Advanced Glycated apoA-IV Loses Its Ability to Prevent the LPS-Induced Reduction in Cholesterol Efflux-Related Gene Expression in Macrophages

Abstract

We addressed how advanced glycation (AGE) affects the ability of apoA-IV to impair inflammation and restore the expression of genes involved in cholesterol efflux in lipopolysaccharide- (LPS-) treated macrophages. Recombinant human apoA-IV was nonenzymatically glycated by incubation with glycolaldehyde (GAD), incubated with cholesterol-loaded bone marrow-derived macrophages (BMDMs), and then stimulated with LPS prior to measurement of proinflammatory cytokines by ELISA. Genes involved in cholesterol efflux were quantified by RT-qPCR, and cholesterol efflux was measured by liquid scintillation counting. Carboxymethyllysine (CML) and pyrraline (PYR) levels, determined by Liquid Chromatography-Mass Spectrometry (LC-MS/MS), were greater in AGE-modified apoA-IV (AGE-apoA-IV) compared to unmodified-apoA-IV. AGE-apoA-IV inhibited expression of interleukin 6 (Il6), TNF-alpha (Tnf), IL-1 beta (Il1b), toll-like receptor 4 (Tlr4), tumor necrosis factor receptor-associated factor 6 (Traf6), Janus kinase 2/signal transducer and activator of transcription 3 (Jak2/Stat3), nuclear factor kappa B (Nfkb), and AGE receptor 1 (Ddost) as well as IL-6 and TNF-alpha secretion. AGE-apoA-IV alone did not change cholesterol efflux or ABCA-1 levels but was unable to restore the LPS-induced reduction in expression of Abca1 and Abcg1. AGE-apoA-IV inhibited inflammation but lost its ability to counteract the LPS-induced changes in expression of genes involved in macrophage cholesterol efflux that may contribute to atherosclerosis.

Figure 1

Pyrraline (PYR) and carboxymethyllysine (CML) content in ApoA-IV submitted to in vitro advanced glycation. ApoA-IV was incubated for 24 h, at 37°C with different concentrations of glycolaldehyde (GAD). After digestion, samples were analyzed by LC-MS/MS (n = 9). One-way ANOVA with Tukey's post-test was utilized to compare groups (data from 3 independent experiments; mean ± SD; ^∗p < 0.0001).

Figure 2

TNF-alpha and IL-6 secretion and Tnf, Il6, Il1b, and Ccl2 mRNA expression by macrophages treated with unmodified or AGE-apoA-IV and further stimulated with LPS. Bone marrow-derived macrophages (BMDMs) were overloaded with acetylated LDL (50 μg/mL) and incubated with unmodified or AGE-apoA-IV (n = 9). Control incubations were kept in DMEM/FAFA alone. After washing, macrophages were stimulated for 24 h with 1 μg/mL of LPS. TNF-alpha and IL-6 secretion were quantified by ELISA (a and b). Gene expression (c–f) was determined by RT-qPCR as described in Material and Methods section. Data are representative of 3 independent experiments (mean ± SD; ^∗p < 0.05).

Figure 3

Tlr4, Traf6, Myd88, Nfkb, Rela, Jak2, and Stat3 expression by macrophages treated with unmodified or AGE-apoA-IV and further stimulated with LPS. Bone marrow-derived macrophages (BMDMs) were overloaded with acetylated LDL (50 μg/mL) and incubated with unmodified or AGE-apoA-IV. After washing, macrophages were stimulated for 24 h with 1 μg/mL of LPS (n = 9). Gene expression was determined by RT-qPCR as described in Material and Methods section. Data are representative of 3 independent experiments (mean ± SD, ^∗p < 0.05).

Figure 4

Ager and Ddost expression by macrophages treated with unmodified or AGE-apoA-IV and further stimulated with LPS. Bone marrow-derived macrophages (BMDMs) were overloaded with acetylated LDL (50 μg/mL) and incubated with unmodified or AGE-apoA-IV. After washing, macrophages were stimulated for 24 h with 1 μg/mL of LPS (n = 9). Gene expression was determined by RT-qPCR as described in Material and Methods section. Data are representative of 3 independent experiments (mean ± SD, ^∗p < 0.05).

Figure 5

ABCA-1 expression and ¹⁴C-cholesterol efflux in macrophages treated with unmodified and AGE-apoA-IV (a). Bone marrow-derived macrophages (BMDM) were overloaded with acetylated LDL (50 μg/mL) and incubated with unmodified or AGE-apoA-IV for 48 h. After washing, macrophages were stimulated for 24 h with 1 μg/mL of LPS. The ABCA-1 protein level was determined by immunoblot (n = 3) (b). BMDMs were overloaded with acetylated LDL (50 μg/mL) and ¹⁴C-cholesterol for 48 h. After maintenance in equilibrium medium, cells were incubated with unmodified or AGE-apoA-IV as cholesterol acceptors (n = 6). Cholesterol efflux was calculated as ¹⁴C-cholesterol in medium/¹⁴C-cholesterol in medium +¹⁴C-cholesterol in cells × 100. Basal efflux was determined in incubations with DMEM containing fatty acid-free albumin that was subtracted from those with apoA-IV. Data are representative of 3 independent experiments (mean ± SD) and were compared by the Student t test.

Figure 6

Abca1, Abcg1, Scarb1, Nr1h3, and Nr1h2 mRNA expression in macrophages treated with unmodified or AGE-apoA-IV and further stimulated with LPS. Bone marrow-derived macrophages (BMDMs) were overloaded with acetylated LDL (50 μg/mL) and incubated with unmodified or AGE-apoA-IV. After washing, macrophages were stimulated for 24 h with 1 μg/mL of LPS (n = 9). Gene expression was determined by RT-qPCR as described in Material and Methods section. Data are representative of 3 independent experiments (mean ± SD, ^∗p < 0.05) and were compared by ANOVA and Tukey's post-test.