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Circulation Research. 2006;98:e2-e10
Published online before print December 22, 2005, doi: 10.1161/01.RES.0000201285.87524.9e
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(Circulation Research. 2006;98:e2.)
© 2006 American Heart Association, Inc.

UltraRapid Communications

Adherent Platelets Recruit and Induce Differentiation of Murine Embryonic Endothelial Progenitor Cells to Mature Endothelial Cells In Vitro

Harald Langer, Andreas Eberhard May, Karin Daub, Ulrich Heinzmann, Peter Lang, Michael Schumm, Dietmar Vestweber, Steffen Massberg, Tanja Schönberger, Iris Pfisterer, Antonis K. Hatzopoulos, Meinrad Gawaz

From the Medizinische Klinik III (H.L., A.E.M., K.D., T.S., M.G.), Eberhard Karls Universität Tübingen, Germany; GSF-Research Center for Environment and Health (U.H.), Institute of Pathology, Munich; Clinic for Pediatrics (P.L., M.S.), Eberhard Karls Universität Tübingen, Germany; Institute of Cell Biology (D.V.), ZMBE, University of Münster, Germany; Deutsches Herzzentrum München (S.M.), Technische Universität München, Germany; and GSF-Research Center for Environment and Health (I.P., A.K.H.), Institute for Clinical Molecular Biology and Tumor Genetics, Munich, Germany and Vanderbilt University Medical Center, Division of Cardiovascular Medicine, Nashville, Tenn.

Correspondence to Meinrad Gawaz, MD, Medizinische Klinik III, Universitätsklinikum Tübingen, Otfried-Müllerstr.10, 72076 Tübingen, Germany. E-mail meinrad.gawaz{at}med.uni-tuebingen.de


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The homing and differentiation mechanisms of endothelial progenitor cells (EPCs) at sites of vascular lesions are unclear. To investigate whether platelets play a role in the recruitment and differentiation of EPCs, we made use of a robust mouse embryonic EPC (eEPC) line that reliably differentiates to a mature endothelial phenotype. We found that platelets stimulate chemotaxis and migration of these murine eEPCs. Further, the substantial adhesion of murine eEPCs on immobilized platelets that occurs under dynamic flow conditions is inhibited by neutralizing anti–P-selectin glycoprotein ligand-1 and anti–VLA-4 (ß1-integrin) monoclonal antibodies but not by anti-CD11b (aM-integrin; macrophage antigen-1). Coincubation of murine eEPCs with platelets for 5 days induced differentiation of EPCs to mature endothelial cells as verified by positive von Willebrand factor immunofluorescence and detection of Weibel Palade bodies through electron microscopy. We conclude that platelets may play a critical part in the capture and subsequent differentiation of murine eEPCs at sites of vascular lesions, revealing a possible new role of platelets in neoendothelization after vascular injury.


Key Words: endothelial progenitor cells • platelets • endothelization • vascular lesion • adhesion


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Vascular endothelium represents a barrier between circulating blood cells and the subendothelial matrix. Discontinuity of this barrier (eg, at sites of vascular injury or rupture of an atherosclerotic plaque) exposes various components of the subendothelial matrix, including collagen, fibronectin, or von Willebrand factor (vWF).1 Platelets rapidly adhere to vascular lesions, resulting in thrombus formation.1,2 If this process is controlled, platelets passivate vascular injury and initiate the healing process.1 However, uncontrolled platelet-mediated thrombus formation leads to acute thrombotic occlusion or plaque progression.2 However, the underlying mechanisms of platelet-mediated vascular remodeling and healing of lesions is poorly understood so far.

Endothelial progenitor cells (EPCs) are a circulating, bone marrow–derived cell population of large nonleukocyte cells that appear to participate in vascular repair and homeostasis. In response to cytokine stimulation and ischemia, these cells are mobilized from the bone marrow, home to the ischemic tissue, and contribute to neovascularization and angiogenesis.3–6 Furthermore, EPCs are regarded to have a key role in the maintenance of vascular integrity and to act as "repair" cells in response to endothelial injury.7,8 Several groups have raised colonies in vitro from CD34+ cells, which resemble mature endothelial cells, implying that EPCs are capable of promoting endothelization.9 Recruitment of EPCs toward vascular lesions has been regarded as a critical initial step in atherosclerosis and a result of the actions of various cardiovascular risk factors.10 Current data suggest that decrease in circulating EPCs contributes not only to impaired angiogenesis but also to the progression of atherosclerosis,11 and patients at risk for coronary artery disease have a decreased number of circulating EPCs with impaired activity.9,12–14

However, the mechanisms that regulate recruitment of EPCs toward vascular lesions and initiate neoendothelization are not understood. In general, recruitment of circulating cells is a well-controlled process that involves chemotaxis, tethering, adhesion, and migration of cells into the subendothelial tissues. Several adhesion molecules have been described to regulate adhesion and migration of EPCs, including P-selectin glycoprotein ligand (PSGL)-1 and ß1- and ß2-integrins.15–17

Adherent platelets secrete potent chemokines at sites of vascular injury and display several ligands for adhesion receptors on their surface, and therefore, they represent potential mediators of progenitor cell homing.18 In this study, we investigated the capacity of platelets to induce chemotaxis, adhesion, and migration of EPCs and provide evidence that platelets may also contribute to the differentiation of EPCs into endothelial cells.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Reagents
RB40.34 (function-blocking anti-mouse P-selectin) and 4RA10 (function-blocking anti-mouse PSGL-1) were raised as described previously.19,20 A goat anti-human polyclonal antibody (monoclonal antibody [mAb]) directed against vWF (C-20), which cross-reacts with mouse vWF, and N6,2'-O-dibutyryladenosine3':5'-cyclic monophosphate (cAMP) were purchased from Santa Cruz Biotechnology. Phycoerythrin or fluorescein isothiocyanate (FITC)–conjugated mAb rat anti-mouse CD162 (PSGL-1; clone 2PH1), CD49d (VLA-4; {alpha}-chain; clone 9C10), CD11b (clone M1/70), and blocking mAb anti-mouse CD29 (ß1-integrin; clone 18) were purchased from Pharmingen. mAb anti-CD41 (GPIIb; clone P2) was from Beckmann Coulter. For immunofluorescence microscopy, a goat polyclonal antibody to mouse vWF (clone C20; Santa Cruz Biotechnology), an Alexa Fluor rabbit anti-goat IgG (Molecular Probes), and rhodamine phalloidin (R415; Molecular Probes) were used. A rabbit antibody to human vWF was from Dako Cytomation GmbH. Secondary Cy3-labeled sheep anti-rabbit antibody and bovine collagen type I were purchased from Sigma. Tissue Tek for histological fixation of specimen was from Sukura.

Cell Culture of Murine Embryonic EPCs and Human CD34+ Cells
For our studies, we used the murine embryonic EPC (eEPC) line T17b. The cells were isolated from the egg cylinders with adjacent yolk sacs of mice embryos at embryonic days 7.5 to 8.5 as described in detail previously.21 A major advantage of this cell line is the robust growth properties in culture, the stable morphological phenotype, and the absence of difficulties to isolate and maintain a purified lineage of endothelial precursor cells. The characterization of the eEPCs has been described previously.15,21 The cells are cultured in flasks precoated with 0.1% gelatin. The culture medium consists of DMEM supplemented with 20% heat-inactivated FCS, 0.1 mmol/L ß-mercaptoethanol, 1 mmol/L nonessential amino acids, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L L-glutamine (Invitrogen). For immunofluorescence microscopy, the mature mouse endothelial cell line mouse heart endothelial cell clone 5-T (MHEC5-T) served as positive control and was cultured as described previously.22 Human CD34+ cells were isolated from human cord blood and cultured as described previously.23

Isolation of Platelets and Preparation of Platelet Clots
Human platelets were isolated as described previously.24 Briefly, venous blood was drawn from the antecubital vein of healthy volunteers and collected in acid-citrate-dextrose buffer. After centrifugation at 430g for 20 minutes, platelet-rich plasma was removed, added to Tyrodes–HEPES buffer (2.5 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L KCl, 2.5 mmol/L NaHCO3, 0.36 mmol/L NaH2PO4, 5.5 mmol/L glucose, and 1 mg/mL BSA, pH 6.5), and centrifuged at 900g for 10 minutes. After removal of the supernatant, the resulting platelet pellet was resuspended in Tyrodes–HEPES buffer (pH 7.4 supplemented with 1 mmol/L CaCl2 and 1 mmol/L MgCl2). Murine platelets were isolated from pathogen-free C57 BL/6J mice (Charles River Laboratories, Wilmington, Mass) as described.25 In some experiments, a platelet clot was generated through addition of 2U/mL thrombin to platelet-rich plasma. The forming clot was harvested with sterile instruments and used for subsequent experiments.

Migration and Chemotaxis of eEPCs
To evaluate the effect of platelets on chemotaxis and migration of EPCs, a trans-well culture system was used.26 In brief, isolated platelets were allowed to adhere to the bottom of 24-well culture plates and stimulated with a-thrombin 1U/mL for 60 minutes. T17b cells (100 000/mL) were added on top using trans-well culture inserts (5.0 µm pore size; Costar) that allowed physical separation of T17b cells from platelets. After 24 hours, the trans-well insert was removed, and migrated T17b cells were stained with crystal-violet and densitometrically quantified.

Platelet Adhesion to eEPCs Under Static and Dynamic Conditions
Static Adhesion
To evaluate eEPC/platelet adhesion under static conditions, isolated platelets (2x108/mL) were allowed to adhere to 96-well plates for 2 hours. Subsequently, T17b cells were added and incubated for 60 minutes. After 3 gentle washing steps with Tyrodes buffer, residual adherent eEPCs were counted by direct phase contrast microscopy. In a second adhesion assay, T17b (1x105/mL) cells were incubated for 30 minutes with isolated platelets (2x108/mL) or buffer as control under agitation (1000 rpm) using a conventional platelet aggregometer. Thereafter, cell suspension was washed 3 times with Tyrodes buffer, and eEPCs were incubated with FITC-conjugated anti-CD41 or control anti-CD11b mAb. The percentage of CD41-positive T17b cells was analyzed by flow cytometry and was used as index of platelet/eEPC adhesion.

Dynamic Adhesion
Adhesion experiments under flow conditions were basically performed as described previously.27 In brief, glass coverslips were coated with collagen type I (5 µg/mL) as described previously28 and used in a flow chamber (Oligene). Thereafter, isolated platelets (2x108/mL) were allowed to adhere to collagen for 30 minutes. In some experiments, adherent platelets were pretreated with anti-CD62P mAb (5 µg/mL) before eEPCs were perfused over immobilized platelets. In other experiments, eEPCs (T17b) were preincubated with anti-CD29, anti-CD162, or anti-CD11b (5 µg/mL). Perfusion was performed with T17b cells resuspended in Tyrodes–HEPES buffer (pH 7.4 supplemented with 1 mmol/L CaCl2 and 1 mmol/L MgCl2) at shear rates of 2000 s–1 (high shear). All experiments were recorded in real time on videotape and evaluated offline using Capimage software (Zeintl).

Scratch Assay
eEPCs were grown to confluence in a 24-well plate. Subsequently, the cell monolayer was wounded with a plastic pipette tip generating a gap of &1-mm width, and cells were incubated with isolated platelets resuspended in medium (0.1 to 1x108/mL) or growth medium alone as control. Migrated cells were counted after 24 hours. Thereafter, wells were incubated with an anti-vWF antibody (5 µg/mL) and a secondary Alexa Fluor 488–conjugated rabbit anti-goat antibody, as described above, and analyzed by immunofluorescence microscopy, as described below. To detect specific mediators of this platelet-mediated migration, blocking mAbs to platelet-derived growth factor-AB (Promega), CD40L (Biozol), CD11b (clone M1/70; Pharmingen), and CD54 (Beckmann Coulter), using concentrations of 5 µg/mL for each substance, were used. Activation protein tumor necrosis factor-{alpha} (Tebu-bio; 20 ng/mL) served as positive control.

Scanning and Transmission Electron Microscopy
For scanning electron microscopy (SEM), T17b cells were cultivated on coverslips in the absence or presence of platelets (2x108 cells/mL) for 24 hours. Thereafter, the cells were washed 2 times with PBS, and the coverslips were fixed and examined using a field emission scanning electron microscope (JSM-6300F; Jeol Ltd.). For transmission electron microscopy, eEPCs were grown to 70% to 80% confluence and coincubated with isolated platelets (2x108/mL) for 5 days in culture medium. Subsequently, cells were washed and fixed using a buffer consisting of glutaraldehyde (2.5%) and tannin (0.02%) in a sodium cacodylate buffer, pH 7.4, before electron microscopy was performed.27

Reverse Transcription–Polymerase Chain Reaction
After incubation of T17b cells with buffer alone, cAMP (0.5 mmol/L), or isolated platelets (4x108/mL) for 5 days, the 6-well plates were carefully washed with PBS and analyzed by RT-PCR as described.29 Briefly, total RNA was extracted using the RNeasy Mini Kit (Qiagen). Contaminating DNA was removed by DNase using the Qiagen RNase-free DNase Set. RT-PCR was performed using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) and Taq Polymerase (GIBCO BRL). Annealing temperature was 94°C for 30 seconds, extension at 72°C for 30 seconds, with a final 7-minute extension at 72°C. The primer sequences were as follows: c-kit forward 5'-ATA AAG GGT TCA GTG TGT TAC GTG G-3', reverse 5'-TTG GAA AGG TGC AAG AGT GTA GC-3'; CD31 (platelet-endothelial cell adhesion molecule-1 [PECAM-1]) forward 5'-GTT GTC ATT GGA GTG GTC ATC G-3', reverse 5'-CCT TCC GTT CTC TTG GTG AGG-3'; ß-actin (internal control), forward 5'-ACC TTC AAC ACC CCA GCC ATG-3', reverse 5'-GCT CGG TCA GGA TCT TCA TGA GG-3'. The primer sequences for flk-1, tie-2 and aldolase were as follows: flk-1 5'-gga ATT CAG GCA TTG TAC TGA GAG-3', 5'-cgga TCC AAG TTG GTC TTT TCC TG-3'; tie-2 5'-CAA CAG CGT CTA TCG GAC TCC-3', 5'-GAA AAG GCT GGG TTG CTT GAT C-3'; aldolase, 5'-AGC TGT CTG ACA TCG CTC ACC G-3', 5-CAC ATA CTG GCA GCG CTT CAAG-3'. Lowercase letters indicate nucleotides added for cloning purposes.

Immunofluorescence Microscopy
eEPCs were coincubated with medium or platelets for 5 days on chamber slides and processed for immunofluorescence microscopy. The mouse endothelial cell line MHEC5-T was used as positive control. Between each incubation step, cells were gently washed with PBS. T17b cells were fixed with 2% formaldehyde solution for 20 minutes. Afterward, cells were washed with 2% glycine, permeabilized with 0.2% Triton X-100, and incubated with PBS containing a goat anti-mouse vWF antibody (5 µg/mL) for 30 minutes. Unspecific binding was prevented by BSA (3%; 1 hour). Subsequently, an Alexa Fluor rabbit anti-goat antibody (5 µg/mL) was added for another 30 minutes. Furthermore, rhodamine phalloidin (5 µg/mL; detection of cytoskeleton) was applied for 30 minutes. Accordingly, CD34+ cells were coincubated with isolated platelets (2x108/mL) or medium as control for 10 days. For labeling, a rabbit anti-human vWF Ab (Dako Cytomation GmbH) and a secondary sheep anti-rabbit Ab (Sigma) were used. Samples were analyzed by standard and confocal immunofluorescence microscopy.

Data Presentation and Statistics
Comparisons between group means were performed using Student t test or ANOVA analysis. Data are presented as mean±SD. P<0.05 was considered statistically significant.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Platelets Induce Chemotaxis and Migration of eEPCs
Adherent platelets secrete various chemotactic compounds on activation.18 Thus, we evaluated the effect of platelets on eEPCs chemotaxis in a trans-well chamber culture system. Chemotaxis of eEPCs (T17b cells) toward adherent platelets was substantially increased compared with controls (P<0.01; Figure 1). Next, we studied the effect of platelets on eEPC migration. T17b cells were grown to confluence in 24-well plates, and the cell monolayers were wounded in a linear fashion with a pipette tip. Thereafter, the wounded eEPC monolayer was incubated for an additional 12 hours in the absence or presence of platelets (0.1 to 1x108 cells/mL medium), and the number of eEPCs that migrated into the denuded area was evaluated (Figure 2). We found that eEPC migration was significantly increased, depending on platelet number (Figure 2). At a platelet concentration of 1x108/mL, eEPC migration was enhanced by &2.5-fold (P<0.01) compared with control, indicating that platelets stimulate migration of eEPCs. To detect the specific mediators of platelet-induced migration, blocking antibodies were used, as indicated in figure legends. By addition of blocking anti–platelet-derived growth factor-AB, a substantial reduction of migration was found (Figure 2D and 2E).


Figure 1
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  		Figure 1. Chemotactic effects of platelets on eEPCs. Isolated platelets were allowed to adhere to the bottom of a trans-well system and stimulated with {alpha}-thrombin (1U/mL) for 60 minutes, as indicated in Materials and Methods. T17b cells (1x105/mL) were applied to the upper chamber of the system. After 24 hours, the migrated T17b cells were stained with crystal violet. A shows photographs of eEPCs that have migrated toward the stimulated platelets as visualized by positive staining (right panel). No migration took place in the control group without platelets (left panel). B, Densitometric quantification shows significantly enhanced migration toward platelets compared with control. *P<0.01.


Figure 2
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  		Figure 2. Effect of platelets on eEPC migration. A, A confluent monolayer of eEPCs was wounded with a plastic pipette and coincubated with increasing concentrations of isolated platelets (0.1 to 1x108/mL). After 24 hours, the number of migrated EPCs was counted by using a defined frame that was projected to each photograph. *P<0.01 compared with control. B and C, For subsequent experiments, migration was assessed after 24 hours using a platelet concentration of 1 x108/mL. B shows representative photomicrographs of migration experiments. C, Mean and SD of 6 independent eEPC migration experiments. *P<0.01 compared with EPCs alone (without platelets). D, After wounding of the eEPC monolayer, isolated platelets (1x108/mL) were added without blocking mAbs (5 µg/mL each) for 24 hours. *P<0.05; n=4. E shows representative photomicrographs of these migration experiments with blocking mAbs.

eEPCs Adhere to Platelets Under Static and Dynamic Flow Conditions
Platelets play a critical role in the recruitment and adhesion of circulating leukocytes to the vascular wall.18 To test whether EPCs bind to platelets, isolated platelets (2x108/mL) were allowed to adhere to 96-well plates for 2 hours. Subsequently, EPCs were added to the wells, and adhesion of EPCs on platelets was evaluated as described in Materials and Methods. Under these static conditions, EPCs showed an &4-fold enhanced adhesion to platelets compared with the plastic surface control (P<0.01; Figure 3A and 3B). In similar fashion, a flow cytometric adhesion assay showed enhanced coadhesion of platelets with EPCs under stirring conditions (Figure 3C). Furthermore, adhesion of isolated platelets to eEPCs could be visualized by scanning electron microscopy (data not shown).


Figure 3
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  		Figure 3. Platelet–eEPC interaction under static conditions. A, 96-well plates were incubated with or without freshly isolated platelets to achieve adherent platelet layers as described in Materials and Methods. T17b cells (1x105/mL) were allowed to adhere to these plates. After 30 minutes, the plates were gently washed twice, and adherent eEPCs were quantified by using a defined frame that was projected to each photograph. The mean and SD of 5 independent experiments is shown. *P<0.05 compared with control (plastic). B, Representative phase contrast images of adherent eEPCs. C, Cultured T17b cells (1x105/mL) were incubated with freshly isolated platelets (2x108/mL) for 30 minutes under agitation using a conventional aggregometer. After washing with Tyrodes buffer, T17b cells were incubated with FITC-labeled anti-CD41 mAb and analyzed by flow cytometry. The analysis of 5 independent experiments (mean±SD of the mean fluorescence) is shown. *P<0.05 vs buffer control.

Next, we evaluated the determinants that mediate eEPC adhesion to platelets under arterial flow conditions. In a parallel plate flow chamber, eEPCs were perfused over platelets immobilized on collagen at a wall shear rate of 2000 s–1 as described.27 eEPCs substantially adhered to immobilized platelets under flow conditions but not to immobilized collagen type I (P<0.01; Figure 4A). Interestingly, rolling of eEPCs on immobilized platelets was almost completely inhibited in the presence of blocking anti-CD62P (P-selectin) or anti-CD162 (PSGL-1) antibodies but not by anti-CD29 (ß1-integrin; Figure 4). In contrast, firm adhesion of eEPCs to platelets was attenuated by both anti-CD62P/anti-CD162 and anti-CD29 mAbs (Figure 4A). Virtually no inhibition of adhesion of eEPCs on immobilized platelets was found in the presence of a blocking anti-CD11b (anti-integrin {alpha}M, macrophage antigen-1 {alpha}) mAb (Figure 4A). Flow cytometric analysis of eEPCs showed substantial cell surface expression of CD162 (PSGL-1) and of CD49d (VLA-4) on T17b cells (data not shown), confirming previously published results.15 Together, these data indicate that adhesion of eEPCs onto immobilized platelets occurs under dynamic flow conditions. The adhesion of eEPCs to immobilized platelets is specifically regulated by selectins (PSGL-1/P-selectin; rolling) and integrins (ß1-integrin; firm adhesion). To test whether these findings can be transferred from the murine eEPC model to adult progenitor cells, dynamic adhesion of adult human CD34+ cells isolated from cord blood was evaluated. Compared with collagen control, significantly more CD34+ cells adhered to a platelet-covered collagen surface (Figure 4B).


Figure 4
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  		Figure 4. Adhesion of eEPCs to immobilized platelets under arterial shear conditions. Coverslips were precoated with collagen I (5 µg/mL) and additionally preincubated with or without freshly isolated platelets (2x108/mL) to achieve adherent platelet layers as described in Materials and Methods. Resuspended T17b cells (5x104/mL) were perfused over these coverslips in the presence or absence of blocking mAbs (5 µg/mL) as indicated. After 5 minutes, adherent eEPC were quantified by offline counting. The mean and SD of 4 independent experiments is shown. *P<0.05 compared with control (collagen without platelets). B, Coverslips were precoated with collagen I (5 µg/mL) and additionally preincubated with or without freshly isolated platelets (2x108/mL) to achieve adherent platelet layers as described in Materials and Methods. Resuspended human CD34+ cells (5x104/mL) were perfused over these coverslips. After 5 minutes, adherent CD34+ cells were quantified by offline counting. The mean and SD of 4 independent experiments is shown. *P<0.05 compared with control (collagen without platelets).

Platelets Induce Differentiation of eEPCs Into Mature Endothelial Cells
Next we asked whether platelets stimulate differentiation of eEPCs into mature endothelial cells. eEPCs have been shown to differentiate into mature endothelial cells on stimulation with cAMP.15 As shown in Figure 2, platelets stimulate migration and change the morphology of eEPCs (Figure 5). Migrating eEPCs show enhanced expression of vWF as visualized by immunofluorescence microscopy (Figure 5A), implying that in the presence of platelets, eEPCs alter their phenotype toward mature endothelial cells. To further elucidate the effects of platelets in eEPC differentiation, eEPCs were cultured in the presence of platelets for 5 days and analyzed by light and fluorescence microcopy. We found that a noticeable amount of eEPCs acquired an endothelium-like shape with a 3- to 5-fold larger, spindle-shaped appearance with cellular protrusions (Figure 5B). In contrast, no change in the phenotype of eEPCs was noted in the absence of platelets.


Figure 5
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  		Figure 5. Effect of platelets on eEPC migration and morphology. A, A confluent monolayer of eEPCs was wounded with a plastic pipette and coincubated with isolated platelets (2x108/mL). After 24 hours, at the edge of the wound, migrating eEPCs showed enhanced expression of vWF as verified by immunofluorescence microscopy. B, T17b cells were left untreated (right panel, magnification x80) or coincubated with platelets (left panel, magnification x40) as described in Materials and Methods. After 5 days, photomicrographs were taken to assess cell morphology. < indicates original morphology of the cells. <- indicates modified cell morphology after coincubation with platelets.

Immunofluorescence verified that eEPCs transform into mature endothelial cells in the presence of platelets. We found that eEPCs coincubated in the presence of platelets for 7 to 10 days showed enhanced vWF expression compared with untreated cells (Figure 6A). Moreover, eEPCs cocultured with platelets revealed a typical endothelial cell-like cytoskeleton rearrangement, as verified by staining with phalloidin. As a representative control of the differentiated endothelial phenotype, we used the mouse endothelial cell line MHEC5-T (Figure 6A). Transmission electron microscopy showed the formation and presence of Weibel Palade bodies in eEPCs cocultured in the presence of platelets similar to mature endothelial cells (Figure 6B). Additionally, RT-PCR analysis of native eEPCs showed virtually no expression of the mature endothelial cell marker CD31 (PECAM-1) and a distinct signal for the stem cell marker c-kit (Figure 6). However, after incubation of EPCs with isolated platelets for 5 days, CD31 mRNA expression increased, whereas c-kit decreased substantially (cAMP-induced differentiation served as a positive control15). In a second RT-PCR analysis, the endothelial cell markers flk-1 (vascular endothelial growth factor receptor), vascular endothelial-cadherin, and thrombomodulin were increased after incubation with isolated platelets or cAMP. Consistent with previous results, expression of Tie-2 was not enhanced after incubation with cAMP,15 nor with platelets. To test, whether these findings can be transferred from the murine eEPC model to adult human progenitor cells, differentiation of human CD34+ cells by platelets was evaluated. After 10 days of coincubation, a change in morphology of CD34+ cells could be observed with regard to increased size and spindle-shaped morphology (Figure 6D). Furthermore, these cells were found to be positive for vWF (Figure 6D). Moreover, these cells displayed substantial amounts of cytoskeleton, characteristic for endothelial cells, but not progenitor cells, as verified by phalloidin staining.


Figure 6
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  		Figure 6. Differentiation of eEPCs by platelets. A, T17b cells were left untreated or incubated in 6-well plates with platelets. After 5 days, platelets were removed and eEPCs were incubated with FITC-conjugated goat anti-mouse vWF antibody (5 µg/mL) for 30 minutes. After washing, an Alexa Fluor rabbit anti-goat IgG was added for an additional 30 minutes. For detection of cell cytoskeleton, eEPCs were incubated with rhodamine phalloidin (5 µg/mL) for another 30 minutes. Then eEPCs were washed and analyzed using fluorescence microscopy at 568 to 590 nm and 505 to 530 nm, respectively. The mouse endothelial cell line MHEC5-T served as a positive control. B, Electron microscopic analysis of T17b cells after incubation with medium or platelets for 5 days. The mouse endothelial cell line MHEC5-T served as a control. Analysis revealed Weibel Palade bodies (indicated with <-, 311x61 nm; magnification x80 000) in eEPCs treated with platelets, which show comparable appearance to those in the mouse endothelial cells MHEC5-T (320x89 nm; magnification x80 000). T17b cells incubated with medium alone showed no Weibel-Palade-bodies. C, RT-PCR analysis of the stem cell marker c-kit and of the marker for mature endothelial cells CD31 (PECAM-1) in T17b cells treated with medium, cAMP (0.5 mmol/L), or isolated platelets (4x108/mL), as indicated for 5 days. The image shown is representative for 2 independent experiments. ß-Actin mRNA was analyzed as internal control. D, RT-PCR analysis of the markers for mature endothelial cells thrombomodulin, flk-1 (vascular endothelial growth factor receptor), vascular endothelial-cadherin, and Tie-2 in T17b cells treated with medium, cAMP (0.5 mmol/L), or isolated platelets (4x108/mL) as indicated for 5 days. The image shown is representative for 2 independent experiments. Aldolase mRNA was analyzed as internal control. E, CD34+ cells (5x104) were coincubated with isolated platelets (2x108/mL) in 96-well plates for 10 days. Compared with untreated CD34+ cells (arrows, top panel), cells coincubated with platelets developed an endothelial spindle-shaped phenotype (arrows, top panel). vWF immunostaining and DAPI staining of CD34+ cells (bottom panel) and progenitor cells after 10 days coculture with platelets (bottom panel).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In this study, we have shown that platelets regulate chemotaxis, adhesion, and differentiation of murine EPCs (eEPCs). In brief, the major findings of the present study are: (1) platelets induce chemotaxis and migration of a mouse eEPC line (T17b); (2) murine eEPCs specifically adhere to immobilized platelets via PSGL-1 and VLA-4 under high-shear dynamic flow conditions; and (3) platelets induce differentiation of murine eEPCs and maturation into endothelial cells as evidenced by specific immunological (vWF) and morphological (Weibel Palade bodies) endothelial markers. The findings imply that platelets are critical for recruitment of circulating murine eEPCs to areas of vascular or ischemic lesions. Interaction of these eEPCs with activated platelets may play a prominent physiological role for vascular or tissue remodeling. A disturbed platelet interaction with EPCs may in turn be associated with defective vascular and tissue repair mechanisms involved in pathologies such as atherosclerosis and myocardial infarction.

The maintenance of endothelial integrity is of crucial importance for preventing atherosclerotic plaque erosion and rupture, causing myocardial infarction and sudden cardiovascular death.30 Recently, circulating EPCs have been shown to incorporate into sites of neovascularization4,31,32 and to home to sites of endothelial denudation.8,33 EPCs recruited at site of vascular lesions accelerate re-endothelization and lesion repair.34 However, the mechanisms that regulate EPC homing to vascular or tissue lesions are incompletely understood.

Platelets accumulate within seconds to sites of vascular injury and release a variety of potent chemotactic factors that induce recruitment of circulating blood cells toward sites of vessel injury.35 Adherent platelets express various receptors (eg, P-selectin) on their surface that regulate adhesion of neutrophils and mononuclear cells.35 Previous studies indicated that platelet-derived microparticles could influence number and biological functions of stem cells.36,37 Thus, we hypothesized that platelets mediate chemotaxis and adhesion of murine eEPCs. As reported previously, ß1-integrin CD49d (VLA-4) and PSGL-1 are the major adhesion receptors present on the cell surface of EPCs38,39 and mediate progenitor cell homing to the endothelial monolayer of bone marrow microvessels via binding to their counterpartner P-selectin and vascular cell adhesion molecule-1. A subset of cord blood–derived CD42+ cells, which are enriched in primitive CD38+ cells, demonstrate reduced rolling attributable to partial expression of a nonfunctional form of PSGL-1.40 Neutralizing the ß1-integrin VLA-4 blocks homing of human CD34+ cells to the bone marrow of fetal sheep.41 In the present study, we show that murine eEPCs adhere to immobilized platelets under flow conditions similar to arterial shear stress rates. We demonstrate that rolling of these eEPCs onto immobilized platelets is mediated by PSGL-1 and that VLA-4 is required for firm adhesion.

Recruitment and incorporation of EPCs require a coordinated sequence of multistep adhesive and signaling events, including chemoattraction, adhesion, and migration, and finally the differentiation to endothelial cells. Preliminary data suggest that EPCs have the capacity to regenerate the injured endothelial monolayer.23 However, the mechanisms that induce differentiation of EPCs into mature endothelial cells remain unexplained. Platelets store a variety of potent growth factors within their granules, including vascular endothelial growth factor-A,42 that are released into the microenvironment of activated platelets. We found that in the microenvironment of platelets, murine eEPCs migrate and adhere to immobilized platelets and start to differentiate into mature endothelial cells, as evidenced by expression of vWF and formation of Weibel Palade bodies, organelles that are specific for mature endothelial cells.

In this study, we made use of a mouse eEPC line. A hindering factor for a systematic analysis of EPC homing and differentiation mechanisms is the fact that adult EPCs are difficult to isolate and maintain and vary substantially between individuals with respect to functional properties. The major advantages of eEPCs are their robust growth properties in culture and practical genetic manipulation. The eEPCs express early endothelial markers, differentiate to mature endothelial cells, form vascular tubes in vitro, and build blood vessels after transplantation during embryogenesis.21 In addition, the homing of eEPCs in hypoxic tumors, their participation in tumor vessel formation,43 as well as their stimulation of angiogenesis in chronic and acute ischemia44 render eEPCs a relevant model system to study the biology of EPCs. To evaluate, if the central findings of this study derived from the murine progenitor cell model can be transferred to human progenitor cells, key experiments were performed using adult human CD34+ cells. We show that adult CD34+ cells adhere to platelets under high shear rates and that platelets induce differentiation of CD34+ cells into endothelial cells. Nevertheless, further studies using human bone marrow–derived stem cells have to be performed to confirm the exact mechanisms of this interaction within the human system.

In conclusion, we provide first evidence that platelets mediate recruitment of murine eEPCs and stimulate differentiation of these eEPCs into endothelial cells. If these findings can be transferred to the human system, the mechanism described in the present study may play a central physiological role for maintenance of vessel integrity and neoendothelization of vascular lesions. Further, an altered interaction of platelets and EPCs may play a critical role in atheroprogression.


*    Acknowledgments
 
The study was supported by grants of the Deutsche Forschungsgemeinschaft {Graduiertenkolleg (GK 794) "Zellbiologische Mechanismen immunassoziierter Prozesse" and "Vaskulare Medizin" (GRK 438) and MA 2186/3-1} to M.G. and S.M.; and the Schwerpunktprogramm "Angiogenese" to A.K.H. We acknowledge the excellent technical assistance of Heike Runge, Sandra Kerstan, and Kirsten Langenbrink.


*    Footnotes
 
Original received July 17, 2005; first resubmission received August 25, 2005; second resubmission received November 7, 2005; revised second resubmission received December 3, 2005; accepted December 14, 2005.


*    References
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 *References
 

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