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Cholesterol Efflux Capacity of Apolipoprotein A-I Varies with the Extent of Differentiation and Foam Cell Formation of THP-1 Cells

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ABSTRACT

Apolipoprotein A-I (apoA-I), the main protein component of high-density lipoprotein (HDL), has many protective functions against atherosclerosis, one of them being cholesterol efflux capacity. Although cholesterol efflux capacity measurement is suggested to be a key biomarker for evaluating the risk of development of atherosclerosis, the assay has not been optimized till date. This study aims at investigating the effect of different states of cells on the cholesterol efflux capacity. We also studied the effect of apoA-I modification by homocysteine, a risk factor for atherosclerosis, on cholesterol efflux capacity in different states of cells. The cholesterol efflux capacity of apoA-I was greatly influenced by the extent of differentiation of THP-1 cells and attenuated by excessive foam cell formation. N-Homocysteinylated apoA-I indicated a lower cholesterol efflux capacity than normal apoA-I in the optimized condition, whereas no significant difference was observed in the cholesterol efflux capacity between apoA-I in the excessive cell differentiation or foam cell formation states. These results suggest that cholesterol efflux capacity of apoA-I varies depending on the state of cells. Therefore, the cholesterol efflux assay should be performed using protocols optimized according to the objective of the experiment.

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Isoelectric focusing for apoA-I and apoA-I treated with homocysteine thiolactone. (a) Representative isoelectric focusing patterns of apoA-I and N-Hcy apoA-I. Purified apoA-I was incubated with or without 10 mmol/L of homocysteine thiolactone at 37°C for 6 hours. After dialysis against PBS, the samples were analyzed by isoelectric focusing followed by western blotting using anti-apoA-I antibody. (b) Histogram represents the percentage of N-Hcy apoA-I to total apoA-I using CS analyzer. The values were indicated by mean + SD (n = 3, ∗P < 0.05).
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fig6: Isoelectric focusing for apoA-I and apoA-I treated with homocysteine thiolactone. (a) Representative isoelectric focusing patterns of apoA-I and N-Hcy apoA-I. Purified apoA-I was incubated with or without 10 mmol/L of homocysteine thiolactone at 37°C for 6 hours. After dialysis against PBS, the samples were analyzed by isoelectric focusing followed by western blotting using anti-apoA-I antibody. (b) Histogram represents the percentage of N-Hcy apoA-I to total apoA-I using CS analyzer. The values were indicated by mean + SD (n = 3, ∗P < 0.05).

Mentions: Using various stages of cells regulated differentiation and foam cell formation, the effect of N-homocysteinylation on cholesterol efflux capacity of apoA-I was evaluated. To confirm the extent of N-homocysteinylation of apoA-I, apoA-I incubated with HcyT was tested using an isoelectric focusing. Compared to normal apoA-I, N-Hcy apoA-I indicates higher isoelectric point (pI) depending on the number of attached homocysteine compounds (Figure 6(a)). The ratio of N-Hcy apoA-I to total apoA-I on the isoelectric focusing pattern was 51.9 ± 5.9% (Figure 6(b)). Cholesterol efflux capacities of apoA-I and N-Hcy apoA-I reached those peaks at day 2 and gradually decreased depending on the periods of the treatment with PMA (Figure 7(a)). In case of foam cell formation, a significant decrease was observed in cholesterol efflux capacities of apoA-I and N-Hcy apoA-I according to the acLDL loading (Figure 7(b)). In addition, the cholesterol efflux capacity of N-Hcy apoA-I was significantly lower than that of intact apoA-I by differentiation and foam cell formation at day 1 and day 2, respectively; however no significant difference was observed after day 3 (Figure 7).


Cholesterol Efflux Capacity of Apolipoprotein A-I Varies with the Extent of Differentiation and Foam Cell Formation of THP-1 Cells
Isoelectric focusing for apoA-I and apoA-I treated with homocysteine thiolactone. (a) Representative isoelectric focusing patterns of apoA-I and N-Hcy apoA-I. Purified apoA-I was incubated with or without 10 mmol/L of homocysteine thiolactone at 37°C for 6 hours. After dialysis against PBS, the samples were analyzed by isoelectric focusing followed by western blotting using anti-apoA-I antibody. (b) Histogram represents the percentage of N-Hcy apoA-I to total apoA-I using CS analyzer. The values were indicated by mean + SD (n = 3, ∗P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: Isoelectric focusing for apoA-I and apoA-I treated with homocysteine thiolactone. (a) Representative isoelectric focusing patterns of apoA-I and N-Hcy apoA-I. Purified apoA-I was incubated with or without 10 mmol/L of homocysteine thiolactone at 37°C for 6 hours. After dialysis against PBS, the samples were analyzed by isoelectric focusing followed by western blotting using anti-apoA-I antibody. (b) Histogram represents the percentage of N-Hcy apoA-I to total apoA-I using CS analyzer. The values were indicated by mean + SD (n = 3, ∗P < 0.05).
Mentions: Using various stages of cells regulated differentiation and foam cell formation, the effect of N-homocysteinylation on cholesterol efflux capacity of apoA-I was evaluated. To confirm the extent of N-homocysteinylation of apoA-I, apoA-I incubated with HcyT was tested using an isoelectric focusing. Compared to normal apoA-I, N-Hcy apoA-I indicates higher isoelectric point (pI) depending on the number of attached homocysteine compounds (Figure 6(a)). The ratio of N-Hcy apoA-I to total apoA-I on the isoelectric focusing pattern was 51.9 ± 5.9% (Figure 6(b)). Cholesterol efflux capacities of apoA-I and N-Hcy apoA-I reached those peaks at day 2 and gradually decreased depending on the periods of the treatment with PMA (Figure 7(a)). In case of foam cell formation, a significant decrease was observed in cholesterol efflux capacities of apoA-I and N-Hcy apoA-I according to the acLDL loading (Figure 7(b)). In addition, the cholesterol efflux capacity of N-Hcy apoA-I was significantly lower than that of intact apoA-I by differentiation and foam cell formation at day 1 and day 2, respectively; however no significant difference was observed after day 3 (Figure 7).

View Article: PubMed Central - PubMed

ABSTRACT

Apolipoprotein A-I (apoA-I), the main protein component of high-density lipoprotein (HDL), has many protective functions against atherosclerosis, one of them being cholesterol efflux capacity. Although cholesterol efflux capacity measurement is suggested to be a key biomarker for evaluating the risk of development of atherosclerosis, the assay has not been optimized till date. This study aims at investigating the effect of different states of cells on the cholesterol efflux capacity. We also studied the effect of apoA-I modification by homocysteine, a risk factor for atherosclerosis, on cholesterol efflux capacity in different states of cells. The cholesterol efflux capacity of apoA-I was greatly influenced by the extent of differentiation of THP-1 cells and attenuated by excessive foam cell formation. N-Homocysteinylated apoA-I indicated a lower cholesterol efflux capacity than normal apoA-I in the optimized condition, whereas no significant difference was observed in the cholesterol efflux capacity between apoA-I in the excessive cell differentiation or foam cell formation states. These results suggest that cholesterol efflux capacity of apoA-I varies depending on the state of cells. Therefore, the cholesterol efflux assay should be performed using protocols optimized according to the objective of the experiment.

No MeSH data available.