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N-acetylcysteine attenuates atherosclerosis progression in aging LDL receptor deficient mice with preserved M2 macrophages and increased CD146

  • Qingyi Zhu
    Affiliations
    Center for Precision Medicine and Division of Cardiovascular Medicine, Department of Medicine, University of Missouri School of Medicine, Columbia, MO, USA

    Department of Cardiology, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China
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  • Yichao Xiao
    Affiliations
    Center for Precision Medicine and Division of Cardiovascular Medicine, Department of Medicine, University of Missouri School of Medicine, Columbia, MO, USA
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  • Meng Jiang
    Affiliations
    Center for Precision Medicine and Division of Cardiovascular Medicine, Department of Medicine, University of Missouri School of Medicine, Columbia, MO, USA
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  • Xuanyou Liu
    Affiliations
    Center for Precision Medicine and Division of Cardiovascular Medicine, Department of Medicine, University of Missouri School of Medicine, Columbia, MO, USA
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  • Yuqi Cui
    Affiliations
    Center for Precision Medicine and Division of Cardiovascular Medicine, Department of Medicine, University of Missouri School of Medicine, Columbia, MO, USA
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  • Hong Hao
    Affiliations
    Center for Precision Medicine and Division of Cardiovascular Medicine, Department of Medicine, University of Missouri School of Medicine, Columbia, MO, USA
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  • Gregory C. Flaker
    Affiliations
    Center for Precision Medicine and Division of Cardiovascular Medicine, Department of Medicine, University of Missouri School of Medicine, Columbia, MO, USA
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  • Qiming Liu
    Affiliations
    Department of Cardiology, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China
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  • Shenghua Zhou
    Affiliations
    Department of Cardiology, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China
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  • Zhenguo Liu
    Correspondence
    Corresponding author. Division of Cardiovascular Medicine, Center for Precision Medicine, Department of Medicine, University of Missouri School of Medicine, 1 Hospital Drive, CE306, Columbia, MO, 65212, USA.
    Affiliations
    Center for Precision Medicine and Division of Cardiovascular Medicine, Department of Medicine, University of Missouri School of Medicine, Columbia, MO, USA
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Open AccessPublished:August 12, 2022DOI:https://doi.org/10.1016/j.atherosclerosis.2022.08.008

      Highlights

      • -Long-term NAC treatment attenuated atherosclerosis progression in aging LDLR-/- mice on a normal diet.
      • - NAC treatment did not reverse atherosclerotic lesions.
      • NAC treatment increased CD146 level in atherosclerotic lesions.
      • NAC treatment preserved M2 population and M2 polarization in aging LDLR−/− mice.
      • NAC treatment increased CD146 level in atherosclerotic lesions.

      Abstract

      Background and aims

      Inflammation and reactive oxygen species (ROS) are important to the pathogenesis of atherosclerosis. The effect of antioxidants on atherosclerosis is inconsistent, and sometimes controversial. We aimed to test the hypothesis that attenuation of atherosclerosis by N-acetylcysteine (NAC) depends on NAC treatment timing and duration.

      Methods

      Male LDL receptor deficient (LDLR−/−) mice were fed a normal diet (ND) and divided into controls (on ND for 24 months), models 1–2 (at age of 9 months, starting NAC treatment for 3 or 6 months), and model 3 (at age of 18 months, starting NAC treatment for 6 months). To determine if hyperlipidemia compromises NAC treatment outcome, mice were fed a high fat diet (HFD) starting at age of 6 weeks and treated with NAC starting at 9 months of age for 6 months.

      Results

      NAC treatment for 6 months, not for 3 months, significantly attenuated atherosclerosis progression, but did not reverse atherosclerotic lesions, in aging LDLR−/− mice on ND. NAC had no effect on atherosclerotic lesions in mice on HFD. NAC treatment significantly decreased aortic ROS production, and the levels of inflammatory cytokines in serum and aorta of aging LDLR−/− mice with increased CD146 level. Bone marrow transplantation study with GFP-positive bone marrow cells showed that NAC treatment preserved M2 population and M2 polarization in the aorta of LDLR−/− mice.

      Conclusions

      Early and adequate NAC treatment could effectively attenuate inflammation and atherosclerosis progression with preserved M2 population and increased CD146 level in aging LDLR−/− mice without extreme hyperlipidemia.

      Graphical abstract

      Keywords

      1. Introduction

      Atherosclerosis is a chronic inflammatory disease and one of the major causes of morbidity and mortality globally, despite aggressive risk stratifications including smoking cessation, optimal control of lipid, blood pressure, and diabetes [
      • Virani S.S.
      • Alonso A.
      • Aparicio H.J.
      • et al.
      Heart disease and stroke statistics-2021 update: a report from the American heart association.
      ]. Therapies with antioxidants including vitamin E and C or β-carotene failed to achieve significant clinical benefits in patients with cardiovascular diseases (CVD) including atherosclerosis [
      • Bjelakovic G.
      • Nikolova D.
      • Gluud L.L.
      • et al.
      Antioxidant Supplements for Prevention of Mortality in Healthy Participants and Patients with Various Diseases.
      ]. New therapies including interleukin-1β monoclonal antibody canakinumab and anti-inflammatory drug colchicine could significantly reduce the rate of major adverse cardiovascular events (MACE) including nonfatal myocardial infarction, nonfatal stroke, and cardiovascular death [
      • Thompson P.L.
      • Nidorf S.M.
      Colchicine: an affordable anti-inflammatory agent for atherosclerosis.
      ,
      • Ridker P.M.
      • Everett B.M.
      • Thuren T.
      • et al.
      Antiinflammatory therapy with canakinumab for atherosclerotic disease.
      ,
      • Nidorf S.M.
      • Fiolet A.T.L.
      • Mosterd A.
      • et al.
      Colchicine in patients with chronic coronary disease.
      ]. However, canakinumab therapy did not decrease all-cause mortality, and was associated with a significant increase in the incidence of fatal infection (including sepsis) [
      • Ridker P.M.
      • Everett B.M.
      • Thuren T.
      • et al.
      Antiinflammatory therapy with canakinumab for atherosclerotic disease.
      ], and high cost. While colchicine is a generic drug and decreases the rate of MACE, colchicine therapy is associated with a significant increase in death from non-cardiovascular causes [
      • Nidorf S.M.
      • Fiolet A.T.L.
      • Mosterd A.
      • et al.
      Colchicine in patients with chronic coronary disease.
      ]. Thus, alternative options are needed to attenuate atherosclerosis.
      N-acetylcysteine (NAC) has been traditionally considered an antioxidant although it is more like an anti-inflammatory agent and could effectively attenuate reactive oxygen species (ROS) production and reduce inflammation [
      • Park J.H.
      • Kang S.S.
      • Kim J.Y.
      • et al.
      The antioxidant N-acetylcysteine inhibits inflammatory and apoptotic processes in human conjunctival epithelial cells in a high-glucose environment.
      ]. NAC treatment delays cellular senescence of endothelial cells from atherosclerotic patients [
      • Voghel G.
      • Thorin-Trescases N.
      • Farhat N.
      • et al.
      Chronic treatment with N-acetyl-cystein delays cellular senescence in endothelial cells isolated from a subgroup of atherosclerotic patients.
      ], and improves coronary and peripheral endothelium-dependent vasodilation in human subjects with or without atherosclerosis [
      • Andrews N.P.
      • Prasad A.
      • Quyyumi A.A.
      N-acetylcysteine improves coronary and peripheral vascular function.
      ]. NAC treatment also inhibits foam cell formation induced by oxidized low-density lipoprotein (LDL) and suppresses matrix-degrading capacity of foam cells [
      • Sung H.J.
      • Kim J.
      • Kim Y.
      • et al.
      N-acetyl cysteine suppresses the foam cell formation that is induced by oxidized low density lipoprotein via regulation of gene expression.
      ]. In young apolipoprotein E-deficient (apoE−/−) mice [
      • Shimada K.
      • Murayama T.
      • Yokode M.
      • et al.
      N-acetylcysteine reduces the severity of atherosclerosis in apolipoprotein E-deficient mice by reducing superoxide production.
      ,
      • Ivanovski O.
      • Szumilak D.
      • Nguyen-Khoa T.
      • et al.
      The antioxidant N-acetylcysteine prevents accelerated atherosclerosis in uremic apolipoprotein E knockout mice.
      ] and LDL receptor deficient (LDLR−/−) mice [
      • Cui Y.
      • Narasimhulu C.A.
      • Liu L.
      • et al.
      N-acetylcysteine inhibits in vivo oxidation of native low-density lipoprotein.
      ,
      • Song G.
      • Zong C.
      • Zhang Z.
      • et al.
      Molecular hydrogen stabilizes atherosclerotic plaque in low-density lipoprotein receptor-knockout mice.
      ] on an atherogenic high fat diet (HFD), NAC significantly decreases the progression of atherosclerosis. However, it is unclear if NAC could attenuate the progression of atherosclerosis or reverse the course of atherosclerosis in aging mice. This is an important question since atherosclerosis and related coronary artery disease (CAD) significantly contribute to the morbidity and mortality in the elderly [
      • Virani S.S.
      • Alonso A.
      • Aparicio H.J.
      • et al.
      Heart disease and stroke statistics-2021 update: a report from the American heart association.
      ].
      Macrophages are critical to the pathogenesis of atherosclerosis [
      • Yuan X.M.
      • Ward L.J.
      • Forssell C.
      • et al.
      Carotid atheroma from men has significantly higher levels of inflammation and iron metabolism enabled by macrophages.
      ], and an increase in macrophage polarization to proinflammatory macrophages (M1) or a decrease in anti-inflammatory macrophages (M2) plays an important role in inflammation and atherosclerotic progression [
      • Park K.
      • Li Q.
      • Evcimen N.D.
      • et al.
      Exogenous insulin infusion can decrease atherosclerosis in diabetic rodents by improving lipids, inflammation, and endothelial function.
      ]. The present study was to test the hypothesis that attenuation of atherosclerosis progression by NAC depends on the timing and duration of NAC treatment in aging LDLR−/− mice in association with preserved M2 macrophage polarization. The objectives were: 1) to demonstrate the time-dependent effect of NAC on atherosclerosis progression by defining the optimal starting time and duration for NAC treatment in aging LDLR−/− mice; 2) to determine if atherosclerotic lesions could be reversed with NAC treatment in aging LDLR−/− mice; 3) to define the impact of HFD on NAC therapeutic outcome on atherosclerosis; and 4) to evaluate the effect of NAC on ROS production and inflammation, as well as macrophage population and polarization in aorta.

      2. Materials and methods

      2.1 Animal model and study design

      All animal experiments were performed in accordance with the “Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health”. The experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Missouri School of Medicine, Columbia, Missouri, USA (Protocol Number 10118). Male LDLR−/− and age-matched wild-type (WT) C57BL/6 mice (from Jackson Laboratory) were fed a normal diet (ND), and randomly divided into experimental groups with 5–12 mice in each group to determine the optimal starting time and duration of NAC treatment: controls: mice on ND for 24 months; models 1 and 2: at age of 9 months, starting NAC treatment for 3 and 6 months, respectively; model 3: at age of 18 months, starting NAC treatment for 6 months.
      To evaluate the effect of hyperlipidemia on the outcome of NAC treatment, LDLR−/− mice were fed a HFD (TD.88,137 from Harlan-Teklad, USA, containing 17% anhydrous milk fat, 0.2% cholesterol with the total fat of 21% by weight, and 42% kcal from fat), starting at age of 6 weeks, and treated with NAC starting at 9 months of age for 3 or 6 months, with age-matched LDLR−/− mice on a HFD without NAC treatment as control. All animals were housed in a temperature-controlled facility with free access to water. Mice in the NAC treatment groups received NAC (1 mg/ml in drinking water) for 3 or 6 months. By the end of each time point, mice were weighed and sacrificed. Blood and aorta were collected for serum lipid analysis, ROS production and cytokine measurements. Aortas were processed for atherosclerotic lesion analysis as described [
      • Andres-Manzano M.J.
      • Andres V.
      • Dorado B.
      Oil red O and hematoxylin and eosin staining for quantification of atherosclerosis burden in mouse aorta and aortic root.
      ]. Another 3 groups of mice (12 months of age) including WT mice, LDLR−/− mice with a ND treated with NAC (LDLR−/− + NAC) or without NAC (LDLR−/−) for 3 months, were used for measurements and analysis of inflammatory gene and anti-inflammatory gene expressions, and macrophages.

      2.2 Lipid measurement

      Serum lipid profiles including total cholesterol (TC), high-density lipoprotein cholesterol (HDL), LDL cholesterol (LDL) and triglyceride (TRG) levels in each mouse were determined using an Alere Cholestech LDX® Analyzer following manufacturer's instructions.

      2.3 Quantification of atherosclerotic lesions

      After perfusion with sterile phosphate-buffered saline (PBS), whole aortas were dissected from the aortic valve to the site 5 mm after the iliac artery bifurcation. Both left and right carotid arteries were included in the preparation. The aortic and carotid arterial preparations were fixed with 4% paraformaldehyde for at least 48h at 4 °C. After removing adventitial fat under a dissection microscope, the preparations underwent Oil-Red-O staining as described [
      • Andres-Manzano M.J.
      • Andres V.
      • Dorado B.
      Oil red O and hematoxylin and eosin staining for quantification of atherosclerosis burden in mouse aorta and aortic root.
      ]. The lesions of aortic root in the 12 month old and 15 month old mice were also quantitatively analyzed as described [
      • Andres-Manzano M.J.
      • Andres V.
      • Dorado B.
      Oil red O and hematoxylin and eosin staining for quantification of atherosclerosis burden in mouse aorta and aortic root.
      ]. Lesions of aortic root in the 24 month old mice on a ND and mice on a HFD were not analyzed since the aortic lesions were extensive and could adequately reflect the overall burden of atherosclerotic lesions. The lesions were recorded with a digital camera and analyzed using Image J software that converted the staining areas into quantitative data.

      2.4 qRT-PCR

      Total RNA was obtained using Trizol preparation agents (Sigma-Aldrich) from the aorta of each mouse and prepared using RNeasy columns (Qiagen) with DNase I treatment (Thermo Scientific) for qRT-PCR analysis. cDNA was synthesized with 1 μg of total RNA in each sample using RevertAid RT Kit (Thermo Scientific). qRT-PCR was performed using Applied Biosystems QuanStudio™ 3 Real-Time PCR System with the primers listed in Supplementary Table 1.

      2.5 ELISA

      The levels of serum monocyte chemoattractant protein-1 (MCP-1), C-reactive protein (CRP), interleukin 6 (IL-6), IL-1β, and TNF-α were determined using ELISA kits as per manufacturer's protocol (R&D systems, MN).

      2.6 Immunofluorescence (IF) staining

      For macrophages, CD146, and α-smooth muscle actin (α-SMA) staining, the aortic rings and roots were collected and prepared for cryostat sectioning. Aortic root cryostat cross sections (7 μm) were made within 48 h after tissue collection with the chamber temperature of −21 °C as described [
      • Kumar A.
      • Accorsi A.
      • Rhee Y.
      • et al.
      Do's and don'ts in the preparation of muscle cryosections for histological analysis.
      ]. IF staining of the preparations was performed by incubation with anti-α-SMA-FITC antibody (#F-3777 from Sigma, dilution factor of 1:500), anti-CD146-AF647 antibody (#134718 from Biolegend, dilution factor of 1:200), anti-CD80 antibody (#66406-1-Ig from Proteintech, dilution factor of 1:100), anti-cleaved-Caspase 3 antibody (#AF7022 from Affinity Biosciences, dilution factor of 1:400), anti-F4/80 antibody (#ab100790 from Abcam, dilution factor of 1:100), anti-CD206-AF647 antibody (#141712 from Biolegend, dilution factor of 1:2000, and DAPI (#D1306 from Invitrogen, dilution factor of 1:100), followed by anti-mouse second antibody AF488 (#S0017 from Affinity Biosciences) at a dilution factor of 1:500, anti-rabbit second antibody AF647 (#S0013 from Affinity Biosciences) at a dilution factor of 1:500, and anti-rabbit second antibody AF594 (#A11072 from Invitrogen) at a dilution factor of 1:200. The preparations were examined with a Leica TCP SP8 confocal microscope. Both fluorescence intensity and cell counts were used to quantify the population of M2 macrophage. F4/80 positive cells were counted as monocytes/macrophages, and F4/80- and CD206-double-positive cells as M2 macrophages. The macrophages and M2 macrophages were counted from 4 random fields for each section with 3 sections for each mouse, and the average cell numbers for each mouse were used for analysis (total cell number and the M2% of the total F4/80 positive cells).

      2.7 Flow cytometry analysis of macrophages in aorta

      Cells were prepared from the aorta of aging mice for multicolor flow cytometric analysis of M1 and M2 macrophages in aorta after elimination of red blood cells (RBC) with RBC lysis buffer using LSR Fortessa™ X-20 (BD Bioscience, CA, USA). Cells in aorta were obtained and prepared using enzyme digestion for flow cytometry analysis for macrophages and M1 and M2 as described [
      • Gjurich B.N.
      • Taghavie-Moghadam P.L.
      • Galkina E.V.
      Flow cytometric analysis of immune cells within murine aorta.
      ]. Living cells were gated using yellow LIVE/DEAD™ Fixable Dead Cell Stain Kits (1:1,000, Invitrogen, Carlsbad, CA, US). CD45-positive cells were identified as leukocytes. F4/80-positive cells were defined as monocytes/macrophages in the aorta. M1 macrophages were identified as F4/80+/CD80+/CD206- cells, and M2 as F4/80+/CD80-/CD206+ cells. Anti-F4/80 AF488 (#123120, dilution factor of 1:125), and anti-CD206 PE (#141706, dilution factor of 1:125) were from Biolegend (San Diego, CA, U.S). Anti-CD80 BV786 (#740888, at 1:125) was from BD Pharmingen (San Jose, CA, US). For analysis of GFP-positive macrophages (see below), anti-F4/80 AF594 (#123140, dilution factor of 1:125) was used since anti-F4/80 AF488 shares the same color as GFP. Data analysis was performed using FlowJo software.

      2.8 ROS detection

      ROS level in the cryostat aortic cross sections was measured with a confocal microscopy using dihydroethidium (DHE) dye as described [
      • Lau Y.S.
      • Tian X.Y.
      • Mustafa M.R.
      • et al.
      Boldine improves endothelial function in diabetic db/db mice through inhibition of angiotensin II-mediated BMP4-oxidative stress cascade.
      ]. Adipose tissue was removed from the aorta under a dissection microscope as described [
      • Mohanta S.
      • Yin C.
      • Weber C.
      • et al.
      Aorta atherosclerosis lesion analysis in hyperlipidemic mice.
      ]. A 6 mm-long aortic tissue distal to the bifurcation of the subclavian artery was obtained from each mouse. Aortic cryostat cross sections were prepared as described [
      • Kumar A.
      • Accorsi A.
      • Rhee Y.
      • et al.
      Do's and don'ts in the preparation of muscle cryosections for histological analysis.
      ], and incubated with DHE (5 μmol/L, Invitrogen, Carlsbad, CA, USA) at 37 °C for 15 min in normal saline. After washing with normal saline, the preparations were examined using a laser scanning confocal microscope (Leica TCP SP8, Leica Microsystems). The fluorescence intensity was measured with 515 nm excitation and 585 nm emission, and the images were analyzed using Image J software. To detect DNA damages from oxidative stress in the lesions, the aortic cryostat cross sections were incubated with 8-oxoguanine antibody (dilution factor of 1:200, #GTX41980 from GeneTex) at 37 °C for 1.5 h, and followed by anti-HRP second antibody and diaminobenzidine (to show the brown color).

      2.9 Bone marrow transplantation

      GFP-positive bone marrow (BM) cells were prepared from mice with global GFP expression and resuspended in PBS with 2% FBS (2 × 106 cells/100μl) for BM transplant (BMT). The recipient mice (12-week male WT C57BL/6 mice and age-matched LDLR−/− mice) were given 4 × 106 GFP-positive BM cells in a volume of 0.2 ml via tail vein after sub-lethal irradiation with 11 Gy as described [
      • Flomerfelt F.A.
      • Gress R.E.
      Bone marrow and fetal liver radiation chimeras.
      ]. BMT efficiency was evaluated with flow cytometry 4 weeks after BMT, and the LDLR−/− mice were treated with NAC (1 mg/ml in drinking water) for 1 month after BM repopulation with GFP-positive BM cells (4 weeks after BMT).

      2.10 Statistics analysis

      The data were expressed as mean ± standard error (X±SE) and analyzed using two-independent-sample t-test (two-sided) for two groups of data or one way ANOVA (analysis of variance) followed by post hoc LSD test or Dunnett's test for three or more groups of data. Pearson's correlation coefficient was used to evaluate the association between individual serum cytokine level and the burden of aortic atherosclerosis. SPSS 20.0 statistical software was used for the data analysis. A p < 0.05 was considered statistically significant.

      3. Results

      3.1 NAC treatment time-dependently attenuated atherosclerosis progression, but did not reverse atherosclerotic lesions

      Long-term NAC treatment for up to 6 months had no significant effect on body weight in LDLR−/−mice fed either ND or HFD. As expected, mice on an HFD had a significantly higher body weight than those on a ND (Supplementary Table 2). Total cholesterol (TC), LDL, and triglyceride (TRG) levels in LDLR−/− mice on either ND or HFD were all significantly higher than that in age-matched WT mice. NAC treatment did not significantly modify the serum lipid profiles, except TRG levels, in LDLR−/− mice on HFD that was significantly decreased with NAC treatment (Supplementary Table 3).
      Visible atherosclerotic plaques were present in the aorta at 9 month old LDLR−/− mice on an ND with the total lesion area of 6.55 ± 1.25% of aorta, while there was no atherosclerotic lesion in age-matched WT mice as expected (Fig. 1A). The lesion area was significantly increased to 15.36 ± 2.07% and 21.93 ± 1.80% in LDLR−/− mice on an ND at 12 months and 15 months of age, respectively (Fig. 1B and C). When LDLR−/− mice on an ND were treated continuously with NAC starting at age 9 months, there was no change in atherosclerotic lesion area after 3 months of treatment (Fig. 1B). However, a significant reduction in lesion area was observed after 6 months of NAC treatment (15.21 ± 2.15% vs 21.93 ± 1.80%, p = 0.026, Fig. 1C). There was no difference in lesion area in mice with NAC treatment between age 12 months and 15 months. When starting to treat the mice at age 18 months, however, 6 months of NAC treatment had no significant effect on the progression of atherosclerotic lesions (LDLR−/− 29.93 ± 2.20% vs LDLR−/− + NAC 32.95 ± 2.12%, p = 0.701, Fig. 1D). When LDLR−/− mice were a HFD, a significant amount of atherosclerotic plaque was present in aorta at 9 months of age in LDLR−/− mice with the total lesion area of 68.75 ± 3.23% of the whole inner surface of aorta. NAC treatment for 6 months starting at age 9 months had no significant effect on aortic atherosclerotic lesion progression (HFD: 87.57 ± 1.24% vs HFD + NAC: 88.24 ± 3.25%, p = 0.852, Fig. 1E).
      Fig. 1
      Fig. 1NAC treatment time-dependently attenuated atherosclerosis progression, but did not reverse atherosclerotic lesions.
      Oil-red-O-staining showed that aortic atherosclerotic plaques were present in LDLR−/− mice on ND at 9 months of age, while no atherosclerotic lesion was present in age-matched WT mice (A). The lesion area was significantly increased in LDLR−/− mice on ND at 12 months (B) and 15 months of age (C). When treated with NAC starting at age 9 months, there was no change in atherosclerotic lesion area after 3 months of treatment (B). A significant reduction in lesion area was observed after 6 months of NAC treatment (C). When starting at age 18 months, a 6-month NAC treatment had no significant effect on atherosclerotic lesion progression (D). In LDLR−/− mice on HFD, NAC treatment for 6 months starting at age 9 months had no effect on atherosclerotic lesion progression (E). Representative images of cross-sections and quantification of atherosclerotic lesions in aortic roots are shown in F, G and H. There was no difference in the lesion area in aortic root cross sections between LDLR−/− mice with or without 3-month NAC treatment when started at 9 months of age. However, a 6-month NAC treatment significantly attenuated the atherosclerotic progression with reduced lesion area. Immuno-histological analysis showed a significant reduction of α-SMA level in the aorta of aging LDLR−/− mice on ND after 3 months of NAC treatment. However, NAC treatment had no effect on the level of α-SMA in the aorta of mice at the age of 15 months (I and J). CD146 level was significantly increased in the aortic root cross sections in aging LDLR−/− mice on ND after 3 months and 6 months of NAC treatment (K and L). M: month; NAC: N-acetylcysteine; LDLR−/−: LDLR−/− mice; LDLR−/− + NAC: LDLR−/− mice treated with NAC. Data are presented as mean ± SE. *p < 0.05, n = 5–12. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
      Analysis of the atherosclerotic lesions in aortic root cross sections using Oil-Red-O staining also showed that NAC treatment for 6 months, not for 3 months, significantly attenuated the progression of atherosclerosis in LDLR−/− mice on ND. There was no difference in the average lesion area in the aortic cross sections between LDLR−/− mice with or without 3 months of NAC treatment when started at 9 months of age (LDLR−/− 0.027 ± 0.016 mm2 vs LDLR−/− + NAC 0.033 ± 0.012 mm2, p = 0.779, n = 6, Fig. 1F and G). Similarly, the percentage of lesion area in the aortic root cross sections was the same for LDLR−/− mice on ND with or without 3 months of NAC treatment starting at 9 months of age (Fig. 1H). However, NAC treatment for 6 months starting at 9 months of age significantly attenuated the atherosclerotic progression with reduced lesion area (LDLR−/− 0.145 ± 0.031 mm2 vs LDLR−/− + NAC 0.063 ± 0.016 mm2, p = 0.042, Fig. 1F), and the percentage of lesion size (LDLR−/− 35.60 ± 6.10% vs. LDLR−/− + NAC 17.27 ± 3.58%, p = 0.027, n = 6, Fig. 1H) in the aortic root cross sections in mice on ND when starting NAC treatment at age of 9 months.
      Immuno-histological analysis showed a significant amount of α-SMA was present in the aorta of aging LDLR−/− mice on ND that was significantly increased after 3 months of NAC treatment. However, there was no significant difference in the level of α-SMA in the aorta in mice at age 15 months with or without NAC treatment (Fig. 1I and J). CD146 was detectable in the aortic root cross sections in aging LDLR−/− mice on ND that was substantially increased after 3 months and 6 months of NAC treatment (Fig. 1K and L).

      3.2 NAC treatment effectively prevented ROS production in aorta of aging LDLR−/− mice

      To determine the potential role of ROS production in the progression of aortic atherosclerotic lesions, aortic ROS level was evaluated. DHE assay showed that aortic ROS level was significantly increased in aging LDLR−/− mice at 9 months, 12 months, and 15 months of age on ND compared with age-matched WT mice. NAC treatment for either 3 months or 6 months effectively blocked the increase in ROS production in the aorta of aging LDLR−/− mice (Fig. 2A). Immuno-histological analysis showed that 8-oxoguanine level was also significantly increased in the aorta of aging LDLR−/− mice on ND as compared with age-matched WT control. NAC treatment effectively attenuated the level of 8-oxoguanine in the aorta of aging LDLR−/− mice (Fig. 2B).
      Fig. 2
      Fig. 2NAC treatment effectively prevented ROS production in the aorta of aging LDLR−/− mice.
      Representative fluorescent images and quantification of ROS formation in the aorta are shown for each group. DHE staining showed that aortic ROS level was significantly increased in aging LDLR−/− mice on ND compared with age-matched WT mice. NAC treatment for 3 months or 6 months effectively blocked the increase in ROS production in the aorta of aging LDLR−/− mice. Of note, no data was available for the group with NAC treatment at the time point of 9 months since this time point was the baseline before NAC treatment (A). Immuno-histological analysis showed that 8-oxoguanine level was also significantly increased in the aorta of aging LDLR−/− mice on ND as compared with the age-matched WT control. NAC treatment effectively suppressed the level of 8-oxoguanine in the aorta of aging LDLR−/− mice (B). DHE: dihydroethidium staining; BF: bare field; M: month; NAC: N-acetylcysteine; WT: wild-type mice; LDLR−/−: LDLR−/− mice; LDLR−/− + NAC: LDLR−/− mice treated with NAC. Data are presented as mean ± SE, n = 6.

      3.3 NAC treatment significantly decreased inflammatory cytokines in serum and aorta of aging LDLR−/− mice

      ELISA analysis showed that the levels of serum CRP, MCP-1 and IL-6 were significantly increased in LDLR−/− mice on ND at the age of 15 months compared with age-matched WT mice. At the age of 12 months, only the levels of serum MCP-1 and IL-6 were significantly increased in LDLR−/− mice over age-matched WT mice. NAC treatment effectively prevented the increase of serum CRP, IL-6, and MCP-1 in LDLR−/− mice at the age of 15 months (with 6 months of NAC treatment). However, NAC treatment only prevented the rise of serum MCP-1 levels at the age of 12 months (with 3 months of NAC treatment) (Fig. 3A–C). Interestingly, the levels of serum IL-1β and TNF-α were not increased significantly in LDLR−/− mice at the age of 12 months and 15 months compared to age-matched WT mice, although there was a trend of increase in their serum levels. NAC treatment had no significant effect on serum IL-1β and TNF-α levels, although there was a trend of reduction in their serum levels after 6 months of NAC treatment (Supplementary Figs. 1A and B). Pearson's correlation coefficient showed that there was a significant correlation between aortic atherosclerotic lesion area and serum levels of CRP and IL-6 in LDLR--/ mice, while no correlation between lesion size and serum MCP-1 levels (Fig. 3A–C).
      Fig. 3
      Fig. 3NAC treatment significantly decreased inflammatory cytokines in serum and aorta of aging LDLR−/− mice.
      ELISA analysis showed that the levels of serum CRP, MCP-1 and IL-6 were significantly increased in LDLR−/− mice on D at the age of 15 months compared with age-matched WT mice. At the age of 12 months, only the levels of serum MCP-1 and IL-6 were significantly increased in LDLR−/− mice over age-matched WT mice. NAC treatment for 6 months prevented the increase in serum CRP (A), IL-6 (B), and MCP-1 (C) in LDLR−/− mice at the age of 15 months, while 3 months of NAC treatment only prevented the rise of serum MCP-1 levels (C). There was a positive correlation between atherosclerotic lesion area and serum levels of CRP (A) and IL-6 (B) in LDLR−/− mice, while no correlation between lesion size and serum MCP-1 levels (C). qRT-PCR analysis demonstrated that the expression levels of TNF- α, IL-6 and MCP-1 in the aorta were significantly increased in LDLR−/− mice at the age 12 months on ND compared with age-matched WT mice, and this was blocked with 3 months of NAC treatment. Expression of IL-10 was significantly decreased in the aorta of aging LDLR−/− mice on ND compared with age-matched WT mice. NAC treatment substantially enhanced IL-10 expression in the aorta of aging LDLR−/− mice (D). M: month; WT: wild-type mice; NAC: N-acetylcysteine; LDLR−/−: LDLR−/− mice; LDLR−/− + NAC: LDLR−/− mice treated with NAC. Data are presented as mean ± SE, n = 8–10.
      qRT-PCR analysis demonstrated that the expression levels of inflammatory cytokines Tnf-α, Il-6, and Mcp-1 in the aorta were significantly increased in LDLR−/− mice at the age 12 months on ND compared with age-matched WT mice and they were all effectively blocked with 3 months of NAC treatment. On the other hand, expression of anti-inflammatory cytokine Il-10 was significantly decreased in the aorta of aging LDLR−/− mice on ND compared with age-matched WT mice. NAC treatment substantially enhanced Il-10 expression in the aorta of aging LDLR−/− mice (Fig. 3D).

      3.4 NAC treatment preserved M2 population in aorta of aging LDLR−/− mice

      Macrophage populations in the aorta of aging mice were evaluated using flow cytometry analysis. To avoid high auto-fluorescent background and non-specific staining, a viability dye was used, and only single cells were gated. Leukocytes in the preparations were identified as CD45 positive cells (Supplementary Fig. 2). There was no difference in the M1 population (defined as F4/80+/CD80+/CD206- cells) between WT mice and age-matched LDLR−/− mice without or with NAC treatment (Fig. 4B). However, the M2 population (F4/80+/CD80-/CD206+) in the aorta was significantly decreased in aging LDLR−/− mice on ND compared with age-matched WT mice and was effectively reversed with NAC treatment (Fig. 4C). Decreased M2 population was also observed using immunostaining of F4/80-AF594+ and CD206-AF647+ in the cross sections of aortic root of aging LDLR−/− mice (Fig. 4D and G). The percentage of average M2 cells by cell counts was significantly higher in 12-month-old LDLR−/− mice treated with NAC for 3 months than those without NAC treatment (Fig. 4E and F). The immunofluorescence intensity for M2 in the aorta was also significantly higher in aging LDLR−/− mice with NAC treatment than without NAC treatment (Fig. 4G). There were minimal amounts of macrophages in the aorta of WT mice (data not shown).
      Fig. 4
      Fig. 4NAC treatment preserved M2 population in the aorta of aging LDLR−/− mice.
      Macrophage populations in the aorta of aging mice were evaluated using flow cytometry analysis using CD80 and CD206 (A). There was no difference in M1 population (F4/80+/CD80+/CD206- cells) between WT and LDLR−/− mice without or with NAC treatment (B). M2 population (F4/80+/CD80-/CD206+) in the aorta was significantly decreased in aging LDLR−/− mice (12 months of age) on ND compared with age-matched WT mice, and this was effectively reversed with 3 months of NAC treatment (C). Similar findings on M2 population in the aorta of aging LDLR−/− mice were observed using immunostaining of F4/80-AF594+/CD206-AF647+ (D–F). Immuno-histological analysis of the aortic root cross sections of aging LDLR−/− mice also showed that CD80 level remained the same with or without NAC treatment (H and I). However, NAC treatment significantly decreased the overall level of caspase 3 in the aorta of aging LDLR−/− mice on ND (H and J). No significant difference in caspase-3 level was observed in CD80-positive cells (H and K). WT: wild-type mice; NAC: N-acetylcysteine; LDLR−/−: LDLR−/− mice; LDLR−/− + NAC: LDLR−/− mice treated with NAC. Data are presented as mean ± SE, n = 6–10.
      Immuno-histological analysis of the aortic root cross sections of aging LDLR−/− mice (12 months old) also showed that CD80 level was the same in the aorta of mice with or without NAC treatment (Fig. 4H and I). However, NAC treatment significantly decreased the overall level of caspase-3 in the aorta of aging LDLR−/− mice on a ND (Fig. H and J). Interestingly, there was no significant difference in caspase-3 level in CD80-positive cells in aorta (Fig. 4H and K).

      3.5 NAC treatment decreased M1/M2 ratio during macrophage polarization in aorta of LDLR−/− mice

      Bone marrow (BM)-derived monocytes significantly contribute to macrophage population and atherosclerotic plaque formation in aorta [
      • Moore K.J.
      • Sheedy F.J.
      • Fisher E.A.
      Macrophages in atherosclerosis: a dynamic balance.
      ]. To determine if NAC treatment could attenuate monocyte accumulation and macrophage polarization in the aorta of LDLR−/− mice, BMT with GFP-positive BM cells was performed in LDLR−/− mice with and without NAC treatment and in age-matched WT mice. At 2 weeks after BMT, over 84% of cells in peripheral blood were GFP-positive (Supplementary Fig. 3A), confirming successful BMT with GFP-positive BM cells. GFP-positive monocytes/macrophages in aorta were analyzed using flow-cytometry (Supplementary Fig. 3B). GFP-positive cells were selected first, and CD45/F4/80-double positive cells were defined as monocytes/macrophages in aorta. CD80+/CD206--cells were defined as M1 and CD80-/CD206+-cells as M2 (Fig. 5A). GFP-positive macrophage population in the aorta was significantly increased (almost doubled) in aging LDLR−/− mice on ND compared with the age-matched WT mice. There was no difference in the GFP-positive macrophage population in the aorta between NAC-treated aging LDLR−/− mice and those without NAC treatment (Fig. 5B). M1/M2 ratio of GFP-positive macrophages in the aorta was also significantly increased in LDLR−/− mice on ND compared with age-matched WT mice. NAC treatment effectively prevented the increase of the M1/M2 ratio in the aorta of LDLR−/− mice (Fig. 5C).
      Fig. 5
      Fig. 5NAC treatment decreased M1/M2 ratio during macrophage polarization in the aorta of LDLR−/− mice.
      Flow-cytometry analysis showed that, using bone marrow transplantation with GFP-positive bone marrow cells, CD80+/CD206--cells were defined as M1 and CD80-/CD206+-cells as M2 (A). The population of GFP-positive macrophage (CD45+/F4/80+) in aorta was significantly increased in aging LDLR−/− mice (12 months of age) on ND compared with the age-matched WT mice. No difference in GFP-positive macrophage population in aorta was observed between NAC-treated aging LDLR−/− mice and the ones without NAC treatment (B). The M1/M2 ratio of GFP-positive macrophages in the aorta was significantly increased in LDLR−/− mice compared with age-matched WT mice. NAC treatment for 3 months effectively prevented the increase of the M1/M2 ratio in LDLR−/− mice (C). WT: wild-type mice; NAC: N-acetylcysteine; LDLR−/−: LDLR−/− mice; LDLR−/− + NAC: LDLR−/− mice treated with NAC. Data are presented as mean ± SE, n = 8–10.

      4. Discussion

      ROS and oxidative stress are critical to the development and progression of atherosclerosis [
      • Yang X.
      • Li Y.
      • Li Y.
      • et al.
      Oxidative stress-mediated atherosclerosis: mechanisms and therapies.
      ,
      • Kattoor A.J.
      • Pothineni N.V.K.
      • Palagiri D.
      • et al.
      Oxidative stress in atherosclerosis.
      ]. Many cardiovascular risk factors, including hyperlipidemia, hypertension, diabetes mellitus, obesity, and smoking, are associated with enhanced ROS generation [
      • Forstermann U.
      • Xia N.
      • Li H.
      Roles of vascular oxidative stress and nitric oxide in the pathogenesis of atherosclerosis.
      ]. However, despite strong pre-clinical evidence showing significant protective effects of antioxidants against atherosclerosis [
      • Ivanovski O.
      • Szumilak D.
      • Nguyen-Khoa T.
      • et al.
      The antioxidant N-acetylcysteine prevents accelerated atherosclerosis in uremic apolipoprotein E knockout mice.
      ,
      • Song G.
      • Zong C.
      • Zhang Z.
      • et al.
      Molecular hydrogen stabilizes atherosclerotic plaque in low-density lipoprotein receptor-knockout mice.
      ], most clinical trials with antioxidants failed to achieve significant clinical benefits (and may be harmful) in patients with cardiovascular diseases (CVD) especially atherosclerosis [
      • Bjelakovic G.
      • Nikolova D.
      • Gluud L.L.
      • et al.
      Antioxidant Supplements for Prevention of Mortality in Healthy Participants and Patients with Various Diseases.
      ]. In the present study, we demonstrated that: 1) long-term NAC treatment (6 months), not short-term (3 months), significantly attenuated atherosclerosis progression, but did not reverse atherosclerotic lesion, in aging LDLR−/− mice on ND; 2) long-term NAC treatment (6 months) had no effect on atherosclerosis progression in aging LDLR−/− mice on HFD; 3) NAC treatment had no beneficial effect on atherosclerosis progression in aging LDLR−/− mice on ND when advanced atherosclerotic lesions were present; 4) NAC treatment significantly decreased aortic ROS production, and inflammatory cytokines in serum and aortas of aging LDLR−/− mice; and 5) NAC treatment preserved M2 population and M2 polarization and increased the level of CD146 in the aorta of aging LDLR−/− mice.
      Atherosclerosis in human subjects is a dynamic process involving the progression of early lesions to advanced plaques. Early atherosclerotic lesions in coronary arteries usually start in the second decade of life in humans [
      • Lusis A.J.
      • Atherosclerosis
      ], and slowly progress to the stage of clinical significance in the 40s and 50s [
      • Otsuka F.
      • Kramer M.C.
      • Woudstra P.
      • et al.
      Natural progression of atherosclerosis from pathologic intimal thickening to late fibroatheroma in human coronary arteries: a pathology study.
      ]. It is challenging to study the pathogenesis of atherosclerosis since the commonly used laboratory mice do not develop atherosclerosis under normal conditions. There are currently two mouse models that develop spontaneous atherosclerosis: apolipoprotein E deficient (apoE−/−) mice and LDLR−/− mice [
      • Oppi S.
      • Luscher T.F.
      • Stein S.
      Mouse models for atherosclerosis research-which is my line?.
      ]. Visible atherosclerotic lesions could be present at the ages of 10–12 weeks in ApoE−/− mice on regular diet, and at 3–4 weeks in LDLR−/− mice on HFD with severely elevated serum lipid levels [
      • Emini Veseli B.
      • Perrotta P.
      • De Meyer
      GRA
      Animal models of atherosclerosis.
      ]. Thus, the courses of atherosclerotic lesion development in these two models are not compatible with that in human subjects. However, aging LDLR−/− mice (9 months and older) without HFD could develop spontaneous atherosclerosis with serum LDL levels (about 200–300 mg/dl, Supplementary Table 3), similar to atherosclerotic patients. In the present study, we observed that significant atherosclerotic lesions occurred at 9 months of age in LDLR−/− mice on ND (equivalent to middle-aged humans of about 38–47 years). Thus, the aging LDLR−/− mouse model with ND was comparable with the course of development and progression of atherosclerosis in human subjects.
      In the present study, we observed that NAC treatment significantly attenuated the progression of atherosclerosis in aging LDLR−/− mice only when mice were treated continuously for 6 months and on ND, and not effective when the treatment duration was 3 months or on HFD. NAC treatment could not reverse pre-existing atherosclerotic lesions. The data also showed that 6 months of NAC treatment had no significant effect on serum lipid profiles except TG level that was decreased with NAC treatment. In a previous study, we observed that NAC treatment significantly decreased atherosclerotic lesion burden in young LDLR−/− mice (6–8 weeks) on HFD [
      • Cui Y.
      • Narasimhulu C.A.
      • Liu L.
      • et al.
      N-acetylcysteine inhibits in vivo oxidation of native low-density lipoprotein.
      ]. These data suggest that NAC treatment on atherosclerosis could be effective in aging LDLR−/− mice only under specific conditions: early treatment with adequate treatment duration without extremely high lipid levels. The findings from the present study may provide an explanation for the disappointing outcomes on clinical studies with antioxidants since most of the study patients are over 50 years old with advanced atherosclerotic lesions [
      • Kattoor A.J.
      • Pothineni N.V.K.
      • Palagiri D.
      • et al.
      Oxidative stress in atherosclerosis.
      ]. Thus, a randomized study with the appropriate study population with age of 35–50 years is needed to confirm the findings from animal studies.
      Although the exact mechanisms for the development and progression of atherosclerosis are complex and not fully defined, inflammation and related oxidative stress play a critical role in the pathogenesis of atherosclerosis. The levels of inflammatory cytokines including TNF-α, IL-1β, and IL-6 are significantly increased in hyperlipidemic states [
      • Tahir A.
      • Martinez P.J.
      • Ahmad F.
      • et al.
      An evaluation of lipid profile and pro-inflammatory cytokines as determinants of cardiovascular disease in those with diabetes: a study on a Mexican American cohort.
      ]. These cytokines could activate local and systemic inflammatory responses with increased ROS production and oxidative stress and accelerate the development of atherosclerosis [
      • Galkina E.
      • Ley K.
      Immune and inflammatory mechanisms of atherosclerosis.
      ,
      • Zhang L.
      • Connelly J.J.
      • Peppel K.
      • et al.
      Aging-related atherosclerosis is exacerbated by arterial expression of tumor necrosis factor receptor-1: evidence from mouse models and human association studies.
      ]. Transcription of TNF-α, MCP-1 and IL-6 in the aorta was decreased, with significant reduction of their mRNA levels with NAC treatment in aging LDLR−/− mice. Interestingly, NAC treatment significantly decreased the levels of serum IL-6 and CRP in aging LDLR−/− mice only when treated for 6 months, and reduced serum MCP-1 level after treatment for 3 months or longer. One of the interesting findings in the present study was that there was a significant positive correlation between serum levels of IL-6 and CRP and aortic atherosclerotic burden. It is known that both IL-6 and CRP are established markers of systemic vascular inflammation and atherothrombosis [
      • Bermudez E.A.
      • Rifai N.
      • Buring J.
      • Manson J.E.
      • Ridker P.M.
      Interrelationships among circulating interleukin-6, C-reactive protein, and traditional cardiovascular risk factors in women.
      ]. IL-6 plays an important role in inflammation and immune response, and induces the hepatic synthesis of CRP [
      • Tanaka T.
      • Narazaki M.
      • Kishimoto T.
      IL-6 in inflammation, immunity, and disease.
      ]. IL-6 is associated with atherosclerosis in both animal studies and human subjects [
      • Huber S.A.
      • Sakkinen P.
      • Conze D.
      • Hardin N.
      • Tracy R.
      Interleukin-6 exacerbates early atherosclerosis in mice.
      ,
      • Akita K.
      • Isoda K.
      • Sato-Okabayashi Y.
      • et al.
      An interleukin-6 receptor antibody suppresses atherosclerosis in atherogenic mice.
      ,
      • Lindmark E.
      • Diderholm E.
      • Wallentin L.
      • Siegbahn A.
      Relationship between interleukin 6 and mortality in patients with unstable coronary artery disease: effects of an early invasive or noninvasive strategy.
      ], and has been considered an important therapeutic target for inflammation and atherosclerosis and related coronary artery disease [
      • Swerdlow D.I.
      • Holmes M.V.
      • Kuchenbaecker K.B.
      • et al.
      The interleukin-6 receptor as a target for prevention of coronary heart disease: a mendelian randomisation analysis.
      ,
      • Cai T.
      • Zhang Y.
      • Ho Y.-L.
      • et al.
      Association of interleukin 6 receptor variant with cardiovascular disease. Effects of interleukin 6 receptor blocking therapy: a phenome-wide association study.
      ,
      • Ridker P.M.
      • Devalaraja M.
      • Baeres F.M.M.
      • et al.
      RESCUE Investigators: IL-6 inhibition with ziltivekimab in patients at high atherosclerotic risk (RESCUE): a double-blind, randomised, placebo-controlled, phase 2 trial.
      ]. Oral NAC treatment has been shown to decrease serum levels of CRP and IL-6 in patients with end-stage renal disease or rheumatoid arthritis [
      • Saddadi F.
      • Alatab S.
      • Pasha F.
      • Ganji M.R.
      • Soleimanian T.
      The effect of treatment with N-acetylcysteine on the serum levels of C-reactive protein and interleukin-6 in patients on hemodialysis.
      ,
      • Hashemi G.
      • Mirjalili M.
      • Basiri Z.
      • et al.
      A pilot study to evaluate the effects of oral N-acetyl cysteine on inflammatory and oxidative stress biomarkers in rheumatoid arthritis.
      ]. Large randomized clinical studies are needed to determine if NAC could decrease serum levels of IL-6 and CRP in patients with atherosclerosis and potential mechanism(s).
      Macrophages are one of the important sources for inflammatory cytokines [
      • Arango Duque G.
      • Descoteaux A.
      Macrophage cytokines: involvement in immunity and infectious diseases.
      ]. M1 is considered pro-inflammatory and M2 anti-inflammatory [
      • Park K.
      • Li Q.
      • Evcimen N.D.
      • et al.
      Exogenous insulin infusion can decrease atherosclerosis in diabetic rodents by improving lipids, inflammation, and endothelial function.
      ], thus the M1/M2 ratio is an important determinant for the direction of inflammatory response [
      • Cole S.L.
      • Dunning J.
      • Kok W.L.
      • et al.
      M1-like monocytes are a major immunological determinant of severity in previously healthy adults with life-threatening influenza.
      ]. Macrophages are also important in the development and progression of atherosclerosis and the stability of atherosclerotic plaques [
      • Cochain C.
      • Zernecke A.
      Macrophages in vascular inflammation and atherosclerosis.
      ]. The data from the present study indeed showed that pro-inflammatory cytokines (CRP, MCP-1, IL-6) were significantly increased, while the anti-inflammatory cytokine IL-10 was significantly decreased in aging LDLR−/− mice in association with significantly increased ROS production and an increased M1/M2 ratio largely due to the decreased M2 population in the aorta. BMT studies showed that the increased M1/M2 ratio in the aorta of aging LDLR−/− mice was predominantly due to decreased M2 polarization. NAC treatment effectively reversed the changes in the expressions of pro-inflammatory and anti-inflammatory cytokines, ROS production, and macrophage polarization in the aorta of aging LDLR−/− mice. Interestingly, NAC treatment has no effect on the migration of monocytes from circulation into aorta in aging LDLR−/− mice nor on the M1 population in the aorta. It is unclear why the M1 population was not significantly changed in the aorta of aging LDLR−/− mice. The data from the present study showed that CD80 level remained the same in mice with and without NAC treatment. Although NAC treatment significantly decreased the overall level of caspase-3 in the aortic lesions of aging LDLR−/− mice, the caspase-3 level was not changed in CD80-positive cells in mice with NAC treatment, suggesting that NAC might have minimal effect on M1 macrophages. It is very important and interesting to define the mechanism(s) on the selective effect of NAC on M2 macrophages in future studies.
      One of the interesting findings in the present study was that NAC treatment significantly increased the level of CD146 in the aortic atherosclerotic lesions. CD146 is an adhesion molecule with a molecular weight of 113 kDa and is expressed in many types of cells including vascular endothelial cells, smooth muscle cells, pericytes, lymphocytes, some cancer cells, and intraplaque macrophages [
      • Shih I.M.
      The role of CD146 (Mel-CAM) in biology and pathology.
      ,
      • Ouhtit A.
      • Gaur R.L.
      • Abd Elmageed Z.Y.
      • Fernando A.
      • et al.
      Towards understanding the mode of action of the multifaceted cell adhesion receptor CD146.
      ]. The role of CD146 in the development and progression of atherosclerosis has been under investigation. Early studies have revealed that CD146 expression is significantly increased within human atherosclerotic lesions, and the soluble form of CD146 is considered as a biomarker of subclinical atherosclerosis [
      • Qian Y.N.
      • Luo Y.T.
      • Duan H.X.
      • et al.
      Adhesion molecule CD146 and its soluble form correlate well with carotid atherosclerosis and plaque instability.
      ,
      • Dogansen S.C.
      • Helvaci A.
      • Adas M.
      • et al.
      The relationship between early atherosclerosis and endothelial dysfunction in type 1 diabetic patients as evidenced by measurement of carotid intima-media thickness and soluble CD146 levels: a cross sectional study.
      ]. Another study has demonstrated that CD146 is involved in the formation of macrophage foam cells and their retention within the atherosclerotic plaque, thus potentially contributing to the development of atherosclerosis [
      • Luo Y.
      • Duan H.
      • Qian Y.
      • et al.
      Macrophagic CD146 promotes foam cell formation and retention during atherosclerosis.
      ]. However, a recent study using CD146-and ApoE double-deficient mouse model has shown that CD146 deficiency enhances the formation of atherosclerotic plaque possibly through the inhibition of p38-MAPK-mediated signaling pathway(s) [
      • Blin M.G.
      • Bachelier R.
      • Fallague K.
      • et al.
      CD146 deficiency promotes plaque formation in a mouse model of atherosclerosis by enhancing RANTES secretion and leukocyte recruitment.
      ]. In the present study, we observed that CD146 level was significantly increased in the aortic lesions of aging LDLR−/− mice with NAC treatment. Further studies are needed to determine if an increased expression of CD146 could be a critical mechanism for the beneficial effect of NAC on the progression of atherosclerosis using CD146 deficient mouse model.
      NAC has been an established drug since the 1960s and is on the World Health Organization's List of 40 “Essential Medicines” and is available as an inexpensive generic drug. NAC readily enters cells, and the sulfhydryl group within NAC molecule directly scavenges ROS, modulates the cellular redox state both intracellularly and extracellularly, regulates cytokine synthesis (anti/pro-inflammatory effect), and inhibits some important inflammation-related signaling pathways (like NF-kB) [
      • Salamon S.
      • Kramar B.
      • Marolt T.P.
      • et al.
      Medical and dietary uses of N-acetylcysteine.
      ], leading to reduced ROS production and a decreased level of oxidative stress. It is known that native LDL itself is not atherogenic and becomes atherogenic when converted to oxidized LDL [
      • Parthasarathy S.
      • Litvinov D.
      • Selvarajan K.
      • et al.
      Lipid peroxidation and decomposition--conflicting roles in plaque vulnerability and stability.
      ]. NAC has been shown to block the in vitro biotransformation of native LDL to oxidized LDL, and attenuate in vivo oxidation of native LDL and ROS formation from oxidized LDL [
      • Cui Y.
      • Narasimhulu C.A.
      • Liu L.
      • et al.
      N-acetylcysteine inhibits in vivo oxidation of native low-density lipoprotein.
      ]. Thus, NAC could decrease the progression of atherosclerotic lesions through multiple mechanisms including (but not limited to) suppression of pro-inflammatory cytokine production, preservation of M2 polarization, inhibition of LDL biotransformation to oxidized LDL, and direct ROS scavenging.
      Major limitations: 1) only male mice were used in the study; 2) no detailed mechanisms on how NAC could reduce inflammatory cytokines were investigated; 3) no data to determine if similar findings from the mouse model could be present in human subjects; 4) only one time point data on the mRNA levels of cytokines in the aorta and no serum IL-10 levels were available due to limited availability of tissue samples; 5) no quantitative analysis was conducted for foam cells in the atherosclerotic lesions; and 6) no detailed mechanisms of how NAC treatment could preserve M2 population and increase the level of CD146 in the mouse model is showed.

      4.1 Conclusions

      We demonstrated that a 6-month, not 3-month, NAC treatment significantly decreased the progression of atherosclerosis in aging LDLR−/− mice on ND in association with decreased pre-inflammatory cytokine production and preserved M2 polarization, as well as increased CD146 level. However, NAC treatment could not reverse pre-existing atherosclerotic lesion, nor attenuate the progression of atherosclerosis in aging LDLR−/− mice on a HFD. These data suggest that the timing and duration of NAC treatment, as well as serum lipid level and disease stage, are critical factors for NAC therapeutic outcomes on atherosclerosis. NAC treatment was only effective when applied early over a long period of time, with a reasonable control of serum lipid level.

      Financial support

      This work was partially supported by US NIH grants to ZL ( HL124122 and HL094650 ).

      CRediT authorship contribution statement

      Qingyi Zhu: Formal analysis, Investigation, Data curation, Writing – original draft. Yichao Xiao: Investigation, Software. Meng Jiang: Investigation. Xuanyou Liu: Investigation, Data curation. Yuqi Cui: Project administration. Hong Hao: Validation. Gregory C. Flaker: Writing – review & editing. Qiming Liu: Resources. Shenghua Zhou: Supervision. Zhenguo Liu: Conceptualization, Methodology, Supervision, Writing – review & editing, Funding acquisition.

      Declaration of interests

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

      Appendix A. Supplementary data

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