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Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, TaiwanDivision of Cardiovascular Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei, Taiwan
Schizophrenia patients taking atypical antipsychotic drugs have high risk to suffer from metabolism disorders.
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Olanzapine accelerates the progression of atherosclerosis by deregulating hepatic lipid metabolism and aortic inflammation.
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Atypical antipsychotic drugs activate PPARγ, the key transcription factor for CD36 expression by inducing ROS pathway.
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Atypical antipsychotic drugs exacerbate atherosclerosis by activating NOX-ROS-PPARγ-CD36 pathway.
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This study establishes the relationship between atypical antipsychotic agents, cholesterol metabolism and atherosclerosis.
Abstract
Background
Clinical reports indicate that schizophrenia patients taking atypical antipsychotic drugs suffer from metabolism diseases including atherosclerosis. However, the mechanisms underlying the detrimental effect of atypical antipsychotic drugs on atherosclerosis remain to be explored.
Methods
In this study, we used apolipoprotein E-deficient (apoe−/−) hyperlipidemic mice and apoe−/−cd36−/− mice to investigate the underlying mechanism of atypical antipsychotic drugs on atherosclerosis and macrophage-foam cells.
Results
In vivo studies showed that genetic deletion of cd36 gene ablated the pro-atherogenic effect of olanzapine in apoe−/− mice. Moreover, in vitro studies revealed that genetic deletion or siRNA-mediated knockdown of cd36 or pharmacological inhibition of CD36 prevented atypical antipsychotic drugs-induced oxLDL accumulation in macrophages. Additionally, olanzapine and clozapine activated NADPH oxidase (NOX) to generate reactive oxygen species (ROS) which upregulated the activity of peroxisome proliferator-activated receptor γ (PPARγ) and subsequently elevated CD36 expression. Inhibition of NOX activity, ROS production or PPARγ activity suppressed CD36 expression and abolished the detrimental effects of olanzapine and clozapine on oxLDL accumulation in macrophages.
Conclusion
Collectively, our results suggest that atypical antipsychotic drugs exacerbate atherosclerosis and macrophage-foam cell formation by activating the NOX-ROS-PPARγ-CD36 pathway.
Neurovascular unit dysfunction and blood-brain barrier hyperpermeability contribute to schizophrenia neurobiology: a theoretical integration of clinical and experimental evidence.
]. Schizophrenia is characterized by a persistent and disabling psychotic disorder with common symptoms including delusions, hallucinations and disorganized thoughts [
]. The etiology of schizophrenia is not fully understood. The dopamine hypothesis of schizophrenia is one of the most discussed theories in psychiatry [
]. Atypical antipsychotic drugs such as olanzapine and clozapine targeting to dopaminergic signal transduction have been prescribed for treating schizophrenia and found to have beneficial effect on relieving the symptoms of schizophrenia [
]. With clinical importance, growing evidence indicates that patients who take atypical antipsychotic agents including olanzapine and clozapine are associated with the increased risk of metabolic syndromes including obesity, diabetes and hyperlipidemia; however, the underlying mechanism is still elusive [
Effects of second-generation antipsychotics on selected markers of one-carbon metabolism and metabolic syndrome components in first-episode schizophrenia patients.
]. Recently, we reported that olanzapine accelerates the progression of atherosclerosis by deregulating hepatic lipid metabolism and aortic inflammation in hyperlipidemia apolipoprotein E-deficient (apoe−/−) mice [
]. Nevertheless, little is known about the involving cellular and molecular mechanism(s) underlying the pro-atherogenic effect of atypical antipsychotic drugs. To this end, studies delineating the mechanisms of atypical antipsychotic drugs on dysregulation of cholesterol metabolism are warranted.
Atherosclerosis results from the interaction between oxidized low-density lipoprotein (oxLDL), leukocytes, vascular cells, and the elements of the arterial wall, which accelerates the development of complex lesions and ultimately leads to acute clinical complications [
]. In particular, the accumulation of the lipid-laden macrophage-foam cells in the vessel plays a crucial role in the initiation and the progression of atherosclerotic lesions [
]. Apart from its role in the regulation of cholesterol metabolism, CD36 is involved in the key events of all stages of atherosclerosis including endothelial dysfunction, monocyte recruitment, monocyte/macrophage differentiation and thrombosis formation [
]. The expression of CD36 is tightly regulated by transcription factor peroxisome proliferator activated receptor-γ (PPARγ) in response to the stimuli such as atherogenic factors, inflammatory cytokines or clinical drugs [
]. However, the significance of PPARγ-CD36 signaling pathway in the detrimental effect of atypical antipsychotic drugs on atherosclerosis is limited.
Given the impact of atypical antipsychotic agents in the pathogenesis of metabolic diseases, we aimed to characterize the molecular mechanisms of atypical antipsychotic drugs in the formation of macrophage-foam cells and progression of atherosclerosis. In this study, we first investigated the involvement of CD36 in the olanzapine-mediated acceleration of atherosclerosis; and second, we determined whether olanzapine activates PPARγ to upregulate the expression of CD36 in macrophage-foam cells. Our third aim was to explore the role of NADPH oxidase (NOX)-reactive oxygen species (ROS) signaling in the atypical antipsychotic drug-mediated activation of PPARγ-CD36 pathway in macrophage-foam cells. Here, we provide additional new information for understanding the adverse effect of atypical antipsychotic drugs involved in the cholesterol metabolism of macrophages and atherosclerosis.
2. Materials and methods
2.1 Reagents
Olanzapine, clozapine and PPARγ transcription factor assay kit was from Cayman Chemical (Ann Arbor, MI, USA). Mouse antibody for p47-phox (Santa Cruz Biotechnology Cat# sc-17,844, RRID: AB_627987) and HO-1 (Santa Cruz Biotechnology Cat# sc-136,960, RRID: AB_2011613); control siRNA and NOX2 siRNA were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antibodies for CD36 (Abcam Cat# ab133625, RRID: AB_2716564), PPARγ (Abcam Cat# ab209350, RRID: AB_2890099) and 4-HNE (Abcam Cat# ab46545, RRID: RRID: AB_722490); rat antibody for F4/80 (Abcam Cat# ab6640, RRID: RRID: AB_1140040) and malondialdehyde (MDA) assay kit were obtained from Abcam (Cambridge, MA, USA). Mouse antibodies for α-tubulin (Sigma-Aldrich Cat# T9026, RRID: RRID: AB_477593) and NAC and APO were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse antibodies for GPx (R&D systems Cat# AF3798, RRID: RRID: AB_2112108) and pro-inflammatory cytokine ELISA kits were obtained from R&D systems (Minneapolis, MN, USA). Dil-oxLDL was purchased from Biomedical Technologies (Stoughton, MA, USA). Dihydroethidine (DHE) and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Molecular Probes (Eugene OR, USA). The EnzyChrom NADP+/NAD(P)H assay kit was obtained from BioAssay Systems (Hayward, CA, USA). Lipofectamine® RNAiMAX reagent was obtained from Thermo Fisher Scientific (Lafayette, CO, USA).
2.2 Mice
This study conformed to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, eighth edition, 2011), and all animal experiments were approved by the Animal Care and Utilization Committee of National Yang-Ming University (No. 1051224). Wild-type (WT) C57BL/6 mice were purchased from the National Laboratory Animal Center, National Science Council (Taipei, Taiwan); apoe−/− mice and cd36−/− with C57BL/6 background were purchased from Jackson laboratory (Bar Harbor, Maine). The apoe−/−cd36−/− mice were generated by cross-breeding apoe−/− and cd36−/− mice and the polymerase chain reaction (PCR) of genomic DNA was performed to confirm apoe−/− and cd36−/− genotypes. PCR was performed with the primers: CD36 (forward): 5′- TTG AAG TGC TGA TCC TTT CAG A-3′, CD36 (reverse): 5′- TGT TTG TTT CAC CAC ACT GGA-3′, CD36 (mutant): 5′- CGC CTT CTT GAC GAG TTC-3′, apoE (forward): 5′- GCC TAG CCG AGG GAG AGC CG-3′, apoE 5′- TGT GAC TTG GGA GCT CTG CAG C-3′, apoE 5′- GCC GCC CCG ACT GCA TCT-3′ at 95 °C for 2 min, followed by 30 cycles at 94 °C for 30 s, 57 °C for 30 s, 72 °C 90 s and additional extension at 72 °C for 10 min.
The apoe−/− mice and apoe−/−cd36−/− mice at the age of 4 months received orally administered olanzapine (3 mg/kg/day, n = 8) or oil (vehicle control, n = 8) for 4 weeks. All experimental mice were fed with a chow diet and sacrificed by CO2 at 5-month age. At the end of experiments, hearts and aortas were collected and subjected to further experiments.
2.3 Cell culture
Mouse mononuclear cells were isolated from the femurs bone marrow by Percol density gradient centrifugation and then cultured in MEMα supplemented with M-CSF (50 ng/ml), penicillin (100 U/ml)/streptomycin (100 μg/ml) and 10% FBS to differentiate into bone marrow-derived macrophages (BMDMs) for 5 days in a humidified incubator at 37 °C, 95% air/5% CO2. Murine J774.A1 macrophages (ATCC, TIB-67) and BMDMs were used for in vitro study in this investigation. J774.A1 macrophages were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml).
2.4 Histological examination
Hearts were harvested from mice were fixed with 4% paraformaldehyde and dehydrated serially by concentrations of alcohol (30%, 50%, 70%, 90% and 100%), following by xylene for 60 min. Then tissues were immersed in paraffin under 60 °C for overnight and embedded in paraffin. The tissue blocks were sectioned in 8 μm thickness. For histological examination, the heart sections were subjected to hematoxylin and eosin (H&E) staining. For the quantification of atherosclerotic lesions, 50 serial sections from the aortic sinus of each mouse were collected. A total of 10 to 12 sections sampled from every 4 consecutive sections were deparaffinized and subjected to hematoxylin and eosin staining. The photomicrographs of atherosclerotic lesions at aortic sinus were taken under a Motic TYPE 102 M microscope (Motic Images Plus 2.0, Xiamen, China).
2.5 Western blot analysis
Aortas or macrophages were lysed in lysis buffer (50 mmol/L Tris pH 7.5, 5 mmol/L EDTA, 300 mmol/L NaCl, 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin and 10 μg/mL aprotinin) and proteins of lysates were then transferred onto a PVDF membrane and blocked in 5% skim milk for 1 h. The blots were incubated with various specific primary antibodies, followed by the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies. The protein bands were visualized by using an enzyme-linked chemiluminescence detection kit (PerkimElmer, Waltham, MA, USA), and the band density was evaluated using the TotalLab 1D (Newcastle Upon Tyne, UK).To ensure equal loading, α-tubulin was evaluated as internal control in each experiment.
2.6 Immunohistochemistry
The deparaffinized sections were incubated with retrieval buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 10 min at 37 °C. The sections were blocked with 2% BSA for 60 min at 37 °C and incubated with primary antibody 2 h at 37 °C and then the HRP-conjugated secondary antibody for overnight at 4 °C. Antigenic sites were visualized by adding 3,3-diaminobenzidine and observed under a Nikon TE2000-U microscope (Tokyo) with an image analysis system QCapture Pro 6.0 (QImaging, BC, Canada).
2.7 Dil-oxLDL internalization assay
Macrophages were pretreated with olanzapine (120 ng/ml) or clozapine (120 ng/ml) for 2 h and then with Dil-oxLDL (10 μg/ml) in the presence of olanzapine or clozapine for additional 18 h at 37 °C. Cells were lysed and cellular lysates were analyzed using fluorometry (Molecular Devices, Sunnyvale, CA, USA) with excitation and emission wavelengths of 514 and 550 nm. Images were digitally captured under a Nikon TE2000-U microscope with an image analysis system.
2.8 Measurement of PPARγ activity
After treatment with olanzapine, the PPARγ activity of aortas and macrophages was assessed by using ELISA assay kit (Cayman Chemical, MI, USA) according to the manufacturer's protocol. A specific dsDNA sequence containing the peroxisome proliferator response element was coated onto the bottom of wells of a 96 well plate. The nuclear extracts of aortas and macrophages were then added into wells and incubated for 1 h at 37 °C. PPARγ was detected by the addition of a specific primary antibody against PPARγ, followed by the corresponding HRP-conjugated secondary antibody. The antigenic reaction was visualized by adding tetramethylbenzidine substrate and the absorbance was measured at 450 nm.
2.9 Lipid peroxidation assay
The levels of MDA, a product of lipid peroxidation, in the aortas of olanzapine-treated apoe−/− mice were determined by assay kits according to the manufacturer's instructions and the results serve as a biomarker for oxidative stress.
2.10 Determination of NADPH oxidase activity
Macrophages were pretreated with or without a ROS scavenger, NAC (10 mM) or a NOX inhibitor, APO (50 μM) for 2 h and then treated with olanzapine (120 ng/ml) or clozapine (120 ng/ml) for 15 min. Cellular lysates were subjected to the NOX activity assay by using the EnzyChrom NADP+/NADPH assay kit according to manufacturer's protocol.
2.11 Measurement of intracellular ROS levels
Macrophages were preincubated with 10 μM DHE or 20 μM DCFH-DA at 37 °C for 30 min. The DHE or DCFH-DA containing cell medium was removed and replaced with fresh medium. Cells were then incubated with olanzapine (120 ng/ml) or clozapine (120 ng/ml) for 15 min. The fluorescence intensity in cellular lysates was analyzed using fluorometry at 530 nm excitation and 620 nm emission for ethidium (ETH) and 488 nm excitation and 530 nm emission for DCF, respectively. Images were photomicrographed under a Nikon TE2000-U microscope with an image analysis system.
2.12 Transfection assay
Macrophages were transfected with control siRNA or NOX2 siRNA (12.5, 25, 50 and 100 nM) by use of lipofectamine® RNAiMAX reagent (7.5 μl/1 ml medium) for 24 h and then incubated with testing reagents for the indicated times.
2.13 Statistical analysis
Results are presented as mean ± SEM. Data from in vitro studies were evaluated by non-parametric tests. For comparisons of data of two groups, Mann-Whitney U test was used. For comparisons of data of more than two groups, one-way analysis of variance (ANOVA) was performed followed by the Mann-Whitney U test. Data from in vivo studies were evaluated by parametric tests. Two-way ANOVA followed by LSD test was used for multiple comparisons. SPSS v20.0 (SPSS Inc., Chicago, IL) was used for analysis. Differences were considered statistically significant at P < 0.05.
3. Results
3.1 CD36 is a crucial regulator in olanzapine-exacerbated atherosclerosis, hyperlipidemia and inflammatory response in apoE−/− mice
We first investigated the functional significance of CD36 in olanzapine-exacerbated atherosclerosis using apoe−/− and apoe−/−cd36−/− mice. Male apoe−/− and apoe−/−cd36−/− mice at 16 week-old were received daily oral treatment with olanzapine (OLZ, 3 mg/kg/day) for 4 weeks. As compared with vehicle group, olanzapine increased aortic atherosclerotic lesions in apoe−/− mice but not in apoe−/−cd36−/− mice (Fig. 1A ). As well, genetic deletion of cd36 prevented the olanzapine-induced increase in the serum levels of total cholesterol, non-HDL-c, HDL-c and triglycerides in the apoe−/− background (Fig. 1B). Moreover, compared with vehicle group, olanzapine increased the levels of TNF-α, IL-6 and MIP-2 but not IL-1β in aortas of apoe−/− mice. However, the upregulation of TNF-α, IL-6 and MIP-2 by olanzapine were attenuated in the aortas of apoe−/−cd36−/− mice aortas (Fig. 1C). Moreover, the protein expression of F4/80 (the murine macrophage marker) in atherosclerotic aortas was increased in olanzapine-treated apoe−/− mice, but not in olanzapine-treated apoe−/−cd36−/− mice (Fig. 2A and B ). Collectively, these findings suggest that CD36 plays an important role in olanzapine-worsened atherosclerosis, hyperlipidemia and inflammation.
Fig. 1Genetic deletion of cd36 ablated the olanzapine-induced acceleration of atherosclerosis in apoe−/− mice. Male apoe−/− and apoe−/−cd36−/−mice at 4-month-old were orally administered with olanzapine (OLZ, 3 mg/kg/day) or vehicle (oil) for 4 weeks. (A) Atherosclerotic lesions at aortic roots were stained with H&E. Scale bar = 300 μm. (B) Serum levels of total cholesterol, non-high-density lipoprotein cholesterol (non-HDL-c), HDL cholesterol (HDL-c), and triglycerides. (C) ELISA of aortic levels of TNF-α, IL-1β, IL-6, and MIP-2. Data are mean ± SEM from 8 mice. *P < 0.05 vs. vehicle-treated apoe−/− mice; #P < 0.05 vs. OLZ-treated apoe−/− mice.
Fig. 2Genetic deletion of cd36 attenuates olanzapine-exacerbated inflammation and macrophage accumulation in aortas of apoe−/− mice. Male apoe−/− mice at 4-month-old were orally administered with olanzapine (OLZ, 3 mg/kg/day) or vehicle (oil) for 4 weeks. (A) Western blot analysis of protein levels of CD36, F4/80 and α-tubulin in aortas from apoe−/− and apoe−/−cd36−/− mice. (B) Immunostaining was performed with normal rabbit IgG, anti-CD36 or anti-F4/80 antibody (a macrophage marker). Cell nuclei were stained with hematoxylin. Scale bar = 50 μm. Data are mean ± SEM from 8 mice. *P < 0.05 vs. vehicle-treated apoe−/− mice; #P < 0.05 vs. OLZ-treated apoe−/− mice.
3.2 CD36 plays a key role in olanzapine- and clozapine-aggravated oxLDL accumulation in macrophage-foam cells
We next examined the role of CD36 in olanzapine-mediated pro-atherogenic effect on the formation of foam cells. As shown in Fig. 3, treatment with olanzapine (120 ng/ml) or clozapine (120 ng/ml) increased the oxLDL-induced lipid accumulation in WT BMDMs but not in cd36−/− BMDMs (Fig. 3A). Similar results were found in murine J774.A1 macrophages that pretreatment with SSO (a CD36 inhibitor, 50 μM) or transfection with CD36 siRNA (50 nM) blunted the function of CD36 and prevented olanzapine- and clozapine-worsened oxLDL accumulation in J774.A1 cells (Fig. 3B and C). These results indicate that CD36 is critical for atypical antipsychotic drug-mediated dysregulation of cholesterol metabolism of macrophage-foam cells.
Fig. 3CD36 mediates the harmful effect of atypical antipsychotic drugs on oxLDL-induced lipid accumulation in macrophages. (A) WT and cd36−/− BMDMs were pretreated with olanzapine (OLZ, 120 ng/ml) or clozapine (CLO, 120 ng/ml) for 2 h. (B) J774.A1 macrophages were pretreated with or without a CD36 inhibitor (sulfo-N-succinimidyl oleate; SSO, 50 μM) for 2 h and then OLZ or CLO treatment for 2 h. (C) J774.A1 cells were transfected with control siRNA or CD36 siRNA (50 nM) for 24 h and then treated with OLZ or CLO (120 ng/ml) for 2 h. After incubation with OLZ or CLO, cells were then incubated with Dil-oxLDL (10 μg/ml) for an additional 24 h. The representative fluorescent images of Dil-oxLDL-induced lipid accumulation and quantitative data of fluorescence intensity. Scale bar = 50 μm. Data are the mean ± SEM from five independent experiments. (A) *P < 0.05 vs. WT vehicle group; #P < 0.05 vs. WT Dil-oxLDL; $P < 0.05 vs. WT Dil-oxLDL + OLZ group or WT Dil-oxLDL + CLO group; (B) and (C) *P < 0.05 vs. vehicle group; #P < 0.05 vs. Dil-oxLDL; $P < 0.05 vs. Dil-oxLDL + OLZ group or Dil-oxLDL + CLO group.
]. Thus, we determined whether olanzapine-induced upregulation of CD36 in aortas is mediated through the activation of PPARγ. Treatment with olanzapine for 4 weeks increased the activity of PPARγ but not protein expression in atherosclerotic aortas of apoe−/− mice (Fig. 4A and B ). Moreover, pretreatment of GW9662 (a PPARγ inhibitor, 2 μM) abolished olanzapine- or clozapine-induced PPARγ activation and up-regulation of CD36 protein (Fig. 4C and D). These results suggest that atypical antipsychotic drugs upregulate CD36 protein expression by inducing PPARγ activation.
Fig. 4PPARγ mediates the induction of CD36 expression by antipsychotic drugs. A and B apoe−/− and apoe−/−cd36−/− mice at 4 months old were orally administered with olanzapine (OLZ, 3 mg/kg/day) or vehicle (oil) for 4 weeks. (A) Western blot analysis of protein levels of PPARγ and α-tubulin in aortas. (B) PPARγ activity of aortas isolated from apoe−/− and apoe−/−cd36−/− mice. (C) and (D) J774.A1 cells were pretreated with or without GW9662 (a PPARγ inhibitor, 2 μM) for 2 h and then olanzapine (OLZ, 120 ng/ml) or clozapine (CLO, 120 ng/ml) for 18 h. PPARγ activity and protein levels of CD36 and α-tubulin were assessed. Data are mean ± SEM from 8 mice. *P < 0.05 vs. the vehicle-treated group; #P < 0.05 vs. OLZ or CLO alone group.
3.4 Activation of NOX-ROS-PPARγ-CD36 signaling pathway contributes to the atypical antipsychotic drugs-exacerbated oxLDL accumulation in macrophage-foam cells
ROS plays an important role in PPARγ activation, CD36 expression, foam cell formation and atherosclerosis progression [
Reactive oxygen species (ROS) mediate p300-dependent STAT1 protein interaction with peroxisome proliferator-activated receptor (PPAR)-γ in CD36 protein expression and foam cell formation.
]. Therefore, we investigated whether olanzapine increased the oxidative stress in atherosclerotic aortas of apoe−/− mice and subsequently increased PPARγ activity. As shown in Fig. 5, olanzapine increased lipid peroxidation levels and NOX2 activity in the aortas of apoe−/− mice (Fig. 5A and B). As well, the levels of p47 (a subunit of NOX in phagocytes) and 4-HNE (the product of lipid peroxidation in cells) were increased in the aortas of olanzapine-treated apoe−/− mice (Fig. 5C). Moreover, olanzapine increased GPx and HO-1 protein expression, two anti-oxidative proteins in response to oxidative stress challenge, in the aortas of apoe−/− mice (Fig. 5C). To confirm the in vivo findings, J774.A1 macrophages were used as in vitro model to delineate the potential significance of ROS in the detrimental effect of atypical antipsychotic drugs in the formation of macrophage-foam cells. Our results demonstrated that both olanzapine and clozapine increased NOX activity as early as 5 min, reaching a peak at 15 min after treatment (Fig. 5D). In parallel, ROS production was also increased with atypical antipsychotic drugs treatment as early as 5 min, with a peak at 15 min in macrophages (Fig. 5E and F). Moreover, pretreatment with NAC (a ROS scavenger, 10 mM), or APO (a ROS inhibitor, 50 μM) ablated the olanzapine- or clozapine-induced increase in NOX2 activity (Fig. 6A ), the production of superoxide and hydrogen peroxide (Fig. 6B and C), and the protein expression of p47, 4-HNE, GPx and HO-1 (Fig. 6D and E).
Fig. 5Atypical antipsychotic drugs increase oxidative stress in apoe−/− mice and in macrophages. Aortas were collected from male apoe−/− mice at 4-month-old were orally administered with vehicle (oil) or olanzapine (OLZ, 3 mg/kg/day) for 4 weeks. (A) The aortic level of lipid peroxidation, (B) the NOX activity and (C) western blot analysis of protein levels of p47, 4-HNE, HO-1, GPx and α-tubulin in apoe−/− mice. D to F J77A1.A1 macrophages were treated with OLZ or CLO (120 ng/ml) for the indicated times. (D) The NOX activity was analyzed by the NADP+/NADPH assay, and the levels of intracellular ROS were evaluated by (E) the intensity of red fluorescent DHE and (F) green fluorescent DCF, respectively. Data are mean ± SEM from 8 mice. *P < 0.05 vs. vehicle-treated apoe−/− mice or vehicle-treated macrophages.
Fig. 6Inhibition of the NOX-ROS pathway abolishes the atypical antipsychotic drug-induced increase in the NOX activity, ROS production and oxidative stress. J774.A1 cells were pretreated with or without an NOX inhibitor, apocynin (APO, 50 μM) or a ROS scavenger, N-acetylcysteine (NAC, 10 mM) for 2 h and then treated olanzapine (OLZ, 120 ng/ml) or clozapine (CLO, 120 ng/ml) for 15 min. The NOX activity was assayed by the NADP+/NADPH assay, and the levels of intracellular ROS were evaluated by the intensity of red fluorescent DHE and green fluorescent DCF, respectively. (A) The fold of change in NOX activity. (B) Representative fluorescent microscopy images of superoxide and c hydrogen peroxide. (D) and (E) Western blot analysis of protein levels of p47, 4-HNE, HO-1, GPx and α-tubulin. Data are the mean ± SEM from five independent experiments. *P < 0.05 vs. vehicle group; #P < 0.05 vs. OLZ-treated group or CLO-treated group.
We then determined the causal relationship between the NOX-ROS signaling and atypical antipsychotic drug-mediated dysregulation of cholesterol metabolism in macrophage-foam cells. The NOX activity or ROS production was depleted by pretreatment with APO (50 μM) or NAC (10 mM), respectively and our results showed that APO and NAC attenuated the olanzapine- or clozapine-induced increase in PPARγ activity (Fig. 7A ) and CD36 expression (Fig. 7B), as well as Dil-oxLDL-induced lipid accumulation (Fig. 7C). Additionally, pretreatment with siRNA (12.5, 25, 50 and 100 nM), which was designed to affect gene knockdown of NOX2, the primary source of ROS generation in macrophages, significantly decreased the expression of NOX2 protein (Fig. 7D). Furthermore, pretreatment with NOX2 siRNA (50 nM) prevented the olanzapine- or clozapine-induced increase in PPARγ activity, CD36 expression, and the Dil-oxLDL-mediated lipid accumulation (Fig. 7D-G). These results indicate that the activation of NOX-ROS signaling is essential for the atypical antipsychotic drug-induced dysregulation of cholesterol metabolism in macrophages. Collectively, our findings indicate that the activation of NOX-ROS-PPARγ-CD36 signaling pathway is required for the atypical antipsychotic drug-mediated impairment of cholesterol metabolism in macrophage-foam cells and atherosclerosis (Fig. 8).
Fig. 7Inhibition of the NOX-ROS signaling diminishes the atypical antipsychotic drug-induced increase in PPARγ activation, CD36 expression, and oxLDL accumulation in macrophages. Cells were pretreatment with or without an NOX inhibitor, apocynin (APO, 50 μM) or a ROS scavenger, N-acetylcysteine (NAC, 10 mM) and OLZ (120 ng/ml) or CLO (120 ng/ml) for 2 h before treating with Dil-oxLDL (50 μg/ml) for 24 h. (A) The level of PPARγ activity. (B) Western blot analysis of protein levels of CD36 and α-tubulin. (C) The quantitative results and representative fluorescent images of lipid accumulation. (D) J774.A1 cells were transfected with indicated concentrations of NOX2 siRNA for 24 h. Western blot analysis of protein levels of NOX2 and α-tubulin. E-G Cells were transfected with or without NOX2 siRNA (50 nM) for 24 h, followed by indicated treatments. (E) The level of PPARγ activity. (F) Western blot analysis of protein levels of CD36 and α-tubulin. (G) The Representative fluorescent images and the quantitative results of intracellular lipid accumulation in macrophages. Data are the mean ± SEM from five independent experiments. Data are the mean ± SEM from five independent experiments. *P < 0.05 vs. vehicle group; #P < 0.05 vs. OLZ-treated group or CLO-treated group.
Fig. 8The proposed molecular mechanisms by which atypical antipsychotic drugs activate the NOX-ROS-PPARγ-CD36 pathway to promote the formation of foam cells and accelerate the progression of atherosclerosis. In macrophages, olanzapine or clozapine elicits the activation of the NOX-ROS pathway, which in turn increases PPARγ activity and upregulates the expression of CD36, resulting in an increase in oxLDL internalization and ultimately leading to the excessive lipid accumulation in macrophages. Consequently, such the pro-atherogenic action may stimulate the inflammatory response, exacerbate the dysregulation of lipid metabolism and accelerates the progression of atherosclerosis in apoe−/− mice.
Effects of second-generation antipsychotics on selected markers of one-carbon metabolism and metabolic syndrome components in first-episode schizophrenia patients.
]. However, how atypical antipsychotic drug triggers the metabolic disturbances and disease progression remains elusive. In this study, we used hyperlipidemia mouse models apoe−/− mice and apoe−/−cd36−/− mice, as well as macrophage culture system to investigate the molecular mechanism underlying the pro-atherogenic effect of atypical antipsychotic drugs. We found that chronic treatment with olanzapine for four weeks worsened the hyperlipidemia, inflammation and atherosclerosis in apoe−/− mice, which was in agreement with our previous findings that atypical antipsychotic drug accelerates the progression of atherosclerosis [
]. Nevertheless, our knowledge about the molecular mechanism behind the pro-atherogenic effect of olanzapine is less detailed. Notably, our findings showed that genetic disruption of cd36 ablated the deleterious effect of olanzapine on the key events of atherogenesis including hyperlipidemia and inflammation in apoe−/− mice, suggesting the central role of CD36 in the pro-atherogenic action of atypical antipsychotic drugs. In vitro study further confirmed this notion that inhibition of CD36 function by pharmacological inhibitor and genetic manipulation abolished the clozapine- and olanzapine-aggravated increase in the intracellular lipid accumulation in macrophage-foam cells. Moreover, our results showed that atypical antipsychotic drugs increased NOX activity and ROS production, leading to activation of PPARγ and upregulation of CD36, which were blunted by treatment with the ROS scavenger NAC, NOX inhibitor APO or NOX2 siRNA, suggesting that the NOX/ROS signaling is essential for clozapine and olanzapine to activate PPARγ and induce CD36 expression. Collectively, we characterized the involvement of NOX-ROS-PPARγ-CD36 signaling in the atypical antipsychotic drug-mediated dysregulation of cholesterol metabolism in macrophage-foam cells and the acceleration of atherosclerosis.
The macrophage foam cells accumulated in the intima of the aorta is a key hallmark and plays a crucial role in the initiation and progression of atherosclerosis [
Absence of CD36 protects against atherosclerosis in ApoE knock-out mice with no additional protection provided by absence of scavenger receptor a I/II.
], however, the underlying molecular mechanism in olanzapine-exacerbated hyperlipidemia and atherosclerosis is unclear. To this end, we created the apoe−/−cd36−/− mice and used pharmacological and molecular approaches to explore the role of CD36 in the detrimental effect of atypical antipsychotic drugs on the cholesterol metabolism of foam cells and atherosclerosis. Our findings indicate that olanzapine failed to increase the size of atherosclerotic plaque in apoe−/−cd36−/− mice as compared to that in apoe−/− mice, suggesting the crucial role of CD36 in the pro-atherogenic effect of atypical antipsychotic drugs. In vitro results further confirmed this notion by the evidence that genetic deletion of cd36 prevented the olanzapine- or clozapine-worsened the oxLDL-induced lipid accumulation in macrophages. In view of its function, the up-regulation of CD36 by atypical antipsychotic drugs observed in this study is likely to promote foam cell formation and accelerate the progression of atherosclerosis.
PPARγ is a master transcription factor for the expression of target genes involved in regulating lipid metabolism [
]. Our study identifies PPARγ as the key transcriptional factor for atypical antipsychotic drug-induced up-regulation of CD36, and atherosclerotic progression, which is agreement with the findings by Brandl et al. and Su et al. that PPARγ pathway may contribute to olanzapine-induced metabolic disorders [
No evidence for a role of the peroxisome proliferator-activated receptor γ (PPARG) and adiponectin (ADIPOQ) genes in antipsychotic-induced weight gain.
]. This notion is also supported by our in vitro observations that inhibition of PPARγ activity attenuates the up-regulation of CD36 and the formation of foam cells by olanzapine and clozapine. These findings are consistent with those in studies of activation of PPARγ-CD36 signaling by protease inhibitors of human immunodeficiency virus promotes atherosclerotic lesion formation [
HIV protease inhibitors promote atherosclerotic lesion formation independent of dyslipidemia by increasing CD36-dependent cholesteryl ester accumulation in macrophages.
]. The possible explanation for the discrepancy between our results and those of above studies is that PPARγ cooperates liver X receptor (LXRα) to increase the expression of cholesterol efflux-related genes and induces anti-inflammatory M2 macrophage polarization and therefore leads to the retardation of atherosclerosis [
]. Moreover, PPARγ agonists have been are used in clinical practice for treating type 2 diabetes [24, 37]. However, the long-term use of PPARγ agonists has been challenged due to adverse effects such as increased risk of metabolic syndrome and cardiovascular diseases [
], which is in line with our findings that PPARγ activation mediates the pro-atherogenic effect of atypical antipsychotic drugs. Collectively, our results suggest olanzapine may aggravate atherosclerosis and promote foam cell formation by increasing the PPARγ-CD36 pathway-dependent oxLDL uptake; in contrast, decreasing the LXRα-ABCA1 pathway-mediated cholesterol efflux. In view of the complex nature in the regulation of cholesterol metabolism of macrophages, further investigation delineating the detail mechanism underlying the effects of atypical antipsychotic drugs on the biology of macrophage-foam cells and atherosclerosis are warranted.
Oxidative stress is the imbalance between the generation of ROS by NOX and the detoxification of ROS by antioxidant defense system [
]. Ample evidence suggests that the NOX-ROS signaling pathway contributes to pathological events in all stages of atherosclerosis and targets ROS pathway with antioxidants is an effective therapeutic strategy for preventing oxidative stress-mediated metabolic events [
]. Our data confirmed the involvement of NOX-derived ROS in the detrimental effect of atypical antipsychotic drugs in the formation of foam cells and atherosclerosis. Also, we demonstrated that olanzapine and clozapine increase the production of superoxide and hydrogen peroxide in macrophages and olanzapine increases the protein expression of p47 and lipid peroxidation, which leads to an increase in the production of MDA and 4-HNE in aortas of apoe−/− mice. These results are in agreement with the findings by Heiser et al. that olanzapine and clozapine increase the circulating level of ROS in rats [
]. Inhibition of ROS production by apocynin and NAC abolished the deleterious effect of atypical antipsychotic drugs on the formation of macrophage-foam cells. Moreover, our results indicate that the NOX-ROS pathway is the upstream signaling for the activation of PPARγ-CD36 pathway in macrophages. On the other hand, we also found that the levels of antioxidant enzymes HO-1 and GPx were increased after administration of atypical antipsychotic drugs which is consistent with the findings reported by Tsai et al. that atypical antipsychotic drugs increase the serum level of GPx [
]. Conceivably, these findings suggest that the antioxidants may have the therapeutic value in preventing the unfavorable effect of atypical antipsychotic drugs on metabolic diseases in patients with schizophrenia. Nonetheless, determining the detailed mechanism underlying the ROS-mediated detrimental effects by atypical antipsychotic drugs requires further investigations and clinical trials.
However, our study has several limitations. First, we only used a global CD36 deletion model to investigate the role of CD36 in the atypical anti-psychotic drug-induced dysregulation of cholesterol metabolism of macrophage-foam cells and atherosclerosis progression. Nevertheless, our in vitro data from macrophages cannot be directly extrapolated to the in vivo situation without an in vivo macrophage specific CD36 null mice or CD36 null hypomorphic apoe−/− mice by bone marrow transplantation. Thus, additional studies to delineate the specific role of macrophage CD36 in the pro-atherogenic effect of atypical anti-psychotic drugs are warranted. Second, our study is restricted to male mice. However, it has been reported that female schizophrenia patients taking clozapine or olanzapine represent higher risk for metabolic dysfunction and cardiovascular outcomes than males [
Prevalence of the metabolic syndrome in patients with schizophrenia: baseline results from the clinical antipsychotic trials of intervention effectiveness (CATIE) schizophrenia trial and comparison with national estimates from NHANES III.
]. To this end, further investigations defining the role of gender difference in atypical antipsychotic drug-mediated dysregulation of cholesterol metabolism and atherosclerosis are required.
In conclusion, congruous data are observed in the dysregulation of ROS and lipid metabolism in macrophage and in mouse model by atypical antipsychotic drugs. Moreover, our results provide new evidence for better understanding of the multifaceted properties of atypical antipsychotic drugs on the cholesterol metabolism in macrophage-foam cells and atherosclerosis that olanzapine and clozapine deregulate cellular redox status, promote the PPARγ-CD36-mediated lipoprotein internalization, and increase inflammatory response, all of which exacerbate the accumulation of macrophage-foam cells in aortas and hastens the progression of atherosclerosis. Additionally, we established the relationship between atypical antipsychotic agents, cholesterol metabolism of macrophages and atherosclerosis, which broadens the clinical significance of atypical antipsychotic drugs in metabolic disorders.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
This study was supported by grants from the Ministry of Science and Technology of Taiwan (106-2320-B-002-057-MY3, 106-2320-B-002-056, 106-2811-B-002-146, 108-2811-B-002-542, 109-2811-B-002-566, 110-2811-B-002-534 and 108-2320-B-002-032-MY3).
CRediT authorship contribution statement
Conceptualization, Song-Kun Shyue and Tzong-Shyuan Lee; Methodology, Chia-Hui Chen, Shr-Jeng Jim Leu, Chiao-Po Hsu, Ching-Chian Pan; Formal analysis, Chia-Hui Chen and Ching-Chian Pan; Investigation, Chia-Hui Chen, Shr-Jeng Jim Leu, Chiao-Po Hsu; Data curation, Chia-Hui Chen and Chiao-Po Hsu; Writing—original draft preparation, Song-Kun Shyue and Tzong-Shyuan Lee; Supervision, Tzong-Shyuan Lee; Funding acquisition, Tzong-Shyuan Lee. All authors have read and approved to the published version of the manuscript.
References
Maksymetz J.
Moran S.P.
Conn P.J.
Targeting metabotropic glutamate receptors for novel treatments of schizophrenia.
Neurovascular unit dysfunction and blood-brain barrier hyperpermeability contribute to schizophrenia neurobiology: a theoretical integration of clinical and experimental evidence.
Effects of second-generation antipsychotics on selected markers of one-carbon metabolism and metabolic syndrome components in first-episode schizophrenia patients.
Reactive oxygen species (ROS) mediate p300-dependent STAT1 protein interaction with peroxisome proliferator-activated receptor (PPAR)-γ in CD36 protein expression and foam cell formation.
Absence of CD36 protects against atherosclerosis in ApoE knock-out mice with no additional protection provided by absence of scavenger receptor a I/II.
No evidence for a role of the peroxisome proliferator-activated receptor γ (PPARG) and adiponectin (ADIPOQ) genes in antipsychotic-induced weight gain.
HIV protease inhibitors promote atherosclerotic lesion formation independent of dyslipidemia by increasing CD36-dependent cholesteryl ester accumulation in macrophages.
Prevalence of the metabolic syndrome in patients with schizophrenia: baseline results from the clinical antipsychotic trials of intervention effectiveness (CATIE) schizophrenia trial and comparison with national estimates from NHANES III.
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