Hyperhomocysteinemia promotes inflammatory monocyte generation and accelerates atherosclerosis in transgenic cystathionine beta-synthase-deficient mice

Abstract

Background: Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. Monocytes display inflammatory and resident subsets and commit to specific functions in atherogenesis. In this study, we examined the hypothesis that HHcy modulates monocyte heterogeneity and leads to atherosclerosis.

Methods and results: We established a novel atherosclerosis-susceptible mouse model with both severe HHcy and hypercholesterolemia in which the mouse cystathionine beta-synthase (CBS) and apolipoprotein E (apoE) genes are deficient and an inducible human CBS transgene is introduced to circumvent the neonatal lethality of the CBS deficiency (Tg-hCBS apoE(-/-) Cbs(-/-) mice). Severe HHcy accelerated atherosclerosis and inflammatory monocyte/macrophage accumulation in lesions and increased plasma tumor necrosis factor-alpha and monocyte chemoattractant protein-1 levels in Tg-hCBS apoE(-/-) Cbs(-/-) mice fed a high-fat diet. Furthermore, we characterized monocyte heterogeneity in Tg-hCBS apoE(-/-) Cbs(-/-) mice and another severe HHcy mouse model (Tg-S466L Cbs(-/-)) with a disease-relevant mutation (Tg-S466L) that lacks hyperlipidemia. HHcy increased monocyte population and selective expansion of inflammatory Ly-6C(hi) and Ly-6C(mid) monocyte subsets in blood, spleen, and bone marrow of Tg-S466L Cbs(-/-) and Tg-hCBS apoE(-/-) Cbs(-/-) mice. These changes were exacerbated in Tg-S466L Cbs(-/-) mice with aging. Addition of l-homocysteine (100 to 500 micromol/L), but not l-cysteine, maintained the Ly-6C(hi) subset and induced the Ly-6C(mid) subset in cultured mouse primary splenocytes. Homocysteine-induced differentiation of the Ly-6C(mid) subset was prevented by catalase plus superoxide dismutase and the NAD(P)H oxidase inhibitor apocynin.

Conclusions: HHcy promotes differentiation of inflammatory monocyte subsets and their accumulation in atherosclerotic lesions via NAD(P)H oxidase-mediated oxidant stress.

Figure 1. Plasma levels of Hcy and Met, and organ weight in transgenic CBS deficient mice

Tg-hCBSapoE^−/− Cbs mice were fed a HF diet at 8 weeks of age for an additional 8 weeks. The Tg-S466L Cbs and Tg-hCBS apoE^−/−Cbs mice were fed a rodent chow. A, B & C, Plasma levels of Hcy and Met. D, Body weight, E, F, G and H. heart, liver, spleen and kidney weights relative to tibia lengths, respectively. Values are mean±SD. P values from independent t test. n=10–12, Hcy, homocysteine; Met, methionine, HF, high fat.

Figure 2. HHcy accelerated atherosclerotic lesions, enhanced MC/Mφ and Ly-6C positive MC/Mφ accumulation in the lesion in Tg-hCBS apoE^−/− Cbs mice

Mice were fed a HF diet at 8 weeks of age for an additional 8 weeks. A. Photomicrographs of mouse aortic sinus cross sections stained with oil red O and counterstained with hematoxylin. B. Photomicrographs of mouse aortic sinus cross sections immunostained with MOMA-2 (MC/Mφ marker, green), Ly6C (red), DAPI (blue). Merge (yellow to orange) shows the accumulation of Ly-6C positive MC/Mφ in the lesion. C, D, E & F. Quantitative analysis of lesions in the aortic sinuses. Atherosclerotic lesion area was defined as the neointimal region between the lumen and IEL (C), MC/Mφ area (D), Ly-6C positive area (E), and Ly-6C positive MC/Mφ area (F). Correlation analysis of Hcy levels with lesion area, MC/Mφ area, Ly-6C positive area, and Ly-6C positive MC/Mφ area were shown in scattered dot graphs. Each data point represents one mouse. Values are mean ± SD, n=8–9. MC, monocytes; Mφ, macrophages; IEL, internal elastic lamina.

Figure 3. HHcy increased plasma TNF-α and MCP-1 levels in Tg-hCBS apoE^−/− Cbs mice

EDTA anticoagulant plasma was harvested from mice fed a HF diet at 8 weeks of age for an additional 8 weeks. TNF-α and MCP-1 levels in plasma were assessed by ELISA. Correlation analysis of Hcy levels with plasma TNF-α and MCP-1 levels were shown in scattered dot graphs. Each data point represents one mouse. Values are mean ± SD. P values from independent t test or correlation analysis. n=10–12.

Figure 4. HHcy increased monocyte population in peripheral blood, spleen and BM in transgenic Cbs mice independent of hyperlipidemia

Peripheral blood, spleen and BM cells were isolated from 6 and 8 month old Tg-S466L Cbsmice (A&B) and 15 month old Tg-hCBS apoE^−/− Cbs mice (C&D), stained with antibodies against CD11b and Ly6C, and analyzed by flow cytometry. A & C. Representative dot plots depicting nucleated cells (gate i, eliminated red blood cell content) and MNC cells (gate ii). Histograms describe the percent of CD11b⁺ MNC/monocytes in gate ii. Shaded and open curve area represent data from the non-antibody negative control and CD11b antibody staining groups, respectively, B & D. CD11b⁺ MNC/monocyte percentage in gate i. The percent of CD11b⁺ MNC/monocytes in gate i was calculated by multiplying CD11b⁺ MNC/monocyte event percent in gate ii with the MNC percent in gate i. Values are mean ± SD. n=6. Independent t test was used. BM, bone marrow, SSC, side-scatter light; FSC, forward-scatter light; MNC, mononuclear cells.

Figure 5. Characterization of monocyte subsets in transgenic Cbs mice

Peripheral blood, spleen and BM cells were isolated from 6 and 8 month old Tg-S466L Cbs mice (A&B) and 15 month old Tg-hCBS apoE^−/− Cbs mice (C&D), stained with antibodies against CD11b and Ly6C, and analyzed by flow cytometry. MNC were gated in gate ii as described in Figure 4, and divided into three subsets based on CD11b and Ly-6C expression: CD11b⁺Ly-6C^hi, CD11b⁺Ly-6C^mid and CD11b⁺Ly-6C^low. A & C. Representative dot plots depicting Ly-6C and CD11b expression in gate ii MNC. B & D. Ly-6C subsets in gate ii CD11b⁺ MNC/monocyte. Values are mean ± SD. n=6. Independent t test was used. SSC, side-scattered light; FSC, forward-scatter light; MNC, mononuclear cells.

Figure 6. Ly-6Cmonocyte survival and differentiation in cultured primary mouse splenocytes

Splenocytes were isolated from 2 month old C57B/L6 wild-type mice and cultured. Cells were treated with rIFNγ, L-Hcy or L-Cys at indicated doses for 24 hr for survival study, or 48 hr during a 72 hr culture for differentiation study. Cells were stained with antibodies against CD11b and Ly6C, and analyzed by flow cytometry. MNC were defined by gate ii and further divided into three subsets based on Ly-6C expression levels. A. Representative dot plots depicting the distribution of monocyte subsets of CD11b⁺Ly-6C^low, CD11b⁺Ly-6C^mid and CD11b⁺Ly-6C^hi cells. B. CD11b⁺Ly-6Cmonocyte subset distribution. Schematic designs are inserted to describe the experimental strategies. Data are representative of three separate experiments and are shown as mean ± SEM. * compared with the control group at the same time point. ^# compared with the L-Hcy treatment with identical concentrations. MNC, mononuclear cells.

Figure 7. Anti-oxidants abolished Ly-6C^mid monocyte differentiation induced by Hcy in cultured primary mouse splenocytes

Splenocytes were isolated from 2 month old C57B/L6 wild-type mice. Cells were cultured for 24 hr and then treated with SOD-PEG (250U/mL) plus CAT-PEG (250U/mL), uric acid (60μg/mL), apocynin (100μM), allopurinol (30μg/mL), or L-NAME (1mM) for 1 hr before exposure to L-Hcy (500μM), or treated with 50 μM L-Hcy in the presence of 25μM adenosine for an additional 48 hr. Cells were then stained with antibodies against CD11b and Ly6C, and analyzed by flow cytometry. MNC were defined by gate ii and further divided into three monocyte subsets based on CD11b and Ly-6C expression. A. Representative dot plots depicting the distribution of monocyte subsets of CD11b⁺Ly-6C^low, CD11b⁺Ly-6C^mid and CD11b⁺Ly-6C^hi cells. B & C. CD11b⁺Ly-6C^mid monocyte subsets. Data are representative of 3 independent experiments. Data are expressed as mean ± SEM. *compared with the parallel no L-Hcy control. ^#compared with500 μM L-Hcy treatment without other inhibitors. PEG, Polyethylene glycol; SOD, superoxide dismutase; CAT, Catalase; MNC, mononuclear cells.