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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 *

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 °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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Related in: MedlinePlus

CCL2 suppressed the binding (4 °C) and association (37 °C) of HDL to/with ECs, and HDL cellular internalization.A, binding of 125I-HDL to ECs at 4 °C. HCAECs and HUVECs were incubated with the indicated concentration of 125I-HDL (0, 5, 10, 15, and 20 μg/ml) for 1 h, in the absence (total binding) or in the presence of a 40-fold excess of unlabeled HDL (nonspecific binding) at 4 °C. Then the cells were solubilized in 0.1 n NaOH for 60 min, and the protein concentration and radioactivity were measured in the lysate. Radioactivity was measured by using a counter from PerkinElmer Life Sciences, and the measurements were normalized to the protein content determined by the Bradford protein assay (Bio-Rad). Specific binding was calculated by subtracting the values of the unspecific binding from those of the total binding. B, dose response of CCL2 on HDL binding. Cells were incubated with 10 μg/ml 125I-HDL at 4 °C for 1 h after treating with increasing doses of CCL2 (0, 20, 40, and 80 ng/ml) for 18 h. Other procedures were the same as A. C, time course of CCL2 on HDL binding. Cells were incubated with 10 μg/ml 125I-HDL at 4 °C for 1 h after treating with CCL2 at 40 ng/ml for the indicated times (0, 12, 18, and 24 h). Other procedures were the same as A. D, association of 125I-HDL with ECs at 37 °C. HCAECs and HUVECs were incubated with the indicated concentrations of 125I-HDL (0, 5, 10, 15, and 20 μg/ml) for 1 h in the absence or in the presence of a 40-fold excess of unlabeled HDL at 37 °C. Other procedures were the same as A. E, dose response of CCL2 on HDL association. Cells were incubated with 10 μg/ml 125I-HDL at 37 °C for 1 h after treating with increasing doses of CCL2 (0, 20, 40, and 80 ng/ml) for 18 h. Other procedures were the same as A. F, time course of CCL2 on HDL association. Cells were incubated with 10 μg/ml 125I-HDL at 37 °C for 1 h after treating with CCL2 at 40 ng/ml for the indicated times (0, 12, 18, and 24 h). Other procedures were the same as A. G, effect of CCL2 on HDL internalization. Live ECs were incubated with 10 μg/ml Alexa 488-HDL (green)-labeled protein and DIL-HDL(red)-labeled phospholipid for 1 h at 37 °C after treating with 40 ng/ml CCL2 for18 h. The cells were fixed and imaged using a confocal microscope (LSM780) (×20). H, quantitative analysis of HDL internalization induced by CCL2. I, HDL perinuclear localization. Cells were incubated with Alexa 488-HDL (green) together with Alexa 594-transferrin (red) after treating with 40 ng/ml CCL2 for 18 h, and partial co-localization (yellow) was assessed. Cells were fixed and imaged using a confocal microscope (LSM780) (×20). The results are represented as the mean ± S.D. of at least three individual experiments performed in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the untreated cells.
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Figure 1: CCL2 suppressed the binding (4 °C) and association (37 °C) of HDL to/with ECs, and HDL cellular internalization.A, binding of 125I-HDL to ECs at 4 °C. HCAECs and HUVECs were incubated with the indicated concentration of 125I-HDL (0, 5, 10, 15, and 20 μg/ml) for 1 h, in the absence (total binding) or in the presence of a 40-fold excess of unlabeled HDL (nonspecific binding) at 4 °C. Then the cells were solubilized in 0.1 n NaOH for 60 min, and the protein concentration and radioactivity were measured in the lysate. Radioactivity was measured by using a counter from PerkinElmer Life Sciences, and the measurements were normalized to the protein content determined by the Bradford protein assay (Bio-Rad). Specific binding was calculated by subtracting the values of the unspecific binding from those of the total binding. B, dose response of CCL2 on HDL binding. Cells were incubated with 10 μg/ml 125I-HDL at 4 °C for 1 h after treating with increasing doses of CCL2 (0, 20, 40, and 80 ng/ml) for 18 h. Other procedures were the same as A. C, time course of CCL2 on HDL binding. Cells were incubated with 10 μg/ml 125I-HDL at 4 °C for 1 h after treating with CCL2 at 40 ng/ml for the indicated times (0, 12, 18, and 24 h). Other procedures were the same as A. D, association of 125I-HDL with ECs at 37 °C. HCAECs and HUVECs were incubated with the indicated concentrations of 125I-HDL (0, 5, 10, 15, and 20 μg/ml) for 1 h in the absence or in the presence of a 40-fold excess of unlabeled HDL at 37 °C. Other procedures were the same as A. E, dose response of CCL2 on HDL association. Cells were incubated with 10 μg/ml 125I-HDL at 37 °C for 1 h after treating with increasing doses of CCL2 (0, 20, 40, and 80 ng/ml) for 18 h. Other procedures were the same as A. F, time course of CCL2 on HDL association. Cells were incubated with 10 μg/ml 125I-HDL at 37 °C for 1 h after treating with CCL2 at 40 ng/ml for the indicated times (0, 12, 18, and 24 h). Other procedures were the same as A. G, effect of CCL2 on HDL internalization. Live ECs were incubated with 10 μg/ml Alexa 488-HDL (green)-labeled protein and DIL-HDL(red)-labeled phospholipid for 1 h at 37 °C after treating with 40 ng/ml CCL2 for18 h. The cells were fixed and imaged using a confocal microscope (LSM780) (×20). H, quantitative analysis of HDL internalization induced by CCL2. I, HDL perinuclear localization. Cells were incubated with Alexa 488-HDL (green) together with Alexa 594-transferrin (red) after treating with 40 ng/ml CCL2 for 18 h, and partial co-localization (yellow) was assessed. Cells were fixed and imaged using a confocal microscope (LSM780) (×20). The results are represented as the mean ± S.D. of at least three individual experiments performed in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the untreated cells.

Mentions: We next determined the effect of CCL2 on HDL uptake by ECs. This effect was characterized by determining 125I-HDL binding at 4 °C and cell association at 37 °C in live HCAECs and HUVECs. The specific binding of HDL at 4 °C and association at 37 °C to/with ECs were increased with the increasing doses of 125I-HDL (Fig. 1, A and D). ECs were treated with increasing doses of CCL2 (0, 20, 40, and 80 ng/ml) for 18 h or with increasing times (0, 12, 18, and 24 h) at a fixed dose of 40 ng/ml CCL2. CCL2 decreased 125I-HDL binding in HCAECs and HUVECs from 20.2 to 19.5% at 20 ng/ml up to 52.8 and 47.7% at 80 ng/ml (Fig. 1B) and from 19.2 to 13.9% at 12 h up to 51.0 and 43.2% at 24 h (Fig. 1C), respectively. Similarly, CCL2 also decreased 125I-HDL cell association in HCAECs and HUVECs from 6.1 to 16.6% at 20 ng/ml up to the maximum values of 36.6 and 42.7% at 80 ng/ml (Fig. 1E) and from 13.2 to 25.1% at 12 h up to the maximum values of 60.6 and 65.0% at 24 h (Fig. 1F), respectively. Because of the significance of the observed results, all subsequent experiments were performed with cells incubated with 40 ng/ml CCL2 for 18 h. We found TNF-α (10 ng/ml for 24 h) incubated with HCAECs could produce the maximum concentration of endogenous CCL2 (16,988 ± 276 pg/ml) but could not significantly inhibit the 125I-HDL binding and association to/with ECs (data not shown).


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 suppressed the binding (4 °C) and association (37 °C) of HDL to/with ECs, and HDL cellular internalization.A, binding of 125I-HDL to ECs at 4 °C. HCAECs and HUVECs were incubated with the indicated concentration of 125I-HDL (0, 5, 10, 15, and 20 μg/ml) for 1 h, in the absence (total binding) or in the presence of a 40-fold excess of unlabeled HDL (nonspecific binding) at 4 °C. Then the cells were solubilized in 0.1 n NaOH for 60 min, and the protein concentration and radioactivity were measured in the lysate. Radioactivity was measured by using a counter from PerkinElmer Life Sciences, and the measurements were normalized to the protein content determined by the Bradford protein assay (Bio-Rad). Specific binding was calculated by subtracting the values of the unspecific binding from those of the total binding. B, dose response of CCL2 on HDL binding. Cells were incubated with 10 μg/ml 125I-HDL at 4 °C for 1 h after treating with increasing doses of CCL2 (0, 20, 40, and 80 ng/ml) for 18 h. Other procedures were the same as A. C, time course of CCL2 on HDL binding. Cells were incubated with 10 μg/ml 125I-HDL at 4 °C for 1 h after treating with CCL2 at 40 ng/ml for the indicated times (0, 12, 18, and 24 h). Other procedures were the same as A. D, association of 125I-HDL with ECs at 37 °C. HCAECs and HUVECs were incubated with the indicated concentrations of 125I-HDL (0, 5, 10, 15, and 20 μg/ml) for 1 h in the absence or in the presence of a 40-fold excess of unlabeled HDL at 37 °C. Other procedures were the same as A. E, dose response of CCL2 on HDL association. Cells were incubated with 10 μg/ml 125I-HDL at 37 °C for 1 h after treating with increasing doses of CCL2 (0, 20, 40, and 80 ng/ml) for 18 h. Other procedures were the same as A. F, time course of CCL2 on HDL association. Cells were incubated with 10 μg/ml 125I-HDL at 37 °C for 1 h after treating with CCL2 at 40 ng/ml for the indicated times (0, 12, 18, and 24 h). Other procedures were the same as A. G, effect of CCL2 on HDL internalization. Live ECs were incubated with 10 μg/ml Alexa 488-HDL (green)-labeled protein and DIL-HDL(red)-labeled phospholipid for 1 h at 37 °C after treating with 40 ng/ml CCL2 for18 h. The cells were fixed and imaged using a confocal microscope (LSM780) (×20). H, quantitative analysis of HDL internalization induced by CCL2. I, HDL perinuclear localization. Cells were incubated with Alexa 488-HDL (green) together with Alexa 594-transferrin (red) after treating with 40 ng/ml CCL2 for 18 h, and partial co-localization (yellow) was assessed. Cells were fixed and imaged using a confocal microscope (LSM780) (×20). The results are represented as the mean ± S.D. of at least three individual experiments performed in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the untreated cells.
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Figure 1: CCL2 suppressed the binding (4 °C) and association (37 °C) of HDL to/with ECs, and HDL cellular internalization.A, binding of 125I-HDL to ECs at 4 °C. HCAECs and HUVECs were incubated with the indicated concentration of 125I-HDL (0, 5, 10, 15, and 20 μg/ml) for 1 h, in the absence (total binding) or in the presence of a 40-fold excess of unlabeled HDL (nonspecific binding) at 4 °C. Then the cells were solubilized in 0.1 n NaOH for 60 min, and the protein concentration and radioactivity were measured in the lysate. Radioactivity was measured by using a counter from PerkinElmer Life Sciences, and the measurements were normalized to the protein content determined by the Bradford protein assay (Bio-Rad). Specific binding was calculated by subtracting the values of the unspecific binding from those of the total binding. B, dose response of CCL2 on HDL binding. Cells were incubated with 10 μg/ml 125I-HDL at 4 °C for 1 h after treating with increasing doses of CCL2 (0, 20, 40, and 80 ng/ml) for 18 h. Other procedures were the same as A. C, time course of CCL2 on HDL binding. Cells were incubated with 10 μg/ml 125I-HDL at 4 °C for 1 h after treating with CCL2 at 40 ng/ml for the indicated times (0, 12, 18, and 24 h). Other procedures were the same as A. D, association of 125I-HDL with ECs at 37 °C. HCAECs and HUVECs were incubated with the indicated concentrations of 125I-HDL (0, 5, 10, 15, and 20 μg/ml) for 1 h in the absence or in the presence of a 40-fold excess of unlabeled HDL at 37 °C. Other procedures were the same as A. E, dose response of CCL2 on HDL association. Cells were incubated with 10 μg/ml 125I-HDL at 37 °C for 1 h after treating with increasing doses of CCL2 (0, 20, 40, and 80 ng/ml) for 18 h. Other procedures were the same as A. F, time course of CCL2 on HDL association. Cells were incubated with 10 μg/ml 125I-HDL at 37 °C for 1 h after treating with CCL2 at 40 ng/ml for the indicated times (0, 12, 18, and 24 h). Other procedures were the same as A. G, effect of CCL2 on HDL internalization. Live ECs were incubated with 10 μg/ml Alexa 488-HDL (green)-labeled protein and DIL-HDL(red)-labeled phospholipid for 1 h at 37 °C after treating with 40 ng/ml CCL2 for18 h. The cells were fixed and imaged using a confocal microscope (LSM780) (×20). H, quantitative analysis of HDL internalization induced by CCL2. I, HDL perinuclear localization. Cells were incubated with Alexa 488-HDL (green) together with Alexa 594-transferrin (red) after treating with 40 ng/ml CCL2 for 18 h, and partial co-localization (yellow) was assessed. Cells were fixed and imaged using a confocal microscope (LSM780) (×20). The results are represented as the mean ± S.D. of at least three individual experiments performed in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the untreated cells.
Mentions: We next determined the effect of CCL2 on HDL uptake by ECs. This effect was characterized by determining 125I-HDL binding at 4 °C and cell association at 37 °C in live HCAECs and HUVECs. The specific binding of HDL at 4 °C and association at 37 °C to/with ECs were increased with the increasing doses of 125I-HDL (Fig. 1, A and D). ECs were treated with increasing doses of CCL2 (0, 20, 40, and 80 ng/ml) for 18 h or with increasing times (0, 12, 18, and 24 h) at a fixed dose of 40 ng/ml CCL2. CCL2 decreased 125I-HDL binding in HCAECs and HUVECs from 20.2 to 19.5% at 20 ng/ml up to 52.8 and 47.7% at 80 ng/ml (Fig. 1B) and from 19.2 to 13.9% at 12 h up to 51.0 and 43.2% at 24 h (Fig. 1C), respectively. Similarly, CCL2 also decreased 125I-HDL cell association in HCAECs and HUVECs from 6.1 to 16.6% at 20 ng/ml up to the maximum values of 36.6 and 42.7% at 80 ng/ml (Fig. 1E) and from 13.2 to 25.1% at 12 h up to the maximum values of 60.6 and 65.0% at 24 h (Fig. 1F), respectively. Because of the significance of the observed results, all subsequent experiments were performed with cells incubated with 40 ng/ml CCL2 for 18 h. We found TNF-α (10 ng/ml for 24 h) incubated with HCAECs could produce the maximum concentration of endogenous CCL2 (16,988 ± 276 pg/ml) but could not significantly inhibit the 125I-HDL binding and association to/with ECs (data not shown).

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