Relationship of impairment induced by intracellular S-adenosylhomocysteine accumulation with DNA methylation in human umbilical vein endothelial cells treated with 3-deazaadenosine

Abstract

The aim of this study was to estimate the relationship of endothelial dysfunction induced by intracellular S-adenosylhomocysteine (SAH) accumulation and DNA methylation in human umbilical vein endothelial cells (HUVEC). The isolated HUVEC were incubated with 3-deazaadenosine (DZA) to induce experimental intracellular SAH accumulation. The impairment of HUVEC function was assessed by changes in morphology and proliferative ability. The expression of DNA methyltransferase-1 (DNMT1) and the atherosclerosis related genes [oestrogen receptor-alpha (ER-alpha), extracellular superoxide dismutase (EC-SOD) and monocyte chemoattractant protein-1 (MCP-1)] were analysed using quantitative real-time PCR. Global DNA methylated status was measured using the cytosine extension assay. The methylated patterns of ER-alpha, EC-SOD and MCP-1 genes were determined with methylation-specific PCR. We found that DZA administration increased intracellular SAH levels progressively and simultaneously decreased Hcy content in medium. Moreover, the supplementation induced HUVEC apoptosis, inhibited proliferation ability and DNMT1 mRNA expression (P < 0.05) and furthermore reduced global DNA methylation status (P < 0.05). Correlation analysis showed the presence of a negative correlation between intracellular SAH concentration, proliferative ability, and expression of ER-alpha, EC-SOD, and DNMT1 (r = -0.89, -0.86, -0.92 and -0.88 respectively, P < 0.001); and a positive correlation with MCP-1 expression and DNA [(3)H]-dCTP incorporation (r = 0.89 and 0.93 respectively, P < 0.001). Our results showed that endothelial dysfunction induced by intracellular SAH accumulation is mediated by regulating the expression of atherosclerosis related genes in HUVEC, which is not related with gene promoter methylated patterns, but may be associated with altered global DNA hypomethylated status. These findings suggest that SAH can act as the potential molecular biological marker in the promotion of atherogenesis.

Figure 1

The levels of total homocsteine (tHcy) in culture medium (a) and intracellular S-adenosylhomocysteine (SAH) (b) in HUVEC incubated with 0 (white), 5 (light grey), 10 (deep grey) and 20 (black) μmol/l 3-deazaadenosine (DZA) for 24–72 h. Values are means for three determinations for each time point, with standard deviations represented by vertical bars. *P<0.05, **P<0.01. Mean value was significantly different from that for HUVEC at the same culture time incubated without DZA (Student’s t-test).

Figure 2

Examples of HUVEC incubated without (a, c) or with (b, d) 20 μmol/l 3-deazaadenosine (DZA) for 72 h showing the changes of ultrastructural organization (Bar = 2.5 μm) detected with electronmicroscope (a, b) and lipid droplets accumulation (Bar = 100 μm) with oil red O dyeing (c, d). Images are representative of fields from three experiments.

Figure 3

TUNEL analysis of apoptosis presence in HUVEC treated with 20 μmol/l 3-deazaadenosine (DZA) for 0 (a), 24 (b), 48 (c) and 72 h (d) on a confocal microscope. Images are representative of fields from the same experiment (Bar = 200 μm).

Figure 4

The relative ER-α (a), EC-SOD (b), MCP-1 (c) and DNMT1 (d) mRNA expression normalized for corresponding β-actin levels in HUVEC incubated with the tested 3-deazaadenosine (DZA) for 72 h detected with quantitative real-time PCR (qRT-PCR) as our previous method (Yu et al. 2007). Values are means for three determinations for each time point, with standard deviations represented by vertical bars. *P<0.05, **P<0.01. Mean value was significantly different from that for HUVEC at the same culture time incubated without DZA (Student’s t-test).

Figure 5

The global DNA methylated status in HUVEC incubated with 0 (white), 5 (light grey), 10 (deep grey) and 20 (black) μmol/l 3-deazaadenosine (DZA) for 24–72 h analysed with cytosine extension assay (Pogribny et al. 1999). Values are means for three determinations for each time point, with standard deviations represented by vertical bars. *P<0.05, **P<0.01. Mean value was significantly different from that for HUVEC at the same culture time incubated without DZA (Student’s t-test).

Figure 6

The promoter methylation of ER-α (a), MCP-1 (b), EC-SOD (c) in HUVEC incubated with/without the tested 3-deazaadenosine (DZA) for 72 h detected with methylation specific PCR (MSP) (Herman et al. 1996). Images are representative of fields from three experiments. U, unmethylation primer; M, methylation primer.