Homocysteine injures vascular endothelial cells by inhibiting mitochondrial activity

Abstract

The aim of the present study was to investigate the role of homocysteine (Hcy) in the pathogenesis of pulmonary embolism (PE) and the associated molecular mechanisms in human umbilical vein endothelial cells (HUVECs). Hcy contents were detected with high-performance liquid chromatography. Apoptosis was detected by flow cytometry using Annexin-V staining. Cytochrome c oxidase (COX) activity was assessed with an enzyme activity assay, and the expression levels of COX 17 were determined by western blot analysis. Intracellular reactive oxygen species levels were measured using a microplate reader with a fluorescence probe. The results demonstrated that, compared with the control group, the serum Hcy levels were significantly elevated in the PE group, suggesting that Hcy may be an indicator for PE. Following treatment with Hcy, the apoptosis rate was markedly elevated in HUVECs. Moreover, Hcy decreased COX activity and downregulated the expression of COX 17 in HUVECs. Furthermore, Hcy increased the ROS levels in these endothelial cells. However, all the above-mentioned physiopathological changes induced by Hcy in HUVECs could be restored by folic acid. In conclusion, the results of the present study demonstrated that Hcy inhibited COX activity, downregulated COX 17 expression, increased intracellular ROS levels and enhanced apoptosis in endothelial cells.

Keywords: apoptosis; cytochrome c oxidase; homocysteine; pulmonary embolism; vascular endothelial cells.

Figure 1.

Serum Hcy levels are elevated in patients with PE. The serum Hcy levels were determined in the control and PE groups using high-performance liquid chromatography. *P<0.05, vs. the control group. Hcy, homocysteine; PE, pulmonary embolism.

Figure 2.

Hcy induces apoptosis in HUVECs. HUVECs were first incubated with Hcy at 0, 0.01, 0.1, and 1 mM for 24 h. The apoptotic levels were then assessed sby flow cytometry with Annexin-V staining, and the apoptosis rates were calculated accordingly. *P<0.05, vs. the control group. Hcy, homocysteine; HUVECs, human umbilical vein endothelial cells.

Figure 3.

Hcy decreases COX activity and COX 17 expression levels in HUVECs. HUVECs were treated with Hcy at 0, 0.01, 0.1, and 1 mM for 24 h. (A) COX activity was assessed using a COX activity assay kit. (B) The protein expression levels of COX 17 were detected by western blot analysis. *P<0.05, vs. the control group. COX, cytochrome c oxidase; Hcy, homocysteine; HUVECs, human umbilical vein endothelial cells.

Figure 4.

Hcy increases the intracellular ROS levels in HUVECs. HUVECs were incubated with 0, 0.01, 0.1 and 1 mM Hcy for 24 h. The intracellular ROS levels were then determined. *P<0.05, vs. the control group. ROS, reactive oxygen species; Hcy, homocysteine; HUVECs, human umbilical vein endothelial cells.

Figure 5.

Folic acid alleviates the physiopathological changes induced by Hcy. HUVECs were treated with 1 mM Hcy and/or 100 µmol/l folic acid for 24 h, and then the (A) apoptosis rate, the (B) COX activity levels, the (C and D) COX 17 expression levels and the (E) intracellular ROS levels were determined in these cells. *P<0.05, vs. the control group; ^#P<0.05, vs. the Hcy group. ROS, reactive oxygen species; COX, cytochrome c oxidase; Hcy, homocysteine; HUVECs, human umbilical vein endothelial cells.