H007-treated hamsters fed a Paigen diet for 2 weeks. D. FPLC lipoprotein profiles from pooled plasma (n = 8) of vehicle- or IMM-H007-treated WT mice fed a chow diet for 2 weeks. E, F: Fecal neutral sterol secretion of chow diet-fed (E) and Paigen diet-fed (F) mice treated for 2 weeks. Data are mean ± SEM. *P < 0.05, **P < 0.01 versus control.

Fig. 5.

IMM-H007 promotes cholesterol efflux in vitro via ABCA1. J774 cells (A) and THP-1 cells (B) were used to evaluate cholesterol efflux to apoB-depleted serum (PEG-HDL) from vehicle- or IMM-H007-treated mice. ABCA1-mediated efflux was upregulated with 0.3 mM cAMP in J774 cells and with 10 μM 9-cis-retinoic acid in THP-1 cells, and it was determined as the proportion of the cholesterol inhibited by 20 μM probucol. In J774 cells, SR-BI-mediated efflux was determined as the proportion of cholesterol efflux inhibited by 1 μM BLT-1. Other modes of efflux were calculated as total efflux minus ABCA1- and SR-BI-mediated efflux. Plasma from vehicle- and IMM-H007-treated chow diet-fed WT mice (n = 8) was obtained 2 weeks after treatment; PEG-HDL was prepared by removal of apoB lipoproteins with polyethylene glycol and diluted to 2.5% in medium as the efflux acceptor. C, D: ABCA1-mediated efflux was defined as the percent cholesterol efflux to apoA-I (10 μg/ml, 4 h incubation) from vehicle- versus IMM-H007-treated cells that were also treated with cAMP [J774 cells (C)] or 9-cis-retinoic acid [THP-1 cells (D)]. E: SR-BI-mediated efflux from vehicle- versus IMM-H007-treated J774 cells by PEG-HDL isolated from vehicle-treated mice was defined as percent cholesterol efflux from cells treated with cAMP minus the percent cholesterol efflux from comparably treated cells additionally preincubated with BLT-1 (1 μM) for 2 h. F: Aqueous diffusion-mediated cholesterol efflux to methyl-β-cyclodextrin (1 mg/ml, 30 min incubation) from vehicle- versus IMM-H007-treated J774 cells. Data are mean ± SEM, n = 3. *P < 0.05, **P < 0.01 versus control. NS, nonsignificant.

Fig. 6.

IMM-H007 enhances HDL-mediated RCT in vivo. ³H-cholesterol and acLDL-loaded J774 macrophages were injected into apoE^−/− mice fed a Paigen diet and treated with vehicle, probucol (200 mg/kg), or the indicated doses of IMM-H007 for 2 weeks (n = 5), and the amount of ³H-tracer in plasma (A), bile acid (B), liver (C), and feces (D) was determined by scintillation counting. Data are mean ± SEM, except in (A), in which legibility has been optimized by showing only mean values for 50 and 100 mg/kg IMM-H007. In those groups, all SEMs were less than 25% of mean values. *P < 0.05, **P < 0.01 versus control.

Fig. 7.

IMM-H007 alleviates atherosclerotic plaque development in apoE^−/− mice. apoE^−/− mice (n = 10–12) fed a Paigen diet were treated with vehicle, probucol (200 mg/kg), or the indicated doses of IMM-H007 for 10 weeks. A: Plaques (arrows) in aortic arches and thoracic aortas of representative apoE^−/− mice are shown. En face Oil Red O stained aortas (B) and cryosections of mouse aortic roots (C) from vehicle and treated apoE^−/− mice are shown. Mean atherosclerotic lesion areas of aorta (D) and aortic root (E) were determined using ImageJ software; each data point represents an individual animal. The horizontal lines denote the mean of each group. Scale bars: 1 cm (B) and 20 μm (C).