Interaction of platelets with endothelial progenitor cells in the experimental atherosclerosis: Role of transplanted endothelial progenitor cells and platelet microparticles.

Biol. Cell (2015) 107, 189–204

DOI: 10.1111/boc.201400071

Research article

Interaction of platelets with endothelial progenitor cells in the experimental atherosclerosis: Role of transplanted endothelial progenitor cells and platelet microparticles Nicoleta Alexandru1 , Eugen Andrei, Emanuel Dragan and Adriana Georgescu1 Institute of Cellular Biology and Pathology ‘Nicolae Simionescu’ of the Romanian Academy, Bucharest, Romania

Background information. Recent studies suggest that endothelial progenitor cells (EPCs) and platelets have an important role in repair following vascular injury. Although evidence suggest that platelets are essential in EPC attracting, homing and differentiation to the injury site; however, the platelet effects on EPC function in atherosclerosis have received less attention. In this context, we followed the consequences of circulating EPCs and platelet microparticles (PMPs) administration on platelet–EPC interaction in atherosclerosis and the involved mechanisms. The experiments were performed on Golden Syrian hamsters divided in five equal groups: control (C), hypertensive– hypercholesterolemic (HH), HH treated with EPCs (HH–EPCs) or PMPs (HH–PMPs) and HH treated with EPCs and PMPs (HH–EPCs–PMPs). Results. Compared with C group, EPCs isolated from HH and HH–PMPs groups presented a reduction of endothelial nitric oxide synthase and vascular endothelial growth factor expressions and an increase in thrombospondin-1 expression and inflammatory molecule secretion: interleukin 8 (IL)-8, myeloperoxidase (MPO) and plasminogen activator inhibitor-1 (PAI-1). EPC administration had beneficial effects, the obtained results being similar with those from the C group, while the combination with PMPs did not improve the EPC influences. Static coincubation of EPCs from HH and HH–PMPs with analogous platelets resulted in an increased EPC adhesion/migration, and IL-8, monocyte chemotactic protein-1, regulated on activation, normal T expressed and secreted, MPO and PAI-1 release, explained by the platelet hyperaggregability induced by pronounced distribution of vasodilator-stimulated phosphoprotein and filamentous actin, and the secretion of proinflammatory factors: IL-1β, -6, -8, CD40 ligand. EPC therapy alone revealed an impaired platelet–EPC interaction directly correlated with the reduction of inflammatory markers and platelet aggregability. Moreover, in a dynamic flow system, EPCs and platelets from HH and HH–PMPs exhibited weakened interplay abilities, while EPC transplantation reinforces them. Conclusions. The present study demonstrates that HH animals revealed functional impairment of EPCs and platelets, which correlate with their reduced contribution to re-endothelialisation at the injury site, although in vitro exposure to immobilised platelets promotes their adhesion and migration. EPC administration alone recovers EPC/platelet functions and consolidates their interaction under dynamic flow conditions. These findings disclose new advances in understanding the platelet–EPC interaction and its role in the vascular repair.

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´ e´ Franc¸aise des Microscopies and Societ ´ e´ de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd 2015 Societ

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N. Alexandru and others

Introduction Atherosclerosis, an inflammatory process that selectively affects arteries, is highly prevalent in human, and its thrombo-occlusive complications (stroke and myocardial infarction) are becoming major causes of morbidity and mortality in the industrialised world (Liu et al., 2009). In the atherogenic process, there is a balance between two ongoing events, endothelial damage and repair. The failure of endothelial repair process is intimately linked to atherosclerotic inflammation and lesion formation. Under physiological conditions, the integrity of endothelial monolayer is maintained by replication of adjacent cells; however, in the conditions of increased endothelial injury, the regeneration of injured endothelium is assisted by circulating endothelial progenitor cells (EPCs) homing into the artery wall (Xu, 2006). Common conditions predisposing to atherosclerosis such as hyperlipidaemia, hypertension and diabetes are significantly associated with reduced number and impaired functionality of EPCs (Du et al., 2012; Georgescu et al., 2012). Moreover, a number of reports provide evidence that reduced levels of EPCs independently predict early subclinical atherosclerosis, disease progression, occurrence of cardiovascular events, death from cardiovascular causes and prognosis after ischemic stroke (Schmidt-Lucke et al., 2005; Werner et al., 2005; Fadini et al., 2006; Yip et al., 2008). The contribution of EPCs to the restoration of endothelial monolayer induced the possibility to use EPCs as a novel preventative and/or treatment strategy for atherosclerosis (Liu et al., 2009). It was demonstrated that EPCs generated in vitro from peripheral blood mononuclear cells (PBMCs) have potential therapeutic applications in the vascular tissue engineering and cell-based methods (Werner 1 To

whom correspondence should be addressed (email: [email protected] and [email protected]) Key words: Animal models, Cardiovascular risk factors, Endothelial progenitor cells, Platelets. Abbreviations: BSA, bovine serum albumin; CD40L, CD40 ligand; eNOS, endothelial nitric oxide synthase; EPCs, endothelial progenitor cells; F-actin, filamentous actin; IL, interleukin; MCP-1, monocyte chemotactic protein-1; MNCs, mononuclear cells; MPO, myeloperoxidase; PAI-1, plasminogen activator inhibitor-1; PFA, paraformaldehyde; PMPs, platelets microparticles; PBMCs, peripheral blood mononuclear cells; PRP, platelet-rich plasma; RANTES, regulated on activation, normal T expressed and secreted; SDF-1alfa, stromal-cellderived factor-1; TF, tissue factor; TSP-1, thrombospondin-1; VASP, vasodilatorstimulated phosphoprotein; VEGF, vascular endothelial growth factor; VEGF-R2, vascular endothelial growth factor receptor 2

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et al., 2006; Yoder et al., 2007; Co et al., 2008; Miglionico et al. 2008; Larsen et al., 2012). Functional studies in atherosclerotic apolipoprotein E deficient mice (Wassmann et al., 2006) and also in hypertensive–hypercholesterolemic (HH) hamster (Alexandru et al., 2013) indicate that EPC administration can improve the arterial dysfunction by increasing contraction and relaxation, and reduce the atherosclerosis development by decreasing lipid, macrophage, microparticle and EPC content of arterial wall. Recently, it has shown the useful role of direct infusion of progenitor cells in a murine model of atherosclerosis, where it was found that it delays atherosclerotic plaque progression and decreases inflammatory mediators (Tousoulis et al., 2013). Increasing data indicate that the platelet adhesion on vascular wall is the first step following vascular injury. Moreover, platelet activation results in shape change and secretion. Shape change is mediated by a rapid, precise, remodelling of the resting platelet cytoskeleton and the assembly of filamentous actin (F-actin) mediated by vasodilator-stimulated phosphoprotein (VASP, an actin-binding protein involved in negatively regulating secretory and adhesive events in these cells) (Falet et al. 2005; Wentworth et al., 2006). Activated platelets secret inflammatory mediators such as stromal-cell-derived factor-1 (SDF-1α), regulated on activation, normal T expressed and secreted (RANTES), monocyte chemotactic protein-1 (MCP-1), interleukins (such as interleukin (IL)-1β, -6, -8) and CD40 ligand (CD40L), inducing a lowgrade inflammation of the endothelium and vessel wall, which contributes to atherosclerosis development (Lievens von Hundelshausen, 2011; Reinhart, 2013).Furthermore, other studies demonstrated that adherent platelets contribute to the progenitor cell recruitment to arterial thrombi in vitro and in vivo and induce their subsequent differentiation toward an endothelial phenotype or into foam cells depending on conditions (Daub et al., 2006; de Boer et al., 2006; Massberg et al., 2006; Stellos et al., 2008; Badimon et al., 2012). Accordingly, we designed the present study based on our previous papers (Alexandru et al., 2011, 2013; Georgescu et al., 2012, 2013) to explore the interaction between platelets and circulating EPCs and possible mechanisms involved in this crosstalk in the experimental induced atherosclerosis.

EPC therapy and platelets in atherosclerosis

Additionally, we investigated the effects of PBMCderived EPC administration, alone or in correlation with platelet microparticles (PMPs) and also of PMPs transplantation alone, in the platelet–EPC interaction.

Results NOS, VEGF and TSP-1 protein expression on EPCs

For demonstrating the EPC dysfunction in the hypertension–hyperlipidaemia conditions, the immunofluorescence staining for eNOS (red), VEGF (red) and TSP-1 (green) was applied (Figure 1). The results illustrate that the HH and HH–PMPs groups are characterised by the substantial reduction in eNOS and VEGF expression compared with the C group, whereas EPC administration prevents their decrease (Figure 1, P 0.05). In contrast, TSP1 expression was upregulated in EPCs from HH, HH–PMPs and HH–EPCs–PMPs groups, and in the HH–EPCs group was similar to that in the C group (Figure 1, P 0.05). These findings suggest that EPC treatment in the experimental atherosclerosis improves the EPC functions in these conditions, whereas PMP administration has a negative impact on their activities.

Chemokines and thromboinflammatory factors secreted by EPCs

To investigate whether EPCs release chemokines, known to promote inflammatory or atherogenic processes (Zhang et al., 2009), we examined the secretion of IL-8, MCP-1 and RANTES by circulating ECPs obtained from the experimental models applying the ELISA method. Compared with the C group, IL-8 concentration in EPC supernatants was higher for almost all animal groups: HH (1.26-fold), HH– PMPs (1.48-fold) and HH–EPCs–PMPs (1.33fold) (Table 1, P 0.05). The concentration measured in HH–EPCs group was similar to the value in the C group. Compared with the HH group, we recorded decreased values of the IL-8 concentration by 1.29fold in the HH–EPCs group, and slightly enhanced values by 1.18-fold in the HH–PMPs group and 1.05-fold in the HH–EPCs–PMPs group.

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Research article For MCP-1 and RANTES, our results also show an increase in concentration levels in EPC supernatant from HH, HH–PMPs, HH–EPCs–PMPs groups and similar values for the HH-EPCs group, when we compared them with the C group (unpublished data). Thromboinflammatory factors such as myeloperoxidase (MPO), plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF) can serve as biomarkers and mediators of the thromboinflammatory cascade during plaque formation and plaque rupture (Zhang et al., 2009). Therefore, using the same method, we measured the MPO, PAI-1 and TF release by circulating EPCs obtained from hamster groups, after a 24-h incubation period. Compared with the C group, for all others groups, the MPO release in EPC supernatant was increased 3.71-fold (for HH), 2.17-fold (for HH–EPCs), 5.41-fold (for HH–PMPs) and 3.29-fold (for HH–EPCs–PMPs) (Table 1, P 0.05). Compared with the HH group, the MPO levels were reduced to 1.71-fold and 1.13-fold for HH–EPCs and HH–EPCs–PMPs groups, respectively, while in the HH–PMPs group the levels were increased to 1.46-fold. The analysis of PAI-1 concentration in EPC supernatants obtained from HH, HH–PMPs and HH– EPCs–PMPs groups revealed a 1.92-fold, 2.21fold and 1.6-fold augmentation compared with C group (Table 1, P 0.05). In samples from the HH-EPCs group, the PAI-1 values were comparable to those in the C group, and reduced to 1.89-fold when these were compared with the values from the HH group. Also, the PAI-1 levels were diminished 1.20-fold at the HH–EPCs–PMPs group compared with the HH group. Conversely, the PAI-1 concentration displayed a slight increase 1.15-fold for the HH–PMPs group compared with the HH group (Table 1). The measurement of TF concentration in EPC supernatants derived from HH, HH-PMPs and HHEPCs-PMPs groups shows an increase in values compared with those from the C group (unpublished data). The above results indicate that the EPC administration in hypertension associated with hypercholesterolemia reduces the levels of proinflammatory molecules secreted by circulating EPCs, while PMP


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Figure 1 Immunofluorescence staining of EPCs Sorted EPCs adherent to a collagen-coated surface were fixed, permeabilised and immunostained with anti-eNOS, -VEGF and -TSP-1 antibodies, and then detected using PE-conjugated anti-rabbit IgG and FITC-conjugated anti-mouse IgG antibodies. From left to right, Panel 1: Immunofluorescence detection of eNOS on the EPC surface (red). Panel 2: Immunofluorescence detection of TSP-1 (green) on the EPC surface. Panel 3: Immunofluorescence detection of VEGF on the EPC surface (red). Scale bare is 100 µm. Images show the distribution of the same proteins in EPCs derived from C group in top panels, HH group in the second panels, HH-EPCs group in the third panels, HH-PMPs group in the fourth panels and HH-EPCs-PMPs group in the bottom panels. The images are representative for three independent experiments.

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Table 1 Chemokine and thromboinflammatory factor release in EPC supernatants derived from all experimental hamster groups after 24 h incubation (n = 5 for each investigated molecule)

IL-8 (pg/mL) MPO (ng/mL) PAI-1 (ng/mL)

C

HH

HH–EPCs

HH–PMPs

HH–EPCs—PMPs

19.574 ± 1.36 0.224 ± 0.053 0.095 ± 0.008

24.597 ± 1.26 (*P 0.05) 0.83 ± 0.287 (*P 0.05) 0.182 ± 0.03 (*P 0.05)

19.105 ± 0.42 0.487 ± 0.11 (*P 0.05) 0.096 ± 0.01

29.042 ± 0.51 (*P 0.05) 1.213 ± 0.28 (*P 0.05) 0.210 ± 0.02 (*P 0.05)

25.929 ± 0.43 (*P 0.05) 0.738 ± 0.05 (*P 0.05) 0.152 ± 0.01 (*P 0.05)

Data are means ± SEM. The statistical significance, noticeably different, was represented as *P values versus control C group; n = the number of animals.

administration induces a general augmentation of secreted molecules. Adhesion and migration of EPCs to platelets in a static and dynamic system

Platelets play a critical role in the recruitment and adhesion of circulating EPCs toward injured vessel wall (Daub et al., 2006). To evaluate how platelets influence EPC functions, and whether EPC administration has an impact on platelet– EPC interactions in atherosclerosis, we coincubated EPCs isolated from all experimental groups with analogous platelets in a static and dynamic system. Regarding static adhesion system, using direct phase contrast microscopy, we found that the binding of EPCs to platelets was increased in HH, HH–PMPs and HH–EPCs–PMPs groups compared with the C group 6.26-fold, 7.73-fold and 4.29- fold, respectively (n = 3, Figure 2, P 0.001). For the HH–EPCs group, the number of adherent EPCs was similar to the C group. Next, we analysed chemokines (IL-8, MCP-1 and RANTES) and thromboinflammatory factors (MPO and PAI-1) released by EPCs after interaction with immobilised platelets. Compared with the C group, the measured values by the ELISA method were higher especially in supernatant obtained from HH, HH-PMPs and HH-EPCs-PMPs groups (Table 2, P 0.001). For the HH–EPCs group, the detected concentrations were similar or not significantly different from those found at the C group. In the second adhesion assay, the dynamic flow system, the binding of EPCs to platelets was evaluated by flow cytometry as percentage corresponding to KDR+ and CD41+ cells from 50,000 events. The results showed a decreased coadhesion of EPCs to platelets in HH, HH–PMPs, and HH–EPCs–PMPs groups compared with the C group (Table 3, P 0.05). For experiments realised on cells obtained from

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the HH-EPCs group, the percentage was similar to those from the C group (Table 3). The results showed that immobilised-activated platelets stimulated EPC adhesion in hypertension associated with hypercholesterolemia conditions and at PMP administration. This behaviour is similar to the situation in which the activated platelets stimulate EPC homing at sites of endothelial vascular injury and favour their differentiation into endothelial cells in in vivo conditions. In this way, EPCs could secrete many vasoactive and angiogenic factors that may modulate vascular thrombosis and haemostasis and might influence platelet functions. Interesting, the platelet binding to EPCs under agitation condition was reduced in HH, HH–PMPs and HH–EPCs–PMPs groups compared with the C group, suggesting that the direct interaction between these blood cells in circulation is affected. This decrease in binding may be explained by the dynamic changes in the expression of cell surface markers, as well as by the upregulation of platelet and EPC inhibiting factors, throughout the process of atherosclerosis. EPC administration induced the behaviour of EPCs like in the C group, and in combination with PMPs was not more efficient than EPC treatment only. Chemotaxis and migration of EPCs induced by platelets

Adherent platelets become activated and secrete or expose multiple inflammatory factors including growth factors, chemokines and cytokines (Jennings, 2009). Thus, we evaluated the effect of isolated platelets from each experimental group on EPC chemotaxis and migration in a transwell chamber culture system. Using direct phase contrast microscopy, we found that compared with the C group, the transmigrated EPC number toward analogous adherent platelets was increased 4.81-fold


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Figure 2 The interaction of EPCs with platelets in a static adhesion system (A) Twenty-four-well plates precoated with collagen I (10 µg/mL) were incubated with or without freshly isolated platelets in order to achieve adherent platelets layers and also with EPCs as described in Materials and Method section. (B) EPCs (1 × 105 cells) were incubated with freshly isolated platelets (5 × 106 cells) immobilised on collagen I precoated plates, and the adherent EPCs were counted for each experimental group: C, HH, HH–EPCs, HH–PMPs, HH–EPCs–PMPs. (C) The mean and SD of three independent experiments for all investigated groups are shown. * P < 0.05 as compared with control. Plts, platelets.

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Table 2 Chemokine and thromboinflammatory factor release in EPC supernatants after static adhesion (n = 4 for each investigated molecule from all experimental groups) C IL-8 (pg/mL) MCP-1 (pg/mL) RANTES (pg/mL) MPO (ng/mL) PAI-1 (ng/mL)

8.524 338.45 10.99 0.415 0.356

HH ± ± ± ± ±

0.52 45.78 1.52 0.13 0.045

HH–EPCs

11. 579 649.85 14.278 0.710 1.015

± ± ± ± ±

0.47 (*P 0.05) 61.85 (*P 0.05) 0.36 (*P 0.05) 0.41 (*P 0.05) 0.33 (*P 0.05)

8.890 397.89 9.196 0.524 0.601

± ± ± ± ±

HH–PMPs 0.63 43.68 0.69 0.13 0.09

15.106 754.65 10.605 1.346 1.664

± ± ± ± ±

HH–EPCs–PMPs 0.62 (*P 0.05) 61.12 (*P 0.05) 0.48 0.11 (*P 0.05) 0.49 (*P 0.05)

13.176 554.94 6.090 0.751 1.607

± ± ± ± ±

0.42 (*P 0.05) 51.05 (*P 0.05) 0.62 0.28 (*P 0.05) 0.58 (*P 0.05)


Table 3 The quantification of platelet–EPC interaction in a dynamic flow system by flow cytometry analysis Hamster groups (n = 4)

Percent of PE- and FITC-labelled cells representing the binding platelets to EPCs (%)

Control HH HH—EPCs HH—PMPs HH–EPCs–PMPs

7.873 5.973 7.416 5.237 6.476

± ± ± ± ±

1.042 1.204 (*P 0.05) 0.86 1.245 (*P 0.05) 1.575


in condition of hypertension associated with hypercholesterolemia, 7.63-fold in the HH–PMPs group and 1.61-fold in the HH–EPCs–PMPs group (Figure 3, n = 3, P 0.05). For the HH-EPCs group, the number was similar to that from the C group. Subsequently, by using the ELISA method on EPC medium collected from transwell culture inserts, we measured the IL-8, MCP-1, RANTES, MPO and PAI-1 levels released by EPCs after migration to adherent platelets. Compared with the C group, the values measured were higher especially in EPC medium obtained from HH, HH–PMPs and HH– EPCs–PMPs groups (Table 4, P 0.05). For the HH–EPCs group, the detected concentrations were similar to those from the C group. To detect the specific mediators of EPC migration induced by platelets, we assessed the levels of chemokines released by platelets after interaction with EPC in remained medium from well plates by applying the ELISA method. For MCP-1 concentrations, we obtained the following values: 342.19 ± 34.61 pg/mL for the C group, 439.44 ± 35.92 pg/mL

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for the HH group, 559.07 ± 16.14 pg/mL for the HH–PMP group and 389.015 ± 27.17 pg/mL for the HH–EPC–PMP group. These values were significantly decreased for the C group. In the samples from the HH–EPCs group, MCP-1concentration of 345.07 ± 79.85 pg/mL was similar to that measured for the C group (n = 3). For RANTES concentrations, the values acquired in platelet supernatants were as follows: 4.556 ± 0.53 pg/mL for the C group, 7.065 ± 0.18 pg/mL for the HH group, 5.020 ± 0.73 pg/mL for the HH–EPC group, 10.402 ± 0.97 pg/mL for the HH-PMPs group and 5.697± 0.456 pg/mL for the HH—EPCs–PMPs group (n = 3). These results indicate that platelets stimulate the EPC migration, and this is augmented in hypertension associated with hypercholesterolemia and also after PMP administration that induces the platelet hyperactivity. Moreover, EPC treatment is correlated with the reduction in platelet activation and consequently with the impairment of EPC chemotaxis and migration. Platelet aggregation mediated by VASP and F-actin

VASP is a critical protein involved in the remodelling of actin cytoskeleton in platelets and plays an important role in the regulation of its adhesive events being also involved in the platelet aggregation and secretion (Wentworth et al., 2006). Notably, through its anticapping activity, VASP promotes filopodia formation by allowing linear actin polymerisation (Pula et al., 2006). In this context, we examined the VASP and actin organisation in platelets isolated from the five experimental groups. Figure 4 shows that collagen-induced platelet aggregation was increased and accompanied by filopodia formation at HH and HH–PMPs groups, compared with platelets isolated from the C group,


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Figure 3 Chemotactic effects of platelets on EPCs Isolated platelets (5 × 106 cells) from hamster groups were allowed to adhere to the bottom of a transwell system coated with collagen (10 µg/mL) for 2 h, as indicated in Materials and Method section. Sorted EPCs (1 × 105 /mL) were applied to the upper chamber of the system. After 24 h, the migrated EPCs were stained with Hoechst 33258 solution (2 µg/mL). (A) Representative pictures for three independent experiments with EPCs that have migrated toward the adherent platelets, visualised by positive staining. (B) The quantification of EPC number on the transwell culture inserts. (C) The quantification of transmigrated EPCs in the bottom of the transwell system. Investigated hamster groups: C, HH, HH–EPCs, HH–PMPs, HH–EPCs–PMPs. *P 0.05.

while distribution of VASP and F-actin was less pronounced in platelets obtained from HH–EPCs. When we combined EPC with PMP treatment, the F-actin expression was reduced compared with that observed at HH and HH–PMPs groups, whereas the expression of VASP was diminished only when compared with the HH group. The analysis of immunohistochemistry colour images revealed that the combination of hypertension– hyperlipidaemia had a significant impact on the cytoskeletal structure in platelets and also induced the

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platelet aggregation, whereas EPC administration had a beneficial effect on these changes (data are not shown). Proinflammatory factors secreted in the activated platelet supernatants

The activation of platelets results in the release of various cytokines, such as ILs and CD40L, which further stimulate the endothelium and promote interactions that facilitate firm leukocyte adhesion to endothelial-adhered platelets or directly to the

Research article


Table 4 Chemokine and thromboinflammatory factor release in EPC supernatants of investigated hamster groups after migration and adhesion (n = 4 for each investigated molecule) Control IL-8 (pg/mL) MCP-1 (pg/mL) RANTES (pg/mL) MPO (ng/mL) PAI-1 (ng/mL)

9.970 228.85 4.578 0.411 0.940

HH ± ± ± ± ±

0.37 47.17 0.44 0.53 0.13

12.525 530.74 6.109 1.795 2.281

HH–EPC ± ± ± ± ±

0.47 (*P 0.05) 56.35 (*P 0.05) 0.59 (*P 0.05) 1.27 (*P 0.05) 0.47 (*P0.05)

10.256 253.74 4.629 0.477 0.949

± ± ± ± ±

HH–PMP 0.42 40.41 0.55 0.05 0.65

16.785 620.74 6.405 1.968 3.249

± ± ± ± ±

HH–EPC–PMP 0.67 (*P 0.05) 53.33 (*P 0.05) 0.40 (*P 0.05) 0.35 (*P 0.05) 0.42 (*P 0.05)

13.425 422.39 5.338 0.516 2.697

± ± ± ± ±

0.41 (*P 0.05) 37.20 (*P 0.05) 0.85 (*P 0.05) 0.25 (*P 0.05) 1.09 (*P 0.05)


Figure 4 Immunofluorescence staining of F-actin and VASP in platelets Hamster platelets adherent to a collagen-coated surface were fixed, permeabilised and immunostained with 1:100 anti-VASP and 1 µg/mL FITC-Phalloidin antibodies. VASP was then detected using PE-conjugated anti-rabbit IgG antibody. Images show the distribution of F-actin (the second column), VASP (the third column) and their superimposition (the fourth column) from three independent experiments. The phase-contrast images of the platelets are also shown (the first column). Platelets derived from C group are presented in the top panels, HH group in the second panels, HH–EPCs group in the third panels, HH–PMPs group in the fourth panels and HH–EPCs–PMPs group in the bottom panels (scale bar: 30 µm).

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Figure 5 Proinflammatory factors released by platelets The secretion of IL-1β, -6, -8 and CD40L was measured after the centrifugation of platelets obtained from all experimental hamster groups. The data for each analysed factor are presented as means ± SEM. The statistical significance (P 0.05) noticeably different was represented as *P values (for comparisons with C group) and as **P (for comparisons with HH group); n = 5 (the number of investigated animals).

endothelium, supporting plaque formation (Badimon et al., 2012). Moreover, these factors might be able to exert putative effects on EPC functions. Therefore, we measured the concentrations of IL-1β, -6, -8 and CD40L in the supernatants obtained from the hamster group derived platelets. Compared with the C group, the levels of IL-1β were higher especially in the platelet supernatants isolated from HH, HH–PMPs and HH–EPCs– PMPs groups (the enhancements were 2.14-fold, 2.54-fold and 1.45-fold, respectively), whereas for the HH–EPCs group, the IL-1beta levels were slightly increased: 1.26-fold. Compared with the HH group, the IL-1β concentrations were decreased 1.69-fold for the HH–EPCs group and 1.48-fold for the HH–EPCs–PMPs group, whereas for the HH– PMPs group were increased 1.19-fold (Figure 5, P 0.05). The measurements of IL-6 concentrations in the platelet supernatants isolated from HH, HH–PMPs and HH–EPCs–PMPs groups revealed a significant augmentation compared with the C group : 3.02fold, 3.11-fold and 2.74-fold, respectively (Figure 5, P 0.05). In the samples from the HH–EPCs group, IL-6 concentrations were slightly increased to

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1.65-fold compared with the C group. Moreover, compared with the HH group, we recorded decreased values for IL-6 in the platelet supernatants from the HH–EPCs and HH–EPCs–PMPs groups by 1.83fold and 1.11-fold, respectively. Compared with the C group, the IL-8 concentrations were enhanced in the platelet supernatants from HH, HH–PMPs and HH–EPCs–PMPs groups: 2.03-fold, 2.24-fold and 1.60-fold, respectively (Figure 5, P 0.05). For the HH–EPCs group, the concentration of IL-6 was similar to those in the C group. Compared with the HH group, HH–EPCs and HH–EPCs–PMPs groups displayed reduced levels of IL-8: 1.90-fold and 1.27-fold, respectively. As shown in Figure 5, the concentrations of IL-6 in the platelet supernatants generated from the HH–PMPs group were insignificantly modified compared with the HH group. The assessment of CD40L presence in the platelet supernatants shows that compared with the C group, the values for CD40L were increased 1.96-fold in the HH group, 2.18-fold in the HH–PMPs group and 1.17-fold in the HH–EPCs–PMPs group. The concentrations measured in the HH–EPCs group were similar to the values in the C group. Compared with the HH group, in HH–EPCs and HH–EPCs– PMPs groups, the values for CD40L were reduced to 1.92-fold and 1.67-fold. In platelets isolated from HH–PMPs group, the values for CD40L were enhanced a 1.11-fold compared with the HH group (Figure 5). The above results indicate that elevated levels of proinflammatory factors released by platelets in hypertension associated with hypercholesterolemia are reduced by EPC treatment and are increased after PMP administration.

Discussion EPCs have been considered to be a predictive marker for cardiovascular diseases and vascular dysfunction. Increasing data suggest that EPCs play an important role in the re-endothelialisation of injured blood vessels, a process that is critical for slowing or reversing the progression of atherosclerosis (Liu et al., 2009). In the present study, we investigated the effects of EPC and PMP transplantation alone or simultaneously on the interaction between platelets and circulating EPCs and possible mechanisms involved in this


cross-talk in the experimental induced atherosclerosis. The contribution of EPCs to re-endothelialisation was analysed by the presence of VEGF and eNOS on circulating EPCs isolated from five different experimental animal models characterised previously by our group (Alexandru et al., 2011, 2013; Georgescu et al., 2012, 2013). The results show that EPCs derived from HH and HH–PMPs groups exhibit an altered production of VEGF and eNOS compared with the C group, while EPC administration improves their expression. Cui et al. (2011) demonstrated in a rat model that the transplantation of EPCs increases eNOS and can repair balloon-caused carotid artery injury. Recently, it has been shown that PI3K/Akt/eNOS signalling pathway is involved in the EPC-mediated re-endothelialisation after arterial injury (Yang et al., 2012). Moreover, it was demonstrated that the statins, estrogens and erythropoietins could enhance re-endothelialisation or augment neovascularisation by upregulating eNOS expression of EPCs (Kietadisorn et al., 2012). The attachment of circulating EPCs to denuded arterial wall is considered to be a critical event for re-endothelialisation, and in this process TSP-1 is a key molecule in the adhesion to extracellular matrices. Accumulating evidences show that TSP-1 represents an important link in the accelerated development of atherosclerotic lesions in diabetes (Stenina et al., 2003; Ii et al., 2006). Therefore, we examined the TSP-1 protein distribution on EPCs and we found that hamsters HH treated or not with PMPs exhibited an increased expression of TSP-1 compared with the C group. These findings explain the reduced contribution of EPCs to repair in conditions of induced atherosclerosis, but the full TSP-1 implications in this process remain to be further investigated. The similar upregulation of TSP-1 expression on EPCs has been reported in diabetic EPCs and it was associated with impaired EPC adhesion activity (Ii et al., 2006). Inflammatory activation is a critical component of atherogenesis and endothelial dysfunction (Zhang et al., 2009). Correspondingly, we studied the release of proinflammatory mediators (IL-8, MCP-1 and RANTES) by EPCs, which are involved in the growth and formation of the atherosclerotic plaque, and also in the secretion of thromboinflammatory factors (MPO, PAI-1 and TF) with a role in the thrombus formation and plaque rupture. The levels of the above-investigated molecules were increased in the

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Research article medium obtained from EPCs isolated from HH and HH–PMPs groups, suggesting that dysfunctional EPCs have some proatherogenic effects. The treatment with EPCs reduced the values for these factors close to those in the C group, indicating that EPCbased administration can attenuate the inflammation in atherosclerosis. In combination with PMPs, the EPC treatment was not effective in decreasing the levels of these molecules, one explanation being that administrated EPCs would be vulnerable to inflammatory milieu. Our results are supported by those obtained from the study of Zhang et al. (2009), which demonstrated that cultured EPCs besides a cocktail of angiogenic factors also secrete a combination of detrimental factors that could affect plaque growth and rupture or promote a proinflammatory environment in the vasculature. The mobilisation, chemotaxis, adhesion and interaction with vascular cells are essential steps of progenitor cell-mediated tissue repair (Sopova et al., 2012). Since platelets circulate near the vessel wall and are the first cells to tether and subsequently adhere to subendothelium exposed by vascular injury, their interaction with EPCs is a central mechanism for directing and ‘homing’ EPCs to vascular injury sites (Leshem-Lev et al., 2010). The significant interaction between activated platelets and EPCs has been shown under both static and flow conditions and it appears to be mediated by P-selectin–P-selectin glycoprotein ligand-1 binding, α 4 integrin by β 1 and β 2 integrins (de Boer et al., 2006; Langer et al., 2006; Lev et al., 2006; AbouSaleh et al., 2009). In our study, in a static adhesion system, we assessed in vitro effect of immobilised platelets on the capacity of circulating EPCs to adhere and migrate. Our results showed that activated platelets stimulated EPC adhesion and migration, and these are augmented in the hypertension associated with hypercholesterolemia accompanied or not with PMP administration. This could be explained by the fact that in these conditions of inflammation, platelets are more reactive that in normal state and release many factors that influence these properties. Interesting, in a dynamic flow system, the platelet binding to EPCs under agitation condition was reduced in HH, HH–PMPs and HH–EPCs–PMPs groups compared with the C group, indicating that direct interactions are affected, but more experiments are required to elucidate the involved precise mechanisms.


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In a recent study on in patients with acute coronary syndrome, it has been shown that platelets form coaggregates with circulating CD34+ progenitor cells (Stellos et al., 2013). EPC administration makes that circulating EPCs to have a similar behaviour with those from the C group, because in this model platelet activation was reduced. Results in connection with the fact that platelets stimulate the chemotaxis and migration of mouse embryonic EPCs have been reported in vitro (Langer et al., 2006), but nothing has been described concerning platelet–EPC interaction in HH conditions. Additionally, our data reveal that the levels of chemokines and thromboinflammatory factors released by EPCs after migration and adhesion were higher in supernatant collected from cells derived from HH, HH–PMPs and HH–EPCs–PMPs groups, indicating that EPCs have an inflammatory potential. Also, EPC treatment had a beneficial effect in reducing these levels. In order to decipher the mechanisms by which platelets affect EPC functions, we focused on the platelet adhesion, process controlled by filopodia formation, a step of primary importance in regulating platelet activity in haemostasis and thrombosis (Pula et al., 2006). Thus, we examined the distribution of F-actin and VASP, some of the regulators of filopodia in platelets. The results illustrated platelet aggregability in HH and HH–PMPs groups, reflected by the increased distribution of these proteins and filopodia formation. An improvement of platelet function was presented in the group treated with EPCs compared with the HH group. These findings support the previous studies of our group which have shown that the platelet activation is enhanced in hypertension associated with hypercholesterolemia combined or not with PMP administration, while EPC therapy had a beneficial effect (Alexandru et al., 2011, 2013). Adherent platelets become activated and secrete or expose multiple inflammatory factors including growth factors, chemokines, cytokines and coagulation factors (Langer et al., 2006; Jennings, 2009). Moreover, the immediate presence of platelets in the atherosclerotic lesions renders them a potential checkpoint regulator of downstream events (Langer and Gawaz, 2008). Thus, we measure some proinflammatory mediators (IL-1β, -6, -8 and CD40L) released in platelet supernatants with major role in the atherosclerosis development (Lievens von Hundelshausen, 2011). We have found elevated levels of these molecules in

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activated platelets isolated from the HH group compared with the C group, and much higher in HHPMPs group compared with the HH group. These results confirm the microparticle contribution to inflammatory milieu in atherosclerosis through their action on platelet secretion. Moreover, EPC administration reduced the values of these mediators close to those in the C group, suggesting that EPC-based therapy attenuated the inflammation at the site of atherosclerotic lesion. The simultaneous administration of EPCs and PMPs induced a decrease in investigated molecules levels, but not as effective as the therapy with EPCs only. These results complete our previously work that disclosed an increased secretion of several cytokine/chemokines and growth factors (SDF-1α, MCP-1, RANTES, VEGF, PF4 and PDGF) by activated platelets obtained from HH and HH– PMPs groups compared with the C group (Alexandru et al., 2013). Taken together, these data provide novel insights into mechanisms involved in the communication between EPCs and platelets in experimental atherosclerosis, demonstrating that platelets have an important role in regulating EPC functions. Moreover, circulating EPC-based therapy has a beneficial impact on this relationship in hypertension associated with hypercholesterolemia, improving both platelet and EPC function. Furthermore, the administration of EPCs in combination with PMPs did not induce improvements on obtained effects when given only EPCs. Understanding of platelet–EPC interaction provides us new insights into the mechanisms of vascular homeostasis and possible new therapeutic targets supporting vascular repair. However, further studies are needed to define pathways that underlie this crosstalk between EPCs and platelets.

Materials and Methods Hamster models of atherosclerosis and transplant

The experiments were performed on platelets isolated from the blood of Golden Syrian hamsters (3 months old, n = 120) divided in five equal groups: (1) control, C (fed with a standard hamster diet); (2) HH (fed with standard diet enriched with 3% cholesterol, 15% butter and 8% NaCl, for 4 months); (3) HH treated with EPCs, HH–EPCs (injected via the retro-orbital plexus with 1 × 105 EPCs from C group in volume 300 µL, in one dose per month during diet-induced atherosclerotic process); (4) HH treated with PMPs, HH–PMPs (injected via the retro-orbital plexus with 1 × 105 PMPs from HH group in volume 300 µL, in one dose per month during diet-induced atherosclerotic

Research article


process) and (5) HH treated with EPCs and PMPs, HH–EPCs– PMPs (injected with 1 × 105 /mL EPCs from C hamsters and 1 × 105 /mL PMPs from HH hamsters in maximum of 300 µL volume, associated with specific HH diet for 4 month) (Alexandru et al., 2011, 2013; Georgescu et al., 2012, 2013). The reason of retro-orbital sinus injection with EPCs was to prevent atherosclerosis process and with PMPs was that to follow the acceleration of atherosclerosis in HH model. To investigate the possible competition or help between PMPs and EPCs, we designed another animal group, HH–EPCs–PMPs hamster. At 16 weeks, after the beginning of the experiment, the hamsters were sacrificed for biochemical and functional assays. All the protocols were approved by the Ethics Committees from Institute of Cellular Biology and Pathology ‘Nicolae Simionescu’ and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996). Also, the research on these animals was made in accordance with procedures defined in the Council Directive 86/609/EEC of 24 November 1986.

PMP sorting

PMPs were sorted from platelet-free plasma according to the method reported by Georgescu et al. (2009, 2012), using specific antibodies integrin αIIb (M148) PE (for CD41), annexin V FITC (for PS) and also the MoFlo flow cytometer (Dako) equipped with high-speed cell sorter. The number of PMPs was adjusted at 1×105 /mL in PBS. Platelet isolation

Hamsters were slightly anesthetised with ether, and blood was collected from the retro-orbital plexus. Platelets were separated according to the method reported by Alexandru et al. (2011, 2013). Briefly, the procedure consists of the collection of venous blood in ACD buffer (2.73% citric acid, 4.48% trisodium citrate and 2% glucose) and centrifugation at 400×g for 10 min. PRP obtained was spun down at 600×g for 10 min, and the platelets suspended in calcium-free Hepes buffer (pH 7.0) supplemented with 1% bovine serum albumin (BSA) and 0.15 U/mL apyrase. Phase contrast microscopy of the pellets showed that these were not aggregated, and the preparation was devoid of erythrocytes and leukocytes.

EPC isolation and sorting

PBMCs were fractionated using Histopaque-1077 by densitygradient centrifugation as described by Georgescu et al. (2012). Brief protocol: layer of 1 mL whole blood onto 3 mL Histopaque centrifuge at 400×g for 30 min, and aspirate upper layer to within 0.5 cm of opaque interface containing the mononuclear cells (MNCs). Discard upper layer and transfer the opaque interface into clean tube. Add 10 mL isotonic phosphate-buffered saline (PBS) solution containing 2% fetal bovine serum (FBS), mix, centrifuge at 256×g for 10 min, aspirate supernatant and discard, repeat three times the washing and afterward resuspend the MNCs pellet in 10 mL PBS. EPCs were sorted from MNCs by flow cytometry (MoFlo equipped with high-speed cell sorter), using the specific antibodies for CD34, vascular endothelial growth factor receptor 2 (VEGF-R2) (KDR or Flk-1) and adjusting them at the same number of 1 × 105 /mL in PBS. Importantly, in our previous paper, we showed that early EPCs (CD133+ , CD34+ , VEGF-R2+ ) are very rare in the peripheral blood compared with late EPCs (CD34+ and VEGFR2+ ) (Georgescu et al., 2012). There are some other studies that demonstrated that in the peripheral circulation of adults, more mature EPCs are found (positive for CD34 and VEGFR-2) that obviously have lost CD133 (Yin et al., 1997). For this reason, in our current study, we decided to use late or mature EPCs. Notably, the monthly blood collection did not affect the health status of animals. Preparation of platelet-free plasma, the source for circulating PMPs

Plasma PMPs were separated according to the method reported by Georgescu et al. (2009, 2012). Briefly, the procedure consists of the collection of venous blood in 0.138 M trisodium citrate 9/1 (vol/vol), centrifugation at 1000×g for 15 min at 15°C and separation of platelet-rich plasma (PRP). PRP was centrifuged at 2500×g for 15 min at 15°C and the platelet-free plasma obtained was centrifuged again at 13,000×g for 5 min at 15°C allowing the collection of PMPs in the supernatant.

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Immunofluorescence detection of EPC proteins

The EPCs (1 × 105 cells in PBS buffer) sorted as described above were added in 96-well plates coated with collagen type I (50 µg/mL), and incubated overnight at 37°C and 5% CO2 . Subsequently, the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature, treated with 0.1% Triton X-100/PBS, for 10 min, and incubated in 3% BSA in PBS at room temperature for 30 min (Wan et al., 2010). After incubation with the primary antibodies against endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) (1:200, Santa Cruz) for 2 h, the treatment with 0.1% Triton X-100/PBS for 10 min, washing with PBS and incubation with 1% BSA in PBS, at room temperature, for 30 min, EPCs were incubated with the secondary antibodies: PE-labelled anti-rabbit IgG (for eNOS and VEGF detection) and FITC-labelled anti-mouse IgG (for TSP-1) for 1 h. Images were captured with a 20× objective of fluorescence microscope (Axio Vert.A1 Fl, software Axio Vision Rel 483SE64-SP1; Carl Zeiss) and were processed with Adobe Photoshop (Adobe System). Paracrine activity of EPCs

To measure the paracrine activity of EPCs, cells were obtained from peripheral blood of the hamsters and sorted at the same number (1 × 105 cells/well) as described above in a culture plate with Dulbecco’s modified Eagle medium (1‰). After incubation at 37°C and 5% CO2 for 24 h, the supernatants were collected and the levels of chemokines and thromboinflammatory mediators were measured by the quantitative enzyme-linked immunosorbent assay (ELISA) (Zhang et al., 2009). Binding of EPCs to platelets in a static adhesion system

The isolated platelets (5 × 106 cells) were allowed to adhere to 24-well plates coated with collagen type I (10 µg/mL) for 2 h, followed by blocking with 2% BSA in PBS (Langer et al., 2006, 2007). Subsequently, after removing supernatants, sorted EPCs (1 × 105 cells in PBS buffer supplemented with 2% FBS)


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were added and incubated for 60 min. Furthermore, mediums were collected, centrifuged at 13,000 rpm for 10 min to remove the EPCs and the supernatants were kept for the quantification of chemokines and thromboinflammatory mediators by ELISA. On the plate, after washing with Tyrode’s buffer, the residual adherent EPCs were counted by direct phase contrast microscopy. Binding of EPCs to platelets in a dynamic flow system

In a second adhesion assay, the binding of platelets to EPCs was determined in mixed cell populations of 50:1 ratio, using a duallabelling technique with antibodies directed against plateletCD41-(PE) and EPC-KDR-(FITC). Sorted EPCs (1 × 105 cell) were incubated for 30 min with isolated platelets (5 × 106 cell) under agitation (1000 rpm) at 37°C. Thereafter, the cell suspensions were washed with Tyrodes buffer and incubated with FITC-conjugated anti-KDR and PE-conjugated anti-CD41. The percentage of dual KDR- and CD41-positive events was analysed by flow cytometry and used as index of platelet/EPC adhesion (Langer et al., 2006). Chemotaxis and migration of EPCs

A transwell culture system (Boyden migration assay) was used as described by Langer et al. (2006). In brief, isolated platelets (5 × 106 cells) were allowed to adhere to the bottom of 24-well culture plates coated with collagen type I (10 µg/mL) for 2 h. Sorted EPC cells (1 × 105 cell/mL) in basal medium were added on top using transwell culture inserts (5.0 µm pore size; Costar) that allowed physical separation from platelets. After 24 h of incubation at 37°C and 5% CO2 , the transwell inserts were removed and the lower side of the filters were washed with PBS and fixed with 2% PFA. For quantification, cell nuclei were stained with Hoechst 33258 solution (2 µg/mL). Migrating cells into the lower chamber were counted manually under a microscope (Lev et al., 2006). In addition, the remaining medium with ECPs from transwell culture inserts was centrifuged at 18,320×g for 10 min, and the remaining medium with platelets from well plates was centrifuged at 1730×g for 10 min. Also, the supernatants were collected for the quantitative ELISA. Localisation of F-actin and VASP on platelets by immunofluorescence

Platelets isolated as described above were stimulated by adhesion to collagen-coated surface (Pula et al., 2006). Coverslips were coated with 100 µg/mL collagen overnight, followed by nonspecific binding saturation with 2% BSA for 1 h and the addition of platelet suspensions (1.5 × 108 cells) for 90 min. Reactions were stopped by the addition of 4% PFA in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 , 2 mM KH2 PO4 ). The platelet permeabilisation and immunolabelling were performed as described by Pula et al. (2005). Briefly, platelets were permeabilised with 0.05% Triton X-100/PBS for 10 min, and then incubated overnight with primary antibodies for VASP detection (rabbit anti-VASP in 1% BSA/PBS) followed by the secondary antibody (PE-labelled anti-rabbit IgG). As for the F-actin detection, the above permeabilised platelets were incubated with FITC-Phalloidin in 1% BSA/PBS for 1 h. Subsequently, the coverslips were mounted onto slides using DPX Mountant solution and viewed on a fluorescence microscope in combination with phase contrast microscope (Microscope Zeiss Axio Observer Z1).

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Images were captured with a 63×/0.75 Korr objective and were processed using an Adobe Photoshop (Adobe System). Proinflammatory factor measurement in platelet supernatants

Platelet supernatant was obtained according to the method described by Dernbach et al. (2008). Isolated platelets as above were resuspended in Hepes buffer (1 × 106 cells/mL) and then activated by the centrifugation at 10,000×g for 10 min. The supernatants thus obtained were used to measure the concentration of the following molecules: IL-1β, -6, -8 and CD40L by the ELISA method using the specific kits according to the manufacturer’s instructions (R&D Systems). Briefly, samples were added in each well of a 96-well microtiter plate coated with chemokine-specific antibodies and incubated for 1 or 2 h at room temperature. After washing and adding of conjugate, substrate and stop solution, the optical density at 450 nm was measured using a spectrophotometer (TECAN, InfiniteM200PRO). Reagents

The standard chemicals, reagents and the specific antibodies were purchased from Sigma Chemical, Santa Cruz Biotechnology (www.scbt.com) and R&D Systems. All others reagents used were of analytical grade. Data analysis

The statistical evaluation of results was performed using one-way analysis of variance and Student’s t-test. Data were considered statistically significant when P < 0.05. For the flow cytometry experiments, a software based on auto and manual compensation was used (Summit 4.3 Software; DakoCytomation).

Author contribution N.A. had made substantial contributions to conception and design of the study, performed the experiments and data acquisition, analysis and interpretation of data and wrote the manuscript. E.A. carried out the ELISA assays, had contribution to the acquisition of the data for flow cytometry and performed the statistical analysis. E.D. effectively participated at the realisation of the experimental models. A.G. participated in designing of the study, performed the experiments, made substantial contributions to the acquisition of the data, helped to draft the manuscript and revised it critically for important intellectual content. All authors read and approved the final manuscript. Funding This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS – UEFISCDI, project ID PNII-CT-ERC2012-1 (6ERC-like/July 18, 2012), by the Romanian Academy and by the Project ID:143/SMIS CSNR 2667, European Regional Development Fund (ERDF) co-financed investment in Research,


Technology Development and Innovation for Competitiveness.

Acknowledgements The authors gratefully acknowledge and appreciate the dedicated work and help of the technicians Marilena Isachi, Marcela Toader (biochemistry) and Safta Nae (experimental models). Conflict of interest statement The authors have declared no conflict of interest. References Abou-Saleh, H., Yacoub, D., Theor ´ et, ˆ J.-F., Gillis, M.A., Neagoe, P.E., Labarthe, B., Theroux, P., Sirois, M.G., Tabrizian, M., Thorin, E. and ´ Merhi, Y. (2009) Endothelial progenitor cells bind and inhibit platelet function and thrombus formation. Circulation 120, 2230–2239 Alexandru, N., Popov, D., Dragan, E., Andrei, E. and Georgescu, A. (2011) Platelet activation in hypertension associated with hypercholesterolemia: effects of irbesartan. J. Thromb. Haemost. 9, 173–184 Alexandru, N., Popov, D., Dragan, E., Andrei, E. and Georgescu, A. (2013) Circulating endothelial progenitor cell and platelet microparticle impact on platelet activation in hypertension associated with hypercholesterolemia. PLoS ONE 8, e52058 Badimon, L., Padro, ´ T. and Vilahur, G. (2012) Atherosclerosis, platelets and thrombosis in acute ischaemic heart disease. Eur. Heart J. Acute Cardiovasc. Care 1, 60–74 Co, M., Tay, E., Lee, C.H., Poh, K.K., Low, A., Lim, J., Lim, I.H., Lim, Y.T. and Tan, H.C. (2008) Use of endothelial progenitor cell capture stent (Genous Bio-Engineered R Stent) during primary percutaneous coronary intervention in acute myocardial infarction: intermediate- to long-term clinical follow-up. Am. Heart J. 155, 128–132 Cui, B., Huang, L., Fang, Y., Guo, R., Yin, Y. and Zhao, X. (2011) Transplantation of endothelial progenitor cells overexpressing endothelial nitric oxide synthase enhances inhibition of neointimal hyperplasia and restores endothelium-dependent vasodilatation. Microvasc. Res. 81, 143–150 Daub, K., Langer, H., Seizer, P., Stellos, K., May, A.E., Goyal, P., Bigalke, B., Schonberger, T., Geisler, T., Siegel-Axel, D., ¨ Oostendorp, R.A.J., Lindemann, S., Gawaz, M. (2006) Platelets induce differentiation of human CD34+ progenitor cells into foam cells and endothelial cells. FASEB J. 20, 2559–2561 deBoer, H.C., Verseyden, C., Ulfman, L.H., Zwaginga, J.J., Bot, I., Biessen, E.A., Rabelink, T.J. and vanZonneveld, A.J. (2006) Fibrin and activated platelets cooperatively guide stem cells to a vascular injury and promote differentiation towards an endothelial cell phenotype. Arterioscler. Thromb. Vasc. Biol. 26, 1653–1659 Dernbach, E., Randriamboavonjy, V., Fleming, I., Zeiher, A.M., Dimmeler, S. and Urbich, C. (2008) Impaired interaction of platelets with endothelial progenitor cells in patients with cardiovascular risk factors. Basic Res. Cardiol. 103, 572–581 Du, F., Zhou, J., Gong, R., Huang, X., Pansuria, M., Virtue, A., Li, X., Wang, H. and Yang, X.F. (2012) Endothelial progenitor cells in atherosclerosis. Front. Biosci. (Landmark Ed) 17, 2327–2349 Fadini, G.P., Coracina, A., Baesso, I., Agostini, C., Tiengo, A., Avogaro, A. and deKreutzenberg, S.V. (2006) Peripheral blood CD34+KDR+ endothelial progenitor cells are determinants of

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Received: 29 September 2014; Accepted: 6 March 2015; Accepted article online: 12 March 2015

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