Stromal cell-derived factor-1α (SDF-1α/CXCL12) is a member of the CXC chemokine family, which was originally isolated from murine bone marrow stromal cells and characterized as a pre-B-cell growth stimulating factor.1 CXCR4, a 7-transmembrane-spanning G protein-coupled receptor, is the receptor for SDF-1α and is also a coreceptor for human immunodeficiency virus type 1 infection.2 SDF-1α/CXCR4 interaction plays an important role during embryogenesis in hematopoiesis, vascular development, cardiogenesis, and cerebellar development.
Endothelial progenitor cells (EPCs) have been isolated from peripheral blood and showed to be incorporated into sites of physiological and pathological neovascularization in vivo.3,4 More recently, it has been suggested that circulating EPCs may also be a marker of endothelial function and cardiovascular risk. Endothelial dysfunction is usually one of the earlier markers of atherosclerosis, and predisposes one to vasoconstriction and thrombosis.5 EPCs may exert an important function as an endogenous repair mechanism to maintain the integrity of the endothelial monolayer by replacing denuded parts of the artery. The maintenance of endothelial monolayer may prevent thrombotic complication and atherosclerotic lesion development. These beneficial properties of EPCs are attractive for cell therapy that targets the endothelial regeneration. However, various risk factors for coronary artery disease, such as aging, diabetes, hypercholesterolemia, hypertension, and smoking, affect the number and functional activity of EPCs in healthy volunteers6 and in patients with coronary artery disease.7
Recent studies have shown that SDF-1α plays an important role in the regulation of a variety of cellular functions of EPCs such as cell migration, proliferation, and survival.8,9 Despite the apparently important role of CXCR4/SDF-1α in EPC migration, proliferation, survival, and angiogenesis, relatively little is known about the signal transduction pathways that mediate these effects in EPCs. Studies in many cell types have implicated both PI3K/Akt and MAPK/ERK signal transduction pathways in the control of directional cell migration and the sensing of chemoattractant gradients by the cell.10-13 In this study, we aimed to investigate the effect of SDF-1α on migration of EPCs and the functional role of PI3K/Akt and MAPK/ERK signal transduction pathways in SDF-1α-induced migration of EPCs.
Isolation of Mononuclear Cells and Cell Culture
EPCs were cultured according to a previously described technique.8 Briefly, peripheral blood mononuclear cells (PBMNCs) were isolated from healthy volunteers by density gradient centrifugation with Ficoll separating solution (Cedarlane Laboratories, Hornby, Canada). After purification with three washing steps, 1 × 107 PBMNCs were plated on six-well plates coated with fibronectin (Chemicon, Temecula, CA). Cells were cultured in endothelial basal medium (EBM)-2 (Clonetics, Walkersville, MD) with single aliquots of EGM-2MV containing 5% fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), fibroblast growth factor-2, epidermal growth factor, insulin-like growth factor, and ascorbic acid. After 4 days in culture, nonadherent cells were removed by washing with phosphate-buffered saline (PBS), new medium was applied and culture was maintained through 7 days.
CD34+ cells from isolated human peripheral blood mononuclear cells were positively selected using the MiniMACS immunomagnetic separation system according to the manufacturer's instructions as recently described.14
Fluorescent chemical detection of EPCs was performed on attached mononuclear cells after 7 days in culture. Direct fluorescent staining was used to detect dual binding of fluorescein isothiocyanate (FITC)-labeled Ulex europaeus agglutinin (UEA)-1 (Sigma Chemical Co., St. Louis, MO) and 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine (DiI)-labeled acetylated low-density lipoprotein (ac-LDL; Molecular Probe, Eugene, OR). Cells were first incubated with acLDL at 37°C and later fixed with 2% paraformaldehyde for 10 minutes. After washing, the cells were reacted with UEA-1 (10 μg/mL) for 1 hour. After the staining, samples were viewed with an inverted fluorescent microscope and further demonstrated by laser scanning confocal microscope. Cells demonstrating double-positive fluorescence were identified as differentiating EPCs.15 Three independent experiments were performed, and three independent investigators evaluated the number of EPCs per well by counting five randomly selected high-power fields (×200) with an inverted fluorescent microscope.
Fluorescence-Activated Cell Sorting
Fluorescence-activated cell sorting (FACS) detection of EPCs was performed after 7 days in culture. The procedure of FACS staining was described previously.15 In brief, a total of 2-3 × 105 cells were resuspended with 200 μL of PBS containing 0.5% bovine serum albumin (BSA) and incubated for 20 minutes at 4°C with anti-vascular endothelium (VE)-cadherin (Chemicon), phycoerythrin-conjugated monoclonal antibodies against kinase insert domain-containing receptor (KDR; R&D, Minneapolis, MN), CD34, AC133 (Miltenyi Biotec, Auburn, CA), and CXCR4 (R&D). A FITC-conjugated antimouse antibody was added for staining with VE-cadherin. Isotype-identical antibodies served as controls. After staining, the cells were fixed in 2% paraformaldehyde. Quantitative FACS was performed on a FACStar flow cytometer. Three independent experiments were performed, and the groups represent the mean of the three separate experiments.
Reverse-Transcription Polymerase Chain Reaction
Total cellular RNA was prepared using Absolutely RNA reverse-transcription (RT) polymerase chain reaction (PCR) Miniprep Kit from Invitrogen (Carlsbad, CA). Briefly, EPCs were lysed with lysis buffer, and RNA was purified following the instruction of the manufacturer. The purified RNA was suspended in diethyl pyrocarbonate-treated water. RNA was converted into cDNA using murine leukemia virus reverse transcriptase (Gibco BRL Life Technologies). The transcribed cDNA was then used for PCR amplification to estimate the expression of CXCR4. Two specific primers matching the published sequences were used to identify and amplify CXCR4 (sense primer: 5′-CTT CTA CCC CAA TGA CTT GTG G -3′; antisense primer: 5′-AAT GTA GTA AGG CAG CCA ACA G-3′). The reaction mixtures were heated at 94°C for 5 minutes, followed by 25 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. Subsequently, PCR products were electrophoresed through 1.8% agarose gel and visualized by ethidium bromide. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as a reference.
After 7 days of incubation, culture medium was removed and replaced with EBM-2 without any supplement 12 hours before migration assay. EPC migration was evaluated by using a modified Boyden chamber assay. SDF-1α (PeproTech, Rehovof, Israel) was diluted to appropriate concentrations in EBM-2 supplemented with 0.1% BSA, and 200 μL of the final dilution was placed in the lower compartment of a Boyden chamber. Then 3 × 104 cells were suspended in 50 μL of EBM-2 supplemented with 0.1% BSA and placed in the upper compartment. After incubation for 5 hours at 37°C in 5% CO2 condition, the cells that had not migrated were removed from the upper surface of the filters using cotton swabs, and those that migrated to the lower surface of the filters were fixed in methanol and stained with 4′,6-diamidino-2-phenylindole (DAPI; Roche Applied Science, Indianapolis, IN). Migration was determined by counting the cell number with a microscope at 100× magnification. Five visual fields were chosen randomly for each assay. The average number of the migrating cells in five fields was taken as the cell migration number of the group. Six independent experiments were examined, and the groups represent the mean of the six separate experiments.
Western Blot Analysis
After 7 days of culture, EPCs were deprived of serum for 12 hours to render the cells quiescent. Cellular proteins were prepared and separated by 10% sodium dodecyl sulfate polyacrylamide gel and electrotransferred to a polyvinylidene difluoride membrane. The membranes were blocked in blocking solution (Tris-buffered solution) containing 0.1% (v/v) Tween 20 and 5% (v/v) BSA for 1 hour and then incubated overnight with rabbit polyclonal anti-phospho-Akt-Ser473 (1:500), anti-Akt (1:1000), anti-phospho-ERK1/2 (1:500), anti-ERK1/2 (1:1000), anti-phospho-eNOS-Ser1177 (1:500) antibodies (Cell Signaling Technology, Inc., Beverly, MA), or anti-β-actin polyclonal antibody (1:1000). The membranes were washed extensively in Tris-buffered saline containing 0.1% (v/v) Tween 20 before incubation for 1 hour with a secondary antirabbit antibody conjugated to horseradish peroxidase (1:5000). Protein was then visualized using enhanced chemiluminescence solution from Amersham and x-ray film.
Data were expressed as means ± SEM for three to six individual experiments. Statistical analysis between two groups was performed using unpaired Student's t test; comparisons between multiple groups were made by one-way analysis of variance. Probability values were considered significant at P < 0.05.
Characterization of EPCs
After 7 days of culture, ex vivo expanded EPCs derived from peripheral blood of healthy human volunteers exhibited spindle-shaped morphology (Fig. 1). EPCs were also characterized as adherent cells double positive for Dil-Ac-LDL-uptake and lectin binding by using LSCM (laser scanning confocal microscope) (Fig. 2). A total of 93.8 ± 4.5% of adherent cells showed uptake of Dil-Ac-LDL and lectin binding after 7 days cultured in our study. They were additionally confirmed by demonstrating the expression of well-established cell surface markers like VE-cadherin (77.3 ± 6.1%), KDR (78.7 ± 9.7%), CD34 (31.3 ± 4.2%), and AC133 (18.7 ± 5.0%) by FACS.
CXCR4 Expression in EPCs
To investigate CXCR4 expression in CD34+ cells and EPCs, we first did an RT-PCR. As shown in Figure 3A, CXCR4 was well expressed in day 7 cultured EPCs, whereas it was poorly expressed in freshly isolated human peripheral blood CD34+ cells.
To further investigate CXCR4 expression, we did FACS analysis. It elucidated that 54.7 ± 4.5% of day 7 cultured EPCs express CXCR4, whereas only 4.0 ± 1.0% of CD34+ cells showed CXCR4 expression (Fig. 3B).
SDF-1α Induces Concentration-Dependent EPCs Migration
The effects of SDF-1α on EPCs migration were analyzed in a modified Boyden chamber assay (Fig. 4). SDF-1α induced EPCs migration in a concentration-dependent manner and the maximum migration was observed when 100 ng/mL SDF-1α was applied in the assay conditions.
SDF-1α Induces CXCR4-Dependent EPC Migration
To investigate the role of CXCR4 in SDF-1α-induced migration, EPCs at 7 days were pretreated for 30 minutes with 5 μg/mL AMD3100 (Sigma; CXCR4-specific peptide antagonist) before migration assay. As shown in Figure 4C, SDF-1α-induced EPCs migration was inhibited by AMD3100, indicating that SDF-1α-induced EPCs migration was specifically via its receptor CXCR4.
PI3K/Akt/eNOS Pathway is Involved in SDF-1α-Induced EPCs Migration
To evaluate the functional roles of PI3K/Akt and MAPK/ERK signal transduction pathways in SDF-1α-induced migration, EPCs at 7 days were pretreated for 30 minutes with PI3K-specific inhibitors (LY294002 and wortmannin) and the MEK-specific inhibitor PD98059 (Cell Signaling Technology) before migration assay. As shown in Figure 4C, the pretreatment of EPCs with 20 μM LY294002 or 100 nM wortmannin nearly completely blocked SDF-1α-induced EPC migration, whereas 20 μM PD98059 had no significant effect on SDF-1α-induced migration, suggesting that PI3K/Akt signal transduction pathway, but not MAPK/ERK signal transduction pathway, is required for SDF-1α-induced migration of EPCs.
Akt-mediated endothelial cell migration is dependent on subsequent phosphorylation of eNOS on Ser1177 and NO generation.16 To further study the molecular mechanisms beyond SDF-1α-mediated migration, EPCs at 7 days were pretreated for 30 minutes with 1mM eNOS inhibitor (NG-nitro-arginine methyl ester, L-NAME). As shown in Figure 4C, L-NAME also significantly attenuated SDF-1α-induced EPCs migration.
Taken together, these results demonstrate that PI3K/Akt/eNOS signal transduction pathway plays a crucial role in SDF-1α-induced EPCs migration.
SDF-1α Treatment Activates PI3K/Akt/eNOS Pathway
Because SDF-1α-induced migration of EPCs was regulated by the PI3K/Akt/eNOS signal transduction pathway, we examined the effect of SDF-1α on Akt and eNOS phosphorylation in EPCs. EPCs were stimulated with 100 ng/mL SDF-1α for several different times and immunoblots were performed with a phosphospecific Akt antibody directed at the Ser473 phosphorylation Site and phosphospecific eNOS antibody directed at Ser.1177 As illustrated in Figure 5A, SDF-1α treatment of EPCs stimulated a time-dependent Akt phosphorylation. The phospho-Akt level reached a maximum 10 to 15 minutes after 100 ng/mL SDF-1α treatment, and the significantly enhanced Akt phosphorylation could still be observed 60 minutes after SDF-1α treatment, whereas no significant change in total Akt expression was observed over the course of the experiment. SDF-1α induced eNOS phosphorylation was similar to Akt phosphorylation.
To further address the effect of SDF-1α on Akt phosphorylation, EPCs treated with several concentration of SDF-1α for 15 minutes with or without pretreatment with AMD3100. As revealed in Figure 5B, stimulation with SDF-1α led a concentration-dependent phosphorylation of Akt. SDF-1α-induced Akt phosphorylation was nearly completely inhibited with AMD3100, a CXCR4-specific peptide antagonist.
SDF-1α Treatment Activates MAPK/ERK1/2 Pathway
To further study cell signaling mediated by SDF-1α, we also investigated whether SDF-1α could activate MAP kinase ERK1/2 by Western blot. As shown in Figure 6A, SDF-1α also could activate both ERK1 and ERK 2. The phospho-ERK1/2 level reached a maximum 5 minutes after 100 ng/mL SDF-1α treatment, whereas no significant change in total ERK1/2 expression was observed over the course of the investigation. SDF-1α also induced ERK1/2 phosphorylation in a concentration-dependent manner (Fig. 6B).
We showed that 20 μM PD98059 had no significant effect on SDF-1α-induced migration. Therefore, to rule out an involvement of ERK1/2 in SDF-1α-induced migration of EPCs, we should investigate whether 20 μM PD98059 could inhibit ERK1/2 phosphorylation induced by SDF-1α. As shown in Figure 6B, SDF-1α-induced ERK1/2 phosphorylation was blocked by 20 μM PD98059.
The chemokine SDF-1α and its cognate receptor CXCR4 have an unusually wide tissue distribution and a high degree of homology (>90%) between different species in comparison to other chemokine/receptor family members, underscoring the importance of these two molecules during embryonic development and tissue homeostasis in postnatal organisms. In the present study, we demonstrated that endothelial progenitor cells express CXCR4 by RT-PCR and flow cytometric analysis, and the percentage of EPCs expressing CXCR4 was 13-fold higher compared with that of freshly isolated peripheral blood-derived CD34+ cells. Various factors may be involved in CXCR4 upregulation during incubation, including cell-surface adhesion, cell-cell contact between CD34+ and low-density mononuclear cells, growth factor production, and adhesion molecule interactions.14 Kahn and his colleagues have reported that CXCR4 overexpression in human CD34+ progenitors using a lentiviral gene transfer technique helps navigate these cells to the murine bone marrow and spleen in response to SDF-1α signaling.17 Cells overexpressing CXCR4 will show significant increases in SDF-1α-mediated chemotaxis and actin polymerization.
It is generally believed that SDF-1α mediates many disparate processes exclusively via a single cell surface receptor known as chemokine receptor CXCR4. But recently, Burns et al18 reported that an alternate receptor, CXCR7, which could also bind with high affinity to SDF-1α. Unlike many other chemokine receptors, ligand activation of CXCR7 does not cause Ca2+ mobilization or cell migration. Expression of CXCR7 provides cells with a growth and survival advantage and increased adhesion properties. In present study, we showed that SDF-1α-induced migration of EPCs was CXCR4 dependent as confirmed by the total inhibition by AMD3100, a CXCR4-specific peptide antagonist.
Given the low numbers of circulating progenitor cells, chemoattraction may be of utmost importance to allow for recruitment of reasonable numbers of progenitor cells to the ischemic or injured tissue. SDF-1α binding to CXCR4 leads to increased tyrosine phosphorylation of the receptor, leading to CXCR4 dimerization and internalization. Downstream targets of SDF-1α/CXCR4 interactions are known to mediate various cellular processes such as chemotactic migration. Indeed, SDF-1α has been proven to stimulate recruitment of progenitor cells to the ischemic tissue.8,19 In this study, from a functional standpoint, we showed that SDF-1α treatment induced a concentration dependent migration of EPCs, which was consistent with the result obtained by Yamaguchi.8
Akt is a known downstream effector of the PI3K-dependent signaling cascade. MAPKs are a family of protein kinases and consist of ERKs, p38s, and JNKs.20 In general, ERKs involve in cell growth and survival, whereas p38s or JNKs participates in cell death or apoptosis.21 CXCR4 is a Gi-coupled receptor, and studies have shown that SDF-1α, after binding to CXCR4, causes mobilization of calcium, decrease of cyclic AMP within the cells, and activation of multiple signal transduction pathways, including PI3K, phospholipase C-γ/protein kinase C, and MAP kinases ERK1/2.22-25 Both PI3K/Akt and MAPK/ERK signal transduction pathways have been shown to mediate the cell migration induced by chemokines or cytokines in different cell types. Recently, studies in EPCs have demonstrated that statins, estrogen, VEGF, and EPO could improve migration, proliferation, and prevent senescence or apoptosis of EPCs, in which the activation of the PI3K/Akt signal transduction pathway may play an important role.26-29 Wang and his colleagues have reported that PI3K/Akt, but not MAPK/ERK, is required for SDF-1α-mediated migration of hematopoietic progenitor cells and primary marrow CD34+ cells.11 However, in COS-7 cell, MAPK signal transduction pathway has been shown to regulate cell migration.12 Moreover, some studies have showed that both PI3K/Akt and MAPK/ERK signal transduction pathways are involved in the regulation of SDF-1α-mediated migration.13,24 To investigate the functional roles of PI3K/Akt and MAPK/ERK signaling cascades in SDF-1α-induced cell migration of EPCs, we did migration array with PI3K-specific inhibitors (LY294002 and wortmannin) or MEK-specific inhibitor (PD98095). The results showed that PI3K inhibitors, LY294002 and wortmannin, nearly totally blocked SDF-1α-induced EPC migration. In contrast, MEK inhibitor PD98059 had no significant effect on SDF-1α-induced EPC migration, indicating that PI3K/Akt activation, but not ERK activation, is required for SDF-1α-induced EPC migration. Therefore, it seems that the cell signal transduction pathways involved in SDF-1α/CXCR4-mediated cell migration are cell-type dependent.
Many growth factors and hormones have been shown to regulate cell proliferation, migration, and angiogenesis, including the activation of eNOS activity, via the PI3K/Akt signaling pathway. Activation of Akt has been shown to stimulate phosphorylation of eNOS at Ser1177 and then increases endothelial NO production, which leads to subsequent cells growth and migration. Our data also demonstrated that eNOS activation was required for SDF-1α-mediated EPC migration.
To further study signal transduction pathways mediated by the SDF-1a/CXCR4 interaction, we also investigated whether SDF-1a could activate PI3K/Akt/eNOS and MAPK/ERK signal transduction pathways. Western blot analysis revealed that SDF-1a treatment stimulated a time and concentration dependent Akt, eNOS, ERK1/2 phosphorylation.
Athough, in present study, ERK1/2 phosphorylation is not required for SDF-1α-induced migration of EPCs, we believe that it may play an important role in mediating EPC proliferation and survival. The functional role of ERK1/2 and Akt activation in cell proliferation and survival of EPCs are currently under investigation in our laboratory.
The chemotactic effect of SDF-1α on EPCs via PI3K/Akt/eNOS pathway is important for potential cell therapy.
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