Specific Kv1.3 blockade modulates key cholesterol-metabolism-associated molecules in human macrophages exposed to ox-LDL.

Cholesterol-metabolism-associated molecules, including scavenger receptor class A (SR-A), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), CD36, ACAT1, ABCA1, ABCG1, and scavenger receptor class B type I, can modulate cholesterol metabolism in the transformation from macrophages to foam cells. Voltage-gated potassium channel Kv1.3 has increasingly been demonstrated to play an important role in the modulation of macrophage function. Here, we investigate the role of Kv1.3 in modulating cholesterol-metabolism-associated molecules in human acute monocytic leukemia cell-derived macrophages (THP-1 macrophages) and human monocyte-derived macrophages exposed to oxidized LDL (ox-LDL). Human Kv1.3 and Kv1.5 channels (hKv1.3 and hKv1.5) are expressed in macrophages and form a heteromultimeric channel. The hKv1.3-E314 antibody that we had generated as a specific hKv1.3 blocker inhibited outward delayed rectifier potassium currents, whereas the hKv1.5-E313 antibody that we had generated as a specific hKv1.5 blocker failed. Accordingly, the hKv1.3-E314 antibody reduced percentage of cholesterol ester and enhanced apoA-I-mediated cholesterol efflux in THP-1 macrophages and human monocyte-derived macrophages exposed to ox-LDL. The hKv1.3-E314 antibody downregulated SR-A, LOX-1, and ACAT1 expression and upregulated ABCA1 expression in THP-1 macrophages and human monocyte-derived macrophages. Our results reveal that specific Kv1.3 blockade represents a novel strategy modulating cholesterol metabolism in macrophages, which benefits the treatment of atherosclerotic lesions.

After differentiation, THP-1 macrophages or HMDMs were exposed to 100 µg/ml ox-LDL in the presence of the hKv1.3-E314 antibody at varying concentrations of 37.5, 75, or 300 nM for 2 h. THP-1 macrophages or HMDMs were exposed to 100 µg/ml ox-LDL for 24 h to accelerate foam cell formation with less toxicity or apoptosis ( 33,34 ). Meanwhile, to observe the action of the hKv1.3-E314 antibody during the transformation, THP-1 macrophages or HMDMs were exposed to 100 µg/ml ox-LDL for up to 36 or 48 h in the presence of the 300 nM hKv1.3-E314 antibody.
In the experiments, THP-1 macrophages and HMDMs exposed to the hKv1.3-E314 antibody alone and 100 µg/ml ox-LDL alone were cultured for 24 h.

Immunofl uorescent staining
THP-1 macrophages were fi xed and blocked with a solution containing 1% BSA and 10% goat serum (Invitrogen, Carlsbad, CA). Fixed cells were incubated with the hKv1.3-E314 antibody or the hKv1.5-E313 antibody and then with the FITC-conjugated secondary anti-rabbit goat antibody (Alomone, Israel). Nuclear chromatin was stained with DAPI (eBioscience, San Diego, CA). Negative control was prepared by the primary antibody preincubated with an excess of corresponding antigenic peptides. Cell samples were imaged with a Nikon A1si confocal laser microscope (Nikon, Tokyo, Japan).

Cholesterol content and effl ux analysis
Cells were counterstained with hematoxylin and oil red O (ORO) following the routine procedure. Cells with a lipid droplet area no less than the width of the nucleus were designated ORO positive (ORO+). The ORO+ cells were counted ( 35 ).
HPLC was conducted as follows. Briefl y, cells were sonicated and lysed before triglycerides and proteins were eliminated from cell lysates. Dissolved in a solution of n-hexane and isopropanol (4:1, V/V), free cholesterol (FC) was extracted. One aliquot sample was treated with cholesterol esterase to obtain total cholesterol (TC). Samples were dried through a vacuum degasser and dissolved in a mobile phase containing isopropanol:n-heptane:acetonitrile (35:12:52, v/v). TC and FC were measured by a chromatographer system (VARIAN Prostar 210). Cholesterol ester (CE) was calculated through the subduction of FC from TC.
Percentage of cholesterol effl ux was measured by liquid scintillation counting. Treated THP-1 macrophages or HMDMs were labeled with 1.0 µCi/ml [ 3 H]cholesterol. ApoA-I (10 µg/ml), HDL 2 (50 µg/ml), or HDL 3 (50 µg/ml) was also added to media. The percentage of cholesterol effl ux was calculated by dividing media-derived radioactivity by the sum of the radioactivity in media and cells: [media counts/ (media counts + cellular counts)] × 100%. system or network to maintain cellular cholesterol homeostasis in macrophages. It is clinic-promising to modulate the expression of these cholesterol-metabolism-associated molecules in macrophages.

Ethics statement
Our experiment involving fresh plasma and peripheral blood mononuclear cells of normolipidemic volunteers was approved by volunteers and Wuhan Blood Centre (authorizations: 2010-8) and conformed to the Declaration of Helsinki.

Antibody generation, LDL isolation, and oxidization
The antibody targeting the E314 peptide of human Kv1.3 pore region (named the hKv1.3-E314 antibody; China Patent Application Number of the E314 peptide: 201110044416.x) was previously generated and used as a specifi c blocker of hKv1.3 channels ( 26 ). In addition, we generated the antibody targeting the E313 peptide of human Kv1.5 pore region (named the hKv1.5-E313 antibody; China Patent Application Number of the E313 peptide: 201110293643.6) as a specifi c blocker of hKv1.5 channels following the same strategy as described previously (29)(30)(31).
Native LDLs (densities ranging from 1.006 to 1.063 g/ml) were isolated from fresh plasma of normolipidemic volunteers by sequential preparative ultracentrifugation according to published standard protocols ( 32 ). Then LDLs were oxidized with 10 µM CuSO 4 to obtain ox-LDL.

Cell culture
THP-1 cells were purchased from American Type Culture Collection (ATCC) and maintained in RPMI 1640 medium supplemented with 10% FBS at 37°C. To induce monocyteto-macrophage differentiation, THP-1 cells were cultured in the presence of 160 nM phorbol 12-myristate 13-acetate for 72 h.
Peripheral blood mononuclear cells were isolated from peripheral blood samples of normal volunteers by Ficoll density gradient centrifugation and cultured in RPMI 1640 medium with 10% FBS at 37°C in 5% CO 2 for 7 days to induce differentiation into HMDMs. in THP-1 macrophages and THP-1-derived foam cells. In the transformation from macrophages to foam cells, hKv1.3 or hKv1.5 expression showed no signifi cant difference. The hKv1.3-E314 antibody or the hKv1.5-E313 antibody specifi cally recognizes human Kv1.3 or Kv1.5 channels and binds to plasma membrane in THP-1 macrophages By Western blotting and immunofl uorescent staining, we confi rmed specifi city and plasma membrane binding of both the antibodies (the hKv1.3-E314 antibody and the hKv1.5-E313 antibody) that we had generated in THP-1 macrophages. The hKv1.3-E314 antibody or the hKv1.5-E313 antibody, respectively, recognized 64 kDa or 75 kDa protein, whereas both the antibodies preincubated with corresponding antigenic peptides were unable to recognize identical molecular weight proteins (supplementary Fig. IIA, B). Immunofl uorescent staining results indicated that only plasma membrane was stained with green fl uorescence in THP-1 macrophages (supplementary Fig. IIC, D).

The hKv1.3-E314 antibody reduces cholesterol content in THP-1 macrophages and HMDMs exposed to ox-LDL and enhances apoA-I-mediated cholesterol effl ux
We had a direct-viewing of cholesterol content in THP-1 macrophages and HMDMs exposed to 100 µg/ml ox-LDL in the presence or absence of the hKv1.3-E314 antibody by ORO staining. When THP-1 macrophages and HMDMs were exposed to 100 µg/ml ox-LDL, lipid droplets increased ( Fig. 1C, K ). In the presence of the 300 nM hKv1.3-E314 antibody, lipid droplets in THP-1 macrophages and HMDMs decreased markedly ( Fig. 1D, L ). The amount of ORO+ cells increased when THP-1 macrophages and

Real-time quantitative RT-PCR
Total cellular RNA was isolated, and cDNA was synthesized by reverse transcription reaction. Real-time quantitative PCR was performed with SYBR ® Premix Ex Taq TM (Takara, Japan) using Applied Biosystems StepOne Realtime PCR System. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. Fold changes in mRNA expression level normalized to GAPDH were calculated by the comparative Ct method formula 2 Ϫ ⌬ ⌬ Ct . The sequences of the PCR primers are listed in Table 1 .

Statistics
All data are presented as the means ± SEM. SPSS 13.0 software was used for statistical analysis. Direct comparisons between two groups were made using unpaired t -test. Data from more than two groups were available for ANOVA. P < 0.05 was considered as statistically signifi cant.  The hKv1.3-E314 antibody downregulates SR-A, LOX-1, and ACAT1 expression and upregulates ABCA1 expression in THP-1 macrophages and HMDMs exposed to ox-LDL By real-time PCR and Western blotting, we assayed the mRNA and protein level of cholesterol-metabolismassociated molecules in THP-1 macrophages and HMDMs exposed to 100 µg/ml ox-LDL in the absence or presence of the hKv1.3-E314 antibody, which include SR-A, CD36, LOX-1, ACAT1, ABCA1, ABCG1, and SR-B I. Some of these molecules were downregulated or upregulated.

Human
Compared with SR-A and LOX-1 expression levels in THP-1 macrophages or HMDMs exposed to 100 µg/ml ox-LDL alone, which were elevated in THP-1 macrophages and in HMDMs, the mRNA and protein levels of SR-A and LOX-1 were downregulated in the presence of the hKv1.3-E314 antibody in a concentration-dependent manner. The SR-A and LOX-1 expressions did not change with ox-LDL exposure time ranging from 24 h to 48 h. The mRNA and protein level of CD36 were elevated in HMDMs but not in THP-1 macrophages. There was no HMDMs were exposed to 100 µg/ml ox-LDL ( Fig. 1G, O), and the amount decreased signifi cantly in the presence of the 300 nM hKv1.3-E314 antibody ( Fig. 1H, P, Q ).
By HPLC, TC, FC, and CE in treated THP-1 macrophages and HMDMs were quantifi ed. In THP-1 macrophages and HMDMs, there were signifi cant decreases of TC and CE in the presence of the hKv1.3-E314 antibody in a concentrationdependent manner, compared with the absence of the hKv1.3-E314 antibody. Increases of FC were also observed in the presence of the hKv1.3-E314 antibody, whereas TC decreased. The homeostasis of FC, TC, and CE did not alter with ox-LDL exposure times ranging from 24 h to 48 h ( Table 2 ).
THP-1 macrophages and HMDMs exposed to 100 µg/ml ox-LDL. The ACAT1 expression did not alter with ox-LDL exposure time ranging from 24 h to 48 h ( Fig. 4 ).
Of all the molecules mediating cholesterol effl ux, including ABCA1, ABCG1, and SR-B I, only ABCA1 expression signifi cant alteration of CD36 expression level in cells preincubated with various concentrations of the hKv1.3-E314 antibody ( Fig. 3 ).
The hKv1.3-E314 antibody also downregulated, in a concentration-dependent manner, ACAT1 expression in Percentage of CE and FC in THP-1 macrophages and HMDMs exposed to 100 µg/ml ox-LDL in the absence (macrophages and macrophages exposed to 100 µg/ml ox-LDL or the 300 nM hKv1.3-E314 antibody alone) or presence of the hKv1.3-E314 antibody at a varying concentrations of 37.5, 75, or 300 nM. In the presence of the 300 nM hKv1.3-E314 antibody, macrophages were exposed to 100 µg/ml ox-LDL, respectively, for 24, 36, or 48 h. HPLC was performed to determine TC, FC, and CE. Data represent the means ± SEM of three independent experiments ( n = 3). * P > 0.05, **P < 0.01 versus macrophages exposed to 100 µg/ml ox-LDL alone. There was no signifi cant difference in percentage of cholesterol ester when macrophages were exposed to 100 µg/ml ox-LDL for 24, 36, or 48 h in the presence of the 300 nM hKv1.3-E314 antibody.

Fig. 2.
Percentage of cholesterol effl ux from THP-1 macrophages or HMDMs exposed to 100 µg/ml ox-LDL in the absence (macrophages and macrophages exposed to 100 µg/ml ox-LDL or the 300 nM hKv1.3-E314 antibody alone) or presence of the hKv1.3-E314 antibody at varying concentrations of 37.5, 75, or 300 nM. In the presence of the 300 nM hKv1.3-E314 antibody, macrophages were exposed to 100 µg/ml ox-LDL for 24, 36,  . *P < 0.05, **P < 0.01, and P > 0.05 versus macrophages exposed to 100 µg/ml ox-LDL alone. There was no signifi cant difference in percentage of apoA-I or HDL-mediated cholesterol effl ux when macrophages were exposed to 100 µg/ml ox-LDL for 24, 36, or 48 h in the presence of the 300 nM hKv1. 3-E314 antibody. in mouse macrophages (37)(38)(39). Herein our data provide supportive evidence that the complex is present in THP-1 macrophages. Owing to the homogeneous structural features of the entire voltage-gated potassium channel superfamily and the conservation of drug-binding sites of the Kv1.3 and Kv1.5 channels ( 40,41 ), the coexistence makes it diffi cult to discriminate the dominance of hKv1.3 or hKv1.5 channels in THP-1 macrophages, at which possible pharmaceutical targets would be aimed.
We generated two antibodies directed against the extracellular peptides of hKv1.3 or hKv1.5 pore region, which can specifi cally block hKv1.3 or hKv1.5 channels, respectively, named the hKv1.3-E314 antibody and the hKv1.5-E313 antibody. These antibodies are not able to cross-react to other closely related Kv1 channels but can react with themselves. The hKv1.3-E314 antibody signifi cantly inhibited outward delayed rectifi er potassium currents in THP-1 macrophages in a concentration-dependent manner, whereas the hKv1.5-E313 antibody failed to show an inhibiting tendency at the concentration identical to the hKv1.3-E314 antibody, which indicates that hKv1.3-containing subunit regulates the permeability of the heteromultimeric channel.
Initially, when the heteromultimeric Kv1.3 channel was blocked, cholesterol content decreased pronouncedly was upregulated in THP-1 macrophages and HMDMs exposed to 100 µg/ml ox-LDL in the absence or presence of the hKv1.3-E314 antibody in a concentration-dependent manner. In line with ABCA1 mRNA level, ABCA1 protein level was signifi cantly elevated compared with its level in THP-1 macrophages exposed to 100 µg/ml ox-LDL alone. The hKv1.3-E314 antibody at a varying concentration caused a 1.5-to 3-fold increase in THP-1 macrophages and a 1.3 to 3.1-fold increase in HMDMs. And the ABCA1 expression did not alter with ox-LDL exposure time ranging from 24 h to 48 h ( Fig. 5 ).

DISCUSSION
Our study confi rms that blockade of Kv1.3 prevents foam cell formation. We have, using a novel antibody-based approach, provided the fi rst evidence for some of the molecular changes that contribute to this effect.
Outward delayed rectifi er potassium currents are elicited and elevated when membranes are depolarized accompanied by macrophage activation ( 19,36 ). The currents were long thought to be carried by the Kv1.3 channel ( 19,20 ). However, in recent years, the Kv1.3 and Kv1.5 channels have been identifi ed, forming a heteromultimeric complex Fig. 3. mRNA and protein expression levels of SR-A, LOX-1, and CD36 in THP-1 macrophages and HMDMs exposed to 100 µg/ml ox-LDL in the absence (macrophages and macrophages exposed to 100 µg/ml ox-LDL or the 300 nM hKv1.3-E314 antibody alone) or presence of the hKv1.3-E314 antibody at varying concentrations of 37.5, 75, or 300 nM. In the presence of the 300 nM hKv1.3-E314 antibody, macrophages were exposed to 100 µg/ml ox-LDL for 24, 36, or 48 h. mRNA and protein levels of SR-A, LOX-1, and CD36 were assayed by real-time quantitative RT-PCR and Western blotting. A and C: mRNA levels of SR-A, LOX-1, and CD36 in THP-1 macrophages and HMDMs. B and D: Protein levels of SR-A, LOX-1, and CD36 in THP-1 macrophages and HMDMs (n = 3). *P < 0.05, **P < 0.01, and P > 0.05 versus macrophages exposed to 100 µg/ml ox-LDL alone. There was no signifi cant difference in mRNA and protein levels when macrophages were exposed to 100 µg/ml ox-LDL for 24, 36, or 48 h in the presence of the 300 nM hKv1.3-E314 antibody. caused a signifi cant reduction of cholesterol accumulation in macrophages by downregulating SR-A, LOX-1, and ACAT1 expression and upregulating ABCA1 expression, of which ACAT1 and ABCA1 are considered to be candidate targets for the treatment of atherosclerotic lesions due to their roles in macrophage cholesterol metabolism ( 18,(52)(53)(54)(55). ACAT1 inhibition or downregulation was shown to be atheroprotective in animal models ( 18,(56)(57)(58). ABCA1 acts as the primary gatekeeper for eliminating excess tissue cholesterol and represents the first and rate-controlling step in reverse cholesterol transport ( 59,60 ). The paramount importance of ABCA1 is exemplifi ed by Tangier disease, which is characterized by loss-of-function mutations in the ABCA1 gene ( 61 ), and is highlighted by an ABCA1deficient or ABCA1-overexpression mouse model, which disabled or enhanced removal of intracellular free cholesterol ( 54,62 ). However, mere ACAT inhibition is not an effective strategy for ameliorating atherosclerosis and may promote atherogenesis, which was validated by the ACAT Intravascular Atherosclerosis Treatment Evaluation (ACTIVATE) study ( 63 ). Moreover, the ACTIVATE study and a clinical trial about apoA-I Milano suggest that ACAT1 inhibitors when used in combination with those compounds, which increase reverse cholesterol transport in THP-1 macrophages and HMDMs exposed to ox-LDL, which was generally consistent with Lei's result by another Kv1.3 blocker ( 25 ). Furthermore, a novel and interesting fi nding emerged that apoA-I-mediated cholesterol effl ux from THP-1 macrophages and HM-DMs was greatly enhanced. These fi ndings enabled us to investigate the underlying mechanism in human macrophages, including THP-1 macrophages and HMDMs.
In this study, we presented an expression pattern of key cholesterol-metabolism-associated molecules in THP-1 macrophages or HMDMs preincubated with various concentrations of the hKv1.3-E314 antibody. The mRNA and protein expression of SR-A and LOX-1 was downregulated, thereby terminating the positive feedback of ox-LDL uptake. ACAT1 downregulation also resulted in the reduction of cholesterol ester synthesis. The downregulation of ACAT1 produced a large amount of free cholesterol in macrophages, which is harmful to cells and facilitates plaque destabilization (42)(43)(44)(45). Surplus FC effl ux from macrophages could be mediated by ABCA1 upregulation interacting with lipid-free apoA-I (46)(47)(48), unlike ABCG1 and SR-B I interacting with mature HDL ( 47,(49)(50)(51).
The expression pattern represents a comprehensive system or network modulating cholesterol infl ux, synthesis, and effl ux. The modulating system or network Fig. 4. mRNA and protein expression levels of ACAT1 in THP-1 macrophages and HMDMs exposed to 100 µg/ml ox-LDL in the absence (macrophages and macrophages exposed to 100 µg/ml ox-LDL or the 300 nM hKv1.3-E314 antibody alone) or presence of the hKv1.3-E314 antibody at varying concentrations of 37.5, 75, or 300 nM. In the presence of the 300 nM hKv1.3-E314 antibody, macrophages were exposed to 100 µg/ml ox-LDL for 24, 36, or 48 h. mRNA and protein levels of ACAT1 were assayed, respectively, by real-time quantitative RT-PCR and Western blotting. A and C: mRNA levels of ACAT1 in THP-1 macrophages and HMDMs,. B and D: Protein levels of ACAT1 in THP-1 macrophages and HMDM cells (n = 3). *P < 0.05, **P < 0.01, and P > 0.05 versus macrophages exposed to 100 µg/ml ox-LDL alone. There was no signifi cant difference in mRNA and protein levels when macrophages were exposed to 100 µg/ml ox-LDL for 24, 36, or 48 h in the presence of the 300 nM hKv1.3-E314 antibody. may benefi t the treatment of atherosclerosis (63)(64)(65)(66). ACAT1 downregulation and ABCA1 upregulation by specifi c Kv1.3 blockade in macrophages conform to the strategy.
Although our study presented attractive results that the hKv1.3-E314 antibody can prevent foam cell formation, for the sake of clinical practice, it is necessary to investigate the effect of the antibody on foam cells after exposure to ox-LDL through further research.
In previous studies, Kv1.3 has been validated to play a key role in the modulation of pathogenic T subset function ( 24,40,(67)(68)(69). Moreover, selective Kv1.3 blockade can reach equilibrium between effi cacy and safety in animal models, revealing no systemic toxicity ( 68,69 ). Increasing evidence shows that a specifi ed pathogenic T subset can aggravate atherosclerosis ( 70,71 ). These results encourage us to investigate an atheroprotective effect of the hKv1.3-E314 antibody on T lymphocytes in our ongoing efforts.
Overall, specifi c Kv1.3 blockade exerts an atheroprotective effect in vitro, which signifi es potential value in the treatment of atherosclerotic lesions as a novel strategy. In the future, the monoclonal antibody derived from the E314 peptide of human Kv1.3 pore region will be used in in vivo studies due to its specifi city and long circulating biological half-life ( 72,73 ).  5. mRNA and protein expression levels of ABCA1, ABCG1, and SR-B I in THP-1 macrophages and HMDMs exposed to 100 µg/ml ox-LDL in the absence (macrophages and macrophages exposed to 100 µg/ml ox-LDL or the 300 nM hKv1.3-E314 antibody alone) or presence of the hKv1.3-E314 antibody at varying concentrations of 37.5, 75, or 300 nM. In the presence of the 300 nM hKv1.3-E314 antibody, macrophages were exposed to 100 µg/ml ox-LDL for 24, 36, or 48 h. mRNA and protein levels of ABCA1, ABCG1, and SR-B I were assayed by real-time quantitative RT-PCR and Western blotting. A and C: mRNA levels of ABCA1, ABCG1, and SR-B I in THP-1 macrophages and HMDMs,. B and D: Protein levels of ABCA1, ABCG1, and SR-B I in THP-1 macrophages and HMDMs, (n = 3). *P < 0.05, **P < 0.01, and P > 0.05 versus macrophages exposed to 100 µg/ml ox-LDL alone. There was no signifi cant difference in mRNA and protein levels when macrophages were exposed to 100 µg/ml ox-LDL for 24, 36, or 48 h in the presence of the 300 nM hKv1.3-E314 antibody.