Published online before print December 17, 2007, doi: 10.1161/CIRCULATIONAHA.107.714691
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(Circulation. 2008;117:206-215.)
© 2008 American Heart Association, Inc.
Molecular Cardiology |
Platelet-Derived Stromal Cell–Derived Factor-1 Regulates Adhesion and Promotes Differentiation of Human CD34+ Cells to Endothelial Progenitor Cells
From Medizinische Klinik III (K.S., H.L., K.D., T.S., A.G., T.G., B.B., I.M., I.S., P.S., B.F.K., D.S.-A., A.E.M., S.L., M.G.) and Kinderklinik (M.S.), Eberhard Karls-Universität Tübingen, Tübingen, Germany.
Correspondence to Meinrad Gawaz, MD, Medizinische Klinik III, Universitätsklinikum Tübingen, Otfried-Müller Straße 10, 72076 Tübingen, Germany. E-mail meinrad.gawaz{at}med.uni-tuebingen.de
Received May 10, 2007; accepted October 22, 2007.
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Abstract |
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Background— Peripheral homing of progenitor cells in areas of diseased organs is critical for tissue regeneration. The chemokine stromal cell–derived factor-1 (SDF-1) regulates homing of CD34+ stem cells. We evaluated the role of platelet-derived SDF-1 in adhesion and differentiation of human CD34+ cells into endothelial progenitor cells.
Methods and Results— Adherent platelets express substantial amounts of SDF-1 and recruit CD34+ cells in vitro and in vivo. A monoclonal antibody to SDF-1 or to its counterreceptor, CXCR4, inhibits stem cell adhesion on adherent platelets under high arterial shear in vitro and after carotid ligation in mice, as determined by intravital fluorescence microscopy. Platelets that adhere to human arterial endothelial cells enhance the adhesion of CD34+ cells on endothelium under flow conditions, a process that is inhibited by anti-SDF-1. During intestinal ischemia/reperfusion in mice, anti-SDF-1 and anti-CXCR4, but not isotype control antibodies, abolish the recruitment of CD34+ cells in microcirculation. Moreover, platelet-derived SDF-1 binding to CXCR4 receptor promotes platelet-induced differentiation of CD34+ cells into endothelial progenitor cells, as verified by colony-forming assays in vitro.
Conclusions— These findings imply that platelet-derived SDF-1 regulates adhesion of stem cells in vitro and in vivo and promotes differentiation of CD34+ cells to endothelial progenitor cells. Because tissue regeneration depends on recruitment of progenitor cells to peripheral vasculature and their subsequent differentiation, platelet-derived SDF-1 may contribute to vascular and myocardial regeneration.
Key Words: platelets • arteries • microcirculation • platelet-derived factors • revascularization
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Introduction |
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Regeneration of tissue or organs depends on recruitment and accumulation of a small population of self-renewing stem cells.1 Several organs (eg, myocardium and liver) may regenerate by attracting progenitor cells that circulate in peripheral blood after mobilization from their niches in response to stimuli related to tissue/organ damage.1 Accumulating evidence suggests that bone marrow–derived circulating progenitor cells contribute to vascular repair or remodeling under physiological or pathological conditions, respectively.2 Bone marrow–derived CD34+ stem cells support the integrity of the vascular endothelium.3,4 In patients with acute myocardial infarction, the ability of CD34+ cells to differentiate into endothelial cells influences functional improvement and infarct size reduction, which suggests that administration of progenitor cells could be a promising therapy to salvage ischemic damage.5 Recruitment and incorporation of endothelial progenitor cells requires a coordinated sequence of multistep adhesive and signaling events that includes chemoattraction, migration, adhesion, and finally, differentiation to endothelial cells.2 To extravasate to target tissue, circulating progenitor cells must be recruited and must firmly arrest on vascular endothelium within the microcirculation of peripheral organs; however, to date, the molecular determinants of progenitor cell adhesion to endothelium are incompletely understood.
Clinical Perspective p 215
It is well known that platelets recruit circulating leukocytes toward the inflamed endothelial monolayer.6 Activated platelets adhere to endothelial cells, secrete a variety of potent proinflammatory and mitogenic mediators, and thereby activate endothelial cells and change their chemotactic and adhesive properties.7 Enhanced platelet/endothelium adhesion occurs in the microcirculation of inflamed tissue and during reperfusion of ischemic organs.6,8
The chemokine stromal cell–derived factor-1 (SDF-1, CXCL12) and its receptor, CXCR4, regulate homing or trafficking of bone marrow–derived cells to bone marrow or circulation, respectively.9 The SDF-1 counterreceptor CXCR4 was detected on CD34+ cells purified from bone marrow, peripheral blood, and cord blood.10,11 SDF-1 plays a central role in the homing of circulating CD34+ cells in peripheral tissue such as ischemic myocardium,12 but the mechanisms of its action remain obscure. SDF-1 is also involved in recruitment of stem cells to the liver and to the site of vascular injury.13,14 Moreover, SDF-1 is expressed in atherosclerotic plaques, and its protein expression is upregulated in the heart early after myocardial infarction.15 SDF-1 mRNA and protein expression are enhanced primarily in the infarct zone of myocardium.15
Recently, we showed that platelets recruit bone marrow–derived progenitor cells to arterial thrombi in vivo by involving platelet P-selectin and glycoprotein IIb integrin and that activated platelets secrete SDF-1, which supports the migration and accumulation of murine embryonic endothelial progenitor cells into the platelet-rich thrombus in vivo.16 Furthermore, hematopoietic cytokines, through graded deployment of SDF-1 from platelets, support mobilization and recruitment of CXCR4+ vascular endothelial growth factor receptor (VEGFR) 1–positive (VEGFR1+) hemangiocytes.17 Nevertheless, the exact role of platelet-derived SDF-1 in the direct adhesion of CD34+ cells via their CXCR4 receptor on platelets has not been shown thus far. We recently reported that platelets regulate differentiation of human CD34+ cells into mature endothelial cells18; however, the impact of platelet-derived SDF-1 on the ability of progenitor cells to form endothelial colonies has not been elucidated.
In the present study, we analyzed the role of platelet-derived SDF-1 in adhesion of CD34+ cells on platelets adherent to collagen or to endothelial cells in vitro and after carotid ligation in mice or after ischemia/reperfusion injury in the intestinal microcirculation of mice in vivo. Moreover, we subsequently determined the impact of platelet-derived SDF-1 on platelet-induced differentiation of CD34+ cells into endothelial progenitor cells.
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Methods |
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Methods are described in detail in the online Data Supplement.
Isolation and Culture of Platelets, Human Arterial Endothelial Cells, and Human CD34+ Cells
Human platelets were isolated as described previously.19 Human arterial endothelial cells (HAECs) were isolated and passaged according to techniques described previously.20 Human CD34+ cells were isolated either from human cord blood or from bone marrow and cultured as described previously.18
Flow Cytometry
Expression of SDF-1 was determined on isolated platelets and HAECs with 1-color flow cytometry as described previously.21 CD34+ cells, HAECs, and progenitor cell–derived endothelial cells were tested for expression of CD146, ICAM-1 (intercellular adhesion molecule-1), VE-cadherin (vascular endothelial cadherin), PECAM-1 (platelet/endothelial cell adhesion molecule-1), CD34, CD18, and CD45.
Adhesion Assays Under Static and Dynamic Conditions
Adhesion of CD34+ cells to immobilized platelets and to HAECs was performed under static and dynamic conditions (flow chamber) as described previously.19
Carotid Ligation in Mice and Intravital Microscopy
To evaluate the effect of the platelet-derived SDF-1 on progenitor cell recruitment in vivo, the common carotid artery of wild-type C57BL/6J mice was injured by ligation, and dichlorofluorescein (DCF)-stained CD34+ cells were injected intravenously and visualized with intravital fluorescence microscopy as described previously.22
Intestinal Ischemia/Reperfusion Model in Mice
Fluorescent progenitor cells were infused after intestinal ischemia/reperfusion injury and visualized in the postischemic microcirculation by intravital fluorescence microscopy as described previously.8
Colony-Forming Unit Assay
To analyze the effect of SDF-1 and CXCR4 on platelet-induced CD34+ cell formation of endothelial progenitor cell colonies, isolated platelets were coincubated with CD34+ progenitor cells as described previously (see the Data Supplement).18
Reverse Transcription–Polymerase Chain Reaction
On differentiation of CD34+ progenitor cells to endothelial progenitor cell colonies, endothelial cells were further cultivated in culture flasks and analyzed for expression of mRNA for endothelial nitric oxide synthase, CD34, PECAM-1 (CD31), tie-2, flk-1(VEGFR-2), and β-actin by reverse transcription–polymerase chain reaction as described previously.19,23
Statistical Analysis
Data are presented as mean±SEM. For pairwise comparisons between anti-SDF-1 and control IgG1 or between anti-CXCR4 and control IgG2b, we applied a 2-tailed unpaired t test. For multiple comparisons between 3 or more groups, we applied an ANOVA analysis test with a subsequent Scheffé post hoc analysis. All tests were 2-tailed, and statistical significance was considered for probability values <0.05. All statistical analyses were performed with SPSS version 13 for Windows (SPSS Inc, Chicago, Ill).
The authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.
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Results |
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Platelet Surface Expression of SDF-1 Is Increased on Platelet Activation
To analyze whether surface expression of SDF-1 is enhanced on platelet activation, including adhesion on collagen, we evaluated the surface expression of SDF-1 on resting and activated washed platelets. Resting platelets do not show substantial surface expression of SDF-1 (Figure 2A); however, on activation with ADP (20 µmol/L), platelets degranulate and show surface expression of significant amounts of SDF-1 (resting platelets versus ADP-activated platelets: mean fluorescence intensity [MFI]±SEM 49±1.9 versus 74±8.6, P=0.047; n=3; Figure 1A). Similar results were obtained on activation of platelets with thrombin-related activating peptide (10 µmol/L; resting platelets versus platelets activated with thrombin-related activating peptide: mean MFI±SEM 49±1.9 versus 71.62±5.7, P=0.02; n=3; Figure 1A). Similarly, adhesion of platelets to immobilized collagen for 15 to 30 minutes resulted in enhanced surface expression of SDF-1 (resting platelets versus adherent platelets: mean MFI±SEM 49±1.9 versus 71.47±6.3, P=0.032; n=3; Figure 1A and 1B). Expression of platelet-bound P-selectin (CD62P) served as a positive control (Figure 1A), whereas isotype control IgG1 was used as a negative control. Thus, the degree of platelet-associated SDF-1 was dependent on the extent of platelet activation.
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Platelet-Derived SDF-1 and Immobilized SDF-1 Mediates Adhesion of CD34+ Cells Under Static Conditions
Next, we investigated whether platelet-derived SDF-1 recruits circulating progenitor cells. We found that human CD34+ cells adhere to immobilized platelets but not to immobilized collagen type I alone, which represents the major extracellular matrix component of the injured arterial wall (P<0.001; Figure 2A and 2B). Adhesion of CD34+ cells to immobilized platelets was significantly attenuated in the presence of a blocking monoclonal antibody (mAb) to SDF-1 or to its counterreceptor CXCR4 but not in the presence of the respective isotype control mAb, which indicates that platelet-derived SDF-1 is critical for progenitor cell–platelet interaction (P=0.002 or P=0.001, respectively; Figure 2A and 2B). As a control, the adhesion of CD34+ cells over immobilized platelets was not markedly attenuated in the presence of a blocking mAb to glycoprotein Ib, which is abundantly expressed on the surface of platelets, or to its counterreceptor Mac-1 (CD11b), which is expressed on CD34+ cells (Figure 2B).
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To verify the role of the SDF-1–CXCR4 axis on adhesion of CD34+ cells, we tested the adhesion of CD34+ cells on immobilized SDF-1 under static conditions. A blocking mAb to CXCR4 reduced the adhesion of progenitor cells on immobilized SDF-1 (P=0.021; Figure 2C and 2D).
Platelet-Derived SDF-1 Recruits CD34+ Cells Under High Arterial Shear In Vitro and at Sites of Vascular Injury In Vivo
To verify our findings under high shear conditions similar to arterial flow, we conducted perfusion experiments of CD34+ cells over adherent platelets on collagen in a parallel-plate flow chamber at a wall shear rate of 2000 s–1 (Figure 3A). A remarkable number of perfused CD34+ cells quickly turned into rolling and later into firm adherent cells over immobilized platelets (Figure 3B and 3C; Data Supplement Movie I). Preincubation of immobilized platelets or CD34+ cells with a neutralizing mAb to SDF-1 or CXCR4, respectively, attenuated both rolling and firm adhesion of CD34+ cells to immobilized platelets compared with respective control mAb (control IgG versus anti-SDF-1: rolling P=0.006, firm adhesion P=0.001; control IgG2b versus anti-CXCR4: rolling P=0.001, firm adhesion P=0.003; Figure 3B and 3C; Data Supplement Movie I).
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To further verify our findings in vivo, the common carotid artery of C57BL/6J mice was injured by ligation, and DCF-stained CD34+ cells were injected intravenously. As reported previously, the first response to vascular injury is platelet adhesion to exposed subendothelium,6 a process that contributes to the recruitment of murine progenitor cells to the injured carotid artery.16 Therefore, we addressed the biological relevance of platelet-derived SDF-1 for the recruitment of human CD34+ cells in injured carotid arteries in vivo. Preinfusion of a mAb to SDF-1 or preincubation of CD34+ cells with a mAb to CXCR4 resulted in decreased tethering (data not shown) and firm adhesion of progenitor cells to the injured vessel wall 5 and 30 minutes after carotid ligation compared with respective isotype control mAb (control IgG1 versus anti-SDF-1: 5 minutes P=0.0016, 30 minutes P=0.0014; control IgG2b versus anti-CXCR4: 5 minutes P=0.028, 30 minutes P=0.0084; Figure 4A, 4B, and 4C; Data Supplement Movie II). These data clearly demonstrate that platelet-derived SDF-1 critically regulates the adhesion of CD34+ cells in areas of platelet accumulation after vascular injury.
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Platelets Adherent to Endothelial Cells Support Recruitment of CD34+ Cells Toward Endothelium via Platelet-Derived SDF-1
Interaction of circulating progenitor cells with the endothelial monolayer of the vasculature of peripheral organs is critical for homing. Previously, we and others showed that platelets activate endothelial cells and support adhesion of leukocytes to the endothelium.24–26 Thus, we investigated whether platelets support recruitment of CD34+ cells to cultured HAECs. We found that cultured nonactivated endothelial cells do not support firm adhesion of CD34+ cells under arterial flow conditions (Figure 5A and 5B). However, when monolayers of HAECs were activated with tumor necrosis factor-
/interferon-
or interleukin-1β, adhesion of CD34+ cells was significantly enhanced (P=0.005 or P=0.016, respectively). Coincubation of HAECs with washed platelets caused a significant elevation of adherent CD34+ cells on the endothelial surface compared with resting HAECs (P<0.001; Figure 5A and 5B) and compared with HAECs stimulated with tumor necrosis factor-/interferon- or interleukin-1β (P=0.008 for both), which indicates that platelets play a critical role in recruitment of circulating CD34+ cells to the endothelium. Pretreatment of nonactivated or chemokine-activated (interleukin-1β) endothelial cells with an anti-SDF-1 mAb did not cause any significant reduction in adhesion of CD34+ (data not shown). In contrast, anti-SDF-1 significantly decreased both rolling and firm adhesion of CD34+ cells to platelets adherent to endothelium (P<0.05 and P<0.001, respectively; Figure 5C and 5D; Data Supplement Movie III).
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Flow cytometric analysis of SDF-1 expression on endothelial cells (whether activated with tumor necrosis factor-/interferon- or interleukin-1β or not) revealed that low levels of SDF-1 were constitutively expressed on the surface of both resting and activated endothelial cells. This indicates that although low levels of SDF-1 are expressed on the surface of endothelial cells, which are not influenced after chemokine activation, primarily or exclusively platelet-derived SDF-1 regulates CD34+ cell recruitment to endothelial monolayers.
Platelet-Derived SDF-1 Recruits CD34+ Progenitor Cells in Mouse Small Intestinal Microcirculation After Ischemia/Reperfusion Injury
Enhanced platelet/endothelium adhesion occurs in microcirculation (both venules and arterioles) of inflamed tissue and during reperfusion of ischemic organs.6,8 To evaluate the biological significance of platelet-derived SDF-1 in the regulation of peripheral homing of progenitor cells in vivo, fluorescent CD34+ cells were infused before intestinal ischemia/reperfusion injury and visualized in the postischemic microcirculation by intravital fluorescence microscopy. Although CD34+ cells adhered to both venules and arterioles, an increased adhesion of progenitor cells was observed in venules compared with arterioles (data not shown). Preinfusion of a mAb to SDF-1 or preincubation of CD34+ cells with a mAb to CXCR4 virtually blocked the adhesion of progenitor cells in arterioles (data not shown) and in venules (Figure 6A and 6B; Data Supplement Movie IV).
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Platelet-Derived SDF-1 Promotes Differentiation of CD34+ Cells Into Endothelial Progenitor Cells
Recently, we reported that adherent platelets cause CD34+ cell differentiation to endothelial cells.19 To further evaluate the molecular requirements of platelet-dependent differentiation of progenitor cells, CD34+ cells were coincubated with isolated platelets as described previously. Isolated CD34+ cells were allowed to adhere to immobilized platelets, immobilized fibronectin, or collagen and were cultivated in endothelial cell growth medium as described previously.19 CD34+ cells formed colonies on immobilized platelets similar to immobilized fibronectin, which indicates differentiation into endothelial cells (Figure 7A and 7B). After 5 days, the morphology of initially round CD34+ cells on immobilized platelets evolved into adherent spindle-shaped cells that were positive for von Willebrand factor and revealed typical cytoskeletal rearrangement as demonstrated by phalloidin staining (Figure 7C). In contrast, virtually no colonies were formed on collagen, which is the major extracellular matrix protein (Figure 7A and 7B). The number of colonies formed on immobilized platelets was significantly higher than in fibronectin or collagen wells (mean number of colonies±SEM: platelets versus fibronectin versus collagen 12±0.84 versus 8.9±0.7 versus 1±1, P<0.001; Figure 7A and 7B).
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Next, we evaluated the effect of SDF-1 on platelet-mediated formation of endothelial colonies. We found that in the presence of a neutralizing anti-SDF-1 or anti-CXCR4, the formation of endothelial progenitor cell colonies was significantly inhibited compared with respective isotype control mAb (mean number of colonies±SEM: control IgG1 versus anti-SDF-1 11.5±0.86 versus 3.25±0.75, P<0.001; control IgG2b versus anti-CXCR4 11.5±0.5 versus 3±0.58, P=0.002; Figure 7A and 7B). Similarly, anti-SDF-1 attenuated the expression of the endothelium-specific marker CD146 compared with IgG control, which reflects the lower number of endothelial colonies (Figure 7D). Together, these data clearly indicate that platelet-derived SDF-1 plays a crucial role in recruitment and favors differentiation of human CD34+ cells into endothelial progenitor cells.
Verification of CD34+ cell differentiation into endothelial progenitor cells was performed with von Willebrand factor immunofluorescence staining and flow cytometry. Specifically, surface expression of CD146, ICAM-1, VE-cadherin, PECAM-1, CD34, CD18, and CD45 was tested on progenitor cells by flow cytometry, as described in Methods. CD34+ cells and HAECs were used as a negative and positive control, respectively. When CD34+ cells were cultivated to form colony-forming units, they exhibited similar endothelial surface markers (such as CD146, CD144, or CD31) as primary endothelial cell cultures cultivated from human arteries (Figure 8A). Polymerase chain reaction analysis showed that CD34+ cell–derived endothelial progenitor cells exhibited positive signals for endothelial nitric oxide synthase, Tie-2, and VEGFR-2, similar to those signals obtained from arterial endothelial cells (Figure 8B). Next, we analyzed whether CD34+ cell–derived endothelial progenitor cells could be activated to express activation-dependent surface markers such as ICAM-1 (CD54) and CD106. We found that stimulation of CD34+ cell–derived endothelial progenitor cells with tumor necrosis factor-/interferon- cytokines resulted in enhanced expression of CD54 and CD106 similar to the activation profile obtained when HAECs were used (Figure 8C).
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Discussion |
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The major findings of the present study are as follows: (1) Expression of SDF-1 on the surface of adherent platelets is increased and mediates rolling and firm adhesion of CD34+ progenitor cells on immobilized platelets under flow conditions in vitro and at sites of vascular injury in vivo. (2) Platelets adherent on endothelial cells support the recruitment of human CD34+ cells toward endothelium in vitro under high arterial shear and in microcirculation of mice after ischemia/reperfusion injury via platelet-derived SDF-1. (3) Platelet-derived SDF-1 promotes differentiation of CD34+ cells into endothelial progenitor cells.
These findings imply that activated platelets and platelet-membrane bound SDF-1 are critically involved in the recruitment of circulating CD34+ progenitor cells at sites of vascular injury and in the microcirculation of ischemic tissue (eg, myocardium), where enhanced platelet adhesion and aggregate formation occur.8,20 Interaction of platelets with CD34+ cells in peripheral organs is regulated by platelet-derived SDF-1, which may play an important role in structural and functional repair mechanisms in several diseased organs (eg, heart, liver, and brain) in which substantial accumulation of platelets has been documented during ischemia/reperfusion.13
Circulating progenitor cells have been shown to instigate new vessel formation via angiogenesis and neovascularization but also have the potential to provide ongoing vascular and tissue repair by homing to sites of vascular or tissue damage.27 Bone marrow–derived stem cells, including hematopoietic CD34+ stem cells, can exhibit tremendous cellular differentiation in numerous organs. Bone marrow–derived stem cells may also promote structural and functional repair in several organs, such as the heart, liver, or brain. CD34+ cells have been described to be recruited to the ischemic myocardium, where they differentiate into cardiac and vascular cells and restore cardiac function.28 Thus, the identification of cellular mediators and tissue-specific chemokines that facilitate selective recruitment of bone marrow–derived stem and progenitor cells to specific organs is a critical step for the development of new strategies to accelerate cardiovascular regeneration and tissue revascularization. However, the mechanisms that recruit circulating progenitor cells toward vascular lesions and regulate repair mechanisms of ischemic peripheral organs are still incompletely understood.28
Recent studies have identified specific molecular signals, such as SDF-1/CXCR4, required for the interaction of bone marrow–derived stem cells and damaged host tissues. SDF-1, the ligand for CXCR4, plays a crucial role in the trafficking of CXCR4-positive circulating bone marrow–derived cells into diseased organs. SDF-1 is expressed by various endothelial cells29 and fibroblasts/osteoblasts of various organs, including heart,15 skeletal muscle,30 liver,13 brain,31 and kidney.32 Moreover, SDF-1 secretion is increased during tissue damage such as myocardial infarction33 and hind-limb ischemia,4 which suggests that SDF-1 may play a pivotal role in chemoattraction of the CXCR4+ cells necessary for organ/tissue regeneration.29,34,35
Platelets are the first circulating blood cells that interact with the injured vessel wall or that adhere to inflamed endothelium of ischemic tissue.6 Our previous16 and present data imply that CD34+ cells bind to these platelets and initiate endothelial microvascular repair. Activated platelets release a variety of potent proinflammatory and chemotactic factors (eg, interleukin-1β)26,34 and growth factors (platelet-derived growth factor)36 that in turn activate endothelium and support recruitment of circulating blood cells.22 Thus, we investigated whether platelet/endothelium interaction and platelet-derived SDF-1 regulate adhesion of CD34+ cells to the endothelium. The present data show that platelet-derived SDF-1 mediates adhesion of CD34+ cells onto immobilized platelets and endothelium; however, only a small percentage of CD34+ cells initially adherent on immobilized platelets gave rise to endothelial colony cells. Thus, we conclude that most of the platelet-derived SDF-1–dependent recruitment of CD34+ cells constitutes non–endothelial cell–forming CD34+ cells. At present, we do not know the pathophysiological significance of CD34+ cells that do not differentiate into endothelial cells in the presence of platelets. Nevertheless, because substantial platelet adhesion to the endothelium occurs in the microcirculation of ischemic tissue during reperfusion,37 the present findings indicate that platelet-dependent CD34+ cell homing in peripheral organs is critically involved in the regeneration of vascular lesions or ischemic tissue. Moreover, SDF-1 activates the integrins lymphocyte function-associated antigen-1 (LFA-1), very late antigen (VLA)-4, and VLA-5 on immature human CD34+ cells, which play a role in transendothelial/stromal migration and engraftment and in arrest on vascular endothelium.38,39 However, the impact of platelets is not limited to their role in cell recruitment. Recently, we showed that platelets induce differentiation of CD34+ cells into endothelial cells.19 Thus, platelets also may have a major impact on tissue regeneration and angiogenesis through induction of the differentiation of progenitor cells. Herein, we have described that simultaneous with cell adhesion, platelet-derived SDF-1 favors generation of endothelial progenitor cells from CD34+ cells. In the present study, we proved that blocking the SDF-1–CXCR4 axis resulted in reduced formation of endothelial progenitor cell colonies, but we were unable to show the exact mechanism responsible for platelet-derived SDF-1–dependent endothelial progenitor cell colony formation. Decreased adhesion onto platelets and/or blockage of CXCR4 activation could explain to some extent the decreased formation of endothelial progenitor cell colonies in the presence of anti-SDF-1 or anti-CXCR4. Further studies are needed to determine the exact mechanism responsible for the platelet-bound SDF-1–dependent differentiation of CD34+ cells to endothelial progenitor cells.
Homing of progenitor cells in peripheral tissue is a multistep cascade that includes initial adhesion to activated endothelium or exposed matrix, transmigration through the endothelium, and invasion of the target tissue. Because platelets are the first circulating blood cells that adhere to vascular lesions and that accumulate in the microcirculation within ischemic tissue, platelet-derived SDF-1 appears to be a key factor that regulates trafficking of stem and progenitor cells to ischemic tissue. Local delivery of SDF-1 can enhance progenitor cell recruitment and neovascularization.15 Although platelet/progenitor cell interaction may play a physiological role in repair mechanisms of damaged organs, understanding of the underlying molecular determinants may offer new strategies to support vascular repair and tissue regeneration of ischemic organs.
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Acknowledgments |
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We thank Jadwiga Kwiatkowska for providing outstanding technical assistance.
Sources of Funding
This study was supported in part by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg GK794, MA121/2-1, Li849/3-1 to Dr Lindemann) and the Wilhelm Sander Foundation, Novartis Foundation, the Karl & Lore Klein Foundation, and the Karl Kuhn Foundation (to Dr Gawaz).
Disclosures
None.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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1. Ratajczak MZ, Zuba-Surma E, Kucia M, Reca R, Wojakowski W, Ratajczak J. The pleiotropic effects of the SDF-1–CXCR4 axis on organogenesis, regeneration and tumorigenesis. Leukemia. 2006; 20: 1915–1924.[CrossRef][Medline] [Order article via Infotrieve]
2. Urbich C, Dimmeler S. Endothelial progenitor cells: characterization and role in vascular biology. Circ Res. 2004; 95: 343–353.
3. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275: 964–967.
4. Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, Magner M, Isner JM, Asahara T. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med. 1999; 5: 434–438.[CrossRef][Medline] [Order article via Infotrieve]
5. Numaguchi Y, Sone T, Okumura K, Ishii M, Morita Y, Kubota R, Yokouchi K, Imai H, Harada M, Osanai H, Kondo T, Murohara T. The impact of the capability of circulating progenitor cell to differentiate on myocardial salvage in patients with primary acute myocardial infarction. Circulation. 2006; 114 (suppl I): I-114–I-119.[Medline] [Order article via Infotrieve]
6. Massberg S, Gruner S, Konrad I, Garcia Arguinzonis MI, Eigenthaler M, Hemler K, Kersting J, Schulz C, Muller I, Besta F, Nieswandt B, Heinzmann U, Walter U, Gawaz M. Enhanced in vivo platelet adhesion in vasodilator-stimulated phosphoprotein (VASP)-deficient mice. Blood. 2004; 103: 136–142.
7. Gawaz M, Langer H, May AE. Platelets in inflammation and atherogenesis. J Clin Invest. 2005; 115: 3378–3384.[CrossRef][Medline] [Order article via Infotrieve]
8. Massberg S, Enders G, Leiderer R, Eisenmenger S, Vestweber D, Krombach F, Messmer K. Platelet-endothelial cell interactions during ischemia/reperfusion: the role of P-selectin. Blood. 1998; 92: 507–515.
9. Aiuti A, Webb IJ, Bleul C, Springer T, Gutierrez-Ramos JC. The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood. J Exp Med. 1997; 185: 111–120.
10. Lataillade JJ, Clay D, Dupuy C, Rigal S, Jasmin C, Bourin P, Le Bousse-Kerdiles MC. Chemokine SDF-1 enhances circulating CD34(+) cell proliferation in synergy with cytokines: possible role in progenitor survival. Blood. 2000; 95: 756–768.
11. Broxmeyer HE, Orschell CM, Clapp DW, Hangoc G, Cooper S, Plett PA, Liles WC, Li X, Graham-Evans B, Campbell TB, Calandra G, Bridger G, Dale DC, Srour EF. Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist. J Exp Med. 2005; 201: 1307–1318.
12. Abbott JD, Huang Y, Liu D, Hickey R, Krause DS, Giordano FJ. Stromal cell-derived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury. Circulation. 2004; 110: 3300–3305.
13. Kollet O, Shivtiel S, Chen YQ, Suriawinata J, Thung SN, Dabeva MD, Kahn J, Spiegel A, Dar A, Samira S, Goichberg P, Kalinkovich A, Arenzana-Seisdedos F, Nagler A, Hardan I, Revel M, Shafritz DA, Lapidot T. HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver. J Clin Invest. 2003; 112: 160–169.[CrossRef][Medline] [Order article via Infotrieve]
14. Schober A, Knarren S, Lietz M, Lin EA, Weber C. Crucial role of stromal cell-derived factor-1alpha in neointima formation after vascular injury in apolipoprotein E-deficient mice. Circulation. 2003; 108: 2491–2497.
15. Askari AT, Unzek S, Popovic ZB, Goldman CK, Forudi F, Kiedrowski M, Rovner A, Ellis SG, Thomas JD, DiCorleto PE, Topol EJ, Penn MS. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet. 2003; 362: 697–703.[CrossRef][Medline] [Order article via Infotrieve]
16. Massberg S, Konrad I, Schurzinger K, Lorenz M, Schneider S, Zohlnhoefer D, Hoppe K, Schiemann M, Kennerknecht E, Sauer S, Schulz C, Kerstan S, Rudelius M, Seidl S, Sorge F, Langer H, Peluso M, Goyal P, Vestweber D, Emambokus NR, Busch DH, Frampton J, Gawaz M. Platelets secrete stromal cell-derived factor 1alpha and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo. J Exp Med. 2006; 203: 1221–1233.
17. Jin DK, Shido K, Kopp HG, Petit I, Shmelkov SV, Young LM, Hooper AT, Amano H, Avecilla ST, Heissig B, Hattori K, Zhang F, Hicklin DJ, Wu Y, Zhu Z, Dunn A, Salari H, Werb Z, Hackett NR, Crystal RG, Lyden D, Rafii S. Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+ hemangiocytes. Nat Med. 2006; 12: 557–567.[CrossRef][Medline] [Order article via Infotrieve]
18. Daub K, Langer H, Seizer P, Stellos K, May AE, Goyal P, Bigalke B, Schonberger T, Geisler T, Siegel-Axel D, Oostendorp RA, Lindemann S, Gawaz M. Platelets induce differentiation of human CD34+ progenitor cells into foam cells and endothelial cells. FASEB J. 2006; 20: 2559–2561.
19. Langer H, May AE, Daub K, Heinzmann U, Lang P, Schumm M, Vestweber D, Massberg S, Schonberger T, Pfisterer I, Hatzopoulos AK, Gawaz M. Adherent platelets recruit and induce differentiation of murine embryonic endothelial progenitor cells to mature endothelial cells in vitro. Circ Res. 2006; 98: e2–e10.
20. Gawaz M, Neumann FJ, Ott I, Schiessler A, Schomig A. Platelet function in acute myocardial infarction treated with direct angioplasty. Circulation. 1996; 93: 229–237.
21. Gawaz M, Neumann FJ, Schomig A. Evaluation of platelet membrane glycoproteins in coronary artery disease: consequences for diagnosis and therapy. Circulation. 1999; 99: e1–e11.
22. Massberg S, Brand K, Gruner S, Page S, Muller E, Muller I, Bergmeier W, Richter T, Lorenz M, Konrad I, Nieswandt B, Gawaz M. A critical role of platelet adhesion in the initiation of atherosclerotic lesion formation. J Exp Med. 2002; 196: 887–896.
23. May A, Kalsch T, Massberg S, Herouy Y, Schmidt R, Gawaz M. Engagement of glycoprotein IIb/IIIa (alpha(IIb)beta3) on platelets upregulates CD40L and triggers CD40L-dependent matrix degradation by endothelial cells. Circulation. 2002; 106: 2111–2117.
24. Gawaz M, Neumann FJ, Dickfeld T, Reininger A, Adelsberger H, Gebhardt A, Schomig A. Vitronectin receptor (alpha(v)beta3) mediates platelet adhesion to the luminal aspect of endothelial cells: implications for reperfusion in acute myocardial infarction. Circulation. 1997; 96: 1809–1818.
25. Gawaz M, Neumann FJ, Dickfeld T, Koch W, Laugwitz KL, Adelsberger H, Langenbrink K, Page S, Neumeier D, Schomig A, Brand K. Activated platelets induce monocyte chemotactic protein-1 secretion and surface expression of intercellular adhesion molecule-1 on endothelial cells. Circulation. 1998; 98: 1164–1171.
26. Gawaz M, Brand K, Dickfeld T, Pogatsa-Murray G, Page S, Bogner C, Koch W, Schomig A, Neumann F. Platelets induce alterations of chemotactic and adhesive properties of endothelial cells mediated through an interleukin-1-dependent mechanism: implications for atherogenesis. Atherosclerosis. 2000; 148: 75–85.[CrossRef][Medline] [Order article via Infotrieve]
27. Xu Q. The impact of progenitor cells in atherosclerosis. Nat Clin Pract Cardiovasc Med. 2006; 3: 94–101.[CrossRef][Medline] [Order article via Infotrieve]
28. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientific foundations of cardiac repair. J Clin Invest. 2005; 115: 572–583.[CrossRef][Medline] [Order article via Infotrieve]
29. Ceradini DJ, Kulkarni AR, Callaghan MJ, Tepper OM, Bastidas N, Kleinman ME, Capla JM, Galiano RD, Levine JP, Gurtner GC. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat Med. 2004; 10: 858–864.[CrossRef][Medline] [Order article via Infotrieve]
30. Ratajczak MZ, Majka M, Kucia M, Drukala J, Pietrzkowski Z, Peiper S, Janowska-Wieczorek A. Expression of functional CXCR4 by muscle satellite cells and secretion of SDF-1 by muscle-derived fibroblasts is associated with the presence of both muscle progenitors in bone marrow and hematopoietic stem/progenitor cells in muscles. Stem Cells. 2003; 21: 363–371.
31. Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature. 1998; 393: 595–599.[CrossRef][Medline] [Order article via Infotrieve]
32. Schrader AJ, Lechner O, Templin M, Dittmar KE, Machtens S, Mengel M, Probst-Kepper M, Franzke A, Wollensak T, Gatzlaff P, Atzpodien J, Buer J, Lauber J. CXCR4/CXCL12 expression and signalling in kidney cancer. Br J Cancer. 2002; 86: 1250–1256.[CrossRef][Medline] [Order article via Infotrieve]
33. Wojakowski W, Tendera M, Michalowska A, Majka M, Kucia M, Maslankiewicz K, Wyderka R, Ochala A, Ratajczak MZ. Mobilization of CD34/CXCR4+, CD34/CD117+, c-met+ stem cells, and mononuclear cells expressing early cardiac, muscle, and endothelial markers into peripheral blood in patients with acute myocardial infarction. Circulation. 2004; 110: 3213–3220.
34. Kawamoto A, Gwon HC, Iwaguro H, Yamaguchi JI, Uchida S, Masuda H, Silver M, Ma H, Kearney M, Isner JM, Asahara T. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation. 2001; 103: 634–637.
35. Korbling M, Estrov Z. Adult stem cells for tissue repair: a new therapeutic concept? N Engl J Med. 2003; 349: 570–582.
36. Massberg S, Vogt F, Dickfeld T, Brand K, Page S, Gawaz M. Activated platelets trigger an inflammatory response and enhance migration of aortic smooth muscle cells. Thromb Res. 2003; 110: 187–194.[CrossRef][Medline] [Order article via Infotrieve]
37. Moers A, Nieswandt B, Massberg S, Wettschureck N, Gruner S, Konrad I, Schulte V, Aktas B, Gratacap MP, Simon MI, Gawaz M, Offermanns S. G13 is an essential mediator of platelet activation in hemostasis and thrombosis. Nat Med. 2003; 9: 1418–1422.[CrossRef][Medline] [Order article via Infotrieve]
38. Peled A, Kollet O, Ponomaryov T, Petit I, Franitza S, Grabovsky V, Slav MM, Nagler A, Lider O, Alon R, Zipori D, Lapidot T. The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood. 2000; 95: 3289–3296.
39. Peled A, Grabovsky V, Habler L, Sandbank J, Arenzana-Seisdedos F, Petit I, Ben-Hur H, Lapidot T, Alon R. The chemokine SDF-1 stimulates integrin-mediated arrest of CD34(+) cells on vascular endothelium under shear flow. J Clin Invest. 1999; 104: 1199–1211.[Medline] [Order article via Infotrieve]
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The online-only Data Supplement, consisting of an expanded Methods section and movies, is available with this article at http://circ.ahajournals.org/ cgi/content/full/CIRCULATIONAHA.107.714691/DC1.
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