Differential chemokine receptor expression regulates functional specialization of endothelial progenitor cell subpopulations
- 210 Downloads
Postnatal vasculogenesis is mediated by endothelial progenitor cells (EPCs) which consist of subpopulations with different functional capacities. Our goal was to profile chemokine receptor expression on relevant subsets of EPCs and to characterize their role for effector functions. CD34+/CD133+/VEGFR2+ EPCs were characterized by high expression of chemokine receptors CXCR4, CX3CR1, BLT1, and low level expression of CXCR2 and CCR2, while primordial CD34−/CD133+/VEGFR2+ EPCs express these chemokine receptors at comparably low levels. Migration assays revealed that SDF-1, fractalkine, and LTB4 significantly increase migration of CD34−/CD133+/VEGFR2+ EPCs, while SDF-1 was the only potent agonist of migration of CD34+/CD133+/VEGFR2+ EPCs. SDF-1, fractalkine, and LTB4 trigger significant increase adhesion of CD34+/CD133+/VEGFR2+ EPCs, while in CD34−/CD133+/VEGFR2+ EPCs SDF-1 and fractalkine are equipotent agonists and LTB4 triggers a smaller though still significant increase in adhesion. Differential expression of specific chemokine receptors is an important regulator in terms of migration and adhesion of biologically relevant EPC-subpopulations, which may have implications for cell therapeutic strategies for treatment of ischemic vascular disease.
KeywordsEndothelial progenitor cell subpopulations Adhesion Migration Chemokines
Circulating endothelial progenitor cells (EPCs) are recruited from the flowing blood system to sites of ischemia and endothelial damage, where they mediate re-endothelialization and neovascularization by incorporating into the endothelium and/or mediating paracrine effects [1, 2, 12, 14, 16, 21, 24, 32, 34]. A growing body of evidence from clinical studies shows enhancement of blood flow and left-ventricular function in patients with coronary and peripheral artery disease by transplantation of culture-expanded EPCs or bone marrow-derived cells [4, 5, 7, 16, 27]. EPCs originate from the bone marrow and selected non-bone marrow sources and comprise a heterogeneous family of angiogenic precursors that can be characterized by expression of surface markers such as CD34, CD133, and vascular endothelial growth factor receptor-2 (VEGFR-2 or KDR in humans), by uptake of acetylated low-density lipoproteins (DiIAc-LDL), by binding of endothelial-specific lectins such as Ulex europaeus, and by generation of colony forming units (CFU) in culture assays [2, 12, 14, 24, 32, 34]. Based on surface marker expression patterns, EPCs are thought to consist of a more mature CD34+/CD133−/VEGFR2+ subset with limited biological activity and a more immature CD34+/CD133+/VEGFR2+ subpopulation with higher functional potency . Recently, we have identified a new CD34−/133+/VEGFR2+ EPC-subpopulation, which is a precursor of “classical” CD34+/133+/VEGFR2+ EPCs, and functionally more potent than these with respect to homing and vascular repair .
Chemokines are a superfamily of chemoattractant cytokines that orchestrate the recruitment of circulating cells into the vasculature via binding to G protein-coupled, 7-transmembrane spanning receptors resulting in rapid adhesion to the endothelium and subsequent migration to areas of need within the vessel wall [6, 20, 38]. Clinically, chemokines play a causal role in many inflammatory pathologies including atherosclerosis [6, 20, 33, 38]. Regarding EPCs, most data are currently available for stromal cell-derived factor-1 (SDF-1, CXCL12) and its receptor CXCR4, which are not only important mediators of EPC mobilization and recruitment in vitro and in vivo [14, 26, 39], but are also associated with improvement in cardiac function after myocardial infarction  and might modulate cardiac regeneration and neovascularisation via new signaling pathways . Moreover, CXCR2 and its ligands CXCL1 and CXCL7 were recently shown to be involved in homing of EPCs to sites of arterial injury and for endothelial recovery [13, 14]. In addition, roles for CCR5 and CCR2 were described in mediating homing of EPCs to tumor neovasculature [14, 30]. To date, the chemokine receptor expression on subsets of EPCs and its role for EPC effector function is unknown.
Therefore, it was the aim of this study to investigate chemokine receptor expression patterns on biologically important EPC-subpopulations and to determine the functional relevance of specific chemokines for migration and adhesion of EPC-subsets. We focussed on chemokine receptors CXCR4, CX3CR1, BLT1, CCR2, CXCR2 and their specific ligands SDF-1, fractalkine, LTB4, MCP-1, and IL-8, respectively, as these have been shown to play important roles in the regulation of cell trafficking in atherogenesis [6, 8, 14, 20, 38].
Isolation of circulating human EPC-subpopulations
All studies were approved by the local ethics committee and in accordance with institutional guidelines. Human mononuclear cells (MNCs) were isolated from peripheral blood of healthy volunteers (n = 7, age 32 ± 1.7 years, 5 male, 2 female) by density gradient centrifugation using Biocoll (Biocoll Separating Solution; Biochrom) as previously described [9, 12, 34, 36]. CD34−/CD133+/VEGFR2+ and CD34+/CD133+/VEGFR2+ EPC-subpopulations were isolated by 2-step magnetic bead purification according to the manufacturer’s instructions (Miltenyi Biotec). EPC purity was determined by flow cytometry as previously described . Tissue culture reagents including fetal bovine serum (FBS) were from Gibco. Dulbecco’s phosphate buffered saline (PBS) with and without calcium was obtained from Sigma.
Culture and staining of EPCs
When indicated, bead-purified CD34−/CD133+/VEGFR2+ and CD34+/CD133+/VEGFR2+ EPC-subpopulations were cultured as previously described  on fibronectin-coated (Sigma) dishes in endothelial basal medium (EBM, CellSystems) with supplements (1 μg/mL hydrocortisone, 3 μg/mL bovine brain extract, 30 μg/mL gentamycin, 50 μg/mL amphotericin B, 10 μg/mL hEGF, 20% fetal calf serum) at 37°C in a 5% CO2 atmosphere for up to 4 days. Staining with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-labeled acetylated LDL (DiI-Ac-LDL; Harbor Bio-Products) and counterstaining with fluorescein isothiocyanate-labeled lectin (BS1-Lectin; Sigma) was performed as previously described [9, 12, 34, 36]. Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole). Double-positive cells were counted manually in three or more representative high power fields (hpf) by two blinded observers (Nikon Eclipse E600, 10× magnification). All measurements were performed in triplicates.
Fluorescence-activated cell-sorter (FACS) analysis was performed as previously described in detail [9, 12, 34, 36]. In brief, viable human EPC were analyzed for CD133 (Miltenyi), CD34 (Becton-Dickinson), and a biotinylated goat monoclonal antibody against the extracellular domain of human KDR (R&D Systems). Isotype-matched antibodies served as controls in every experiment (Pharmingen). For analysis of chemokine receptor expression of EPC-subpopulations the following fluorescent antibodies were used: CXCR4, CX3CR1, BLTR1, CCR2, and CXCR2 (Pharmingen, R&D). Flow cytometry was performed using a Becton–Dickinson FACSCalibur set and Cell Quest Pro software to detect fluorescence, forward scatter, and side scatter.
Adhesion assays were carried out as previously described . Human bead-purified EPC-subpopulations (CD34−/CD133+/VEGFR2+ and CD34+/CD133+/VEGFR2+, 1 × 105 per condition) were allowed to adhere on fibronectin (Sigma) coated plates for 10 min in presence or absence of 100 nM of stromal-derived factor (SDF-1), fractalkine (CX3CL1), or leukotriene B4 (LTB4). Dishes were vigorously washed vigorously three times with changes of 1× PBS, and adherent cells were counted using a Nikon TS 100 microscope after DAPI-stain.
The migratory capacity of EPC-subpopulations was assessed by modified Boyden chambers assays as previously described . In brief, bead-purified EPC-subpopulations (105 cells in 500 μl EBM without supplements) were placed in the upper chamber (HTS Fluoroblock, 8 μm pore size, BD Biosciences, Heidelberg, Germany) which was transferred to a 24-well plate containing EBM without supplements and 100 nM SDF-1, Fractalkine, or LTB4. After 12 h EPC-transmigration into the lower chamber was measured by ELISA (OD520 nM) according to the manufacturer’s instructions (Calbiochem).
Data are expressed as mean ± standard error of the mean (SEM). Continuous variables were tested for normal distribution with the Kolmogorov–Smirnov test and compared using a one-way ANOVA test. Means between two categories were compared with the use of a two-tailed, unpaired Student’s t test. The one-way ANOVA test was used for comparisons of categorical variables. Univariate and non-parametric bivariate correlations were performed using the Spearman rank correlation coefficient. Multiple linear regression was performed where indicated to identify covariants influencing the prediction of EPC changes in peripheral blood. Statistical significance was assumed when the null hypothesis could be rejected at p < 0.05. Statistical analysis was performed using SigmaStat.
Chemokine receptor expression profile on subpopulations of EPCs
Chemokine-triggered migration of EPC-subpopulations
Chemokine-triggered adhesion of EPC-subpopulations
The present study demonstrates differential expression of chemokine receptors on biologically important subsets of EPCs with high expression levels of CXCR4, CX3CR1, and BLT1 on more mature CD34+/CD133+/VEGFR2+ EPCs, whereas only SDF-1 mediates migration while adhesion is triggered by SDF-1, fractalkine, and LTB4. Primordial CD34−/CD133+/VEGFR2+ EPCs are characterized by low level basal chemokine receptor expression with SDF-1, fractalkine, and LTB4 significantly triggering migration and adhesion.
We have recently identified a circulating CD34−/CD133+/VEGFR2+ EPC-subpopulation which is a precursor of more mature CD34+/133+/VEGFR2+ EPCs and displays higher homing capacities to sites of limb ischemia in human volunteers than “classical” CD34+/133+/VEGFR2+ EPCs . Moreover, CD34−/CD133+/VEGFR2+ EPCs are upregulated in unstable human coronary lesions and, in a mouse model of endothelial denudation, rapidly home to sites of ischemia and vascular injury, where they more potently promote endothelial regeneration and lesion reduction than CD34+/CD133+/VEGFR2+ EPCs . Our present data extend these prior studies by showing that CD34−/CD133+/VEGFR2+ and CD34+/CD133+/VEGFR2+ subpopulations of EPCs display differential chemokine receptor profiles which translate into differential chemokine-triggered functional activities with respect to adhesion and migration. These results are consistent with a growing body of evidence from other cellular systems suggesting the existence of a functional specialization within the chemokine receptor network with some receptors coupling more efficiently to adhesion pathways, while others are more effectively linked to pathways mediating migration and chemotaxis [3, 9, 10, 11, 35]. This division of labor among chemokine receptors increases the efficiency of cellular trafficking and, in case of immune cells, is believed to enhance the opulence of host protection . Future studies are needed to dissect the relevant intracellular signaling pathways specifically responsible for adhesion or migration for each chemokine receptor type expressed on EPC-subpopulations.
As in particular CD34−/CD133+/VEGFR2+ EPCs emerge as key players for cardiovascular regeneration within the EPC-superfamily , regulation of this cellular subset in different pathophysiologies gains increasing importance. Very recently, Jung et al.  found that patients with diabetes and vascular disease have lower numbers of CD34−/CD133+/VEGFR2+ EPCs and a larger proportion of apoptotic EPCs, while numbers of CD34+/CD133+/VEGFR2+ EPCs did not differ between patients with and without diabetes. These findings might contribute to decreased functional capacity of EPCs and increased cardiovascular complications associated with diabetes mellitus, especially factoring that SDF-1 is increased in healthy individuals compared to supernatants derived from patients with diabetes . Recently, an alteration in cytokine levels in the bone marrow microenvironment of diabetes mice has been showed contributing to the dysfunction of hematopoetic stem cells . Keymel and co-workers  showed an age-dependant inverse relationship of EPC function ex vivo which may lead to an accelerated vascular remodeling due to chronic impairment of endothelial maintenance . One mechanism for this has been shown concerning advanced glycation end products which depress function of EOCs via p38 and ERK 1/2 mitogen-activated protein kinase pathways . Seeger and co-workers  showed afterwards that inhibition of p38 MAP kinase improves number and functional capacities reducing atherosclerotic disease progression. Further, numbers of CD34− EPCs but not CD34+ EPCs were higher in overweight adolescents in association with increases in systolic blood pressure, hsCRP, HbA1c, and decreased HDL . Therefore, CD34− EPCs might possibly be useful as biomarkers for occult vascular damage indicating a higher risk for future cardiovascular events in obese teenagers, which will have to be elucidated in future investigations.
With respect to atherosclerosis, the role of EPCs is currently unclear with several studies pointing to an important anti-atherosclerotic and protective effect of EPCs, while some studies suggest an increase in lesion formation associated with EPC administration [18, 35]. The role of EPC-subpopulations and their specific chemokine receptor repertoire for atherogenesis is currently unknown and will have to be investigated in the future.
We conclude that differential expression of specific chemokine receptors is an important regulator of migration and adhesion of biologically relevant EPC-subpopulation, which may have implications for cell therapeutic strategies for the treatment of ischemic vascular disease.
This study was supported by the Deutsche Forschungsgemeinschaft (KFO 196) and a HOMFOR grant of the University of the Saarland. The excellent technical work of Claudia Schormann is greatly appreciated.
Conflict of interest
No conflicts of interest to disclose.
- 1.Aicher A, Rentsch M, Sasaki K, Ellwart JW, Fändrich F, Siebert R, Cooke JP, Dimmeler S, Heeschen C (2007) Nonbone marrow-derived circulating progenitor cells contribute to postnatal neovascularization following tissue ischemia. Circ Res 100:581–589. doi:10.1161/01.RES.0000259562.63718.35 CrossRefPubMedGoogle Scholar
- 2.Asahara T, Murohara T, Sullivan A, Silver M, van der Z.R, Li T, Witzenbichler B, Schatteman G, and Isner JM (1997) Isolation of putative progenitor endothelial cells for angiogenesis. Science 275:964–967Google Scholar
- 4.Assmus B, Schächinger V, Teupe C, Britten M, Lehmann R, Döbert N, Grünwald F, Aicher A, Urbich C, Martin H, Hoelzer D, Dimmeler S, Zeiher AM (2002) Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI). Circulation 106:3009–3017. doi:10.1161/01.CIR.0000043246.74879.CD CrossRefPubMedGoogle Scholar
- 5.Assmus B, Honold J, Schächinger V, Britten MB, Fischer-Rasokat U, Lehmann R, Teupe C, Pistorius K, Martin H, Abolmaali ND, Tonn T, Dimmeler S, Zeiher AM (2006) Transcoronary transplantation of progenitor cells after myocardial infarction. N Engl J Med 355:1222–1232. doi:10.1056/NEJMoa051779 CrossRefPubMedGoogle Scholar
- 7.Erbs S, Linke A, Adams V, Lenk K, Thiele H, Diederich KW, Emmrich F, Kluge R, Kendziorra K, Sabri O, Schuler G, Hambrecht R (2005) Transplantation of blood-derived progenitor cells after recanalization of chronic coronary artery occlusion: first randomized and placebo-controlled study. Circ Res 97:756–762. doi:10.1161/01.RES.0000185811.71306.8b CrossRefPubMedGoogle Scholar
- 13.Hristov M, Zernecke A, Bidzhekov K, Liehn EA, Shagdarsuren E, Ludwig A, Weber C (2007) Importance of CXC chemokine receptor 2 in the homing of human peripheral blood endothelial progenitor cells to sites of arterial injury. Circ Res 100:590–597. doi:10.1161/01.RES.0000259043.42571.68 CrossRefPubMedGoogle Scholar
- 15.Jung C, Fischer N, Fritzenwanger M, Thude H, Ferrari M, Fabris M, Brehm BR, Barz D, Figulla HR (2009) Endothelial progenitor cells in adolescents: impact of overweight, age, smoking, sport and cytokines in younger age. Clin Res Cardiol 98:179–188. doi:10.1007/s00392-008-0739-5 CrossRefPubMedGoogle Scholar
- 16.Kaur S, Kumar TR, Uruno A, Sugawara A, Jayakumar K, Kartha CC (2009) Genetic engineering with endothelial nitric oxide synthase improves functional properties of endothelial progenitor cells from patients with coronary artery disease: an in vitro study. Basic Res Cardiol 104:739–749. doi:10.1007/s00395-009-0039-x CrossRefPubMedGoogle Scholar
- 23.Orlandi A, Chavakis E, Seeger F, Tjwa M, Zeiher AM, Dimmeler S (2010) Long-term diabetes impairs repopulation of hematopoietic progenitor cells and dysregulates the cytokine expression in the bone marrow microenvironment in mice. Basic Res Cardiol 105:703–712. doi:10.1007/s00395-010-0109-0 CrossRefPubMedGoogle Scholar
- 27.Schächinger V, Erbs S, Elsässer A, Haberbosch W, Hambrecht R, Hölschermann H, Yu J, Corti R, Mathey DG, Hamm CW, Süselbeck T, Assmus B, Tonn T, Dimmeler S, Zeiher AM, REPAIR-AMI Investigators (2006) Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl J Med 355:1210–1221. doi:10.1056/NEJMoa060186 CrossRefPubMedGoogle Scholar
- 28.Schuh A, Liehn EA, Sasse A, Hristov M, Sobota R, Kelm M, Merx MW, Weber C (2008) Transplantation of endothelial progenitor cells improves neovascularization and left ventricular function after myocardial infarction in a rat model. Basic Res Cardiol 103:69–77. doi:10.1007/s00395-007-0685-9 CrossRefPubMedGoogle Scholar
- 29.Seeger FH, Sedding D, Langheinrich AC, Haendeler J, Zeiher AM, Dimmeler S (2010) Inhibition of the p38 MAP kinase in vivo improves number and functional activity of vasculogenic cells and reduces atherosclerotic disease progression. Basic Res Cardiol 105:389–397. doi:10.1007/s00395-009-0072-9 CrossRefPubMedGoogle Scholar
- 31.Sun C, Liang C, Ren Y, Zhen Y, He Z, Wang H, Tan H, Pan X, Wu Z (2009) Advanced glycation end products depress function of endothelial progenitor cells via p38 and ERK 1/2 mitogen-activated protein kinase pathways. Basic Res Cardiol 104:42–49. doi:10.1007/s00395-008-0738-8 CrossRefPubMedGoogle Scholar
- 35.Walenta K, Werner C, Böhm M, Friedrich EB (2010) Promises and pitfalls of endothelial progenitor cells in cardiovascular disease. Stem Cells (in review)Google Scholar
- 39.Yamaguchi J, Kusano KF, Masuo O, Kawamoto A, Silver M, Murasawa S, Bosch-Marce M, Masuda H, Losordo DW, Isner JM, Asahara T (2003) Stromal cell-derived factor 1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization. Circulation 107:1322–1328. doi:10.1161/01.CIR.0000055313.77510.22 CrossRefPubMedGoogle Scholar