Pioglitazone Suppresses CXCR7 Expression To Inhibit Human Macrophages Chemotaxis Through PPARγ

Patients with type 2 diabetes mellitus(T2DM) are at high risk of fatal and non-fatal macrovascular events, including coronary arteriosclerosis and/or atherosclerosis. Atherosclerosis is the major cause of death in subjects with T2 DM.Large-vessel atherosclerosis frequently accompanies the development of diabetes mellitus, and both these conditions share many common genetic and environmental risk factors. In clinical practice, thiazolidinedione drugs(TZDs), and pioglitazone in particular, significantly lowers risk of cardiovascular events among a broad population of patients with T2 DM. Pioglitazone, as a agonist of peroxisome proliferator-activated receptor γ(PPARγ) not only reduce hyperglycemia and insulin resistance and conversion to T2 DM, but also lower blood pressure, increase high density lipoprotein cholesterol(HDL-C) and adiponectin concentrations, and reduce plasma levels of a wide range of inflammatory markers in vitro and in vivo. However,pioglitazone exhibiting anti-atherosclerotic effects with the underlying mechanisms still remains unclear. Thus, a better understanding of antiatherogenic mechanisms of pioglitazone would not only provide insight into the pleiotropic actions, but may also help to identify novel targets for cardiovascular treatment.Monocyte and macrophage accumulation into local vessel walls drives atherosclerosis progression and leads to the atherosclerotic mass, which represents several potential targets in exploring the atherosclerosis therapy.The pathophysiology of atherosclerosis, especially the chemotaxis of macrophage in atherosclerotic lesions is mainly controlled by chemokines. In nearly 40 human chemokines, the majority were pro-atherogenic 。 However, conflicting results are reported for the pro- or anti-atherogenic effects of stromal cell-derived factor-1(SDF-1) in atherosclerosis。For a while, CXCR4 has been recognized as the only specific receptor responsible forSDF-1 until that CXCR7 has been characterized.whether CXCR7 acts as a signaling or non-signaling “decoy” receptor remains a debate.On the one hand, several studies suggested that CXCR7 is a decoy receptor and functions as a scavenger to remove extracellular SDF-1 or via hetero-dimerization with CXCR4 indirectly regulating CXCR4 signal transduction。On the other hand,CXCR7 was demonstrated to trigger SDF-1-mediated cancer cell or neuron migration.During monocyte-to-macrophage differentiation, CXCR7 is induced while CXCR4 is Downregulated。In addition, CXCR7 is primarily responsible for SDF-1and I-TAC stimulation of pro-inflammatory signaling pathways, leading to enhanced macrophage phagocytosis and thus potentially contributing to atherogenesis.Furthermore, the up-regulated CXCR7 receptor during monocyte-to-macrophage differentiation is specific responder in macrophage chemotaxis。On the other hand,PPARγ agonists, including pioglitazone and rosiglitazone, decrease chemokine receptors expression in proinflammatory macrophage, including CCR1, CCR2, CCR3,CCR5 and CX3CR1. Intrigued by these findings, we were interested in exploring a potential effect of pioglitazone on macrophage CXCR7 expression and function.MATERIALS AND METHODS1. Patients: From May 2012 to September 2013, 24 consecutive patients with T2 DM who were eligible for surgical carotid endarterectomy were enrolled from a single center. Patients were divided into two groups according to onset pioglitazone or non-TZDs therapy. Human atherosclerotic plaques were removed in the surgery.Blood samples were collected at 24 h before the surgery, and centrifuged at 1500 g for15 min. The serum was decanted and stored at-80 ℃ until assayed. Serum pioglitazone was quantified using an internally standardized high-performance liquid chromatography(HPLC) assay with UV detection as described previously。This study was approved by the institutional ethics committee of Second Hospital of Jilin University(Changchun, China), and written informed consent was obtained from every patient. The study was undertaken in full accordance with the Declaration of Helsinki, and other bioethical principles.2. Cell preparation, culture and stimulation: Peripheral blood mononuclear cells(PBMCs) were isolated by density gradient centrifugation using Ficoll-Hypaque as an established protocol. The investigation conforms to the principles outlined in the Declaration of Helsinki for use of human tissue or subjects, and was approved by the institutional ethics committee of Second Hospital of Jilin University(Changchun,China), and written informed consent was obtained from every subject. Isolated PBMCs were washed twice and resuspended in PBS containing 0.5% bovine serum albumin(BSA)(Sigma). Untouched human primary monocytes were purified by negative selection using Pan Monocyte Isolation Kit(Miltenyi Biotec). Subsequently,the purity of isolated monocytes was above 95%, as confirmed by fluorescence activated cell sorter analysis. Isolated monocytes were cultured in RPMI 1640 culture medium(GBICO) supplemented with 10%(v/v) fetal bovine serum(FBS)(Hy Clone Lab, Inc.), penicillin(100 U/m L) and streptomycin(100 μg/m L). To obtain polarized human macrophage, monocytes were differentiated into macrophage by treatment of the cells with the IFN-γ(100 ng/ml) and LPS(100 ng/ml) for 72 h. Cultures were maintained in an incubator at 37 oC in an atmosphere of 5% CO2 and 95% air。3. Cell viability: Cell viability was determined using Cell Counting Kit 8(CCK8, Dojindo) according to manufacturer’s protocol. In brief, cells(5×104cells/well) were plated into 96-well plates containing 100 μL of the growth medium under indicated treatment at 37 °C in 5% CO2 for up to 72 h. Cell viability was evaluated by measuring the absorbance at 570 nm.4. Real-time PCR: Total RNA was isolated using Trizol reagent according to the manufacturer’s protocol, and 2 μg of total RNA was converted to c DNA by Super Script TM III First-Strand Synthesis System for RT-PCR(Invitrogen, Life Technologies). Brilliant SYBR-Green was combined with c DNA and corresponding primers specific for indicated genes and real-time PCR amplification was performed using a Stratagene Mx3000 P system(Cedar Creek). Template c DNA was denatured at95 oC for 10 min followed by 40 cycles(denaturation for 1 min at 95 oC, annealing for1 min at 60 oC, and extension for 2 min at 72 o C). The expression levels of target genes were measured using comparative Ct method(dd Ct) normalized againstGAPDH.5. Flow Cytometry: Macrophages were collected by gentle scrapping, washed with ice-cold phosphate buffered saline containing 1% BSA. The cells were then Fc-blocked by treatment with 1μg of human Ig G/105 cells for 30 min at 4 oC. Next,the cells were incubated at 4 oC for 30 min with monoclonal antibodies against CXCR7(Allophycocyanin(APC), clone 11G8, R&D systems), CXCR4(APC, clone12G5, R&D systems) and CXCR3(APC, clone 49801, R&D systems) following the manufacturers’ instructions. Finally, the cells were washed and analyzed on a FACSCalibur flow cytometer(Becton Dickinson).CXCR7, CXCR4 and CXCR3 expression levels are referred to as mean fluorescence intensity.6. Western Blot: To detect the protein expression of CXCR7.7. Enzyme-linked immunosorbent assay(ELISA): MCP-1, IL-6, CCL18 and IL-10 were measured in the supernatants using ELISA according to the manufacturer’s instruction(e Bioscience)8. si RNAs transfection: Human PPARγ, PPARα, CXCR7, CXCR4 and non-targeting(scrambled) si RNAs were obtained from Qiagen. Transfection of macrophage was performed using Transmessenger Transfection Reagent(Qiagen) as described by the manufacturer.9. Chemotaxis assay: Chemotaxis assays were performed using 6.5-mm Transwell tissue culture inserts with a 5 μm pore size(Corning). Macophages were suspended at 5×106 cells/ml in RPMI 1640 with 0.5% BSA, and 100 μl of cell suspension was added to an insert in a well. The lower compartment was placed with600 μL of medium containing indicated concentrations of SDF-1, I-TAC or TC14012,the plates were then incubated for 180 min. Cells were fixed, and stained with the Three Step Stain Set(Richard-Allan Scientific). Macrophages that had remained in the upper chamber were removed by wiping the filters with cotton swaps.Macrophages found on the bottom of filters were counted as cells that had carried out chemotaxis. The migrant cells were counted in five randomly selected high-power fields(400×) per well. The chemotaxis index(CI) was calculated as the number of cells that migrated to the sample medium divided by the number of cells that migratedto the control medium. For the stimulation or inhibition assay, cells were pretreated with PPARγ agonist Rosiglitazone(Sigma) and antagonist T0070907(Selleck Chemicals) and then loaded into the upper chamber.The pretreatment time of pioglitazone was 24 h and pioglitazone was present during chemotaxis assay.10. Statistical analysis: Data are provided as mean ± standard deviation values.Statistical differences between groups were analyzed by Student t-test and Mann Whitney-test and were considered significant when P<0.05. 2-tailed Spearman correlation coefficients between two genes were calculated from real-time PCR ΔCt data. All reported P values were two-sided. All analyses were performed with Graph Pad Prism 5.0 software.RESULTSIn vitro results1. Differentiated macrophage were treatmented with different concentration of pioglitazone,which is 0.05μM, 0.2μM, 1μM, and then compare with the non-pioglitazone group.The result is that pioglitazone markedly reduced m RNA and protein levels of CXCR7 in differentiated macrophage in a-dose dependent way.Otherwise,we set five different time point(0、1、6、12、24hours) in order to detect whether pioglitazone can effect the expresstion of CXCR7.Our data shows that Time course experiments for CXCR7 m RNA and protein levels revealed that pioglitazone(1 μM) significantly inhibited CXCR7 m RNA expression levels by approximately3-fold(versus 0 hour) at 12 and 24 hours.2. In addition to differentiated macrophages, we also analyze the CXCR7 m RNA expression profile during monocyte-to-macrophage differentiation, the result showed that the expression of CXCR7 was induced during monocyte-to-macrophage differentiation in the presence of IFN-γ and LPS, and pioglitazone suppressed the CXCR7 expression during this process. Both real-time PCR and FACS analysis revealed that both CXCR4 and CXCR3 expression levels were markedly lower versus CXCR7 levels and remained unchanged after pioglitazone treatment, suggesting that pioglitazone did not alter CXCR4 or CXCR3 expression in macrophage.3. PPARα and PPARγ were successfully suppressed by transfection of specific si RNAs, but not by scramble control si RNAs. In PPARγ knockdown macrophage,pioglitazone failed to inhibit CXCR7 expression, whereas silencing PPARα did not affect pioglitazone-induced CXCR7 suppression. To further prove that pioglitazone indeed inhibit the expression of CCR7 by PPAR,we used PPAR γ antagonist T0070907 and PPAR γ agonist rosiglitazone, our consequence illustrated that PPARγ antagonist T0070907 dose dependently abolished pioglitazone-downregulated CXCR7 expression in macrophage. On the other hand, pioglitazone-induced CXCR7 downregulation in macrophage was strongly amplified by PPARγ agonist rosiglitazone in a dose-dependent manner.4. SDF-1, I-TAC and TC14012 significantly induced macrophage chemotaxis in the absence of pioglitazone, while macrophage chemotaxis was blocked when treated with pioglitazone.5. To further confirm CXCR7 the crucial role in macrophage chemotaxis,CXCR7 was silenced in differentiated macrophage using si RNA methods. CXCR7 m RNA expression was significantly suppressed by transfection of CXCR7-selective si RNAs compared with transfection of scramble control si RNAs. Notably,transfection of CXCR7 si RNA did not modify CXCR4 or CXCR3 expression. As predicted, silencing CXCR7 abolished the effect of SDF-1, I-TAC and TC14012 on chemotaxis of macrophage.6. To determine whether pioglitazone activating PPARγ was essential in macrophage chemotaxis inhibition mediated by CXCR7 downregulation, we used both PPARγ agonist and antagonist in differentiated macrophage exposed to SDF-1,I-TAC or TC14012 under the treatment of pioglitazone(24 hours). As predicted,PPARγ antagonist T0070907 abolished pioglitazone-inhibited macrophage chemotaxis, whereas PPARγ agonist rosiglitazone enhanced pioglitazone-mediated macrophage chemotaxis inhibition.7. Cell viability was evaluated by CCK8,our data showed that the cell viability of macrophage was not affected by pioglitazone concentration from 0.01 μ M to10μM.Meanwhile, Several macrophage differentiation markers expressions includingM1 markers such as MCP-1 and IL-6, as well as M2 markers such as chemokine(C-C motif) ligand18(CCL18) and IL-10 were analyzed. As shown that, both the m RNA and protein levels of MCP-1, IL-6, CCL18, and IL-10 were not influenced by pioglitazone treatment.In vivo results1. We used real-time PCR analysis in RNA samples isolated from atherosclerotic lesions from pioglitazone or non-TZDs-treated patients was performed.Baseline characters of patients from the two cohorts were comparable. Median time of pioglitazone medication was 2 months. Intriguingly, pioglitazone treatment significantly reduced CXCR7 、 SDF-1 and I-TAC expression in plaques of pioglitazone-treated patients when compared with non-TZDs-treated patients. On the contrary, the expression of PPARγ was markedly increased in plaques of pioglitazone-treated patients.2. Serum concentration of pioglitazone negatively correlated with CXCR7 expression levels in carotid atherosclerotic lesions, suggesting that pioglitazone is capable of suppressing CXCR7 expression in vivo.CONCLUSION1. Pioglitazone inhibits CXCR7 expression in differentiated macrophage via PPARγ but not PPARαactivation.2. Downregulation of CXCR7 is responsible for macrophage chemotaxis inhibition by pioglitazone.3. Pioglitazone-mediated CXCR7 suppression and macrophage chemotaxis inhibition are not related to cell viability or differentiation.4. Pioglitazone therapy-induced PPAR γ activation suppresses CXCR7 expression in human carotid atherosclerotic lesions.