Protein kinase R-like endoplasmic reticulum kinase and glycogen synthase kinase-3α/β regulate foam cell formation
2014; Elsevier BV; Volume: 55; Issue: 11 Linguagem: Inglês
10.1194/jlr.m051094
ISSN1539-7262
AutoresCameron S. McAlpine, Geoff H. Werstuck,
Tópico(s)Phagocytosis and Immune Regulation
ResumoEvidence suggests a causative role for endoplasmic reticulum (ER) stress in the development of atherosclerosis. This study investigated the potential role of glycogen synthase kinase (GSK)-3α/β in proatherogenic ER stress signaling. Thp1-derived macrophages were treated with the ER stress-inducing agents, glucosamine, thapsigargin, or palmitate. Using small-molecule inhibitors of specific unfolded protein response (UPR) signaling pathways, we found that protein kinase R-like ER kinase (PERK), but not inositol requiring enzyme 1 or activating transcription factor 6, is required for the activation of GSK3α/β by ER stress. GSK3α/β inhibition or siRNA-directed knockdown attenuated ER stress-induced expression of distal components of the PERK pathway. Macrophage foam cells within atherosclerotic plaques and isolated macrophages from ApoE−/− mice fed a diet supplemented with the GSK3α/β inhibitor valproate had reduced levels of C/EBP homologous protein (CHOP). GSK3α/β inhibition blocked ER stress-induced lipid accumulation and the upregulation of genes associated with lipid metabolism. In primary mouse macrophages, PERK inhibition blocked ER stress-induced lipid accumulation, whereas constitutively active S9A-GSK3β promoted foam cell formation and CHOP expression, even in cells treated with a PERK inhibitor. These findings suggest that ER stress-PERK-GSK3α/β signaling promotes proatherogenic macrophage lipid accumulation. Evidence suggests a causative role for endoplasmic reticulum (ER) stress in the development of atherosclerosis. This study investigated the potential role of glycogen synthase kinase (GSK)-3α/β in proatherogenic ER stress signaling. Thp1-derived macrophages were treated with the ER stress-inducing agents, glucosamine, thapsigargin, or palmitate. Using small-molecule inhibitors of specific unfolded protein response (UPR) signaling pathways, we found that protein kinase R-like ER kinase (PERK), but not inositol requiring enzyme 1 or activating transcription factor 6, is required for the activation of GSK3α/β by ER stress. GSK3α/β inhibition or siRNA-directed knockdown attenuated ER stress-induced expression of distal components of the PERK pathway. Macrophage foam cells within atherosclerotic plaques and isolated macrophages from ApoE−/− mice fed a diet supplemented with the GSK3α/β inhibitor valproate had reduced levels of C/EBP homologous protein (CHOP). GSK3α/β inhibition blocked ER stress-induced lipid accumulation and the upregulation of genes associated with lipid metabolism. In primary mouse macrophages, PERK inhibition blocked ER stress-induced lipid accumulation, whereas constitutively active S9A-GSK3β promoted foam cell formation and CHOP expression, even in cells treated with a PERK inhibitor. These findings suggest that ER stress-PERK-GSK3α/β signaling promotes proatherogenic macrophage lipid accumulation. Atherosclerosis is an inflammatory disease within the walls of medium and large arteries (1Ross R. Mechanisms of disease - atherosclerosis - an inflammatory disease.N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19243) Google Scholar). It is the leading cause of cerebrovascular and cardiovascular diseases, which together account for a third of all deaths in Western societies (1Ross R. Mechanisms of disease - atherosclerosis - an inflammatory disease.N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19243) Google Scholar, 2Glass C.K. Witztum J.L. Atherosclerosis: the road ahead.Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2636) Google Scholar). Multiple risk factors contribute to the initiation and progression of atherosclerosis including diabetes mellitus, hypertension, obesity, dyslipidemia, a sedentary life style, and smoking (3Yusuf S. Hawken S. Ounpuu S. Dans T. Avezum A. Lanas F. McQueen M. Budaj A. Pais P. Varigos J. INTERHEART Study Investigators et al.Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study.Lancet. 2004; 364: 937-952Abstract Full Text Full Text PDF PubMed Scopus (8439) Google Scholar). One of the hallmark features of every stage of atherogenesis, from the fatty streak to the complex plaque, is the presence of lipid-laden macrophages known as foam cells. Intimal macrophage/foam cells accumulate lipids from LDL and modified LDL particles and secrete a variety of inflammatory cytokines. In advanced plaques, foam cells undergo apoptosis, thereby contributing to the formation of a highly thrombotic, lipid rich, necrotic core [reviewed by Moore and Tabas (4Moore K.J. Tabas I. Macrophages in the pathogenesis of atherosclerosis.Cell. 2011; 145: 341-355Abstract Full Text Full Text PDF PubMed Scopus (1814) Google Scholar)]. The molecular events that promote the initiation and development of atherosclerosis are poorly understood. A better understanding of the signaling networks that regulate foam cell formation and atherosclerotic plaque development may lead to the identification of novel therapeutic targets. The endoplasmic reticulum (ER) is the organelle responsible for the proper folding, modification, and processing of secretory, transmembrane, and ER resident proteins. If the processing capacity of the ER is overwhelmed, unfolded or misfolded proteins begin to accumulate, a condition known as ER stress. The accumulation of misfolded proteins triggers the initiation of the unfolded protein response (UPR), which is composed of three signaling cascades regulated by ER transmembrane proteins [reviewed Schroder and Kaufman (5Schröder M. Kaufman R. The mammalian unfolded protein response.Annu. Rev. Biochem. 2005; 74: 739-789Crossref PubMed Scopus (2438) Google Scholar)]. The activation of protein kinase R-like endoplasmic reticulum kinase (PERK), inositol requiring enzyme (IRE) 1, and activating transcription factor (ATF) 6 coordinate the attenuation of protein translation and the increased expression of cellular chaperones, as the cell attempts to reattain ER homeostasis. If these early, adaptive mechanisms are not successful at alleviating the stress, downstream components of the UPR will initiate proapoptotic pathways to eliminate the cell. ER stress and UPR activation have been associated with the progression and development of atherosclerotic plaques. Multiple cardiovascular risk factors including hyperglycemia (6Khan M.I. Pichna B.A. Shi Y. Bowes A.J. Werstuck G.H. Evidence supporting a role for endoplasmic reticulum stress in the development of atherosclerosis in a hyperglycaemic mouse model.Antioxid. Redox Signal. 2009; 11: 2289-2298Crossref PubMed Scopus (55) Google Scholar, 7McAlpine C.S. Bowes A.J. Khan M.I. Shi Y. Werstuck G.H. Endoplasmic reticulum stress and glycogen synthase kinase-3 beta activation in apolipoprotein E-deficient mouse models of accelerated atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 82-91Crossref PubMed Scopus (46) Google Scholar), hyperhomocysteinemia (7McAlpine C.S. Bowes A.J. Khan M.I. Shi Y. Werstuck G.H. Endoplasmic reticulum stress and glycogen synthase kinase-3 beta activation in apolipoprotein E-deficient mouse models of accelerated atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 82-91Crossref PubMed Scopus (46) Google Scholar, 8Werstuck G.H. Lentz S.R. Dayal S. 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Phosphorylation at residue serine 21 of α and serine 9 of β is indicative of inhibition, while phosphorylation at tyrosine 279 of α and tyrosine 216 of β is associated with kinase activation. GSK3α/β activity can also be regulated by altering its subcellular localization (22Meares G.P. Jope R.S. Resolution of the nuclear localization mechanism of glycogen synthase kinase-3 - functional effects in apoptosis.J. Biol. Chem. 2007; 282: 16989-17001Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 23Bijur G.N. Jope R.S. Proapoptotic stimuli induce nuclear accumulation of glycogen synthase kinase-3 beta.J. Biol. Chem. 2001; 276: 37436-37442Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Although GSK3α and GSK3β share 90% homology within the kinase domain, the enzymes have been shown to have both distinct as well as common substrates (24Kerkela R. Kockeritz L. MacAulay K. Zhou J. Doble B.W. Beahm C. Greytak S. Woulfe K. Trivedi C.M. Woodgett J.R. et al.Deletion of GSK-3 beta in mice leads to hypertrophic cardiomyopathy secondary to cardiomyoblast hyperproliferation.J. Clin. Invest. 2008; 118: 3609-3618Crossref PubMed Scopus (187) Google Scholar, 25Doble B.W. Patel S. Wood G.A. Kockeritz L.K. Woodgett J.R. Functional redundancy of GSK-3 alpha and GSK-3 beta in Wnt/beta-catenin signaling shown by using an allelic series of embryonic stem cell lines.Dev. Cell. 2007; 12: 957-971Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 26MacAulay K. Doble B.W. Patel S. Hansotia T. Sinclair E.M. Drucker D.J. Nagy A. Woodgett J.R. Glycogen synthase kinase 3alpha-specific regulation of murine hepatic glycogen metabolism.Cell Metab. 2007; 6: 329-337Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Recent evidence suggests that the role of GSK3α/β in cell metabolism extends to ER stress and the activation of proatherogenic pathways. In cultured cells, conditions of ER stress activate GSK3β (27Song L. De Sarno P. Jope R. Central role of glycogen synthase kinase-3 beta in endoplasmic reticulum stress-induced caspase-3 activation.J. Biol. Chem. 2002; 277: 44701-44708Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 28Chen Y.Y. Chen G. Fan Z. Luo J. Ke Z.J. GSK3 beta and endoplasmic reticulum stress mediate rotenone-induced death of SK-N-MC neuroblastoma cells.Biochem. Pharmacol. 2008; 76: 128-138Crossref PubMed Scopus (47) Google Scholar). In vivo studies have also demonstrated a role for GSK3α/β in the regulation of NF-κB (29Hoeflich K.P. Luo J. Rubie E.A. Tsao M.S. Jin O. Woodgett J.R. Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation.Nature. 2000; 406: 86-90Crossref PubMed Scopus (1222) Google Scholar). Furthermore, our group and others have shown that inhibition of GSK3α/β is associated with attenuated atherosclerotic development and reduced hepatic steatosis in different mouse models (7McAlpine C.S. Bowes A.J. Khan M.I. Shi Y. Werstuck G.H. Endoplasmic reticulum stress and glycogen synthase kinase-3 beta activation in apolipoprotein E-deficient mouse models of accelerated atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 82-91Crossref PubMed Scopus (46) Google Scholar, 30Bowes A.J. Khan M.I. Shi Y. Robertson L. Werstuck G.H. Valproate attenuates accelerated atherosclerosis in hyperglycemic ApoE-deficient mice: evidence in support of a role for endoplasmic reticulum stress and glycogen synthase kinase-3 in lesion development and hepatic steatosis.Am. J. Pathol. 2009; 174: 330-342Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 31Choi S.E. Jang H.J. Kang Y. Jung J.G. Han S.J. Kim H.J. Kim D.J. Lee K.W. Atherosclerosis induced by a high-fat diet is alleviated by lithium chloride via reduction of VCAM expression in ApoE-deficient mice.Vascul. Pharmacol. 2010; 53: 264-272Crossref PubMed Scopus (37) Google Scholar). However, the mechanisms by which ER stress modulates GSK3α and/or β, and how GSK3α/β regulates proatherogenic processes, remain unresolved. In this study, we present evidence showing that ER stress-induced GSK3α/β does not regulate the adaptive components of UPR signaling but instead acts as a modulator of distal, proapoptotic elements of the PERK signaling pathway. Moreover, we demonstrate that inhibition of PERK-GSK3α/β signaling attenuates macrophage lipid biosynthesis and uptake, lipid accumulation, and foam cell formation induced by ER stress. Thp-1 human monocytes were cultured in Roswell Park Memorial Institute 1640 medium (Invitrogen) containing 10% fetal bovine serum at 37°C and 5% CO2. Monocytes were differentiated into macrophages by exposing the cells to 100 nM PMA for 72 h. Thioglycolate-elicited peritoneal macrophages were isolated from 8-week-old female C57BL6 mice or ApoE−/− mice and cultured in DMEM (Life Technologies) containing 10% fetal bovine serum. Cultured cells were treated with 1 μM thapsigargin (Thaps), 5 mM glucosamine (GLN), or 600 μM PA coupled with 4% BSA for 18 h. Enzymatic activity was inhibited by pretreating the cells with indicated inhibitors and concentrations for 2 h. GSK3α/β activity was inhibited with 4 μM CT99021 (cat#13122, Cayman Chemical). PERK was inhibited with 3 μM GSK2606414 (cat#516535, Millipore), IRE1 was inhibited with 6 μM IRE1 Inhibitor III (cat#412512, Millipore), and ATF6 was inhibited with 250 μM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF; cat#A8456, Sigma Aldrich). Adenovirus infections were performed 72 h prior to the experiments using a multiplicity of infection (MOI) of 10 of either an empty adenovirus vector (Ad-CMV-Null) or adenovirus encoding constitutively active GSK3β (Ad-CMV-S9A-GSK3β). GSK3α/β siRNA was purchased from Cell Signaling (cat#6301), and all siRNA experiments were conducted using antibody-free media; 50 nM scramble (control) or GSK3α/β siRNA was transfected into cells using Lipofectamine (cat#11668-019, Invitrogen) for 8 h and then treated, as indicated, 24 h later. Total RNA was isolated from cultured cells using an RNeasy mini kit (Qiagen). RNA was quantified by measuring the absorbance at 260 nm, and RNA purity was verified by calculating the ratio of the absorbance at 260 and 280 nm (A260/A280). cDNA was reverse transcribed from 1 μg of RNA using Superscript II Reverse Transcriptase (Invitrogen). Real-time PCR was performed on the StepOne Plus (Applied Biosystems) using iQ SYBR Green Supermix (Bio-Rad), 1 μg cDNA, and 500 nM forward and reverse primers. See supplementary Table I for primer sequences and amplified product size. Total protein lysates were solubilized in kinase buffer containing 50 mM Tris HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 1% Triton ×100, 10 mM DTT, 1 mM benzamidine, 1 mM PMSF, and PhosSTOP Phosphatase Inhibitor (Roche). Protein lysates (15 μg) were diluted in SDS-PAGE gel loading buffer, resolved by SDS-PAGE, and transferred to nitrocellulose membranes. Blots were probed with primary antibodies against phospho-S51-EIF2α antibody (cat#3398, Cell Signaling), KDEL (cat#SPA-827, Assay Designs), protein disulphide isomerase (PDI; cat#SPA-891, Assay Designs), CHOP (cat#sc-7351, Santa Cruz Biotechnology), β-catenin (cat#9581, Cell Signaling), GSK3α/β (cat#5676, Cell Signaling), or β-actin (cat#A3854, Sigma-Aldrich). After incubation with the appropriate primary and HRP-conjugated secondary antibodies (Life Technologies), membranes were developed using the Immobilon Western chemiluminescent HRP substrate (Millipore). Protein band intensities were quantified and normalized to β-actin. Five-week-old female ApoE−/− (B6.129P2-ApoEtm1Unc) mice (n = 24), purchased from Jackson Labs (stock #002050), were placed on a high-fat diet (HFD) containing 21% milk fat and 0.2% cholesterol (TD97363, Harlan Teklad). After 1 week, half of the mice (n = 12) were switched to an HFD supplemented with 625 mg/kg sodium valproate (VPA), while the other half of the mice (n = 12) remained on unsupplemented HFD. All mice had unrestricted access to both food and water throughout the study. Mice were euthanized at 24 weeks of age for atherosclerotic plaque analysis or 8 weeks of age for analysis of peritoneal macropahges, and blood and tissues were collected for further analysis. The McMaster University Animal Research Ethics Board approved all procedures. Mice were euthanized, and vasculature was flushed with 1× PBS and perfusion-fixed with 10% neutral buffered formalin. Liver and heart, including the aortic root, were removed and embedded in paraffin. Serial sections (4 μm thick) of aortic root were collected on precoated glass slides. Sections were stained with primary antibodies against KDEL, CHOP (cat#sc-575, Santa Cruz), or Mac3 (cat#553322, Becton Dickson Co.). Serial sections were stained with preimmune IgG, in place of primary antibodies, to control for nonspecific staining. Images were captured with an Olympus microscope and a 12.5 megapixel DP71 digital camera. Immunofluorescence was quantified using ImageJ 1.43 software. Briefly, 12 aortic sections from each animal (n = 6 to 7 mice per treatment group), representing the entire length of the lesion, were stained and imaged. Staining intensity above background was determined over a fixed threshold. The staining intensity of the 12 aortic sections from each animal was averaged to provide a staining intensity for each animal. Data shown are average staining intensities for each animal within the group. Total GSK3α/β activity was determined from 250 μg total cell protein (supplementary Fig. I). For isoform-specific analysis, GSK3α or GSK3β were immunoprecipitated from 600 μg total cell protein in kinase buffer using a monoclonal antibody specific for GSK3β (cat#610202, BD Transductions) or GSK3α (cat#07-389, Cell Signaling) and Ultra Link immobilized Protein A Plus (Pierce). Kinase activity was measured by monitoring the incorporation of 32P onto phospho-glycogen synthase peptide-2 (pGS-2; Upstate Biotech). Briefly, either cell lysate or immunoprecipitated GSK3α or β was combined with 15 μM pGS-2 and 0.5 μCi/μl [γ32P]ATP in a reaction mixture containing 20 mM MOPS, 50 μM EDTA, 0.25 mM Mg acetate, 5 mM MgCl2, 5 mM β-glycerol phosphate, 1 mM EGTA, 0.25 mM Na3VO4, 0.2 mM DTT, and 35 μM ATP in a total volume of 40 μl. As background controls, a subset of samples were incubated with 0.5 μM CT99021. After 60 min at room temperature, samples were placed on ice, then spotted onto Whatman P81 phosphocellulose paper (GE Healthcare) and washed 3× with 0.75% o-phosphoric acid and once with acetone. 32P incorporation onto the substrate was determined by scintillation counting, and total counts minus background are reported. Esterified and unesterified cholesterol levels were determined in macrophages using a Cholesterol Quantitation Kit (cat#MAK043, Sigma-Aldrich) according to the manufacturer's instructions. Briefly, lipids were extracted from 1 × 106 cells with chloroform/isopropanol/IGEPAL CA-630 (7:11:0.1). Lipids were incubated with a cholesterol probe and either with (total cholesterol) or without (free cholesterol) cholesterol esterase for 60 min at 37°C. Esterified cholesterol levels were determined by the difference between total and free cholesterol levels. The absorbance of the sample was determined at 570 nm (A570) and compared with a known standard (supplementary Fig. I). Protein concentrations were determined in the organic phase using a Bradford assay (Bio-Rad). Lipid uptake was determined by treating cells as indicated and then supplementing media with Alexa fluor 488-AcLDL (7 μg/ml) (Life Technologies) for 2 h at 37°C and 5% CO2. To observe lipid droplets, cells were grown or differentiated onto glass coverslips and stained with Oil Red O (0.5% w/v) dissolved in isopropanol/PBS (4:3) followed by 4',6-diamidino-2-phenylindole (DAPI). Coverslips were mounted onto slides in Crystal-mount media. All images were captured using an Olympus microscope and a 12.5 megapixel DP71 digital camera. Oil Red O and Alexa fluor 488-AcLDL was quantified using ImageJ 1.43 software. Briefly, each biological experiment and treatment was conducted a minimum of four times. From each of these biological replicates, a minimum of five images, each containing ∼200 cells, were captured. The stained area over background as well as cell number were quantified. Data from each image of a biological replicate were combined providing an average stained area per cell with a minimum of 1,000 cells. Data shown are average stained areas per cell from at least four biological replicates (minimum 4,000 cells). All experiments are representative of at least three independent biological experiments. All data are expressed as mean ± SD. An unpaired Student's t-test or one-way ANOVA test was used, as appropriate, to determine statistical significance. A value of P < 0.05 was considered statistically significant. Thp-1 human monocytic cells were differentiated into macrophages by exposure to 100 nM PMA for 72 h. The small-molecule GSK3α/β inhibitor CT99021 was used to directly inhibit GSK3α/β activity (32Bain J. Plater L. Elliott M. Shpiro N. Hastie C.J. Mclauchlan H. Klevernic I. Arthur J.S.C. Alessi D.R. Cohen P. The selectivity of protein kinase inhibitors: a further update.Biochem. J. 2007; 408: 297-315Crossref PubMed Scopus (2105) Google Scholar). To confirm inhibition, GSK3α and GSK3β were immunoprecipitated from Thp-1 macrophage lysates, and kinase activity was determined in the presence or absence of 0.5 μM CT99021 (33Kim A.J. Shi Y. Austin R.C. Werstuck G.H. Valproate protects cells from ER stress-induced lipid accumulation and apoptosis by inhibiting glycogen synthase kinase-3.J. Cell Sci. 2005; 118: 89-99Crossref PubMed Scopus (234) Google Scholar) (supplementary Fig. IIA). GSK3α/β inhibition was verified indirectly by monitoring the accumulation of β-catenin in cells treated with 4 μM CT99021 (supplementary Fig. IIB). To determine the impact of GSK3α/β inhibition on ER stress-induced chaperone expression, macrophages were pretreated for 2 h in the presence or absence of 4 μM CT99021 and then challenged with ER stress-inducing agents, including 1 μM Thaps, 5 mM GLN, or 600 μM PA, for 18 h. Neither ER stress nor GSK3α/β inhibition reduced Thp-1 macrophage cell viability below 80% (supplementary Fig. III). Total RNA was isolated, and quantitative real-time PCR was performed. The expression levels of the cellular chaperones and foldases, glucose-related protein (GRP) 78, GRP94, calreticulin, and PDI, were determined (Fig. 1). These components of the adaptive ER stress response were significantly upregulated by Thaps, GLN, and PA (Fig. 1). GSK3α/β inhibition did not alter GRP78, GRP94, calreticulin, or PDI expression (Fig. 1). Consistent with these findings, siRNA-directed knockdown of GSK3α/β did not alter the ability of Thaps, GLN, or PA to increase GRP78 protein levels (supplementary Fig. IVA–C). These results suggest that GSK3α/β activity is not required for early, adaptive UPR signaling. We next investigated the three branches of UPR and the potential role of GSK3α/β in each of these signaling pathways. Initially, the effect of ER stress on GSK3α/β activation was determined. ER stress induced by Thaps, GLN, and PA significantly increased GSK3α/β activity in Thp-1 macrophages (Fig. 2A). Macrophages were then exposed to inhibitors of each of the three UPR signaling pathways. Inhibition of the PERK, but not IRE or ATF6, significantly attenuated ER stress-induced GSK3α/β activity (Fig. 2A and supplementary Fig. V). Activated PERK phosphorylates the eukaryotic initiation factor (eIF) 2α at serine 51. This phosphorylation event results in the attenuation of general protein translation and the specific upregulation of ATF4 and CHOP. Immunoblot analysis of protein lysates from macrophages challenged with Thaps, GLN, or PA shows the expected ER stress-induced phosphorylation of eIF2α, indicative of the activation of the PERK signaling pathway (Fig. 2B, C). P-eIF2α levels were unaffected by GSK3α/β inhibition suggesting that GSK3α/β does not affect PERK activity directly. However, ER stress-induced CHOP and ATF4 expression were blocked by GSK3α/β inhibition and siRNA knockdown (Fig. 2B, D–F, and supplementary Fig. IVA–D). These results indicate that GSK3α/β plays a role in the regulation of downstream components of the PERK branch of the UPR. Having identified a role for GSK3α/β in PERK signaling in vitro, we then asked if CHOP expression in macrophages within the atherosclerotic plaque could be attenuated by GSK3α/β inhibition. Five-week-old female ApoE−/− mice were placed on an HFD containing 21% milk fat and 0.2% cholesterol for 20 weeks. A subset of mice were given an HFD supplemented with VPA (625 mg VPA/kg body weight), a small molecule shown to inhibit GSK3α/β both in vitro and in vivo (7McAlpine C.S. Bowes A.J. Khan M.I. Shi Y. Werstuck G.H. Endoplasmic reticulum stress and glycogen synthase kinase-3 beta activation in apolipoprotein E-deficient mouse models of accelerated atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 82-91Crossref PubMed Scopus (46) Google Schola
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