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(Circulation. 2004;110:3472-3479.)
© 2004 American Heart Association, Inc.

Molecular Cardiology

Functional Interplay Between the Macrophage Scavenger Receptor Class B Type I and Pitavastatin (NK-104)

Jihong Han, PhD; Michael Parsons, MD; Xiaoye Zhou, MD; Andrew C. Nicholson, DVM, PhD; Antonio M. Gotto, Jr, MD, DPhil; David P. Hajjar, PhD

From the Center of Vascular Biology and Department of Pathology (J.H., X.Z., A.C.N., D.P.H.) and the Department of Medicine (M.P., A.M.G.), Weill Medical College of Cornell University, New York, NY.

Correspondence to Jihong Han, PhD, Center of Vascular Biology/Department of Pathology, Weill Medical College of Cornell University, 1300 York Ave, New York, NY 10021. E-mail jhan{at}med.cornell.edu

Received February 2, 2004; de novo received March 25, 2004; revision received September 3, 2004; accepted September 14, 2004.

	Abstract

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Background— Scavenger receptor class B type I (SR-BI), a receptor for high-density lipoprotein (HDL), plays an important role in the bidirectional cholesterol exchange between cells and HDL particles and the atherosclerotic lesion development. Enhancement of SR-BI expression significantly reduces, whereas lack of SR-BI expression accelerates, the atherosclerotic lesion development in proatherogenic mice. Statins, a class of inhibitors for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, significantly suppress de novo cholesterol synthesis and reduce the incidence of coronary heart disease. Statins also display multiple pleiotropic effects independently of cholesterol synthesis in the vascular cells. Here, we investigated the effects of pitavastatin (NK-104), a newly synthesized statin, on macrophage SR-BI expression.

Methods and Results— We found that pitavastatin significantly increased SR-BI mRNA and protein expression in a macrophage cell line in a concentration- and time-dependent manner. It also increased SR-BI expression in both mouse peritoneal and human monocyte-derived macrophages. Associated with increased SR-BI expression, pitavastatin enhanced macrophage HDL binding, uptake of [¹⁴C]cholesteryl oleate/HDL, and efflux of [³H]cholesterol to HDL. Pitavastatin abolished the inhibition of macrophage SR-BI expression by cholesterol biosynthetic intermediates. It also restored SR-BI expression inhibited by lipopolysaccharide and tumor necrosis factor- through its inactivation of the transcription factor nuclear factor-B.

Conclusions— Our data demonstrate that pitavastatin can stimulate macrophage SR-BI expression by reduction of cholesterol biosynthetic intermediates and antiinflammatory action and suggest additional pleiotropic effects of statins by which they may reduce the incidence of coronary heart disease.

Key Words: statins • macrophages • scavenger receptors • lipoproteins

	Introduction

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High-density lipoprotein (HDL) performs various functions.¹ In a pathway defined as reverse cholesterol transport, HDL provides an acceptor for the removal of cholesterol from peripheral tissues and carries the cholesterol to other lipoproteins for hepatic uptake and secretion.² This process is an important mechanism in the removal of cholesterol from sites of lipid deposition. Conversely, HDL also provides steroidogenic and other tissues with a source of cholesterol.

The bidirectional flux of cholesterol across the plasma membrane requires the specific receptor for HDL binding.³ Scavenger receptor class B type I (SR-BI), a member of the family of class B scavenger receptors including CD36 and lysosomal integral membrane protein-II (LIMP-II), has been shown to have high affinity for HDL binding and moderate affinity for other lipoproteins, such as native LDL, acetylated LDL, oxidized LDL, and anionic phospholipid vesicles.⁴ In various cell types, SR-BI can bind HDL reversibly and mediate cholesterol efflux and cholesteryl ester uptake.^5,6 A direct relationship has been observed between the flux rate of cholesterol through HDL and the levels of SR-BI expression.⁵

SR-BI is highly expressed in several tissues and cell types, including monocyte/macrophages.⁴ It has been thought to play an important role in the development of human atherosclerotic lesions, because high expression of SR-BI protects proatherogenic mice against lesion development, whereas disruption of SR-BI accelerates the onset of atherosclerosis.^7,8 SR-BI expression can be affected by monocyte/macrophage differentiation and by several molecules, such as probucol, polyunsaturated fatty acids, cellular cholesterol levels, testosterone, lipopolysaccharide (LPS), oxidized LDL, and the cytokines tumor necrosis factor- (TNF-) and interferon- (IFN-).⁴

Statins, potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, significantly reduce total and LDL cholesterol levels in the blood and decrease the incidence of coronary heart disease in patients.⁹ Other pleiotropic effects of statins in the vessel wall include¹⁰ (1) increasing endothelial NO synthase (eNOS) activity and tissue-type plasminogen activator (tPA); (2) decreasing plasminogen activator inhibitor-1 (PAI-1), endothelin-1, proinflammatory cytokine (interleukin [IL]-1ß, IL-6) expression, and LDL oxidation; (3) reducing migration and proliferation of smooth muscle cells; and (4) reducing matrix metalloproteinases (MMPs), inducible NO synthetase (iNOS), and monocyte chemoattractant 1 (MCP-1).

Pitavastatin (NK-104, CAS 147526-32-7; monocalcium bis [(3R,5S,6E)-7-(2-cyclopropyl-4-(4-fluorophenyl)-3-quiolyl)-3,5-dihydroxy-6-heptenoate] is a recently developed HMG-CoA reductase inhibitor. It significantly reduces serum total and LDL cholesterols and triglycerides, whereas it moderately increases HDL cholesterol.¹¹ Recently, we reported that this statin significantly decreased the oxidized LDL receptor CD36.¹² Downregulation of macrophage CD36 expression by pitavastatin was defined through a novel mechanism, because it can suppress peroxisome proliferator–activated receptor- (PPAR-) expression while increasing phosphorylation of PPAR-. Here, we have investigated the impact of pitavastatin on macrophage SR-BI expression in an attempt to more fully define its role in cholesterol metabolism. We demonstrate that pitavastatin can increase SR-BI expression through its influence on cholesterol biosynthetic intermediates and the inflammatory response.

	Methods

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Cell Lines and Reagents
J774 cells, a murine macrophage cell line (ATCC), were cultured in RPMI 1640 medium containing 10% FCS, 50 µg/mL each of penicillin and streptomycin, and 2 mmol/L glutamine. Cells were switched to serum-free medium and received treatments when the confluence was 85%.

To collect peritoneal macrophages, mice were injected with 3 mL of 4% thioglycolate and maintained with access to water and normal chow for 5 days. Mice were euthanized by decapitation, after which peritoneal macrophages were collected from the mouse abdomen by lavage with PBS. Cells were cultured in complete RPMI 1640 medium for 3 hours, and all floating cells were removed. Adhesive cells continued to be cultured with complete RPMI medium for 2 additional days and were treated as indicated.

To isolate human monocytes, blood was drawn from a healthy donor and then mixed (5:1) with dextran sedimentation mixture (15 g dextran, MW 500 000, 15 g D-glucose, and 4.5 g NaCl in 500 mL distilled water and filtered through a 0.2-µm filter). The mixture was incubated for 60 minutes at room temperature to sediment red cells. The upper plasma layer containing lymphocytes was collected and centrifuged for 10 minutes at 1000 rpm. The cell pellet was resuspended with serum-free RPMI medium and dispensed on top of Ficoll-Paque at room temperature. After centrifugation for 20 minutes at 2250 rpm, cells at the interface were carefully collected and washed twice with serum-free RPMI medium. Cells were then suspended in RPMI medium containing 20% FCS in FCS-precoated dishes. After 4 hours in culture, floating cells were removed, and the remaining cells were allowed to complete the monocyte/macrophage differentiation process for 7 days more.

Rabbit polyclonal anti-human/mouse SR-BI antibody was purchased from Novus Biologicals Inc. Anti–nuclear factor (NF)-B p65 and anti–IB- antibodies were purchased from Santa Cruz Biotechnology Inc. NF-B DNA binding activity assay kits were purchased from Active-Motif, Inc. Chemicals were obtained from Sigma-Aldrich Co.

Isolation of Total RNA, Purification of Poly(A⁺) RNA, and Northern Blot
Cells were lysed in RNAzol B (Tel-Test, Inc), chloroform was extracted, and total RNA was precipitated in isopropanol. After it had been washed with 80% and 100% ethanol, the dried pellet of total RNA was dissolved in distilled water and quantified. The poly(A⁺) RNA was purified from 100 µg of total RNA by use of the PolyATtract mRNA Isolation System III (Promega).

Poly(A⁺) RNA was loaded on 1% formaldehyde agarose gel. After electrophoresis, Poly(A⁺) RNA was transferred to a Zeta-probe GT Genomic Tested Blotting Membrane (Bio-Rad Laboratories) in 10x SSC by capillary force overnight. The blot was UV-crosslinked for 2 minutes, then prehybridized with Hybrisol I (Oncor, Inc) for 30 minutes before the addition of a ³²P-randomly primed labeling probe for mouse SR-BI or GAPDH. After overnight hybridization, the membrane was washed for 2x 20 minutes with 2x SSC and 0.2% SDS, and for 2x 20 minutes with 0.2x SSC and 0.2% SDS at 55°C. The blot was autoradiographed by exposure to a x-ray film (X-Omat AR, Kodak). The semiquantitative assay of autoradiograms was assessed by densitometric scanning with a UMAX UC630 flatbed scanner attached to a Macintosh IIci (Apple Computer) running NIH Image software. The probe for mouse SR-BI was generated by reverse transcription–polymerase chain reaction (RT-PCR) based on the published sequence.¹³ The sequences of 5'- and 3'- oligonucleotides used were TCGGCGTTGTCATGATCCTC (121-141) and GGTTCATAAAAGCACGCTGG (551-571), respectively.

Analysis of HDL Binding, Efflux of Free Cholesterol, and Influx of Cholesteryl Ester
HDLs (1.063 to 1.210 g/mL) were isolated from normal human plasma by sequential ultracentrifugation, dialyzed against PBS containing 0.3 mmol/L EDTA, sterilized by filtration through a 0.22-µm filter, and stored under N₂ gas at 4°C. Protein content was determined by the methods of Lowry.¹⁴

To perform the binding of HDL to macrophages, HDL was fluorescein-conjugated with a reactive succinimidyl-ester of carboxyl-fluorescein by use of a labeling kit purchased from Princeton Separations. After treatment, macrophages (1x10⁶ cells/sample) were washed twice with cold PBS and then incubated with 20 µg/mL of labeled HDL in serum-free medium for 2 hours at 4°C. After washing twice with PBS, cells were subjected to fluorescence-activated cell sorting (FACS) (Coulter FACScan) to determine the binding of HDL to macrophages.

Efflux of free cholesterol from macrophages and influx of cholesteryl ester to macrophages after treatments were conducted according to a previous description.¹⁵

Analysis of SR-BI, NF-B p65, and IB- Expression by Western Blotting and SR-BI Surface Expression by FACS
After treatment, macrophages were washed twice with cold PBS, then scraped and lysed in ice-cold lysis buffer (50 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxychlorate, 1 mmol/L PMSF, 50 mmol/L sodium fluoride, 1 mmol/L sodium orthovanadate, 50 µg/mL aprotinin, and 50 µg/mL leupeptin). Lysate was sonicated for 20 cycles, then microcentrifuged for 15 minutes at 4°C, and the supernatant was transferred to a new test tube. After the content had been determined, proteins were loaded and separated on 12% SDS-PAGE and transferred onto nylon-enhanced nitrocellulose membrane. The membranes were blocked with a solution of 0.1% Tween 20/PBS (PBS-T) containing 5% fat-free milk for 1 hour, then incubated with either rabbit polyclonal anti–SR-BI (1:2000), anti–NF-B (1:200), or anti–IB- (1:200) antibody, respectively, for 2 hours at room temperature, followed by a washing for 3x 10 minutes with PBS-T buffer. The blot was reblocked with PBS-T containing 5% milk followed by incubation with horseradish peroxidase conjugated goat anti-rabbit IgG for another hour at room temperature. After washing 3x10 minutes with PBS-T, the membrane was incubated for 1 minute in a mixture of equal volumes of Western blot chemiluminescence reagents 1 and 2 and then exposed to film before development.

To analyze SR-BI surface expression, human monocyte-derived macrophages were scraped and washed twice with PBS containing 1% FCS. Approximately 1x10⁶ cells from each sample were blocked for 30 minutes at room temperature with PBS containing 5% goat serum. After the washing, cells were incubated with rabbit anti–SR-BI antibody (1:100) for 1 hour at room temperature. Cells were then incubated with goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated IgG (1:50) for 45 minutes. After a washing with PBS, cells were subjected to flow cytometric evaluation.

Extraction of Nuclear Proteins and Electrophoretic Mobility Shift Assay of NF-B DNA Binding Activity
After treatments, cells were washed twice with cold PBS and then resuspended in 400 µL of cold buffer A (mmol/L: 10 HEPES, pH 7.9, 10 KCl, 0.1 EDTA, 0.1 EGTA, 1 DTT, and 0.5 PMSF). After 15 minutes’ incubation on ice, cell suspensions were added to 25 µL of 10% NP-40 and vortexed vigorously for 10 seconds. The supernatant was removed after spinning for 30 seconds at 13 000 rpm. The pellet was resuspended in 100 µL cold buffer C (20 mmol/L HEPES, pH 7.91, 400 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L PMSF, 10 µg/mL leupeptin, and 10 µg/mL aprotinin) and kept on ice for 15 minutes. The mixture was spun again for 15 minutes at 13 000 rpm with a microfuge, and the supernatant was collected as nuclear proteins. Nuclear proteins were divided into aliquots and kept at –80°C.

To determine the DNA binding activity of NF-B, the corresponding oligo probe was end-labeled with -[³²P]-ATP by T4 polynucleotide kinase and purified with a MicroSpin G-25 column. Reaction of nuclear proteins (8 µg/sample) and ³²P-labeled oligo probe was completed according to the manufacturer’s instructions. Reaction mixture was loaded on precooled, 5% polyacrylamide (38:2) native gel. The complex of nuclear protein–DNA probe was separated from the unbound probe by electrophoresis and detected by radiography after the gel was air-dried.

Determination of Secreted TNF- in Cultured Medium by ELISA
Macrophages were cultured in 60-mm dishes until 90% confluence. Cells were washed with serum-free medium and then remained in the serum-free medium with treatments. The cultured medium was collected at different times after treatment and spun for 5 minutes with a Microfuge. The supernatant was saved, and TNF- was detected by ELISA (the assay kit was purchased from BD Biosciences). The secreted TNF- in culture medium was calculated on the basis of a standard curve of given TNF- concentrations and expressed as ng/mL medium, with triplicate assays for each sample.

Data Analysis
All experiments were repeated at least 3 times, and representative results are presented. Data were analyzed by paired t test in an assay of cholesterol efflux, influx of cholesteryl oleate, and TNF- secretion.

	Results

Top Abstract Introduction Methods Results Discussion References

 
To determine whether the novel statin pitavastatin (NK-104) can alter macrophage SR-BI expression, J774 cells were treated with pitavastatin at different concentrations overnight, and SR-BI expression was determined by Western blot (Figure 1A). Compared with control (cells treated with the solvent that dissolved pitavastatin, DMSO), we found that pitavastatin significantly induced SR-BI protein expression. The maximal induction was obtained with 10 µmol/L of pitavastatin (3-fold). To determine whether the induction of SR-BI protein by pitavastatin also occurred at the transcriptional level, we analyzed the RNA transcript by Northern blot and found that SR-BI mRNA was also concentration-dependently increased by pitavastatin (Figure 1B).

View larger version (30K): [in this window] [in a new window]   Figure 1. Statins induce SR-BI expression in macrophage cell line J774 cells. J774 macrophage cell line was cultured in complete RPMI medium. At 90% confluence, cells were switched to serum-free medium and received treatments. After treatments, cells were collected to extract cellular proteins or total RNA as described in Methods. Cell lysate (20 µg/sample) was used to analyze SR-BI protein expression by Western blot. Total RNA (100 µg) was used to isolate poly(A+) RNA and detect SR-BI mRNA expression by Northern blot. A, Effect of pitavastatin on SR-BI protein expression. Macrophages were treated with indicated concentrations of pitavastatin for 16 hours. B, Effect of pitavastatin on macrophage SR-BI mRNA expression. Time of treatment was 12 hours. C, Time course of pitavastatin-induced macro-phage SR-BI protein expression. Cells were treated with 10 µmol/L of pitavastatin for times indicated. D, Macrophages were treated with 10 µmol/L of simvastatin (Sim), pravastatin (Pra), and rosuvastatin (Ros), respectively, for 16 hours.

To study the dynamic changes of SR-BI expression in response to pitavastatin treatments, macrophages were treated with 10 µmol/L pitavastatin for various times and analyzed for SR-BI protein expression by Western blot (Figure 1C). The induction of SR-BI expression by pitavastatin was observed as early as 1 hour after treatment, suggesting a rapid response. Maximal induction occurred after 12 hours of treatment, and it was maintained for 24 hours after treatment.

We also determined whether other well-established statins could alter SR-BI protein expression in macrophages. Simvastatin, pravastatin, and rosuvastatin also significantly induced macrophage SR-BI expression (Figure 1D).

The functional consequences of increased SR-BI expression in response to pitavastatin were evaluated by determination of HDL binding to macrophages, efflux of free cholesterol to HDL from macrophages, and influx of cholesteryl oleate/HDL to macrophages. Figure 2 demonstrates that pitavastatin significantly increased HDL binding to macrophages (Figure 2A), and it also enhanced the efflux of free cholesterol to HDL from macrophages and uptake of cholesteryl oleate/HDL by macrophages (Figure 2B).

View larger version (26K): [in this window] [in a new window]   Figure 2. Effects of pitavastatin on macrophage HDL binding, efflux of cholesterol, and uptake of cholesteryl oleate/HDL. A, Macrophages were treated with 5 or 10 µmol/L pitavastatin overnight. Binding of HDL was determined as described in Methods. B, Effect of pitavastatin on macrophage cholesterol efflux and cholesteryl ester influx. For cholesterol efflux assay, cells (in 12-well plates) were previously labeled with 150 nCi/mL of [3H]cholesterol and 50 µg/mL of acetylated LDL in medium for 8 hours before treatment with 5 µmol/L pitavastatin overnight. After that, cells were washed once with PBS and added with 2 mL/well of serum-free medium containing 50 µg /mL of HDL. Radioactivity in medium was determined and normalized by cellular protein content. For influx of cholesteryl oleate/HDL, cells in 12-well plates were treated with 5 µmol/L pitavastatin overnight; after washing with PBS, cells were added to serum-free medium containing 100 nCi/mL of [14C]cholesteryl oleate and 100 µg/mL of HDL and incubated at 37°C. At indicated times, medium was removed. Cells were washed with PBS and then lysed in 0.2N NaOH. Cellular lysate was used to detect radioactivity and protein content. *P<0.05 vs control (n=4).

To investigate the physiological relevance of statins on macrophage SR-BI expression, we isolated peritoneal macrophages from mice and treated them with pitavastatin overnight. SR-BI expression was determined by Western blot (Figure 3A). Pitavastatin significantly induced peritoneal macrophage SR-BI expression in a concentration-dependent manner. The stimulative effect of pitavastatin on macrophage SR-BI expression was further demonstrated by increased SR-BI surface expression in human monocyte-derived macrophages that were previously treated with 1 and 5 µmol/L of pitavastatin (Figure 3B).

View larger version (26K): [in this window] [in a new window]   Figure 3. Pitavastatin induced SR-BI expression by primary macrophages. A, Peritoneal macrophages were isolated from mice as described in Methods. Cells were cultured in completed RPMI medium and treated with indicated concentrations of pitavastatin for 16 hours. Cellular proteins were extracted, and SR-BI expression was determined by Western blot. B, Human monocytes were isolated from healthy donor’s blood and cultured for 1 week to differentiate macrophages. Human macrophages were then treated with 1 and 5 µmol/L of pitavastatin for 20 hours. Macrophage SR-BI surface protein was detected by FACS as described in Methods.

Inhibition of mevalonate synthesis by statins may also prevent the synthesis of other important isoprenoid intermediates of the cholesterol biosynthetic pathway,¹⁰ ie, farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). We reasoned that pitavastatin could induce macrophage SR-BI expression through its effect on these intermediates. To test this hypothesis, we examined the effects of mevalonate on macrophage SR-BI expression. Mevalonate decreased macrophage SR-BI protein expression only at a relative high concentration (50 µmol/L), and such inhibition was abolished by pitavastatin (Figure 4A). Another intermediate (FPP) for cholesterol biosynthesis also showed moderate inhibitory effects on macrophage SR-BI expression, but the effects were seen at much lower concentrations (1 and 5 µmol/L, Figure 4B). GGPP, a key intermediate to geranylgeranylate proteins involving in cell signaling cascades, was also influenced by pitavastatin action. Figure 4C showed that GGPP moderately suppressed macrophage SR-BI expression. This action was blocked by pitavastatin, and similar findings were observed with ubiquinone (Coenzyme Q₁₀, Figure 4D). In addition, we observed that inhibition of geranylgeranylation (by a geranylgeranyl transferase I inhibitor, GGTI-287), but not inhibition of farnesylation (by a farnesyl transferase inhibitor, FTI-277), significantly induced macrophage SR-BI expression (data not shown), suggesting that pitavastatin induced macrophage SR-BI expression through inhibition of small G proteins of the Rho family rather than the Ras family. These results suggest that cholesterol biosynthetic intermediates may play an important role in the regulation of macrophage SR-BI expression.

View larger version (40K): [in this window] [in a new window]   Figure 4. Pitavastatin restored macrophage SR-BI expression after inhibition by cholesterol biosynthetic intermediates. Confluent J774 macrophages were treated with mevalonate (10 and 50 µmol/L, A), farnesyl-pyrophosphate (FPP, 1 and 5 µmol/L, B), geranylgeranyl-pyrophosphate (GG-PP, 1 and 5 µmol/L, C), and coenzyme Q10 (CoQ10, 2 and 5 µmol/L, D), respectively, in presence and absence of pitavastatin (10 µmol/L) for 16 hours in serum-free medium. Cell lysate was extracted and analyzed for SR-BI expression by Western blot.

It was shown previously that LPS and TNF- suppress SR-BI expression in different cell types with unclear mechanisms.¹⁶ In our studies, we treated macrophages with LPS or TNF- in the presence or absence of inhibitors for NF-B, a transcriptional activator of many proinflammatory proteins. Both LPS and TNF- suppressed more than 50% of SR-BI expression. Although NF-B inhibitors alone did not show effects on SR-BI expression, they significantly blocked the inhibitory actions of LPS and TNF- on SR-BI expression (Figure 5). These data indicate that LPS and TNF- can decrease SR-BI expression through their activation of NF-B. LPS more likely inhibits macrophage SR-BI expression through its stimulation of TNF- production in macrophages, because the secretion of TNF- in response to LPS treatment was rapid. After 2 hours of treatment with 200 ng/mL LPS, we detected that the concentration of TNF- in serum-free cultured medium was 32.5±2.3 ng/mL, compared with undetectable levels of TNF- in control cells even after 10 hours of incubation.

View larger version (31K): [in this window] [in a new window]   Figure 5. NF-B inhibitors blocked inhibition of macrophage SR-BI expression by LPS and TNF-. A, Macrophages were treated with LPS (200 ng/mL) or NF-B inhibitor (Bay 11-7083 and Bay 11-7085, 5 µmol/L in each) or LPS plus NF-B inhibitor for 16 hours. Cell lysate was extracted, and SR-BI expression was determined by Western blot. B, Macrophages were treated with TNF- or NF-B inhibitor (Bay 11-7083, Bay 11-7085, NF-B SN50) or TNF- plus NF-B inhibitor at indicated concentration for 16 hours. Cell lysate was used to assess macrophage SR-BI expression by Western blot.

To determine whether the induction of macrophage SR-BI expression by pitavastatin may have occurred as a result of some antiinflammatory action, we first determined whether pitavastatin could block the inhibitory actions of LPS and TNF- on SR-BI expression. We found that LPS and TNF- significantly decreased SR-BI expression; pitavastatin partially blocked LPS action but totally overwhelmed the TNF- action on macrophage SR-BI expression (Figure 6).

View larger version (30K): [in this window] [in a new window]   Figure 6. Pitavastatin restored macrophage SR-BI expression after inhibition by LPS and TNF-. Confluent macrophages in serum-free medium were treated with various concentrations of LPS (A) or TNF- (B) in presence or absence of 5 µmol/L pitavastatin for 16 hours. Cell lysate was extracted and was then used to assess SR-BI expression by Western blot.

Finally, to investigate this possibility further, we determined whether pitavastatin could alter NF-B DNA binding activity using electrophoretic mobility shift assay. Figure 7A demonstrates that pitavastatin reduced more than 50% of NF-B DNA binding activity in J774 macrophages. We further determined whether pitavastatin was able to regulate expression of NF-B complex proteins. NF-B p65 expression was moderately decreased, but IB- expression was significantly increased, by pitavastatin (Figure 7B). The opposite effects of pitavastatin on these 2 proteins could lead to the decreased NF-B DNA binding activity.

View larger version (51K): [in this window] [in a new window]   Figure 7. Pitavastatin inhibited NF-B DNA binding activity. Confluent J774 macrophages were treated with pitavastatin at indicated concentrations for 12 hours. Nuclear proteins were extracted to assess NF-B DNA binding activity by electrophoretic mobility shift assay, and whole cellular proteins were used to assess NF-B p65 and IB- expression by Western blot, respectively. A, Effect of pitavastatin on NF-B DNA binding activity: 8 µg of nuclear proteins from each sample was used to conduct DNA-protein reactions as described in Methods. Samples a to c were PMA-treat Jurkat nuclear extract supplied in assay kits. In reaction of sample b, an 100-fold excess of unlabeled probe was added to reaction system. In reaction of sample c, an 100-fold excess of unlabeled mutant probe was added to reaction system. B, Effect of pitavastatin on NF-B p65 and IB- expression: 40 µg of cellular proteins from each sample was loaded on 12% SDS-PAGE to separate and detect NF-B p65 and IB- expression by Western blot.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
LDL cholesterol levels are correlated with the incidence of coronary heart disease. Both the reduction of dietary cholesterol and statin therapy to reduce de novo synthesis of cholesterol have had significant clinical benefits.⁹ Clinical benefits of statin therapy have been identified in addition to their lipid-lowering actions, suggesting that these pharmacological agents have numerous pleiotropic effects.¹⁰ In this study, we observed that statins can increase macrophage SR-BI expression through cholesterol biosynthetic intermediates and antiinflammatory action. Tsuruoka et al¹⁷ consistently demonstrated a similar observation in human keratinocytes by simvastatin.

The effects of pitavastatin on SR-BI expression through the influence on cholesterol biosynthesis are consistent with our previous report that lipid loading of macrophages can reduce SR-BI expression.¹⁵ In addition to macrophages, reducing intracellular cholesterol levels by ß-cyclodextran demonstrates a reverse relationship between SR-BI expression and the cellular cholesterol pool in an adrenal cell line (Y1-BS1).¹⁸ It is noteworthy that although macrophages express HMG-CoA reductase,¹⁹ pitavastatin moderately reduces macrophage cholesterol levels (10% to 15% decreases have been observed compared with control after overnight incubation with 10 µmol/L of pitavastatin; data not shown). The cellular impact of pitavastatin on levels of cholesterol biosynthetic intermediates in macrophages is unclear. Cells that have been treated with these intermediates have decreased SR-BI expression (20% to 30%, Figure 4). Furthermore, cotreatment with pitavastatin and these intermediates can induce SR-BI expression to a degree comparable to that by pitavastatin alone, suggesting that an additional mechanism is involved in the regulation of macrophage SR-BI expression by pitavastatin.

Antiinflammatory effects of statins include the inhibition of leukocyte-endothelium interactions, the reduction of the expression of adhesion molecules such as intercellular adhesion molecule-1, and the suppression of the production of proinflammatory cytokines (IL-1ß, IL-6, and cyclooxygenase-2).¹⁰ The effects of statins on TNF- are controversial. Lovastatin inhibits LPS-induced expression of TNF- in rat primary astrocytes, microglia, and macrophages,²⁰ but it synergizes antiproliferative activity in MmB16 melanoma cells and L1210 leukemia cells,²¹ antitumor effects against Ha-ras–transformed murine tumor,²² and induction of E-selectin, intercellular adhesion molecule-1, and vascular adhesion molecule-1 in endothelial cells with TNF-.²³ Although atorvastatin suppresses TNF- production in Th1 cells,²⁴ it works cooperatively with TNF- to stimulate the generation of PAI-1 in PMA- or TGF-ß1/1,25-dihydroxyvitamin D₃–differentiated HL-60 cells.²⁵ Atorvastatin has no effect on TNF- production in familial hypercholesterolemia patients.²⁶ Rosuvastatin decreases TNF- expression in apoE*3-Leiden transgenic female mice,²⁷ whereas cerivastatin has no effect on human macrophage TNF- production.²⁸ In contrast, Kiener et al²⁹ reported that atorvastatin, lovastatin, and simvastatin can stimulate the production of TNF- in human monocytes. To determine whether pitavastatin increased macrophage SR-BI expression by altering TNF- levels, we first assessed the amount of TNF- in culture medium after pitavastatin treatment. Consistent with some of the above-described observations, we did not see changes in TNF- levels by pitavastatin in our cells up to 10 hours of treatment (data not shown); however, after treatment for 14 hours, we observed a moderate increase in TNF-. For instance, TNF- levels were 0.064±0.051, 0.136±0.054, and 3.153±1.18 ng/mL in control and 1- and 5-µmol/L pitavastatin–treated samples, respectively (significantly different from control at P<0.05). Greater increases in TNF- levels were observed with longer treatments of cells, and the induction was concentration-dependent (data not shown). We also observed that pitavastatin failed to block LPS-induced TNF- production (data not shown). We believe that these data suggest that induction of macrophage SR-BI expression by pitavastatin is not through its effect on macrophage TNF- production. Compared with the rapid increase in SR-BI expression, pitavastatin increased TNF- at a very slow rate. This action does not negatively affect SR-BI induction, because we observed that pitavastatin significantly blocked the inhibition of SR-BI expression by TNF- and LPS. These data also suggest the existence of other mechanism(s) by which pitavastatin can induce SR-BI expression.

NF-B is a transcriptional factor that regulates many genes, including proinflammatory cytokines.³⁰ Our data showing that NF-B inhibitors restore macrophage SR-BI expression that is initially inhibited by LPS or TNF- (Figure 5) imply that NF-B might be involved in the induction of SR-BI expression by pitavastatin. Indeed, we observed that pitavastatin functioned as an NF-B inhibitor to restore SR-BI expression that was initially inhibited by LPS or TNF- (Figure 6). Statins have been reported to reduce NF-B activity in several cell types.^31,32 We consistently detected that pitavastatin inhibited NF-B activity in macrophages and that this inhibitory action was because of increased IB- expression and decreased NF-B p65 expression (Figure 7). We interpret our results to indicate that the impact of pitavastatin on NF-B activity may play a more important role in addition to its effects on cholesterol biosynthetic intermediates in its induction of macrophage SR-BI expression.

Finally, PPAR-, a transcriptional factor in the regulation of fatty acid metabolism, diabetes, inflammation, and atherosclerosis,³³ plays a critical role in the regulation of CD36 expression. However, its role in the regulation of SR-BI expression is controversial. Although some PPAR- ligands have been reported to increase SR-BI expression³⁴ and stimulate HMG-CoA reductase activity in macrophages,³⁵ we have observed that most PPAR- ligands do not have any effect on macrophage SR-BI expression. In contrast, we observed that ciglitazone can have strong inhibitory effects on macrophage SR-BI expression (unpublished observations). Previously, we reported that pitavastatin decreased macrophage PPAR- expression at both the RNA and protein levels. This statin can increase mitogen-activated protein kinase activity and the level of phosphorylated PPAR-, a negative regulator for its target genes.¹² Therefore, we believe that the induction of macrophage SR-BI expression by pitavastatin does not occur primarily through PPAR- but rather through cholesterol biosynthetic intermediates and NF-B activity. These findings suggest additional pleiotropic effects of this during atherosclerotic foam cell development.

	Acknowledgments

 
This work was supported by a sponsored research agreement with Kowa Co, Ltd, Tokyo, Japan, and the Abercrombie Foundation (Dr Gotto). Dr Parsons was supported, in part, by a National Institutes of Health (NIH) T32 training grant in vascular biology (NIH HL-07423, awarded to Dr Hajjar). Drs Gotto and Hajjar are also consultants to the Kowa Co in the area of atherosclerosis research, and they have received a sponsored research grant to study the impact of NK-104 on atherosclerosis.

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