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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Circ Res. Author manuscript; available in PMC 2013 June 22.
Published in final edited form as:
PMCID: PMC3412115
NIHMSID: NIHMS386879

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

Abstract

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 Ly6Chigh 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 (Ly6Chigh+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

Introduction

Numerous clinical studies have established hyperhomocysteinemia (HHcy) as an independent risk factor for cardiovascular diseases (CVD) in the general population, similar to hypercholesterolemia or smoking. We have previously proposed that HHcy accelerates atherosclerosis by inhibiting endothelial cell (EC) growth, post-injury reendothelialization, endothelial-dependent vessel relaxation and HDL biosynthesis.16 Recently, we and others demonstrated that HHcy increases plasma levels of the pro-inflammatory cytokine TNFα and monocyte chemotactic protein (MCP)-1 in transgenic cystathionine β-synthase (CBS)/apolipoprotein E (ApoE)-deficient mice (Tg-hCBS ApoE−/− Cbs−/−)7 and that homocysteine (Hcy) induces proinflammatory chemokine expression in human aortic ECs and monocytes (MC).8, 9 However, the causative role of HHcy in systemic and vessel wall inflammation has not been studied and the effect of HHcy on athrogenesis needs to be confirmed.

Previous HHcy atherogenesis studies were all performed in ApoE−/− mice. To avoid the potential confounding effects of ApoE deficiency, it is necessary to validate the effect of HHcy on atherogenesis in a different atherosclerosis accessible animal model free of ApoE depletion. In this study, we generated the Ldlr−/− Cbs−/+ mice, because both ApoE−/− and Ldlr−/− mice have been wildly used animal models of atherosclerosis.

Despite its established strong association with CVD, the causative role of HHcy in CVD remains controversial. Several secondary prevention trials of Hcy-lowering therapy were reported to have no benefit on combined endpoints of cardiovascular events.10 However, Hcy-lowering has been found to be beneficial in reducing cardiovascular risk in CBS deficient patients11 and in preventing the recurrence of stroke.1216 In the HOPE 2 Trial, Hcy-lowering reduced the risk of overall stroke.17 Such benefit was also found in a large population-based cohort study, showing that Hcy-lowering due to folic acid fortification significantly reduced stroke mortality in the general population.18 Some of the negative findings probably reflects limitations related to statistic power, relatively low levels of plasma Hcy levels prior to the treatment (plasma Hcy=10–15µM), co-medication confounders, irreversible disease stage, and difficulty of nutritional control in selected patient populations. Therefore, it is important to develop a well-controlled animal model of Hcy-lowering therapy for severe HHcy to confirm the causative effect of HHcy and dissect the underlying mechanisms.

Recent advances in immunology have identified functional subsets of MC, which exhibit distinct pathophysiological roles.19, 20 Mouse MC subsets can be distinguished by differential expression of an inflammatory MC marker Ly-6C.21 Ly-6Chigh and Ly-6Cmiddle MC subsets have been linked with inflammatory disease, including hypercholesterolemia and atherosclerosis, and named as inflammatory MC subsets.19 We recently reported that severe HHcy elevated circulating Ly-6Chigh+middle MC independent of hyperlipidemia, pointing out a new proatherogenic mechanism of HHcy.7 However, how HHcy modulates MC subset function and its differentiation in the vessel wall has not been studied.

In this study, we created Ldlr−/− Cbs−/+ mice, established HHcy-inducing and Hcy-lowering strategies, and assessed the causative role of HHcy on atherogenesis, MC differentiation, and relevant inflammation systemically and in the vessel wall.

Methods

(details in supplemental material)

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 EGFPBM 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 EGFPBM 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).

Results

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

Table 1
A. Vitamin content in mouse diets. Basal vitamin requirement is determined by the “Guidelines for adequate nutrition of mice suggested by National Laboratory Animal Nutrition Committee (24 in Reference). We designed HF diet containing sufficient ...

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

The HF+HM diet induced severe HHcy and hyperlipidemia in the EGFPBM 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

Figure 1
HHcy increased atherosclerosis and BM-derived cells in the lesion and aortas of EGFPBM LDLr−/− CBS−/+ mice

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 EGFPBM 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 EGFPBM Ldlr−/− Cbs−/+ mice

Chimeric EGFPBM 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 EGFPBM 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 EGFPBM 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-6Chigh inflammatory MC subset in the blood of EGFPBM 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-6Chigh, Ly-6Cmiddle and Ly-6Clow) (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-6Chigh and Ly-6Cmiddle 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-6Clow 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-6Chigh population in the blood (Figure 2C&D).

Figure 2
Severe HHcy increased total MC and inflammatory MC subsets in the blood, spleen and BM of LDLr−/− CBS−/+ mice

HHcy caused accumulation of BM-derived and tissue-origin MC and Mϕ in the aortas of EGFPBM 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).

Figure 3
HHcy promoted BM-derived and resident inflammatory MC differentiation and increased MC heterogeneity in the aorta of EGFPBM LDLr−/− CBS−/+ mice

HHcy increased both BM-derived and resident inflammatory MC subsets, and promoted MC heterogeneity in the aorta of EGFPBM 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-6Chigh+middleF4/80), R2 the differentiating MC (Ly-6Chigh+middleF4/80+), R3 the differentiated Mϕ (Ly-6Clow F4/80+), and R4 the Ly-6ClowF4/80MC, 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-6Clow 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-6Chigh+middleF4/80+ and Ly-6Chigh+middleF4/80 (R1 and R2) cells. The Ly-6ClowF4/80+ and Ly-6ClowF4/80 (R3 and R4) were less responsive to HHcy. Vitamin treatment completely prevented the induction of inflammatory and differentiating MC (R1 and R2), and Ly-6Clow 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 EGFPBM 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).

Figure 4
HHcy-induced systemic inflammation was prevented by vitamin treatment in EGFPBM LDLr−/− CBS−/+ mice

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-6Chigh MC population by 143%, 149% and 173%, and CD11b+Ly-6Cmiddle 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-6Chigh MC, but did not change CD11b+Ly-6Cmiddle MC population (Figure 5B&C). L-Hcy increased TNF-α-producing CD11b+Ly-6Cmiddle+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-6Clow residential MC, and reduced dual cytokine-producing Q1 cells (Figure 5D&E).

Figure 5
L-Hcy promoted rIFNγ-induced inflammatory MC formation and pro-inflammatory cytokines production in primary mouse splenocytes
Figure 6
Folic Acid and antioxidant reagents prevented L-Hcy-induced inflammatory MC differentiation in primary mouse splenocytes

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

L-Hcy (500µM) increased Ly-6Chigh and Ly-6Cmiddle subsets by 191% and 147%, respectively. Folic acid (100µM) did not change the populations of Ly-6Chigh and Ly-6Cmiddle subsets (101% and 94%), but prevented the increase of Ly-6Chigh and Ly-6Cmiddle 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-6Chigh to 72% and had no effect on Ly-6Cmiddle subsets (100% of the control group). The combination of SOD and catalase prevented Hcy-induced Ly-6Chigh (93% of the control) and Ly-6Cmiddle (99% of the control) subsets. Apocynin (100µM), an antioxidant,36 reduced the Hcy-induced Ly-6Chigh subset to 105% and Ly-6Cmiddle subset to 114%, FA and antioxidants did not change Ly-6Clow 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.

Figure 7
Folic Acid and antioxidant reduced L-Hcy-induced superoxide anion production in inflammatory MC

Discussion

In this study, we examined the causative role and mechanism of HHcy in atherogenesis and MC differentiation by using a sophisticated experimental model system consisting of a novel Ldlr−/− Cbs−/+ mouse line and a set of relevant diets for HHcy-inducing and Hcy-lowering. We used a group of state-of-the-art technologies including GFP traced BM transplantation, FACS analysis of MC subsets in the vessel wall, intracellular cytokine staining, and in vitro MC differentiation in mouse primary splenocytes. We report five major findings here: 1) severe HHcy accelerates atherosclerosis independent of apoE deficiency, 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 leading to pro-inflammatory cytokine production and systemic inflammation, and 5) Hcy promotes inflammatory MC subset differentiation via oxidative-stress in primary splenocytes.

The CBS deficient mouse was established by Dr. Nobuyo Maeda and colleagues.22 Plasma Hcy levels are doubled in CBS−/+ mice (Hcy 6–14 µM), as compared to CBS+/+ mice (Hcy 3–8 µM) on a regular rodent chow.22,4 However, if challenged with a high vitamin containing HM diet, CBS−/+ mice developed severe HHcy (plasma Hcy 140 µM), while the CBS+/+ mice had moderate HHcy (plasma Hcy 40 µM).4 In this study, we designed a HF+HM diet containing low but sufficient vitamins, which induced aggressive severe HHcy (plasma Hcy 244 µM) in the Ldlr−/− Cbs−/+ mice. These data suggests that CBS activity is largely reduced in CBS−/+ mice, which can’t catabolize excessive substrate (methionine) provided by HM diet.

The Ldlr−/− Cbs−/+ mouse is a valid disease model for HHcy-induced pathology, in which atherosclerosis can be examined independent of apoE deficiency. In addition, we lowered folate/B vitamin content to basic nutritional requirement levels (Table 1A) in the HF and HF+HM diet. This strategy does not only ensure adequate nutrition of mice 31 and eradicates metabolic disturbances that may be caused by the folate-free diet previously used by the others to induce HHcy,37 but also increases the sensitivity to Hcy interventions. Plasma Hcy is increased to 244µM in mice fed a HF+HM diet; this is reduced to 46µM utilizing folate-emphasized combination therapy at clinically tested dosages (Figure 1A).32, 33

Our data provides strong evidence supporting the causative role of HHcy in atherogenesis because Hcy-lowering therapy completely prevented HHcy-induced atherosclerotic lesion (Figure 1, ,22 & 4). This is consistent with Hcy-lowering studies in CBS deficient human in which lower plasma Hcy significantly reduced cardiovascular events.11 Our preventive strategy by supplement therapeutic vitamins (HV) in the HHcy diet (HM) clearly demonstrated the benefit of preventive Hcy-lowering therapy. Studies are underway to determine whether Hcy-lowering can reverse CV outcomes in disease models in light of the clinical therapy condition.

Vitamin treatment increased serum folate level by 3.9-fold, but did not significantly change B6/B12 vitamin levels (Table 1B). These data suggest that folate modulation may be sufficient to prevent the induction of atherosclerotic lesion formation and inflammatory MC differentiation in HHcy, whereas B6/B12 supplement may not be critical for the regression of atherosclerosis and MC differentiation.

Vitamin therapy reduced the HHcy-induced expansion of all three MC subsets to basal levels in the spleen and BM, and CD11b+Ly-6Chigh population, but not CD11b+Ly-6Cmid/low MC in the blood. These data suggest HHcy stimulates all MC subset replenishment in the spleen and BM, and selectively promotes the release of CD11b+Ly-6Chigh MC to the blood (Figure 2). Because blood CD11b+Ly-6Chigh MC levels respond to the treatment and correspond to atherosclerotic lesion size, it is a valid therapeutic readout for cardiovascular intervention.

We found that HHcy-accelerated atherosclerotic lesion is comprised predominately of BM-derived MC/Mϕ, and enhanced by inflammatory MC in Ldlr−/− Cbs−/+ mice (Figure 1 & 3). This is consistent with our previous observation in Tg-hCBS Cbs−/− mice,7 extending the theory that MC accumulation in HHcy increases proportionately with lesion size.38 The accumulation of blood origin GFP+ MC/Mϕ in the lesion is likely related to Hcy-induced endothelial activation and MC transmigration, which are under active investigation in our laboratory.

Under steady-state conditions, CD11b+Ly-6Chigh BM MC are released into the peripheral blood and are thought to become CD11b+Ly-6Cmiddle MC before they form CD11b+Ly-6Clow MC.19, 20, 39 By flow cytometry analysis of whole aortic cell suspensions, we found that HHcy increases the aortic accumulation of all subsets of MC and Mϕ of both GFP+ and GFP cells (Figure 3). Importantly, the most dramatic increased populations are Ly-6Chigh+middle MC (8- and 8.5-fold increase for GFP F4/80+ and F4/80, and 18.5- and 5.8-fold for GFP+ F4/80+ and F4/80 cells, respectively) and CD11b+Ly-6G+ Mϕ (12- and 9-fold for GFP and GFP+ cells, respectively). These data indicate that Ly-6Chigh+middle MC is the most responsive MC to be recruited (GFP+) into and expanded (both GFP and GFP+) in the vessel wall, which are further differentiated into Mϕ and contribute to vessel wall inflammation and atherosclerosis. Vitamin treatment completely reversed inflammatory and differentiating MC induction, for both GFP and GFP+ cells, reduced GFP differentiated Mϕ, but had no effect on GFP+ differentiated Mϕ, which is Ly-6Clow/F4/80+ and has less inflammatory potential. It is likely that BM-origin inflammatory MC and Mϕ are more responsive to vitamin therapy, but the BM-origin differentiated Mϕ which has less inflammatory potential is not responsive. These data provide strong evidence supporting the causative role of HHcy in promoting recruitment and differentiation of inflammatory MC and Mϕ and maturation of both BM- and tissue-origin cells in the vessel wall. Our studies support the notion that Hcy-lowering therapy reverses MC differentiation and tissue-origin Mϕ differentiation, but not BM-origin Mϕ differentiation in the vessel wall.

We and others have suggested that HHcy induces systemic inflammation 7, 37 and cytokine production.40 Here, we demonstrate that HHcy increases plasma pro-inflammatory cytokine (IL-6 and TNFα), which can be produced by MC, and chemokine (MCP-1) levels, which were prevented by vitamin treatment (Figure 4). Further, we show that L-Hcy (100–500µM) induces the differentiation of inflammatory MC subsets, which are responsible for Hcy-induced TNF-α and IL-6 production in a MC differentiation model in primary mouse splenocytes (Figure 5). These data provide strong evidence supporting a causative role for HHcy in inducing systemic inflammation by promoting Ly-6Chigh+middle MC differentiation.

Finally, we demonstrate that HHcy-induced inflammatory MC differentiation and superoxide anion production can be inhibited by folic acid, antioxidant SOD+catalase or apocynin (Figure 6 & 7). These data suggest that HHcy-induced inflammatory MC differentiation is mediated, in large part, through oxidative stress. Future studies should be important to identify mechanisms underlying Hcy-induced superoxide anion production in MC.

In summary, we provide direct evidence supporting a new theory that the pro-inflammatory effects of HHcy modulate MC recruitment and differentiation, thereby contributing to atherogenesis. HHcy enriches MC in the spleen and BM, and selectively promotes the release of CD11b+Ly-6Chigh MC to the blood, which contributes to systemic inflammation and vessel wall MC accumulation. HHcy facilitates inflammatory Ly-6Chigh+middle MC recruitment and differentiation, and Mϕ differentiation/maturation of both BM- and tissue-origin in the vessel wall, thereby accelerating atherogenesis. The observation that vitamin therapy largely decreases plasma Hcy levels in parallel with declines in inflammatory markers, circulating and aortic inflammatory MC differentiation, and atherosclerotic lesion formation, establishes that HHcy is a cause, but not only a bystander or marker for systemic inflammation and atherosclerosis, at least in mice.

Our data also indicate that managing plasma folate levels is associated with Hcy-lowering and complete protection against atherosclerosis and inflammatory MC differentiation.

Conclusion

These results suggest that HHcy promotes atherosclerosis by facilitating inflammatory MC differentiation via oxidant stress.

Novelty and significance

What is known?

  • Hyperhomocysteinemia (HHcy) is a potent independent rick factor of cardiovascular disease (CVD), and is associated with atherosclerosis in human and animal models.
  • Benefit of homocysteine (Hcy)-lowering therapy in human is inconclusive.
  • Relevant and sensitive animal model of HHcy and Hcy-lowering therapy is needed.
  • Monocytes (MC) contribute to systemic inflammation and atherosclerosis.
  • We reported that HHcy increased inflammatory MC in the peripheral tissues [bone marrow (BM), blood and spleen] in mice.

What new information does this article contribute?

  • A novel mouse model of HHcy and atherosclerosis (LDLr−/− CBS−/+ mice)
  • New sets of diet with relevant vitamin levels for inducing severe HHcy and lowering Hcy levels.
  • Both BM- and tissue-origin MC can generate inflammatory MC in the vessel wall and contribute to atherosclerosis.
  • HHcy is a cause for atherosclerosis, inflammatory MC generation and systemic inflammation in mice.
  • Hcy promotes inflammatory MC generation via oxidative-stress signaling in cultured primary splenocytes.

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.

Supplementary Material

01

Acknowledgments

Funding Sources: This work was supported in part by NIH Grants HL67033, HL77288, HL82774 and HL11076 (HW); HL94451 and HL108910 (XFY); and HL57299 (WDK).

Abbreviations

Ab
antibody
ApoE
apolipoprotein E
BM
Bone marrow
CAT
catalase
CBS
cystathionine β-synthase
CVD
cardiovascular diseases
Cys
cysteine
DHE
dihydroethidium
EC
endothelial cell
EGFP
enhanced green fluorescent protein
FA
folic acid
FBG
fasting blood glucose
FSC
forward scatter
Hcy
homocysteine
HF
high fat
HHcy
hyperhomocysteinemia
High
Ly-6Chigh subset
HM
high methionine
HV
high vitamin
IEL
internal elastic Lamina
LDLr
low-density lipoprotein receptor
Low
Ly-6Clow subset
mAbs
monoclonal antibodies
MC
monocyte
MCP
monocyte chemotactic protein
Mid
Ly-6Cmiddle subset
MNC
mononuclear cells
O2
Superoxide anion
PEG
polyethylene glycol
rIFNγ
interferon-γ
SOD
superoxide dismutase
SSC
side-scatter
TC
total cholesterol
TG
triglyceride

Footnotes

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