Pitavastatin Inhibits Remnant Lipoprotein-Induced Macrophage Foam Cell Formation Through ApoB48 Receptor–Dependent Mechanism
Objective— Atherogenic remnant lipoproteins (RLPs) are known to induce foam cell formation in macrophages in vitro and in vivo. We examined the involvement of apoB48 receptor (apoB48R), a novel receptor for RLPs, in that process in vitro and its potential regulation by pitavastatin.
Methods and Results— THP-1 macrophages were incubated in the presence of RLPs (20 mg cholesterol/dL, 24 hours) isolated from hypertriglyceridemic subjects. RLPs significantly increased intracellular cholesterol ester (CE) and triglyceride (TG) contents (4.8-fold and 5.8-fold, respectively) in the macrophages. Transfection of THP-1 macrophages with short interfering RNA (siRNA) against apoB48R significantly inhibited RLP-induced TG accumulation by 44%. When THP-1 macrophages were pretreated with pitavastatin (5 μmol/L, 24 hours), the expression of apoB48R was significantly decreased and RLP-induced TG accumulation was reduced by 56%. ApoB48R siRNA also inhibited TG accumulation in THP-1 macrophage induced by β–very-low-density lipoprotein derived from apoE−/− mice by 58%, supporting the notion that apoB48R recognizes and takes-up RLPs in an apoE-independent manner.
Conclusions— RLPs induce macrophage foam cell formation via apoB48R. Pitavastatin inhibits RLP-induced macrophage foam cell formation. The underlying mechanism involves, at least in part, inhibition of apoB48R-dependent mechanism. Our findings indicate a potential role of apoB48R in atherosclerosis.
Clinical studies have revealed that remnant lipoproteins (RLPs), which are produced by hydrolysis of chylomicrons (CMs) and very-low-density lipoproteins (VLDL), are closely related to atherosclerosis, independent of high-density lipoprotein and low-density lipoprotein (LDL).1,2 There is also increasing evidence that RLPs play a causative role in atherogenesis, and we recently reported that they induced monocyte-endothelial interaction and vascular smooth muscle cell proliferation.3,4 However, the effect(s) of RLPs on cellular mechanism(s) during atherogenesis have not been fully elucidated.
Atherogenesis involves the appearance of lipid-loaded foam cells derived from macrophages in the arterial intima. RLPs from hypertriglyceridemic VLDL and CMs cause rapid lipid accumulation, and induce foam cell formation in macrophages, whereas normal VLDL and LDL do not.5 Thus, inhibition of macrophage foam cell formation induced by RLPs may contribute to the prevention of atherosclerosis. It has been reported that mechanisms independent of apolipoprotein (apo) E and LDL receptor family are involved in lipid accumulation in macrophages.6 Recently, apoB48 receptor (apoB48R) was shown to be involved in the uptake of triglyceride-rich lipoproteins (TRLs) and contribute to atherogenesis,7 although the role of apoB48R in this process remains unclear. Herein, we report for the first time to our knowledge the dominant role of apoB48R in RLP-induced foam cell formation by selective downregulation of apoB48R with short interfering RNA (siRNA).
Recently, 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitor, or statin, has been suggested to have beneficial effects for the prevention of atherosclerosis, independent of its LDL cholesterol-lowering effect.8 We found that statins reduced monocyte adhesion to endothelial cells via inhibition of Rho GTPase pathway.3,9 In the present study, treatment with pitavastatin lowered RLP-induced macrophage foam cell via, at least in part, inhibition of the expression of apoB48R in THP-1 macrophages. Our results suggest that apoB48R pathway might be a novel therapeutic target for atherogenesis, particularly in hypertriglyceridemic patients. Further, pitavastatin may exert a beneficial effect on atherogenesis by suppressing this pathway.
Reagents and Cell Culture
Pitavastatin was kindly provided by Kowa Pharmaceutical Company, Tokyo, Japan. Phorbol-12-myristate-13-acetate (PMA), C3 exoenzyme, farnesyl-pyrophosphate (FPP), and geranylgeranyl-pyrophosphate (GGPP) were purchased from Wako. RPMI 1640, DMEM, and FBS were obtained from GIBCO BRL. Antibodies used in the present study were as follows: mouse anti-RhoA monoclonal antibody (Santa Cruz Biotechnology), rabbit anti-GAPDH antibody (Sigma), HRP-conjugated goat anti-mouse IgG, and HRP-conjugated goat anti-rabbit IgG (Cal-tag). Rabbit anti-human apoB48R antibody was a generous gift from Dr Sandra H. Gianturco and Dr William A. Bradley, The University of Alabama at Birmingham.
Human monocytic THP-1 cells were obtained from American Type Culture Collection and maintained in RPMI 1640 medium supplemented with 10% FCS, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mmol/L l-glutamine in a humidified atmosphere of 5% CO2 at 37°C. To obtain THP-1 macrophages, THP-1 cells were plated in 35-mm dishes (1×107 cells/dish) and incubated with PMA at a concentration of 200 nmol/L for 4 days. Human peripheral blood monocytes were isolated from healthy volunteers using Ficoll-Paque (Pharmacia Biotech) and a monocyte-negative isolation kit (Miltenyi Biotech). To examine cell viability, cells were stained with 4′, 6-Diamidino-2-phenylindole dihydrochloride (DAPI; 200 ng/mL) (Dojindo), and viable cells were counted at the required time point using a hemacytometer.
EDTA plasma was obtained from 24 patients with hypertriglyceridemia who showed an elevated RLP concentration (>7.5 mg cholesterol/dL) 4 hours after eating breakfast. They had no cardiovascular diseases or diabetes and had not taken cardiovascular medicine or antioxidants. The protocol of this study complied with the guidelines for the conduct of research involving human subjects by the Committee on Human Research at Tokyo Medical and Dental University. RLPs were isolated from plasma samples using an RLP-C Kit, as described previously,10 then dialyzed overnight against 5 L of PBS containing 50 μmol/L EDTA (pH 7.4) and sterilized using a 0.22-μm filter unit (Millipore). The prepared RLPs were analyzed by SDS-PAGE in a 5% to 20% linear gradient gel (Funakoshi) and visualized with a silver stain reagent (Daiichi). Densitometry showed that apoB48 comprised only 5% to 7% of total apoB in RLPs, as we and others reported previously.3,11 β-VLDL (density<1.006 g/mL) was isolated from freshly prepared plasma taken from apoE−/− mice (obtained from Jackson Laboratory, Bar Harbor, Me, and fed a normal diet) at 8 weeks of age by ultracentrifugation.
Oil Red O Staining
THP-1 macrophages were seeded into multi-well slides (Nunc) at a concentration of 1×106 cells per well. The cells were washed 3 times with phosphate-buffered saline, fixed with formaldehyde, and stained with oil red O. Lipid accumulation was observed under a microscope.
Cellular Lipid Analysis
To determine intracellular lipid contents, THP-1 macrophages were removed from the culture plates and washed twice with phosphate-buffered saline. Then, intracellular lipids were extracted using isopropanol/hexane. Cholesterol ester, triglyceride (TG), and protein mass were determined enzymatically.
Cells were disrupted in a lysis buffer and total cell lysates prepared. An equal amount of protein (10 μg) was subjected to SDS-PAGE, after which immunoblotting was performed using the indicated antibodies. Immunoreactive proteins were detected using an enhanced chemiluminescence advance (Amersham Pharmacia Biotech). For a RhoA translocation assay, cell lysates from the membranes and cytosol fractions were prepared as described previously.9
Transfection of siRNA Against ApoB48R
siRNA was designed to target the coding sequence of human apoB48R cDNA. The target sequences were directed to the single-strand region according to the predicted secondary RNA structure and sequences of the form (AA/CA)N19 with GC contents of <70% were selected from this region.12 Nineteen nucleotide RNAs followed by TT/TG were selected, then chemically synthesized and gel-purified. Sequences corresponding to the siRNA were nucleotides 1060 to 1079 for apoB48R coding region (GenBank accession number AF141332). Nonrelevant 19 RNAs were used to generate the control siRNA. Double-stranded siRNAs were generated and transfected into THP-1 macrophages, as described previously.13 Transfection efficiency was evaluated using BLOCK-IT Fluorescent dsRNA (Invitrogen);14 64.3±13.8% of THP-1 macrophages were positive for fluorescein isothiocyanate 48 hours after transfection.
RhoA Pull-Down Assay
RhoA pull-down assay was performed using Rho activation kit (Upstate) following the manufacturer’s protocol.15
Results are presented as the mean±SD. Data were analyzed using analysis of variance (ANOVA), with a value of P<0.05 considered significant.
RLPs Induce Foam Cell Formation in THP-1 Macrophages
We investigated whether RLPs could induce macrophage foam cell formation. First, THP-1 cells were incubated in the presence of PMA at a concentration of 200 nmol/L for 4 days for full differentiation. After the medium was replaced with fresh medium with or without RLPs, THP-1 cells were incubated for an additional 24 hours. Oil red O staining showed that treatment with RLPs induced foam cell formation in PMA-treated THP-1 macrophages (Figure 1). Intracellular TG and cholesterol ester contents in the cells were also significantly increased 24 hours after the addition of RLPs in a dose-dependent manner (Figure 1).
Expression of Remnant Receptors During Differentiation in THP-1 Macrophages
To elucidate the mechanism(s) by which RLPs induce macrophage foam cell formation, we examined the expression levels of representative remnant receptors and related proteins with immunoblotting.16 During the differentiation process, immunoreactive LDL receptors nearly disappeared. In contrast, CD36, a scavenger receptor not present in THP-1 monocytes, appeared after PMA treatment, indicating that THP-1 monocytes differentiated into macrophages (Figure 2A). LDL receptor-related protein (LRP) was not detected throughout the differentiation process (data not shown). Interestingly, the expression level of apo48R, which is present in monocytes, remained unchanged throughout the differentiation process (Figure 2A) and was not downregulated by incubation with RLPs (Figure 2B).
siRNA Against ApoB48R Decreases RLP-Induced Foam Cell Formation in THP-1 Macrophages
To elucidate the specific role of apoB48R in RLP-induced foam cell formation, we introduced siRNA against apoB48R into THP-1 macrophages. Transfection of THP-1 macrophages with apoB48R siRNA reduced the expression of apoB48R 48 hours after transfection in an apoB48R siRNA dose-dependent manner. However, apoB48R siRNA did not affect the expression of GAPDH (Figure 3A) or that of CD36 (data not shown). When THP-1 macrophages were transfected with apoB48R siRNA, RLP-induced foam cell formation was significantly reduced (Figure 3B). Next, THP-1 macrophages were incubated with β-VLDL derived from apoE−/− mice, a model ligand of apoE-devoid remnant lipoproteins, because it has been reported that apoB48R takes-up TRLs in an apoE-independent fashion.7,17 ApoB48R siRNA also reduced macrophage foam cell formation induced by β-VLDL. ApoB48R siRNA was more effective compared with the case of RLP-induced foam cell formation (Figure 3C). These results indicate that apoB48R plays a role, at least in part, in the taking up of RLPs, and that the suppression of apoB48R expression may reduce RLP-induced foam cell formation.
Pitavastatin Inhibits RLP-Induced Foam Cell Formation in THP-1 Macrophages
To examine the effect of pitavastatin on RLP-induced foam cell formation, THP-1 macrophages were incubated with or without pitavastatin for 24 hours. THP-1 lipid contents, very low at baseline, were not significantly affected by treatment with pitavastatin (data not shown). Further, when THP-1 macrophages were pretreated with pitavastatin before RLP treatment, foam cell formation, detected by oil red O staining, was significantly inhibited, and the increments of intracellular TG and cholesterol ester content by RLPs were also significantly reduced by pretreatment with pitavastatin in a dose-dependent manner (Figure 4A).
Pitavastatin Suppresses ApoB48R Expression in THP-1 Macrophages Through RhoA-Dependent Mechanism
Next, we examined whether pitavastatin modulates the expression levels of apoB48R in the THP-1 macrophages. The expression level of apoB48R was significantly decreased after 24 hours of incubation with pitavastatin in a dose-dependent manner (Figure 5A). Statins are known to inhibit the mevalonate pathway, resulting in inhibition of the activity of RhoA, a Rho GTPase family member. Many studies have reported that statins exert their bioactive effects via inhibition of RhoA, independent of a lipid-lowering effect. Thus, we examined whether RhoA is involved in the reduction of apoB48R expression by pitavastatin. When THP-1 macrophages were pretreated with GGPP before pitavastatin treatment, reduction of apoB48R expression by pitavastatin was almost restored. In contrast, FPP was not so effective in recovering apoB48R expression compared with GGPP (Figure 5B). To examine whether pitavastatin treatment had an effect on RhoA activity, the GTP-binding capacity of RhoA was measured in THP-1 macrophages. As shown in Figure 5C, the amount of GTPγS-bound RhoA was reduced when THP-1 macrophages were treated with pitavastatin. Pitavastatin also decreased membrane translocation of RhoA in THP-1 macrophages (data not shown). In addition, C3 exoenzyme, a specific RhoA inhibitor, reduced apoB48R expression in THP-1 macrophages (Figure 5D) and inhibited RLP-induced foam cell formation (Figure 5E). Pitavastatin treatment did not affect the expression of LDL receptor and LRP in THP-1 macrophages (data not shown). Although pitavastatin suppressed CD36 expression by 15% at 5 μmol/L (data not shown), the reduction rate was much lower than that of apoB48R.
Pitavastatin Suppresses ApoE-Deficient β-VLDL Uptake by THP-1 Macrophages
To further confirm whether the inhibition of RLP-induced foam cell formation by pitavastatin is dependent on apoB48R, we examined the effect of pitavastatin on the uptake of β-VLDL derived from apoE−/− mice. THP-1 macrophages were incubated with apoE-deficient β-VLDL in the presence or absence of pitavastatin, and then lipid accumulation was evaluated. These β-VLDL induced macrophage foam cell formation, although to a lesser extent when compared with native RLPs. Consistent with the effect of pitavastatin on apoB48R expression, pretreatment with pitavastatin reduced lipid accumulation in THP-1 macrophages (Figure 4B), supporting the notion that this compound may serve to reduce lipid accumulation even in the absence of apoE-dependent pathway.
Pitavastatin Suppresses ApoB48R Expression in Human Peripheral Blood Macrophages
To demonstrate the involvement of apoB48R in RLP uptake and its potential regulation by pitavastatin in more pathophysiological conditions, we conducted experiments using human peripheral blood macrophages. Western blot analysis showed that pitavastatin treatment for 24 hours significantly reduced apoB48R expression and lipid accumulation induced by RLPs in human peripheral blood macrophages (Figure I, available online at http://atvb.ahajournals.org), which were similar to the effects seen in THP-1 macrophages.
It is known that excessive RLPs enter vascular walls, where they are taken-up by macrophages and induce foam cell formation in those cells. TRL remnants are found in atherosclerotic plaques;18 however, the mechanisms by which RLPs induce foam cell formation have not been fully elucidated. The LDL receptor, known to bind RLPs, nearly disappear during the differentiation of monocytes into macrophages, whereas apoB48R is constantly present during the differentiation process, in striking contrast to LDL receptor families. Because apoB48R is detected in the reticulo-endothelial system, but not in hepatic cells, we focused on this unique remnant receptor and examined its role in RLP-induced macrophage foam cell formation. ApoB48R is found in human atherosclerotic plaques,7 suggesting its involvement in the uptake of TRLs by macrophages. ApoB48R binds the apoB48 of dietary TRL (CM and CM remnant) or the apoB48-equivalent domain of apoB100 in hypertriglyceridemic VLDL.17 Dietary TRLs, which lack the C-terminal domain of apoB100 that binds to the LDL receptor, cannot bind to the LDL receptor via apoB48, the major apoB species formed in the intestine. Thus, apoB48R may account for, at least in part, the observed direct macrophage uptake of TRLs in vivo and for foam cell formation seen in humans with elevated TRLs.19,20
The present results showed that gene silencing of apoB48R by siRNA directly leads to a successful inhibition of RLP-induced foam cell formation. Because apoB48R is an apoE-independent receptor in human and murine monocyte-macrophages,21,22 we conducted experiments using β-VLDL devoid of apoE, which were also able to induce macrophage foam cell formation. Interestingly, the effect of apoB48R siRNA was more prominent in the case of β-VLDL treatment. Taken together, our findings indicate that apoB48R is involved in the uptake of RLP and foam cell formation, independent of an apoE-mediated pathway. Further, because the expression levels of apoB48R do not diminish after the cells accumulate extensive lipids, macrophages may allow further uptake of these lipoproteins by this pathway, as is the case with scavenger receptors. The relatively low affinity of nascent VLDL for apoB48R may account for the fact that they do not induce monocyte foam cell formation.
We also showed that pitavastatin inhibits RLP-induced foam cell formation. The expression levels of remnant receptors in THP-1 macrophages in the presence of pitavastatin were examined. Neither LDL receptor nor LRP were detected in those macrophages after treatment, indicating that pitavastatin does not affect the differentiation process once THP-1 cells have fully differentiated into macrophages. In the present study, the expression level of apoB48R was significantly reduced by treatment with pitavastatin and it also significantly reduced foam cell formation induced by RLP or β-VLDL loading.
Further, the effect of pitavastatin on other receptors related to RLP uptake was also examined. Pitavastatin did not affect LDL receptor and LRP. CD36, a scavenger receptor considered to be involved in atherogenic lipoprotein uptake, is also reported to be modulated by pitavastatin.23 However, the effect of pitavastatin on CD36 was relatively small at the concentrations used in the present study, compared with its effect on apoB48R. It has been reported that statins have no effect on acyl CoA: cholesterol acyltransferase (ACAT) activity24 and downregulate rather than upregulate ABCA1 in macrophages in vitro.25 Considering that the gene silencing of apoB48R by siRNA significantly reduced RLP-induced foam cell formation (Figure 3), we believe that apoB48R is involved in the uptake of RLPs and that pitavastatin decreased RLP uptake by macrophages at least in part by modulating apoB48R-dependent mechanism, although yet-unknown mechanism(s) may be involved in this process.
It is known that statins downregulate GGPP, an isoprenoid intermediate. Isoprenoids are important for post-translational modification of proteins, such as the small GTP-binding proteins. It has been reported that many of the cholesterol-independent effects of statins may be mediated by the inhibition of Rho GTPase. Thus, in the present study, we investigated whether apoB48R expression is modulated by RhoA-dependent pathway. The pitavastatin-mediated reduction of apoB48R expression was restored in the presence of GGPP, which bypass the HMG-CoA reductase-RhoA pathway. Moreover, C3 exoenzyme, a specific RhoA inhibitor, reduced apoB48R expression. Therefore, it may be possible that RhoA plays a role as a positive regulator of apoB48R expression and that pitavastatin decreases apoB48R expression by inhibiting the geranylgeranylation of RhoA.
The regulation of apoB48R expression remains unclear, although peroxisome proliferator-activated receptors were recently reported to inhibit apoB48R expression.26 As shown in Figure 2, apoB48R was not regulated by sterol content or the state of differentiation of human and murine monocyte-macrophages. Our results are the first to show that pitavastatin suppresses apoB48R via a RhoA-dependent mechanism, independent of sterol content or state of differentiation. Further, we found that pitavastatin decreased the expression of apoB48R in human peripheral blood macrophages and RLP-induced foam cell formation. Based on these findings, we considered that this mechanism also potentially occurs in vivo.
In conclusion, our data indicate that foam cell formation induced by RLP is, at least in part, mediated by an apoB48R-dependent pathway. Therefore, apoB48R may be a novel therapeutic target for atherogenesis, particularly in hypertriglyceridemic patients.
The authors gratefully acknowledge support (10178102) and special coordination funds from the Ministry of Education, Science, Technology, and Culture of Japan. We also thank Dr Gianturco and Dr Bradley for providing the rabbit anti-human apoB48R antibody. The authors express their sincere appreciation to Naoko Tomie, Keiko Takahashi, and Noriko Nitta for their technical assistance.
- Received March 23, 2004.
- Accepted November 4, 2004.
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