Fibrin and Activated Platelets Cooperatively Guide Stem Cells to a Vascular Injury and Promote Differentiation Towards an Endothelial Cell Phenotype
Objective— Bone marrow-derived progenitor cells play a role in vascular regeneration. However, their homing to areas of vascular injury is poorly understood. One of the earliest responses to an injury is the activation of coagulation and platelets. In this study we assessed the role of hemostatic components in the recruitment of CD34+ cells to sites of injury.
Methods and Results— Using an ex vivo injury model, representing endothelial cell (EC) injury or vessel denudation, we studied homing of CD34+ under flow. Platelet aggregates facilitated initial tethering and rolling of CD34+ cells through interaction of P-selectin expressed by platelets and P-selectin glycoprotein ligand-1 (PSGL-1), expressed by CD34+ cells. Ligation of PSGL-1 activated adhesion molecules on CD34+ cells, ultimately leading to firm adhesion of CD34+ cells to tissue factor-expressing ECs or to fibrin-containing thrombi formed on subendothelium. We also demonstrate that fibrin-containing thrombi can support migration of CD34+ cells to the site of injury and subsequent differentiation toward a mature EC phenotype. Additionally, intravenously injected CD34+ cells homed in vivo to denuded arteries in the presence of endogenous leukocytes.
Conclusions— We provide evidence that hemostatic factors, associated with vascular injury, provide a regulatory microenvironment for re-endothelialization mediated by circulating progenitor cells.
The structural and functional integrity of the endothelium is determined by the balance between endothelial injury and repair. Prolonged exposure to cardiovascular risk factors leads to oxidative stress, elevated endothelial cell (EC) turnover and ultimately EC death. Increasing evidence indicates that circulating endothelial progenitor cells (EPC) contribute to re-endothelialization.1–4
Consequently, EPC function may play a critical role in the maintenance of the integrity of endothelium5 and the protection against atherosclerosis. EPCs include different populations of CD34+ cells, either CD133 or vascular endothelial growth factor receptor-2 (VEGFR-2) positive, or CD14+/CD34low cells.6 Whereas the importance of EPCs for vascular maintenance and repair is only recently appreciated, the exact molecular mechanisms that lead to the actual arrest of EPCs at the site of injury are not clear.
Injury of the endothelium coincides with the activation of the coagulation system and of platelets.7,8 Because platelets and fibrin are known to be potent adhesive substrates for leukocytes,9 we hypothesized that these hemostatic factors might also facilitate homing of EPCs to the injury. Moreover, activated platelets release cytokines such as vascular endothelial growth factor (VEGF) and fibroblast growth factor and lipid mediators such as sphingosin-1-phosphate, known to enhance angiogenesis and neovascularization.10 Therefore, we also studied the contribution of hemostatic factors in EPC migration and differentiation toward an EC phenotype. Our data delineate the concept that the hemostatic clot may provide local cues for initial tethering, firm adhesion, migration, and differentiation of EPCs, ultimately leading to repair of vascular injury.
All materials and methods are detailed in the Methods section in the online data supplement available at http://atvb.ahajournals.org
In a Model of Activated EC, CD34+ Use Thrombi to Escape From Flow
First, we studied mild EC injury, such as hypoxia, in which ECs are activated but not apoptotic. Ischemia induces release of tumor necrosis factor-α (TNFα),11 which activates ECs, leading to expression of tissue factor (TF),12 deposition of fibrin, and platelet adhesion.8 In our model system, this situation is mimicked by incubation of an EC monolayer with TNFα for 6 hours.
CD34+ cells were perfused over a monolayer of nonactivated human microvascular EC (HMEC) or stimulated with TNFα. Adhesion to nonstimulated HMEC was almost absent (6±8/mm2) but increased mildly after TNF stimulation (16±16/mm2, Figure 1A). When TNF-stimulated HMECs were pre-perfused for 5 minutes with low-molecular-weight heparin (LMWH)-anticoagulated platelet-rich plasma (PRP), irregularly shaped platelet thrombi formed in time, which rounded up during the washing phase. Perfusion of CD34+ cells over these thrombi resulted in a very distinct performance. They attached and rolled further downstream along the thrombus. Subsequently, the cells detached from the thrombus and attached firmly to the endothelial monolayer located downstream the platelet aggregate (Figure 1B). The presence of platelet thrombi promoted a dramatic increase in the number of firmly attached CD34+ cells (265±194/mm2) (Figure 1A).
To investigate the role of the P-selectin/PSGL-1 pathway, antibodies were used to block P-selectin expressed by thrombi or PSGL-1 expressed by CD34+ cells. In both cases, CD34+ cells were unable to tether, roll, and adhere to the thrombi (10±19/mm2 and 25±26/mm2, respectively), showing the critical role for the P-selectin/PSGL-1 interactions in the initial homing of CD34+ cells to thrombi localized on stimulated ECs (Figure 1A). Platelet aggregate formation was more pronounced at the inlet of the perfusion channel (Figure 1B; panel a) and progressively decreased in number and size more downstream the channel (panels b to e). This explains the large standard deviation (SD) for total adhesion of CD34+ cells per mm2 (Figure 1A), which was nevertheless significantly higher than adhesion to surfaces without platelet aggregates (P<0.001). Calculation of the surface area of thrombi and the number of firmly attached CD34+ cells per microscopic field revealed a positive correlation (panel f, P=0.001).
In a Model of Vascular Denudation, CD34+ Cells Adhere to Fibrin-Rich Thrombi
A surgical or traumatic injury or ischemia-induced apoptosis may lead to endothelial denudation of a vessel. Denudation results in exposure of endothelial cell matrix (ECM) proteins to flowing blood, which leads to adhesion and aggregate formation of platelets and fibrin formation after activation of the coagulation cascade by TF in the ECM.13
To test the capacity of platelets and fibrin to support adhesion of CD34+ cells, thrombi of different composition were generated on TF-rich ECM (Figure 2A): (I) ECM alone; (II) ECM with fibrin; (III) ECM with a monolayer of platelets; (IV) combination of II+III; (V and VI) ECM pre-perfused with LMWH-anticoagulated whole blood, leading to the formation of thrombi in and on a network of fibrin fibers. LMWH allows the formation of surface-bound procoagulant thrombin, whereas thrombin released in solution is inhibited instantaneously by antithrombin III present in the plasma.14 Subsequently, the different surfaces were perfused with isolated CD34+ cells. Total adhesion of CD34+ cells to ECM increased in the following order: I, II, III, IV, and V/VI (Figure 2A, 2B). When larger thrombi were formed (Figure 2A VI), clusters of CD34+ cells were located downstream the thrombi.
Next, we assessed the capacity of the adhesive surfaces in supporting firm/shear-resistant adhesion of CD34+ cells under flow. After 5 minutes of perfusion, shear stress was increased (2 dyne/cm2) and the adhered CD34+ cells that moved ≥1.0 μm/sec (white bars) were defined as rolling cells (Figure 2B). On ECM alone (I), the small amount of CD34+ cells that tethered kept rolling. On ECM+fibrin (II), none of the CD34+ cells were defined as rolling (black bars), whereas on ECM+platelets (III) 75% of the CD34+ cells were classified as rolling. The presence of fibrin decreased the rolling percentage: 46% on ECM+fibrinogen+platelets (IV) and 21% on ECM with an LMWH whole blood-derived clot (V). Taken together, these data indicate that adhesion of CD34+ cells was most efficient on the most physiological thrombus, containing both fibrin and platelets, formed from flowing LMWH-anticoagulated whole blood (V).
P-Selectin Induces Expression of Adhesion Molecules on CD34+ Cells
In analogy to neutrophils,15 ligation of P-selectin with PSGL-1 may activate/upregulate cellular adhesion molecules (CAMs) on CD34+ cells. Fluorescence-activated cell sorter (FACS) analysis showed that 78.5% of the CD34+ cells express PSGL-1 (Figure IA, gray peak, available online at http://atvb.ahajournals.org) above background level (black line). Soluble P-selectin/Fc chimera (P-sel/Fc) showed dose-dependent binding to CD34+ cells (Figure IB), although the fluorescent intensity was heterogeneously distributed. Control human IgG showed no binding (Figure IC). As a measure of CAM activation, P-selectin–inducible binding of CD34+ cells to immobilized fibrinogen was studied. P-sel/Fc (10 μg/mL) induced CD34+ adhesion to fibrinogen (expressed as 100%) (Figure 3), whereas P-selectin–inducible fibrinogen binding was inhibited for 77% by pre-incubation of the CD34+ cells with an antibody against PSGL-1. Human IgG (10 μg/mL) did not induce fibrinogen binding and CD34+ did not bind to noncoated wells (not shown). To characterize the CAMs in more detail, function-blocking antibodies were added to P-selectin-primed CD34+ cells. Fibrinogen binding was partly inhibited by an antibody against MAC-1 and was completely blocked with an antibody against αvβ5, whereas an antibody against αvβ3 and nonspecific mouse IgG had no effect. These data indicate that priming CD34+ cells with P-selectin activates/upregulates CAMs, which promote binding to immobilized fibrinogen.
Platelet Products Attract CD34+ Cells
Because CD34+ cells adhere at, or close to, fibrin and activated platelets, these local conditions may also affect migration of the cells. To investigate this, a Boyden chamber was used, in which the lower compartment contained fibrin, platelets, or fibrin plus platelets. HEPES buffer containing thrombin+hirudin was used as negative control and VEGF as positive migration factor. As shown in Figure 4 control, fibrin or platelets alone did not induce migration of the CD34+ cells. In contrast, the combination of fibrin+platelets promoted CD34+ cell migration even to a greater extent than VEGF. These data indicate that platelets and/or platelet products, maximally released in the presence of fibrin, can provide a potent stimulus for the migration of CD34+.
Platelet-Containing Fibrin Clots Drive CD34+ Cells Toward an EC Phenotype
To investigate the potential of thrombus-derived factors in supporting EC differentiation, isolated CD34+ cells (500 000 cells/well) were culture on fibrin ± platelets in serum-free medium. In this way, fibrin ± platelet products are the only supplements to the culture medium.
After 9 days in culture, only 30±5% of the cell input could be recovered when cultured on a fibrin clot, whereas 85±5% of the cells could be recovered from fibrin+platelets (data not shown). FACS analysis was performed on gated cells (red dots, R1, excluding cells debris and platelets) and showed that of the fibrin-recovered cells 92.0% were positive for CD34 (Figure 5C); this accounted for 85.8% when cells were cultured on fibrin+platelets (Figure 5G). The fact that fibrin+platelets yielded &3 times more cells suggests that platelets provide survival factor(s), which was confirmed with annexin-V binding: in the presence of platelets 11.2% of the recovered CD34+ cells were apoptotic (purple dots, Figure 5G), whereas in the absence of platelets 49.5% of the CD34+ cells were apoptotic (Figure 5C). Apoptotic cells were localized in the left population on forward/side scatter plots (Figure 5D and 5H), enabling gating the viable cells (blue dots, R3) and excluding the platelets. Next, expression of EC markers was assessed on viable cells (in R3). Results are expressed as percentage of cells that exceed background staining obtained with isotype-matched controls. For comparison, the same EC markers were analyzed on freshly isolated CD34+ cells and CD34+-derived mature ECs (Table).
Culturing the cells on fibrin increased the percentage of VEGFR-2–positive cells from 0.2% on freshly isolated CD34+ cells to 3.0% when cultured in the presence of fibrin and to 7.9% in the presence of fibrin+platelets. VEGFR-2–positive cells were also positive for CD34 (not shown). Uptake of DiI-labeled acLDL increased from 15.1% on isolated CD34+ cells to 30.6% on fibrin and 45.4% on fibrin+platelets, whereas 94.9% of the CD34+-derived ECs were positive for DiI-labeled acLDL.
The percentage of cultured CD31-positive cells increased in both conditions to the level of mature ECs, whereas other EC-markers such as the expression CD144, CD105, and FGFR-1 were hardly affected. These data show that the microenvironment of a hemostatic clot provides survival factors for CD34+ cells and commitment toward a more EC-like phenotype.
In Vivo Homing of CD34+ to a Vascular Injury
To investigate whether homing of CD34+ cells actually occurs in a natural environment, in vivo homing of human CD34+ to denuded mouse carotid arteries was investigated according to Schober et al.16
Homing of calcein-labeled CD34+ cells could only be observed in the wire-injured carotid artery, 24 hours after injection. Figure IIA to IID (available online at http://atvb.ahajournals.org) shows a representative image recorded using confocal laser scanning microscopy (CLSM) in which in total 6 cells were detected in stacked images covering a 3-dimensional microscopic field with an area of 70×70×17 μm (please see http://atvb.ahajournals.org). The size (&5 μm in diameter) and morphology of the homed cells was identical to calcein-labeled CD34+ cells before injection (Figure IIE). A rotating projection of Figure II is available at http://atvb.ahajournals.org. A 2-dimensional scan of calcein-labeled CD34+ cells and their environment (panels B, C, D, E) show that nonlabeled leukocytes were located in close proximity to homed CD34+ cells, indicating that also mouse leukocytes show a homing tendency to sites of injury. CD34+ cells could not be observed in injured arteries 2 hours after injection (data not shown) or in noninjured contralateral arteries (Figure 6A). These data indicate that in an in vivo situation, in the context of endogenous leukocytes, circulating CD34+ cells are able to home to sites of endothelial injury.
CD34+ progenitor cells have been shown to contribute to re-endothelialization at sites of vascular injury.17–21 However, as the number of CD34+ in the circulation is relatively low, the actual homing process must be very efficient. The occurrence of an injury coincides with the activation of the coagulation system and activation of platelets.7,8 Therefore, we hypothesized that this hemostatic response might offer the necessary cues for EPCs to home to sites of vascular injury. To investigate this hypothesis, a well-established ex vivo injury model was used, mimicking activated ECs or a denuded vessel and thus representing different stages of EC injury. Based on our results, we postulate a new model for EPC homing and differentiation.
Our data show that direct binding of CD34+ to nonstimulated, and even stimulated ECs, and to subendothelium was limited under flow, implicating that CD34+ cells are inert to (dys)functional ECs and to exposed ECM. Furthermore, we show that platelets act as an intermediate factor to tether EPCs, indicating that platelets are a prerequisite for the initial step of the homing process of CD34+ cells to a vascular injury.
Functional blockage of P-selectin expressed by adherent platelets, or PSGL-1, expressed by CD34+ cells abrogated binding of the CD34+ cells to platelet thrombi, which underscores a major role for the P-selectin/PSGL-1 pathway.
Engraftment of human bone marrow by CD34+ cells is also mediated by P-and E-selectin expressed by bone marrow ECs.22 Our findings show that in the case of a vascular injury, platelet thrombi accumulated in the vicinity of the injury, provide the necessary P-selectin for the deceleration of circulating CD34+ cells. These results are in agreement with a report by Zernecke et al, who showed that platelets and SDF-1α act in concert to recruit progenitor cells after injury.23
The degree of EC injury determines the composition of the thrombus: on activated ECs, mainly platelet aggregates are formed, which are hardly enforced by fibrin fibers, leading to erosion of the platelet aggregates during the washing phase (not shown). On ECM, however, thrombi are formed in and on a network of fibrin fibers. Firm adhesion of CD34+ cells only occurred when the CD34+ cells encountered activated ECs, located downstream the platelet aggregates or when fibrin had formed on exposed ECM. Our data suggest that ligation of PSGL-1 on CD34+ cells activates/upregulates cellular adhesion molecules, thereby increasing the binding activity of CD34+ cells toward fibrin and stimulated ECs. Priming the CD34+ cells with soluble P-selectin induced fibrinogen-binding through expression of integrins, because binding could be inhibited by antibodies directed against MAC-1 (αMβ2) and αvβ5, but not αvβ3.
Our results furthermore show that the CD34+ cells are able to migrate toward thrombus-released factors that are maximally secreted in the presence of both platelets and fibrin.
The microenvironment of the platelet-rich fibrin clot is also most supportive for the survival and differentiation of CD34+ cells toward a more EC-like phenotype, as evidenced by the enhanced expression of VEGFR-2 and other EC-markers. Data published by Pelosi et al indicate that CD34+ cells coexpressing VEGFR-2 constitute a functional subpopulation that has the capacity to migrate and differentiate into ECs.24 However, our perfusion data imply that the capacity of the CD34+ cells to bind P-selectin is rate-limiting for the initial stage of the multistep process of EPC recruitment and thus to the ultimate contribution of EPCs to EC repair. Furthermore, once CD34+ cells are immobilized on the injured vessel wall, they become subject to fluid shear stress, which enhances their VEGFR-2 expression, proliferation, and tube formation.25,26 Our differentiation assay was performed under static conditions and, therefore, may be considered as suboptimal. Nevertheless, these suboptimal conditions did lead to EC differentiation, illustrating the potency of the platelet-rich fibrin clot and the high degree of plasticity of CD34+ cells.
Platelets elaborate an array of factors that are involved in wound healing, of which many factors play a role in the biology of EPCs as well. For instance, the potent growth factor VEGF, which is highly accumulated in procoagulant regions,27 not only leads to recruitment of EPCs into the circulation28,29 but also stimulates the differentiation of CD34+ cells into ECs.30 Our article describes for the first time to our knowledge the possible synergistic role of hemostatic factors in EPC biology, not only as a substrate for homing but also as source for platelet-derived angiogenic factors.
The proposed homing mechanism of EPCs bears many similarities with other cell adhesion cascades, all initiated by a primary tethering event mediated by P-selectin on platelets and their carbohydrate ligands.31 This has been observed for many leukocyte subtypes including T lymphocytes,32 NK cells,33 neutrophils,34 or monocytes.35 Our in vivo data show that in a normal organism, recruitment of CD34+ cells is injury specific and does take place even in the presence of mature leukocytes, which probably use similar ligands for their adhesion.
Taken together, our data have identified the hemostatic clot as contributor to vascular repair mechanisms mediated by EPCs under physiological conditions.
Characterization of the factors involved in this complex process may contribute to the development of innovative strategies to augment EPC-mediated vascular regeneration in pathophysiological conditions.
The authors thank F.A. Prins (Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands) for assistance with confocal laser scanning microscopy.
Source of Funding
This study was supported in part by the Netherlands Heart Foundation (grant NHS 2002B157).
Original received October 12, 2005; final version accepted March 31, 2006.
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