European Review for Medical and Pharmacological Sciences 2020; 24: 7002-7014 KLF7 promotes macrophage activation by activating the NF-κB signaling pathway in epicardial adipose tissue in patients with coronary artery disease W.-H. HUANG1, Y.-J. XUE1,2, Y.-J. ZHOU1, X.-G. WU1, X.-X. WU1, X.-X. ZHANG1, X.-X. DONG1, Y.-T. WEI2,3 1Medicine Department, Shihezi University, Shihezi, Xinjiang, China 2Department of Thoracic and Cardiovascular Surgery, The First Affiliated Hospital of Shihezi University School of Medicine, Shihezi, China 3Jining First People’s Hospital, Jining, China Wenhua Huang and Yajun Xue contributed equally to this work Abstract. – OBJECTIVE: Inflammatory ac- potential therapeutic target for cardiovascular cumulation in epicardial adipose tissue (EAT) diseases such as CAD. may influence the formation and development of coronary artery disease (CAD). EAT macro- Key Words: phages exhibit M1 polarization and the secre- KLF7, Macrophage, NF-κB signaling pathway, Coro- tion of a large number of inflammatory factors in nary artery disease. CAD patients. Emerging data demonstrate that Krüppel-like factor-7 (KLF7), contributes to the regulation of adipocyte differentiation and the secretion of adipose tissue inflammation. How- Introduction ever, the function of KLF7 in EAT inflammation still remains to be uncovered. This study aims to Coronary artery disease (CAD) is the leading investigate the role of KLF7 in macrophage acti- vation in EAT. cause of death worldwide, resulting in 9.48 mil- 1 PATIENTS AND METHODS: The levels of in- lion deaths in 2016 . Endothelial dysfunction is terleukin-6 (IL-6) and tumor necrosis factor-α the first sign in the development of atherogenesis (TNF-α) in cell supernatant were measured by (AS)2,3, followed by the invasion of pro-inflamma- enzyme-linked immunosorbent assay (ELISA). tory cytokines, such as interleukin-6 (IL-6) and The mRNA expression levels were measured tumor necrosis factor-α (TNF-α). This occurs in by Real Time-PCR. The protein expression level was detected by Western blot. the epicardial adipose tissue (EAT) and the artery RESULTS: The expression of inflammatory wall around the coronary arteries. factors and KLF7 were markedly increased in EAT evolves from brown adipose tissues, it is CAD EAT than non-CAD EAT. KLF7 is highly ex- in close proximity to the coronary arteries4 and pressed in human THP-1-derived macrophages shares the same origin with omental adipose tis- induced by inflammatory stimuli, such as LPS. sue. As a result, EAT can secrete pro-inflamma- The knockdown of KLF7 inhibited the release tory cytokines and adipokines5. The tissue is also of inflammatory factors and significantly de- creased the expression of KLF7 in human THP- an active endocrine organ that secretes substan- 6 7 1-derived macrophages stimulated by LPS. More- tial TNF-a and IL-6 via paracrine or vasocrine over, transfection with KLF7-siRNA caused the effects. It is able to infiltrate the myocardium and marked inhibition of LPS-induced phosphoryla- coronary artery walls because of the lack of fascia tion of JNK-MAPKs and also suppressed the lev- between the coronary arteries and EAT8-10. els of p-p65 and inhibited the activation of p-IκBα. Macrophage activation is an integral process CONCLUSIONS: Taken together, these results indicate that KLF7 enhances macrophage acti- in the development of atherosclerosis. The best vation, mediated by JNK-NF-κB signaling path- described immunocompetent cells within EAT 11,12 ways in EAT. This suggests that KLF7 may be a are macrophages. Some scholars have shown 7002 Corresponding Author: Yutao Wei, MD; e-mail: [email protected] KLF7 promotes macrophage activation in patients with coronary artery disease that EAT macrophages exhibit M1 polarization the approved guidelines for human experimental in CAD patients, and the percentage of these research. cells is higher in CAD than non-CAD patients. EAT is characterized by the increased infiltra- Study Population tion of macrophages, suggesting that they might Thirty patients with CAD who underwent off- represent an important source of inflammation pump coronary artery bypass grafting (CABG) in adipose tissue. As pattern recognition recep- were enrolled in this study. Other 30 patients who tors, Toll-like receptors (TLRs) associate their underwent atrial septal defect repair or valvular signaling pathways with the infiltration of mac- replacement surgery without coronary artery ste- rophages in the EAT of patients with CAD11. nosis were included as the control group. EAT bi- TLRs can initiate the activation of nuclear fac- opsy samples (average weight 0.5-1.0 g) were col- tor-kB (NF-kB) and induce the expression of in- lected from the anterior wall of the left ventricle, flammatory genes. In CADs, TLR-4 is the main which was adjacent to the diseased segment, one TLR isoform involved in EAT inflammation13. hour after the administration of anesthesia. All Kruppel-like factor 7 (KLF7) is a member of samples were immediately stored in an RNA sta- the 18 KLF family that plays different roles in bilizer and then transferred to -80°C before total gene expression and regulation14,15. This ubiq- RNA extraction. Patients with acute myocardial uitous transcription factor is expressed in car- infarction, severe heart failure, active infections, diovascular, digestive, respiratory, and immune hematologic diseases, rheumatologic diseases, systems and is involved in cardiovascular dis- steroid or immunomodulatory drug use, and his- ease, cancer, and inflammatory conditions16. tories of chemotherapy or radiotherapy were ex- KLF7, which localizes in the nucleus, is a mem- cluded. The baseline characteristics and laborato- ber of the pluripotency transcriptional network ry parameters of the patients are shown in Table I. and plays a vital role in transcriptional activa- tion17,18. There has been little research into the Cell Culture role of KLF7 in causing inflammation. In a re- Human monocytic THP-1 cells were pur- cent study, the expression of KLF7 was remark- chased from the cell bank of the Chinese Acad- ably and positively correlated with TLR4 and emy of Sciences in Shanghai, China. THP-1 IL-6 in adipose tissue samples19. However, the cells were cultured in Roswell Park Memorial functional role of KLF7 in macrophage activa- Institute-1640 (RPMI-1640) medium (HyClone, tion in EAT remains unknown. South Logan, UT, USA) containing 10% fetal This study found that KLF7 had a significant bovine serum (FBS; Gibco, Rockville, MD, positive correlation with inflammatory factors in USA), penicillin (100 U/mL), and streptomycin CAD EAT. Moreover, KLF7 protein levels were (100 μg/mL) at 37°C in a humidified atmosphere markedly increased in CAD EAT compared to containing 5% CO2. The THP-1 cells were seed- non-CAD EAT. Our results indicate that knock- ed in 12-well plates at a density of 2x105 cells out of KLF7 decreases LPS-induced expression per well and differentiated into macrophages by of cytokines, including IL-6 and TNF-α by acti- using 100 μg/mL phorbol-12-myristate 13-ac- vating JNK-NF-κB signaling pathways in human etate (Invitrogen, Carlsbad, CA, USA) for 24 THP-1-derived macrophages. These results sug- hours. The cells were then incubated with 20 gest that KLF7-NF-κB may form a positive feed- ng/mL IFN-γ (Gibco, Rockville, MD, USA) and back loop to drive macrophage activation and the 10 ng/mL LPS (Sigma-Aldrich, St. Louis, MO, production of inflammatory factors in EAT. USA) for 24 hours to induce M1 macrophages before their use in experiments. Patients and Methods Real-Time Polymerase Chain Reaction (PCR) Ethical Note The total RNA from CAD EATs, non-CAD All procedures involving human subjects were EATs, and the experimental groups of cells were approved by the medical Ethics Committee at the extracted using TRIzol (Thermo Fisher Scientif- First Affiliated Hospital of Shihezi University ic, Inc., Waltham, MA, USA) in accordance with School of Medicine. Written informed consent the manufacturer’s instructions. The purity and was obtained from all subjects and all associat- concentration of RNAs were measured using a ed methods were conducted in accordance with NanoDrop 1000 spectrophotometer (Thermo 7003 W.-H. Huang, Y.-J. Xue, Y.-J. Zhou, X.-G. Wu, X.-X. Wu, X.-X. Zhang, X.-X. Dong, Y.-T. Wei Fisher Scientific, Waltham, MA, USA). Reverse ed with horseradish peroxidase (HRP) and by transcription was performed using the PrimeS- detection using the Pierce Fast Western Blot cript RT reagent Kit with genomic deoxyribo- Kit (Thermo Fisher Scientific, Waltham, MA, nucleic acid (gDNA) eraser (TaKaRa Bio, Inc., USA). Antibodies used in the study were β-ac- Otsu, Shiga, Japan) with 500 ng of total RNA tin (ZSGBBIO, Beijing, China), KLF7 (Abcam, isolated for each sample. Quantitative Real Cambridge, MA, USA), p-IkBα (S32/S36), Time-PCR (qRT-PCR) was performed with the JNK, p-JNK (T183/ Y185), p-p65 (Cell Sig- QuantiNova SYBR Green PCR kit (Qiagen, naling Technology, Danvers, MA, USA), and Hilden, Germany) using the 7500 Real-Time mouse IgG and rabbit IgG (ZSGBBIO, Beijing, PCR System (Applied Biosystems, Foster City, China). β-actin protein was probed with mice CA, USA). The gene expression levels of beta-ac- antibodies to β-actin (ZSGBBIO, Beijing, Chi- tin, KLF7, TLR4, IL-6, CD68, TNF-α, JNK, and na, TA-09; 1:1000 dilution). KLF7 protein was NF-kB were quantified, and the primer sequenc- probed with rabbit antibodies to KLF7 (Ab- es are listed in Table II. All assays were per- cam, Cambridge, MA, USA, ab129149; 1:1000 formed in triplicate. The relative amounts of the dilution). Phospho-IkBα protein was probed target gene transcripts were calculated using the with mice antibodies to Phospho-IkBα (CST, comparative cycle time method. The relative ex- Danvers, MA, USA, #9246; 1:1000 dilution). pression levels for each tissue and control sam- JNK protein was probed with rabbit antibod- ples were calculated by the comparative critical ies to JNK (CST, Danvers, MA, USA, #9252; threshold method.