Endothelial progenitor cells inhibit platelet function in a P-selectin-dependent manner

Abstract

Background: The role of endothelial progenitor cells (EPCs) in vascular repair is related to their recruitment at the sites of injury and their interaction with different components of the circulatory system. We have previously shown that EPCs bind and inhibit platelet function and impair thrombus formation via prostacyclin secretion, but the role of EPC binding to platelet P-selectin in this process has not been fully characterized. In the present study, we assessed the impact of EPCs on thrombus formation and we addressed the implication of P-selectin in this process.

Methods: EPCs were generated from human peripheral blood mononuclear cells cultured on fibronectin in conditioned media. The impact of EPCs on platelet aggregation and thrombus formation was investigated in P-selectin deficient (P-sel(-/-)) mice and their wild-type (WT) counterparts.

Results: EPCs significantly and dose-dependently impaired collagen-induced whole blood platelet aggregation in WT mice, whereas no effects were observed in P-sel(-/-) mice. Moreover, in a ferric chloride-induced arterial thrombosis model, infusion of EPCs significantly reduced thrombus formation in WT, but not in P-sel(-/-) mice. Furthermore, the relative mass of thrombi generated in EPC-treated P-sel(-/-) mice were significantly larger than those in EPC-treated WT mice, and the number of EPCs recruited within the thrombi and along the arterial wall was reduced in P-sel(-/-) mice as compared to WT mice.

Conclusion: This study shows that EPCs impair platelet aggregation and reduce thrombus formation via a cellular mechanism involving binding to platelet P-selectin. These findings add new insights into the role of EPC-platelet interactions in the regulation of thrombotic events during vascular repair.

Figure 1

Morphological change of PBMCs-derived EPCs in vitro. Adherence, sequential changes and differentiation of PBMCs are observed under inverted optical microscopy. A) At day 0, freshly isolated PBMCs were plated on fibronectin and the majority of cells are non-adherent with a rounded morphology. B) At day 3, the adherent cells appear either as single cells or as irregular colony-like structures. C) At day 5, colonies are better defined and form a central cluster of round cells with elongated spindle-like cells at the periphery. D) At day 10, cells show a flat monolayer of spindle-shaped cells.

Figure 2

Expression of cell surface markers. A) Representative overlay plots showing the expression of cell surface markers, as determined by flow cytometry. Single color immunostaining of freshly isolated PBMCs at day 0 (black plots) and culture-derived EPCs at day 10 (gray plots) was performed with saturating concentrations of mouse anti-human PE-conjugated monoclonal antibodies directed against CD14, CD34 and VEGFR2. Overlay plots are presented as the number of events over the log of associated fluorescence. B) Histogram represents the mean data ± SEM of at least 4 independent experiments.

Figure 3

Effect of EPCs on collagen-induced platelet aggregation. Whole blood was pre-incubated with different concentrations of EPCs in a 4-channel lumi-aggregometer under shear (1,000 rpm) at 37°C. Platelet aggregation was initiated by adding collagen (3 μg/mL) and then monitored for 5 minutes. Representative traces of whole blood platelet aggregation from A) WT and B) P-sel^−/− mice. The mean data ± SEM of 5 independent experiments, summarizing the effects of different EPC concentrations on collagen-induced platelet aggregation in WT and P-sel^−/− mice are presented in C and D, respectively.

Figure 4

Effect of EPCs on thrombus formation. Fresh culture media or EPCs were infused and allowed to circulate for 5 minutes, followed by application of FeCl₃ for 3 minutes and continuously monitoring of carotid blood flow for 20 minutes post-FeCl₃ injury. Thrombus formation in WT and P-sel^−/− mice infused with fresh culture media (Black line, n = 7) or with 500 x 10³ cells/mouse (Green line, n = 7). Insert: Effects of increasing concentrations of EPCs ranging from 125 x 10³ cells/mouse (red line, n = 7), 250 x 10³ cells/mouse (blue line, n = 7), or 500 x 10³ cells/mouse (green line, n = 7) in WT mice.

Figure 5

Histological cross-sections of FeCl₃-injured mouse carotid arteries. A) Representative histological transverse sections of FeCl₃-injured mouse carotid arteries treated with EPCs, PBMCs, or fresh culture media (control) and stained with hematoxylin and eosin. (Magnifications 20X). Arterial thrombus mass was completely occlusive in control-and PBMC-treated mice (panels A & B, n = 4) and partially occlusive in arteries from EPC-treated mice (panel C, n = 4). Panel D represent the injured artery from P-sel^−/− mice treated with 500 x 10³ EPCs (n = 4). B) Histogram represents the mean data ± SEM of cross-sectional area of arterial thrombi expressed as percentage of lumen area.

Figure 6

Fluorescent confocal imaging and immunostaining of FeCl₃-injured mouse carotid arteries. Photomicrographs show fluorescent confocal imaging and immunostaining of mouse carotid arteries after vascular injury. EPCs were labeled with an intracellular fluorescence marker (DiL) 1 hour prior to injection in WT (Panel D) and P-sel^−/− (Panel G) mice and assessed by confocal fluorescence on cryostat cross-sections. Panel A represent the injured arteries from non-labeled PBMC-treated mice used as negative control. The corresponding Differential Interference Contrast (D.I.C) of the identical sections of each carotid is shown on the right (panels B, E & H). (Scale bar = 50 μm). Anti-CD34 immunostaining of the injured arteries in EPC-treated mice show that CD34 positive cells were uniformly distributed within the thrombus and along the vessel wall in WT - but poorly distributed in P-sel^−/− mice confirming the recruitment of EPCs to the luminal aspect of arterial thrombi in vivo (arrows in F and I). The cells in C are CD34 negative. The images are representative of at least 4 independent experiments.