Reduction of Intracellular Chloride Concentration Promotes Foam Cell Formation

Abstract

Background: Previous work has demonstrated that the volume-regulated chloride channel is activated during foam cell formation, and inhibition of chloride movement prevents intracellular lipid accumulation. However, the mechanism explaining how chloride movement promotes foam cell formation is not clear.

Methods and Results: Foam cell formation was determined by Oil Red O staining. Western blotting and co-immunoprecipitation were used to examine protein expression and protein-protein interaction. [Cl⁻]_i was measured using 6-methoxy-N-ethylquinolinium iodide dye. The results showed that [Cl⁻]_i was decreased in monocytes/macrophages from patients with hypercholesterolemia and from apoE^−/− mice fed with a high-fat diet. Lowering [Cl⁻]_i upregulated scavenger receptor A (SR-A) expression, increased the binding and uptake of oxLDL, enhanced pro-inflammatory cytokine production and subsequently accelerated foam cell formation in macrophages from humans and mice. In addition, low Cl⁻ solution stimulated the activation of JNK and p38 mitogen-activated protein kinases. Inhibition of JNK and p38 blocked Cl⁻ reduced medium-induced SR-A expression and lipid accumulation. In contrast, reduction of [Cl⁻]_i promoted the interaction of SR-A with caveolin-1, thus facilitating caveolin-1-dependent SR-A endocytosis. Moreover, disruption of caveolae attenuated SR-A internalization, JNK and p38 activation, and ultimately prevented SR-A expression and foam cell formation stimulated by low Cl⁻ medium.

Conclusions: This data provide strong evidence that reduction of [Cl⁻]_i is a critical contributor to intracellular lipid accumulation, suggesting that modulation of [Cl⁻]_i is a novel avenue to prevent foam cell formation and atherosclerosis. (Circ J 2016; 80: 1024–1033)

Atherosclerosis, which is characterized by the accumulation of lipids in the large arteries, is the primary cause of cardiovascular diseases.¹ The development of atherosclerosis has been regarded as a chronic inflammatory process in response to abnormal lipid metabolism.² During atherogenesis, hyperlipidemia-induced endothelial activation would activate the transmigration of circulating leukocytes into the subintima, where the monocytes become macrophages and these macrophages then transform into foam cells after uptaking modified lipoproteins such as the oxidized and acetylated forms of low-density lipoproteins (oxLDL and acLDL) through macrophage scavenger receptors, including scavenger receptor A (SR-A) and CD36.^3–5 Although foam cell formation has been suggested to be a critical step for atherogenesis, the molecular mechanisms regulating this pathophysiological process are not fully understood.

Chloride (Cl⁻) is the major anion in extracellular fluid.⁶ Recent accumulating evidence using pharmacological inhibitor or genetic deletion strategies have revealed that chloride channels, including the volume-regulated chloride channel and the calcium-activated chloride channel, play a critical role in regulating cell volume, proliferation, differentiation, migration, apoptosis, insulin secretion, neuron excitability and synaptic activation.^7–10 Interestingly, our recent work in macrophages showed that the activity of the volume-regulated chloride channel is increased during foam cell formation in response to oxLDL. Inhibition of the volume-regulated chloride channel obviously prevented lipid accumulation in macrophages.¹¹ These results demonstrated that the volume-regulated chloride channel is an essential regulator of lipid uptake; however, the mechanism explaining how the volume-regulated chloride channel promotes foam cell formation is not clear.

Chloride channel is one of the major routes for chloride transport across the cell membrane. In fact, previous work from our lab and another have revealed that several stimuli, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β and angiotensin II, could activate a ClC-3-dependent chloride efflux in endothelial cells and vascular smooth muscle cells, and thus evoke the decrease of intracellular chloride concentration ([Cl⁻]_i).^6,12 Moreover, we provided the evidence showing that a reduction of [Cl⁻]_i could activate the NFκB pathway and thus promote endothelial cell inflammation,⁶ indicative of the critical role of [Cl⁻]_i in cardiovascular diseases. Notably, during foam cell formation, we also observed that [Cl⁻]_i is decreased following the activation of the volume-regulated chloride channel.¹¹ Therefore, we hypothesized that the reduction of [Cl⁻]_i may be a critical contributor to foam cell formation.

To test this hypothesis, we examined the lipid uptake in response to oxLDL in macrophages under normal or low [Cl⁻]_i. Our results demonstrated that a reduction of [Cl⁻]_i promoted lipid uptake by upregulating SR-A expression through activating the JNK/p38 signaling pathway.

Methods

Ethics Statement

The acquisition of peripheral venous blood was approved by the Medical Research Ethics Committee of Sun Yat-Sen University. Informed consent was obtained from all subjects, and the experiments were conducted according to the principles expressed in the Declaration of Helsinki. All animal experiments were approved by the Sun Yat-Sen University Committee for Animal Research and conformed to the “Guide for the Care and Use of Laboratory Animals” of the National Institute of Health in China.

Materials and Reagents

RPMI 1640 medium and fetal calf serum were purchased from Gibco (Carlsbad, CA, USA). Methylβcyclodextrin (MβCD), genistein, SP600125, SB203580, Oil red O and Hoechst 33258 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Caveolin-1, GAPDH, β-actin, phospho-JNK (Thr183/Tyr185), JNK, IgG antibodies and Protein A/G agarose were from Santa Cruz Biotechnology (Dallas, TX, USA). Phospho-p38 (Thr180/Tyr182) and p38 antibodies were purchased from Cell Signaling Technology (Danver, MA, USA). SR-A and CD36 antibodies and IL-1β, IL-6, and TNF-α Quantikine ELISA Kits were from R&D Systems (Minneapolis, MN, USA).

Cell Culture and Treatment

C57BL/6 mice were obtained from the Animal Experiment Center of Zhongshan Medicine School and housed on a 12:12 h light/dark cycle with free access to water and diet. Mice were anesthetized intraperitoneally with 2% pentobarbital sodium, and thioglycolate-elicited peritoneal macrophages were isolated from mice, as previously described.¹¹ THP-1 cells were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). THP-1 cells were incubated with 100 ng/ml PMA for 24 h to differentiate into macrophages. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 100 U/ml each of penicillin and 100 ug/ml streptomycin at 37℃ in 5% CO₂ and 95% humidity.

Human Blood Collection and Monocyte Isolation

Peripheral venous blood was isolated from healthy adult volunteers and hypercholesterolemia patients from Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University. The baseline classification according to age, sex, body mass index (BMI), total cholesterol, triglyceride and, HDL-c and LDL-c is presented in Table S1. Blood samples were collected into heparin-containing tubes and peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over a Ficoll Histopaque gradient. Monocytes were then isolated from PBMCs using positive selection with CD14 MicroBeads according to the manufacturer’s instructions (Myltenyi Biotec, Germany). Monocytes were induced to differentiate to macrophages for 6 days in the presence of recombinant human M-CSF (25 ng/ml). The cells were then cultured in RPMI 1640 supplemented with L-glutamine, penicillin, streptomycin, and 10% FCS.

Preparation of Chloride-Reduced Medium

Chloride-reduced medium was prepared by replacing chloride with gluconate, as previously described.⁶ Briefly, RPMI 1640 medium lacking NaCl and KCl was initially obtained from Invitrogen (Grand Island, NE, USA). The normal chloride medium was prepared by adding 105 mmol/L NaCl and 5 mmol/L KCl. The chloride-free medium (low Cl⁻) was prepared by adding 105 mmol/L sodium gluconate and 5 mmol/L potassium gluconate (pH=7.2). The osmolarities of the solutions ranged from 303.4 to 310.2 mosmol/kg·H₂O and were measured with a freezing point depression osmometer (OSMOMAT030, Germany).

Measurement of [Cl⁻]_i

Intracellular chloride concentration [Cl⁻]_i was measured using 6-methoxy-N-ethylquinolinium iodide (MEQ), as previously described.⁸ Briefly, MEQ was reduced to its cell-permeable derivative, 6-methoxy-N-ethyl-1,2-dihydroquinoline (dihydro-MEQ). Cells were incubated with 100–150 μmol/L diH-MEQ in a Ringer’s buffer solution containing (mmol/L): 119 NaCl, 2.5 KCl, 1.0 NaH₂PO4, 1.3 MgSO₄, 2.5 CaCl₂, 26 NaHCO₃, 11 glucose, at a pH of 7.4, and at a room temperature in the dark for 30 min. In the cytoplasm, dihydro-MEQ is quickly oxidized to MEQ, which is sensitive to [Cl⁻]_i. Fluorescence of MEQ is quenched collisionally by Cl⁻. The relationship between fluorescence intensity of MEQ and chloride concentration is given by the Stern-Volmer equation: (F_O/F)–1=K_SV [Q]. Where F_O is the fluorescence intensity without halide or other quenching ions; F is the fluorescence intensity in the presence of quencher; [Q] is the concentration of quencher; and K_SV is the Stern-Volmer constant. Fluorescence quenching induced by Cl⁻ was monitored by MetaFluor imaging software (Universal Imaging Systems, Chester, PA, USA) with 350-nm excitation and 435-nm emission wavelengths.

Oil Red O Staining

Macrophages were washed with cold phosphate-buffered saline (PBS) 3 times, fixed for 10 min with 4% ice-cold paraformaldehyde at room temperature, and stained with 0.3% Oil red O solution (dissolved in isopropanol:water, 3:2) for 10 min. The Oil red O solution was removed and the cells were washed with isopropanol for 15s, followed by washing with PBS. Images were captured at 20× magnification by microscopy. Semi-quantitative analysis of Oil red O positive staining was performed by using Image-J software (NIH, MD, USA).

oxLDL Uptake and Binding

For oxLDL binding and uptake assay, oxLDL was labeled with a fluorescent probe, l,l’-dioctadecyl-3,3,3’,3’-tetramethyl-indocarbocyanine perchlorate (DiI), as previously described.¹³ THP-1-derived macrophages after treatment were exposed to DiI-labeled oxLDL for 2 h at 4℃ or for 4 h at 37℃. Then the cells were fixed with 4% paraformaldehyde for 10 min, followed by incubation of Hoechst 33258 for nuclei staining. After washing 3 times with PBS for 5 min, the cells were quickly imaged by a FV500 laser-scanning confocal microscope (Olympus, Tokyo, Japan) keeping the same exposure for every dish. The fluorescence density was analyzed by Image-J software.

Intracellular Cholesterol Measurement

Intracellular cholesterol content was determined as previously described.^11,13 Briefly, THP-1-derived macrophages after treatment were harvested and washed with PBS 3 times, and then isopropyl alcohol was added. Samples were sonicated with 3 times of 10-s bursts to isolate intracellular liquid. A respective amount of 0.1 ml sonicates were mixed with 0.9 ml assay solutions (0.1 U/ml cholesterol oxidase, 0.01 U/ml cholesterol ester hydrolase, 1 U/ml peroxidase, 0.05% Triton X-100, 1 mmol/L sodium cholate, and 0.6 mg/ml β-hydroxyphenylacetic acid, pH7.4) and then incubated at 37℃ for 1 h. The fluorescence of the mixture was measured by a fluorospectrophotometer (RF-5000; Shimadzu Co, Kyoto, Japan). The fluorescence intensity was normalized to protein concentration.

Western Blotting Analysis

Western blotting analysis was performed as previously described.⁶ Briefly, cells were washed 3 times with ice-cold PBS and then lysed with RIPA lysis buffer (Beyotime, Jiangsu, China) containing a protease inhibitor cocktail (Merck, Darmstadt, Germany). Equal proteins were separated by 10% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked in 5% non-fat dry milk diluted with TBST (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.1% Tween 20, pH 7.5) for 1 h at room temperature and incubated with the following primary antibodies at 4℃ overnight: SR-A (1:1,000 dilution), caveolin-1 (1:1,000 dilution), phospho-JNK (1:1,000 dilution), JNK (1:1,000 dilution), GAPDH (1:1,000 dilution); phospho-p38 (1:2,000 dilution), p38 (1:2,000 dilution); and CD36 (1:4,000 dilution). After incubation of appropriate secondary horseradish peroxidase-conjugated antibodies, including HRP-conjugated anti-rabbit, anti-goat or anti-mouse (diluted 1:1,000; Cell Signaling Technology) for 1 h, bands were visualized using a Pierce ECL Plus Substrate (Thermo Scientific, Waltham, MA, USA) and quantified by Image-J software.

Immunoprecipitation

Immunoprecipitation was performed as previously described.¹⁴ Cell lysates were immunoprecipitated with SR-A or caveolin-1 antibody. Normal goat IgG or rabbit IgG (Santa Cruz Biotechnology) was used as a negative control. Immunoprecipitates were collected by protein A/G agarose, washed with lysis buffer 3 times, and subjected to Western bolt analysis with caveolin-1 or SR-A antibody.

Isolation of Lipid Rafts

Lipid raft-enriched membrane fractions were prepared using a modification of a detergent-free method.¹⁵ THP-1-derived macrophages were washed with PBS twice and scraped into 2 ml of lysis buffer (500 mmol/L Na₂ CO₃, 1 mmol/L EDTA, 0.1% protease inhibitor cocktail, pH 11.0). Cell lysates were sonicated on ice with 3 times of 20-s bursts. The homogenate was mixed with 2 ml of 80% sucrose in MES-NaCl buffer (25 mmol/L MES, 150 mmol/L NaCl, 1 mmol/L EDTA, 0.1% protease inhibitor cocktail, pH 6.5) to form 40% sucrose and loaded at the bottom of a 12-ml ultracentrifuge tube. A discontinuous sucrose gradient was generated by layering 4 ml of 35% sucrose and then 4 ml of 5% sucrose (both in MES-NaCl buffer containing 250 mmol/L Na₂ CO₃). The gradient was centrifuged at 39,000 rpms for 16 h at 4℃ using a SW41 Tirotor (Beckman). Samples were removed in 1-ml aliquots to form 12 fractions.

Quantitative Real-Time Polymerase Chain Reaction

Total RNA was isolated from macrophages using Trizol reagent according to the manufacturer’s instructions. Two micrograms of total RNA were reverse transcribed in a total volume of 20 µl, and real-time PCR was performed using SYBR green fluorescence. Samples were run in duplicate with RNA preparations from 3 to 5 independent experiments. Each real-time PCR reaction consisted of 1 µl RT product, 10 µl SYBR Green PCR Master Mix, and 500 nmol/L forward and reverse primers (Table S2). Reactions were carried out on a MyiQ Single Color Real-time PCR Detection System (Bio-Rad) for 40 cycles (95℃ for 10 s, 60℃ for 1 min) after an initial 3-min incubation at 95℃. The fold change in expression of each gene was calculated using the 2^−△△CT method, with 18S rRNA as an internal control.

Confocal Immunofluorescence Microscopy

THP-1-derived macrophages were grown on glass slides in 35-mm dishes. The cells after treatment were washed 3 times with ice-cold PBS, fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 in PBS for 3 min, blocked with 2% BSA in PBS for 1 h and then co-incubated with goat-anti-SR-A and rabbit-anti-caveolin-1 antibodies (1:100 dilution) at 4℃ overnight. After 3 washes with PBS, the cells were co-incubated with the secondary antibodies (anti-goat Cy3-labeled and anti-rabbit FITC-labeled antibodies, 1:100 dilution; Jackson ImmunoResearch Laboratories) for 1 h at 37℃. The nuclei were stained with Hoechst 33258. Co-localization studies were carried out using an Olympus FV500 confocal microscope in multi-tracking mode to prevent interference of the dyes.

Inflammatory Cytokine Measurements

IL-1β, IL-6 and TNF-α concentration in cell cultured medium were determined by ELISA assays as recommend by the manufacturer.

Statistical Analysis

All data were expressed as mean±SEM. Statistical analysis was determined in SPSS 16.0 system (SPSS Inc, Chicago, IL, USA) by an unpaired 2-tailed Student’s t-test or one-way ANOVA followed by the Bonferroni multiple comparison post-hoc test with a 95% confidence interval. P<0.05 was considered to be statistically significant.

Results

Reduction of [Cl⁻]_i Promotes Foam Cell Formation

Compared with the healthy group, the [Cl⁻]_i in monocytes isolated form patients with hypercholesterolemia was significantly decreased (Figure 1A). A similar decrease of [Cl⁻]_i was observed in peritoneal macrophages from apoE^−/− mice after a 12-week high-fat diet (Figure S1A). In addition, oxLDL (50 μg/ml) treatment also induced a decrease of [Cl⁻]_i in human macrophages and THP-1-derived macrophages (Figures 1B,S1B). To investigate whether the alteration of [Cl⁻]_i is involved in regulating foam cell formation, we prepared the cell culture medium with a reduced chloride concentration to decrease intracellular chloride content (Figures 1C,S1C) and measured its effect on lipid accumulation. Low Cl⁻ solution itself could increase lipid accumulation in human macrophages, as evidenced by Oil red O staining and intracellular cholesterol content assay. Moreover, oxLDL-induced lipid accumulation was more pronounced in low Cl⁻ solution compared with that in a normal Cl⁻ condition (Figures 1D–F,S1D–F).

Figure 1.

Reduction of intracellular chloride concentration ([Cl⁻]_i) accelerates lipid accumulation in human monocyte-derived macrophages treated with or without oxidized low-density lipoproteins (oxLDL). (A) [Cl⁻]_i was measured in monocytes isolated from healthy adult volunteers (normal) or hypercholesterolemia patients by using a MEQ fluorescence probe. **P<0.01 vs. normal, n=11. (B) Human monocyte-derived macrophages were exposed to oxLDL (50 μg/ml) for 48 h, [Cl⁻]_i was measured. **P<0.01 vs. control, n=5. (C) [Cl⁻]_i in human macrophages incubated in normal or reduced chloride (Cl⁻) medium. **P<0.01 vs. normal Cl⁻ medium, n=6. (D–F) Human macrophages were incubated in normal Cl⁻ or low Cl⁻ medium in the presence or absence of oxLDL, respectively. After 48 h, the accumulation of red O-positive droplets (D and E, n=5) and intracellular cholesterol levels (F, n=6) were measured as describe in the Methods section. **P<0.01 vs. control, ^##P<0.01 vs. oxLDL alone.

Lowering [Cl⁻]_i Enhances SR-A Expression and Lipid Uptake in Human Macrophages

To investigate whether increased binding or uptake of modified lipoproteins accounts for increased foam cell formation in low Cl⁻ solution, we examined receptor-mediated binding and uptake of fluorescently labeled oxLDL (Dil-oxLDL). Our results showed that the binding and uptake of Dil-oxLDL in macrophages treated with or without oxLDL were both remarkably enhanced under low Cl⁻ solution (Figures 2A,B,S2A,B). To further understand the mechanism by which a low Cl⁻ solution augments the binding and uptake of Dil-oxLDL, the expression of scavenger receptors, SR-A and CD36, were examined. A low Cl⁻ solution increased SR-A protein expression under basal conditions and after oxLDL treatment (Figures 2C,S2C). However, reduction of [Cl⁻]_i had no effect on CD36 expression both at baseline and after oxLDL treatment (Figures 2D,S2D). These results suggested that the increase of SR-A expression may account for the enhanced lipid uptake under low Cl⁻ solution.

Figure 2.

A decrease of intracellular chloride concentration ([Cl⁻]_i) enhances binding and uptake of fluorescently labeled oxidized low-density lipoprotein (Dil-oxLDL) and scavenger receptor A (SR-A) expression in human monocyte-derived macrophages. (A,B) Human macrophages incubated in normal chloride (Cl⁻) or low Cl⁻ medium were treated with or without oxLDL (50 μg/ml) for 48 h. Then, the cells were exposed to DiI-labeled oxLDL for 2 h at 4℃ or for 4 h at 37℃ to assess binding (A) or uptake (B) of Dil-labeled oxLDL, respectively. **P<0.01 vs. control, ^##P<0.01 vs. oxLDL alone, n=5. (C,D) Cells were treated as mentioned above. Both SR-A (C) and CD36 (D) protein expression was examined by Western blotting. **P<0.01 vs. control, ^##P<0.01 vs. oxLDL alone, n=6.

JNK and p38 Kinase Activation Contributes to Low Cl⁻ Solution-Induced SR-A Expression and Lipid Accumulation

Activation of mitogen-activated protein kinases (MAPKs), in particular JNK and p38 kinase, has been demonstrated to be critical for SR-A upregulation,^3,16 so we then assessed the activities of JNK and p38 kinase in low [Cl⁻]_i medium. oxLDL treatment increased the phosphorylation levels of JNK and p38 kinase in macrophages (Figure S3). Reduction of [Cl⁻]_i increased the basal phosphorylation levels of JNK and p38 kinase and further enhanced oxLDL-induced JNK and p38 phosphorylation (Figures 3A,B), indicating [Cl⁻]_i is involved in regulating JNK and p38 kinase activation. Furthermore, inhibitors of JNK (SP600125, 20 µmol/L) and p38 kinase (SB203580, 10 µmol/L) both significantly prevented low Cl⁻ solution-induced SR-A expression, the binding and uptake of Dil-oxLDL, and thus reduced intracellular lipid accumulation (Figures 3C–G,S4,S5). These data suggested that activation of JNK and p38 kinase is required for low Cl⁻ solution-induced SR-A expression and foam cell formation.

Figure 3.

The JNK and p38 MAPK activation is required for low chloride (Cl⁻) solution-induced foam cell formation. (A,B) A decrease of intracellular chloride concentration ([Cl⁻]_i) increased phosphorylation of JNK (A) and p38 MAPK (B) before and after oxidized low-density lipoprotein (oxLDL) treatment. **P<0.01 vs. control, ^##P<0.01 vs. oxLDL alone, n=6. (C–G) THP-1 macrophages were pretreated with a JNK inhibitor, SP600125 (20 µmol/L), or a p38 inhibitor, SB203580 (10 µmol/L), or with both for 3 h prior to Cl⁻-reduced medium treatment for 48 h. Scavenger receptor A expression (C, n=6), fluorescently labeled oxidized low-density lipoprotein (Dil-oxLDL) binding (D, n=4) and uptake (E, n=5), Oil red O staining (F, n=5) and intracellular cholesterol levels (G, n=6) were measured. **P<0.01 vs. control, ^##P<0.01 vs. low Cl⁻+DMSO.

Low Cl⁻ Solution Potentiates Caveolin-1-Dependent SR-A Internalization

Scavenger receptor-A-mediated internalization of lipids into the cells is a key step for intracellular lipid accumulation and foam cell formation. To investigate whether increased SR-A internalization contributes to low Cl⁻ solution-induced foam cell formation, we compared the SR-A endocytosis process in normal Cl⁻ solution with that in low Cl⁻ medium. Immunofluorescence staining revealed that SR-A is mainly located at the cell membrane in normal Cl⁻ solution, under low Cl⁻ medium, however, the fluorescence intensity of SR-A in the cytoplasmic portion was significantly increased (Figure 4A). In addition, continuous sucrose density gradient fractionation showed that SR-A was distributed in lighter density fractions in normal Cl⁻ solution, and there was a reduction of [Cl⁻]_i- induced SR-A redistribution into the heavier fractions (Figure 4B). These findings indicated that a decrease of [Cl⁻]_i enhanced SR-A internalization.

Figure 4.

Lowering intracellular chloride concentration ([Cl⁻]_i) promotes caveolin-1-dependent scavenger receptor A (SR-A) internalization. (A) THP-1 macrophages were pretreated with MβCD (2 mmol/L) or genistein (150 μmol/L) for 3 h, and then incubated in a low chloride (Cl⁻) solution for another 48 h. Co-localization of SR-A and caveolin-1 was examined by immunofluorescence experiments using antibodies against SR-A (red) and caveolin-1 (green). The nuclei were stained with Hoechst 33258. Representative images from 6 independent experiments are shown. (B) Cells were cultured in low Cl⁻ solution in the presence or absence of oxidized low-density lipoproteins (oxLDL) for 48 h. The expression of SR-A and caveolin-1 (Cav-1) in different fractions of lipid rafts was examined. Representative Western blots from 5 independent experiments are shown. (C,D) A decrease of [Cl⁻]_i increased caveolin-1 expression (C) and SR-A/caveolin-1 interaction (D) before and after oxLDL treatment. **P<0.01 vs. control, ^##P<0.01 vs. oxLDL alone, n=6.

Caveolin-1, which serves as a SR-A binding partner, has been demonstrated to play a critical role in regulating SR-A endocytosis.^14,15 Indeed, SR-A colocalizes with caveolin-1 in the cell membrane (Figure 4A). A co-immunoprecipitation assay showed that SR-A and caveolin-1 interact with each other (Figure S6). Reduction of [Cl⁻]_i enhanced caveolin-1 expression (Figure 4C). Moreover, SR-A and caveolin-1 interaction in macrophages was increased in low Cl⁻ medium before and after oxLDL treatment, indicating that lowering [Cl⁻]_i promoted the recruitment of SR-A to the caveolae (Figure 4D). Similar with SR-A, caveolin-1 is also mainly distributed at the cell membrane fractions in normal Cl⁻ solution. A decrease of [Cl⁻]_i induced the translocation of caveolin-1 to the cytoplasma accompanying SR-A (Figures 4A,B). Notably, disruption of caveolae with MβCD or genistein blunted the internalization of both caveolin-1 and SR-A after exposure to reduced Cl⁻ medium (Figures 4A,B). These results demonstrated that increased caveolin 1-dependent internalization of SR-A contributed to the lipid uptake under low Cl⁻ conditions.

Disruption of Caveolae Impairs Low Cl⁻ Solution-Induced JNK and p38 Activation and Foam Cell Formation

Previous work has revealed that caveolin-1-dependent SR-A endocytosis could activate JNK and p38 kinase.¹⁴ To understand whether SR-A endocytosis mediates low Cl⁻ solution-induced JNK and p38 activation, we assessed the phosphorylation of JNK and p38 in low Cl⁻ solution after disruption of caveolae. Our results showed that MβCD and genistein, 2 specific inhibitors of caveolae, decreased the low Cl⁻ solution-induced activation of JNK and p38 MAPK (Figures 5A,B). As expected, MβCD and genistein remarkably attenuated Cl⁻ reduced medium-induced SR-A expression (Figure 5C). Moreover, MβCD and genistein treatment obviously reduced the binding and uptake of Dil-oxLDL, intracellular lipid droplets and cholesterol levels in Cl⁻ reduced medium (Figures 5D–G,S7). These findings indicated that caveolae-dependent SR-A endocytosis is required for low Cl⁻ solution-induced JNK and p38 activation, SR-A upregulation and foam cell formation.

Figure 5.

Disruption of caveolae impairs low chloride (Cl⁻) solution-induced JNK and p38 activation, and lipid accumulation. (A–C) THP-1-derived macrophages were pretreated with MβCD (2 mmol/L) or genistein (150 μmol/L) for 3 h prior to incubation with a low Cl⁻ solution. Phosphorylated and total JNK (A) and p38 (B), and scavenger receptor A (SR-A) expression (C) were analyzed by Western blotting (n=6). (D–G) Cells were treated as shown in (A), fluorescently labeled oxidized low-density lipoprotein (Dil-oxLDL) binding (D, n=4) and uptake (E, n=4), Oil red O staining (F, n=5), and intracellular cholesterol levels (G, n=5) were examined as described in the Methods section. **P<0.01 vs. control, ^##P<0.01 vs. low Cl⁻+DMSO.

Reduction of [Cl⁻]_i Potentiates an Inflammatory Response in Macrophages

Abnormal lipid accumulation in macrophages and their subsequent foam cell formation usually result in chronic inflammation, which marks the initiation of atherosclerosis.^2,17 To investigate whether inflammation is involved in [Cl⁻]_i decrease-induced foam cell formation, several pro-inflammatory cytokines were measured in macrophages treated with or without low Cl⁻ solution. A decrease of [Cl⁻]_i dramatically enhanced the expression of IL-1β, IL-6 and TNF-α in macrophages of mice fed with a high-fat diet (Figures 6A–F). In line with these results, lowering [Cl⁻]_i also increased oxLDL-induced IL-1β, IL-6 and TNF-α expression in human monocyte-derived macrophages (Figures S8A–F).

Figure 6.

Lowering intracellular chloride concentration ([Cl⁻]_i) potentiates inflammatory cytokines production. (A–C) Peritoneal macrophages isolated from high-fat-diet-fed apoE^−/− mice were incubated in normal or reduced chloride (Cl⁻) medium for 48 h. Then, the secretions of interleukin (IL)-1β (A), IL-6 (B) and TNF-α (C) in the culture medium were determined by an ELISA, respectively. (D–F) The mRNA levels of IL-1β (D), IL-6 (E) and TNF-α (F) were examined by quantitative PCR, respectively. All results were from 4 individual mice in each group. **P<0.01 vs. normal diet (ND), ^##P<0.01 vs. high-fat diet (HFD).

Discussion

The data from the present study demonstrated that a reduction of [Cl⁻]_i promotes JNK/p38 MAPKs activation and increases SR-A expression. In addition, a decrease in [Cl⁻]_i enhances the binding of caveolin-1 with SR-A and facilitates caveolae-dependent internalization of SR-A. The integral effects of these processes promote lipid accumulation and thus induce foam cell formation in macrophages.

Cl⁻ movement across the cell plasma membrane has been suggested to play an important role in regulating a variety of physiological processes, including cell volume regulation, cell proliferation and apoptosis, inflammation, and synaptic transmission.^6,7,10 Following Cl⁻ movement, [Cl⁻]_i is dynamically regulated. In fact, previous studies have revealed that [Cl⁻]_i is decreased in T cells undergoing apoptosis, in vascular smooth muscle cells during hypertension and in endothelial cells after activation.^6,18,19 Moreover, blockade of chloride efflux with chloride channel blockers prevents cell apoptosis and inhibits vascular inflammation, indicative of the critical role of [Cl⁻]_i in these processes. Notably, a recent clinic study demonstrated that serum Cl⁻ concentration is an independent predictor of mortality in hypertensive patients, and that low serum Cl⁻ is associated with great mortality risk,²⁰ further supporting the hypothesis that Cl⁻ movement across cell membrane may play an essential role in regulating cardiovascular homeostasis. In the present study, we provided the first clue that intracellular Cl⁻ is an essential modulator for macrophage lipid accumulation and atherogenesis based on the following evidence: (1) [Cl⁻]_i is significantly reduced in monocytes isolated from patients with hypercholesterolemia in macrophages from apoE^−/− mice fed with a high-fat diet; and (2) reduction of [Cl⁻]_i increases SR-A expression, promotes lipid binding and uptake and subsequently potentiates foam cell formation in macrophages.. During foam cell formation, the increase of the cell volume due to the uptake of lipid could activate the volume-regulated chloride channel and subsequently evoke Cl⁻ efflux, which would lead to the decrease of [Cl⁻]_i. Therefore, it is not surprising that the blocking of this chloride efflux with chloride channel blockers could prevent lipid accumulation in macrophages.¹¹ Interestingly, our recent study in RAW264.7 macrophages showed that ClC-3, a candidate of volume-regulated chloride channel, is upregulated after oxLDL treatment. Knockout of ClC-3 prevents foam cell formation and atherogenesis in apoE^−/− mice.¹³ These data indicate that a reduction of [Cl⁻]_i is a link between ClC-3 volume-regulated chloride channel activation and atherosclerosis.

Previous studies have demonstrated that MAPKs, in particular JNK and p38 MAPK, play an important role in regulation SR-A expression.^3,16 Indeed, oxLDL treatment increased the phosphorylation level of both JNK and p38 MAPK. A decrease of [Cl⁻]_i could activate JNK and p38 MAPK under basal conditions and further potentiate oxLDL-induced activation of JNK and p38 MAPK. Inhibition of JNK and p38 MAPKs dramatically blunts low Cl⁻-induced SR-A expression and foam cell formation in macrophages. These results demonstrate that activation of JNK and p38 MAPK contributes to SR-A upregulation under low [Cl⁻]_i conditions.

Scavenger receptor-A is a multifunctional macrophage receptor that is involved in modulating macrophage growth, cell adhesion, phagocytosis, as well as foam cell formation.^15,21,22 SR-A, together with its ligand, could be internalized into the cytoplasm via coated pit-mediated endocytosis and thus enhance lipid uptake.^23,24 Caveolin-1 has been shown to be the binding partner of SR-A and plays an important role in regulating SR-A internalization.^14,15 In this study, we found that low Cl⁻ solution increased caveolin-1 expression both at baseline and after oxLDL treatment. Moreover, our results showed that reduction of [Cl⁻]_i enhances the formation of SR-A/caveolin-1 complex and triggers SR-A internalization. Blockade of caveolae inhibits low Cl⁻-induced SR-A endocytosis, indicating that SR-A internalization is dependent on caveolin-1. Notably, although reduction of [Cl⁻]_i enhances SR-A internalization, SR-A expression in the cell membrane is not decreased, but rather increased under low Cl⁻ conditions. It is not surprised since low Cl⁻ solution can increase SR-A expression. More interestingly, we further observed that disruption of caveolae dramatically inhibits low Cl⁻ solution-induced SR-A expression and lipid accumulation, indicating that caveolae-dependent endocytosis is also required for SR-A expression and foam cell formation. Indeed, disruption of caveolae significantly impaired low Cl⁻ solution-induced activation of JNK and p38. Our data is consistent with a previous study, which demonstrated that caveolae-dependent SR-A endocytosis is a critical contributor to JNK and p38 activation.¹⁴ These findings suggest that caveolae-dependent SR-A endocytosis acts as a bridge between [Cl⁻]_i and JNK/p38 activation, which offers an explanation as to why low Cl⁻-induced caveolae-dependent SR-A internalization could increase SR-A expression.

Foam cell formation can secret abundant amounts of cytokines and chemokines, which further promotes the initiation and development of atherosclerosis.^17,25 Interestingly, the Cl⁻ level has been suggested to play an essential role in regulating inflammation. In cortical thick ascending limb of Henle cells, a low Cl⁻ solution could increase the expression of COX-2, a key enzyme for inflammation response.²⁶ Our previous study also showed that a reduction of [Cl⁻]_i potentiated TNF-α-induced NF-κB activation and vascular inflammation.⁶ Here, we found that lowering [Cl⁻]_i increases the expression of inflammatory genes such as IL-1β, IL-6 and TNF-α in peritoneal macrophages from high-fat-diet-fed apoE^−/− mice and in oxLDL-treated human macrophages. These data suggest that lowering [Cl⁻]_i-induced foam cells are functionally inflammatory form of macrophages.

In conclusion, the findings in this study provide strong evidence that a reduction of [Cl⁻]_i is a critical contributor to foam cell formation and inflammation. A decrease of [Cl⁻]_i facilitates the assembly of a SR-A/caveolin-1 complex and promotes caveolae-mediated SR-A endocytosis, which subsequently activates JNK and p38 and increases lipid accumulation. Therefore, the modulation of [Cl⁻]_i may be a novel strategy to prevent atherosclerosis.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 81525025, 81473206, 81471132, 81273500 and 81230082), and the Natural Science Foundation of Guangdong Province (Nos. 2013010015263, 2014A030313030 and 2014A030310031).

Supplementary Files

Supplementary File 1

Table S1. Clinical parameters according to lipid levels of healthy adult volunteers and hypercholesterolemia patients

Table S2. Primers used for quantitative real-time polymerase chain reaction

Figure S1. Reduction of intracellular chloride concentration ([Cl⁻]_i) accelerates lipid accumulation in THP-1 macrophages treated with or without oxidized low-density lipoproteins (oxLDL).

Figure S2. A decrease of intracellular chloride concentration ([Cl⁻]_i) enhances binding and uptake of fluorescently labeled oxidized low-density lipoproteins (Dil-oxLDL) and scavenger receptor A (SR-A) expression in THP-1 macrophages.

Figure S3. Oxidized low-density lipoproteins (oxLDL) activate JNK and p38 mitogen-activated protein kinases (MAPK).

Figure S4. JNK and p38 mitogen-activated protein kinases (MAPK) activation is required for low chloride (Cl⁻) solution-induced scavenger receptor A (SR-A) gene expression.

Figure S5. Inhibition of JNK and p38 mitogen-activated protein kinases (MAPK) ameliorates oxidized low-density lipoprotein (oxLDL)-induced foam cell formation.

Figure S6. Scavenger receptor A (SR-A) interacts with caveolin-1 in whole cell lysates or in lipid rafts.

Figure S7. Disruption of caveolae attenuates low chloride (Cl⁻) solution-induced lipid accumulation.

Figure S8. Lowering intracellular chloride concentration ([Cl⁻]_i) potentiates inflammatory cytokine production.

Please find supplementary file(s);

http://dx.doi.org/10.1253/circj.CJ-15-1209

References

• 1.    Mo  ZC,  Xiao  J,  Tang  SL,  Ouyang  XP,  He  PP,  Lv  YC, et al. Advanced oxidation protein products exacerbates lipid accumulation and atherosclerosis through downregulation of ATP-binding cassette transporter A1 and G1 expression in apolipoprotein E knockout mice. Circ J 2014; 78: 2760–2770.
• 2.    De Paoli  F,  Staels  B,  Chinetti-Gbaguidi  G. Macrophage phenotypes and their modulation in atherosclerosis. Circ J 2014; 78: 1775–1781.
• 3.    Ricci  R,  Sumara  G,  Sumara  I,  Rozenberg  I,  Kurrer  M,  Akhmedov  A, et al. Requirement of JNK2 for scavenger receptor A-mediated foam cell formation in atherogenesis. Science 2004; 306: 1558–1561.
• 4.    Suzuki  H,  Kurihara  Y,  Takeya  M,  Kamada  N,  Kataoka  M,  Jishage  K, et al. A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature 1997; 386: 292–296.
• 5.    Wang  JG,  Aikawa  M. Toll-like receptors and Src-family kinases in atherosclerosis: Focus on macrophages. Circ J 2015; 79: 2332–2334.
• 6.    Yang  H,  Huang  LY,  Zeng  DY,  Huang  EW,  Liang  SJ,  Tang  YB, et al. Decrease of intracellular chloride concentration promotes endothelial cell inflammation by activating nuclear factor-kappaB pathway. Hypertension 2012; 60: 1287–1293.
• 7.    Guan  YY,  Wang  GL,  Zhou  JG. The ClC-3 Cl- channel in cell volume regulation, proliferation and apoptosis in vascular smooth muscle cells. Trends Pharmacol Sci 2006; 27: 290–296.
• 8.    Zhou  JG,  Ren  JL,  Qiu  QY,  He  H,  Guan  YY. Regulation of intracellular Cl- concentration through volume-regulated ClC-3 chloride channels in A10 vascular smooth muscle cells. J Biol Chem 2005; 280: 7301–7308.
• 9.    Deriy  LV,  Gomez  EA,  Jacobson  DA,  Wang  X,  Hopson  JA,  Liu  XY, et al. The granular chloride channel ClC-3 is permissive for insulin secretion. Cell Metab 2009; 10: 316–323.
• 10.    Wang  XQ,  Deriy  LV,  Foss  S,  Huang  P,  Lamb  FS,  Kaetzel  MA, et al. CLC-3 channels modulate excitatory synaptic transmission in hippocampal neurons. Neuron 2006; 52: 321–333.
• 11.    Hong  L,  Xie  ZZ,  Du  YH,  Tang  YB,  Tao  J,  Lv  XF, et al. Alteration of volume-regulated chloride channel during macrophage-derived foam cell formation in atherosclerosis. Atherosclerosis 2011; 216: 59–66.
• 12.    Matsumura  T,  Sakai  M,  Kobori  S,  Biwa  T,  Takemura  T,  Matsuda  H, et al. Two intracellular signaling pathways for activation of protein kinase C are involved in oxidized low-density lipoprotein-induced macrophage growth. Arterioscler Thromb Vasc Biol 1997; 17: 3013–3020.
• 13.    Tao  J,  Liu  CZ,  Yang  J,  Xie  ZZ,  Ma  MM,  Li  XY, et al. ClC-3 deficiency prevents atherosclerotic lesion development in ApoE(–/–) mice. J Mol Cell Cardiol 2015; 87: 237–247.
• 14.    Zhu  XD,  Zhuang  Y,  Ben  JJ,  Qian  LL,  Huang  HP,  Bai  H, et al. Caveolae-dependent endocytosis is required for class A macrophage scavenger receptor-mediated apoptosis in macrophages. J Biol Chem 2011; 286: 8231–8239.
• 15.    Ben  J,  Zhang  Y,  Zhou  R,  Zhang  H,  Zhu  X,  Li  X, et al. Major vault protein regulates class A scavenger receptor-mediated tumor necrosis factor-alpha synthesis and apoptosis in macrophages. J Biol Chem 2013; 288: 20076–20084.
• 16.    Nikolic  D,  Calderon  L,  Du  L,  Post  SR. SR-A ligand and M-CSF dynamically regulate SR-A expression and function in primary macrophages via p38 MAPK activation. BMC Immunol 2011; 12: 37.
• 17.    Geczy  CL,  Chung  YM,  Hiroshima  Y. Calgranulins may contribute vascular protection in atherogenesis. Circ J 2014; 78: 271–280.
• 18.    Heimlich  G,  Cidlowski  JA. Selective role of intracellular chloride in the regulation of the intrinsic but not extrinsic pathway of apoptosis in Jurkat T-cells. J Biol Chem 2006; 281: 2232–2241.
• 19.    Shi  XL,  Wang  GL,  Zhang  Z,  Liu  YJ,  Chen  JH,  Zhou  JG, et al. Alteration of volume-regulated chloride movement in rat cerebrovascular smooth muscle cells during hypertension. Hypertension 2007; 49: 1371–1377.
• 20.    McCallum  L,  Jeemon  P,  Hastie  CE,  Patel  RK,  Williamson  C,  Redzuan  AM, et al. Serum chloride is an independent predictor of mortality in hypertensive patients. Hypertension 2013; 62: 836–843.
• 21.    Robbins  CS,  Hilgendorf  I,  Weber  GF,  Theurl  I,  Iwamoto  Y,  Figueiredo  JL, et al. Local proliferation dominates lesional macrophage accumulation in atherosclerosis. Nat Med 2013; 19: 1166–1172.
• 22.    Areschoug  T,  Gordon  S. Scavenger receptors: Role in innate immunity and microbial pathogenesis. Cell Microbiol 2009; 11: 1160–1169.
• 23.    Chen  Y,  Wang  X,  Ben  J,  Yue  S,  Bai  H,  Guan  X, et al. The di-leucine motif contributes to class a scavenger receptor-mediated internalization of acetylated lipoproteins. Arterioscler Thromb Vasc Biol 2006; 26: 1317–1322.
• 24.    Liou  J,  Kim  ML,  Heo  WD,  Jones  JT,  Myers  JW,  Ferrell  JE Jr, et al. STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr Biol 2005; 15: 1235–1241.
• 25.    Ketelhuth  DF,  Hansson  GK. Modulation of autoimmunity and atherosclerosis: Common targets and promising translational approaches against disease. Circ J 2015; 79: 924–933.
• 26.    Cheng  HF,  Wang  JL,  Zhang  MZ,  McKanna  JA,  Harris  RC. Role of p38 in the regulation of renal cortical cyclooxygenase-2 expression by extracellular chloride. J Clin Invest 2000; 106: 681–688.