Nicotinamide Prevents Apolipoprotein B-Containing Lipoprotein Oxidation, Inflammation and Atherosclerosis in Apolipoprotein E-Deficient Mice

Abstract

The potential of nicotinamide (NAM) to prevent atherosclerosis has not yet been examined. This study investigated the effect of NAM supplementation on the development of atherosclerosis in a mouse model of the disease. The development of aortic atherosclerosis was significantly reduced (NAM low dose: 45%; NAM high dose: 55%) in NAM-treated, apolipoprotein (Apo)E-deficient mice challenged with a Western diet for 4 weeks. NAM administration significantly increased (1.8-fold) the plasma concentration of proatherogenic ApoB-containing lipoproteins in NAM high-dose (HD)-treated mice compared with untreated mice. However, isolated ApoB-containing lipoproteins from NAM HD mice were less prone to oxidation than those of untreated mice. This result was consistent with the decreased (1.5-fold) concentration of oxidized low-density lipoproteins in this group. Immunohistochemical staining of aortas from NAM-treated mice showed significantly increased levels of IL-10 (NAM low-dose (LD): 1.3-fold; NAM HD: 1.2-fold), concomitant with a significant decrease in the relative expression of TNFα (NAM LD: -44%; NAM HD: -57%). An improved anti-inflammatory pattern was reproduced in macrophages cultured in the presence of NAM. Thus, dietary NAM supplementation in ApoE-deficient mice prevented the development of atherosclerosis and improved protection against ApoB-containing lipoprotein oxidation and aortic inflammation.

Keywords: ATP-binding cassette (ABC) transporters; cardiovascular disease; cytokine; macrophage; niacinamide; vitamin B3.

Figure 1

Analysis of proximal aortic lesions in apolipoprotein (Apo)E-deficient mice treated with different doses of NAM. (a) Representative images of aortic atherosclerotic lesion in 12-week-old mice challenged to a western diet and NAM over 1 month at 2 months of age. (b) Area of proximal aortic lesion quantified from 8 mice per group. Data are expressed as the median (interquartile range) of four consecutive sections throughout the aortic sinus that were obtained every 20 µm when aortic valves became visible. Statistically significant differences among groups for each variable were determined using a nonparametric a Kruskal–Wallis test followed by Dunn’s posttest. Differences were considered significant when p < 0.05. Specifically, * p < 0.05 vs. Untreated group. Abbreviations used: NAM LD, low-dose, NAM-treated mice; NAM HD, high-dose, NAM-treated mice.

Figure 2

Antioxidant effects of NAM on ApoE-deficient mice. Two-month-old male ApoE-deficient mice were challenged with a Western diet and NAM for 1 month. (a) Representative diene formation curves of non-HDL particles. (b) Median diene formation lag time calculated from the oxidation curves (n = 3–4 plasma pools/group). (c) Serum oxLDL concentration (.n = 8 per group). (d) Oxidation curves of human LDL incubated at NAM concentration 0.10 mM and 1.0 mM. This experiment was replicated twice with similar outcomes. Oxidation kinetics were carried using plasma lipoproteins isolated by sequential ultracentrifugation from pools of 2–3 mouse plasmas of each experimental group, panels (a,b), or pooled human plasma, panel (d), as appropriate. In panels (b,c), data are expressed as medians (interquartile ranges). Statistically significant differences among groups for each variable were determined using a nonparametric a Kruskal–Wallis test followed by Dunn’s posttest. Differences were considered significant when p < 0.05. Specifically, * p < 0.05 vs. Untreated group or † p < 0.05 vs. NAM LD-treated mice. Abbreviations used: NAM LD, low-dose, NAM-treated mice; NAM HD, high-dose, NAM-treated mice; OD, optical density.

Figure 3

Anti-inflammatory effects of NAM on ApoE-deficient mice and cultured macrophages. Two-month-old male ApoE-deficient mice were challenged with a Western diet and NAM for 1 month. (a) Plasma concentration of cytokines (TNFα, IL-6, IL-10, and IL-4) in ApoE-deficient mice (n = 5–6). (b) Relative aortic mRNA levels of inflammatory targets. (c) Correlation between the area of proximal aortic lesions and circulating IL-10 levels. (d) Correlation between the area of proximal aortic lesions and Il10 aortic mRNA levels. (e) Immunohistochemical (IHC) analysis of TNFα and IL-10 levels in the aorta. Left panel, bar chart showing the relative (arbitrary units) expression of TNFα and IL-10 in the aortas from different groups (n = 5–6 mice per group). Right panel, representative images of immunohistochemical staining for IL-10 and TNFα in thoracoabdominal aortas. (f) Relative mRNA levels of cytokines assessed in LPS-activated J774A.1 macrophages exposed to different doses of NAM for 24 h. Data are expressed as the median (interquartile range) of 4 independent experiments. In panels (a,b,e), data are presented as medians (interquartile ranges) (n = 5–6 samples/group) and from 4 independent experiments in panel F. Statistically significant differences among groups for each variable were determined using a nonparametric a Kruskal–Wallis test followed by Dunn’s posttest. Differences were considered significant when p < 0.05. Specifically, * p < 0.05 vs. Untreated group or † p < 0.05 vs. NAM HD-treated mice. In panels (c,d), the relationship between parameters was tested using a nonparametric Spearman’s correlation test. Mice of all groups were considered for analysis. Abbreviations used: LPS, Lipopolysaccharide; NAM, nicotinamide; NAM LD, low-dose, NAM-treated mice; NAM HD, high-dose, NAM-treated mice.