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. 2012 Jun 22;111(1):37-49.
doi: 10.1161/CIRCRESAHA.112.269472. Epub 2012 May 24.

Severe hyperhomocysteinemia promotes bone marrow-derived and resident inflammatory monocyte differentiation and atherosclerosis in LDLr/CBS-deficient mice

Daqing Zhang  1 , Pu Fang, Xiaohua Jiang, Jun Nelson, Jodene K Moore, Warren D Kruger, Remus M Berretta, Steven R Houser, Xiaofeng Yang, Hong Wang
Affiliations

Severe hyperhomocysteinemia promotes bone marrow-derived and resident inflammatory monocyte differentiation and atherosclerosis in LDLr/CBS-deficient mice

Daqing Zhang et al. Circ Res. .

Abstract

Rationale: Hyperhomocysteinemia (HHcy) accelerates atherosclerosis and increases inflammatory monocytes (MC) in peripheral tissues. However, its causative role in atherosclerosis is not well established and its effect on vascular inflammation has not been studied. The underlying mechanism is unknown.

Objective: This study examined the causative role of HHcy in atherogenesis and its effect on inflammatory MC differentiation.

Methods and results: We generated a novel HHcy and hyperlipidemia mouse model, in which cystathionine β-synthase (CBS) and low-density lipoprotein receptor (LDLr) genes were deficient (Ldlr(-/-) Cbs(-/+)). Severe HHcy (plasma homocysteine (Hcy)=275 μmol/L) was induced by a high methionine diet containing sufficient basal levels of B vitamins. Plasma Hcy levels were lowered to 46 μmol/L from 244 μmol/L by vitamin supplementation, which elevated plasma folate levels. Bone marrow (BM)-derived cells were traced by the transplantation of BM cells from enhanced green fluorescent protein (EGFP) transgenic mice after sublethal irradiation of the recipient. HHcy accelerated atherosclerosis and promoted Ly6C(high) inflammatory MC differentiation of both BM and tissue origins in the aortas and peripheral tissues. It also elevated plasma levels of TNF-α, IL-6, and MCP-1; increased vessel wall MC accumulation; and increased macrophage maturation. Hcy-lowering therapy reversed HHcy-induced lesion formation, plasma cytokine increase, and blood and vessel inflammatory MC (Ly6C(high+middle)) accumulation. Plasma Hcy levels were positively correlated with plasma levels of proinflammatory cytokines. In primary mouse splenocytes, L-Hcy promoted rIFNγ-induced inflammatory MC differentiation, as well as increased TNF-α, IL-6, and superoxide anion production in inflammatory MC subsets. Antioxidants and folic acid reversed L-Hcy-induced inflammatory MC differentiation and oxidative stress in inflammatory MC subsets.

Conclusions: HHcy causes vessel wall inflammatory MC differentiation and macrophage maturation of both BM and tissue origins, leading to atherosclerosis via an oxidative stress-related mechanism.

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Conflict of interest statement

Conflict-of interest; none

Figures

Figure 1
Figure 1. HHcy increased atherosclerosis and BM-derived cells in the lesion and aortas of EGFPBM LDLr−/− CBS−/+ mice
A. Chimeric EGFPBM LDLr−/− CBS−/+ mice. Male Ldlr−/− Cbs−/+ mice were transplanted with BM cells from EGFP Tg mice immediately after semi-lethal dose of irradiation at 8 wks of age, fed an indicated diet at age of 14 wks for 8 wks, and sacrificed at age of 22 wks. B. Plasma levels of Hcy. C. Representative histograms depicting blood GFP+ cell-chimerism. Mouse blood were collected 6 wks after BMT and analyzed by flow cytometry for GFP frequency measurement. D. Photomicrographs of atherosclerotic lesions. Mouse aortic sinus cross sections were stained with oil-red O, counterstained with hematoxylin, and immunostained with MOMA-2 (MC/Mϕ marker), and DAPI (nuclear). Images were acquired by two-laser Nikon confocal microscopy. E. Quantitative analysis of lesions in the aortic sinuses. Atherosclerotic lesion area was defined as the neointimal region between the lumen and IEL. F. Representative histogram depicting vessel wall GFP+ cells. Pooled aortas were digested. Suspended cells were analyzed by flow cytometry for GFP frequency measurement. The shaded curve area represents cells from control mouse without BMT. G. Quantitative analysis of vessel wall GFP+ cells per aorta. Values represent Mean±SEM (n=7–12 mice), p values from independent t test, *p<0.05 vs HF+HM. HHcy, hyperhomocysteinemia; LDLr, LDL receptor; CBS, cystathionine β-synthase; MC, monocytes; Mϕ, macrophage; high fat; HM, high methionine; HV, high vitamin; IEL, internal elastic Lamina.
Figure 2
Figure 2. Severe HHcy increased total MC and inflammatory MC subsets in the blood, spleen and BM of LDLr−/− CBS−/+ mice
LDLr−/− CBS−/+ mice were fed an indicated diet at age of 14 wks for additional 8 wks, and sacrificed. Peripheral blood, spleen and BM cells were isolated, stained with anti-CD11b & -Ly-6C mAbs, and analyzed by flow cytometry. A. Representative dot plots depicting nucleated cells (gate i) and MNC cells (gate ii). B. Representative histogram and dot plots depicting MC identified as CD11b+MNC, and further divided into three subsets; Ly-6Chigh, Ly-6Cmiddle, and Ly-6Clow. C & D. Quantitative analysis of total MNC and MC (C) and MC subsets (D) in the blood, spleen and BM was shown in bar graphs. Values represent mean±SD (n=6), p values from independent t test. *p<0.05 vs HF+HM. HHcy, hyperhomocysteinemia; HF, high fat; HM, high methionine, HV: high vitamin. MNC, mononuclear cell; MC, monocyte; Low, Ly-6Clow subset; Mid, Ly-6Cmiddle subset; High, Ly-6Chigh subset.
Figure 3
Figure 3. HHcy promoted BM-derived and resident inflammatory MC differentiation and increased MC heterogeneity in the aorta of EGFPBM LDLr−/− CBS−/+ mice
Chimeric EGFPBM LDLr−/− CBS−/+ mice were generated as described in Fig 1A. Mouse aortas were digested with a protease cocktail and pooled (3–5 mice/sample). Suspended aortic cells were stained with violet to exclude dead cells, and then with mAbs against Ly6G (myeloid granulocyte maturation marker), CD11b (MC marker), F4/80 (mature Mϕ marker) and Ly6C (inflammatory MC marker), and assayed by flow cytometry. Two cellular populations, BM-derived (GFP+) and resident (GFP−) cells, were analyzed separately. A & B. Representative dot plots and quantification of aortic suspensions depicting Mϕ (gate i) and MC cells (gate ii). Ratio of Mϕ to MC indicates Mϕ maturation. C & D. Representative contour plots and quantification of MC subsets in the aorta: Both GFP− and GFP+ MC in gate ii were divided into 4 populations: R1, inflammatory MC (Ly-6Chigh+middle F4/80−); R2, differentiating MC (Ly-6Chigh+middle F4/80+); R3, differentiated Mϕ (Ly-6Clow F4/80+); R4, Ly-6Clow MC, respectively. Value represents Mean±SEM (n=10 mice), p values from independent t test, *p<0.05 vs HF+HM diet group. HHcy, hyperhomocysteinemia; HF, high fat; HM, high methionine; HV, high vitamin; MNC, mononuclear cell; MC, monocytes; Mϕ, macrophage.
Figure 4
Figure 4. HHcy-induced systemic inflammation was prevented by vitamin treatment in EGFPBM LDLr−/− CBS−/+ mice
A. Plasma pro-inflammatory cytokine analyses. Chimeric EGFPBM LDLr−/− CBS−/+ mice were generated as described in Fig 1A. Plasma IL-6, TNF-α, and MCP-1 levels were assessed by ELISA. B. Correlation analyses were performed between plasma cytokines and Hcy levels. One data dot represents data from a single mouse. n=9, *p<0.05 vs HF+HM diet, Values represent mean±SEM, p values from independent t test (A) and Spearman correlation analysis (B). HHcy, hyperhomocysteinemia; HF, high fat; HM, high methionine; HV, high vitamin; MC, monocytes; Mϕ, macrophage; MCP-1, monocyte chemoattractant protein-1.
Figure 5
Figure 5. L-Hcy promoted rIFNγ-induced inflammatory MC formation and pro-inflammatory cytokines production in primary mouse splenocytes
A. MC differentiation study. Splenocytes from WT mice were primed with rIFNγ at plating and treated with L-Hcy for 48 hr. Cells were stained with CD11b and Ly-6C mAbs and analyzed by flow cytometry. Gate ii cells are MNC. B & C. Representative dot plots and quantification of MC subsets. Gate ii MNC were selected. MC were defined as CD11b+ MNC and divided into Ly-6Chigh, Ly-6Cmiddle, and Ly-6Clow subsets. D & E. Representative dot plots and quantification of intracellular inflammatory cytokine analysis. Hcy treated mouse splenocytes were stained for surface markers with anti-CD11b (MC marker) and -Ly-6C (inflammatory MC marker) mAbs, and followed with intracellular cytokine staining by incubating with anti-TNF-α and -IL-6, or isotype-matched mAbs after permeablization. MC was divided into two subsets: CD11b+Ly-6Clow MC (Q1) and inflammatory MC CD11b+Ly-6Chigh+middle (Q2), and further each was divided into 3 groups (R1, R2, and R3) based on intracellular cytokine production. Values were normalized with that from isotype stain. Values represent mean±SEM, n=9, p values from independent t test,*p<0.05 vs rIFNγ control. Hcy, homocysteine, Cys, cysteine; MNC, mononuclear cells; MC, monocytes; Low, Ly-6Clow subset; Mid, Ly-6Cmiddle subset; High, Ly-6Chigh subset.
Figure 6
Figure 6. Folic Acid and antioxidant reagents prevented L-Hcy-induced inflammatory MC differentiation in primary mouse splenocytes
A. Splenocytes were primed with rIFNγ (100 U/ml) and treated with FA (100µM), antioxidants PEG-SOD plus PEG-CAT, or apocynin 1 hour before the exposure to L-Hcy (500 µM). Cells were stained with CD11b and Ly-6C mAbs and analyzed by flow cytometry analysis. MNC were selected by low granular content, as reflected in lower side-scatter light (SSC), and larger cell size, as reflected in higher forward scatter light (FSC). MC were defined as CD11b+ MNC and divided into Ly-6Chigh (R3), Ly-6Cmiddle (R2), and Ly-6Clow (R1) subsets. B. Representative dot plots of MC subset analysis. C. Quantification of MC subsets. Data are representative of 3 independent experiments, and expressed as mean±SEM, p values from 2 way ANOVA analysis, * p<0.05 vs no Hcy no inhibitor control; # p<0.05 vs Hcy no inhibitor control; MNC, mononuclear cell; MC, monocytes; Hcy, homocysteine; PEG, polyethylene glycol; FA, folic acid; SOD, superoxide dismutase; CAT, catalase.
Figure 7
Figure 7. Folic Acid and antioxidant reduced L-Hcy-induced superoxide anion production in inflammatory MC
A. Splenocytes were primed with rIFNγ (100 U/ml) and treated with FA (100µM), and antioxidants PEG-SOD (150U/ml) plus PEG-CAT (250U/ml), or apocynin (100µM), 1 hour before the exposure to L-Hcy (500 µM). Cells were stained with DHE and then CD11b and Ly-6C mAbs before flow cytometry analysis. MNCs were selected by low granular content, as reflected in lower side-scatter light (SSC), and larger cell size, as reflected in higher forward scatter light (FSC). MCs were defined as CD11b+ MNC and further divided into Ly-6C+ and Ly-6C− groups. B & C. Representative histograms of superoxide containing DHE+ CD11b+Ly6C+ and CD11b+Ly6C− MNC. D & E. Quantification of DHE+ MC analysis. Data are representative of 3 independent experiments, and expressed as mean±SEM, p values from 2 way ANOVA analysis, * p<0.05 vs no Hcy no inhibitor control; # p<0.05 vs Hcy no inhibitor control; MNC, mononuclear cell; MC, monocytes; Hcy, homocysteine; PEG, polyethylene glycol; FA, folic acid; SOD, superoxide dismutase; CAT, catalase.
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