Cellular hypomethylation is associated with impaired nitric oxide production by cultured human endothelial cells

Abstract

Hyperhomocysteinemia (HHcy) is a risk factor for vascular disease, but the underlying mechanisms remain incompletely defined. Reduced bioavailability of nitric oxide (NO) is a principal manifestation of underlying endothelial dysfunction, which is an initial event in vascular disease. Inhibition of cellular methylation reactions by S-adenosylhomocysteine (AdoHcy), which accumulates during HHcy, has been suggested to contribute to vascular dysfunction. However, thus far, the effect of intracellular AdoHcy accumulation on NO bioavailability has not yet been fully substantiated by experimental evidence. The present study was carried out to evaluate whether disturbances in cellular methylation status affect NO production by cultured human endothelial cells. Here, we show that a hypomethylating environment, induced by the accumulation of AdoHcy, impairs NO production. Consistent with this finding, we observed decreased eNOS expression and activity, but, by contrast, enhanced NOS3 transcription. Taken together, our data support the existence of regulatory post-transcriptional mechanisms modulated by cellular methylation potential leading to impaired NO production by cultured human endothelial cells. As such, our conclusions may have implications for the HHcy-mediated reductions in NO bioavailability and endothelial dysfunction.

Fig. 1

Intracellular AdoHcy (a), and extracellular Hcy (b) and ADMA (c) concentrations in HUVEC incubated in culture medium supplemented with increasing concentrations of ADA for 24 h. Data are presented as means ± SD. (**p <0.01 vs. control, n ≥ 3; *p <0.05 vs. control, n = 6)

Fig. 2

Nitrite levels in HUVEC culture medium measured by the Griess reaction. HUVEC were incubated in culture medium supplemented with increasing concentrations of ADA for 12 or 24 h. Data are presented as means ± SD, and are representative of three different cell lines (*p <0.05 vs. control)

Fig. 3

Nitrite levels in HUVEC culture medium supplemented with 0 and 20 μmol/L concentrations of ADA in the absence and in the presence of L-NNA (1 mmol/L) after 18 h of incubation. Data are presented as means ± SD (*p <0.05 vs. control, n = 3)

Fig. 4

eNOS expression in HUVEC cultured with increasing concentrations of ADA after 24 h of incubation. Data are means ± SD. a1 relative eNOS expression was determined by Western blotting with antibodies against eNOS and β-actin. Densitometry was performed on four blots. A representative blot is shown. (*p <0.05 vs. control). a2 eNOS activity was measured in HUVEC incubated in the absence or presence of 20 μmol/L of ADA after 24 h by measuring the conversion of L-arginine to L-citrulline in cell lysates. Four different experiments were performed each in duplicate. (*p <0.05 paired Student’s t test vs. control). b Real-time quantitative RT-PCR of relative NOS3 mRNA levels of three or more independently prepared cDNA pools representing independent RNA isolations. (*p <0.05 vs. control)