Interleukin‐1β augments the angiogenesis of endothelial progenitor cells in an NF‐κB/CXCR7‐dependent manner

Abstract Endothelial progenitor cells (EPCs) are able to trigger angiogenesis, and pro‐inflammatory cytokines have beneficial effects on angiogenesis under physiological and pathological conditions. C‐X‐C chemokine receptor type 7 (CXCR‐7), receptor for stromal cell‐derived factor‐1, plays a critical role in enhancing EPC angiogenic function. Here, we examined whether CXCR7 mediates the pro‐angiogenic effects of the inflammatory cytokine interleukin‐1β (IL‐1β) in EPCs. EPCs were isolated by density gradient centrifugation and angiogenic capability was evaluated in vitro by Matrigel capillary formation assay and fibrin gel bead assay. IL‐1β elevated CXCR7 expression at both the transcriptional and translational levels in a dose‐ and time‐dependent manner, and blockade of the nuclear translocation of NF‐κB dramatically attenuated the IL‐1β‐mediated up‐regulation of CXCR7 expression. IL‐1β stimulation significantly promoted EPCs tube formation and this effect was largely impaired by CXCR7‐siRNA transfection. IL‐1β treatment stimulated extracellular signal‐regulated kinase 1/2 (Erk1/2) phosphorylation, and inhibition of Erk1/2 phosphorylation partially impaired IL‐1β‐induced tube formation of EPCs but without significant effects on CXCR7 expression. Moreover, blocking NF‐κB had no significant effects on IL‐1β‐stimulated Erk1/2 phosphorylation. These findings indicate that CXCR7 plays an important role in the IL‐1β‐enhanced angiogenic capability of EPCs and antagonizing CXCR7 is a potential strategy for inhibiting angiogenesis under inflammatory conditions.


| INTRODUC TI ON
Endothelial progenitor cells (EPCs)are a kind of endothelial precursor cells that have the potential to differentiate into a mature endothelial cell and contribute to endothelial generation and vessel repair in ischaemic tissues and injured blood vessel endothelium, respectively. 1 EPCs control the angiogenic switch of many physiological and pathologic processes, such as the neovascularization of ischaemic tissues 2-4 and tumorigenesis. 5 Local or systemic transplantation of EPCs derived from bone marrow, 6 peripheral blood 7 or cord blood 8 can enhance ischaemic angiogenesis and improve the function of ischaemic tissues in animals with limb or myocardial ischaemia.
C-X-C chemokine receptor type 7 (CXCR-7) is a seven-transmembrane G-protein-coupled receptor that is widely expressed in the haematopoietic system, 9 cardiac microvessels, brain, 10 kidney 11 and several tumour cell lines. [12][13][14] CXCR7 is a novel alternative receptor for stromal cell-derived factor 1 (SDF-1) and has an approximately 10 times higher binding affinity with SDF-1 than CXCR4. 15 CXCR7 knockout mice died at birth because of ventricular septal defects and semilunar heart valve malformation, and this phenotype can be recapitulated in mice with an endothelium-specific deletion of CXCR7, which indicates that CXCR7 is essential for valve formation, vessel protection, endothelial cell growth and survival. 16 In addition, CXCR7 has been reported to be a co-receptor for human and simian immunodeficiency viruses 17 and to be related to memory B cell differentiation. 18 This receptor also uniquely mediates SDF-1-induced renal progenitor cell survival and adhesion to endothelial cells. 19 CXCR7 can also shape the distribution of the chemokine SDF-1 in the environment by acting as a decoy receptor in zebrafish posterior lateral line development. 20,21 Moreover, CXCR7 was reported to enhance tumour development 13,[22][23][24][25] and metastasis 22,26 and to be up-regulated in tumour-associated vessels, 12 indicating a potential role in tumour angiogenesis. Our previous studies 27,28 also demonstrated that CXCR7 plays a critical role in the SDF-1 promotion of EPC-mediated angiogenesis. CXCR7 mediates EPC adhesion, trans-endothelial migration and tube formation induced by SDF-1 and CXCR4, and exclusively mediates EPC survival. In addition, CXCR7 is able to enhance vascular endothelial growth factor (VEGF) expression levels in an SDF-1-independent manner. 29 All these facts show that CXCR7 plays a critical role in EPC-mediated angiogenesis.
Interleukin-1β (IL-1β) is a potent immunoregulatory and pro-inflammatory cytokine secreted by a variety of activated immune cells. IL-1β can infiltrate solid tumours and has been shown to be a pro-angiogenic factor in solid tumours. 30 IL-1β can up-regulate VEGF expression in tumour cells and augment angiopoietin-1 expression in human endothelial cells. 31 Moreover, Rosell et al 32 found that IL-1β augments the angiogenic responses of murine EPCs in vitro in an Erk1/2-dependent manner. However, inhibiting the Erk1/2 pathway can only partly suppress EPC tube formation. 32 This fact indicates that there might be some other mechanism involved in the IL-1β-mediated angiogenic capability of EPCs. In the present study, we investigated the role of CXCR7 in the IL-1β-promoted angiogenic capability of EPCs.
RNA Extraction Kit (Bioteke), High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), SYBR Green PCR Master in the IL-1β-enhanced angiogenic capability of EPCs and antagonizing CXCR7 is a potential strategy for inhibiting angiogenesis under inflammatory conditions.

| Human cord blood EPC isolation and identification
Human umbilical cord blood samples (20-40 mL) from four healthy newborns (three boys and one girl; gestational age range, University approved all protocols and informed consent was obtained from all the parents of the newborns. EPCs were isolated from human cord blood as previously described. 27 In brief, cord blood FBS. The plate was incubated at 37°C in a humidified environment with 5% CO 2 . After 24 hours, the unattached cells and debris were removed by washing with medium. The medium was changed daily for 7 days and thereafter on alternate days. At day 21, EPCs were characterized using acetylated low-density lipoprotein uptake and a lectin binding assay. First, cells were incubated with DiI-acetylated low-density lipoprotein (Dil-acLDL, final concentration 10 mg/mL) at 37°C for 4 hours and then fixed with 3% paraformaldehyde for 10 minutes. After two washes with phosphate-buffered saline (PBS), the cells were then incubated with Ulex europaeus agglutinin-1 (UEA-1, final concentration 10 mg/mL) for 1 hour. After staining, pictures were taken with a fluorescence microscope (Olympus IX71, Olympus). Double-positive-stained cells were identified as differentiating EPCs. EPCs were further identified by CD133 and vascular endothelial growth factor receptor 2 (VEGFR-2) expression using immunofluorescent staining. In this assay, mouse anti-CD133 antibody and rabbit polyclonal antibody against VEGFR-2 were used. Briefly, EPCs were cultured in 24-well plates to 70%-90% confluence. Lipofectamine 3000 (1.5 μL) and 20 pmol siRNA (CXCR7 siRNA or Ctrl siRNA) were diluted with 25 mL of Opti-MEM, respectively. Diluted Lipofectamine 3000 was added to the siRNA solution and incubated for 5 minutes at room temperature. Next, the siRNA-Lipofectamine mixture was added to the EPCs and incubated for 2 days, and then, the CXCR7 mRNA expression level was evaluated by qRT-PCR. Transfected EPCs were used for the tube formation assay within 1 week after transfection. The membranes were incubated overnight at 4°C with rabbit anti-CXCR7 antibody or rabbit anti-phosphorylated-Erk1/2 after blocking in 5% skim milk for 1 hour. Then, the membranes were washed with Tris-buffered saline with Tween-20 (TBST) three times and incubated with the corresponding HRP-conjugated secondary antibody at room temperature for 1 hour. After three washes with TBST, the bands were visualized using ECL and detected by a Western blot imaging system (Tanon). For total-Erk1/2 detection, the same membrane that was used for phosphorylated Erk1/2 detection was washed with stripping buffer (Signagen Laboratories) for 10 minutes, blocked with 5% skim milk and incubated with an anti-Erk1/2 antibody following the same procedure described above.

| Detecting
To detect the nuclear content of NF-κB and histone H3, the nuclear protein from EPCs was extracted using a nucleoprotein extraction kit (Beyotime) according to the manufacturer's instructions.
The nuclear protein concentration was measured by a BCA Protein Assay Kit (Beyotime).

| Detecting CXCR7 mRNA expression by realtime PCR
Total mRNA was extracted from each different group of EPCs using an RNA Extraction Kit, and the concentration was determined with a Nanodrop2000 (Thermo Scientific). Then, the mRNA was reverse transcribed to cDNA with a High-Capacity cDNA

| Matrigel capillary formation assay
The Matrigel capillary formation assay was performed as previously described. 3

| Fibrin gel bead assay
The tube formation capability of EPCs was evaluated by fibrin gel bead assay according to the protocol of Nakatsu et al 33

| Statistical analysis
Results were obtained from at least three independent experiments and are presented as the means ± SD. Statistical analyses were performed using Original 9.0 software (Original Lab) with Student's t test or one-way ANOVA, followed by post hoc multiple comparisons with the Scheffe test. Statistical significance was set at P < .05.

| Identification of human cord blood EPCs
Endothelial progenitor cells isolated from human cord blood were cultured with EGM-2 in plates coated with fibronectin. Typical cell clusters appeared at day 7, and colonies emerged after 21 days of culture ( Figure 1A). Acetylated low-density lipoprotein uptake and the lectin binding assay showed that these cells can uptake DiI-acLDL and bind with UEA-1 ( Figure 1B), indicating an endothelial capability. Moreover, immunofluorescent staining demonstrated that most of the cells were positive for both CD133 and VEGFR2 ( Figure 1C), which further confirmed that those isolated cells were EPCs. Cells from passages 2-4 were used for the following experiments. IL-1β treatment on CXCR7 expression level. EPCs were treated with 10 ng/mL IL-1β for 0, 0.5, 3, 5, 12 or 24 hours, and CXCR7 expression level was detected by real-time PCR, Western blot and flow cytometry. We found that IL-1β treatment elevated CXCR7 expression level in a time-dependent manner, reaching the peak at 3 hours and slightly regressing thereafter ( Figure 2D-F). In addition, we detected the timecourse regression of CXCR7 expression level after IL-1β deprivation.

| IL-1β up-regulates CXCR7 expression in EPCs
EPCs were treated with IL-1β for 5 hours; then, IL-1β was removed, and CXCR7 expression was detected by flow cytometry at 0, 1, 3 and 12 hours later. The results showed that the CXCR7 expression level in EPCs was fairly maintained for 1 hour after deprivation of IL-1β and then gradually regressed to baseline level within 12 hours ( Figure 2G).

| IL-1β promotes capillary and tube formation in EPCs in a CXCR7-dependent manner
Considering the critical role of CXCR7 in EPC-induced angiogenesis, 27 we further investigated the role of CXCR7 in the IL-1β-mediated angiogenic capability of EPCs via Matrigel capillary formation assay and fibrin gel bead assay. For this purpose, CXCR7 was knocked down via CXCR7 siRNA transfection ( Figure 3A). The Matrigel capillary formation assay showed that EPCs treated with IL-1β formed longer capillaries than control EPCs, and siRNA interference with CXCR7 impaired the tube formation capability of EPCs under basal conditions and blocked IL-1β-stimulated tube formation ( Figure 3B,C), indicating that CXCR7 plays a critical role in EPC tube formation in the presence and absence of IL-1β stimulation. The fibrin gel bead assay showed a similar profile ( Figure 3D,E). IL-1β-treated EPCs formed a greater number of tubelike structures that were also longer and with branching, while CXCR7 knockdown substantially impaired tube-like structure formation in the presence and absence of IL-1β, which indicated that CXCR7 is involved in the enhanced tube formation capability of EPCs under IL-1β treatment.

| Inhibition of NF-κB reduces IL-1β-mediated CXCR7 up-regulation in EPCs
After confirming the role of CXCR7 in IL-1β-enhanced EPC tube formation, we further investigated the mechanism of how IL-1β F I G U R E 3 CXCR7 siRNA transfection impairs EPC tube formation induced by IL-1β. CXCR7 mRNA expression in EPCs transfected with different siRNAs was detected by real-time PCR (A). The effects of siRNA transfection on the capillary formation capability of EPCs with or without 10 ng/mL IL-1β treatment were evaluated by Matrigel capillary formation assay (B), and the relative tube length was evaluated using ImageJ software (C). The effects of siRNA transfection on the tube formation capability of EPCs with or without 10 ng/mL IL-1β treatment were evaluated by fibrin gel bead assay (D), and the relative tube length was also evaluated (E). Data shown in the graphs represent three independent experiments. * P < .05, vs Control (Ctrl); # P < .05, vs EPCs with control siRNA treatment; & P < .05, vs EPCs with CXCR7 siRNA treatment. Scale bar represents 100 μm up-regulates CXCR7 expression. NF-κB is one of the typical downstream signalling molecules of IL-1β. 34 Therefore, we detected the direct effect of IL-β on NF-κB activation in EPCs. The results showed that IL-1β stimulation significantly increased NF-κB translocation from the cytoplasm to the nucleus, indicating that IL-1β treatment induced NF-κB activation in EPCs ( Figure 4A). To determine the role of NF-κB in IL-1β-enhanced CXCR7 expression in EPCs, BAY 11-7082, a specific inhibitor of the nuclear translocation of NF-κB, was used to investigate the direct link between IL-1β stimulation, NF-κB and CXCR7 up-regulation. BAY11-7082 (10 μmol/L) treatment had no significant effects on nuclear NF-κB expression under basal conditions (0.85 ± 0.06 vs 1 ± 0.009, P > .05) but significantly inhibited IL-1βstimulated NF-κB nuclear translocation (1.40 ± 0.03 vs 2.10 ± 0.14, P < .05, Figure 4B). Under the same experimental conditions, BAY11-7082 treatment almost completely abolished the IL-1β-mediated upregulation of CXCR7 at the mRNA level (1.19 ± 0.19 vs 11.86 ± 0.27, P < .05, Figure 4C), and significantly attenuated IL-1β-mediated CXCR7 up-regulation on the cell surface of EPCs ( Figure 4D). These results indicate that IL-1β-stimulated CXCR7 up-regulation is at least partially mediated by IL-1β-stimulated NF-κB nuclear translocation.

| Erk1/2 is involved in IL-1β-induced tube formation of EPCs independent of the NF-κB/ CXCR7 pathway
Erk1/2 is another downstream signal mediator of IL-1β and has been demonstrated to mediate IL-1β-stimulated angiogenesis in mouse EPCs in vitro. 32 Herein, we further investigated whether Erk1/2 is involved in IL-1β-induced capillary formation and CXCR7 up-regulation by using the Erk1/2 inhibitor U0126 and Erk siRNA transfection. Results showed that U0126 (10 μmol/L) treatment significantly impaired IL-1β-promoted EPCs capillary formation ( Figure 5A,B).
This result indicated that Erk1/2 is also involved in IL-1β-induced tube formation in EPCs, which was confirmed by the fibrin gel bead assay ( Figure 5C,D). Moreover, siRNA transfection experiments also reached similar conclusions. Erk1/2 knockdown by Erk1-and Erk2-siRNA transfection ( Figure 5E) impaired the tube-structure formation capability of EPCs in the presence or absence of IL-1β ( Figure 5F,G).

| D ISCUSS I ON
It is well-established that inflammation and angiogenesis are interdependent. 35 37 As a typical inflammatory factor, IL-1β has also been found to be involved in angiogenesis. 38 IL-1β can up-regulate VEGF expression in tumour cells 39 and angiopoietin-1 expression in human endothelial cells. 31 In this study, we demonstrated that IL-1β promotes the angiogenic capability of EPCs via the NF-κB/CXCR7 and Erk1/2 pathway.
Untreated EPCs only have a basal expression of CXCR7, but IL-1β can induce CXCR7 expression in a dose-and time-dependent manner ( Figure 2). CXCR7 knockdown impaired IL-1β-promoted EPC tube formation (Figure 3). These results indicate that CXCR7 plays a critical role in IL-1β-induced EPC angiogenesis, which is consistent with the findings of Watanabe, K. and colleagues in HUVECs. 40 NF-κB is a classical IL-1β-induced downstream signalling molecule that plays critical roles in IL-1β-induced angiogenesis in mesenchymal stromal cells 41 and angiogenic factor expression in temporomandibular disc cells. 34 Moreover, NF-κB and G protein-coupled receptors are closely connected in inflammation and tumorigenesis. 42 Herein, we found that the NF-κB inhibitor BAY 11-7082 can abolish IL-1βinduced CXCR7 expression in EPCs and even decreases the baseline CXCR7 mRNA expression level in EPCs ( Figure 4C). These results indicate that NF-κB plays a critical role in CXCR7 transcription induced Cancer is an angiogenesis-dependent disease, and anti-angiogenic drugs are considered to be potent in cancer therapy. 43 IL-1β can infiltrate and promote angiogenesis in solid tumours, 30 and the mitogen-activated protein kinase (MAPK) pathway also plays a principle role in tumour angiogenesis. 44 Rosell et al 32 found that IL-1β augmented the angiogenic response of murine EPCs in an Erk1/2-dependent manner, which is consistent with our results in this study ( Figure 5).
Moreover, we also demonstrated that NF-κB/CXCR7 and Erk1/2 are two independent pathways mediating EPC angiogenesis induced by IL-1β, which is consistent with the research of Hartmann. 45 The MAPK pathway is an important target for inhibiting tumour progression and angiogenesis, 32 and our results in the present study suggest that antagonizing CXCR7 might be a potent complementary strategy for inhibiting MAPK during anti-angiogenic therapy in cancer.
In some other pathological conditions, such as diabetes, angiogenesis is impaired and improving EPC function has been considered as an effective approach to ameliorate angiogenesis. 46 Our recent study 4 showed that enhancing CXCR7 expression in EPCs via lentiviral transfection can promote EPC angiogenic capacity and ameliorate blood perfusion in diabetic ischaemic hindlimbs.
Considering the potential biosafety risk in lentiviral transfection, finding a safer way to up-regulate CXCR7 expression in EPCs would be beneficial for ischaemic vascular diseases. In the present study, IL-1β was found to substantially up-regulate CXCR7 expression in EPCs in a NF-κB-dependent manner (Figures 2 and   4). In addition to IL-1β, another pro-inflammatory cytokine, lipopolysaccharide (LPS), was also found to be able to increase proliferation, migration and tube formation of choroidal endothelial cells through TLR4/NF-κB-mediated up-regulation of CXCR4 and CXCR7. 47 These results reveal a potential way to enhance CXCR7 expression in EPCs and may provide some enlightenment for the further development of CXCR7 agonists.