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Cell Biology International (2011) 35, 201–208 (Printed in Great Britain)
CXCL8 enhances the angiogenic activity of umbilical cord blood-derived outgrowth endothelial cells in vitro
Takashi Kimura*†, Hirao Kohno*, Yoshikazu Matsuoka*, Mari Murakami*†, Ryusuke Nakatsuka*, Makoto Hase*, Katsuhiko Yasuda‡, Yasushi Uemura*, Yutaka Sasaki*, Shirou Fukuhara† and Yoshiaki Sonoda*1
*Department of Stem Cell Biology and Regenerative Medicine, Graduate School of Medical Science, Kansai Medical University, Moriguchi, Osaka 5708506, Japan, †First Department of Internal Medicine, Kansai Medical University, Moriguchi, Osaka 5708506, Japan, and ‡Department of Obstetrics and Gynecology, Kansai Medical University, Moriguchi, Osaka 5708506, Japan


OECs (outgrowth endothelial cells), also known as late-EPCs (late-endothelial progenitor cells), have a high proliferation potential in addition to in vitro tube formation capability. In ischaemic animal models, injected OECs were integrated into regenerating blood vessels and improved neovascularization. Previous reports have demonstrated the expression of CXCL8 to be up-regulated in ischaemic tissues. It has also been documented that CXCL8 stimulates the angiogenic activity of mature ECs (endothelial cells). Therefore, it has been suggested that CXCL8 plays an important role in neovascularization in ischaemic tissues. However, it is still uncertain whether CXCL8 also stimulates the angiogenic activity of OECs. This study evaluated the effects of CXCL8 on the angiogenic activity of OECs in vitro. OECs were isolated from human UCB (umbilical cord blood)-derived mononuclear cells. Phenotypes of the OECs were assessed by flow cytometry, immunostaining, and real-time RT (reverse transcription)-PCR. The effects of CXCL8 on OECs were investigated by transwell migration assay and capillary tube formation assay on Matrigel. The OEC clones isolated from UCB expressed OEC phenotypes. In addition, CXCL8 receptors (CXCR1 and CXCR2) were expressed on these OEC clones. CXCL8 significantly stimulated the transwell migration and capillary tube formation of OECs. Neutralizing antibody against CXCR2, but not CXCR1, abolished a transwell migration of OECs induced by CXCL8, suggesting the involvement of CXCL8/CXCR2 axis in transwell migration. These results demonstrate that CXCL8 stimulates the angiogenic activity of UCB-derived OECs in vitro.


Key words: CXCR2, CXCL8, outgrowth endothelial cell, transwell migration assay, tube formation assay, vasculogenesis

Abbreviations: bFGF, basic fibroblast growth factor, DiI-Ac-LDL, DiI-acetylated low-density lipoprotein, EBM-2, endothelial cell basal medium-2, ECs, endothelial cells, late EPCs, late endothelial progenitor cells, FBS, fetal bovine serum, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, GFR, growth factor-reduced, HMECs, human microvascular endothelial cells, HUVECs, human umbilical vein endothelial cells, IL-6, interleukin-6, KDR, kinase domain receptor, LDL, low-density lipoprotein, MNCs, mononuclear cells, OECs, outgrowth endothelial cells, RT, reverse transcription, SCF, stem cell factor, SDF-1, stromal cell-derived factor-1, UCB, umbilical cord blood, UEA-1, Ulex europaeus agglutinin-1, VEGF, vascular endothelial growth factor

1To whom correspondence should be addressed (email sonoda@takii.kmu.ac.jp).


1. Introduction

Postnatal neovascularization is an important process in ameliorating tissue damage caused by critical ischaemia (Asahara et al., 2004). The stimulation of vessel growth (neovasculogenesis) and capillary sprouting (angiogenesis) by infusion of circulating bone marrow-derived EPCs (endothelial progenitor cells) has been demonstrated in various animal models of tissue ischaemia (Takahashi et al., 1999; Kalka et al., 2000; Urbich et al., 2003). These EPCs consist of at least two different subpopulations, termed early and late EPCs (Hur et al., 2004). Although both types of EPCs are derived from bone marrow-, cord blood-, and peripheral blood-derived MNCs (mononuclear cells) and express endothelial cell markers, they display different morphologies and growth patterns (Hur et al., 2004; Yoon et al., 2005). Early EPCs exhibit a spindle-like morphology, while late EPCs exhibit a cobblestone-like morphology (Hur et al., 2004). Late EPCs show a late outgrowth potential and are thereby known as OECs (outgrowth endothelial cells). The capillary network formation of early and late EPCs on Matrigel showed a marked difference. While early EPCs failed to form capillary-like structures, OECs successfully displayed tube formation on Matrigel (Hur et al., 2004; Duan et al., 2006). Injected OECs have been shown to integrate into regenerating blood vessels and improve neovascularization of ischaemic hind limbs and ischaemic hearts in animal models (Asahara et al., 1997; Urbich et al., 2004).

Angiogenesis is a multistep process which includes EC (endothelial cell) proliferation, migration and tube formation, which is mediated by various angiogenic factors such as VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor) and CXCL8 (Keeley et al., 2008). CXCL8 is a member of the ELR (glutamic acid-leucine-arginine) motif positive (ELR+) CXC chemokines (Strieter et al., 1995). CXCL8 is expressed in leucocytes, fibroblasts, endothelial cells, and various tumour cells and plays an important role in chemoattraction, inflammation, angiogenesis, tumour growth and metastasis (Koch et al., 1992). The expression levels of angiogenic factors such as VEGF and CXCL8 are up-regulated in tissues after ischaemic injury (Kukielka et al., 1995; Lee et al., 2000). Kocher et al. (2006) suggested that CXCL8 produced after myocardial infarction plays a role in the migration of angioblasts to ischemic cardiac tissue.

Mature ECs produce CXCL8, and the expression of CXCL8 in ECs increases under hypoxic culture conditions (Karakurum et al., 1994). The receptors for CXCL8 are CXCR1 and CXCR2, which bind to CXCL8 with high affinity (Rossi and Zlotnik, 2000). These CXCL8 receptors have been observed on mature ECs (Salcedo et al., 2000; Hristov et al., 2007). CXCL8 directly modulates the proliferation and migration of mature ECs and regulates angiogenesis in vitro and in vivo (Li et al., 2003, 2005). These studies suggested that CXCL8 plays a paracrine role in angiogenesis. Recently, Li et al. (2005) demonstrated that CXCL8, produced by mature ECs, directly regulates the EC function and activates the proliferation and migration of ECs in an autocrine manner. However, He et al. (2005) reported a significantly higher amount of CXCL8 to be released by human OECs than by mature ECs. They also demonstrated that a conditioned medium of OECs stimulated the proliferation of mature ECs, thus providing evidence that CXCL8 is an important mediator of the paracrine mitogenic effects of OECs. However, it is still uncertain whether CXCL8, released from mature ECs and OECs, affects the functions of OECs in both a paracrine and an autocrine manner.

This study used in vitro assay to determine whether OECs express CXCL8 receptors in order to elucidate the angiogenic effects of CXCL8 on OECs including transmigration and tube formation on Matrigel matrix. Our data demonstrated that OECs expressed CXCR1 and CXCR2, and CXCL8 enhanced the transmigration and tube formation of OECs. Furthermore, neutralizing antibody against CXCR2, but not CXCR1, significantly inhibited the augmentation of OEC transmigration by CXCL8. These results indicated that the enhancement of OEC transmigration by CXCL8 is primarily mediated by CXCR2.

2. Materials and methods

2.1. Isolation and culture of OECs

UCB (human umbilical cord blood) samples obtained from normal full-term deliveries with signed informed consent were kindly supplied from the Keihan Cord Blood Bank under approval by the institutional review board of Kansai Medical University. MNCs were prepared from UCB samples, and then, OECs were isolated. Frozen UCB nucleated cells were thawed and suspended in alpha medium with 5% FBS (fetal bovine serum) (HyClone) and were incubated for about 12 h. After incubation, UCB MNCs were isolated using Ficoll Paque Plus (GE Healthcare) density gradient centrifugation. The MNCs were washed twice with PBS(−) (Nacalai Tesque) and resuspended in EGM-2 (endothelial growth medium) (EGM-2 Bullet kit, Lonza), which was composed of EBM-2 (endothelial cell basal medium-2), 2% FBS and growth factors, including VEGF, bFGF and IGF-1. About 2×107 MNCs per well were seeded in a six-well plate coated with rat tail collagen type I (Becton Dickinson Biosciences) and were cultured at 37°C in a fully humidified atmosphere of air flushed with 5% CO2. The first medium change was performed after 2 days of culture, and then, medium change was performed every day. After OEC colonies grew, the cells in colonies were trypsinized and seeded onto a plate coated with rat tail collagen type I and named passage 1. All the following experiments were performed using established OECs within 7 to 20 passages. HUVECs (human umbilical vein endothelial cells) were purchased from KURABO and were used as a control.

2.2. Acetyl-LDL (low-density lipoprotein) uptake and lectin binding

The aforementioned OECs were incubated in a medium containing 5 μg/ml DiI-Ac-LDL (DiI-acetylated low-density lipoprotein) (Biomedical Technologies, Inc.) for 4 h at 37°C. After washing with PBS(–) twice, the cells were reacted with 100 μg/ml FITC-labelled UEA-1 (Ulex europaeus agglutinin-1) lectin (EY Laboratories, Inc.) at room temperature for 15 min. After washing twice with PBS(−), the cells were reacted with 15 μg/ml of Hoechst 33342 (Invitrogen) and were subsequently enclosed with PBS(−) with 50% glycerol in a slide glass. The cells were examined under a fluorescence microscope (BX50, Olympus).

2.3. Flow cytometric analysis

OECs were trypsinized and washed twice with PBS(−). Single cell suspensions were stained with anti-CD31-FITC (Beckman Coulter), anti-CD45-FITC (Beckman Coulter), anti-CD34-FITC (BD Immunocytometry), anti-CXCR2-FITC (Santa Cruz Biotechnology) and anti-CD14-PE (Beckman Coulter) monoclonal antibodies. For the detection of KDR (kinase domain receptor), cells were incubated with biotinylated anti-human KDR (Sigma–Aldrich) and then were subsequently incubated with streptavidin-FITC (DAKO). All flow cytometric analyses were performed using a FACS Calibur (BD Biosciences), as reported (Wang et al., 2003; Kimura et al., 2007).

2.4. Real-time RT (reverse transcription)-PCR analysis

Real-time quantitative RT-PCR analysis of OECs and HUVECs was performed to determine the expression of EPC markers and chemokine receptors. Total RNAs were isolated from OECs and HUVECs using RNeasy Plus Micro kit (Qiagen) according to the manufacturer's instructions. Subsequently, total RNAs were reverse transcribed using the iScript cDNA Synthesis Kit (BioRad Laboratories, Inc.) according to the manufacturer's instructions. The relative expression level of the housekeeping gene, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), was used to normalize the expression of the target genes. Real-time RT-PCR using the DNA Engine Opticon2 (MJ Research) was performed with the cDNA sample, using the iQ SYBR Green Supermix (BioRad Laboratories, Inc.). Primers used for PCR are given (Table 1). After normalization of gene expression with GAPDH, the gene expressions of OECs were compared with those of HUVECs.


Table 1 Primer pairs used for quantitative real time RT-PCR

Gene name Strand Primer sequences Product length (bp)
PAI-1 Forward 5′-GGGCCATGGAACAAGGATGA-3′ 216
Reverse 5′-CTCCTTTCCCAAGCAAGTTG-3′
Tie-2 Forward 5′-GTGACCCCTCCCAGAATCTCA-3′ 194
Reverse 5′-ACTGCACAGCTGGTTCTTCC-3′
vWF Forward 5′-TCTGTGGATTCAGTGGATGCA-3′ 84
Reverse 5′-CGTAGCGATCTCCAATTCCAA-3′
VE/cadherin Forward 5′-TGTGGGCTCTCTGTTTGTTG-3′ 252
Reverse 5′-AATGACCTGGGCTCTGTTTC-3′
CXCR1 Forward 5′-CTGAGCCCCAAGTGGAACGAGACA-3′ 151
Reverse 5′-GCACGGAACAGAAGCTTTATTAGGA-3′
CXCR2 Forward 5′-CAATGAATGAATGAATGGCTAAG-3′ 117
Reverse 5′-AAAGTTTTCAAGGTTCGTCCGTGTT-3′
CXCR4 Forward 5′-ATCCCTGCCCTCCTGCTGACTATTC-3′ 230
Reverse 5′-GAGGGCCTTGCGCTTCTGGTG-3′
GAPDH Forward 5′-ACCACAGTCCATGCCATCAC-3′ 451
Reverse 5′-TCCACCACCCTGTTGCTGTA-3′



2.5. OEC migration assay

Cell migration was assayed using a 24-well transwell cell culture chamber (BD Falcon) with 8.0-μm pore PET filter inserts (BD Falcon). OECs (4×104 cells/well) suspended in EBM-2 medium containing 1% FBS were applied to the filter inserts. Various concentrations of test cytokines were added to the same medium in the lower chamber. Twenty-four hours after incubation at 37°C in a 5% CO2 incubator, cells on the upper side of the filter were scraped, and the migratory cells on the lower side were fixed, stained with haematoxylin, and examined under an inverted microscope (×40) (CK2, Olympus). Cells migrating to the lower side of the filters were quantified by counting the stained cells in four random areas per well.

In migration inhibition studies, OECs (4×104 cells/well) were incubated with neutralizing anti-CXCR1 antibody (10 μg/ml, R&D Systems), anti-CXCR2 antibody (20 μg/ml, R&D Systems) or both anti-CXCR1 antibody and anti-CXCR2 antibody for 30 min at 37°C. Thereafter, a migration assay was performed with CXCL8 (200 ng/ml) in the presence of neutralizing antibodies.

2.6. Tube formation assay

Tube formation was assayed on GFR (growth factor-reduced)-Matrigel (BD Biosciences). For reconstitution of a basement membrane, GFR-Matrigel was diluted 2-fold with cold EBM-2 (without FBS) and added to 48-well tissue culture plates (250 μl/well) at 4°C. The 48-well plates were incubated for 1 h at 37°C in a 5% CO2 incubator to allow the Matrigel to solidify. Cultured OECs were trypsinized, counted and resuspended in basal medium (EBM-2). These OECs were deposited on top of the reconstructed basement membrane (4×104 cells/well) in the absence or presence of various cytokines. The plates were incubated for 6 h to allow the formation of tube-like structures. Pictures of eight random fields/well were taken under an inverted microscope (×40). We quantified the total tube length in each well using ImageJ, an image analysis software program developed by the NIH. The number of branches was counted directly using the pictures of eight random fields/well. Each condition was assessed in triplicate.

2.7. Statistical analysis

The data are presented as the mean and S.D. One-way ANOVA (analysis of variance) was used to analyse the data, followed by a Dunnet test for pairwise comparison between the control and each of samples. A random probability value <0.05 was considered statistically significant.

3. Results

3.1. Isolation and characterization of OECs

MNCs from human UCB were seeded in a collagen-coated six-well plate with rat tail collagen type I (BD Biosciences). After 10 days of culture, colonies consisting of cobblestone-like cells appeared (Figure 1A). Each cell showed a polygonal structure and was approximately 50–100 μm in diameter. OECs isolated from a cobblestone-like colony could continuously proliferate and could be re-cultured >20 times (data not shown). These cells took up acetylated LDL and bound UEA-1 lectin (Figure 1B, a–d).

Flow cytometric analysis showed that OEC markers such as CD31, CD34 and KDR were expressed on the OECs. However, CD14 and CD45 antigens were not expressed on the OECs (Figure 2A). In addition, real-time RT-PCR analysis showed the expression of OEC markers such as PAI-1, Tie-2, vWF and VE/cadherin (Figure 2B). These results demonstrated that the isolated cobblestone-like cells had typical characteristics of OECs.

3.2. Stimulation of OEC migration by CXCL8

In order to determine whether CXCL8 stimulates OEC migration, the number of cells that migrated through an insert filter in a lower chamber filled with EBM-2 containing 1% FBS and testing factors was counted after 24 h. As previously reported (Asahara et al., 1999), VEGF strongly stimulated OEC migration (Figure 3A). Although the effects were lower than VEGF, CXCL8 significantly enhanced the migration of OECs (Figure 3B). SCF (stem cell factor), IL-6 (interleukin-6), and SDF-1 (stromal cell-derived factor-1) also significantly stimulated OEC migration (Figure 3B). CXCL8 stimulated OEC migration in a dose-dependent manner, with an optimal effect at 200 ng/ml (Figure 3C).

3.3. Expression of CXCL8 receptors on OECs and the inhibition of OEC migration by neutralizing antibodies to CXCR1 and CXCR2

RT-PCR analysis demonstrated that OECs express CXCL8 receptors (CXCR1 and CXCR2) and SDF-1 receptor (CXCR4) genes (Figure 4A). In addition, flow cytometric analysis demonstrated that OECs expressed CXCL8 receptor (CXCR2) (Figure 4B). Pretreatment of these OECs with the neutralizing antibody against CXCR2 significantly inhibited OEC migration, while the neutralizing antibody against CXCR1 had no effect (Figure 4C). These results indicate that CXCL8 stimulates OEC migration through CXCR2 expressed on OECs.

3.4. Promotional effects of CXCL8 on capillary tube formation by OECs

We subsequently examined whether CXCL8 promotes the formation of tube-like structures, such as branched structures and pseudotubes with enclosed areas, by OECs. OECs formed few tube-like structures on GFR-Matrigel when cultured in EBM-2 medium alone for 6 h (Figure 5A, a). However, they formed more tube-like structures when IL-6, VEGF or bFGF were added to the EBM-2 medium (Figure 5A, b, c, d). Interestingly, CXCL8 enhanced the formation of tube-like structures on GFR-Matrigel (Figure 5A, e). The degree of tube formation was also assessed by total tube length (Figure 5B) and number of branches (Figure 5C). Significant increases in total tube length and number of branches were observed when VEGF or bFGF were added. In addition, CXCL8 significantly augmented the total tube length and the number of branches. These angiogenic effects occurred from 4 h after the addition of these factors and persisted for up to 9 h.

4. Discussion

In order to clarify the angiogenic effects of CXCL8 on OECs, we first isolated a colony of OECs according to the culture method reported by Yoder et al. (2007) with slight modifications. These cells have common features of OECs such as a cobblestone-like colony appearance, uptake of acetylated LDL and binding of UEA-1 lectin (Figure 1). In addition, the isolated OECs expressed cell surface markers and genes (Figure 2) that are compatible with endothelial progenitor cells previously reported by other investigators (Urbich et al., 2004; Yoon et al., 2005). Furthermore, these OECs responded to the chemotactic factors in an in vitro transmigration assay (Figure 3) and formed tube-like structure on GFR-Matrigel (Figure 5).

The expression of CXCL8 receptors (CXCR1 and CXCR2) by cultured mature ECs is controversial. Petzelbauer et al. (1995) reported that they were unable to detect specific CXCL8 binding to cultured HUVECs or CXCL8 receptor mRNA expression by either cultured HUVECs or human dermal microvascular endothelial cells. Furthermore, the direct response of OECs to CXCL8 was not observed in their study. In contrast, Murdoch et al. (1999) demonstrated that HUVECs constitutively express CXCR1 and CXCR2 mRNAs. Salcedo et al. (2000) demonstrated differential expression and responsiveness of CXCR1 and CXCR2 by HMECs (human microvascular endothelial cells) and HUVECs. They reported that HMECs express more CXCL8 receptors than HUVECs. Li et al. (2003) demonstrated that both HUVECs and HMECs express CXCR1 and CXCR2 mRNA and protein.

Although there have been a limited number of studies on CXCL8 receptor expression on OECs, the results still remain controversial. Recently, Smadja et al. (2008) demonstrated CXCR1 and CXCR2 gene expression to be restricted to early EPCs. The present study clearly demonstrated the expression of CXCR1, CXCR2 and CXCR4 mRNAs in our isolated OECs (Figure 4A). In addition, the expression of CXCR2 was confirmed by flow cytometric analysis of OECs (Figure 4B). These results are consistent with those of Yoon et al. (2005) who reported that CXCR1 and CXCR2 mRNA was expressed on early EPCs as well as late EPCs which are similar to OECs.

CXCL8 has been reported to act as a growth and angiogenic factor in mature ECs (Heidemann et al., 2003). Previous studies have demonstrated that EC migration was inhibited by a neutralizing antibody to CXCR2 (Heidemann et al., 2003) and that vascularization was delayed in CXCR2 knockout mice (Devalaraja et al., 2000). In the present study, treatment of OECs with recombinant human CXCL8 resulted in a significant enhancement of cell migration (Figure 3) and capillary tube formation on GFR-Matrigel (Figure 5), thus indicating that CXCL8 exhibits similar angiogenic effects in OECs as observed in mature ECs. Li et al. (2005) observed a reduced migration of mature ECs treated with anti-CXCR2 antibody (but not anti-CXCR1 antibody) in comparison with the controls. Using neutralizing antibodies to CXCR1 and CXCR2, we demonstrated that transmigration of OECs towards CXCL8 was substantially decreased with anti-CXCR2 antibody but not by anti-CXCR1 antibody (Figure 4C), which indicated that the angiogenic effects of CXCL8 on OECs were primarily mediated by CXCR2.

It has been established that mature ECs produce CXCL8 (Karakurum et al., 1994; Ramjeesingh et al., 2003), thereby exerting their angiogenic activities in an autocrine manner (Li et al., 2005). Furthermore, recent studies have reported the production of CXCL8 by OECs, thus suggesting the paracrine role of CXCL8 on angiogenesis by mature ECs (He et al., 2005). These studies suggest that proliferation and angiogenic functions of mature ECs could be modulated by CXCL8 in both an autocrine and a paracrine manner. In the present study, we demonstrated that the angiogenic activity of OECs were significantly enhanced by CXCL8 in vitro. These results indicate that OECs recruited to damaged tissues after ischaemia interact with mature ECs through the CXCL8/CXCR2 axis, which promotes angiogenesis.

In summary, we have demonstrated that (i) OECs express the CXCL8 receptor and (ii) CXCL8 can stimulate OEC migration and tube formation on Matrigel in vitro. These results provide new evidence that CXCL8 plays a significant role in angiogenesis by OECs.

Author contribution

Takashi Kimura performed most of the experiments, collected data and analysed and interpreted the data. Hirao Kohno performed experiments, collected, analysed and interpreted the data and wrote the manuscript. Yoshikazu Matsuoka performed the experiments, collected data and analysed and interpreted the data. Mari Murakami, Ryusuke Nakatsuka, Makoto Hase, Yasushi Uemura, Yutaka Sasaki and Shirou Fukuhara interpreted the data. Katsuhiko Yasuda provided the study materials. Yoshiaki Sonoda designed the research, analysed and interpreted the data and wrote the manuscript.

Acknowledgements

The authors are grateful to the Keihan Cord Blood Bank for providing the samples used in this study.

Funding

This work was supported by Grants-in-Aid for Scientific Research C (grant nos. 19591144 and 21591251) from the Ministry of Education, Science and Culture of Japan, a grant from Haiteku Research Center of the Ministry of Education, a grant from the Science Frontier Program of the Ministry of Education, a grant from the 21st Century Center of Excellence (COE) program of the Ministry of Education, a grant from Strategic Research Base Development program for private universities of the Ministry of Education, a grant from the Promotion and Mutual Aid Corporation for Private Schools of Japan, grants from Kansai Medical University (Research grants B and F), a grant from the Japan Leukemia Research Foundation, a grant from the Mitsubishi Pharma Research Foundation and a grant from the Takeda Science Foundation.

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Received 17 September 2009/8 August 2010; accepted 19 October 2010

Published as Cell Biology International Immediate Publication 19 October 2010, doi:10.1042/CBI20090225


© The Author(s) Journal compilation © 2011 Portland Press Limited


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)