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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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The effect of differentiation on cholesterol efflux capacity of apoA-I. THP-1 cells were treated with 100 ng/mL PMA for different periods (1 to 5 days) and loaded with 3H-cholesterol (1 μCi/mL), acLDL (50 μg protein/mL), and T0901317 (1 μmol/L) for 1 day. The cholesterol efflux capacity of apoA-I (10 μg/mL) was determined under these conditions. The values were indicated by mean + SD (n = 3, ∗P < 0.05 versus day 1, †P < 0.05 versus day 2, and §P < 0.05 versus day 3).
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fig3: The effect of differentiation on cholesterol efflux capacity of apoA-I. THP-1 cells were treated with 100 ng/mL PMA for different periods (1 to 5 days) and loaded with 3H-cholesterol (1 μCi/mL), acLDL (50 μg protein/mL), and T0901317 (1 μmol/L) for 1 day. The cholesterol efflux capacity of apoA-I (10 μg/mL) was determined under these conditions. The values were indicated by mean + SD (n = 3, ∗P < 0.05 versus day 1, †P < 0.05 versus day 2, and §P < 0.05 versus day 3).

Mentions: THP-1 cells differentiated following treatment with PMA for 1 to 5 days. To confirm differentiation of the cells at each stage, the morphology of THP-1 cells was assessed by staining with hematoxylin (Figure 1(a)). Mild morphological changes were observed at day 1.5, and most of these were adendritic cells with approximately 20 μm size. From day 1.5 to day 2, dynamic changes in cells were observed. Cells grew larger and showed the appearance of dendrite formation corresponding to the increase in stimulation. Next, we evaluated the expression of CD11b, an adhesion molecule, on THP-1 cells by flow cytometric analysis (Figure 1(b)). The expression of CD11b was essentially increased in a stimulation with PMA on THP-1 macrophages, while the expression on THP-1 monocytes without PMA treatment was not almost detected (data not shown). Conclusively, expression of CD11b at day 2 was significantly higher than that at day 1 (P < 0.01) and maintained a plateau state (Figure 1(b)). A similar tendency was also observed in the western blotting analysis (Figures 2(a) and 2(b)). The effect of differentiation of THP-1 cells on cholesterol efflux capacity of apoA-I was evaluated on the condition that the period of foam cell formation was fixed at 1 day. The cholesterol efflux capacities were the highest and the second highest at day 2 and day 1 of differentiation, respectively, and gradually deceased after day 3 (Figure 3). Although the period of foam cell formation was fixed for 1 day, total 3H-cholesterol taken up by the cells before efflux was largely different according to the differentiation periods, the lowest level at day 1, a peak at day 2, and gradual decrease after day 3 (Supplementary Figure  2(a)). In addition, 3H-cholesterol mass in the medium after efflux also showed a similar tendency to total 3H-cholesterol taken up by the cells (Supplementary Figure  2(b)). Cholesterol efflux capacities expressed in percentage were largely different from the actual efflux of 3H-cholesterol mass. In contrast, ABCA1 expression was the highest at day 1 and gradually reduced in parallel with the duration of PMA treatment (Supplementary Figure  3).


Cholesterol Efflux Capacity of Apolipoprotein A-I Varies with the Extent of Differentiation and Foam Cell Formation of THP-1 Cells
The effect of differentiation on cholesterol efflux capacity of apoA-I. THP-1 cells were treated with 100 ng/mL PMA for different periods (1 to 5 days) and loaded with 3H-cholesterol (1 μCi/mL), acLDL (50 μg protein/mL), and T0901317 (1 μmol/L) for 1 day. The cholesterol efflux capacity of apoA-I (10 μg/mL) was determined under these conditions. The values were indicated by mean + SD (n = 3, ∗P < 0.05 versus day 1, †P < 0.05 versus day 2, and §P < 0.05 versus day 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5120203&req=5

fig3: The effect of differentiation on cholesterol efflux capacity of apoA-I. THP-1 cells were treated with 100 ng/mL PMA for different periods (1 to 5 days) and loaded with 3H-cholesterol (1 μCi/mL), acLDL (50 μg protein/mL), and T0901317 (1 μmol/L) for 1 day. The cholesterol efflux capacity of apoA-I (10 μg/mL) was determined under these conditions. The values were indicated by mean + SD (n = 3, ∗P < 0.05 versus day 1, †P < 0.05 versus day 2, and §P < 0.05 versus day 3).
Mentions: THP-1 cells differentiated following treatment with PMA for 1 to 5 days. To confirm differentiation of the cells at each stage, the morphology of THP-1 cells was assessed by staining with hematoxylin (Figure 1(a)). Mild morphological changes were observed at day 1.5, and most of these were adendritic cells with approximately 20 μm size. From day 1.5 to day 2, dynamic changes in cells were observed. Cells grew larger and showed the appearance of dendrite formation corresponding to the increase in stimulation. Next, we evaluated the expression of CD11b, an adhesion molecule, on THP-1 cells by flow cytometric analysis (Figure 1(b)). The expression of CD11b was essentially increased in a stimulation with PMA on THP-1 macrophages, while the expression on THP-1 monocytes without PMA treatment was not almost detected (data not shown). Conclusively, expression of CD11b at day 2 was significantly higher than that at day 1 (P < 0.01) and maintained a plateau state (Figure 1(b)). A similar tendency was also observed in the western blotting analysis (Figures 2(a) and 2(b)). The effect of differentiation of THP-1 cells on cholesterol efflux capacity of apoA-I was evaluated on the condition that the period of foam cell formation was fixed at 1 day. The cholesterol efflux capacities were the highest and the second highest at day 2 and day 1 of differentiation, respectively, and gradually deceased after day 3 (Figure 3). Although the period of foam cell formation was fixed for 1 day, total 3H-cholesterol taken up by the cells before efflux was largely different according to the differentiation periods, the lowest level at day 1, a peak at day 2, and gradual decrease after day 3 (Supplementary Figure  2(a)). In addition, 3H-cholesterol mass in the medium after efflux also showed a similar tendency to total 3H-cholesterol taken up by the cells (Supplementary Figure  2(b)). Cholesterol efflux capacities expressed in percentage were largely different from the actual efflux of 3H-cholesterol mass. In contrast, ABCA1 expression was the highest at day 1 and gradually reduced in parallel with the duration of PMA treatment (Supplementary Figure  3).

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.


Related in: MedlinePlus