PAR-1 Activation on Human Late Endothelial Progenitor Cells Enhances Angiogenesis In Vitro With Upregulation of the SDF-1/CXCR4 System
Objectives— The importance of PAR-1 in blood vessel development has been demonstrated in knockout mice. As endothelial progenitor cells (EPCs) are involved in postnatal vasculogenesis, we examined whether they express PAR-1 and whether stimulation by the peptide SFLLRN modulates their angiogenic properties.
Methods and Results— EPC expanded from human CD34+ cord blood cells expressed PAR-1. PAR-1 activation induced EPC proliferation in a concentration-dependent manner far more potently than that of human umbilical vein endothelial cells. PAR-1 activation also enhanced actin reorganization, promoting both spontaneous migration in a Boyden chamber assay and migration toward SDF-1 and VEGF. As shown by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), EPC stimulation by SFLLRN significantly enhanced the mRNA expression of SDF-1 and its receptor CXCR-4. PAR-1 activation also increased CXCR4 expression on EPC and induced SDF-1 secretion, leading to autocrine stimulation. PAR-1 stimulation by SFLLRN also increased the formation of capillary-like structures by EPC in Matrigel, and this effect was abrogated by anti-CXCR-4, anti-SDF-1, and MEK inhibitor pretreatment.
Conclusions— Human EPCs express functional PAR-1. PAR-1 activation promotes cell proliferation and CXCR4-dependent migration and differentiation, leading to a proangiogenic effect.
Evidence continues to accumulate on the importance of endothelial progenitor cells (EPCs) in neovascularization of ischemic tissues.1 EPC transplantation enhances vascular development by in situ differentiation and proliferation within ischemic organs.2 Isolated EPC that contribute to postnatal neovascularization are heterogeneous and display variable morphological growth characteristics. EPCs have been described both as spindle-shaped cells with limited proliferation capacity, and as cobblestone-shaped cells with high expansion capacity (also called late EPCs by Hur et al).3,4
Besides its contribution to hemostasis, thrombin is involved in angiogenesis. Mouse models with impaired thrombin generation display altered vascular development.5 The main thrombin receptor on vascular cells, PAR-1, is a protease-activated G protein-coupled receptor specifically cleaved by thrombin at its extracellular N-terminus. The amino-terminal sequence thereby unmasked acts as a tethered ligand, triggering a rapid response that can be reproduced by a specific hexapeptide (SFLLRN). PAR-1−/− knockout mice show partial embryonic lethality,6 pointing to a specific developmental role of PAR-1. Moreover, PAR-1 activation on mature endothelial cells regulates many aspects of endothelial cell biology, such as induction of vascular endothelial growth factor (VEGF) synthesis7 and upregulation of the main VEGF receptor VEGFR-2.8 The thrombin receptor-activating peptide SFLLRN, which acts as an agonist for PAR-1, was also reported to promote capillary network formation in an in vivo Matrigel plug model.9 Interestingly, the antiangiogenic properties of thalidomide have been linked to inhibition of PAR-1 gene expression.10 PAR-1 activation by thrombin promotes tumor progression and metastasis, both effects being related to new capillary formation.11 However, the mechanism underlying the proangiogenic effect of PAR-1 activation is unclear.
In this study we examined the expression and function of PAR-1 by human late EPCs expanded from cord blood CD34+ cells.
An expanded methods section is available online at http://atvb.ahajournals.org.
CD34+-derived EPC from cord blood were obtained by density gradient centrifugation with Histopaque 1077 (Sigma, St. Louis, Mo) as previously described.12 Isolated mononuclear cells were resuspended in endothelial growth medium-2 (EGM-2) (Clonetics) composed of endothelial cell basal medium-2 (EBM-2), 5% fetal bovine serum, and growth factors. Human endothelial cells (HUVECs) were isolated from human umbilical veins and maintained in EGM-2 medium. To assess cell surface antigen expression, we used fluorescence-activated cell sorter (FACS) analysis as previously described.12 PAR-1 expression on the EPC surface was quantified with a calibrator (Qifikit; Dako, Trappes, France) containing a mixture of 5 calibration beads coated with increasing densities of mouse IgG. Details of immunocytochemistry and confocal immunofluorescence staining are given in the supplementary data section (http://atvb.ahajournals.org).
To test the effect of PAR-1 activation on EPC, all the following experiments were performed after a culture period of 16 hours in unsupplemented EBM-2 medium. Cells were then activated with SFLLRN peptide from Stago Recherche (Gennevilliers, France). The MEK inhibitor PD98059 (Calbiochem) was added 15 minutes before the agonists. All assays were performed in triplicate.
Cell Survival Assay
EPC were activated in serum-free EBM-2 medium containing SFLLRN (50, 75, or 100 μmol/L). DNA synthesis was determined by measuring incorporation of 5′-[3H]-thymidine ([3H]-Tdr) (Amersham, Les Ulis, France) with a Betamatic counter (1900 CA Packard) during 4 hours. Results are expressed as the increase in thymidine incorporation over control (EBM-2 without SFLLRN). The values of the SFLLRN-treated samples were subsequently normalized such that the untreated control value was 1.
Cell Proliferation Assay
The effects of various concentrations of SFLLRN peptide or SDF-1 on EPC proliferation were examined by cell counting with a phase-contrast microscope or by measuring cell phosphatase activity based on the release of paranitrophenol (pNPP) (Sigma) at 405 nm (Fluostar optima; BMG labtech, Champigny Sur Marne, France) after 4 days of incubation. EPCs and HUVECs were activated in 10% fetal bovine serum (FBS) EBM-2 medium containing SFLLRN (25, 50, 75, 100, or 150 μmol/L).
Real-Time Quantitative RT-PCR
The theoretical and practical aspects of real-time quantitative RT-PCR on the ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems) are described in detail elsewhere,13 and the primer sequences are given in the supplementary data section.
Enzyme-Linked Immunosorbent Assay Measurement of Secreted SDF-1 and VEGF
Cells were incubated for 24 hours in EBM-2, 5% FBS at 37°C with 75 μmol/L SFLLRN. SDF-1 and VEGF concentrations were measured with Quantikine enzyme-linked immunosorbent assay kits (R&D systems).
In Vitro Capillary-Like Growth Assay
Cells were activated for 4 hours in EBM-2 medium containing 75 μmol/L SFLLRN. Cells were then seeded on Matrigel (3×104 cells/well) and cultured for 18 hours at 37°C with 5% CO2. Capillary-like structures were examined by phase-contrast microscopy and endothelial cell networks formed by EPC were quantified by computer-assisted analysis (VIDEOMET 5.4.0).
Cell Migration Assay
EPC migration was measured by using modified Boyden chambers (Costar, Avon, France) with 8-μm pore-size filters. EPC were seeded at a density of 5×104 per well in 200 μL of migration medium (EBM-2/1% SVF), and were allowed to migrate for 5 hours at 37°C. Recombinant human VEGF or SDF-1 (R&D systems) was diluted in EBM-2 medium supplemented with 1% FBS and placed in the lower chamber of the modified Boyden chamber, in a volume of 600 μL. When checkerboard analysis was used to evaluate chemotaxis and chemokinesis, 0, 1, 10, 100 ng/mL VEGF was added to the upper and/or lower chamber.
Data are shown as means±SD. Significant differences were identified by ANOVA followed by Fisher’s protected least-significant difference test. Intergroup comparisons of PAR-1 density on the EPC surface were based on the Mann and Whitney nonparametric test. All statistical tests were performed using the Stat View software package (SAS, Cary, NC). Differences with P<0.05 were considered significant.
Late EPCs Express PAR-1
When cultured in the presence of specific endothelial growth factors (EGM-2 medium), human cord blood CD34+ cells (purity 86.0±5.7%) yielded small colonies that appeared within 13.5 days (SD: 3.3 days, median: 14 days; 25 cultures). At confluence, EPC exhibited the cobblestone morphology and monolayer growth pattern typical of the endothelial lineage. The endothelial phenotype of expanded EPC (so-called late EPC)4 was further characterized by positive staining for acetylated low-density lipoprotein uptake, expression of endothelial markers such as Tie-2, von Willebrand factor, CD31, and VEGFR-2 (Figure I, available online at http://atvb.ahajournals.org). EPC retained a high proliferative potential and expressed CD133 with low mRNA levels during 40 days of expansion (Figure II, available online at http://atvb.ahajournals.org). Flow cytometry also showed the expression of the thrombin receptor PAR-1 on 96.0±1.4% of expanded EPC, 85.1±6.7% of which were also positive for CD34. Interestingly, 90.2±1.1% of freshly purified CD34+ cells also expressed PAR-1, pointing to early expression of this receptor (Figure IIIA, available online at http://atvb.ahajournals.org). EPC were expanded for 5 weeks after the first passage, corresponding to a total of 45 to 60 days of culture. Mean PAR-1 density measured by means of quantitative flow cytometry was 13 700 sites per cell, a value similar to that found on HUVECs (19 720 sites; P>0.1). PAR-1 density varied strongly among EPC colonies, but the median expression level remained constant throughout the 5-week expansion period (Figure IIIB).
PAR-1 Activation Promotes EPC Survival and Proliferation
Specific PAR-1 activation of EPC was induced by the peptide SFLLRN, which mimics thrombin activation without cleaving the receptor. To explore the effect of PAR-1 activation on EPC viability, the cells were deprived of serum and growth factors (EBM-2 medium) for 16 hours before adding SFLLRN. In these conditions, SFLLRN induced a concentration-dependent increase in late EPC proliferation, as quantified by 3H-thymidine incorporation, with a maximal effect between 20 and 30 days of culture (Figure 1A). Thus, all subsequent experiments were done within the first 30 days of culture. At optimal concentrations (75 and 100 μmol/L), SFLLRN induced markedly stronger 3H-thymidine incorporation by EPCs than by HUVECs (P=0.027 and 0.0009, respectively; Figure 1B). To investigate the involvement of extracellular signal regulated kinase (ERK) phosphorylation in EPC signaling and in the effect of SFLLRN on EPC proliferation, we used the MAPK kinase (MEK) inhibitor PD98059 to inhibit threonine and tyrosine phosphorylation on ERK1 and ERK2. Pretreatment of EPC with PD98059 (10 μmol/L) inhibited SFLLRN-induced EPC proliferation by 85% (Figure 1C). These results suggest that EPC proliferation can be triggered by PAR-1 activation in the absence of other specific growth factors, and that this effect is associated with ERK phosphorylation, as in HUVECs.14
As shown in Figure 1D, SFLLRN also increased the proliferation of late EPCs cultured in serum-containing medium, in a concentration-dependent manner, with a maximal effect at 75 and 100 μmol/L. EPC proliferation was significantly stronger than HUVEC proliferation (P=0.0045, 0.006 and 0.0003 for SFLLRN concentrations of 75, 100, and 150 μmol/L, respectively; Figure 1D). These results were confirmed by measuring pNPP release at optimal SFLLRN concentrations (75 and 100 μmol/L) (Figure 1E).
Effect of PAR-1 Activation on Proangiogenic Cytokine Gene Expression
To examine the transcriptional effect of PAR-1 activation, we used real-time quantitative RT-PCR to measure the mRNA levels of several angiogenic factors, including VEGF isoforms and SDF-1, and their receptors. EPCs contained low basal levels of SDF-1, CXCR-4, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR-1, VEGFR-2, VEGFR-3, and neuropilin-1 mRNA. SFLLRN induced a slight increase in VEGF-A isoform mRNA after 4 hours. No significant increase in the mRNA expression of the VEGF receptors or the co-receptor NRP-1 was observed (Table).
In contrast, SFLLRN markedly increased the mRNA expression of both SDF-1 and its receptor CXCR-4. The increase in SDF-1 mRNA was 12-fold after 4 hours of stimulation, and 7-fold after 8 hours. In parallel, the CXCR4 mRNA level increased significantly on PAR-1 stimulation, to reach a maximum of 4-fold at 8 hours. These effects were totally abrogated by the MEK inhibitor PD98059, suggesting that enhancement of SDF-1/CXCR-4 is dependent on the ERK pathway.
We then examined the effect of PAR-1 activation on SDF-1 and VEGF secretion by EPC. EPC supernatants were collected after incubation with SFLLRN for 24 hours, and cytokine levels were measured with enzyme-linked immunosorbent assay kits. SDF-1 release increased 2-fold at 75 μmol/L SFLLRN (1200±210 pg/106 versus 618±257 pg/106 in untreated controls, P=0.1; n=3). Only trace amounts of VEGF were detected even after 72 hours of activation by SFLLRN.
PAR-1 Activation Induces Actin Cytoskeleton Reorganization and Spontaneous Migration of Late EPCs
Nonactivated EPCs displayed a faint ring of polymerized actin at their periphery when stained with phalloidin (Figure 2A). Addition of 75 μmol/L SFLLRN to EPC for 5 and 30 minutes induced a strong increase in fluorescence intensity, striking reorganization of the actin cytoskeleton, and an increase in stress fiber formation (Figure 2A). The significant increase in F-actin cell content on SFLLRN stimulation was confirmed and quantified by flow cytometry with alexa-phalloidin (Figure 2B). To examine spontaneous migration linked to PAR-1 activation, EPC were treated for 4 hours with increasing concentrations of SFLLRN before being placed in the upper compartment of a Boyden chamber, the lower compartment of which contained EBM-2 medium. SFLLRN (50, 75, and 100 μmol/L) promoted EPC migration through the membrane in a concentration-dependent manner, with a 2-fold increase at the maximal concentration tested (100 μmol/L, P=0.023; Figure 2C). Together, these findings imply that PAR-1 activation supports motility of late EPCs.
SFLLRN Increases SDF-1 EPC Migration Through CXCR-4 Expression
EPCs and HUVECs were treated with increasing SFLLRN concentrations then allowed to migrate toward VEGF or SDF-1. With VEGF, a significant effect was observed with both EPC and HUVEC (Figure 3A) exposed to concentrations of 75 μmol/L or 100 μmol/L (P=0.018 and P=0.008, respectively) but not to the lowest concentration (50 μmol/L). In contrast, all SFLLRN concentrations induced strong migration toward SDF-1 (P<0.001), the effect being far more potent on EPC than on HUVEC (Figure 3B).
To explain the effect of PAR-1 activation on migration toward chemoattractants, we explored the expression of their respective receptors on the EPC surface. In keeping with the mRNA results, flow cytometry showed a 3-fold increase in CXCR-4 protein expression on EPC on SFLLRN 75 μmol/L treatment (Figure 3C, P<0.01). Interestingly, cell-surface VEGFR-2 expression was not significantly affected by SFLLRN treatment, in keeping with the lack of a significant increase in VEGFR-2 mRNA expression. Thus, the motility of EPC toward SDF-1 may result from increased expression of functional CXCR-4. To confirm the role of CXCR-4, we used 12G5, an mAb recognizing an epitope located in the second extracellular loop of CXCR4,15 which inhibited EPC and HUVEC migration toward SDF-1 by >80%. Interestingly, the MEK inhibitor PD98059 also inhibited &80% of SDF-1-directed migration of SFLLRN-activated EPC (Figure 3D). Taken together, these data strongly suggest that SDF-1-induced migration of SFLLRN-activated EPC is mediated by an increase in CXCR4 expression, the molecular pathway of which involves MEK.
The anti-CXCR4 antibody, however, did not inhibit the chemotactic response of endothelial cells toward VEGF, implying that the effect of this growth factor on SFLLRN-activated EPC is not dependent on SDF-1. In the absence of VEGF receptor induction, it is important to discriminate between chemotaxis (directional motility) and chemokinesis (random motility) to explain the effect of VEGF. We therefore tested various concentrations of VEGF in the upper and/or lower wells of the Boyden chamber. With equal concentrations of VEGF below and above the membrane, a significant enhancement of SFLLRN-activated EPC migration was observed, indicating a chemokinetic effect of VEGF. This chemokinesis represented &40% of the migratory effect (Table II, available online at http://atvb.ahajournals.org).
CXCR4/SDF-1 Pathway Blockade Inhibits EPC Tube Formation Induced by PAR-1 Activation
We used a Matrigel model to examine the capacity of SFLLRN-activated EPC to differentiate into capillary-like structures. When EPC were cultured for 16 hours without serum, they formed few capillary-like structures (Figure 4A, left panel), whereas HUVEC were no longer able to form pseudo-tubes. Treatment with SFLLRN (75 μmol/L) promoted EPC organization into branched structures and pseudo-tubes with enclosed areas (network length: 857±160 μm in untreated controls versus 3111±95 μm in SFLLRN-treated cells, P<0.0001) (Figure 4A right panel, and 4B). Given the role of PAR-1 activation in CXCR-4/SDF-1 induction, we explored the involvement of this system in tubule morphogenesis by using blocking anti-CXCR-4 and anti-SDF-1 antibodies and the MEK inhibitor PD98059. The increase in tube formation in Matrigel induced by SFLLRN was blocked by these antibodies, as well as by PD98059, but not by the irrelevant isotypic control antibody (Figure 4B) or by a VEGFR-2 inhibitor (data not shown). To rule out the possibility of SDF-1–mediated proliferation in Matrigel, we checked that SDF-1 concentrations ranging from 10 to 100 ng/mL did not increase expanded EPC numbers (1.024, 1.021, and 0.879-fold increases, respectively, at 10 ng/mL, 50 ng/mL, and 100 ng/mL; untreated control values were 1). The results of these experiments imply that PAR-1 activation enhances EPC organization into pseudovascular structures in vitro through an autocrine mechanism involving the SDF-1/CXCR-4 pathway.
The demonstration of a specific developmental role of PAR-1 in deficient mice, together with the possible involvement of EPC in postnatal vascularization, prompted us to study the role of PAR-1 in EPC biology. We found that PAR-1 activation promoted the 3 steps of angiogenesis, namely proliferation, migration, and differentiation.
Since Asahara first reported the existence of EPC in peripheral blood,1 several studies have highlighted significant heterogeneity among EPC populations. At least 2 types of EPC have been described.4 “Early” EPC appear within 4 to 7 days of culture, are spindle-shaped, and express both endothelial (von Willebrand factor) and monocytic (CD 14) markers, whereas “late” EPC develop after 2 to 3 weeks of culture and have the characteristic of precursor cells committed to the endothelium lineage, with a cobblestone shape and long-term proliferative potential. In the present study, we chose to use a homogeneous population of late EPC expanded from CD34+ endothelial progenitors isolated from human cord blood, known to be rich in stem cells.16 The endothelial phenotype of late EPC was established by means of morphological, cytometric, immunohistochemical, and immunofluorescence methods. We further showed than human EPC, as well as CD34+ cells, expressed the thrombin receptor PAR-1 at their surface, at levels similar to those found on HUVECs. To activate PAR-1, we used the SFLLRN peptide that mimics the N terminal activating peptide of the thrombin receptor, thereby avoiding the effects of thrombin on its other cell receptors, such as thrombomodulin.14 However, SFLLRN has been shown to activate PAR-2, and we cannot therefore exclude a minor contribution of PAR-2 to the observed effects. Nevertheless, we have previously shown that specific activation of PAR-2 by peptide SLIGKV does not have a significant effect on HUVEC proliferation relative to peptide SFLLRN.17
SFLLRN had a strong, concentration-dependent effect on late EPC survival and proliferation during the first 40 days of culture. This effect peaked between 20 and 30 days, in keeping with reports that circulating EPC gradually lose their proliferative potential when expanded in vitro.18 We found that the MAP kinase ERK signaling pathway, which plays a crucial role in HUVEC proliferation in response to SFLLRN,14 was also involved in EPC proliferation. Interestingly, EPC proliferated far more strongly than HUVEC in response to SFLLRN, independently of PAR-1 density (similar in the 2 cell types); this difference is in keeping with the greater sensitivity of EPC to growth factors and with reports that their progenitor properties persist for several weeks of culture.12 The difference in survival between HUVEC and EPC after SFLLRN activation is also compatible with reports that mature endothelial cell are less resistant than late EPC to apoptosis.4,19 Together, these findings suggest that late EPC from cord blood are not cells that have detached from vessel walls but rather cells that have differentiated from placental blood stem cells. A major barrier to the development of these cells as an autologous cell-therapy product is their paucity in peripheral blood.20 Our data suggest that SFLLRN peptide could be used to expand EPC ex vivo.
To better characterize the effect of PAR-1 activation on EPC, we quantified the mRNA levels of the main pro-angiogenic cytokines and their receptors by using real-time quantitative RT-PCR. Interestingly, PAR-1 activation induced a marked increase in CXCR-4 and SDF-1 mRNA, associated with CXCR-4 overexpression on the EPC membrane and with SDF-1 release into the culture medium. The SDF-1 protein level determined by enzyme-linked immunosorbent assay was lower than expected from the mRNA expression level. However, independently of SDF-1 binding to CXCR-4, it is possible that a fraction of secreted SDF-1 binds to surface proteoglycans, as described with bone marrow endothelial cells.21
Using a standard Matrigel model developed to mimic vascular tube formation, we found that PAR-1 activation induced human EPC to adopt an “angiogenic” phenotype. This effect involved the SDF-1/CXCR-4 pathway, as it was completely abrogated by anti-CXCR-4 and anti-SDF-1 antibodies as well as the MEK inhibitor. Altogether, our data suggest that SDF-1 and CXCR-4 overexpression results from transcriptional upregulation on PAR-1 activation and directly influences vascular tube formation. Vascular tube formation results from a finely tuned balance between proliferation, migration, and differentiation. We found that SDF-1 had no effect on EPC proliferation, in keeping with evidence showing the proangiogenic activity of SDF-1 does not include an effect on cell proliferation.22
Because migration is essential for EPC homing to ischemic tissues, we explored the influence of PAR-1 activation on EPC migration in Boyden chamber assays. SFLLRN promoted spontaneous EPC migration in a concentration-dependent manner, an effect involving actin cytoskeleton reorganization. We also found that SFLLRN induced EPC migration along a VEGF gradient in a concentration-dependent manner, and even more potently along an SDF-1 gradient. SDF-1 and VEGF are both markedly upregulated in hypoxic tissues, and this may contribute significantly to EPC chemoattraction.23–25 CXCR-4 upregulation is a possible mechanism underlying the migratory response of SFLLRN-treated EPC toward SDF-1. We found that SFLLRN enhanced the expression of CXCR-4 and its unique ligand SDF-1, suggesting that the proangiogenic effect of PAR-1 activation may be mediated by an autocrine mechanism involving SDF-1/CXCR-4. It has also been reported that bFGF, VEGF26 and also the sphingosine 1-phosphate receptor, a G protein-coupled receptor bearing similarities to PAR-1,27 can enhance CXCR-4 expression, making cells more responsive to SDF-1.
PAR-1 activation on HUVEC has been shown to upregulate both VEGF synthesis7 and the expression of the main VEGF receptor VEGFR-2.8 However, we observed no activation of the VEGF/VEGFR-2 pathway and no inhibition of vascular tube formation in vitro in the presence of a VEGFR-2 inhibitor. The mRNA and protein expression of VEGF isoforms and receptors did not increase significantly after SFLLRN treatment. Moreover, VEGF-induced migration was far less potent than SDF-1–induced migration. Thus, PAR-1 enhancement of SDF-1/CXCR-4–mediated angiogenesis, occurring independently of VEGF, may be another specific feature of late EPC.
To our knowledge, this study provides the first experimental proof of PAR-1 expression on EPC. Activation of PAR-1 with peptide SFLLRN confers proangiogenic properties on EPC, an effect mediated by SDF-1/CXCR-4 pathway enhancement. It is conceivable that a lack of PAR-1 activation on EPC might explain the embryonic lethality due to abnormal vascular development in PAR-1−/− knockout mice.
This work was supported by research grants from Institut National de la Santé et de la Recherche Médicale (“Réseau de Recherche sur les Cellules Souches”). D.S. was supported by a grant from Fondation pour la Recherche Médicale. We thank I. Laurendeau, C. Avignon, and A. Lokajczyk for their technical assistance. We thank D. Helley and G. Simonin for cytometric analysis. We are indebted to the nursing services of hôpital Jean Rostand (Ivry-sur-Seine) for providing human umbilical cord blood, and to hôpital Port Royal (Paris) for providing umbilical cords.
- Received April 29, 2005.
- Accepted August 5, 2005.
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