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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

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.


The morphological changes in THP-1 cells. THP-1 cells were treated with 100 ng/mL PMA for different duration (1 to 5 days). After treatment, (a) THP-1 cells were fixed with 3.7% formaldehyde and stained with hematoxylin (scale bars, 20 μm). (b) THP-1 cells were incubated with FITC-conjugated anti-CD11b antibody, and CD11b expression was analyzed using flow cytometry. The percentage of CD11b positive cells which exceeded the maximum fluorescent intensity of the isotype control in each day was indicated. The value at day 1 was defined as 1. The values were indicated by mean + SD (n = 5, ∗∗P < 0.01 versus day 1, †P < 0.05 versus day 1.5).
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fig1: The morphological changes in THP-1 cells. THP-1 cells were treated with 100 ng/mL PMA for different duration (1 to 5 days). After treatment, (a) THP-1 cells were fixed with 3.7% formaldehyde and stained with hematoxylin (scale bars, 20 μm). (b) THP-1 cells were incubated with FITC-conjugated anti-CD11b antibody, and CD11b expression was analyzed using flow cytometry. The percentage of CD11b positive cells which exceeded the maximum fluorescent intensity of the isotype control in each day was indicated. The value at day 1 was defined as 1. The values were indicated by mean + SD (n = 5, ∗∗P < 0.01 versus day 1, †P < 0.05 versus day 1.5).

Mentions: THP-1 cells were incubated with RPMI-1640 medium containing 100 ng/mL of phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) supplemented with 0.2% bovine serum albumin (BSA) in 24-well cell culture plates at a density of 2.5 × 105 cells/well. For preparation of various states of differentiated cells, the incubation time was varied from 1 to 5 days as indicated in Figure 1(a). After removing the supernatant, THP-1 cells were loaded with RPMI-1640 containing acetylated low-density lipoprotein (acLDL) (50 μg protein/mL), T0901317 (1 mmol/L; Enzo Life Sciences), a Liver X receptor (LXR) agonist for promoting expression of ABCA1, 3H-cholesterol (1 μCi/mL; PerkinElmer), and BSA (0.2%). The loading time was also varied over 1 to 5 days to generate different stages of foam cells. THP-1 cells were washed thrice with RPMI-1640 medium containing 0.2% BSA before equilibration with RPMI-1640 supplemented with T0901317 (1 mmol/L) and BSA (0.2%) for 18 hours. After removing the supernatant, cells were incubated with apoA-I (10 μg/mL) and T0901317 (1 mmol/L) in RPMI-1640 for additional 4 hours. Efflux medium was separated from the cells and NaOH (0.1 mol/L) was added directly to the cells for lysis. Both the media and cell lysates were counted for radioactivity using the liquid scintillation counter. Cholesterol efflux capacity was calculated as a percentage of radioactivity in the medium as per the given formula: {3H-cholesterol in medium/(3H-cholesterol in medium + 3H-cholesterol in cells)}  × 100 − percentage of passive diffusion in the case of no acceptor (apoA-I).


Cholesterol Efflux Capacity of Apolipoprotein A-I Varies with the Extent of Differentiation and Foam Cell Formation of THP-1 Cells
The morphological changes in THP-1 cells. THP-1 cells were treated with 100 ng/mL PMA for different duration (1 to 5 days). After treatment, (a) THP-1 cells were fixed with 3.7% formaldehyde and stained with hematoxylin (scale bars, 20 μm). (b) THP-1 cells were incubated with FITC-conjugated anti-CD11b antibody, and CD11b expression was analyzed using flow cytometry. The percentage of CD11b positive cells which exceeded the maximum fluorescent intensity of the isotype control in each day was indicated. The value at day 1 was defined as 1. The values were indicated by mean + SD (n = 5, ∗∗P < 0.01 versus day 1, †P < 0.05 versus day 1.5).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5120203&req=5

fig1: The morphological changes in THP-1 cells. THP-1 cells were treated with 100 ng/mL PMA for different duration (1 to 5 days). After treatment, (a) THP-1 cells were fixed with 3.7% formaldehyde and stained with hematoxylin (scale bars, 20 μm). (b) THP-1 cells were incubated with FITC-conjugated anti-CD11b antibody, and CD11b expression was analyzed using flow cytometry. The percentage of CD11b positive cells which exceeded the maximum fluorescent intensity of the isotype control in each day was indicated. The value at day 1 was defined as 1. The values were indicated by mean + SD (n = 5, ∗∗P < 0.01 versus day 1, †P < 0.05 versus day 1.5).
Mentions: THP-1 cells were incubated with RPMI-1640 medium containing 100 ng/mL of phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) supplemented with 0.2% bovine serum albumin (BSA) in 24-well cell culture plates at a density of 2.5 × 105 cells/well. For preparation of various states of differentiated cells, the incubation time was varied from 1 to 5 days as indicated in Figure 1(a). After removing the supernatant, THP-1 cells were loaded with RPMI-1640 containing acetylated low-density lipoprotein (acLDL) (50 μg protein/mL), T0901317 (1 mmol/L; Enzo Life Sciences), a Liver X receptor (LXR) agonist for promoting expression of ABCA1, 3H-cholesterol (1 μCi/mL; PerkinElmer), and BSA (0.2%). The loading time was also varied over 1 to 5 days to generate different stages of foam cells. THP-1 cells were washed thrice with RPMI-1640 medium containing 0.2% BSA before equilibration with RPMI-1640 supplemented with T0901317 (1 mmol/L) and BSA (0.2%) for 18 hours. After removing the supernatant, cells were incubated with apoA-I (10 μg/mL) and T0901317 (1 mmol/L) in RPMI-1640 for additional 4 hours. Efflux medium was separated from the cells and NaOH (0.1 mol/L) was added directly to the cells for lysis. Both the media and cell lysates were counted for radioactivity using the liquid scintillation counter. Cholesterol efflux capacity was calculated as a percentage of radioactivity in the medium as per the given formula: {3H-cholesterol in medium/(3H-cholesterol in medium + 3H-cholesterol in cells)}  × 100 − percentage of passive diffusion in the case of no acceptor (apoA-I).

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.