Ox-LDL Upregulates CRP Expression Through The IGF2 Pathway In THP-1 Macrophages

BACKGROUNDAtherosclerosis is a chronic inflammatory disease that represents the leading cause of morbidity and mortality throughout developed countries. In fact, inflammatory processes are involved at all stages of atherosclerotic development. Therefore, factors that act to limit inflammation in these processes may be beneficial to impede disease progression. Moreover, an increasing number of studies have reported that oxidized low-density lipoprotein (Ox LDL) plays a significant role in the initiation and progression of atherosclerosis. During atherogenesis progression, circulating monocytes adhere to the intima and differentiate into macrophages. After differentiation, intimal macrophages intake Ox-LDL via scavenger receptors, thereby transforming into foam cells. Hence, foam cell formation due to excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis.The insulin-like growth factor (IGF) system plays a crucial role in growth, and development and has recently been implicated in the development and progression ofatherosclerosis. Members of the IGF system include the IGF ligands insulin-like growth factors 1 and 2 (IGF1 and IGF2, respectively), the cell surface receptors IGF1 and 2 (IGF-1R and IGF-2R, respectively), and six different types of binding proteins (IGFBPs). Both IGF1 and IGF2 interact with IGF-1R, a transmembrane tyrosine kinase that is structurally and functionally related to the insulin receptor. IGF2 also binds to IGF-2R, which functions as a clearance receptor by endocytosis and intracellular degradation of its ligand. IGF1 is regulated by growth hormone secretion from the pituitary, while IGF2 production seems independent of growth hormone control. It is established that IGF1 promotes atherosclerosis by partial regulation of endothelial function; however, the relationship between IGF2 and atherosclerosis has not been established.C-reactive protein (CRP) is an acute-phase reactant protein involved in several host defense mechanisms, as indicated by increased levels during the acute-phase response to tissue injury, infection, and other inflammatory stimuli. The role of CRP in complex chronic diseases is gaining increasing recognition. For example, elevated CRP levels have been identified in obesity, cardiovascular disease, and diabetes. Furthermore, a high basal level of serum CRP is an established prognostic indicator of future atherosclerosis and metabolic abnormalities. However, whether Ox-LDL can influence expression of CRP and IGF2 levels in THP-1 macrophages remains uncertain. In the present study, we demonstrated that CRP expression could be upregulated by Ox-LDL through the IGF2 pathway in THP-1 macrophages.MATERIALS AND METHODSPreparation of Ox-LDLNative LDL (200 mg protein/mL; Sigma-Aldrich, St. Louis, MO, USA) was oxidized by exposure to CuSO4 (5 mM free Cu2+) in phosphate-buffered saline (PBS) at 37℃ for 24 h. Control incubations were treated with 200 mM ethylenediaminetetraacetic acid (EDTA). Freshly prepared Ox-LDL was dialyzed at 37℃ for 48 h against 500 volumes of PBS to remove Cu2+ and then sterilized by passage through a 0.45-mm filter. LDL oxidation was confirmed by measurement of thiobarbituric acid-reactive substances (TBARS) with malonaldehyde bis(dimethyl acetal) (MDA) as the standard. The TBARS content of Ox-LDL was 6.0560.16 vs. 0.3260.15 nmol/100 mg of protein in the native LDL preparation (p<0.01). Protein content was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific Pierce, Rockford, IL, USA) with the use of bovine serum albumin as the standard. The Ox-LDL was maintained in 50 mM Tris-HCl,0.15 M NaCl, and 2.0 mM EDTA at pH 7.4 and was used within 10 days of preparation.Cell CultureHuman monocytic THP-1 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal calf serum under standard culture conditions (5% CO2,37℃). Cells were seeded in 6-or 12-well plates or 60-mm dishes.RNA Isolation and Real-Time Quantitative PCR AnalysisTotal RNA from cultured cells was extracted using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions.Real-time quantitative PCR (qPCR) with SYBR Green detection chemistry was performed on an ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) with the following primers:IGF2 forward,5’-GGAACCCACATT GGCCTGA-3’; IGF2 reverse,5-CCGGCGAGGCAGAATATAACAC-3’; CRP forward,5’-AATGTGAACATGTGG GACTTTGTG-3’; and CRP reverse,5-CGCCAGTTCAGGACATTAGG AC-3’.Melt curve analyses of all qPCR products were performed and found to produce a single DNA duplex. All samples were measured in triplicate, and the mean value was considered for comparative analysis. Quantitative measurements were determined using the △△Ct method, and glyceraldehyde 3-phosphate dehydrogenase expression was used as the internal control.Western Blot AnalysisProteins were extracted from cultured cells using radioimmunoprecipitation assay buffer (Biocolor Ltd., Belfast, Northern Ireland, UK), quantified using the BCA protein assay kit (KeyGen Biotechnologies, Nanjing, China), and then subjected to Western blot analysis (10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis; 30μg protein per lane) using rabbit polyclonal antiCRP antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit polyclonal anti-IGF2 antibodies (Abcam, Cambridge, MA, USA), and rabbit polyclonalβ-actin-specific antibodies (Abcam). The proteins were visualized using a chemiluminescence method (ECL Plus Western Blot Detection System; Amerisham Biosciences, Foster City, CA, USA).Transfection with Small Interfering RNASmall interfering RNAs (siRNAs) against IGF2 (IGF2-siRNA) and an irrelevant 21-nucleotide control as a negative control were purchased from Ribo Targets Ltd. (Cambridgeshire, UK). Cells (2×106/well) were transfected using Lipofectamine 2000 transfection reagent (Invitrogen Corporation) for 48 h. Afterward, qPCR and Western blot analysis were performed.Construction of Recombinant PlasmidsThe PIRES2-EGFPand PCR-XL-TOPO vectors (containing IGF2, which were assembled using chemically synthesized oligos by PCR) were purchased from Invitrogen Corporation. The fragment ofEcoRI-IGF2-IRESEGFP-XhoI was amplified using the templates of the PCR-XL-TOPO and PIRES2-EGFPvectors, respectively. EcoRI-IGF2-IRES-EGFP-XhoI was joined by the two abovementioned segments using overlap PCR. Gel electrophoresis was performed, and the relevant band was excised from the gel, double enzyme-digested byEcoRI/XhoI, incorporated into the pcDNA3.1(+) vector, and then transformed into competent Escherichia coliDH5a cells for further amplification and use. The recombinant plasmid was verified by sequencing and named pcDNA3.1-IGF2. The plasmid transfection process was performed using Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions.Statistical AnalysesData are expressed as means±standard deviations (SDs). Results were obtained by one-way analysis of variance followed by the Student-Newman-Keuls test and Student’sttest using SPSS v13.0 statistical software (SPSS Inc., Chicago, IL, USA). A two-tailed probability (p)value<0.05 was considered statistically significant.RESULTS1、Ox-LDL Upregulated IGF2 Expression in THP-1 MacrophagesTo explore possible changes in RNA expression during macrophage formation, we performed microarray analysis of THP-1 macrophages and THP-1 macrophage-derived foam cells in the presence of Ox-LDL using the Arraystar probe dataset, which included 24,748 long noncoding RNAs and 24,420 coding transcripts. The microarray results showed that IGF1, IGF2, and CRP gene expression levels were increased by 219,620, and 326%, respectively, in THP-1 macrophage-derived foam cells compared to THP-1 macrophages. However, there were no significant alterations in levels of IGF-1R, IGF-2R, and six different types of IGFBPs. Ruidavets et al. demonstrated the ability of IGF1 to temper expression of proinflammatory cytokines, although its protective effect was found to be independent of the activity of inflammatory markers, such as CRP. Therefore, our aim was to explore whether Ox-LDL upregulates CRP expression through the IGF2 pathway in THP-1 macrophages. To this end, we verified the effect of Ox-LDL on IGF2 expression in THP-1 macrophages by qPCR and Western blot analyses. Ox-LDL obviously increased IGF2 messenger RNA (mRNA) and protein expression in THP-1 macrophages in a time-and dose-dependent manner.2、Ox-LDL Upregulated CRP Expression in THP-1 MacrophagesThe complex relationship between the inflammatory response and vascular injury is well established, and vascular repair is of utmost importance to the pathogenesis of atherosclerosis. CRP is not only a strong marker of atherosclerosis but also a modulator that suppresses the activity of local and systemic thrombosis regulatory pathways, whereas Ox-LDL plays a crucial role in the initiation and progression of atherosclerosis. Microarray results showed that CRP expression levels were upregulated in THP-1 macrophage-derived foam cells compared to THP-1 macrophages. Thus, we then investigated the effect of OxLDL on CRP expression in THP-1 macrophages. qPCR and Western blot analyses showed that Ox-LDL obviously increased CRP mRNA and protein expression in a timeand dose-dependent manner in THP-1 macrophages.3、The Role of IGF2 in Ox-LDL-Induced CRP Upregulation in THP-1 MacrophagesPrevious studies have shown that the IGF system plays an important regulatory role in inflammation. Therefore, we suspected that Ox-LDL upregulated CRP expression through the IGF2 pathway in THP-1 macrophages. Treatment with recombinant plasmids overexpressing IGF2 (pcDNA3.1-IGF2) increased IGF2 protein expression by 896% in THP-1 macrophages. Moreover, CRP protein expression was markedly increased by treatment with pcDNA3.1-IGF2 and, conversely, treatment with pcDNA3.1-IGF2 significantly enhanced CRP protein expression in Ox-LDL-stimulated THP-1 macrophages. Next, we investigated the effect of IGF2 siRNA on Ox-LDL-induced CRP upregulation. Treatment with siRNA targeting IGF2 decreased IGF2 protein expression by 90% in comparison with control siRNA in THP-1 macrophages. In addition, Ox-LDL-induced CRP upregulation was completely abolished by siRNA-mediated silencing of IGF2 in THP-1 macrophages.CONCLUSIONS1、Ox-LDL obviously increased IGF2 and CRP mRNA and protein expression in THP-1 macrophages in a time-and dose-dependent manner.2、Treatment with Ox-LDL increased CRP protein expression, and this effect was completely abolished by siRNA-mediated silencing of IGF2 in THP-1 macrophages. Moreover, treatment with pcDNA3.1-IGF2 significantly enhanced CRP protein expression in Ox-LDL-stimulated THP-1 macrophages. CRP expression is upregulated by Ox-LDL through the IGF2 pathway in THP-1 macrophages. Our findings provide new insight into the biological effects and underlying mechanisms of the antiatherogenic effects of Ox-LDL.