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Volume 216, Issue 1, May 2011, Pages 59-66

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doi:10.1016/j.atherosclerosis.2011.01.035 | How to Cite or Link Using DOI   Permissions & Reprints

Alteration of volume-regulated chloride channel during macrophage-derived foam cell formation in atherosclerosis

Liang Hong^{a, 1}, Zhi-Zhong Xie^{a, b, 1}, Yan-Hua Du^a, Yong-Bo Tang^a, Jing Tao^a, Xiao-Fei Lv^a, Jia-Guo Zhou^{a, ,} and Yong-Yuan Guan^{a, ,}

^a Department of Pharmacology, Cardiac and Cerebrovascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, 74 Zhongshan 2 Rd, Guangzhou, 510080, People's Republic of China

^b Institute of Pharmacy and Pharmacology, College of Science and Technology, University of South China, Hengyang, 421001, People's Republic of China

Received 14 July 2010;  revised 28 December 2010;  accepted 24 January 2011.  Available online 2 February 2011.

Abstract

Objective

Volume-regulated Cl⁻ channel (VRCC) plays a critical role in regulation of a variety of physiological functions. However, little is known whether VRCC is involved in atherosclerosis. In this study, we investigated the functions of VRCC during foam cell formation in macrophages.

Methods and results

Treatment of RAW264.7 cells with ox-LDL increased intracellular cholesterol content as well as cell volume. After ox-LDL treatment, the resting [Cl⁻]_i in isotonic solution was decreased. Hypotonic solution reduced [Cl⁻]_i and evoked volume-regulated Cl⁻ current in all the cells, however, the swelling-induced reduction of [Cl⁻]_i and increase of Cl⁻ current were more prominent in ox-LDL treated cells than that in control. The increases of volume-regulated Cl⁻ movement positively correlated with the intracellular cholesterol content. Moreover, in peritoneal macrophages isolated from high-fat diet ApoE^−/− mice, the swelling-induced Cl⁻ movement and current were enhanced compared with those in control group, and their increments positively correlated with atherosclerotic plaque area. Finally, activation of VRCC by hypotonic medium significantly accelerated, whereas, inhibition of VRCC with Cl⁻ channel blockers remarkably attenuated, ox-LDL-induced macrophage-derived foam cell formation.

Conclusion

The activity of VRCC is augmented during macrophage-derived foam cell formation. Activation of VRCC accelerated, whereas, inhibition of VRCC attenuated, ox-LDL-induced lipid accumulation in macrophages, suggesting VRCC is involved in the regulation of foam cell formation.

Keywords: Volume-regulated chloride channel; Atherosclerosis; Foam cell; Macrophage

Article Outline

1.

Introduction

2.

Materials and methods

2.1. Foam cell formation

2.2. Measurements of intracellular cholesterol levels

2.3. Measurement of [Cl⁻]_i

2.4. Measurement of I_Cl,vol and cell membrane capacitance

2.5. Effect of hypotonic medium culture on foam cell formation

2.6. Animal model of atherosclerosis

2.7. Peritoneal macrophages isolation

2.8. Analysis of atherosclerotic plaque area

2.9. Statistical analysis

3.

Results

3.1. Ox-LDL induced foam cell formation and cell volume increase in RAW264.7

3.2. Alteration of [Cl⁻]_i during ox-LDL induced foam cell formation in RAW264.7 cells

3.3. Alteration of I_Cl.vol during ox-LDL induced foam cell formation in RAW264.7 cells

3.4. Alterations of [Cl⁻]_i and I_Cl.vol in macrophages from high-fat diet fed ApoE^−/− mice

3.5. Activation of VRCC by hypoosmotic medium accelerated foam cell formation

3.6. Cl⁻ channel blockers prevented foam cell formation

4.

Discussion

Disclosures

Acknowledgements

Appendix A.

Supplementary data

References

1. Introduction

Atherosclerosis is a complex process which involves the interaction between modified lipoproteins, such as the oxidized low-density lipoproteins (ox-LDL), monocyte-derived macrophages, endothelial cells and vascular smooth muscle cells. Through scavenger receptors, macrophages can take up ox-LDL and other lipids to form foam cells, leading to the fatty streaks characteristic of early atherosclerosis. It is well known that accumulation of cholesterol-loaded macrophage-derived foam cell under arterial intima is a crucial step in the development of atherosclerosis [1].

 

The mechanism underlying the pathogenesis of foam cell formation and atherosclerosis involves multiple factors. It has been suggested that atherogenesis is closely related to the disordered ionic movements across the bio-membrane [2], [3], [4] and [5]. The upregulation of intracellular Ca²⁺ ([Ca²⁺]_i) has been demonstrated in the process of atherosclerosis. Accordingly, the atheroprotective role of amlodipine, a calcium channel blocking agent, has been reported in both clinical research and animal models [2] and [3]. The disordered intracellular K⁺ level was also shown to contribute to the development of atherosclerosis [4] and [5]. Although the potential impacts of these cations on atherosclerosis have been addressed by numerous studies in recent years, little information is provided whether there is an involvement of transmembrane anion transport in the progression of atherosclerosis.

Chloride channels are widely found anion pores and ubiquitously expressed in almost all eukaryotic cells which play critical roles in regulation of diverse physiological functions, including cell cycle and apoptosis, transepithelial transport, skeletal muscle tone, cell volume, synaptic transmission and cellular excitability [6], [7], [8], [9] and [10]. The volume-regulated Cl⁻ channel (VRCC) is one of the most important Cl⁻ channels responsible for the transmembrane Cl⁻ movement, and plays a fundamental role in cell volume regulation in response to cell shape change by various physiological and pathophysiological stimuli [6] and [7]. Recent growing evidence has demonstrated that VRCC is closely associated with the regulation of cell proliferation [8], apoptosis [9], and even vascular remodeling during hypertension [10]. We speculated that cell swelling may occur in macrophages due to uptake of modified LDL during the process of foam cell formation and VRCC may be involved in this process.

The aim of this study, therefore, was to determine whether the alteration of Cl⁻ transmembrane movement via VRCC is involved in the development of atherosclerosis. By measuring intracellular Cl⁻ concentration ([Cl⁻]_i) and VRCC current (I_Cl,vol) in foam cells both in vitro and ex vivo, our results strongly suggested that volume-regulated Cl⁻ movement is augmented during macrophage-derived foam cell formation, and its increment positively correlated with atherosclerotic plaque area in the development of atherosclerosis.

2. Materials and methods

2.1. Foam cell formation

Macrophage-derived foam cell was induced as previously described [11]. LDL was isolated from human plasma by sequential ultracentrifugation, and oxidized to ox-LDL with 5 μmol/L CuSO₄ at 37 °C for 24 h. LDL and ox-LDL were sterilized and stored at 4 °C in dark, and used within 2 weeks. RAW264.7 cells (mouse macrophage cell line) were purchased from the American Type Culture Collection. Cells were grown in RMPI 1640 medium supplemented with 10% fetal calf serum, and were maintained at 37 °C in a humidified incubator in a 95% O₂ plus 5% CO₂ atmosphere.

To study the concentration-dependent and time-dependent effects of ox-LDL on foam cell formation, RAW264.7 macrophages were incubated with 40, 80, or 120 μg/mL ox-LDL for 48 h or 80 μg/mL ox-LDL for 6, 12, 24, or 48 h, respectively. The formation of foam cell was evaluated by the measurement of intracellular cholesterol contents and oil red O staining.

2.2. Measurements of intracellular cholesterol levels

Intracellular cholesterol and cholesterol ester contents were measured as previously described [12]. Briefly, RAW264.7 cells after ox-LDL treatment or peritoneal macrophages from high-fat diet ApoE^−/− mice were harvested into a centrifuge tube and washed with PBS three times. 0.3 mL isopropyl alcohol was added to each tube, and intracellular liquid was extracted by ultrasonication. Respective amount of 0.1 mL sonicates and 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 mM sodium cholate, and 0.6 mg/mL β-hydroxyphenylacetic acid, pH7.4) were transferred into a new tube and incubated at 37 °C for one hour. The fluorescence of mixture was determined by fluorospectrophotometer (RF-5000, Shimadzu Co, Japan). The fluorescence intensity was calibrated by the cells protein concentration.

2.3. Measurement of [Cl⁻]_i

[Cl⁻]_i was measured with 6-Methoxy-N-ethylquinolinium iodide (MEQ) as we described previously [10] and [13]. Briefly, MEQ was first reduced to its cell permeable format dihydro-MEQ, which can be quickly oxidized to MEQ in cytoplasm. Cells were then incubated with 100–150 μM dihydro-MEQ in a Ringer's buffer solution containing (mM):119 NaCl, 2.5 KCl, 1.0 NaH₂PO₄, 1.3 MgSO₄, 2.5 CaCl₂, 26 NaHCO₃, and 11 glucose, pH 7.4 at room temperature in the dark for 30 min. MEQ fluorescence quenching induced by intracellular Cl⁻ was monitored by MataFlour Imaging software (Universal Imaging Systems, Chester, PA) with 350-nm excitation wavelength and 435-nm emission wavelength. 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.

Solutions: The osmolarities of the soultions were measured by a freezing point depression osmometer (OSMOMAT030, Germany). The isotonic bath solution (300 mosmol/kg·H₂O) contained (mM): 111 NaCl, 2.5 KCl, 0.5 MgCl₂, 10 HEPES, 5 glucose, and 70 d-mannitol, pH 7.4 with NaOH. The 230 mosmol/kg·H₂O hypotonic solution was prepared by omitting the d-mannitol from the isotonic solution.

The hypotonic-induced decrease in [Cl⁻]_i (Δ[Cl⁻]_i hypo%) was calculated using the following equation: Δ[Cl⁻]_i hypo% = {[Cl⁻]_i iso − [Cl⁻]_i hypo}/[Cl⁻]_i iso × 100%. Where [Cl⁻]_i.iso and [Cl⁻]_i.hypo is the concentration of intracellular chloride under isotonic and hypotonic solutions.

2.4. Measurement of I_Cl,vol and cell membrane capacitance

Whole-cell patch experiments were performed as we described previously [13]. The isotonic bath solution contained (mM): 107 N-methyl-d-glucamine chloride (NMDG-Cl), 1.5 MgCl₂, 2.5 MnCl₂, 0.5 CdCl₂, 0.05 GdCl₃, 10 glucose, 10 HEPES, and 70 d-mannitol, pH 7.4 with NMDG. The osmolarity of this solution measured by a freezing point depression osmometer (OSMOMAT030, Germany) was 300mosmol/kg·H₂O. A 230 mosmol/kg·H₂O hypotonic bath solution containing 116 mM [Cl⁻]_o was made by omitting the 70 mM d-mannitol from the isotonic solution. To examine the Cl⁻ dependence, Cl⁻ in the hypotonic medium was replaced by equimolar aspartate⁻ to obtain the hypotonic solution containing 39 mM [Cl⁻]_o. The internal pipette solution (300 mosmol/kg·H₂O) contained (mM): 95 CsCl, 20 TEACl, 5 ATP-Mg, 5 EGTA, 5 HEPES, and 80 d-mannitol, pH 7.2 with CsOH. Recordings were started 5 min after the establishment of the whole-cell configuration to allow the equilibration of the pipette solution with cell interior. The currents were elicited with voltage steps from −100 mV to +120 mV in +20 mV increment for 400 ms at an interval of 5 s from a holding potential of −40 mV. Currents were sampled at 5 kHz using pCLAMP8.0 software (Axon Instruments) and filtered at 2 kHz. To minimize the changes of liquid junction potentials, a 3 mM KCl-agar salt bridge between the bath and the Ag-AgCl reference electrode was used. The cell membrane capacitance was calculated by integrating the area under an uncompensated capacitive transient elicited by a 5-mV hyperpolarizing pulse from a holding potential of 0 mV. Membrane potential was measured in the whole-cell current clamp mode and was monitored for 5–10 min following establishment of the whole-cell configuration to ensure stability. All experiments were performed at room temperature (25 °C).

2.5. Effect of hypotonic medium culture on foam cell formation

Hypotonic medium with an osmolarity of 230 mosmol/kg·H₂O was prepared by replacing a quarter of normal RMPI 1640 medium with de-ionized water. A 300 mosmol/kg·H₂O isotonic medium was made by adding 70 mM d-mannitol to the hypotonic solution. Under the condition of continuous treatment with 80 μg/mL ox-LDL, RAW264.7 cells were initially cultured by hypotonic medium for 6, 12, or 24 h respectively, followed by normal RMPI 1640 medium for the remaining time of total 48 h. The cells cultured with isotonic medium were used correspondingly as control. To study the effect of membrane depolarization on foam cell formation, 40 mM K-gluconate was added to the hypotonic medium and the osmolarity of this medium was 295.3 ± 3.6 mosmol/kg·H₂O. Here we used K-gluconate instead of KCl to prevent the alteration of extracellular Cl⁻ concentration. The formation of foam cell was evaluated by the measurement of intracellular cholesterol contents and oil red O staining.

2.6. Animal model of atherosclerosis

Atherosclerosis of ApoE^−/− mice was prepared as described previously [14]. Briefly, 50 male 8-week-old ApoE^−/− mice were fed a western-type high-fat diet (0.15% cholesterol, 20% saturated fat) and were randomly divided into 4 groups as follows: (1) control group with normal diet for 20 weeks, (2) 1 week high-fat diet group (normal diet 19 weeks + high-fat diet 1 week), (3) 7 weeks high-fat diet group (normal diet 13 weeks + high-fat diet 7 weeks), and (4) 14 weeks high-fat diet group (normal diet 6 weeks + high-fat diet 14 weeks). All mice were sacrificed at the same age of 20 weeks. The detailed experimental design was clarified as AppendixBFig. S2A in the supplementary materials. All of the experimental procedures were approved by the Sun Yat-Sen University Committee for Animal Research and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

2.7. Peritoneal macrophages isolation

Peritoneal macrophages were isolated from ApoE^−/− mice as previously described [15]. Briefly, at the end of high-fat diet, mice were anaesthetized and intraperitoneally perfused by 20 mL Hanks’ balanced salt solution (mM: NaCl 137.0, KCl 5.4, Na₂HPO₄ 0.42, KH₂PO₄ 0.44 (NaNCO₃ 4.17, glucose 5.55 and HEPES 10.0, pH 7.4) with a rate of 2 mL/min. Peritoneal fluids were gathered and centrifuged for 10 min at 1000 rp. Pellets were washed and re-centrifuged three times with PBS. Macrophages were resuspended in RPMI-1640 containing 10% fetal calf serum at a concentration of 1 ± 10⁶/mL and incubated in a CO₂ (5%) incubator at 37 °C finally.

2.8. Analysis of atherosclerotic plaque area

At 20 weeks, mice were anesthetized by injection of 10% chloral hydrate. After peritoneal fluids were extracted from apoE^−/− mice, the heart and aorta were perfused with 20 mL PBS at a rate of 2 mL/min. The aorta was dissected from the heart to the iliac bifurcation, the adventitial tissue was cleaned carefully under a dissecting microscope, and fixed in 10% buffered formalin for 24 h. The fixed aorta was opened longitudinally and stained with 0.3% oil red-O for 2 h and then were destained in 78% methanol for 5 min. Plaques were analyzed under the Olympus microscope connected to a digital camera with macro conversion lens. The positive staining area was quantified using Image-Pro Plus 5.0 software. The aortic plaque area of each mouse was obtained by the average of the positive staining areas in six sections from the same animal.

2.9. Statistical analysis

All data were expressed as mean ± SD. Statistical analyses were performed using Student's t test, analysis of variance by ANOVA, or linear correlation analysis. Values of P < 0.05 were considered significant.

3. Results

3.1. Ox-LDL induced foam cell formation and cell volume increase in RAW264.7

Ox-LDL treatment increased the accumulation of oil red O-positive droplets in cultured RAW264.7 cells in concentration-dependent (40–120 μg/mL, Fig. 1A) and time-dependent (0–48 h, Fig. 1B) manners. 80 μg/mL ox-LDL treatment for 48 h remarkably increased the oil red O-positive droplets in RAW264.7 cells. So we then tested the effect of 80 μg/mL ox-LDL on intracellular cholesterol levels in cultured RAW264.7 cells at different time points and our results in AppendixBTable S1 showed that ox-LDL significantly increased the total cholesterol, cholesterol ester, and cholesterol esters/total cholesterol ratio in a time-dependent manner. Concomitantly, the cell volume, as assessed by cell membrane capacitance, was also significantly increased in foam cells after ox-LDL treatment in a time-dependent manner (Fig. 1C).

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Fig. 1. 

Effect of ox-LDL treatment on cell membrane electric capacitance in RAW264.7 macrophages. (A) Concentration-dependent effect of ox-LDL on foam cell formation. RAW264.7 macrophages were incubated with 0, 40, 80, or 120 μg/mL ox-LDL for 48 h. Intracellular lipid accumulation was examined by oil red O staining. Representive results from 4 independent experiments were shown (400×). (B) Time-dependent effect of ox-LDL on foam cell formation. RAW264.7 macrophages were incubated without or with 80 μg/mL ox-LDL for 24 or 48 h and then were stained with oil red O. Representive results from 5 independent experiments were shown (400×). (C) Cell capacitance was progressively increased during foam cell formation. (*P < 0.05, **p < 0.01 vs control, n = 12–15). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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3.2. Alteration of [Cl⁻]_i during ox-LDL induced foam cell formation in RAW264.7 cells

To determine whether disordered Cl⁻ transmembrane movement is involved in atherosclerosis, we examined [Cl⁻]_i during foam cell formation. As shown in Fig. 2A, compared with control, the resting [Cl⁻]_i in isotonic solution was significantly decreased during foam cell formation (from 37.99 ± 1.63 mM of control to 32.86 ± 1.69 mM at 24 h and 30.15 ± 1.13 mM at 48 h after ox-LDL treatment). When cells were perfused with hypotonic solution, [Cl⁻]_i of control RAW264.7 cells was reduced to 34.72 ± 1.54 mM, resulting in a 8.6 ± 1.6% decrease of [Cl⁻]_i as compared with its isotonic control. In ox-LDL treated cells, hypotonic perfusion induced more prominent decrease in [Cl⁻]_i (Fig. 2A). The response of [Cl⁻]_i to hypotonic challenge was time- and concentration- dependently augmented after ox-LDL treatment and was in proportion to the accumulation of intracellular cholesterol levels (Fig. 2A–E).

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Fig. 2. 

Effect of ox-LDL on intracellular Cl⁻ concentration ([Cl⁻]_i) and volume-regulated Cl⁻ movement. (A) Time-dependent effect of 80 μg/mL ox-LDL on Cl⁻ movement. i) Representative traces of [Cl⁻]_i under isotonic and hypotonic solution in RAW264.7 macrophages incubated with or without ox-LDL for 6, 12, 24 and 48 h. ii) Alteration of [Cl⁻]_i under isotonic and hypotonic solution in RAW264.7 cells after treatment with ox-LDL for 6 to 48 h. iii) The percentage alteration of [Cl⁻]_i (Δ[Cl⁻]_i,hypo(%)) by hypotonic solution in RAW264.7 cells after treatment with ox-LDL for 6 to 48 h. (*p < 0.05, **p < 0.01 vs control, n = 6–8). (B) Concentration-dependent effect of ox-LDL on Cl⁻ movement. i) Alteration of [Cl⁻]_i under isotonic and hypotonic solution in RAW264.7 cells after treatment with 0, 40, 80, or 120 μg/mL ox-LDL for 48 h. ii) The percentage alteration of [Cl⁻]_i (Δ[Cl⁻]_i,hypo(%)) by hypotonic solution in RAW264.7 cells after treatment with 0, 40, 80, or 120 μg/mL ox-LDL for 48 h. (*p < 0.05, **p < 0.01 vs control, n = 8).

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3.3. Alteration of I_Cl.vol during ox-LDL induced foam cell formation in RAW264.7 cells

The results of [Cl⁻]_i measurement imply that there is an enhanced volume-regulated Cl⁻ movement across the cell membrane during foam cell formation. We therefore examined I_Cl.vol to further explore whether the alteration of [Cl⁻]_i is mediated by VRCC. In control RAW264.7 cells, the basal whole cell current was very small recorded by patch clamp when the cells were exposed in isotonic solution. After extracellular solution was changed from isotonic to hypotonic solution, a large outward rectifying current was evoked with a reverse potential of −1.2 ± 1.6 mV. The current densities under isotonic and hypotonic solution were −10.2 ± 1.4 and −42.1 ± 3.9 pA/pF at −100 mV and 38.4 ± 3.2 and 201.2 ± 11.6 pA/pF at +100 mV, respectively. When the [Cl⁻]_o in the bath solution was changed from 116 to 39 mM by replacing Cl⁻ with aspartate⁻, the current was significantly decreased, with a change in the reverse potential from −1.2 ± 1.6 mV to 26.5 ± 1.5 mV (Fig. S1). The reverse potentials were very close to Cl⁻ equilibrium potential (E_Cl), in view of the fact that the theoretically calculated E_Cl was 0 mV in 116 mM [Cl]_o, and 28.4 mV in 39 mM [Cl]_o. The results indicated that a native osmotic sensitive I_Cl.vol could be activated by hypotonic challenge in RAW264.7 macrophages.

Next, we investigated whether there is an alteration of I_Cl.vol during foam cell formation. Consistent with the reduction in [Cl⁻]_i, the hypotonic induced I_Cl.vol was time-dependently increased during foam cells formation. At +100 mV, the hypotonic-induced net increase of current density from control cells was 161.7 ± 11.9 pA/pF, which was increased to 210.0 ± 16.1 pA/pF and 224.9 ± 20.5 pA/pF at 24- and 48-h after ox-LDL treatment, respectively (Fig. 3A). The correlation analysis revealed a significant positive correlation between the current densities of I_Cl.vol and the alteration of [Cl⁻]_i, suggesting the increased VRCC activity is involved in mediating transmembrane Cl⁻ movement during foam cell formation (n = 8, r = 0.884, P < 0.05, Fig. 3B).

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Fig. 3. 

Effects of ox-LDL on I_Cl.vol in RAW264.7 macrophages. (A) (i) Representative traces of I_Cl.vol in RAW264.7 macrophages without or with ox-LDL (80 μg/mL) treatment for 6, 12, 24, or 48 h. (ii) Mean current densities at ±100 mV in RAW264.7 macrophages without or with ox-LDL (80 μg/mL) treatment for 6–48 h (*P < 0.05, **p < 0.01 vs control, n = 10–12). (B) Correlation between current densities of I_Cl.vol and alteration in [Cl⁻]_i during foam cell formation (n = 10, r = 0.884, P < 0.01). (C) Effects of cell size and H₂O₂ on chloride current in RAW264.7 cells. Cells were incubated with zymosan for 6 h and then the chloride current was recorded under isotonic solution (p < 0.05 vs control, n = 7). Effect of H₂O₂ on the activation of VRCC was measured under isotonic solution containing 500 μM H₂O₂. (**p < 0.01 vs control, n = 6).

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To test whether the increase of cell size per se contributed to the unregulation of VRCC during foam cell formation, we measured the chloride current in RAW264.7 cells incubated with zymosan, an inert material which can be uptaken by macrophage. After zymosan treatment for 6 h, the cell capacitance was increased from 6.6 ± 0.2 pF to 9.5 ± 0.5 pF (p < 0.01, n = 9). Concomitantly, VRCC was also activated and the current densities were increased from −10.1 ± 1.2 to −16.3 ± 2.3 pA/pF at −100 mV and from 33.8 ± 3.0 to 46.3 ± 3.9 pA/pF at +100 mV, respectively. (Fig. 3C, *p < 0.05 vs control, n = 7). Moreover, we observed the effect of hydrogen peroxide (H₂O₂) on chloride current in RAW264.7 cells because oxidative stress plays an important role in ox-LDL induced foam cell formation. Fig. 3C showed that 500 μM H₂O₂ activated VRCC and augmented the current densities to −34.0 ± 3.5 pA/pF at −100 mV and 87.2 ± 6.8 pA/pF at +100 mV, respectively (Fig. 3C, **p < 0.01 vs control, n = 6). These data indicated that both cell size increase and oxidative stress contributed to the ox-LDL-induced activation of VRCC.

3.4. Alterations of [Cl⁻]_i and I_Cl.vol in macrophages from high-fat diet fed ApoE^−/− mice

To further confirm the alteration of VRCC in foam cell formation is also the case ex vivo, we performed experiments in high cholesterol fed ApoE^−/− mice, a well accepted model for atherosclerosis. The experimental design was shown in AppendixBFig. S2A. Compared with normal diet mice, the intracellular cholesterol contents in isolated peritoneal macrophages and the atherosclerotic plaque area of aorta were significantly increased by high cholesterol diet in a time-dependent manner (AppendixBTable S2, Figs. S2B and S2C).

Parallel to the increased atherosclerotic plaque area and intracellular cholesterol accumulation, the volume-regulated Cl⁻ movement in peritoneal macrophages was also increased. Similar to the results of ox-LDL treated RAW264.7 cells, the resting [Cl⁻]_i in isotonic solution was decreased by high cholesterol diet. When cells were perfused with hypotonic solution, a more prominent reduction in [Cl⁻]_i was induced in peritoneal macrophages from high cholesterol diet atherosclerotic mice than that in normal diet control. Hypotonic perfusion resulted in 5.9 ± 2.1% decrease in [Cl⁻]_i of control cells, which were increased to 6.5 ± 1.5%, 15.2 ± 4.2%, and 20.8 ± 6.9% by 1-, 7-, and 14-week high cholesterol diet, respectively (Fig. 4A). It is clearly that hypotonic-induced reduction in [Cl⁻]_i was progressively augmented along with the time-course of high cholesterol diet.

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Fig. 4. 

Alteration of [Cl⁻]_i and I_Cl.vol in peritoneal macrophages from high-fat diet ApoE^−/− mice. (A) Representative traces of [Cl⁻]_i levels under isotonic and hypotonic solution in peritoneal macrophages from control (n = 15), high-cholesterol diet for 1 week (n = 10), 7 weeks (n = 12), and 14 weeks (n = 13) ApoE^−/− mice. (B) (i) Representative traces of I_Cl.vol in peritoneal macrophages from ApoE^−/− mice with high-cholesterol diet for 0 week, 1 week, 7 weeks, and 14 weeks. (ii) Mean current densities at ±100 mV in peritoneal macrophages from ApoE^−/− mice with normal or high-cholesterol diet. (*P < 0.05, **p < 0.01 vs control, n = 15). (C) Cell membrane electric capacitance in peritoneal macrophages from ApoE^−/− mice with normal or high-cholesterol diet. (*P < 0.05 vs control, n = 15). (E) Correlation between atherosclerotic plaque area (%) and current densities (n = 15, r = 0.892, P < 0.01) or Δ[Cl⁻]_i,hypo (n = 12, r = 0.883, P < 0.01) during the progression of atherosclerosis.

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We further examined the alteration of I_Cl.vol in peritoneal macrophages during the process of atherosclerosis in ApoE^−/− mice. As compared with control of 99.7 ± 6.0 pA/pF at +100 mV, the net increase of current densities induced by hypotonic solution from the 1-, 7-, and 14-week high cholesterol-fed groups were elevated to 100.2 ± 4.3 pA/pF, 117.1 ± 7.4 pA/pF, and 142.0 ± 9.8 pA/pF, respectively (Fig. 4B). Moreover, the value of cell membrane electric capacitance was also elevated in a time-dependent manner along with the development of atherosclerosis, implying an increased cell volume (Fig. 4C).

The correlation analysis revealed that atherosclerotic plaque area (%) was positively correlated to the net increase of current densities (n = 15, r = 0.892, P < 0.01) and [Cl⁻]_i reduction level (n = 12, r = 0.883, P < 0.01) induced by hypotonic challenge (Fig. 4D).

3.5. Activation of VRCC by hypoosmotic medium accelerated foam cell formation

Based on the findings above, it is worthy to determine how the increased I_Cl.vol would affect foam cell formation. To clarify this point, we cultured RAW264.7 cells in hypotonic medium to directly activate VRCC. The results showed that, compared with their time-matched isotonic medium culture controls, the intracellular cholesterol accumulation induced by ox-LDL was progressively increased by hypotonic medium at all of the time points (Fig. 5), suggesting VRCC is involved in the regulation of foam cell formation.

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Fig. 5. 

Effect of hypoosmotic culture on foam cell formation. Activation of VRCC by hypotonic medium significantly increased intracellular cholesterol levels induced by ox-LDL. n = 18 from 6 separate experiments, *P < 0.05, **P < 0.01 vs isosmotic control.

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3.6. Cl⁻ channel blockers prevented foam cell formation

To validate the functional role of VRCC in the regulation of foam cell formation, we further observed the effects of Cl⁻ channel blockers on ox-LDL induced cholesterol accumulation in RAW264.7 cells. Pretreatment with 5-nitro-2,2-dicarboxylic acid (NPPB, 100 μM) or anthracene-9-carboxylate (9-AC, 100 μM), the two Cl⁻ channel blockers, remarkably attenuated ox-LDL induced accumulation of oil red O-positive droplets in cultured RAW264.7 cells. After ox-LDL treatment, the intracellular cholesterol level was increased from 80.2 ± 6.1 μg/mg protein to 170.6 ± 15.1 μg/mg protein (p < 0.05, n = 7). NPPB and 9-AC pretreatment decreased ox-LDL induced accumulation of intracellular cholesterol levels to 97.5 ± 7.2 and 107.2 ± 5.9 μg/mg protein, respectively (Fig. 6A, *p < 0.05 vs control, #p < 0.05 vs ox-LDL only group, n = 5–7).

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Fig. 6. 

Effect of Cl⁻ channel blockers on foam cell formation in RAW264.7 cells. (A) RAW264.7 cells were pretreated with Cl⁻ channel blockers, 5-nitro-2,2-dicarboxylic acid (NPPB, 100 μM) or anthracene-9-carboxylate (9-AC, 100 μM) for 1 h and then 80 μg/mL ox-LDL was added into the culture medium. After 48 h, the accumulation of oil red O-positive droplets (i) and intracellular cholesterol levels (ii) were measured as decribed in Section 2. (*p < 0.05 vs control, #p < 0.05 vs ox-LDL only group, n = 5–7). (B) RAW264.7 cells cultured in isotonic medium, isotonic medium containg 40 mM K-gluconate, or hypotonic medium were treated with 80 μg/mL ox-LDL. 48 h later, the cells were stained with oil red O. Representive results from 5 independent experiments were shown (400×). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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In RAW264.7 cells, the rest membrane potential in isotonic solution is −40.6 ± 3.3 mV. Activation of VRCC under hypotonic solution caused membrane depolarization and shifted the membrane potential to −29.4 ± 2.1 mV (p < 0.05, n = 8). We then examined whether membrane depolarization underlies the effect of VRCC activation on foam cell formation. As shown in Fig. 6B compared to the isotonic group, hypotonic medium promoted the accumulation of oil red O-positive droplets in RAW264.7 cells, however, the isotonic medium containing 40 mM K-gluconate, which depolarized the membrane potential to −18.9 ± 2.7 mV, had no effects on foam cell formation, indicating membrane depolarization does not contribute to the functional role of VRCC in regulation of foam cell formation.

4. Discussion

The results of the present study demonstrated several new findings as following: (1) Along with foam cell formation both in ox-LDL treated RAW264.7 cell line and in peritoneal macrophages isolated from high-fat diet ApoE^−/− mice, volume-regulated Cl⁻ movement via VRCC was significantly increased in a time-dependent manner, and its increment positively correlated with intracellular lipid content. (2) The alteration of I_Cl.vol and [Cl⁻]_i in foam cells significantly correlated to the atherosclerotic plaque area during the development of atherosclerosis. (3) VRCC activation increased ox-LDL induced intracellular lipid accumulation and accelerated foam cell formation. In contrast, inhibition of VRCC with Cl⁻ channel blockers prevented ox-LDL induced foam cell formation. These findings suggested that activation of VRCC is involved in the regulation of foam cell formation.

Recently, growing evidence has suggested that the disordered transmembrane cation transport is involved in the pathogenesis of atherosclerosis. For example, a sustained increase in intracellular Ca²⁺ has been shown to be required for ox-LDL uptake [16] and [17], foam cell formation and atherosclerosis could be retarded by calcium channel blocks [2], [3] and [18]. However, little is known about whether anion transport across the membrane is involved in atherosclerosis. VRCC is one of the most important chloride channel responsible for Cl⁻ transport across cell membrane [6] and [7]. An increase in cell volume is usually a typical trigger for VRCC activation, which will evoke the regulatory volume decrease process (RVD) mainly by efflux of Cl⁻ to restore the cell volume towards normal. Apart from the primary function of volume regulation, a few of studies have demonstrated that VRCC participates in a variety of physiological functions, such as cell proliferation, apoptosis, and also angiogenesis [19] and [20]. Most recent study from our lab suggested that volume regulation of [Cl⁻]_i is closely related to blood pressure regulation and may play an important role in cerebrovascular remodeling in hypertension [8]. Our present study provided a new finding that, for the first time to our knowledge, hypotonicity-induced Cl⁻ efflux and chloride current density were significantly increased both in ox-LDL treated RAW264.7 cell line and in peritoneal macrophages isolated from high-fat diet ApoE^−/− mice, indicating the upregulation of VRCC during foam cell formation.

Interestingly, our present results demonstrated that the cell volume of macrophages became larger along with foam cell formation. Therefore, it is likely that VRCC activity would be activated by the increased cell volume due to lipid uptake when macrophages are transforming into foam cells. To test this hypothesis, we studied the effect of cell size per se on VRCC in RAW264.7 cells preincubated with zymosan, an inert material which can be uptaken by macrophage. Indeed, zymosan treatment increased cell capacitance. The increase of cell size could activate an outward chloride current which was inhibited by hypertonic solution. Apart from the cell size, recent growing evidence has demonstrated that a variety of physiological and pathophysiological stimuli, such as angiotensin II, endothelin-1 and H₂O₂, could activate VRCC independent of the increase of cell volume [21], [22], [23], [24] and [25]. In present study, we also observed that H₂O₂, when applying in isotonic solution, activated VRCC in RAW264.7 cells. Because oxidative stress has been reported to play a critical role in ox-LDL induced foam cell formation, so our data here indicated that both cell size increase and oxidative stress contributed to the ox-LDL-induced upregulation of VRCC. The activity of VRCC has been proposed to be modulated by the physical properties of cell membrane [26] and [27]. Previous studies in endothelial and intestine cells have demonstrated that membrane cholesterol content is involved in the regulation of VRCC. Depletion of membrane cholesterol could increase the activity of VRCC [26] and [27]. These results are seemingly inconsistent with our findings since ox-LDL could increase intracellular cholesterol levels. However, it is noteworthy that recent growing work has suggseted that ox-LDL treatment may induce the internalization of caveolin and the depletion of cholesterol from caveolin-enriched membrane fractions [28] and [29]. Therefore, our results cannot exclude the possibility that depletion of membrane cholesterol levels after ox-LDL treatment may also attribute to the increase of VRCC during foam cell formation.

The correlation of VRCC with foam cell formation was further strengthened by the findings that activation of VRCC by hypotonic medium could further significantly increase intracellular cholesterol accumulation and accelerate foam cell formation, whereas, inhibition of VRCC with Cl⁻ channel blockers remarkably attenuated intracellular cholesterol levels and prevented lipid accumulation in ox-LDL treated RAW264.7 macrophages. These findings suggested that, at least, the activation of VRCC plays a functional role in facilitating foam cell formation. In addition, activation of VRCC with hypotonic solution caused membrane depolarization, however, the high K⁺ solution had no significant effects on foam cell formation, suggesting membrane depolarization does not underlie the functional role of VRCC in regulation of foam cell formation.

In summary, the present study demonstrated that volume-regulated Cl⁻ movement via VRCC is augmented during macrophage-derived foam cell formation, and its increment positively correlates with the intracellular cholesterol content as well as the atherosclerotic plaque area. The increased VRCC activity may exert an important role in the development of atherosclerosis in part by facilitating foam cell formation. Clearly, the recognition of VRCC and its role in foam cells is just the beginning of our understanding of the relationship between VRCC and atherosclerosis. It is of great importance, however, that our present research has shed a new light and filled a significant gap in such knowledge to clarify the role of anion channel in atherosclerosis.

Disclosures

None.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Key Grant No. 30730105, and No. 30873060, No. 30973536). National Basic Research Program of China (973 Project. No. 2009CB521903) and Foundation for the Author of National Excellent Doctoral Dissertation of PR China (No. 200773).

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Appendix A. Supplementary data

 

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Corresponding authors. Tel.: +86 20 87331857; fax: +86 20 87331209.
¹ Both authors contributed equally to this work. Copyright © 2011 Elsevier Ireland Ltd All rights reserved.

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Atherosclerosis
Volume 216, Issue 1, May 2011, Pages 59-66  

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