Abstract 5813: Hyperhomocysteinemia Enhances Bone Marrow-derived and Vessel Resident Ly6C Inflammatory Monocyte Differentiation Contributing to Atherogenesis

Background: We recently reported that hyperhomocysteinemia (HHcy) enriched circulating Ly6C monocytes (MCs) independent of hyperlipidemia. Here we studied the effect and mechanism of HHcy on Ly6C MC function and differentiation in aorta. Methods and Results: We generated a mouse model with cystathionine β -synthase (CBS) and LDLr deficiencies. LDLr − / − CBS − /+ mice (8wk) received BM transplantation from EGFP mice. The chimeric mice were fed a high fat (HF) diet as control (Hcy, 10.1 μ M), HF/high methionine (HM) diet to induce severe HHcy (275.6 μ M), or HF/HM/high vitamin B-6/12 and folic acid diet as Hcy-lowering therapy (39.0 μ M) for another 8wk. Morphological analysis exhibited that severe HHcy accelerated atherosclerosis and increased BM-derived Ly6C MC accumulation in the lesion. FACS analysis on whole aortic cell suspension showed that severe HHcy increased BM-derived and resident putative MC (CD11b + Ly6G − ) to 4872 and 3761 from 1332 and 599 cells/aorta in the control. The putative MC was further divided into 4 distinct subsets, severe HHcy, compared with the control, increased BM-derived and resident Ly6C hi MC (Ly6C hi F4/80 − , 816 vs 43 and 842 vs 115), differentiating MC (Ly6C hi F4/80 + , 558 vs 43 and 784 vs 28), differentiated macrophage (Ly6C low F4/80 − , 1874 vs 766 and 1095 vs 92), and Ly6C low MC (Ly6C low F4/80 − , 1624 vs 480 and 1040 vs 365) in aorta. Plasma Hcy levels were significantly correlated with plasma TNF α (r=0.650), IL-6 (r=0.594) and MCP-1 (r=0.591) levels. Hcy-lowering therapy significantly reduced atherosclerotic lesion, plasma inflammatory cytokine levels and aortic MC differentiation. In vitro, L-Hcy (100 –500 μ M) enhanced IFN γ -induced Ly6C hi+mid MC differentiation, which was abolished by Tiron, SOD/catalase, and NADPH oxidase inhibitor apocynin. L-Hcy (500 μ M) increased the TNFa/IL-6 cytokine-producing Ly6C hi+mid MC by 3-fold in IFN γ -treated cells and significantly enhanced transendothelial migration of IFN γ -treated THP1 cells. Conclusions: Severe HHcy induces systemic inflammation and promotes BM-derived and vessel resident Ly6C inflammatory MC differentiation leading to exacerbated atherosclerosis. NADPH oxidase mediated oxidative stress participates in Ly6C inflammatory MC differentiation induced by Hcy.