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CC-Chemokine Ligand 2 (CCL2) Suppresses High Density Lipoprotein (HDL) Internalization and Cholesterol Efflux via CC-Chemokine Receptor 2 (CCR2) Induction and p42/44 Mitogen-activated Protein Kinase (MAPK) Activation in Human Endothelial Cells *

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ABSTRACT

High density lipoprotein (HDL) has been proposed to be internalized and to promote reverse cholesterol transport in endothelial cells (ECs). However, the mechanism underlying these processes has not been studied. In this study, we aim to characterize HDL internalization and cholesterol efflux in ECs and regulatory mechanisms. We found mature HDL particles were reduced in patients with coronary artery disease (CAD), which was associated with an increase in CC-chemokine ligand 2 (CCL2). In cultured primary human coronary artery endothelial cells and human umbilical vein endothelial cells, we determined that CCL2 suppressed the binding (4 °C) and association (37 °C) of HDL to/with ECs and HDL cellular internalization. Furthermore, CCL2 inhibited [3H]cholesterol efflux to HDL/apoA1 in ECs. We further found that CCL2 induced CC-chemokine receptor 2 (CCR2) expression and siRNA-CCR2 reversed CCL2 suppression on HDL binding, association, internalization, and on cholesterol efflux in ECs. Moreover, CCL2 induced p42/44 mitogen-activated protein kinase (MAPK) phosphorylation via CCR2, and p42/44 MAPK inhibition reversed the suppression of CCL2 on HDL metabolism in ECs. Our study suggests that CCL2 was elevated in CAD patients. CCL2 suppressed HDL internalization and cholesterol efflux via CCR2 induction and p42/44 MAPK activation in ECs. CCL2 induction may contribute to impair HDL function and form atherosclerosis in CAD.

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p42/44 MAPK inhibition reversed CCL2 suppression on HDL binding, association, and internalization and [3H]cholesterol efflux to HDL/apoA1 in HCAECs. HCAECs were preincubated with 10 μm U0126 for 30 min and then treated with 40 ng/ml CCL2 for 18 h. 125I-HDL binding at 4 °C, 125I-HDL association at 37 °C, HDL internalization, and [3H]cholesterol efflux were determined as the method in Figs. 1 and 2. A, effect of U0126 on the decrease of 125I-HDL binding at 4 °C induced by CCL2. B, effect of U0126 on the decrease of 125I-HDL association at 37 °C. C, effect of U0126 on the suppression of the internalization of HDL phospholipid and protein (×20). D, effect of U0126 on the suppression of perinuclear distribution of HDL protein (×63). E, effect of U0126 on the suppression of [3H]cholesterol efflux to HDL. F, effect of U0126 on the decrease of [3H]cholesterol efflux to apoA1. The results are represented as means ± S.D. of at least three individual experiments. *, p < 0.05; ***, p < 0.001 compared with the indicated group.
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Figure 6: p42/44 MAPK inhibition reversed CCL2 suppression on HDL binding, association, and internalization and [3H]cholesterol efflux to HDL/apoA1 in HCAECs. HCAECs were preincubated with 10 μm U0126 for 30 min and then treated with 40 ng/ml CCL2 for 18 h. 125I-HDL binding at 4 °C, 125I-HDL association at 37 °C, HDL internalization, and [3H]cholesterol efflux were determined as the method in Figs. 1 and 2. A, effect of U0126 on the decrease of 125I-HDL binding at 4 °C induced by CCL2. B, effect of U0126 on the decrease of 125I-HDL association at 37 °C. C, effect of U0126 on the suppression of the internalization of HDL phospholipid and protein (×20). D, effect of U0126 on the suppression of perinuclear distribution of HDL protein (×63). E, effect of U0126 on the suppression of [3H]cholesterol efflux to HDL. F, effect of U0126 on the decrease of [3H]cholesterol efflux to apoA1. The results are represented as means ± S.D. of at least three individual experiments. *, p < 0.05; ***, p < 0.001 compared with the indicated group.

Mentions: Next, we determined whether the suppression of CCL2 on the HDL internalization in ECs could be also reversed via p42/44 MAPK activation. In U0126-pretreated HCAECs, CCL2 only decreased 125I-HDL binding and cell association by 25.7 and 29.5%, respectively. However, in non-U0126-pretreated HCAECs, CCL2 decreased 125I-HDL binding and cell association by 47.0 and 50.4% compared with the control (Fig. 6, A and B). Similarly, pretreatment with U0126 also improved the suppression on the internalization of DIL-HDL phospholipid and Alexa 488-HDL protein (Fig. 6C). We also traced the reversed effect of internalized Alexa 488-HDL protein in the perinuclear area by U0126 pretreatment. The HDL protein localized in the perinuclear area, which was suppressed by CCL2, could be effectively reversed after U0126 pretreatment (Fig. 6D).


CC-Chemokine Ligand 2 (CCL2) Suppresses High Density Lipoprotein (HDL) Internalization and Cholesterol Efflux via CC-Chemokine Receptor 2 (CCR2) Induction and p42/44 Mitogen-activated Protein Kinase (MAPK) Activation in Human Endothelial Cells *
p42/44 MAPK inhibition reversed CCL2 suppression on HDL binding, association, and internalization and [3H]cholesterol efflux to HDL/apoA1 in HCAECs. HCAECs were preincubated with 10 μm U0126 for 30 min and then treated with 40 ng/ml CCL2 for 18 h. 125I-HDL binding at 4 °C, 125I-HDL association at 37 °C, HDL internalization, and [3H]cholesterol efflux were determined as the method in Figs. 1 and 2. A, effect of U0126 on the decrease of 125I-HDL binding at 4 °C induced by CCL2. B, effect of U0126 on the decrease of 125I-HDL association at 37 °C. C, effect of U0126 on the suppression of the internalization of HDL phospholipid and protein (×20). D, effect of U0126 on the suppression of perinuclear distribution of HDL protein (×63). E, effect of U0126 on the suppression of [3H]cholesterol efflux to HDL. F, effect of U0126 on the decrease of [3H]cholesterol efflux to apoA1. The results are represented as means ± S.D. of at least three individual experiments. *, p < 0.05; ***, p < 0.001 compared with the indicated group.
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Figure 6: p42/44 MAPK inhibition reversed CCL2 suppression on HDL binding, association, and internalization and [3H]cholesterol efflux to HDL/apoA1 in HCAECs. HCAECs were preincubated with 10 μm U0126 for 30 min and then treated with 40 ng/ml CCL2 for 18 h. 125I-HDL binding at 4 °C, 125I-HDL association at 37 °C, HDL internalization, and [3H]cholesterol efflux were determined as the method in Figs. 1 and 2. A, effect of U0126 on the decrease of 125I-HDL binding at 4 °C induced by CCL2. B, effect of U0126 on the decrease of 125I-HDL association at 37 °C. C, effect of U0126 on the suppression of the internalization of HDL phospholipid and protein (×20). D, effect of U0126 on the suppression of perinuclear distribution of HDL protein (×63). E, effect of U0126 on the suppression of [3H]cholesterol efflux to HDL. F, effect of U0126 on the decrease of [3H]cholesterol efflux to apoA1. The results are represented as means ± S.D. of at least three individual experiments. *, p < 0.05; ***, p < 0.001 compared with the indicated group.
Mentions: Next, we determined whether the suppression of CCL2 on the HDL internalization in ECs could be also reversed via p42/44 MAPK activation. In U0126-pretreated HCAECs, CCL2 only decreased 125I-HDL binding and cell association by 25.7 and 29.5%, respectively. However, in non-U0126-pretreated HCAECs, CCL2 decreased 125I-HDL binding and cell association by 47.0 and 50.4% compared with the control (Fig. 6, A and B). Similarly, pretreatment with U0126 also improved the suppression on the internalization of DIL-HDL phospholipid and Alexa 488-HDL protein (Fig. 6C). We also traced the reversed effect of internalized Alexa 488-HDL protein in the perinuclear area by U0126 pretreatment. The HDL protein localized in the perinuclear area, which was suppressed by CCL2, could be effectively reversed after U0126 pretreatment (Fig. 6D).

View Article: PubMed Central - PubMed

ABSTRACT

High density lipoprotein (HDL) has been proposed to be internalized and to promote reverse cholesterol transport in endothelial cells (ECs). However, the mechanism underlying these processes has not been studied. In this study, we aim to characterize HDL internalization and cholesterol efflux in ECs and regulatory mechanisms. We found mature HDL particles were reduced in patients with coronary artery disease (CAD), which was associated with an increase in CC-chemokine ligand 2 (CCL2). In cultured primary human coronary artery endothelial cells and human umbilical vein endothelial cells, we determined that CCL2 suppressed the binding (4 &deg;C) and association (37 &deg;C) of HDL to/with ECs and HDL cellular internalization. Furthermore, CCL2 inhibited [3H]cholesterol efflux to HDL/apoA1 in ECs. We further found that CCL2 induced CC-chemokine receptor 2 (CCR2) expression and siRNA-CCR2 reversed CCL2 suppression on HDL binding, association, internalization, and on cholesterol efflux in ECs. Moreover, CCL2 induced p42/44 mitogen-activated protein kinase (MAPK) phosphorylation via CCR2, and p42/44 MAPK inhibition reversed the suppression of CCL2 on HDL metabolism in ECs. Our study suggests that CCL2 was elevated in CAD patients. CCL2 suppressed HDL internalization and cholesterol efflux via CCR2 induction and p42/44 MAPK activation in ECs. CCL2 induction may contribute to impair HDL function and form atherosclerosis in CAD.

No MeSH data available.


Related in: MedlinePlus