Increased Systemic and Plaque Inflammation in ABCA1 Mutation Carriers With Attenuation by Statins

Abstract

Objective: We previously demonstrated that subjects with functional ATP-binding cassette (ABC) A1 mutations have increased atherosclerosis, which has been attributed to the role of ABCA1 in reverse cholesterol transport. More recently, a proinflammatory effect of Abca1 deficiency was shown in mice, potentially contributing to atherogenesis. In this study, we investigated whether ABCA1 deficiency was associated with proinflammatory changes in humans.

Approach and results: Thirty-one heterozygous, 5 homozygous ABCA1 mutation carriers, and 21 matched controls were studied. (18)Fluorodeoxyglucose positron emission tomography with computed tomographic scanning was performed in a subset of carriers and controls to assess arterial wall inflammation (target:background ratio). Heterozygous ABCA1 mutation carriers had a 20% higher target:background ratio than in controls (target:background ratio; P=0.008). In carriers using statins (n=7), target:background ratio was 21% reduced than in nonstatin users (n=7; P=0.03). We then measured plasma cytokine levels. Tumor necrosis factor α, monocyte chemoattractant protein-1, and interleukin-6 levels were increased in heterozygous and homozygous ABCA1 mutation carriers. We isolated monocytes from carriers and controls and measured inflammatory gene expression. Only TNFα mRNA was increased in monocytes from heterozygous ABCA1 mutation carriers. Additional studies in THP-1 macrophages showed that both ABCA1 deficiency and lipoprotein-deficient plasma from ABCA1 mutation carriers increased inflammatory gene expression.

Conclusions: Our data suggest a proinflammatory state in ABCA1 mutation carriers as reflected by an increased positron emission tomography-MRI signal in nonstatin using subjects, and increased circulating cytokines. The increased inflammation in ABCA1 mutation carriers seems to be attenuated by statins.

Keywords: ABCA1; HDL; cytokines; dyslipidemia; genetics; lipids; positron-emission tomography.

Figure 1

Increased vessel wall inflammation in heterozygous ABCA1 mutation carriers. Vessel wall inflammation was assessed by PET-CT in controls (n=15), and heterozygous ABCA1 mutation carriers without (n=7) and with statin (n=7) treatment. TBR denotes target to background ratio. Data are presented as mean ± SEM. P-values are indicated.

Figure 2

Pro-inflammatory cytokines in plasma of controls, heterozygous and homozygous ABCA1 mutation carriers. Plasma was isolated from controls, heterozygous ABCA1 mutation carriers not using (ABCA1 het) or using a statin (ABCA1 het statin), and homozygous ABCA1 mutation carriers not using (ABCA1 hom) or using a statin (ABCA1 hom statin). Levels of pro-inflammatory cytokines were assessed using ELISA. A. Tumor Necrosis Factor α (TNFα). B. Monocyte Chemoattractant Protein-1 (MCP-1). C. Interleukin-6 (IL-6). In all graphs, each data point represents one patient or control. The mean is indicated. *P<0.05, **P<0.01, ***P<0.001.

Figure 3

Inflammatory cytokine expression in monocytes from controls and heterozygous ABCA1 mutation carriers. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from heterozygous ABCA1 mutation carriers or controls (n=6 per group). CD14+ monocytes were isolated from PBMCs using magnetic beads and mRNA expression for TNFα, IL-1β, MCP-1, and IL-6 was assessed, and corrected for the housekeeping gene cyclophilin. Data are presented as mean ± SEM. **P<0.01.

Figure 4

Effect of cellular ABCA1 deficiency on inflammatory cytokine expression. THP-1 macrophages were incubated with ABCA1 or scrambled siRNA. After 48 h, cells were treated o/n with or without rHDL. Macrophage RNA was isolated and the mRNA expression of TNFα (A), IL-1β (B), IL6 (C), and inducible nitric oxide synthase (iNOS) (D) was measured and corrected for the housekeeping gene cyclophilin. Data are presented as mean ± SEM. *P<0.05, ***P<0.001.

Figure 5

Effect of apoB-depleted plasma and lipoprotein deficient plasma from ABCA1 mutation carriers on macrophage inflammation. (A) ApoB was peg-precipitated from plasma and apoB-depleted plasma was incubated overnight with THP-1 macrophages (n=13 per group; n=4 for homozygous ABCA1 mutation carriers). (B) Plasma samples (n=10 per group; n=4 for homozygous ABCA1 mutation carriers) were pooled and lipoprotein deficient plasma (LPDS) was obtained after ultracentrifugation and incubated with THP-1 macrophages (n=4 per pool). mRNA expression was corrected for the housekeeping gene cyclophilin. Data are presented as mean ± SEM representing individual samples in panel A, and replicates of pooled samples in panel B. *P<0.05, **P<0.01, ***P<0.001.