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

Background

This study examined the causative role of hyperhomocysteinemia (HHcy) in atherogenesis and its effect on inflammatory monocyte (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 µM) was induced by a high methionine diet containing sufficient basal levels of B vitamins. Plasma Hcy levels were lowered to 46 µM from 244 µM 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 sub-lethal 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 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 pro-inflammatory 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.

Conclusion

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.

Keywords: inflammatory monocyte, atherosclerosis, hyperhomocysteinemia

Ldlr^−/− Cbs^−/+ mouse model and organ development assessment

Mice deficient in both Cbs and Ldlr were obtained by cross breeding CBS^−/+ with Ldlr^−/− mice.^{22, 23}

BMT and chimeric EGFP_BM Ldlr^−/− Cbs^−/+ mice

BMT was performed as previously described with modifications.²⁴

Blood biochemistry analysis

Blood from overnight fasted mice were collected and analyzed for Hcy,²⁵ lipid,²⁶ blood glucose, and vitamin analysis.

Aortic sinus cross-sectioning and lesion characterization

(see supplement).⁷

Aortic cell isolation and flow cytometry analysis

Aortas were collected from chimeric EGFP_BM LDLr^−/− CBS^−/+ mice and digested.²⁷ Cells were stained with antibodies against cell surface markers or superoxide anion marker DHE for flow cytometry analysis.²⁸

Plasma cytokine ELISA analysis

Plasma from overnight fasted mice was collected. Plasma IL-6, TNF-α, and MCP-1 levels were assessed using ELISA kits.

MC differentiation study in primary mouse splenocytes and chemical treatment

Splenocytes were isolated from 2 month old C57B/L6 wild-type mice, primed with low dose recombinant interferon-γ (rIFNγ, 100U/ml) at plating. After 24 hours, the cells were treated with L-Hcy (500µM) or L-Cys (500µM), re-stimulated with LPS (1µg/mL) and brefeldin A (5ng/mL), and followed with antibody (Ab) staining prior to flow cytometry analysis.

For mechanistic study, cells were treated with the folic acid (100µM),²⁹ PEG-CAT (250U/ml) plus PEG-SOD (150U/ml), Apocynin (100µM) 1 hr prior to L-Hcy (500µM) exposure for an additional 48 hr.

Dihydroethidium (DHE) staining and flow cytometry analysis in primary mouse splenocytes

Splenocytes were harvested and incubated with DHE (2×10⁻⁶ M), a superoxide indicator, at 37 °C for 30 minutes and then co-incubated with monoclonal antibodies to CD11b (anti-CD11b, clone M1/70)–PE and Ly-6C (anti-Ly6C, AL21)-FITC (BD Pharmingen™, San Diego, CA) for flow cytometry analysis. Superoxide anion (O₂⁻) containing cells were identified as DHE⁺ cells,²⁸ in both CD11b⁺Ly6C⁻ and CD11b⁺Ly6C⁺ populations.

L-Hcy was freshly prepared as previously described.³⁰ All chemicals not specified above were purchased from Sigma-Aldrich (St. Louis, MO).

MC cytokine intracellular staining

Primary mouse splenocytes were cultured and treated with L-Hcy. Cells were stained for cell surface marker Abs, fixed and permeabilized, and finally incubated with cytokine Abs, TNF-α-PE (MP6-XT22) and IL-6-FITC (MP5-20F3).

Therapeutic diet design

We designed a set of mouse diets containing precisely controlled synthetic nutrition ingredients, with an emphasis on controlling folate levels to the basal normal requirements.³¹

As described in Table 1A, the regular rodent chow contains excessive folate and B12 (7.1 and 0.05 mg/kg diet), which are 4.2- and 5-fold greater than the basal requirement (0.5 and 5µg/kg diet) based on the guidelines for adequate mouse nutrition suggested by the National Laboratory Animal Nutrition Committee.³¹ This lowers the sensitivity to dietary induced HHcy and reduces therapeutic responsiveness in mice. Therefore, we designed a set of mouse diets containing precisely controlled synthetic nutrition ingredients, with a special emphasis on controlling folate levels to the basal requirements. Our control diet, HF (TD08028, Harlan Teklad), contains sufficient vitamins (folate 0.6mg/kg, B12 30µg/kg, B6 8.4mg/kg) and 21% fat. The HF+HM diet (TD08029, Harlan Teklad) is the HF diet with up to 19.56g/kg (2%) methionine, an amino acid precursor of Hcy, added to induce HHcy. The HF+HM+HV (TD08118, Harlan Teklad) is a therapeutic diet that adds B vitamins to the HF+HM diet. It contains 6mg/kg folate, 60µg/kg B12, 16.8mg/kg B6, a 10-fold increase in folate content and a 2 fold increase in B6 and B12 relative to the HF+HM diet. We determined an average food consumption of about 3g diet/day/mouse, B vitamin consumption in mice on HF+HM+HF diet were 720µg folate, 7µg B12 and 2mg B6/kg body weight/day, which are dosages used in previous human clinical trials.^{32, 33}

HF+HM diet induced severe HHcy which was reversed by high vitamin supplementation in EGFP_BM Ldlr^−/− Cbs^−/+ mice

The HF+HM diet induced severe HHcy and hyperlipidemia in the EGFP_BM Ldlr^−/− Cbs^−/+ mice. Plasma Hcy increased from 12.1±6.9 to 244.6±50.4µM in mice fed a HF+HM diet (Figure 1B), comparable to the HHcy observed in subjects in the Framingham studies (Hcy up to 219.84µM).³⁴ The vitamin therapy effectively prevented severe HHcy and reduced plasma Hcy levels to 460±33.4µM. Hyperlipedemia (441 mg/dl. TC) is similar to results seen in the ApoE−/− mice and relevant to severe hyperlipidemia in human.³

HHcy and vitamin supplementation had no effect on organ development, plasma lipids, glucose or B6/B12 vitamin levels, but vitamin supplementation increased plasma folate levels in EGFP_BM Ldlr^−/− Cbs^−/+ mice

The HF+HM diet did not change body weight or heart weight (data not shown), but showed a trend of increasing plasma total cholesterol (TC), triglyceride (TG) and fasting blood glucose (FBG), and reducing vitamin B12 and folate levels (Table 1B). Vitamin treatment with the HF+HM+HV diet improved plasma lipid, elevated B6/B12 vitamin, and significantly increased plasma folate levels from 18.9±4.0ng/ml in the HF+HM group to 73.3±22.9ng/ml (3.9-fold).

HHcy increased atherosclerosis and accumulation of BM-derived MCs in atherosclerotic lesions of chimeric EGFP_BM Ldlr^−/− Cbs^−/+ mice

Chimeric EGFP_BM Cbs^−/+Ldlr^−/− mice were generated by BMT from EGFP Tg mice (Figure 1A). The EGFP-transgenic mice expresse GFP in all cell types. It is well recognized that GFP is not toxic to the cells and is a useful marker to identify and follow the fate of cells transplanted to the recipient mice.³⁵ We traced Bone marrow (BM)-derived cells by transplantation of BM cells from EGFP transgenic mice after sublethal irradiation of the recipient. The mean frequency of GFP⁺ cells in the peripheral nucleated cells was 97.9±1.1% in chimeric EGFP_BM Ldlr^−/− Cbs^−/+ mice, close to that seen in donor EGFP mice (98.2%±1.0)(Figure 1C). By cross-section analysis of the aortic sinus, we found that severe HHcy increased atherosclerotic lesion area and its percentage in the sinus in chimeric EGFP_BM Ldlr^−/− Cbs^−/+ mice fed a HF+HM diet (18.9±6.3×10⁴µm² and 22.2±10.7%) compared with that in the control mice fed a HF diet (7.7±4.9×10⁴µm² and 8.3±4.2%) (Figure 1D & E). Vitamin treatment completely prevented HHcy-induced atherosclerotic lesion, and reduced the lesion area and its percentage to that in the control mice (6.6±3.1×10⁴µm² and 8.1±3.9%). By sequential double immunofluorescence staining with monoclonal antibodies (mAbs) anti-MOMA-2 (MC/Mϕ marker) and anti-GFP (BM origin cell marker) (Figure 1D), we found that BM-derived cells (GFP⁺) dominated the cellular population of the lesion, and largely overlapped with MC/Mϕ marker MOMA2. The vitamin treatment prevented the accumulation of GFP⁺ cells and MC/Mϕ in the lesion. By flow cytometric quantification of single cell suspensions of the aortas, we found that HHcy increased donor BM-origin GFP⁺ population in the vessel wall from 6.7%±1.0 in mice on HF diet to 36.6%±3.2 in mice on HF+HM diet, a 5.5-fold increase, which was reduced to 16.4%±0.9 by vitamin treatment (Figure 1F). Consistently, the absolute cell count of BM-origin GFP⁺ population in the aorta was increased 2.9-fold by HHcy, and completely reduced to the basal level by vitamin treatment (Figure 1G).

HHcy increased total MC in the blood, spleen and BM, elevated all MC subsets in the spleen and BM, and raised Ly-6C^high inflammatory MC subset in the blood of EGFP_BM Ldlr^−/− Cbs^−/+ mice

Mononuclear cells (MNC) were isolated based on their lower granularity and larger cell size (gate ii) (Figure 2A). MC was defined as CD11b⁺MNC and further divided into three subsets using anti-Ly-6C Ab (Ly-6C^high, Ly-6C^middle and Ly-6C^low) (Figure 2B). HHcy increased total MNC by 2- and 1.4-fold in the blood and spleen, MC by 5-, 2- and 1.6-fold in the blood, spleen and BM, respectively (Figure 2C). The highest increased subsets were blood Ly-6C^high and Ly-6C^middle MC, the inflammatory subsets, which were increased by 4.7 and 8.8-fold in the blood, 2.6- and 2.6-fold in the spleen, and 3.0- and 2.0-fold in the BM compared to control mice. MC Ly-6C^low populations were increased by 2.5-, 1.9- and 2.1-fold in the corresponding tissues (Figure 2D). Vitamin treatment prevented the increase of all MC populations in the spleen and BM, but only reversed total MC and the Ly-6C^high population in the blood (Figure 2C&D).

HHcy caused accumulation of BM-derived and tissue-origin MC and Mϕ in the aortas of EGFP_BM Ldlr^−/− Cbs^−/+ mice

The aortic cells were first divided into donor-origin BM-derived GFP⁺ and tissue–origin GFP⁻ populations, then further, based on CD11b (MC marker) and Ly-6G (myeloid granulocyte maturation marker) expression. The gate i CD11b⁺Ly-6G⁺ cells were defined as putative Mϕ and gate ii CD11b⁺Ly-6G⁻ as putative MC (Figure 3A). HHcy increased GFP⁺ Mϕ by 9.7-fold and MC by 4.3-fold, GFP⁻ Mϕ by 12-fold and MC 2.9-fold, and Mϕ maturation rate by 3-fold and 2.9-fold in the GFP⁺ and GFP⁻ populations, respectively (Figure 3B). Vitamin treatment completely abolished the effects of HHcy on Mϕ accumulation and maturation, and significantly reduced MC levels in the aorta for both GFP⁺ and GFP⁻ cells (Figure 3B).

HHcy increased both BM-derived and resident inflammatory MC subsets, and promoted MC heterogeneity in the aorta of EGFP_BM Ldlr^−/− Cbs^−/+ mice

Aortic retrieved cells were divided into the GFP⁺ and GFP⁻ groups and analyzed for MC and Mϕ content using CD11b and Ly6G (Fig 3A). HHcy increased vessel wall GFP⁻ Mϕ and MC by 1233% and 429%, and GFP⁺ Mϕ and MC by 970% and 294%, respectively. Mϕ maturation rate was increased by 300% for both GFP⁻ and GFP⁺ population. These increases were all prevented by vitamin supplementation (Fig 3B). MC cells (gate ii from Figure 3A) were further divided into four phenotypiclly distinct populations using the Ly-6C and F4/80. The R1 cells were defined as inflammatory MC (Ly-6C^high+middleF4/80⁻), R2 the differentiating MC (Ly-6C^high+middleF4/80⁺), R3 the differentiated Mϕ (Ly-6C^low F4/80⁺), and R4 the Ly-6C^lowF4/80⁻MC, respectively (Figure 3C). HHcy increased GFP⁻ and GFP⁺ inflammatory MC from 105±5 to 842±64 and 44±4 to 816±2 cells per aorta, the differentiating MC from 92±44 to 784±46 and 97±42 to 558±83, the differentiated Mϕ from 288±133 to 1095±217 and from 986±198 to 1874±350, and the Ly-6C^low MC from 385±44 to 1040±3 and from 528±53 to 1624±214, respectively. Thus, HHcy had similar effects on vessel wall GFP⁻ cells as on GFP⁺ cells. The more HHcy responsive vessel wall MC populations were Ly-6C^high+middleF4/80⁺ and Ly-6C^high+middleF4/80⁻ (R1 and R2) cells. The Ly-6C^lowF4/80⁺ and Ly-6C^lowF4/80⁻ (R3 and R4) were less responsive to HHcy. Vitamin treatment completely prevented the induction of inflammatory and differentiating MC (R1 and R2), and Ly-6C^low MC (R4) for both GFP⁻ and GFP⁺ cells, and largely reduced the GFP⁻ differentiated Mϕ (R3) but had no effect on the GFP⁺ population.

HHcy induced systemic inflammation in EGFP_BM Ldlr^−/− Cbs^−/+ mice

HHcy increased plasma pro-inflammatory cytokines IL-6 from 52 to 76pg/ml, TNF-α from 80 to118pg/ml, and chemokine MCP-1 from 344 to 671pg/ml, respectively (Figure 4A). Vitamin treatment prevented the induction of MCP-1, TNF-α and IL-6. Further, plasma IL-6, TNF-α and MCP-1 levels positively correlated with Hcy levels (Figure 4B).

L-Hcy promoted inflammatory MC differentiation and pro-inflammatory cytokine production in primary mouse splenocytes

L-Hcy (100, 200 and 500µM) increased inflammatory CD11b⁺Ly-6C^high MC population by 143%, 149% and 173%, and CD11b⁺Ly-6C^middle MC by 130%, 154, and 150%, in a dose-dependent manner in rIFNγ-primed primary mouse splenocytes (Fig 6A,B&C). However, L-Cysteine, a sulfhydryl-containing amino acid control, showed a trend in increasing CD11b⁺Ly-6C^high MC, but did not change CD11b⁺Ly-6C^middle MC population (Figure 5B&C). L-Hcy increased TNF-α-producing CD11b⁺Ly-6C^middle+high inflammatory MC (Q2) from 65±15 to 228±46 per million cells (3.5-fold increase), IL-6-producing Q2 cells from 66±19 to 159±32 (2.4-fold), and dual cytokine-producing Q2 cells from 740±208 to 2121±430 (2.9-fold), but had no effect on single cytokine-producing Q1 population, CD11b⁺Ly-6C^low residential MC, and reduced dual cytokine-producing Q1 cells (Figure 5D&E).

Folic Acid and antioxidant reagents prevented L-Hcy-induced inflammatory MC differentiation in primary mouse splenocytes

L-Hcy (500µM) increased Ly-6C^high and Ly-6C^middle subsets by 191% and 147%, respectively. Folic acid (100µM) did not change the populations of Ly-6C^high and Ly-6C^middle subsets (101% and 94%), but prevented the increase of Ly-6C^high and Ly-6C^middle subsets induced by HHcy (from 191% and 147% to 80% and 104%, respectively). The combination of SOD (150U/ml) and catalase (250U/ml), a superoxide anion scavenger and a hydrogen peroxide metabolizing enzyme, respectively, reduced basal Ly-6C^high to 72% and had no effect on Ly-6C^middle subsets (100% of the control group). The combination of SOD and catalase prevented Hcy-induced Ly-6C^high (93% of the control) and Ly-6C^middle (99% of the control) subsets. Apocynin (100µM), an antioxidant,³⁶ reduced the Hcy-induced Ly-6C^high subset to 105% and Ly-6C^middle subset to 114%, FA and antioxidants did not change Ly-6C^low subset in the presence or absence of Hcy (Figure 6A, B &C).

Folic Acid and antioxidant reagents reduced L-Hcy-induced superoxide anion production in inflammatory MC

L-Hcy (500µM) increased superoxide anion producing cells (the DHE⁺ cells) by 167% (from 10.2% to 17%) in CD11b⁺/Ly-6C⁺ inflammatory MC. (Figure 7A,B&C). Hcy-induced DHE⁺ CD11b⁺/Ly-6C⁺ inflammatory MC was completely reversed to 100%, 102%, and 98% if pretreated with FA (100µM), apocynin (100µM) or SOD (250U/ml) plus SOD (150U/ml). The CD11b⁺/Ly-6C⁻ residential MC had very low detectable superoxide anion (2%). DL-Hcy (500µM) did not increase superoxide production in CD11b⁺/Ly-6C⁻ residential MC. FA and antioxidants did not significantly change the population of DHE⁺ CD11b⁺/Ly-6C⁻ MC.

01

Novelty and significance.

Summary.

This study is designed to address 4 major questions; 1) Does HHcy cause atherosclerosis or systemic inflammation? 2) What is the origin of vessel wall MC? 3) How do MC subsets contribute to systemic inflammation? and 4) what mechanism determine inflammatory MC differentiation? These are significant questions and relevant to human disease. We reported five major finding: (1) severe HHcy accelerates atherosclerosis, (2) severe HHcy promotes MC differentiation of both BM- and tissue-origin in peripheral tissues and in the vessel wall, (3) Hcy-lowering therapy via elevation of plasma folate levels prevents HHcy-induced atherosclerosis and inflammatory MC differentiation, (4) HHcy induces inflammatory MC differentiation which leads to systemic inflammation and atherosclerosis, and (5) Oxidative-stress mediates Hcy-induced inflammatory MC differentiation in primary splenocytes. This study provides new knowledge indicating that HHcy is a cause, but not only a biomarker, for atherosclerosis, vessel wall inflammatory MC differentiation and systemic inflammation, which can be largely prevented by folic acid supplement in mice. Our study provided mechanistic insights for atherosclerosis suggesting that vessel wall inflammatory MC can be generated from BM- or tissue-origin MC via oxidative-stress. The translational implication is that Hcy-lowering therapy can be beneficial in preventing atherosclerosis and systemic inflammation in human HHcy.

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