HYPERHOMOCYSTEINEMIA ACCELERATES ATHEROSCLEROSIS BY INDUCING INFLAMMATORY MONOCYTE DIFFERENTIATION IN A HYPERGLYCEMIC MOUSE MODEL

Abstract

Homocysteine (Hcy) is a thiol amino acid formed upon methionine de - methylation. A number of studies have revealed an association between hyperhomocysteinemia (HHcy), in which plasma Hcy levels exceed 15 µM, and diabetic atherosclerosis [1]. Despite these associations, the mechanisms underlying HHcy - associated diabetic atherosclerosis have not been clearly defined. This study assessed the effect of HHcy on diabetic atherosclerosis and its underlying mechanisms. We established a mouse model with a combination of three metabolic disorders, including HHcy (to mimic human HHcy), hyperglycemic (to mimic type 1 diabetes) and dyslipidemia (to exacerbate ApoE-/- mouse's susceptibility to atherosclerosis). In this mouse model, severe HHcy was developed due to mouse Cbs deficiency (mean plasma Hcy 182 µM) in a noval HHcy mouse model (Tg-hCBS Cbs ApoE-/-) generated by our collaborator [2]. Hyperglycemia was developed by 50 mg/kg streptozotocin (STZ) consecutive injection for 5 days (mean blood glucose 397 mg / dL). Dyslipedimia was introduced by high fat (HF, 21.0 % by weight) diet to accelerate aortic lesion formation in the Tg-hCBS Cbs ApoE-/- mice. An inducible human CBS (hCBS) transgene (Tg) was introduced to circumvent the neonatal lethality due to the mouse Cbs deficiency (Tg-hCBS Cbs-/- ApoE-/- mice). A zinc supplement in water could induce hCBS transgene expression and reverse Hcy level (reduced plasma Hcy from 182 µM to 5 µM, p < 0.001). Severe HHcy aggravated metabolic abnormalities, including increased urine secretion (from 8.35 ± 6.81 g/d/mouse in hyperglycemia only mice to 21.14 ± 5.95 g / d / mouse in hyperglycemic HHcy mice, p=0.042), increased water consumption (from 27.28 ± 9.46 g / d / mouse to 44.20 ± 4.66 g / d / mouse, p = 0.026), increased blood glucose level (from 443.20 ± 107.82 mg / dL to 614.27 ± 199.36 mg/dL, p = 0.031), increased heart weight (from 0.08 ± 0.02 g / cm to 0.11 ± 0.03 g / cm, p = 0.031) (data not shown) and decreased body weight (from -0.05 ± 0.92 g / 2 weeks / mouse to -1.78 ± 2.38 g / 2 weeks / mouse, p = 0.017). Hcy-lowering by zinc water reversed HHcy - induced hyperglycemia (from 614.27 ± 199.36 mg / dL to 440.20 ± 134.37 mg / dL, p = 0.032), increased urine secretion (from 21.14 ± 5.95 g / d / mouse to 0.90 ± 1.24 g / d / mouse, p = 0.042), and increased water consumption (from 44.20 ± 4.66 g / d / mouse to 6.08 ± 1.84 g / d / mouse, p = 0.008) in hyperglycemic mice. Increased atherosclerotic lesions were also found in the aortic roots of hyperglycemic HHcy mice (from 30.38 ± 14.34 % of the lumen area to 48.18 ± 12.07 %, p = 0.040). Increased accumulation of monocytes (MCs) and inflammatory MCs were found in atherosclerotic lesions of hyperglycemic HHcy mice. By sequential double immune - fluorescence staining with monoclonal antibodies (mAbs) anti MOMA - 2 (MC / macrophage [MØ] marker) and anti - Ly6C (inflammatory MC marker), we found that hyperglycemic HHcy mice had the largest area and percentage of MC / MØ (MOMA - 2+), and the largest area and percentage of MOMA - 2+ and Ly6C+ overlapped cells (inflammatory MCs). The zinc water supplement prevented the accumulation of MC / MØ and inflammatory MCs in the HHcy alone mice, as well as inflammatory MCs in the hyperglycemic HHcy mice. Severe HHcy increased total mononuclear cells (MNCs) by 2.1 fold in peripheral blood. Hyperglycemia combined with HHcy increased total MNCs by 1.4 -, 2.3 -, and 3.8 - fold in bone marrow (BM), peripheral blood, and spleen compared with hyperglycemic alone mice, respectively. Severe HHcy significantly increased the MC population in BM, peripheral blood, and spleen compared to Tg - hCBS Cbs+/+ ApoE-/- mice. MC populations were further increased in all 3 tissues isolated from hyperglycemic HHcy mice. In addition, we examined the MC population in age-matched, male, C57BL / 6 wild type mice without dietary, water or any other manipulation. HF diet significantly promoted MC expansion in spleen in Tg - hCBS Cbs+/+ ApoE-/- mice compared to the wild type mice. Furthermore, we characterized MC heterogeneity in the Tg-hCBS Cbs ApoE-/- mice. HHcy increased inflammatory lymphocyte antigen 6Chi (Ly6Chi) MC subset in BM (from 9.38 ± 2.03 % to 13.93 ± 5.61 %, p = 0.037), peripheral blood (from 4.92 ± 2.97 % to 11.84 ± 14.01 %, p = 0.049), and spleen (from 2.23 ± 0.87 % to 4.82 ± 4.21 %, p = 0.048) of the hyperglycemic Tg - hCBS Cbs-/- ApoE-/- mice compared to hyperglycemic Tg - hCBS Cbs+/+ ApoE-/- mice. 5 hour incubation of the MCs with 0.1 µg / ml of lipopolysaccharide (LPS) induced higher percentage of inflammatory cytokine tumor necrosis factor (TNF) - α secretion in all 3 subsets of MCs in the mouse BM (CD11b+Ly6Clow, from 30.8 % to 43.7 %; CD11b+Ly6Cmiddle, from 28.6 % to 38.6 %; CD11b+Ly6Chigh, from 48.3 % to 55.2 %), peripheral blood (CD11b+Ly6Clow, from 18.2 % to 42.4 %; CD11b+Ly6Cmiddle, from 36.0 % to 53.9 %; CD11b+Ly6Chigh, from 53.9 % to 54.4 %), and spleen (CD11b+Ly6Clow, from 23.8 % to 38.9 %; CD11b+Ly6Cmiddle, from 13.6 % to 38.8 %; CD11b+Ly6Chigh, from 40.2 % to 59.4 %). Moreover, M1 MØs increased in the hyperglycemic HHcy mouse BM (from 7.78 % to 10.9 %), peripheral blood (from 8.76 % to 23.17 %), and spleen (from 11.65 % to 13.95 %) compared to the hyperglycemia alone mice, respectively. M2 macrophage was decreased significantly in hyperglycemic HHcy mice in BM (from 4.16 % to 2.24 %), peripheral blood (from 5.88 % to 2.58 %), and spleen (from 10.2 % to 5.58 %) compared to the hyperglycemia alone mice, respectively. Plasma level of Hcy was positively correlated with s - adenosylmethionine (SAM) and s - adenosylhomocysteine (SAH) levels, but negatively correlated with SAM / SAH ratio, an indicator of methylation level, in Tg-hCBS Cbs ApoE-/- mice. In hyperglycemia condition (blood glucose ≥ 300 mg / dL), severe HHcy further decreased SAM / SAH ratio in mouse plasma. Also, CD11b+Ly6Clow MCs were negatively correlated with Hcy levels as well as SAH levels, but positively correlated with SAM levels and SAM / SAH ratio. On the other hand, CD11b+Ly6Cmiddle and CD11b+Ly6Chigh MCs were positively correlated with Hcy and SAH levels, but negatively correlated with SAM level and SAM / SAH ratio. This is the case in all BM, peripheral blood and spleen tissues. It indicates that hypomethylation might be one of the mechanisms underlying HHcy's accelerating effects on atherosclerosis in hyperglycemia condition. In cultured primary mouse splenocytes, L - Hcy (200 µM) with D - glucose (D -Glu) (25 mM), not L - Glu, induced Ly6Cmiddle and Ly6Chigh MC subsets (CD11b+Ly6Cmiddle, from 2.0 % in D - Glu treated group to 2.5 % in L - Hcy and D - Glu combined treated group, p = 0.041; CD11b+Ly6Chigh, from 1.4 % to 1.8 %, p = 0.040). These different Ly6C MC subsets exhibited distinguished superoxide anion production (0.92 ± 0.05 % dihydroethidium [DHE]+ cells in Ly6Clow MCs versus 4.02 ± 0.44 % DHE+ cells in Ly6Cmiddle MCs versus 17.33 ± 1.58 % DHE+ cells in Ly6Chigh MCs, p < 0.001), confirming their different activation status. Adding adenovirus overexpressing DNA methyltransferase1 (DNMT1) to the primary mouse splenocytes could rescue the inflammatory MC production induced by the combination of L-Hcy and D-Glu. We concluded that HHcy promotes differentiation of inflammatory MC and contributes to atherosclerotic lesions in hyperglycemia.