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A novel role for the Krüppel-like factor 14 on macrophage inflammatory response and atherosclerosis development
Cardiovascular Pathology 27 (2017) 1–8
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Cardiovascular Pathology
Original Article
A novel role for the Krüppel-like factor 14 on macrophage inflammatory response and atherosclerosis development Xiao Wei a,1, Ruomei Yang a,1, Chengpan Wang a,1, Xun Jian b, Ling Li c, Hua Liu d, Gangyi Yang a,b, Zhiyong Li a,⁎ a
Department of Endocrinology, Yongchuan Hospital, Chongqing Medical University, 402160, Chongqing, China Department of Endocrinology, the Second Affiliated Hospital, Chongqing Medical University, 400010, Chongqing, China Key Laboratory of Diagnostic Medicine (Ministry of Education) and Department of Clinical Biochemistry, College of Laboratory Medicine, Chongqing Medical University, 400010, Chongqing, China d Department of Pediatrics, University of Mississippi Medical Center, Jackson, MS, USA b c
a r t i c l e
i n f o
Article history: Received 13 June 2016 Received in revised form 10 November 2016 Accepted 10 November 2016 Available online xxxx Keywords: Krüppel-like factor 14 Atherosclerosis Inflammatory Lipid metabolism
a b s t r a c t Genome-wide association studies have shown that Krüppel-like factor 14 (KLF14) is associated with both Type 2 diabetes mellitus and lipid metabolism. However, its role in chronic inflammatory responses and the pathogenesis of atherosclerosis remains unknown. The present study was designed to investigate both in vivo and in vitro the impact of KLF14 on chronic inflammatory responses and atherosclerosis. ApoE KO mice, a well-established animal model of atherosclerosis, had higher expressions of KLF14 in aorta tissues than that in C57BL/6 J mice when fed the high-fat diet (HFD) or standard chow diet. Adenovirus-mediated KLF14 knockdown markedly reduced the circulating levels of proinflammatory cytokines and the formation of atherosclerotic lesions in HFD-fed ApoE KO mice. In the in vitro study, KLF14 overexpression in the RAW264.7 macrophages significantly increased the expressions of inflammatory cytokines, total cholesterol (TC), cholesteryl ester (CE), and the ratio of CE to TC in the cells treated with acetylated low-density lipoproteins (AcLDL). Conversely, KLF14 knockdown remarkably attenuated AcLDL-induced increase in TC, CE, and the ratio of CE to TC as well as the expressions of inflammatory cytokines. Furthermore, up-regulation or down-regulation of KLF14 markedly elevated or inhibited the phosphorylation levels of p38 MAPK and ERK1/2 in AcLDL-stimulated RAW264.7 macrophages, respectively. Importantly, treatment with p38 MAPK or ERK1/2 inhibitor nullified the effects of KLF14 on inflammatory cytokine expressions in the cells. These data demonstrate an important role for KLF14 expression in atherosclerotic lesion formation. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Atherosclerosis is a chronic disease characterized by cholesterol plaque formation within the blood vessel wall, and it is one of the leading causes of mortality in developed countries [1]. Epidemiological studies have revealed that atherosclerosis is strongly associated with the dysfunction of lipoprotein metabolism, obesity, hypertension, abnormalities of blood coagulation and fibrinolysis, and chronic inflammation [2–5]. Therefore, the development of atherosclerosis is a complex multifactorial process caused by multiple genetic and environmental factors [5–7]. Although many studies have addressed possible associations between atherosclerosis and genetic variants, the precise mechanism underlying atherosclerosis remains to be elucidated. Recently, large-scale genome-wide association studies (GWAS) have found that variants near the Krüppel-like factor 14 (KLF14) are strongly
Funding: This work was supported by doctoral Fund of Ministry of Education of China (20105503110002, 20125503110003). ⁎ Corresponding author. Tel.: +86-23-68485216; fax: +86-23-68485005. E-mail addresses: [email protected] (G. Yang), [email protected] (Z. Li). 1 These authors contributed equally to this project.
associated with both Type 2 diabetes mellitus and high-density lipoprotein cholesterol (HDL-C) [8–10]. KLF14 belongs to the specificity protein/Krüppel-like factor (SP/KLF) family which constitutes a group of transcription factors that are present in organisms ranging from yeast to vertebrates [11,12]. The structure of SP/KLF super family proteins is defined by the presence of three highly conserved and homologous Cterminal Cys2His2 zinc finger domain which are responsible for DNA binding and a variable N-terminal domain which is responsible for transcriptional regulation as activators and/or repressors in cell- and promoter-dependent manners by interacting with co-activator or cosuppressor molecules via different types of regulatory domains [13–17]. Very recently, it has been reported that newly identified genetic variants near/in the KLF14 gene are strongly associated with blood lipid levels and atherosclerosis in different populations and implicated in the etiology and susceptibility of atherosclerotic-related phenotypes [8,18–20]. In addition, in an exciting new discovery, the maternally expressed KLF14 was found to act as a master trans-regulator of adipose gene expression in humans [21]. Given that the dysfunction of lipid or lipoprotein metabolism is of great importance to the progression of atherosclerosis and its related diseases [22,23], we hypothesized that the KLF14 may also be associated
http://dx.doi.org/10.1016/j.carpath.2016.11.003 1054-8807/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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with atherosclerosis. To address this hypothesis, we sought to determine whether an increase or decrease in KLF14 expression was sufficient to affect both cholesterol metabolism and atherosclerotic plaque development.
of 10% fetal bovine serum for 24 h. After the addition of 10 μL of CCK-8 dye, cells were incubated at 37 °C for 1 h, and the attenuance of each well was read on a microplate reader (Beckman, Ontario, Canada) at 450 nm. The attenuance of the untreated controls was defined as 100% survival [25].
2. Materials and methods
2.5. Assessment of liver and macrophages lipid content
2.1. Construction and purification of recombinant adenoviruses
Hepatic and macrophage lipid contents were measured by a spectrophotometric procedure (Applygen Technologies Inc., Beijing, China) as previously described [26] and expressed as lipid (mg)/wet liver weight (g) or total protein contents (g protein).
The recombinant adenovirus expressing full-length KLF14 gene (AdKLF14) and shRNAs against KLF14 (Ad-shKLF14) were constructed using the AdEasy system as previously described with slight modification [24]. The sequences of KLF14 were as follows: sense 5′CCGGAATTCATGTCGGCCGCCGTGGCT-3′ and antisense 5′-GCGGTCGA CCTACAGGCAAGCAGTGAAGCT-3′. Two recombinant vectors encoding enhanced green fluorescence protein (Ad-GFP and Ad-shGFP, Clontech, Mountain View, CA, USA) were used as respective controls. Large-scale amplification and purification of recombinant adenoviruses were performed with the ViraBind Adenovirus Purification Kit according to the manufacturer's instructions (Cell Biolabs, San Diego, CA, USA), and the product was stored at −80 °C.
2.6. Histological analysis The frozen cross-sections (10 μm thick) of the proximal aorta embedded in optimal cutting temperature (Sakura Finetechnical Co, Ltd) were stained with Oil Red O, and counterstained with hematoxylin. For analyses of aortic lesions en face, whole aortas were fixed in 10% formalin and stained with oil red O. Percentages of en face Oil Red O positive areas to total aortic areas were calculated. Quantitative analyses were performed by using the Image Pro-Plus software (Media Cybernetics, Silver Spring, MD, USA).
2.2. Animal preparation for the in vivo experiments 2.7. Quantitative real-time polymerase chain reaction (PCR) Male C57BL/6 J and ApoE KO mice were purchased from The Experimental Animal Center of Beijing University of Medical Sciences (Beijing, China) at 7 weeks of age, acclimated for a week, and fed either a standard chow diet (SCD, 18% kcal from fat) or a high-fat western diet consisting of 42% fat and 0.15% cholesterol (HFD, Medicience Ltd., Jiangsu, China) for 12 weeks. Animals were individually housed in a temperature-controlled (24 °C) facility with a 12-h light and 12-h dark cycle. ApoE KO mice were injected with Ad-shKLF14, Ad-shGFP [1×10 9PFU in 200 μl of phosphate -buffered saline (PBS)], or PBS by tail vein once a week starting at week 10 until week 12 of receiving the HFD. After 2 weeks of vector injections, mice were euthanized by an overdose of ketamine (80-mg/kg body weight, Sigma, St. Louis, MO, USA) and Xylazine (16 mg/kg, Merck, Munchen Germany). Blood samples were collected, and tissue samples were freeze clamped in situ with aluminum tongs precooled in liquid nitrogen and stored at −80 °C for subsequent analysis. All animal studies were performed in accordance with the guidelines for the use and care of laboratory animals of the Chongqing Medical University Institutional Animal Care and Use Committee and in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).
Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Purified RNA was used as template for first-strand cDNA synthesis using PrimerScript TM RT reagent Kit (Takara Bio Inc. Otsu, Japan). Quantitative real-time PCR was performed with a SYBR Premix Ex Taq TMII kit (Takara Bio Inc. Otsu, Japan) and a Corbett Rotor Gene 6000 real-time PCR system (Corbett Research, Sydney, Australia) according to the manufacturer's instructions. Gene expressions were analyzed using the comparative Ct method and normalized with β-actin. Forward and reverse primer pairs are as listed in Supplemental Table 1. 2.8. Biochemical parameters and cytokine measurements Plasma triglyceride (TG), TC, low-density lipoprotein cholesterol, and HDL-C concentrations were analyzed enzymatically using an autoanalyzer (Hitachi, Japan). Plasma inflammatory cytokines were determined using enzyme-linked immunosorbent assay kits (USCN Life Science Inc., Wuhan, China). 2.9. Immunoblot analysis
2.3. Cell culture and treatment RAW 264.7 mouse monocyte–macrophages and human monocyte/ macrophage cells (THP-1, American Type Culture Collection, Manassas, VA, USA) were grown in 6-well plates in Dulbecco Modified Eagle medium. RAW264.7 macrophages were pretreated with the indicated concentrations of signaling inhibitors (PD098059, SB203580 and SP600125, Sigma, St. Louis, MO, USA) or 0.5% dimethylsulfoxide (DMSO) (Sigma), then incubated with acetylated low-density lipoproteins (Ac-LDL) and finally infected with Ad-KLF14, Ad-shKLF14, or vehicle in serum-free cell culture medium for the indicated time. Cells were collected with radioimmuno precipitation assay buffer for protein analysis or with Trizol (Invitrogen, Carlsbad, CA, USA) for RNA analysis. 2.4. Cell viability assay Cell viability was determined with Cell Count Kit-8 (CCK-8) (Beyotime, Shanghai, China), according to the previous description. Briefly, approximately 5×103 cells per well were seeded in a 96-well plate and allowed to adhere overnight. On the following day, the cells were treated with palmic acid at increasing concentrations in the presence
Tissues from mice were homogenized. For the in vitro experiments, cells were lysed and sonicated in a lysis buffer (50-mM Tris, 150-mM NaCl, 1% Nonidet P-40, 10% sodium cholate, and 1-mM EDTA and protease inhibitor). The protein concentration was measured with a bicinchoninic acid protein quantification kit (Pierce Biotechnology, Rockford, IL, USA). One microliter aliquots of tissue extracts (80 μg) or cell lysates were separated by SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. Immunoblots were blocked in Tris-buffered saline/Tween 20 and 5% skimmed milk or 5% bovine serum albumin for 1 h at room temperature and incubated with primary antibodies including anti-KLF14 (Abcam Inc., Cambridge, UK); anti-p38 mitogen-activated protein kinase (p38 MAPK), anti-Phospho-p38 MAPK; anti-c-Jun. N-terminal kinase (JNK), antiPhospho-JNK; antiinhibition of extracellular signal-regulated kinase (ERK)1/2 and anti-Phospho-ERK1/2 (Cell Signaling, Beverly, MA, USA) and β-actin (Abcam Inc., Cambridge, UK) overnight at 4 °C. Following three consecutive 5-min washes in Tris-buffered saline-tween, blots were incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz, Dallas, Texas, USA) for 1 h at room temperature. After two washes in Tris-buffered saline-tween and a final wash in
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TBS, the blots were scanned using the enhanced chemiluminescence detection system (Viagene Biotech Inc., Florida, USA), and quantification of antigen–antibody complexes was performed using Quantity One analysis software (Bio-Rad, Hercules, CA, USA). 2.10. Immunohistochemistry Formalin-fixed, paraffin-embedded proximal aorta sections treated with antigen-unmasking reagent according to manufacturer's instruction. 2.11. Statistical analysis Data are presented as means±S.E.M. A one-way Analysis of Variance with a least significant difference post hoc test was used to compare mean values between multiple groups, and a two-sample unpaired Student t test was used for two group comparisons. Pb.05 was considered significant. All statistical analyses were performed using Statistical Product and Service Solutions (SPSS) 19.0 (SPSS graduate pack; SPSS, Chicago, IL, USA). 3. Results 3.1. Increased KLF14 expressions in the aortas samples of ApoE KO mice To determine whether KLF14 expression is altered in animal model with atherosclerosis, we performed real-time RT-PCR and Western blotting analyses in aorta samples obtained from C57BL/6 J mice and ApoE KO mice fed with SCD or HFD. As shown in Fig. 1, both KLF14 mRNA and protein expressions in aorta tissues were markedly higher in HFDfed C57BL/6 J mice compared with their littermates fed with SCD (Pb.01, Fig. 1A–B). However, the expression of KLF14 in ApoE KO mice fed with HFD or SCD was higher than that in C57BL/6 J mice (all Pb.01, Fig. 1A–B). In addition, the cross-sectional plaque area of the aortic sinus stained with immunohistochemistry showed a significant increase in the number of KLF14 immunoreactive cells in ApoE KO mice (Supplemental Fig. 1A–D). These results suggest that KLF14 may be expressed in plaque macrophages and correlated with atherosclerosis. 3.2. Effects of KLF14 knockdown on metabolic parameters in ApoE KO mice fed with HFD To elucidate the physiological role of KLF14 in vivo, we constructed a short hairpin RNA expression vector Ad-shKLF14 specific to mouse KLF14. In HFD-fed ApoE KO mice, Ad-shKLF14 treatment achieved 53% and 48% reductions in the levels of KLF14 mRNA and protein in the aorta compared with Ad-shGFP treatment (both Pb.01, Supplemental Fig. 2A–B). We further assessed the effects of KLF14 knockdown on metabolic parameters in HFD-fed ApoE KO mice. We found that KLF14 knockdown failed to change in body weight (Supplemental Fig. 3A). However, serum HDL-C trended to be higher and TG trended to be lower in Ad-shKLF14-treated mice but did not differ significantly from Ad-shGFP-treated littermates (Supplemental Fig. 3B-D). Notably, KLF14 knockdown in HFD-fed ApoE KO mice led to significant decreases in both serum and hepatic TC (Pb.01, Supplemental Fig. 3F–G), suggesting that KLF14 correlates with cholesterol metabolism. 3.3. KLF14 knockdown inhibits the formation of atherosclerotic lesions in ApoE KO mice To study the impact of KLF14 knockdown on the development of atherosclerosis, ApoE KO mice were fed an HFD for 12 weeks and were treated with Ad-shKLF14 or Ad-shGFP. Atherosclerotic lesions in the aorta were analyzed by en face of the spread total aorta and the cross-sections of the aortic sinus. The en face plaque area of the total aorta stained with oil red O was reduced by 53% in HFD-fed and AdshKLF14-treated ApoE KO mice compared with HFD-fed ApoE KO mice alone (Pb.01, Fig. 2A–B). In addition, the cross-sectional plaque area of
Fig. 1. Increased KLF14 expressions in ApoE KO mice. Eight-week-old C57B5/6 J mice and ApoE KO mice were fed with either an SCD or a high-fat western diet for 12 weeks. After euthanization, the aorta was removed for determining the mRNA and protein expression of KLF14. The expressions of KLF14 were detected by RT-PCR and western blot. (A) The KLF14 mRNA expression (n=10). (B) The KLF14 protein expression (n=10). Data are expressed as mean ± S.E.M. **Pb.01.
the aortic sinus was also reduced by 35% in HFD-fed ApoE KO mice treated with Ad-shKLF14 compared with HFD-fed ApoE KO mice alone (Pb.01, Fig. 2C–D), suggesting that KLF14 knockdown resulted in marked reduction of atherosclerotic lesion. 3.4. KLF14 knockdown reduces the circulating levels of proinflammatory cytokine in HFD- fed ApoE KO mice Since atherosclerosis is a chronic inflammatory process, we next analyzed the circulating levels of proinflammatory cytokines in AdshKLF14-treated ApoE KO mice. We found that Ad-shKLF14 treatment significantly decreased the increasing levels of TNF-α, IL-6, and MCP-1 induced by HFD-feeding in ApoE KO mice (Pb.05 or Pb.01, Fig. 3A–C), suggesting that inflammatory response was attenuated by KLF14 knockdown in these animals. 3.5. Effect of KLF14 knockdown on the AcLDL-induced cholesterol accumulation in RAW264.7 macrophages TC, cholesteryl ester (CE), and the ratio of CE to TC in RAW264.7 cells were increased by AcLDL treatment in a dose-dependent manner (Supplemental Fig. 4A–C). As shown in Supplemental Fig. 4D, 40 μg/ml of Ac-LDL has a minimal cytotoxicity. Therefore, it was selected for subsequent
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Fig. 2. Knockdown of KLF14 reduces atherosclerosis in ApoE KO mice fed with HFD. (A) Representative oil red O staining of en face lesions from entire aorta and (B) quantification of en face lesions area in sterile saline, Ad-shGFP, or Ad-shKLF14-treated ApoE KO mice receiving HFD (n=10 each group); (C) Representative oil red O of cross-sectional lesions from aortic root and (D) quantification of cross-sectional lesions areas in sterile saline, Ad-shGFP, or Ad-shKLF14-treated ApoE KO mice receiving HFD (×100) (n=10 each group). Data are expressed as mean ± S.E.M. **Pb.01.
Fig. 3. KLF14 knockdown inhibits the circulating levels of TNF-α, IL-6, and MCP-1 in the ApoE KO mice receiving HFD. The serum proinflammatory cytokines in sterile saline, Ad-shGFP, or Ad-shKLF14-treated ApoE KO mice receiving HFD were detected by enzyme-linked immunosorbent assay. (A) The level of TNF-α. (B) The level of IL-6. (C) The level of MCP-1. The cytokines levels are shown as the relative ratio to the level of sterile saline treated ApoE KO mice fed with HFD (n=10 each group). Data are expressed as mean ± S.E.M. *Pb.05, **Pb.01.
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cells alone (all Pb.01, Fig. 4A–C). In contrast, Ad-shKLF14 treatment in RAW264.7 cells remarkably inhibited the expressions of KLF14 and attenuated the AcLDL-induced increase in TC, CE, and the ratios of CE to TC (all Pb.01; Fig. 4A–C). 3.6. Effects of KLF14 on the expressions of proinflammatory cytokines in RAW264.7 cells To further investigate whether chronic inflammatory response is involved in KLF14-mediated alteration of cholesterol metabolism in vitro, we examined the mRNA expressions of MCP-1, IL-6, and TNF-α in AdKLF14 or Ad-shKLF14-treated RAW264.7 cells. As expected, treatment of RAW264.7 macrophages with AcLDL significantly increased the mRNA expressions of MCP-1, IL-6, and TNF-α. However, when these cells were treated by Ad-KLF14, the mRNA expressions of MCP-1, IL-6, and TNF-α were further increased compared with those of AcLDLtreated cells alone (all Pb.01, Fig. 5A–C). In contrast, AcLDL-induced mRNA expressions of MCP-1, IL-6, and TNF-α were significantly suppressed in RAW264.7 cells treated with Ad-shKLF14 (all Pb.01, Fig. 5A–C). 3.7. Effects of KLF14 on the activities of MAPKs We next investigated the molecular signal pathway responsible for the antiinflammatory and antiatherogenic effects of KLF14 knockdown. AdKLF14 transfection remarkably elevated the phosphorylation of p38 MAPK and ERK1/2 by 40% and 73.2%, respectively, in AcLDL-stimulated RAW264.7 macrophages as compared to vehicle transfection (Pb.01, Fig. 6A–B). Whereas in the RAW264.7 macrophages treated with both Ad-shKLF14 and AcLDL, the kinases activities of p38 MAPK and ERK1/2 were much lower than those of macrophages treated with control vector. However, there was no significant difference in the levels of phosphorylated JNK in the AcLDL-treated RAW264.7 macrophages in the presence of AdKLF14 or Ad-shKLF14 (Fig. 6C). These data suggest that p38 MAPK and ERK1/2 signaling may, at least in part, be involved in the effect of KLF14 on Ac-LDL-stimulated inflammatory cytokine release in macrophages. 3.8. p38 MAPK and ERK1/2 signaling are crucial for KLF14 to promote inflammatory cytokine release
Fig. 4. Effects of KLF14 on the AcLDL-induced accumulation of cholesterol ester in RAW264.7 macrophages. RAW264.7 macrophages were transfected with Ad-GFP, AdKLF14, Ad-shGFP, or Ad-shKLF14 for 24 h and, then, treated with AcLDL for another 24 h. The levels of TC, cholesterol ester (CE), and the ratio of CE to TC in RAW264.7 macrophages were determined using enzymatic colorimetric methods. (A) The level of TC; (B) The level of choleaterol ester; (C) The ratio of CE to TC in cellular. Data are expressed as means±S.E.M. of three to five independent experiments performed in triplicate. **Pb.01.
experiments. We found that KLF14 gene/protein expression was significantly increased in Ac-LDL-induced THP-1 cells (Supplemental Fig. 5). As shown in Fig. 4A–C, KLF14 overexpression vector transfection in RAW 264.7 macrophages led to significant increases in the expressions of KLF14 mRNA as compared with untransfected controls (all Pb.01; Supplemental Fig. 6). In Ad-KLF14 plus AcLDL-treated RAW264.7 cells, TC, CE, and the ratio of CE to TC were significantly elevated by 28.6%, 36.7%, and 7%, respectively, compared with AcLDL-treated RAW264.7
Finally, we sought to delineate a mechanism of KLF14 on regulating inflammatory cytokine release by using special kinases inhibitors. As shown in Fig. 7, Ad-KLF14 transfection in AcLDL-treated RAW264.7 cells significantly increased the mRNA expressions of MCP-1, IL-6, and TNF-α. However, treatment with a p38 MAPK (PD098059) and ERK1/ 2 (SB203580) inhibitors significantly nullified the ability of Ad-KLF14 to promote inflammatory cytokine expressions including MCP-1, IL-6, and TNF-α (all Pb.01, Fig. 7A–F). Conversely, the inhibitory roles of KLF14 knockdown in the production of MCP-1, IL-6, and TNF-α were further exacerbated in RAW264.7 macrophages incubated with AcLDL (Pb.05 or Pb.01, Fig. 7A–F). Treatment with SP600125, a JNK inhibitor, had no significant effect on both Ad-KLF14 and Ad-shKLF14 induced inflammatory cytokine release in RAW264.7 cells in the presence of AcLDL (Supplemental Fig. 7A–C). These data further suggest that both p38 MAPK and ERK1/2 signals involved in the effect of KLF14 on AcLDLstimulated inflammatory cytokine release. 4. Discussion The increasing prevalence of atherosclerosis is thought to be the result of the interaction of environmental factors with a combination of genetic variants. Atherosclerosis is also a chronic inflammatory disease in which multiple cells and the variants of gene types are involved with complex interactions with each other [27]. Although GWAS have found that variants near KLF14 are strongly associated with HDL-C, it is still unknown whether KLF14 regulates cholesterol metabolism and promotes or inhibits atherosclerosis. In the present study, we focused
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Fig. 5. Effects of KLF14 on the AcLDL-induced TNF-α, IL-6, and MCP-1 expressions in RAW264.7 macrophages. RAW264.7 macrophages were treated with Ad-KLF14 or Ad-shKLF14 for 24 h and then stimulated with 40-ng/ml AcLDL for another 24 h. The mRNA expressions of TNF-α (A), IL-6 (B), and MCP-1 (C) were determined by RT-PCR. Data are expressed as means±S.E.M. of three to five independent experiments performed in triplicate. **Pb.01.
on the role of KLF14 in chronic inflammatory response and atherosclerosis. We first found that on both HFD and SCD, the expression of KLF14 in aortic tissue in ApoE KO mice was higher than that of C57BL/ 6 J mice. In addition, a significant increase of KLF14 immunoreactive cells was found in the plaque area of the aortic sinus of ApoE KO mice. In the vitro study, we also found that KLF14 gene/protein expression was significantly increased in Ac-LDL-treated THP-1 cells, suggesting that KLF14 may be expressed in plaque macrophages. Because ApoE KO mice is a well-established animal model of atherosclerosis, increased expression of KLF14 in these animals may be implicated in the formation of atherosclerosis. However, another report demonstrated that both the mRNA and protein expressions of KLF14 were significantly decreased in the liver of C57BL/6 mice in response to a high-fat diet (HFD). The contrasting expression pattern described in these two reports may reflect the differences in expression profile of KLF14 in different tissues, perhaps by cell- and tissue-dependent manners [28]. With molecular loss- and gain-of-function approaches in vivo or vitro, we demonstrate that KLF14 knockdown prevents the progression of atherosclerosis in ApoE KO mice accompanied with effects on metabolic parameters, such as serum and hepatic TC levels. Our data also suggest that KLF14 knockdown reduced the formation of atherosclerotic lesions in ApoE KO mice, at least in part by suppressing chronic
inflammatory response in macrophages through the inhibition of the p38 MAPK and ERK1/2 pathways. Multiple risk factors are implicated in the pathogenesis of atherosclerosis, including metabolic abnormalities, such as hyperlipidemia, obesity, and diabetes [29]. Our previous study demonstrated that ApoE KO mice fed an HFD developed more severe hypercholesterolemia, markedly elevated low density lipoprotein (LDL) cholesterol levels and modestly increased TG and fasting blood glucose and body weight, suggesting severe metabolic abnormalities and insulin resistance [30]. In the current study, we utilized this animal model of metabolic abnormalities to observe the effects of KLF14 on the lipid profile. We first found that in ApoE KO mice fed with both HFD- and SCD, KLF14 expression in aortic tissue was higher than that in C57BL/6 J mice, and KLF14 knockdown in HFD-fed ApoE KO mice led to a significant decrease in both serum and hepatic TC. These results led us to speculate that KLF14 is associated with cholesterol metabolism and atherosclerosis. To test this hypothesis, we further investigated the effects of KLF14 on the formation of atherosclerotic lesions. As expected, KLF14 knockdown in HFD-fed ApoE KO mice significantly decreased both in the en face plaque area of the total aorta and the cross-sectional plaque area of the aortic sinus, confirming that KLF14 is associated with atherosclerotic lesion formation. Cholesterol homeostasis in cells is regulated by a complex set of mechanisms that include cholesterol synthesis, uptake of LDL, cholesterol
Fig. 6. Effects of KLF14 on the AcLDL-induced activities of MAPKs in RAW264.7 macrophages. RAW264.7 macrophages were treated with Ad-GFP, Ad-KLF14, Ad-shGFP, or Ad-shKLF14 for 24 h and then stimulated with AcLDL for another 24 h. The activities of MAPKs were determined by immunoblotting. (A) P38 MAPK phosphorylation; (B) ERK1/2 phosphorylation; (C) JNK phosphorylation. Data are expressed as means±S.E.M. of three to five independent experiments performed in triplicate. *Pb.05, **Pb.01.
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Fig. 7. p38 MAPK and ERK1/2 are crucial for KLF14 to promote inflammatory cytokine release in macrophages. RAW264.7 macrophages were pretreated with 20-μM special inhibitor (SB203580, PD098059) or DMSO as control for 30 min, then transfected with Ad-GFP, Ad-KLF14, Ad-shGFP, or Ad-shKLF14 for 24 h, and finally incubated with AcLDL or BSA for another 24 h. The mRNA expressions of TNF-α (A–B), IL-6 (C–D), and MCP-1 (E-F) were determined by RT-PCR in macrophages. Data are expressed as means±S.E.M. of three to five independent experiments performed in triplicate. *Pb.05, **Pb.01.
esterification, and cholesterol efflux [31]. During atherogenesis, macrophages take up modified LDL in an unregulated manner, via scavenger receptors, ultimately resulting in the depositions of the excessive CEs and free cholesterol that characterize foam cell phenotypes. The formation of cholesterol-laden macrophages is a prominent feature of atherosclerotic lesions [32]. To determine the involvement of KLF14 in AcLDL-induced cholesterol accumulation and formation of foam cells, RAW264.7 cells, which have been shown to form foam cells by AcLDL treatment, were
incubated with AcLDL and treated with Ad-KLF14 or Ad-shKLF14. We found that KLF14 overexpression in RAW 264.7 macrophages led to significant increases in TC, CE, and the ratio of CE to TC. In contrast, KLF14 knockdown attenuated AcLDL-induced increase in TC, CE, and the ratio of CE to TC in these cells. These results suggest that KLF14 is associated with cholesterol accumulation and the formation of foam cells. It is well established that atherosclerosis is a chronic inflammatory disease. In line with this concept, a range of proinflammatory cytokines
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are robustly associated with atherosclerosis, including TNF-α, IL-1, and IL-6. We next analyzed whether the effect of KLF14 on atherogenesis is associated with chronic inflammatory response. We found that KLF14 knockdown reduced the circulating levels of proinflammatory cytokines in HFD-fed ApoE KO mice and AcLDL-induced mRNA expressions of MCP-1, IL-6, and TNF-α in RAW 264.7 macrophages. These results clearly show that the effect of KLF14 knockdown on atherogenesis is associated with its antiinflammatory properties. A growing body of evidence suggests that alterations of several stress-activated signaling pathways, including JNK, p38 MAPK, and ERK1/2 are associated with the transcripts of some proinflammatory factors and pathogenesis of atherosclerosis [33–37]. Our present study shows that KLF14 overexpression in AcLDL-stimulated RAW264.7 macrophages activates p38 MAPK and ERK1/2 signalings accompanied by increased TNF-α, MCP-1, and IL-6 transcript levels, whereas treatment with p38 MAPK and ERK1/2 inhibitors significantly nullifies the ability of Ad-KLF14 to promote inflammatory cytokine expressions including MCP-1, IL-6, and TNF-α. Analogously, inhibition of ERK1/2 and P38 MAPK activation further exacerbates the effects of KLF14 knockdown on suppression of the productions of IL-6, TNF-α, and MCP-1 in macrophage co-cultured with AcLDL. These findings suggest that KLF14 may have promoted the release of proinflammatory cytokines and inflammatory responses by inducing the activations of these signal molecules. However, the precise mechanisms by which KLF14, either directly or indirectly, regulates the activities of P38 MAPK and ERK1/2 need to be examined by using both the KLF14 KO and transgenic mice. During the submission of our paper, in contrast with our data, Guo et al. reported that KLF14 and its activator, perhexiline, increase ApoAI and HDL-C levels in vivo and improve high density lipoprotein function, and administration of perhexiline inhibits atherosclerosis development in ApoE KO mice [28]. The discrepant findings between Guo et al.'s and our studies might be attributable to the differences of external factors which affect the sensitive tolerance tests used, such as sample size, housing conditions, stress and handling, or methodological differences, including different methods used to measure and/or variation among serum concentrations, mRNA, and protein levels. In addition, the different results between Guo et al.'s and our studies may also be in part due to differences in tissue-specific targeting or in cell- and tissuedependent context. Moreover, unlike ours, their study did not examine the effects of KLF14 on chronic inflammatory response and its potential mechanism in macrophages. However, as with most new discoveries, these findings need to be reproduced. Therefore, the conflicting results between Guo et al.'s and our studies are of particular interest and await further in-depth analysis in future studies. In conclusion, the present study both in vivo and in vitro identified a new transcription factor, KLF14, that may be involved in the pathogenesis of atherosclerosis and chronic inflammatory responses. Our observations provide strong support for KLF14 as a promising novel target for the treatment of atherosclerosis. Conflict of interest None. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.carpath.2016.11.003. References [1] Lusis AJ. Atherosclerosis. Nature 2000;407(6801):233–41. [2] Libby P. Inflammation in atherosclerosis. Nature 2002;420(9):868–74. [3] Young JL, Libby P, Schonbeck U. Cytokines in the pathogenesis of atherosclerosis. Thromb Haemost 2002;88(4):554–67. [4] Steinberg D. Lipoproteins and the pathogenesis of atherosclerosis. Circulation 1987; 76(3):508–14.
[5] Ross R, Harker L. Hyperlipidemia and atherosclerosis. Science 1976;193(4258):1094–100. [6] Aulchenko YS, Ripatti S, Lindqvist I, Boomsma D, Heid IM, Pramstaller PP, et al. Loci influencing lipid levels and coronary heart disease risk in 16 European population cohorts. Nat Genet 2009;41(1):47–55. [7] Goldbourt U, Neufeld HN. Genetic aspects of arteriosclerosis. Arteriosclerosis 1986; 6(6):357–77. [8] Teslovich TM, Musunuru K, Smith AV, Edmondson AC, Stylianou IM, Koseki M, et al. Biological, clinical and population relevance of 95 loci for blood lipids. Nature 2010; 466(7307):707–13. [9] Ohshige T, Iwata M, Omori S, Tanaka Y, Hirose H, Kaku K, et al. Association of new loci identified in European genome-wide association studies with susceptibility to type 2 diabetes in the Japanese. PLoS One 2011;6(10), e26911. [10] Ohshige T, Iwata M, Omori S, Hara K, Yasuda K, Morizono T, et al. A genome- wide association study identifies novel risk loci for type 2 diabetes. Nature 2007; 445(7130):881–5. [11] Zhao C, Meng A. Sp1-like transcription factors are regulators of embryonic development in vertebrates. Dev Growth Differ 2005;47(4):201–11. [12] Fernandez-Zapico ME, Lomberk GA, Tsuji S, Tanaka Y, Hirose H, Kaku K, et al. A functional family-wide screening of SP/KLF proteins identifies a subset of suppressors of KRAS- mediated cell growth. Biochem J 2011;435(2):529–37. [13] Philipsen S, Suske G. A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res 1999;27(15):2991–3000. [14] Kaczynski J, Cook T, Urrutia R. Sp1-and Krüppel-like transcription factors. Genome Biol 2003;4(2):206. [15] Bieker JJ. Kruppel-like factors: three fingers in many pies. J Biol Chem 2001;276(37): 34355–8. [16] Gill G, Pascal E, Tseng ZH, Tjian R. A glutamine-rich hydrophobic patch in transcription factor Sp1 contacts the dTAFII110 component of the drosophila TFIID complex and mediates transcriptional activation. Proc Natl Acad Sci U S A 1994;91(1):192–6. [17] Van Vliet J, Turner J, Crossley M. Human Kruppel-like factor 8: a CACCC-box binding protein that associates with CtBP and represses transcription. Nucleic Acids Res 2000;28(9):1955–62. [18] Small KS, Hedman AK, Grundberg E, Nica AC, Thorleifsson G, Kong A, et al. Identification of an imprinted master trans regulator at the KLF14 locus related to multiple metabolic phenotypes. Nat Genet 2011;43(6):561–4. [19] Chen X, Li S, Yang Y, Yang X, Liu Y, Liu Y, et al. Genome-wide association study validation identifies novel loci for atherosclerotic cardiovascular disease. J Thromb Haemost 2012;10(8):1508–14. [20] Global lipids genetics consortium, discovery and refinement of loci associated with lipid levelsNat Genet 2013;45(11):1274–83. [21] Parker-Katiraee L, Carson AR, Yamada T, Arnaud P, Feil R, Abu-Amero SN, et al. Identification of the imprinted KLF14 transcription factor undergoing human-specific accelerated evolution. PLoS Genet 2007;3(5), e65. [22] Dart AM, Chin-Dusting JP. Lipids and the endothelium. Cardiovasc Res 1999;43(2): 308–22. [23] Massaeli H, Pierce GN. Involvement of lipoproteins, free radicals, and calcium in cardiovascular disease processes. Cardiovasc Res 1995;29(5):597–603. [24] Wang C, Dai J, Yang M, Deng G, Xu S, Jia Y, et al. Silencing of FGF-21 expression promotes hepatic gluconeogenesis and glycogenolysis by regulation of the STAT3SOCS3 signal. FEBS J 2014;281(9):2136–47. [25] Yang M, Dai J, Jia Y, Suo L, Li S, Guo Y, et al. Overexpression of juxtaposed with another zinc finger gene 1 reduces proinflammatory cytokine release via inhibition of stress- activated protein kinases and nuclear factor-κB. FEBS J 2014;281(14): 3193–205. [26] Li X, Yang M, Wang H, Jia Y, Yan P, Boden G, et al. Overexpression of JAZF1 protected ApoE-deficient mice from atherosclerosis by inhibiting hepatic cholesterol synthesis via CREB-dependent mechanisms. Int J Cardiol 2014;177(1):100–10. [27] Rader DJ, Daugherty A. Translating molecular discoveries into new therapies for atherosclerosis. Nature 2008;451(7181):904–13. [28] Guo Y, Fan Y, Zhang J, Lomberk GA, Zhou Z, Sun L, et al. Perhexiline activates KLF14 and reduces atherosclerosis by modulating ApoA-I production. J Clin Invest 2015; 125(10):3819–30. [29] Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med 1999;340(2): 115–26. [30] Li L, Miao Z, Liu R, Yang M, Liu H, Yang G. Liraglutide prevents hypoadiponectinemiainduced insulin resistance and alterations of gene expression involved in glucose and lipid metabolism. Mol Med 2011;17(11–12):1168–78. [31] Delton-Vandenbroucke I, Bouvier J, Makino A, Besson N, Pageaux JF, Lagarde M, et al. Anti-bis(monoacylglycero) phosphate antibody accumulates acetylated LDL-derived cholesterol in cultured macrophages. J Lipid Res 2007;48(3):543–52. [32] Tabas I. Consequences of cellular cholesterol accumulation: basic concepts and physiological implications. J Clin Invest 2002;110(7):905–11. [33] Ricchi M, Odoardi MR, Carulli L, Anzivino C, Ballestri S, Pinetti A, et al. Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis incultured hepatocytes. J Gastroenterol Hepatol 2009;24(5):830–40. [34] Boden G, She P, Mozzoli M, Cheung P, Gumireddy K, Reddy P, et al. Free fatty acids produce insulin resistance and activate the proinflammatory nuclear factorkappaB pathway in rat liver. Diabetes 2005;54(12):3458–65. [35] Higa JK, Liang Z, Williams PG, Panee J. Phyllostachys edulis Compounds inhibit palmitic acidinduced monocyte chemoattractant protein 1 (MCP-1) production. PLoS One 2012;7(9), e45082. [36] Jagavelu K, Tietge UJ, Gaestel M, Drexler H, Schieffer B, Bavendiek U. Systemic deficiency of the MAP kinase-activated protein kinase 2 reduces atherosclerosis in hyper-cholesterolemic mice. Circ Res 2007;101(11):1104–12. [37] Bryk D, Olejarz W, Zapolska-Downar D. Mitogen-activated protein kinases in atherosclerosis. Postepy Hig Med Dosw (Online) 2014;68(2):10–22.
Contents lists available at ScienceDirect
Cardiovascular Pathology
Original Article
A novel role for the Krüppel-like factor 14 on macrophage inflammatory response and atherosclerosis development Xiao Wei a,1, Ruomei Yang a,1, Chengpan Wang a,1, Xun Jian b, Ling Li c, Hua Liu d, Gangyi Yang a,b, Zhiyong Li a,⁎ a
Department of Endocrinology, Yongchuan Hospital, Chongqing Medical University, 402160, Chongqing, China Department of Endocrinology, the Second Affiliated Hospital, Chongqing Medical University, 400010, Chongqing, China Key Laboratory of Diagnostic Medicine (Ministry of Education) and Department of Clinical Biochemistry, College of Laboratory Medicine, Chongqing Medical University, 400010, Chongqing, China d Department of Pediatrics, University of Mississippi Medical Center, Jackson, MS, USA b c
a r t i c l e
i n f o
Article history: Received 13 June 2016 Received in revised form 10 November 2016 Accepted 10 November 2016 Available online xxxx Keywords: Krüppel-like factor 14 Atherosclerosis Inflammatory Lipid metabolism
a b s t r a c t Genome-wide association studies have shown that Krüppel-like factor 14 (KLF14) is associated with both Type 2 diabetes mellitus and lipid metabolism. However, its role in chronic inflammatory responses and the pathogenesis of atherosclerosis remains unknown. The present study was designed to investigate both in vivo and in vitro the impact of KLF14 on chronic inflammatory responses and atherosclerosis. ApoE KO mice, a well-established animal model of atherosclerosis, had higher expressions of KLF14 in aorta tissues than that in C57BL/6 J mice when fed the high-fat diet (HFD) or standard chow diet. Adenovirus-mediated KLF14 knockdown markedly reduced the circulating levels of proinflammatory cytokines and the formation of atherosclerotic lesions in HFD-fed ApoE KO mice. In the in vitro study, KLF14 overexpression in the RAW264.7 macrophages significantly increased the expressions of inflammatory cytokines, total cholesterol (TC), cholesteryl ester (CE), and the ratio of CE to TC in the cells treated with acetylated low-density lipoproteins (AcLDL). Conversely, KLF14 knockdown remarkably attenuated AcLDL-induced increase in TC, CE, and the ratio of CE to TC as well as the expressions of inflammatory cytokines. Furthermore, up-regulation or down-regulation of KLF14 markedly elevated or inhibited the phosphorylation levels of p38 MAPK and ERK1/2 in AcLDL-stimulated RAW264.7 macrophages, respectively. Importantly, treatment with p38 MAPK or ERK1/2 inhibitor nullified the effects of KLF14 on inflammatory cytokine expressions in the cells. These data demonstrate an important role for KLF14 expression in atherosclerotic lesion formation. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Atherosclerosis is a chronic disease characterized by cholesterol plaque formation within the blood vessel wall, and it is one of the leading causes of mortality in developed countries [1]. Epidemiological studies have revealed that atherosclerosis is strongly associated with the dysfunction of lipoprotein metabolism, obesity, hypertension, abnormalities of blood coagulation and fibrinolysis, and chronic inflammation [2–5]. Therefore, the development of atherosclerosis is a complex multifactorial process caused by multiple genetic and environmental factors [5–7]. Although many studies have addressed possible associations between atherosclerosis and genetic variants, the precise mechanism underlying atherosclerosis remains to be elucidated. Recently, large-scale genome-wide association studies (GWAS) have found that variants near the Krüppel-like factor 14 (KLF14) are strongly
Funding: This work was supported by doctoral Fund of Ministry of Education of China (20105503110002, 20125503110003). ⁎ Corresponding author. Tel.: +86-23-68485216; fax: +86-23-68485005. E-mail addresses: [email protected] (G. Yang), [email protected] (Z. Li). 1 These authors contributed equally to this project.
associated with both Type 2 diabetes mellitus and high-density lipoprotein cholesterol (HDL-C) [8–10]. KLF14 belongs to the specificity protein/Krüppel-like factor (SP/KLF) family which constitutes a group of transcription factors that are present in organisms ranging from yeast to vertebrates [11,12]. The structure of SP/KLF super family proteins is defined by the presence of three highly conserved and homologous Cterminal Cys2His2 zinc finger domain which are responsible for DNA binding and a variable N-terminal domain which is responsible for transcriptional regulation as activators and/or repressors in cell- and promoter-dependent manners by interacting with co-activator or cosuppressor molecules via different types of regulatory domains [13–17]. Very recently, it has been reported that newly identified genetic variants near/in the KLF14 gene are strongly associated with blood lipid levels and atherosclerosis in different populations and implicated in the etiology and susceptibility of atherosclerotic-related phenotypes [8,18–20]. In addition, in an exciting new discovery, the maternally expressed KLF14 was found to act as a master trans-regulator of adipose gene expression in humans [21]. Given that the dysfunction of lipid or lipoprotein metabolism is of great importance to the progression of atherosclerosis and its related diseases [22,23], we hypothesized that the KLF14 may also be associated
http://dx.doi.org/10.1016/j.carpath.2016.11.003 1054-8807/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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with atherosclerosis. To address this hypothesis, we sought to determine whether an increase or decrease in KLF14 expression was sufficient to affect both cholesterol metabolism and atherosclerotic plaque development.
of 10% fetal bovine serum for 24 h. After the addition of 10 μL of CCK-8 dye, cells were incubated at 37 °C for 1 h, and the attenuance of each well was read on a microplate reader (Beckman, Ontario, Canada) at 450 nm. The attenuance of the untreated controls was defined as 100% survival [25].
2. Materials and methods
2.5. Assessment of liver and macrophages lipid content
2.1. Construction and purification of recombinant adenoviruses
Hepatic and macrophage lipid contents were measured by a spectrophotometric procedure (Applygen Technologies Inc., Beijing, China) as previously described [26] and expressed as lipid (mg)/wet liver weight (g) or total protein contents (g protein).
The recombinant adenovirus expressing full-length KLF14 gene (AdKLF14) and shRNAs against KLF14 (Ad-shKLF14) were constructed using the AdEasy system as previously described with slight modification [24]. The sequences of KLF14 were as follows: sense 5′CCGGAATTCATGTCGGCCGCCGTGGCT-3′ and antisense 5′-GCGGTCGA CCTACAGGCAAGCAGTGAAGCT-3′. Two recombinant vectors encoding enhanced green fluorescence protein (Ad-GFP and Ad-shGFP, Clontech, Mountain View, CA, USA) were used as respective controls. Large-scale amplification and purification of recombinant adenoviruses were performed with the ViraBind Adenovirus Purification Kit according to the manufacturer's instructions (Cell Biolabs, San Diego, CA, USA), and the product was stored at −80 °C.
2.6. Histological analysis The frozen cross-sections (10 μm thick) of the proximal aorta embedded in optimal cutting temperature (Sakura Finetechnical Co, Ltd) were stained with Oil Red O, and counterstained with hematoxylin. For analyses of aortic lesions en face, whole aortas were fixed in 10% formalin and stained with oil red O. Percentages of en face Oil Red O positive areas to total aortic areas were calculated. Quantitative analyses were performed by using the Image Pro-Plus software (Media Cybernetics, Silver Spring, MD, USA).
2.2. Animal preparation for the in vivo experiments 2.7. Quantitative real-time polymerase chain reaction (PCR) Male C57BL/6 J and ApoE KO mice were purchased from The Experimental Animal Center of Beijing University of Medical Sciences (Beijing, China) at 7 weeks of age, acclimated for a week, and fed either a standard chow diet (SCD, 18% kcal from fat) or a high-fat western diet consisting of 42% fat and 0.15% cholesterol (HFD, Medicience Ltd., Jiangsu, China) for 12 weeks. Animals were individually housed in a temperature-controlled (24 °C) facility with a 12-h light and 12-h dark cycle. ApoE KO mice were injected with Ad-shKLF14, Ad-shGFP [1×10 9PFU in 200 μl of phosphate -buffered saline (PBS)], or PBS by tail vein once a week starting at week 10 until week 12 of receiving the HFD. After 2 weeks of vector injections, mice were euthanized by an overdose of ketamine (80-mg/kg body weight, Sigma, St. Louis, MO, USA) and Xylazine (16 mg/kg, Merck, Munchen Germany). Blood samples were collected, and tissue samples were freeze clamped in situ with aluminum tongs precooled in liquid nitrogen and stored at −80 °C for subsequent analysis. All animal studies were performed in accordance with the guidelines for the use and care of laboratory animals of the Chongqing Medical University Institutional Animal Care and Use Committee and in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).
Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Purified RNA was used as template for first-strand cDNA synthesis using PrimerScript TM RT reagent Kit (Takara Bio Inc. Otsu, Japan). Quantitative real-time PCR was performed with a SYBR Premix Ex Taq TMII kit (Takara Bio Inc. Otsu, Japan) and a Corbett Rotor Gene 6000 real-time PCR system (Corbett Research, Sydney, Australia) according to the manufacturer's instructions. Gene expressions were analyzed using the comparative Ct method and normalized with β-actin. Forward and reverse primer pairs are as listed in Supplemental Table 1. 2.8. Biochemical parameters and cytokine measurements Plasma triglyceride (TG), TC, low-density lipoprotein cholesterol, and HDL-C concentrations were analyzed enzymatically using an autoanalyzer (Hitachi, Japan). Plasma inflammatory cytokines were determined using enzyme-linked immunosorbent assay kits (USCN Life Science Inc., Wuhan, China). 2.9. Immunoblot analysis
2.3. Cell culture and treatment RAW 264.7 mouse monocyte–macrophages and human monocyte/ macrophage cells (THP-1, American Type Culture Collection, Manassas, VA, USA) were grown in 6-well plates in Dulbecco Modified Eagle medium. RAW264.7 macrophages were pretreated with the indicated concentrations of signaling inhibitors (PD098059, SB203580 and SP600125, Sigma, St. Louis, MO, USA) or 0.5% dimethylsulfoxide (DMSO) (Sigma), then incubated with acetylated low-density lipoproteins (Ac-LDL) and finally infected with Ad-KLF14, Ad-shKLF14, or vehicle in serum-free cell culture medium for the indicated time. Cells were collected with radioimmuno precipitation assay buffer for protein analysis or with Trizol (Invitrogen, Carlsbad, CA, USA) for RNA analysis. 2.4. Cell viability assay Cell viability was determined with Cell Count Kit-8 (CCK-8) (Beyotime, Shanghai, China), according to the previous description. Briefly, approximately 5×103 cells per well were seeded in a 96-well plate and allowed to adhere overnight. On the following day, the cells were treated with palmic acid at increasing concentrations in the presence
Tissues from mice were homogenized. For the in vitro experiments, cells were lysed and sonicated in a lysis buffer (50-mM Tris, 150-mM NaCl, 1% Nonidet P-40, 10% sodium cholate, and 1-mM EDTA and protease inhibitor). The protein concentration was measured with a bicinchoninic acid protein quantification kit (Pierce Biotechnology, Rockford, IL, USA). One microliter aliquots of tissue extracts (80 μg) or cell lysates were separated by SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. Immunoblots were blocked in Tris-buffered saline/Tween 20 and 5% skimmed milk or 5% bovine serum albumin for 1 h at room temperature and incubated with primary antibodies including anti-KLF14 (Abcam Inc., Cambridge, UK); anti-p38 mitogen-activated protein kinase (p38 MAPK), anti-Phospho-p38 MAPK; anti-c-Jun. N-terminal kinase (JNK), antiPhospho-JNK; antiinhibition of extracellular signal-regulated kinase (ERK)1/2 and anti-Phospho-ERK1/2 (Cell Signaling, Beverly, MA, USA) and β-actin (Abcam Inc., Cambridge, UK) overnight at 4 °C. Following three consecutive 5-min washes in Tris-buffered saline-tween, blots were incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz, Dallas, Texas, USA) for 1 h at room temperature. After two washes in Tris-buffered saline-tween and a final wash in
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TBS, the blots were scanned using the enhanced chemiluminescence detection system (Viagene Biotech Inc., Florida, USA), and quantification of antigen–antibody complexes was performed using Quantity One analysis software (Bio-Rad, Hercules, CA, USA). 2.10. Immunohistochemistry Formalin-fixed, paraffin-embedded proximal aorta sections treated with antigen-unmasking reagent according to manufacturer's instruction. 2.11. Statistical analysis Data are presented as means±S.E.M. A one-way Analysis of Variance with a least significant difference post hoc test was used to compare mean values between multiple groups, and a two-sample unpaired Student t test was used for two group comparisons. Pb.05 was considered significant. All statistical analyses were performed using Statistical Product and Service Solutions (SPSS) 19.0 (SPSS graduate pack; SPSS, Chicago, IL, USA). 3. Results 3.1. Increased KLF14 expressions in the aortas samples of ApoE KO mice To determine whether KLF14 expression is altered in animal model with atherosclerosis, we performed real-time RT-PCR and Western blotting analyses in aorta samples obtained from C57BL/6 J mice and ApoE KO mice fed with SCD or HFD. As shown in Fig. 1, both KLF14 mRNA and protein expressions in aorta tissues were markedly higher in HFDfed C57BL/6 J mice compared with their littermates fed with SCD (Pb.01, Fig. 1A–B). However, the expression of KLF14 in ApoE KO mice fed with HFD or SCD was higher than that in C57BL/6 J mice (all Pb.01, Fig. 1A–B). In addition, the cross-sectional plaque area of the aortic sinus stained with immunohistochemistry showed a significant increase in the number of KLF14 immunoreactive cells in ApoE KO mice (Supplemental Fig. 1A–D). These results suggest that KLF14 may be expressed in plaque macrophages and correlated with atherosclerosis. 3.2. Effects of KLF14 knockdown on metabolic parameters in ApoE KO mice fed with HFD To elucidate the physiological role of KLF14 in vivo, we constructed a short hairpin RNA expression vector Ad-shKLF14 specific to mouse KLF14. In HFD-fed ApoE KO mice, Ad-shKLF14 treatment achieved 53% and 48% reductions in the levels of KLF14 mRNA and protein in the aorta compared with Ad-shGFP treatment (both Pb.01, Supplemental Fig. 2A–B). We further assessed the effects of KLF14 knockdown on metabolic parameters in HFD-fed ApoE KO mice. We found that KLF14 knockdown failed to change in body weight (Supplemental Fig. 3A). However, serum HDL-C trended to be higher and TG trended to be lower in Ad-shKLF14-treated mice but did not differ significantly from Ad-shGFP-treated littermates (Supplemental Fig. 3B-D). Notably, KLF14 knockdown in HFD-fed ApoE KO mice led to significant decreases in both serum and hepatic TC (Pb.01, Supplemental Fig. 3F–G), suggesting that KLF14 correlates with cholesterol metabolism. 3.3. KLF14 knockdown inhibits the formation of atherosclerotic lesions in ApoE KO mice To study the impact of KLF14 knockdown on the development of atherosclerosis, ApoE KO mice were fed an HFD for 12 weeks and were treated with Ad-shKLF14 or Ad-shGFP. Atherosclerotic lesions in the aorta were analyzed by en face of the spread total aorta and the cross-sections of the aortic sinus. The en face plaque area of the total aorta stained with oil red O was reduced by 53% in HFD-fed and AdshKLF14-treated ApoE KO mice compared with HFD-fed ApoE KO mice alone (Pb.01, Fig. 2A–B). In addition, the cross-sectional plaque area of
Fig. 1. Increased KLF14 expressions in ApoE KO mice. Eight-week-old C57B5/6 J mice and ApoE KO mice were fed with either an SCD or a high-fat western diet for 12 weeks. After euthanization, the aorta was removed for determining the mRNA and protein expression of KLF14. The expressions of KLF14 were detected by RT-PCR and western blot. (A) The KLF14 mRNA expression (n=10). (B) The KLF14 protein expression (n=10). Data are expressed as mean ± S.E.M. **Pb.01.
the aortic sinus was also reduced by 35% in HFD-fed ApoE KO mice treated with Ad-shKLF14 compared with HFD-fed ApoE KO mice alone (Pb.01, Fig. 2C–D), suggesting that KLF14 knockdown resulted in marked reduction of atherosclerotic lesion. 3.4. KLF14 knockdown reduces the circulating levels of proinflammatory cytokine in HFD- fed ApoE KO mice Since atherosclerosis is a chronic inflammatory process, we next analyzed the circulating levels of proinflammatory cytokines in AdshKLF14-treated ApoE KO mice. We found that Ad-shKLF14 treatment significantly decreased the increasing levels of TNF-α, IL-6, and MCP-1 induced by HFD-feeding in ApoE KO mice (Pb.05 or Pb.01, Fig. 3A–C), suggesting that inflammatory response was attenuated by KLF14 knockdown in these animals. 3.5. Effect of KLF14 knockdown on the AcLDL-induced cholesterol accumulation in RAW264.7 macrophages TC, cholesteryl ester (CE), and the ratio of CE to TC in RAW264.7 cells were increased by AcLDL treatment in a dose-dependent manner (Supplemental Fig. 4A–C). As shown in Supplemental Fig. 4D, 40 μg/ml of Ac-LDL has a minimal cytotoxicity. Therefore, it was selected for subsequent
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Fig. 2. Knockdown of KLF14 reduces atherosclerosis in ApoE KO mice fed with HFD. (A) Representative oil red O staining of en face lesions from entire aorta and (B) quantification of en face lesions area in sterile saline, Ad-shGFP, or Ad-shKLF14-treated ApoE KO mice receiving HFD (n=10 each group); (C) Representative oil red O of cross-sectional lesions from aortic root and (D) quantification of cross-sectional lesions areas in sterile saline, Ad-shGFP, or Ad-shKLF14-treated ApoE KO mice receiving HFD (×100) (n=10 each group). Data are expressed as mean ± S.E.M. **Pb.01.
Fig. 3. KLF14 knockdown inhibits the circulating levels of TNF-α, IL-6, and MCP-1 in the ApoE KO mice receiving HFD. The serum proinflammatory cytokines in sterile saline, Ad-shGFP, or Ad-shKLF14-treated ApoE KO mice receiving HFD were detected by enzyme-linked immunosorbent assay. (A) The level of TNF-α. (B) The level of IL-6. (C) The level of MCP-1. The cytokines levels are shown as the relative ratio to the level of sterile saline treated ApoE KO mice fed with HFD (n=10 each group). Data are expressed as mean ± S.E.M. *Pb.05, **Pb.01.
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cells alone (all Pb.01, Fig. 4A–C). In contrast, Ad-shKLF14 treatment in RAW264.7 cells remarkably inhibited the expressions of KLF14 and attenuated the AcLDL-induced increase in TC, CE, and the ratios of CE to TC (all Pb.01; Fig. 4A–C). 3.6. Effects of KLF14 on the expressions of proinflammatory cytokines in RAW264.7 cells To further investigate whether chronic inflammatory response is involved in KLF14-mediated alteration of cholesterol metabolism in vitro, we examined the mRNA expressions of MCP-1, IL-6, and TNF-α in AdKLF14 or Ad-shKLF14-treated RAW264.7 cells. As expected, treatment of RAW264.7 macrophages with AcLDL significantly increased the mRNA expressions of MCP-1, IL-6, and TNF-α. However, when these cells were treated by Ad-KLF14, the mRNA expressions of MCP-1, IL-6, and TNF-α were further increased compared with those of AcLDLtreated cells alone (all Pb.01, Fig. 5A–C). In contrast, AcLDL-induced mRNA expressions of MCP-1, IL-6, and TNF-α were significantly suppressed in RAW264.7 cells treated with Ad-shKLF14 (all Pb.01, Fig. 5A–C). 3.7. Effects of KLF14 on the activities of MAPKs We next investigated the molecular signal pathway responsible for the antiinflammatory and antiatherogenic effects of KLF14 knockdown. AdKLF14 transfection remarkably elevated the phosphorylation of p38 MAPK and ERK1/2 by 40% and 73.2%, respectively, in AcLDL-stimulated RAW264.7 macrophages as compared to vehicle transfection (Pb.01, Fig. 6A–B). Whereas in the RAW264.7 macrophages treated with both Ad-shKLF14 and AcLDL, the kinases activities of p38 MAPK and ERK1/2 were much lower than those of macrophages treated with control vector. However, there was no significant difference in the levels of phosphorylated JNK in the AcLDL-treated RAW264.7 macrophages in the presence of AdKLF14 or Ad-shKLF14 (Fig. 6C). These data suggest that p38 MAPK and ERK1/2 signaling may, at least in part, be involved in the effect of KLF14 on Ac-LDL-stimulated inflammatory cytokine release in macrophages. 3.8. p38 MAPK and ERK1/2 signaling are crucial for KLF14 to promote inflammatory cytokine release
Fig. 4. Effects of KLF14 on the AcLDL-induced accumulation of cholesterol ester in RAW264.7 macrophages. RAW264.7 macrophages were transfected with Ad-GFP, AdKLF14, Ad-shGFP, or Ad-shKLF14 for 24 h and, then, treated with AcLDL for another 24 h. The levels of TC, cholesterol ester (CE), and the ratio of CE to TC in RAW264.7 macrophages were determined using enzymatic colorimetric methods. (A) The level of TC; (B) The level of choleaterol ester; (C) The ratio of CE to TC in cellular. Data are expressed as means±S.E.M. of three to five independent experiments performed in triplicate. **Pb.01.
experiments. We found that KLF14 gene/protein expression was significantly increased in Ac-LDL-induced THP-1 cells (Supplemental Fig. 5). As shown in Fig. 4A–C, KLF14 overexpression vector transfection in RAW 264.7 macrophages led to significant increases in the expressions of KLF14 mRNA as compared with untransfected controls (all Pb.01; Supplemental Fig. 6). In Ad-KLF14 plus AcLDL-treated RAW264.7 cells, TC, CE, and the ratio of CE to TC were significantly elevated by 28.6%, 36.7%, and 7%, respectively, compared with AcLDL-treated RAW264.7
Finally, we sought to delineate a mechanism of KLF14 on regulating inflammatory cytokine release by using special kinases inhibitors. As shown in Fig. 7, Ad-KLF14 transfection in AcLDL-treated RAW264.7 cells significantly increased the mRNA expressions of MCP-1, IL-6, and TNF-α. However, treatment with a p38 MAPK (PD098059) and ERK1/ 2 (SB203580) inhibitors significantly nullified the ability of Ad-KLF14 to promote inflammatory cytokine expressions including MCP-1, IL-6, and TNF-α (all Pb.01, Fig. 7A–F). Conversely, the inhibitory roles of KLF14 knockdown in the production of MCP-1, IL-6, and TNF-α were further exacerbated in RAW264.7 macrophages incubated with AcLDL (Pb.05 or Pb.01, Fig. 7A–F). Treatment with SP600125, a JNK inhibitor, had no significant effect on both Ad-KLF14 and Ad-shKLF14 induced inflammatory cytokine release in RAW264.7 cells in the presence of AcLDL (Supplemental Fig. 7A–C). These data further suggest that both p38 MAPK and ERK1/2 signals involved in the effect of KLF14 on AcLDLstimulated inflammatory cytokine release. 4. Discussion The increasing prevalence of atherosclerosis is thought to be the result of the interaction of environmental factors with a combination of genetic variants. Atherosclerosis is also a chronic inflammatory disease in which multiple cells and the variants of gene types are involved with complex interactions with each other [27]. Although GWAS have found that variants near KLF14 are strongly associated with HDL-C, it is still unknown whether KLF14 regulates cholesterol metabolism and promotes or inhibits atherosclerosis. In the present study, we focused
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Fig. 5. Effects of KLF14 on the AcLDL-induced TNF-α, IL-6, and MCP-1 expressions in RAW264.7 macrophages. RAW264.7 macrophages were treated with Ad-KLF14 or Ad-shKLF14 for 24 h and then stimulated with 40-ng/ml AcLDL for another 24 h. The mRNA expressions of TNF-α (A), IL-6 (B), and MCP-1 (C) were determined by RT-PCR. Data are expressed as means±S.E.M. of three to five independent experiments performed in triplicate. **Pb.01.
on the role of KLF14 in chronic inflammatory response and atherosclerosis. We first found that on both HFD and SCD, the expression of KLF14 in aortic tissue in ApoE KO mice was higher than that of C57BL/ 6 J mice. In addition, a significant increase of KLF14 immunoreactive cells was found in the plaque area of the aortic sinus of ApoE KO mice. In the vitro study, we also found that KLF14 gene/protein expression was significantly increased in Ac-LDL-treated THP-1 cells, suggesting that KLF14 may be expressed in plaque macrophages. Because ApoE KO mice is a well-established animal model of atherosclerosis, increased expression of KLF14 in these animals may be implicated in the formation of atherosclerosis. However, another report demonstrated that both the mRNA and protein expressions of KLF14 were significantly decreased in the liver of C57BL/6 mice in response to a high-fat diet (HFD). The contrasting expression pattern described in these two reports may reflect the differences in expression profile of KLF14 in different tissues, perhaps by cell- and tissue-dependent manners [28]. With molecular loss- and gain-of-function approaches in vivo or vitro, we demonstrate that KLF14 knockdown prevents the progression of atherosclerosis in ApoE KO mice accompanied with effects on metabolic parameters, such as serum and hepatic TC levels. Our data also suggest that KLF14 knockdown reduced the formation of atherosclerotic lesions in ApoE KO mice, at least in part by suppressing chronic
inflammatory response in macrophages through the inhibition of the p38 MAPK and ERK1/2 pathways. Multiple risk factors are implicated in the pathogenesis of atherosclerosis, including metabolic abnormalities, such as hyperlipidemia, obesity, and diabetes [29]. Our previous study demonstrated that ApoE KO mice fed an HFD developed more severe hypercholesterolemia, markedly elevated low density lipoprotein (LDL) cholesterol levels and modestly increased TG and fasting blood glucose and body weight, suggesting severe metabolic abnormalities and insulin resistance [30]. In the current study, we utilized this animal model of metabolic abnormalities to observe the effects of KLF14 on the lipid profile. We first found that in ApoE KO mice fed with both HFD- and SCD, KLF14 expression in aortic tissue was higher than that in C57BL/6 J mice, and KLF14 knockdown in HFD-fed ApoE KO mice led to a significant decrease in both serum and hepatic TC. These results led us to speculate that KLF14 is associated with cholesterol metabolism and atherosclerosis. To test this hypothesis, we further investigated the effects of KLF14 on the formation of atherosclerotic lesions. As expected, KLF14 knockdown in HFD-fed ApoE KO mice significantly decreased both in the en face plaque area of the total aorta and the cross-sectional plaque area of the aortic sinus, confirming that KLF14 is associated with atherosclerotic lesion formation. Cholesterol homeostasis in cells is regulated by a complex set of mechanisms that include cholesterol synthesis, uptake of LDL, cholesterol
Fig. 6. Effects of KLF14 on the AcLDL-induced activities of MAPKs in RAW264.7 macrophages. RAW264.7 macrophages were treated with Ad-GFP, Ad-KLF14, Ad-shGFP, or Ad-shKLF14 for 24 h and then stimulated with AcLDL for another 24 h. The activities of MAPKs were determined by immunoblotting. (A) P38 MAPK phosphorylation; (B) ERK1/2 phosphorylation; (C) JNK phosphorylation. Data are expressed as means±S.E.M. of three to five independent experiments performed in triplicate. *Pb.05, **Pb.01.
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Fig. 7. p38 MAPK and ERK1/2 are crucial for KLF14 to promote inflammatory cytokine release in macrophages. RAW264.7 macrophages were pretreated with 20-μM special inhibitor (SB203580, PD098059) or DMSO as control for 30 min, then transfected with Ad-GFP, Ad-KLF14, Ad-shGFP, or Ad-shKLF14 for 24 h, and finally incubated with AcLDL or BSA for another 24 h. The mRNA expressions of TNF-α (A–B), IL-6 (C–D), and MCP-1 (E-F) were determined by RT-PCR in macrophages. Data are expressed as means±S.E.M. of three to five independent experiments performed in triplicate. *Pb.05, **Pb.01.
esterification, and cholesterol efflux [31]. During atherogenesis, macrophages take up modified LDL in an unregulated manner, via scavenger receptors, ultimately resulting in the depositions of the excessive CEs and free cholesterol that characterize foam cell phenotypes. The formation of cholesterol-laden macrophages is a prominent feature of atherosclerotic lesions [32]. To determine the involvement of KLF14 in AcLDL-induced cholesterol accumulation and formation of foam cells, RAW264.7 cells, which have been shown to form foam cells by AcLDL treatment, were
incubated with AcLDL and treated with Ad-KLF14 or Ad-shKLF14. We found that KLF14 overexpression in RAW 264.7 macrophages led to significant increases in TC, CE, and the ratio of CE to TC. In contrast, KLF14 knockdown attenuated AcLDL-induced increase in TC, CE, and the ratio of CE to TC in these cells. These results suggest that KLF14 is associated with cholesterol accumulation and the formation of foam cells. It is well established that atherosclerosis is a chronic inflammatory disease. In line with this concept, a range of proinflammatory cytokines
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are robustly associated with atherosclerosis, including TNF-α, IL-1, and IL-6. We next analyzed whether the effect of KLF14 on atherogenesis is associated with chronic inflammatory response. We found that KLF14 knockdown reduced the circulating levels of proinflammatory cytokines in HFD-fed ApoE KO mice and AcLDL-induced mRNA expressions of MCP-1, IL-6, and TNF-α in RAW 264.7 macrophages. These results clearly show that the effect of KLF14 knockdown on atherogenesis is associated with its antiinflammatory properties. A growing body of evidence suggests that alterations of several stress-activated signaling pathways, including JNK, p38 MAPK, and ERK1/2 are associated with the transcripts of some proinflammatory factors and pathogenesis of atherosclerosis [33–37]. Our present study shows that KLF14 overexpression in AcLDL-stimulated RAW264.7 macrophages activates p38 MAPK and ERK1/2 signalings accompanied by increased TNF-α, MCP-1, and IL-6 transcript levels, whereas treatment with p38 MAPK and ERK1/2 inhibitors significantly nullifies the ability of Ad-KLF14 to promote inflammatory cytokine expressions including MCP-1, IL-6, and TNF-α. Analogously, inhibition of ERK1/2 and P38 MAPK activation further exacerbates the effects of KLF14 knockdown on suppression of the productions of IL-6, TNF-α, and MCP-1 in macrophage co-cultured with AcLDL. These findings suggest that KLF14 may have promoted the release of proinflammatory cytokines and inflammatory responses by inducing the activations of these signal molecules. However, the precise mechanisms by which KLF14, either directly or indirectly, regulates the activities of P38 MAPK and ERK1/2 need to be examined by using both the KLF14 KO and transgenic mice. During the submission of our paper, in contrast with our data, Guo et al. reported that KLF14 and its activator, perhexiline, increase ApoAI and HDL-C levels in vivo and improve high density lipoprotein function, and administration of perhexiline inhibits atherosclerosis development in ApoE KO mice [28]. The discrepant findings between Guo et al.'s and our studies might be attributable to the differences of external factors which affect the sensitive tolerance tests used, such as sample size, housing conditions, stress and handling, or methodological differences, including different methods used to measure and/or variation among serum concentrations, mRNA, and protein levels. In addition, the different results between Guo et al.'s and our studies may also be in part due to differences in tissue-specific targeting or in cell- and tissuedependent context. Moreover, unlike ours, their study did not examine the effects of KLF14 on chronic inflammatory response and its potential mechanism in macrophages. However, as with most new discoveries, these findings need to be reproduced. Therefore, the conflicting results between Guo et al.'s and our studies are of particular interest and await further in-depth analysis in future studies. In conclusion, the present study both in vivo and in vitro identified a new transcription factor, KLF14, that may be involved in the pathogenesis of atherosclerosis and chronic inflammatory responses. Our observations provide strong support for KLF14 as a promising novel target for the treatment of atherosclerosis. Conflict of interest None. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.carpath.2016.11.003. References [1] Lusis AJ. Atherosclerosis. Nature 2000;407(6801):233–41. [2] Libby P. Inflammation in atherosclerosis. Nature 2002;420(9):868–74. [3] Young JL, Libby P, Schonbeck U. Cytokines in the pathogenesis of atherosclerosis. Thromb Haemost 2002;88(4):554–67. [4] Steinberg D. Lipoproteins and the pathogenesis of atherosclerosis. Circulation 1987; 76(3):508–14.
[5] Ross R, Harker L. Hyperlipidemia and atherosclerosis. Science 1976;193(4258):1094–100. [6] Aulchenko YS, Ripatti S, Lindqvist I, Boomsma D, Heid IM, Pramstaller PP, et al. Loci influencing lipid levels and coronary heart disease risk in 16 European population cohorts. Nat Genet 2009;41(1):47–55. [7] Goldbourt U, Neufeld HN. Genetic aspects of arteriosclerosis. Arteriosclerosis 1986; 6(6):357–77. [8] Teslovich TM, Musunuru K, Smith AV, Edmondson AC, Stylianou IM, Koseki M, et al. Biological, clinical and population relevance of 95 loci for blood lipids. Nature 2010; 466(7307):707–13. [9] Ohshige T, Iwata M, Omori S, Tanaka Y, Hirose H, Kaku K, et al. Association of new loci identified in European genome-wide association studies with susceptibility to type 2 diabetes in the Japanese. PLoS One 2011;6(10), e26911. [10] Ohshige T, Iwata M, Omori S, Hara K, Yasuda K, Morizono T, et al. A genome- wide association study identifies novel risk loci for type 2 diabetes. Nature 2007; 445(7130):881–5. [11] Zhao C, Meng A. Sp1-like transcription factors are regulators of embryonic development in vertebrates. Dev Growth Differ 2005;47(4):201–11. [12] Fernandez-Zapico ME, Lomberk GA, Tsuji S, Tanaka Y, Hirose H, Kaku K, et al. A functional family-wide screening of SP/KLF proteins identifies a subset of suppressors of KRAS- mediated cell growth. Biochem J 2011;435(2):529–37. [13] Philipsen S, Suske G. A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res 1999;27(15):2991–3000. [14] Kaczynski J, Cook T, Urrutia R. Sp1-and Krüppel-like transcription factors. Genome Biol 2003;4(2):206. [15] Bieker JJ. Kruppel-like factors: three fingers in many pies. J Biol Chem 2001;276(37): 34355–8. [16] Gill G, Pascal E, Tseng ZH, Tjian R. A glutamine-rich hydrophobic patch in transcription factor Sp1 contacts the dTAFII110 component of the drosophila TFIID complex and mediates transcriptional activation. Proc Natl Acad Sci U S A 1994;91(1):192–6. [17] Van Vliet J, Turner J, Crossley M. Human Kruppel-like factor 8: a CACCC-box binding protein that associates with CtBP and represses transcription. Nucleic Acids Res 2000;28(9):1955–62. [18] Small KS, Hedman AK, Grundberg E, Nica AC, Thorleifsson G, Kong A, et al. Identification of an imprinted master trans regulator at the KLF14 locus related to multiple metabolic phenotypes. Nat Genet 2011;43(6):561–4. [19] Chen X, Li S, Yang Y, Yang X, Liu Y, Liu Y, et al. Genome-wide association study validation identifies novel loci for atherosclerotic cardiovascular disease. J Thromb Haemost 2012;10(8):1508–14. [20] Global lipids genetics consortium, discovery and refinement of loci associated with lipid levelsNat Genet 2013;45(11):1274–83. [21] Parker-Katiraee L, Carson AR, Yamada T, Arnaud P, Feil R, Abu-Amero SN, et al. Identification of the imprinted KLF14 transcription factor undergoing human-specific accelerated evolution. PLoS Genet 2007;3(5), e65. [22] Dart AM, Chin-Dusting JP. Lipids and the endothelium. Cardiovasc Res 1999;43(2): 308–22. [23] Massaeli H, Pierce GN. Involvement of lipoproteins, free radicals, and calcium in cardiovascular disease processes. Cardiovasc Res 1995;29(5):597–603. [24] Wang C, Dai J, Yang M, Deng G, Xu S, Jia Y, et al. Silencing of FGF-21 expression promotes hepatic gluconeogenesis and glycogenolysis by regulation of the STAT3SOCS3 signal. FEBS J 2014;281(9):2136–47. [25] Yang M, Dai J, Jia Y, Suo L, Li S, Guo Y, et al. Overexpression of juxtaposed with another zinc finger gene 1 reduces proinflammatory cytokine release via inhibition of stress- activated protein kinases and nuclear factor-κB. FEBS J 2014;281(14): 3193–205. [26] Li X, Yang M, Wang H, Jia Y, Yan P, Boden G, et al. Overexpression of JAZF1 protected ApoE-deficient mice from atherosclerosis by inhibiting hepatic cholesterol synthesis via CREB-dependent mechanisms. Int J Cardiol 2014;177(1):100–10. [27] Rader DJ, Daugherty A. Translating molecular discoveries into new therapies for atherosclerosis. Nature 2008;451(7181):904–13. [28] Guo Y, Fan Y, Zhang J, Lomberk GA, Zhou Z, Sun L, et al. Perhexiline activates KLF14 and reduces atherosclerosis by modulating ApoA-I production. J Clin Invest 2015; 125(10):3819–30. [29] Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med 1999;340(2): 115–26. [30] Li L, Miao Z, Liu R, Yang M, Liu H, Yang G. Liraglutide prevents hypoadiponectinemiainduced insulin resistance and alterations of gene expression involved in glucose and lipid metabolism. Mol Med 2011;17(11–12):1168–78. [31] Delton-Vandenbroucke I, Bouvier J, Makino A, Besson N, Pageaux JF, Lagarde M, et al. Anti-bis(monoacylglycero) phosphate antibody accumulates acetylated LDL-derived cholesterol in cultured macrophages. J Lipid Res 2007;48(3):543–52. [32] Tabas I. Consequences of cellular cholesterol accumulation: basic concepts and physiological implications. J Clin Invest 2002;110(7):905–11. [33] Ricchi M, Odoardi MR, Carulli L, Anzivino C, Ballestri S, Pinetti A, et al. Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis incultured hepatocytes. J Gastroenterol Hepatol 2009;24(5):830–40. [34] Boden G, She P, Mozzoli M, Cheung P, Gumireddy K, Reddy P, et al. Free fatty acids produce insulin resistance and activate the proinflammatory nuclear factorkappaB pathway in rat liver. Diabetes 2005;54(12):3458–65. [35] Higa JK, Liang Z, Williams PG, Panee J. Phyllostachys edulis Compounds inhibit palmitic acidinduced monocyte chemoattractant protein 1 (MCP-1) production. PLoS One 2012;7(9), e45082. [36] Jagavelu K, Tietge UJ, Gaestel M, Drexler H, Schieffer B, Bavendiek U. Systemic deficiency of the MAP kinase-activated protein kinase 2 reduces atherosclerosis in hyper-cholesterolemic mice. Circ Res 2007;101(11):1104–12. [37] Bryk D, Olejarz W, Zapolska-Downar D. Mitogen-activated protein kinases in atherosclerosis. Postepy Hig Med Dosw (Online) 2014;68(2):10–22.