Figure 1.
Homocysteine (Hcy) induced cell viability changes in and LDH release from human umbilical vein endothelial cells (HUVECs).
A. MTT assay of cell viability changes as a function of Hcy concentrations. B. Measurement of LDH in media (LDH release) after treatment with concentrations of Hcy. C. Intracellular concentrations of Cu after 24 hrs exposure to concentrations of Hcy in cultures. D. Intracellular concentrations of Hcy after 24 hrs exposure to concentrations of Hcy in cultures. Each data point was obtained from three independent experiments and each experiment contains triplicate samples for each treatment. Values are means ±S.E.M. * or # significantly different from control group and from each other (p<0.05).
Figure 2.
Effects of Cu (5 µM) pretreatment on Hcy-induced decrease in cell viability and LDH release.
A. MTT assay of cell viability changes. B. Measurement of LDH in media (LDH release). C. Intracellular concentrations of Cu. D. Intracellular concentrations of Hcy. Each data point was obtained from three independent experiments and each experiment contains triplicate samples for each treatment. Values are means ±S.E.M. *, # or † significantly different from control group and from each other (p<0.05).
Figure 3.
Hcy induced redistribution of cellular Cu.
A. Measurement of cellular Cu distribution after treatment with concentrations of Hcy by HPLC-ICP-MS. B. Hcy-induced redistribution of Cu between high (1st) and low (4th) molecular weight fractions as evaluated by changes in percentage of Cu in each fraction. C. Distribution of Hcy in each fraction. Each data point was obtained from three independent experiments and each experiment contains triplicate samples for each treatment. Values are means ±S.E.M. * or # significantly different from control group and from each other for different fractions (p<0.05).
Figure 5.
Effect of Hcy on mitochondrial membrane potential (ΔΨm) in HUVECs.
A. JC-1 assay of mitochondrial membrane potential changes as a function of Hcy concentrations. B. The effect of Cu pretreatment on Hcy-induced mitochondrial membrane potential changes. Bar: 100 µm. Each data point was obtained from three independent experiments and each experiment contains triplicate samples for each treatment. Values are means ±S.E.M. * significantly different from control group (p<0.05).
Figure 6.
Enzymatic assay for changes in cytochrome c oxidase (CCO) activity.
A. Changes in the CCO activity as a function of Hcy concentrations. B. The effect of Cu pretreatment on Hcy-induced changes in the CCO activity. The treatment protocol and labels are the same as described for Figure 2. Each data point was obtained from three independent experiments and each experiment contains triplicate samples for each treatment. Values are means ±S.E.M. * significantly different from control group (p<0.05).
Figure 7.
Western blot analysis of Hcy-induced changes in COX17 protein level.
A. Changes in the COX17 protein level as a function of Hcy concentrations. B. The effect of Cu pretreatment on Hcy-induced changes in the COX17 protein level. The treatment protocol and labels are the same as described for Figure 2. Semiquantitative analyses based on the density changes of each protein on the blot were obtained from 6 independent blots. Values are means ±S.E.M. * or # significantly different from control group and from each other (p<0.05).
Figure 4.
Mass spectrometric analysis of complex formation between Cu and Hcy.
A. Chromatogram of Cu and Hcy mixture. B. ESI mass spectrum of Cu and Hcy mixture. The histogram identifies homocysteine (136.0422), Cu-homocysteine (197.9637) homocystine (269.0620), and Cu-homocystine (330.9833), as shown in the schematic structures (Table 1).
Table 1.
Chemical formula of homocysteine, Cu-homocysteine complex, homocystine and Cu-homocystine complex.