HDL and CER-001 Inverse-Dose Dependent Inhibition of Atherosclerotic Plaque Formation in apoE-/- Mice: Evidence of ABCA1 Down-Regulation

Abstract

Objective: CER-001 is a novel engineered HDL-mimetic comprised of recombinant human apoA-I and charged phospholipids that was designed to mimic the beneficial properties of nascent pre-ß HDL. In this study, we have evaluated the dose-dependent regulation of ABCA1 expression in vitro and in vivo in the presence of CER-001 and native HDL (HDL3).

Methods and results: CER-001 induced cholesterol efflux from J774 macrophages in a dose-dependent manner similar to natural HDL. A strong down-regulation of the ATP-binding cassette A1 (ABCA1) transporter mRNA (- 50%) as well as the ABCA1 membrane protein expression (- 50%) was observed at higher doses of CER-001 and HDL3 compared to non-lipidated apoA-I. In vivo, in an apoE-/- mouse "flow cessation model," in which the left carotid artery was ligatured to induce local inflammation, the inhibition of atherosclerotic plaque burden progression in response to a dose-range of every-other-day CER-001 or HDL in the presence of a high-fat diet for two weeks was assessed. We observed a U-shaped dose-response curve: inhibition of the plaque total cholesterol content increased with increasing doses of CER-001 or HDL3 up to a maximum inhibition (- 51%) at 5 mg/kg; however, as the dose was increased above this threshold, a progressively less pronounced inhibition of progression was observed, reaching a complete absence of inhibition of progression at doses of 20 mg/kg and over. ABCA1 protein expression in the same atherosclerotic plaque was decreased by-45% and-68% at 50 mg/kg for CER-001 and HDL respectively. Conversely, a-12% and 0% decrease in ABCA1 protein expression was observed at the 5 mg/kg dose for CER-001 and HDL respectively.

Conclusions: These data demonstrate that high doses of HDL and CER-001 are less effective at slowing progression of atherosclerotic plaque in apoE-/- mice compared to lower doses, following a U-shaped dose-response curve. A potential mechanism for this phenomenon is supported by the observation that high doses of HDL and CER-001 induce a rapid and strong down-regulation of ABCA1 both in vitro and in vivo. In conclusion, maximally efficient HDL- or CER-001-mediated cholesterol removal from atherosclerotic plaque is achieved by maximizing macrophage-mediated efflux from the plaque while minimizing dose-dependent down-regulation of ABCA1 expression. These observations may help define the optimal dose of HDL mimetics for testing in clinical trials of atherosclerotic burden regression.

Fig 1. Effect of dose-response of CER-001 and HDL3 in atherosclerotic plaque progression in ligatured carotid of apoE^-/- mice.

The left carotid of apoE^-/- mice (n = 12) was ligatured, fed with HCD and treated (retro-orbital injection) every second day with different concentrations of CER-001 or HDL3 (8 infusions). The carotids were lipid extracted and cholesterol concentrations were determined by HPLC. Panel A; unesterified cholesterol. Panel B; total cholesterol. Panel C; protein level of ABCA1 was measured in the ligatured carotids using Western blot analysis as described in material and methods section. The data represent the means of ABCA1 expression from at least 5 different carotids. Representative Western-blot for carotid analysis was resolved with anti-ABCA1 antibody (1/1000 dilution) and anti-Calnexin antibody (1/1000). * p<0.05, ** p<0.01, ***p<0.005, ****p<0.001, *****p<0.0001

Fig 2. ABCA1 mRNA and protein decrease in J774 macrophages following CER-001 and HDL3 incubation.

Panel A; ABCA1 mRNA level in J774 macrophages was determined in presence of different concentrations of CER-001, HDL3 or delipidated apoA-I for 24h. Panel B; Kinetic of ABCA1 mRNA level at a fixed concentration (250μg/mL) of CER-001, HDL3 or delipidated apoA-I. The qPCR data represent the means of triplicate determinations from a single experiment that is representative of three such experiments. Panel C; J774 macrophages were treated for 24 h in presence of CER-001, HDL3 or delipidated apoA-I at 250μg/mL (time 0). Compounds were removed and replaced with fresh medium for 24 h (time 24) and ABCA1 mRNA level was determined by qPCR. Panel D; The relative membrane protein expression of ABCA1 was measured by Western blot analysis for macrophages treated for 6h with CER-001, HDL3 or apoA-I at 250 μg/ml. cAMP was used for 24h at 300μM. Membrane protein loading for each sample was verified by Western blot using rabbit anti-Calnexin (1/1000 dilution). Membrane fractions were obtained after ultracentrifugation as described in Material & Method section. The data represent the means of triplicate determinations from a single experiment. * p>0.05.

Fig 3. cAMP stimulated-ABCA1 specific efflux decrease in J774 macrophages following CER-001 and HDL3 incubation.

J774 macrophages were incubated with cAMP to increase the ABCA1 expression and then CER-001 and HDL3 at 25 μg/ml were added in the cell culture medium and the specific cAMP cholesterol efflux was determined. apoA-I (25 μg/ml) was used as reference for specific ABCA1-cholesterol efflux in the experiment. ** p<0.01, ***p<0.001.