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.