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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.

No MeSH data available.


CCL2 induced p42/44 MAPK (MAPK-ERK1/2) phosphorylation via CCR2 in HCAECs. Starved HCAECs were treated with either increasing concentrations of CCL2 (0–80 ng/ml) for 10 min or with 40 ng/ml CCL2 for the indicated times (0–60 min). Proteins were extracted from the cultured cells, and the changes in phosphorylation of p42/44 MAPK were analyzed by Western blotting as described under “Experimental Procedures.” The quantitative analysis of protein was performed using ImageJ software. First, the ratios of phosphorylated MAPK/total MAPK at each time point were calculated. Second, the percentage of phosphorylation at 5, 10, 15, 30, and 60 min was calculated as the ratio of phosphorylated MAPK at each time point/the ratio of zero time point. A, p-p42/44 protein expression of dose-effect induced by CCL2. B, p-p42/44 protein expression of time effect induced by CCL2. C, p42/44 MAPK inhibitor could block the phosphorylation of p42/44 MAPK induced by CCL2 via CCR2. Cells transiently expressing control siRNAs (siRNA-NC) or CCR2 siRNAs (siRNA-CCR2) were pretreated with 10 μm U0126 (a p42/44 MAPK inhibitor) for 30 min and then induced by 40 ng/ml CCL2 for 18 h. p-p42/44 protein expression were measured by Western blotting. The experiment was performed three times. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the untreated cells or the indicated groups.
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Figure 5: CCL2 induced p42/44 MAPK (MAPK-ERK1/2) phosphorylation via CCR2 in HCAECs. Starved HCAECs were treated with either increasing concentrations of CCL2 (0–80 ng/ml) for 10 min or with 40 ng/ml CCL2 for the indicated times (0–60 min). Proteins were extracted from the cultured cells, and the changes in phosphorylation of p42/44 MAPK were analyzed by Western blotting as described under “Experimental Procedures.” The quantitative analysis of protein was performed using ImageJ software. First, the ratios of phosphorylated MAPK/total MAPK at each time point were calculated. Second, the percentage of phosphorylation at 5, 10, 15, 30, and 60 min was calculated as the ratio of phosphorylated MAPK at each time point/the ratio of zero time point. A, p-p42/44 protein expression of dose-effect induced by CCL2. B, p-p42/44 protein expression of time effect induced by CCL2. C, p42/44 MAPK inhibitor could block the phosphorylation of p42/44 MAPK induced by CCL2 via CCR2. Cells transiently expressing control siRNAs (siRNA-NC) or CCR2 siRNAs (siRNA-CCR2) were pretreated with 10 μm U0126 (a p42/44 MAPK inhibitor) for 30 min and then induced by 40 ng/ml CCL2 for 18 h. p-p42/44 protein expression were measured by Western blotting. The experiment was performed three times. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the untreated cells or the indicated groups.

Mentions: First, we found that CCL2 treatment from 20 μg/ml up to the maximum of 40 μg/ml and from 5 min up to the maximum of 10 min resulted in an increase of p42/44 MAPK phosphorylation (Fig. 5, A and B). Additionally, this increase of p42/44 MAPK phosphorylation induced by CCL2 could be both blocked by pretreatment with siRNA-CCR2 and U0126, a specific pharmacological inhibitor of p42/44 MAPK (Fig. 5C), suggesting that p42/44 MAPK could be activated by CCL2 via CCR2 induction.


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 *
CCL2 induced p42/44 MAPK (MAPK-ERK1/2) phosphorylation via CCR2 in HCAECs. Starved HCAECs were treated with either increasing concentrations of CCL2 (0–80 ng/ml) for 10 min or with 40 ng/ml CCL2 for the indicated times (0–60 min). Proteins were extracted from the cultured cells, and the changes in phosphorylation of p42/44 MAPK were analyzed by Western blotting as described under “Experimental Procedures.” The quantitative analysis of protein was performed using ImageJ software. First, the ratios of phosphorylated MAPK/total MAPK at each time point were calculated. Second, the percentage of phosphorylation at 5, 10, 15, 30, and 60 min was calculated as the ratio of phosphorylated MAPK at each time point/the ratio of zero time point. A, p-p42/44 protein expression of dose-effect induced by CCL2. B, p-p42/44 protein expression of time effect induced by CCL2. C, p42/44 MAPK inhibitor could block the phosphorylation of p42/44 MAPK induced by CCL2 via CCR2. Cells transiently expressing control siRNAs (siRNA-NC) or CCR2 siRNAs (siRNA-CCR2) were pretreated with 10 μm U0126 (a p42/44 MAPK inhibitor) for 30 min and then induced by 40 ng/ml CCL2 for 18 h. p-p42/44 protein expression were measured by Western blotting. The experiment was performed three times. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the untreated cells or the indicated groups.
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Figure 5: CCL2 induced p42/44 MAPK (MAPK-ERK1/2) phosphorylation via CCR2 in HCAECs. Starved HCAECs were treated with either increasing concentrations of CCL2 (0–80 ng/ml) for 10 min or with 40 ng/ml CCL2 for the indicated times (0–60 min). Proteins were extracted from the cultured cells, and the changes in phosphorylation of p42/44 MAPK were analyzed by Western blotting as described under “Experimental Procedures.” The quantitative analysis of protein was performed using ImageJ software. First, the ratios of phosphorylated MAPK/total MAPK at each time point were calculated. Second, the percentage of phosphorylation at 5, 10, 15, 30, and 60 min was calculated as the ratio of phosphorylated MAPK at each time point/the ratio of zero time point. A, p-p42/44 protein expression of dose-effect induced by CCL2. B, p-p42/44 protein expression of time effect induced by CCL2. C, p42/44 MAPK inhibitor could block the phosphorylation of p42/44 MAPK induced by CCL2 via CCR2. Cells transiently expressing control siRNAs (siRNA-NC) or CCR2 siRNAs (siRNA-CCR2) were pretreated with 10 μm U0126 (a p42/44 MAPK inhibitor) for 30 min and then induced by 40 ng/ml CCL2 for 18 h. p-p42/44 protein expression were measured by Western blotting. The experiment was performed three times. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the untreated cells or the indicated groups.
Mentions: First, we found that CCL2 treatment from 20 μg/ml up to the maximum of 40 μg/ml and from 5 min up to the maximum of 10 min resulted in an increase of p42/44 MAPK phosphorylation (Fig. 5, A and B). Additionally, this increase of p42/44 MAPK phosphorylation induced by CCL2 could be both blocked by pretreatment with siRNA-CCR2 and U0126, a specific pharmacological inhibitor of p42/44 MAPK (Fig. 5C), suggesting that p42/44 MAPK could be activated by CCL2 via CCR2 induction.

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.