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CORRESPONDENCE Stefanie Dimmeler: Dimmeler{at}em.uni-frankfurt.de
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Abbreviations used: ß2–/–, ß2-integrin–deficient; EPC, endothelial progenitor cell; HUVEC, human umbilical vein endothelial cell; MNC, mononuclear cell; vWF, von Willebrand factor.
The term vasculogenesis was originally introduced to describe the de novo formation of new vessels from angioblasts during embryonic development (1). Accumulating evidence suggests that vasculogenesis, mediated by circulating bone marrow–derived endothelial progenitor or hematopoietic stem cells, plays an important role in postnatal neovascularization of adult ischemic tissues (2–7). Human endothelial progenitor cells (EPCs) were initially characterized by the expression of the VEGF receptor 2 (VEGF R2; Flk-1) and a hematopoietic marker such as CD133 (6). EPCs are mobilized from the bone marrow during ischemia (8, 9) or exogenously by stimulation with cytokines such as VEGF and contribute to neovascularization of ischemic tissues (4, 8, 10) or tumors (11). Infusion of EPCs or isolated hematopoietic progenitor cells (e.g., murine Sca-1+/Lin– cells) augmented neovascularization of ischemic myocardium and limbs and improved left ventricular function after myocardial ischemia (12–15). EPCs are preferentially recruited to sites of ischemia and incorporated into vascular structures (2, 4, 8, 12, 16). The mechanisms of EPC homing to sites of ischemia are still unclear. Because integrins are mediating the homing of transplanted hematopoietic stem cells to the bone marrow (17) as well as the recruitment of inflammatory cells to sites of inflammation, we investigated the contribution of integrins and especially of ß2-integrins for homing and neovascularization capacity of EPCs and hematopoietic stem cells to areas of ischemia.
Recruitment of inflammatory cells requires a coordinated sequence of multistep adhesive and signaling events, including selectin-mediated rolling, leukocyte activation by chemokines, integrin-mediated firm adhesion and diapedesis (18–22). During firm adhesion of leukocytes to the endothelium, members of the ß2-integrin family, LFA-1 (
Lß2, CD11a/CD18), Mac-1 (Mß2, CD11b/CD18), and p150,95 (Xß2, CD11c/CD18), as well as ß1-integrins on leukocytes interact with endothelial counterligands such as ICAM-1, VCAM-1, and surface-associated fibrinogen. Mac-1 also regulates leukocyte adhesion to provisional matrix substrates including fibrinogen, which is deposited at sites of inflammation and injury upon increased vascular permeability and damage (19, 20, 23). Because ß2-integrins are strongly expressed on EPCs, we studied the role of the ß2-integrins for homing and neovascularization capacity of peripheral blood–derived cultivated human EPCs, bone marrow–derived murine hematopoietic Sca-1+/Lin– as well as VEGF R2+/Lin– progenitor cells. Our results show that ß2-integrins mediate the adhesive interactions of EPCs to mature endothelial cells and to extracellular matrix proteins and are critical for chemokine-induced transendothelial migration of EPCs in vitro. In a mouse model of hind limb ischemia, using murine Sca-1+/Lin– hematopoietic progenitor cells from ß2-integrin–deficient (ß2–/–) mice, we demonstrate that ß2-integrins are involved in the homing of hematopoietic progenitor cells to sites of ischemia and are critical for their neovascularization capacity. Alternately, preactivation of the ß2-integrins on EPCs by activating antibodies significantly augments the in vivo neovascularization capacity of EPCs, indicating a new therapeutic approach to promote homing of EPCs.
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–prestimulated HUVECs. Addition of an inhibitory ß2-integrin antibody significantly blocked EPC adhesion to HUVECs (Fig. 2 B). VLA–4 as well as Vß3-integrin can also mediate intercellular adhesive interactions by binding to VCAM-1 and PECAM-1, respectively (23, 29). Therefore, a synthetic VLA-4 inhibitor and a cyclic RGD peptide (established inhibitor of the Vß3- and Vß5-integrin) were engaged in these studies. However, these inhibitors did not significantly inhibit EPC adhesion to HUVECs (Fig. 2 B). An inhibitory VLA-4 antibody (clone HP2/1), as well as an inhibitory ß1-integrin antibody (clone 6S6), also had no effect on the adhesion of EPCs to HUVECs (unpublished data). These results demonstrate that EPC adhesion to endothelial cells is predominantly mediated by ß2-integrins expressed on EPCs. Endothelial ICAM-1 and extracellular matrix-associated fibrinogen are established ligands for the ß2-integrins (30–33). Therefore, we investigated whether EPCs are capable of binding to immobilized recombinant human ICAM-1 and fibrinogen via ß2-integrins. Indeed, stimulation with either Mn2+ or an activating ß2-integrin antibody (KIM185) induced adhesion of EPCs to immobilized human ICAM-1 and fibrinogen (Fig. 2, C and D). Adhesion induced by both stimuli was completely abolished in the presence of an inhibitory ß2-integrin antibody (Fig. 2, C and D). In contrast, an inhibitory ß1-integrin antibody had no effect on the adhesion of EPCs to human ICAM-1 (unpublished data). These results demonstrate that EPCs bind to fibrinogen and endothelial ICAM-1 in a ß2-integrin–dependent manner.
Role of ß2-integrins for transmigration of EPCs
We investigated the involvement of ß2-integrins in the transendothelial migration of EPCs in a transwell transmigration assay. Chemoattraction of EPCs by MCP-1, SDF-1, and VEGF significantly increased the transmigration rate of EPCs through HUVEC monolayers (Fig. 3). Addition of an inhibitory ß2-integrin antibody (anti-CD18) significantly reduced EPC transmigration, whereas an inhibitory ß1-integrin antibody (anti-CD29) and RGD peptides had no effect (Fig. 3). Moreover, an inhibitory VLA-4 antibody did not affect chemokine-induced transendothelial migration of EPCs (unpublished data). Thus, ß2-integrins, but not ß1-integrins, mediate chemokine and VEGF-induced transendothelial migration of EPCs (Fig. 3).
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–pretreated mature endothelial cells was significantly blocked by ß2-blocking antibodies (unpublished data).
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Moreover, histological evaluation of ischemic hind limbs of athymic mice 14 d after cell infusion revealed a significantly lower capillary density in mice receiving ß2–/– Sca-1+/Lin– bone marrow cells compared with mice receiving wild-type cells (Fig. 5 C). Furthermore, the number of incorporated male Sca-1+/Lin– cells in female recipients was determined by fluorescence in situ hybridization for the murine Y-chromosome of the infused male cells. The number of incorporated Y-chromosome positive cells was significantly lower for the infusion of ß2–/– cells as compared with the infusion of wild-type cells (Fig. 5, D and E).
Similar results were obtained when using isolated VEGF R2+/Lin– bone marrow–derived cells. The majority of VEGF R2+/Lin– cells expressed CD18 (89 ± 9.6%). Moreover, VEGF R2+/Lin– cells derived from CD18–/– mice cells showed a significantly reduced capacity to augment blood flow after ischemia as compared with WT cells (WT 180 ± 15% of untreated control mice; CD18–/– cells: 125 ± 7% of untreated control mice). These results indicate that the ß2-integrins are involved in the homing of progenitor cells to ischemic tissues and their neovascularization capacity.
Activation of the ß2-integrins improves in vivo homing and neovascularization capacity of EPCs
Because ß2-integrins are involved in the homing of EPCs, we investigated whether preactivation of ß2-integrins may improve homing and neovascularization capacity of human EPCs in the mouse model of hind limb ischemia. Ex vivo–expanded human EPCs isolated from peripheral blood were pretreated with the ß2-integrin–activating antibody (KIM 185), which was shown before to enhance ß2-integrin–dependent adhesion of EPCs to endothelial ICAM-1 or fibrinogen (Fig. 2), and were subsequently infused into athymic mice. To be able to detect an increase in progenitor cell–mediated neovascularization, we used a reduced number of EPCs (105 cells), which is lower than previously published numbers (5 x 105 EPCs; reference 25), to yield a 50% improvement of neovascularization as compared with untreated mice.
Preincubation of the EPCs with the activating ß2-integrin antibody resulted in a significantly enhanced neovascularization capacity of infused EPCs in comparison with control antibody-treated EPCs as assessed by laser Doppler imaging (Fig. 6 A). Incorporation of human EPCs was detected by confocal microscopy using antibodies directed against human HLA and the endothelial marker protein vWF (Fig. 6, B–D). Incorporation of ß2-activating antibody-treated EPCs into the ischemic muscle was increased in comparison with control antibody-treated EPCs (Fig. 6, B and C). Moreover, the numbers of capillaries and small arterioles (20–50 µm) were significantly augmented in mice treated with preactivated EPCs (Fig. 6, E and F; capillary density: EPC + control mAb: 0.77 ± 0.10 capillaries per myocyte; EPC + activating mAb: 1.32 ± 0.09; P = 0.003). Thus, an external activation of the ß2-integrins by an activating antibody before infusion is capable of improving the neovascularization capacity of EPCs.
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Increasing evidence suggests that ß2-integrins are not only expressed on differentiated leukocytes but also on hematopoietic stem/progenitor cells (34, 35). Our in vitro data suggest that ß2-integrins expressed on EPCs mediate homing functions such as endothelial adhesion and transmigration. Moreover, ß2-integrins contribute to the in vivo homing of bone marrow–derived progenitor cells to ischemic tissue. In line with these data, it has been reported previously that ß2-integrins mediate adhesion and transmigration of hematopoietic stem/progenitor cells (36–38). In a recent paper assessing in vivo homing of embryonic EPCs derived from cord blood, the circulating cells arrested within tumor microvessels extravasated into the interstitium and incorporated into neovessels, suggesting that adhesion and transmigration are involved in the recruitment of EPCs to sites of tumor angiogenesis (39). Thus, it is conceivable to speculate that ex vivo–expanded adult EPCs and hematopoietic stem/progenitor cells may engage similar pathways for recruitment to sites of ischemia and incorporation into newly forming vessels.
Our in vivo data provide the first evidence for a direct participation of ß2-integrins in neovascularization processes and particularly in stem/progenitor cell–mediated, ischemia-induced vasculogenesis. Adamis and colleagues previously highlighted the role of ß2-integrins for corneal and choroidal angiogenesis induced by injury (40, 41). In both studies, ß2–/– mice displayed a reduced inflammation-associated angiogenic response after injury and these effects were associated with reduced inflammatory cell infiltrates (40, 41). Yet, no incorporation of leukocytes into new vessels was reported in these studies. Moreover, the same group demonstrated that, in the case of retinal ischemia, leukocyte–endothelial cell interactions contribute to the development of ischemia by inducing vascular obliteration via Fas ligand–mediated endothelial cell apoptosis (42). In contrast with these findings, our data provide evidence that, during hind limb ischemia, intravenous infusion of bone marrow hematopoietic progenitor cells leads to incorporation of the transplanted cells in newly formed vessels and to improvement of neovascularization in an at least partially ß2-integrin–dependent manner. As opposed to EPCs, infusion of inflammatory cells, such as monocytes/macrophages, had only a slight if any effect on the neovascularization of ischemic limbs in the model of hind limb ischemia in athymic mice (25). Thus, our results support a novel direct function of ß2-integrins in progenitor cell–induced vasculogenesis during ischemia, which is distinct from the indirect role of ß2-integrins in the inflammation-associated angiogenesis described by Adamis and colleagues (40, 41). Because the ß2–/– mice display no defect in the mobilization of progenitor cells (43), the neovascularization defect in the ß2–/– mice in the model of hind limb ischemia is most conceivably mediated by a homing defect of progenitor cells into ischemic tissue.
Interestingly, our present data indicate that the recruitment of hematopoietic progenitor cells to sites of ischemia is mediated at least in part by different mechanisms compared with the homing of infused cells into the bone marrow of lethally irradiated recipient mice, which is predominantly mediated via 4ß1 (17, 43). In this context, ß2-integrins only act in a synergistic manner together with the 4ß1-integrin. Our finding that ß2-integrin deficiency does not completely block homing and neovascularization improvement after infusion of Sca-1+/Lin– bone marrow cells suggests that other mechanisms may additionally be involved in these processes. We cannot exclude that 4ß1-integrin partially compensates for the lack of ß2-integrin during in vivo homing of Sca-1+/Lin– bone marrow cells. Interestingly, the homing of inflammatory cells during pneumonia or myocardial ischemia in ß2–/– mice is mediated by the 4ß1-integrin (44, 45). Moreover, the initial cell arrest of embryonic progenitor cell homing during tumor angiogenesis was suggested to be mediated by E- and P-selectin and P-selectin glycoprotein ligand-1 (39). Yet, it is important to underscore that this work was performed with embryonic EPCs, whereas we used adult EPCs and bone marrow stem/progenitor cells. It is likely that different cell types may use distinct mechanisms for homing to sites of ischemia. In addition, it is well established that interactions of selectins with selectin–ligands mediate the rolling of cells on the surface of endothelial cells as the initial step of homing (21). Further studies are needed to elucidate a potentially synergistic role of other adhesion molecules and their counterligands for the multistep recruitment process of adult endothelial progenitor and stem cells to ischemic tissue.
Regardless of potentially additive mechanisms involved in the recruitment of stem/progenitor cells to areas of ischemia, our data clearly demonstrate that preincubation of EPCs with a ß2-integrin–activating antibody markedly enhanced the incorporation of transplanted EPCs in vessels and the neovascularization of ischemic limbs. The peripheral blood–derived EPCs used in the present paper are already used in clinical trials to improve neovascularization in patients with ischemic heart diseases (15). Thus, our results could have important clinical implications as they disclose a mechanism to enhance homing of EPCs and, thereby, improve neovascularization capacity of infused EPCs.
In summary, the present paper demonstrates for the first time a critical role of ß2-integrins in vitro and in vivo for homing and neovascularization capacity of endothelial progenitor and hematopoietic progenitor cells. Moreover, our results show that activation of ß2-integrins appears to be a feasible and promising tool to improve the efficacy of EPC-induced neovascularization. A better understanding of the homing mechanisms of EPCs may lead to the development of new therapeutic strategies for improvement of vasculogenesis in patients with ischemic diseases.
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Sca-1+/Lin– cells were purified from BM MNCs from wild-type and CD18–/– mice by negative selection using a cocktail of biotinylated antibodies to lineage markers (Lineage cell depletion kit, mouse; Miltenyi Biotec) for 10 min at 4°C followed by antibiotin microbeads for 15 min (Miltenyi Biotec). The Lin– BM cells were incubated with anti–Sca-1 microbeads (Miltenyi Biotec) for 15 min and Sca-1+/Lin– BM cells were collected (7). To obtain VEGFR2+ Lin– cells, Lin– cells were incubated with biotinylated Flk-1 antibodies (DSB-X biotin protein labeling kit; Molecular Probes; antibody was obtained from BD Biosciences) for 30 min at 4°C followed by antibiotin microbeads for 15 min.
HUVECs were purchased from Cambrex and cultured in endothelial basal medium supplemented with 1 µg/ml hydrocortisone, 12 µg/ml bovine brain extract, 50 µg/ml gentamycin, 50 ng/ml amphotericin-B, 10 ng/ml epidermal growth factor, and 10% FCS until the third passage. After detachment with trypsin, cells (4 x 105 cells) were grown in 6-cm cell culture dishes or 96-well plates for at least 18 h as described previously (47).
Oligonucleotide microarrays, FACS
10 µg of total RNA was hybridized to the HG-U95Av2 microarray (9670 human genes; Affymetrix, Inc.). The standard protocol used for sample preparation and microarray processing is available from Affymetrix, Inc. Expression data were analyzed using Microarray Suite version 5.0 (Affymetrix, Inc.) and GeneSpring version 4.2 (Silicon Genetics). 3 x 105 human EPCs, peripheral blood, or isolated Sca-1+/Lin– cells were incubated for 30 min at 4°C with FITC- or PE-labeled antibodies (anti-CD11a, -CD11b, -CD11c, -CD18, –Sca-1, and –c-kit; BD Biosciences; anti-vWF was obtained from Acris) or CBRM1/5-antibody (BD Biosciences) for 30 min at 37°C. The mAb24 antibody (provided by N. Hogg, Cancer Research UK London Research Institute, London, England, UK) was incubated for 30 min at 4°C and detected with a secondary FITC-labeled goat anti–mouse antibody (DakoCytomation). Surface expression was quantified using a FACS Calibur (BD Biosciences).
Adhesion, transmigration experiments
Cell–cell adhesion
Cell–cell adhesion was performed as described previously (48, 49). Confluent HUVEC monolayers were stimulated with TNF- (Sigma-Aldrich) for 24 h. Ex vivo–expanded EPCs were stained with Cell Tracker green-CMFDA (Molecular Probes) and were resolved in adhesion buffer (150 mM NaCl, 20 mM Hepes, 2 mM MgCl2, 0.05% BSA, pH 7.4). A total of 105 EPCs/well (in 100 µl adhesion buffer) was added to the HUVEC monolayers in the absence or presence of blocking monoclonal ß2-integrin antibodies (clone IB4; Qbiogene; or mAb 60.3; J. Harlan, University of Washington, Seattle, WA), murine isotype control antibodies (Qbiogene), inhibitory agents cyclic RGD peptide or VLA-4 inhibitor (4-[{2-methyl-phenyl}aminocarbonyl]aminophenyl)acetyl-fibronectin-CS-1 fragment (1980–1983). After 20 min of incubation (37°C), the plates were washed twice with adhesion buffer at room temperature to remove nonadherent cells. Adherent cell tracker green-labeled EPCs were quantified in triplicates on a fluorescence plate reader (Fluostat; BMG Lab Technologies).
Cell–matrix adhesion
Cell–matrix adhesion was performed as described previously (48, 49). 96-well plates were coated overnight (4°C) with 10 µg/ml human fibrinogen (Enzyme Research Laboratories) or soluble recombinant human ICAM-1 (Bender MedSystems) and blocked with 1% (wt/vol) BSA for 1 h at room temperature. Ex vivo–expanded human EPCs in adhesion buffer were seeded at 1.2 x 105 cells/well in 100 µl in the absence or presence of 2 mM MnCl2 or activating human ß2-integrin antibody (clone KIM185; M. Robinson, Celltech Ltd., Slough, England, UK) and were incubated with blocking ß2-integrin mAb (clone IB4 or mAb 60.3) or murine isotype control antibodies (Qbiogene) for 20 min at 37°C. After removal of nonadherent cells by two washing steps, adhesion was quantified in triplicates by counting adherent cells in five randomly selected fields per well (magnification, 20; Axiovert 100; Carl Zeiss MicroImaging, Inc.).
Transmigration
Transendothelial migration was performed as described previously (50) using 6.5-mm transwell filters with 8-µm pore size (Costar). After inserts were coated with 0.2% gelatin (Sigma-Aldrich), HUVECs were seeded on transwell filters and cultivated for 48 h before the experiments were performed in a humidified atmosphere (37°C, 5% CO2). At the beginning of the experiment, 600 µl of migration assay medium (serum-free RPMI 1640 in the absence or presence of MCP-1, SDF1, or VEGF; R&D Systems) was added to the lower compartment of the transwell system. EPCs (5 x 105 in 100 µL) were added to the top compartment in the presence or absence of 30 µg/ml of blocking anti–ß2-integrin antibody (mAb 60.3), anti–ß1-integrin antibody (clone 6S6; Chemicon), anti–4-integrin antibody (clone HP2.1; Immunotech), murine isotype control antibodies (Qbiogene), or RGD peptides. After 18 h at 37°C, the number of cells transmigrated to the bottom compartment was quantified in duplicates with a cell counter (CASY-Counter; Schärfe-System). All inserts were fixed and stained to confirm the confluence of the endothelial monolayer.
Animal experiments
Mice.
8-wk-old ß2–/– mice and their age-matched wild-type littermates (either 129/Sv or C57BL/6J) were generated as described previously (51). All mice were genotyped by Southern blot analysis (51) and maintained under pathogen-free conditions. Athymic NMRI nude mice (6–8 wk) were obtained from The Jackson Laboratory. The animal experiments were approved from the Regional Board of Land Hessen, Germany.
Model of hind limb ischemia
The proximal femoral artery, including the superficial and the deep branch as well as the distal saphenous artery, were ligated. In transplantation experiments, progenitor cells were intravenously injected in nude mice 24 h after induction of limb ischemia. Human EPCs were pretreated with 20 µg/ml activating ß2-integrin antibody (clone KIM 185) or isotype control antibody for 30 min at 37°C and washed twice to remove unbound antibodies before injection (105 EPCs/mouse). In some experiments, sex-mismatched murine Sca-1+/Lin– or Flk-1+/Lin– bone marrow cells from male ß2–/– or wild-type mice were used. After 2 wk, we calculated the ischemic (right) versus normal (left) limb blood flow ratio using a Laser Doppler blood flow imager (Moor Instruments).
Histology.
The capillary density and the number and size of conductant vessels in the semimembraneous and adductor muscles were determined using 8-µm cryosections. Endothelial cells were identified with the panendothelial marker MECA-32 followed by donkey anti–rat IgG Alexa488 or CD31-FITC (BD Biosciences). Injected human EPCs were identified by costaining for HLA-ABC (allophycocyanin labeled; BD Biosciences) and vWF (Acris). Male murine BM-derived cells were identified by fluorescence in situ hybridization for the murine Y-chromosome (Cy3-labeled probe: Cambio; reference 7). Nuclei were stained with Sytox (Molecular Probes). Images were obtained by confocal microscopy (LSM 510; Carl Zeiss MicroImaging, Inc.).
Statistical analysis
Continuous variables are expressed as mean ± SD or SEM. Comparisons between groups were analyzed by Student's t test (two-sided) or analysis of variance with Bonferroni adjustment for experiments with more than two subgroups (SPSS 11.5 software). p-values <0.05 were considered as statistically significant.
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Acknowledgments |
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This work was supported by the Forschergruppe 501 (He 3044/2-2 to C. Heeschen) and the Alfried Krupp-Stiftung (to S. Dimmeler). K. Sasaki was in part supported by the Japan Heart Foundation/Bayer Yakuhin Research Grant Abroad. The work of K. Scharffetter-Kochanek was funded in party by the Collaborative Research Center SFB497-C7, Ulm.
The authors have no conflicting financial interests.
Submitted: 12 July 2004
Accepted: 19 November 2004
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5 per group). (D and E) Incorporation of Y-chromosome positive injected murine Sca-1+/Lin– cells isolated from male wild-type or CD18–/– mice in female recipients after hind limb ischemia (n = 4 per group; n = 10 images per mouse were analyzed). Representative images (E) stained for the panendothelial marker MECA-32 (FITC, green), nuclei (Sytox; blue), and Y-chromosome (Cy3-labeled murine probe, red). Arrows indicate Y-chromosome positive cells.