Fig 2: Endothelial dysfunction was investigated in rabbits by vascular ultrasound in vivo by studying the flow-induced vascular reactivity by infusion of a high volume of saline. The effect of Ad-ABCG1-GFP and of control adenovirus (Ad-GFP) in atherosclerotic rabbits was compared to the effect of Ad-GFP in healthy control rabbits. The relative change in diameter of the left carotid artery compared to baseline (%) is demonstrated after 1–2 min (directly after infusion) and 10 min after infusion. *indicates P<0.05 compared to Ad-GFP-ABCG1. The means ±SEM of at least 6 animals are shown.

Fig 3: Neutrophil Abca1/g1 deficiency does not affect NETosis in atherosclerotic plaques of Ldlr-/- mice. Ldlr-/- mice were transplanted with bone marrow (BM) from Abca1fl/flAbcg1fl/fl (control) or S100A8CreAbca1fl/flAbcg1fl/fl (S100A8-Cre-Abcdko) mice and fed Western-type diet (WTD) for 8 weeks. (A–B) Ly6G+ neutrophils (A) and Ly6G-CD11b+ monocytes (B) were isolated from BM and Abca1 and Abcg1 mRNA expression was assessed. n = 5. *P < 0.05, by t-test. (C) Mice were sacrificed and hearts were isolated, sectioned, and neutrophils were stained in atherosclerotic lesions of the aortic root using myeloperoxidase (MPO). Lesions were also stained for citrullinated histones 2, 8, and 17 (3HCit). NETs show an overlap of MPO and 3HCit staining. Representative pictures are shown. Scale bars: 25 µm.

Fig 4: miR-33b reduces cellular cholesterol efflux and serum HDL-C levels.(a). Western blotting for ABCA1 and ABCG1 proteins in peritoneal macrophages from WT and KI+/+ mice. Representative images are shown. ß-actin was used as the loading control. (b). Cholesterol efflux to apoA-I and HDL-C in peritoneal macrophages from WT and KI+/+ mice (n = 6 each). Values are mean ± s.e.m. ***p < 0.001 (c). Mean plots of HPLC analysis for serum cholesterol in WT and KI+/+ mice (n = 4 and 5, respectively).

Fig 5: Effect of the carboxyl-terminal deletion mutant apoA-I[?(185–243)] bound to rHDL particles on ABCG1-mediated cholesterol efflux from J774 mouse macrophages following treatment with AICAR.J774 mouse macrophages were labeled with [14C]cholesterol (A) or [14C]cholesterol and 30 µg/ml acLDL (B) for 24 h and then incubated with 1 mM AICAR for 24 h. At the end of this incubation period the cells were incubated with rHDL containing WT apoA-I or apoA-I[?(185–243)], at a concentration of 1 µM apoA-I, for 4 h. The net AICAR-induced (ABCG1-mediated) [14C]cholesterol efflux is calculated as the difference in percent of cholesterol efflux between untreated and AICAR-treated cells. Values are the means ± SD from three independent experiments performed in duplicate. **, p < 0.01 vs WT apoA-I; ***, p < 0.001 vs WT apoA-I. (C) Western blot analysis, performed as described in “Materials and Methods”, showing the increase in ABCG1 protein levels after AICAR treatment of J774 macrophages. ABCG1 protein levels of HEK293 cells transfected with empty vector (mock) or with ABCG1-expressing plasmid are also shown for comparison.