Skip to main content

Early outgrowth cells versus endothelial colony forming cells functions in platelet aggregation

Abstract

Background

Endothelial progenitor cells (EPCs) have been implicated in neoangiogenesis, endothelial repair and cell-based therapies for cardiovascular diseases. We have previously shown that the recruitment of EPCs to sites of vascular lesions is facilitated by platelets where EPCs, in turn, modulate platelet function and thrombosis. However, EPCs encompass a heterogeneous population of progenitor cells that may exert different effects on platelet function. Recent evidence suggests the existence of two EPC subtypes: early outgrowth cells (EOCs) and endothelial colony-forming cells (ECFCs). We aimed at characterizing these two EPC subtypes and at identifying their role in platelet aggregation.

Methods

EOCs and ECFCs were generated from human peripheral blood mononuclear cells (PBMCs) seeded in conditioned media on fibronectin and collagen, respectively. The morphological, phenotypical and functional characteristics of EOCs and ECFCs were assessed by optical and confocal laser scanning microscopes, cell surface markers expression, and Matrigel tube formation. The impact of EOCs and ECFCs on platelet aggregation was monitored in collagen-induced optical aggregometry and compared with PBMCs and human umbilical vein endothelial cells (HUVECs). The levels of the anti-platelet agents’ nitric oxide (NO) and prostacyclin (PGI2) released from cultured cells as well as the expression of their respective producing enzymes NO synthases (NOS) and cyclooxygenases (COX) were also assessed.

Results

We showed that EOCs display a monocytic-like phenotype whereas ECFCs have an endothelial-like phenotype. We demonstrated that both EOCs and ECFCs and their supernatants inhibited platelet aggregation; however ECFCs were more efficient than EOCs. This could be related to the release of significantly higher amounts of NO and PGI2 from ECFCs, in comparison to EOCs. Indeed, ECFCs, like HUVECs, constitutively express the endothelial (eNOS)—and inducible (iNOS)—NOS isoforms, and COX-1 and weakly express COX-2, whereas EOCs do not constitutively express these NO and PGI2 producing enzymes.

Conclusion

The different morphological, phenotypic and more importantly the release of the anti-aggregating agents PGI2 and NO in each EPC subtype are implicated in their respective roles in platelet function and thus, may be linked to the increased efficiency of ECFCs in inhibiting platelet aggregation as compared to EOCs.

Background

Endothelial progenitor cells (EPCs) are believed to play a significant role in vascular biology through their implication in vascular repair, cell therapy, and regenerative medicine [14]. Definitive proof of the existence of circulating EPCs was provided in 1997 [5], showing that human peripheral blood contains CD34+ and vascular endothelial growth factor receptor 2+ (VEGFR2+) progenitor cells, which can attach to fibronectin, acquire certain characteristics of mature endothelial cells (ECs) in vitro, and contribute to neoangiogenesis in vivo. Since then, numerous cells have been employed to improve cardiovascular function, including EPCs, bone marrow mononuclear cells, myocardial stem cells, mesenchymal stem cells, and embryonic stem cells [6, 7]. Among these, EPCs have emerged as important modulators in vascular biology and hemostasis. However, controversies still exist as to their therapeutic and diagnostic use [810]. One important aspect of EPC biology is their interaction with vascular and blood cells, which can largely influence their functional properties. More specifically, the interaction of EPCs with platelets provides the critical signal to ensure their migration and homing at sites of vascular injury and their differentiation into ECs [1117]. Conversely, EPCs affect platelet function [1822] and modulate endothelial repair [2327], which add new insights to the importance of EPCs in vascular hemostasis.

In our attempt to elucidate the functional importance of EPC interactions with platelets, we have previously shown that a heterogeneous population of EPCs, derived from PBMCs after 10 days of culture, modulates the function of platelets [18], through binding to platelets via P-selectin [28]. This interaction impairs the function of platelets through an increase in cyclooxygenase-2 (COX-2) expression and prostacyclin (PGI2) release. Recent experimental evidence has established that cells originally defined as EPCs and used for potential therapy are not true endothelial progenitors [8, 2931]. Indeed, recent analyses have revealed that, depending on how EPCs are isolated and cultured, the EPC population can either consist of a minimally proliferative myeloid-monocytic cell population (early EPCs, monocytic EPCs, EOCs, or cultured angiogenic cells) or consist primarily of a highly proliferative non-myeloid EC population (late EPCs, ECFCs, or late outgrowth cells) [9, 3234]. In this regard, we have been characterizing and studying the well-defined EOC subtype and showed that they produce matrix metalloproteinase-9 and reactive oxygen species that influence their pro-angiogenic [23] and anti-platelet functions [19], respectively. On the other hand, ECFCs are considered highly proliferative cells that incorporate at sites of vascular lesions, differentiate into ECs and contribute to endothelial repair [9, 32, 33, 35]. Their anti-platelet properties could be of great importance in vascular hemostasis and repair. Accordingly, this study was designed to describe the characteristics of EOCs and ECFCs, and to compare their effect on platelet function by defining the underlying vasoactive substances implicated in this process. The major findings are that ECFCs are more efficient in inhibiting platelet aggregation than EOCs. This was related to an increase in the production of the potent anti-platelet agents PGI2 and NO by ECFCs, which induce significantly stronger inhibitory effects of platelet aggregation.

Methods

Culture and characterization of human EOCs and ECFCs

This study was carried out according to a protocol accepted by the Montreal Heart Institute ethical committee in agreement with the Declaration of Helsinki. Informed consent was obtained from healthy volunteers aged between 20 and 60 years and medication free over 10 days prior to blood sampling. Ficoll–Paque (GE Healthcare, Piscataway, NJ, USA) density gradient centrifugation was used to isolate peripheral blood mononuclear cells (PBMCs) from 100 mL of peripheral blood, as previously described [18]. To generate EOCs, 1 × 106 PBMCs per cm2 were seeded on 6-well fibronectin-coated tissue culture plates (BD Biosciences, Mississauga, ON, Canada) in complete endothelial growth media EGM-2 (Lonza Inc., Burlington, ON, Canada) at 37 °C in an atmosphere of 5 % CO2. Following 3 days of culture, the medium was changed to remove non-adherent cells. Additional culturing of adherent cells up to 7 days was allowed to obtain EOCs [23, 36]. ECFCs were generated from PBMCs seeded at a density of 5 × 106 mononuclear cells per cm2 in 6-well collagen-coated tissue culture plates (BD Biosciences). Culturing of ECFCs was performed in EGM-2 supplemented with 10 % FBS. The medium was changed daily for 7 days and every other day thereafter until the appearance of endothelial cell colonies (2–4 weeks), which were cultured until the formation of a confluent monolayer of highly proliferative EPCs [3638]. Cells were used between passages three and six. Characterization of EOCs and ECFCs was compared to mature human umbilical vein endothelial cells (HUVECs) (Lonza Inc.) cultured in complete EGM2 with 10 % FBS on 0.2 % gelatin-coated flasks.

Confocal microscopy

EOCs and ECFCs were incubated with DiI-labeled acetylated low density lipoprotein (DiI-Ac-LDL) (Invitrogen, Carlsbad, CA, USA) for 2 h followed by 4 % paraformaldehyde fixation and subsequent 1-hour incubation with FITC-labeled Ulex-lectin (Sigma-Aldrich, St-Louis, MO, USA). Nuclear staining was done with TO-PRO-3 (Invitrogen). Cells were visualized under an LSM 510 confocal microscope (Zeiss, Oberkochen, Germany) at 63× objective magnification [18].

Matrigel tube formation assay

EPC’s potential to form tube-like structures was compared to that of HUVECs on Matrigel-coated 24-well plates [39]. Growth factor reduced Matrigel (BD Biosciences) (200 μL) was incubated for 30 min at 37 °C in a 5 % CO2 atmosphere to allow polymerization and formation of a gel-like surface. Cells were harvested with trypsin–EDTA 0.05 % (Gibco, Burlington, ON, Canada), washed and resuspended in complete EGM-2 at 200 × 103 per 300 μL per well. Following 24-h incubation at 37 °C in a 5 % CO2 atmosphere, cells were viewed under an inverted microscope and images were taken at 10× objective magnification.

Cell surface marker expression

Phenotypic characterization of EOCs and ECFCs was performed by flow cytometry and compared to PBMCs and HUVECs [18]. Cells were harvested by washing cells with phenol red-free and serum-free basal RPMI 1640 medium followed by trypsinization with 0.05 % trypsin–EDTA (Gibco) for 15 min at 37 °C [38]. Cells were subsequently blocked with normal mouse serum for 15 min, washed and incubated for 30 min with PE-labeled fluorescent human monoclonal antibodies against the monocytic marker CD14 (R&D Systems, Minneapolis, MN, USA), the leukocytic marker CD45 (AbD Serotec, Oxford, UK), the progenitor marker CD34 (BD Biosciences), the angiogenic marker VEGFR2 (R&D Systems), and the endothelial markers CD31 (BD Biosciences) and CD144 (BD Biosciences). Cells were then fixed with paraformaldehyde for 30 min and analyzed on an Altra flow cytometer (Beckman Coulter, Mississauga, ON, Canada). Cells were gated by their characteristic forward and side scatter properties.

Platelet aggregation

Platelets were isolated from 100 mL peripheral blood of healthy volunteers, as previously described [18, 19]. Washed platelets resuspended in HBSS–Hank’s buffer were then adjusted to 250 × 106/mL. Platelet aggregation was performed in a four-channel optical aggregometer (Chronolog Corp., Havertown, PA, USA) under shear (1000 rpm) at 37 °C. A volume of 400 μL of the washed platelet preparation was incubated with 100 μL of PBMC, EOC, and ECFC cells or their supernatants at 37 °C for 5 min prior to the assay. Platelet aggregation was then induced with 1 μg/mL collagen (Chronolog Corp.), and traces were recorded until stabilization of platelet aggregation was reached. Controls were performed by incubating platelets with complete EGM-2 culture medium.

Western blot

EOCs and ECFCs were harvested using trypsin–EDTA 0.05 % (Gibco), centrifuged at 500×g for 10 min then resuspended in PBS 1× and sonicated. Protein content was assessed by the Bradford assay method, mixed with the appropriated volume of 4× Laemmli loading buffer and heated for 5 min at 95 °C. Protein lysates (40 μg) were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5 % non-fat milk in TBS-Tween-20 for 1 h. Membranes were then incubated overnight with primary antibodies (1:1000) against eNOS and iNOS (Cell Signaling, Beverly, MA, USA), and against COX-1 and COX-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Following washing steps, membranes were labeled with horseradish peroxidase-conjugated secondary antibody for 1 h, washed and bound peroxidase activity was detected by enhanced chemiluminescence (Perkin Elmer Life Sciences, Waltham, MA, USA).

PGI2 release assay

A commercial radioimmunoassay (RIA) PGI2 kit was used to assess prostacyclin release (Assay Designs, San Diego, CA, USA) according to manufacturer instructions. Levels of 6-keto-PGF1, the stable metabolite of PGI2, were assessed in EOC and ECFC culture supernatants and compared to PBMC and HUVEC supernatants.

NO release assay

A commercial fluorometric NO assay kit was used to assess nitric oxide release (Cayman Chemicals, Ann Arbor, MI, USA) according to manufacturer instructions. Levels of total nitrate and nitrite (NO2 and NO3 ) were assessed in EOC and ECFC supernatants and net nitrate (NO2 ) concentrations were calculated and compared to PBMC and HUVEC supernatants.

Statistical analysis

Results are presented as mean ± SEM of at least three independent experiments. Statistical comparisons were done using either paired student’s t test or a one-way ANOVA followed by a Dunnett’s-t-test for comparison against a single group. Data with p < 0.05 were considered statistically significant.

Results

EOCs and ECFCs are morphologically and phenotypically different

Different culture techniques were required to generate each EPC subtype. EOCs were generated from PBMCs cultured for 7 days on fibronectin-coated plates whereas ECFCs were obtained following longer culture periods (2–4 weeks) on collagen-coated plates. The morphology observed for each cell subtype differs greatly. EOCs form a heterogeneous population of round and elongated cells whereas ECFCs form a cobblestone-like monolayer of homogenous appearance (Fig. 1a). EOCs display an immature endothelial cell character since they still resemble the PBMC population from which they derive following 7 days of culture. On the other hand, ECFCs seem to acquire an endothelial-like phenotype resembling HUVECs. Furthermore, EOCs are short-lived cells which do not survive past 7 days whereas ECFCs are highly proliferative cells which can be passaged and kept in culture for months (data not shown). Both EOCs and ECFCs bind Ulex-lectin and internalize DiI-Ac-LDL, both endothelial cell characteristics (Fig. 1b).

Fig. 1
figure 1

Characterization of PBMC-derived EOCs and ECFCs. a Representative optical microscopy images of EOCs and ECFCs taken at 10× magnification using an inverted light microscope. EOCs display a heterogeneous population of round and elongated cells on fibronectin following 7 days of culture. ECFCs show a homogeneous population forming a cobblestone-like monolayer on collagen following 21 days of culture. b Representative confocal microscopy images showing EOCs and ECFCs triple staining for DiI-Ac-LDL uptake (red), Ulex-lectin binding (green) and TO-PRO-3 nuclear staining (blue) taken at 63× magnification. c Representative optical microscopy images showing the tube-like structure formation potential of EOCs and ECFCs compared to HUVECs on a Matrigel surface taken at 10× magnification using an inverted microscope

EOCs and ECFC tube formation potential

We show that EOCs and ECFCs are also functionally different. EOCs do not form tube-like structures, whereas ECFCs, similarly to HUVECs, form well-structured tubes on Matrigel (Fig. 1c).

EOCs and ECFC cell surface marker expression

EOCs continue to express the leukocytic and monocytic cell surface markers CD45 and CD14, respectively, after 7 days of culture, whereas ECFCs lose these markers throughout their differentiation process. Moreover, EOCs strongly express the angiogenic marker VEGFR2. Unlike EOCs, ECFCs express the progenitor cell marker CD34 and the endothelial cell marker CD144. All cell populations showed uniform expression of the platelet-endothelial cell adhesion molecule CD31 (Fig. 2).

Fig. 2
figure 2

Expression of cell surface markers by EOCs and ECFCs. Flow cytometric analysis of cell surface marker expression profile of EOCs (gray) and ECFCs (white) compared to PBMCs (black) and HUVECs (dark gray) using mouse anti-human PE-conjugated monoclonal antibodies against CD14, CD45, VEGFR2, CD34, CD144 and CD31. Histogram represents the mean data ± SEM of percent cell surface marker expression (n ≥ 3)

EOCs and ECFCs inhibit platelet aggregation

In order to better understand the influence of EPCs on platelets, we sought to investigate the effect of each EPC subtype on platelet aggregation. We show that both EOCs and ECFCs and their respective supernatants inhibit platelet aggregation, in comparison to PBMCs (Fig. 3). However, we noticed an increased efficiency of the supernatants in inhibiting platelet aggregation in comparison to their respective cell samples for both EOCs and ECFCs. We suggest that this may be due to the release of vasoactive agents which play an important role in platelet aggregation. Furthermore, we demonstrated that ECFCs are more efficient at inhibiting platelet aggregation than EOCs.

Fig. 3
figure 3

Effect of PBMCs, EOCs and ECFCs on platelet aggregation. a Representative traces of platelet aggregation. Platelets were incubated with complete EGM-2 medium (control) or in the presence of 4 × 106/mL PBMC, EOC and ECFC cells or their respective supernatants for 5 min at 37 °C prior to aggregation initiation with 1 μg/mL of collagen. b Histogram represents the mean data of percent platelet aggregation ± SEM (n ≥ 3, ** p < 0.01 vs control)

ECFCs release more NO and PGI2 than EOCs

To further understand the increased efficiency of ECFCs compared to EOCs in inhibiting platelet aggregation, we compared the NOS and COX expression profiles in EOCs, ECFCs, PBMCs, and HUVECs. We found that ECFCs, similarly to HUVECs, constitutively express eNOS, iNOS, and COX-1, but to a lesser extent COX-2. In contrast, EOCs do not constitutively express these NO and PGI2 producing enzymes (Fig. 4a). Using radioimmunoassay and fluorometric techniques, we assessed PGI2 and NO release, respectively. We found that EOCs release minimal amounts of NO (60 pmol) and PGI2 (300 pg/mL) whereas ECFCs, similarly to HUVECs, release significantly higher levels of both NO (90 pmol) and PGI2 (5000 pg/mL) (Fig. 4b,c). The higher expression of NOS and COX enzymes in ECFCs account for the significantly higher levels of NO and PGI2 released by these cells in comparison to EOCs.

Fig. 4
figure 4

Expression of NOS and COX and release of NO and PGI2 by EOCs and ECFCs. a Left: NOS and COX protein expression profile in PBMCs, EOCs, ECFCs and HUVECs. Representative blots of cell lysates analyzed for eNOS, iNOS, COX-1 and COX-2 by SDS-PAGE. Right: Histogram represents the mean of data ± SEM expressed as arbitrary units of optical density of blots on the left (n ≥ 3); (b, c) supernatants from 4 × 106/mL PBMCs, EOCs, ECFCs and HUVECs were assessed for PGI2 release by radioimmunoassay of 6-keto-PGF concentration (pg/mL) (b) and for NO release by nitrate/nitrite fluorometric assay for nitrate concentration (pmol) (c). Histograms represent the mean concentration ± SEM of nitrate and PGI2 (n ≥ 3, *p < 0.05 vs PBMCs, ***p < 0.001 vs PBMCs)

Discussion

EPCs have gained much attention in the past decade for their therapeutic role; however, no clear consensus has been achieved to date as to the origin and specific function of these cells. Indeed, the term EPCs represents a complex assortment of progenitor cells that play different role on neoangiogenesis and vascular repair. The characterization and critical re-evaluation of EPC phenotypes are therefore essential for their efficiency as therapeutic and diagnostic tools (for review Fadini et al. [9]). Different conditions such as source, media, incubation times and culture surfaces have yielded different subtypes of EPCs. However, recent evidence suggests the existence of two subtypes of EPCs according to their time-dependent appearance. Based on new findings and on standardized isolation and differentiation protocols [30, 34, 3638], we have first successfully differentiated two subtypes of EPCs from PBMCs: EOCs and ECFCs, each distinct in their morphology, phenotype, and function. As it has previously been argued, we have found that EOCs and ECFCs differ in morphology and phenotype since EOCs possess monocytic-like characteristics, whereas ECFCs are rather endothelial-like cells. In addition, EOCs neither proliferate nor form tube-like structures, whereas ECFCs are highly proliferative and form structured and defined tubes on Matrigel. These data confirm previous studies showing that EOCs lack the ability to incorporate into the neovasculature, but rather influence neovascularization through the release of paracrine factors, whereas ECFCs can be incorporated in new vessel formation [36, 40]. However, the two EPC subtypes seem to act synergistically in vascular repair [4042]. Therefore, we aimed at studying the role of EOCs and ECFCs in platelet function as the former cannot form new vessels but may considerably influence platelets by releasing a multitude of vasoactive substances, whereas the latter may indirectly influence platelet function while incorporating at sites of vascular lesions.

EPCs and platelet interactions occur at sites of vascular lesions in order to promote vascular repair. Being the first cells to be recruited at sites of vascular damage, platelets become activated to release a multitude of paracrine factors (SDF-1) and to express cell surface adhesion molecules (P-selectin), which allow for the recruitment and differentiation of EPCs [14, 15, 43]. In turn, EPCs also modulate the function of platelets by releasing paracrine factors [18, 19], such as PGI2 and NO, which are the two main endothelium-derived vasodilation substances that display potent anti-platelet properties [44]. In the present study, we showed that both EOCs and ECFCs inhibit platelet aggregation. However, ECFCs are significantly more efficient than EOCs at inhibiting this process. Interestingly, the supernatants from both EOCs and ECFCs inhibit platelet aggregation more efficiently than their respective cells. Next, we compared the levels of PGI2 and NO released by EOCs and ECFCs, as these two substances are the major endothelium-derived vasodilation agents that play protective and antithrombotic roles in platelet reactivity. We showed that EOCs release smaller quantities of both PGI2 and NO in comparison to ECFCs. This could be related to the presence of a higher quantity of NO and PGI2 in the supernatants, which accounts for their increased efficiency in inhibiting platelet function as compared to the cells. In this connection, COX-1 and COX-2 are the enzymes involved in the production of prostaglandins, thromboxane and PGI2. Nitric oxide synthases (eNOS, iNOS and nNOS) are the enzymes involved in NO production, among which eNOS and iNOS are mainly responsible for NO production by the endothelium. Different levels of expression and regulation of the COX and NOS enzymes determine the levels of PGI2 and NO released by different vascular cell types. Interestingly, we found that ECFCs, similarly to HUVECs, constitutively express eNOS, iNOS and COX-1, but to a lesser extent COX-2. Surprisingly, EOCs do not constitutively express these NO and PGI2 producing enzymes. This could be related in part to a regulatory mechanism that occurs at this early stage of the differentiation process, where EOCs, like other NO and PGI2 producing cells, need to be triggered to upregulate COX and NOS enzymes. However, further investigation is needed to clarify this issue. In the other hand, the pattern expression of COX and NOS in our ECFC population was close to those found in EPCs generated in our previous study [18]. This could explain why ECFCs release considerably higher amounts of the anti-aggregating agents PGI2 and NO than EOCs, thereby accounting for their enhanced capacity in inhibiting platelet aggregation. However, the mechanism by which EOCs and their supernatants inhibit platelet aggregation remains to be determined. In addition, further research is required to understand the intracellular mechanisms underlying the differential influence of EOCs and ECFCs on platelet function.

Conclusions

Endothelial progenitor cells are regarded as promising therapeutic tools for vascular repair, tissue engineering, and regenerative medicine. However, the clinical usefulness of these cells is hindered by the existence of diverse subtypes of EPCs which exert diverse therapeutic potential. EPCs generated from the culture of PBMCs have been identified as EOCs and ECFCs depending on the culture conditions. In this study, we determined the characteristics of EOCs and ECFCs, and we defined their impact on platelet aggregation, the essential function in both vascular injury and vascular repair. We found that EOCs and ECFCs display distinct morphological, phenotypical, and functional characteristics. In addition, we demonstrated that both EOCs and ECFCs inhibit platelet aggregation, however; the inhibitory effect of ECFCs was more pronounced than EOCs. These inhibitory effects seem to be mediated by the secretion of the anti-aggregating agents PGI2 and NO by EOCs and, to a greater extent by ECFCs, thus accounting for their enhanced capacity in inhibiting platelet aggregation. These findings enforce our current understandings of different subsets of EPCs and provide novel insights into the differential role played by EOCs and ECFCs in modulating platelet function during vascular repair. This knowledge would benefit the management of atherothrombosis during acute coronary syndromes and following percutaneous coronary interventions. Ultimately, this may lead to the development of novel strategies for EPC-derived antithrombotic therapies and vascular regeneration in patients with cardiovascular disease.

Abbreviations

EPCs:

endothelial progenitor cells

ECs:

endothelial cells

EOCs:

early outgrowth cells

ECECs:

endothelial colony forming cells

PBMCs:

peripheral blood mononuclear cells

HUVECs:

human umbilical vein endothelial cells

VEGFR2:

vascular endothelial growth factor receptor 2

PGI2 :

prostacyclin

NO:

nitric oxide

COX:

cyclooxygenase

eNOS:

endothelial nitric oxide synthase

iNOS:

inducible nitric oxide synthase

References

  1. Wassmann S, Werner N, Czech T, Nickenig G. Improvement of endothelial function by systemic transfusion of vascular progenitor cells. Circ Res. 2006;99(8):e74–83.

    Article  CAS  PubMed  Google Scholar 

  2. Larsen K, Cheng C, Tempel D, Parker S, Yazdani S, den Dekker WK, Houtgraaf JH, de Jong R, Swager-ten Hoor S, Ligtenberg E, et al. Capture of circulatory endothelial progenitor cells and accelerated re-endothelialization of a bio-engineered stent in human exvivo shunt and rabbit denudation model. Eur Heart J. 2012;33(1):120–8.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Kong D, Melo LG, Gnecchi M, Zhang L, Mostoslavsky G, Liew CC, Pratt RE, Dzau VJ. Cytokine-induced mobilization of circulating endothelial progenitor cells enhances repair of injured arteries. Circulation. 2004;110(14):2039–46.

    Article  CAS  PubMed  Google Scholar 

  4. Griese DP, Ehsan A, Melo LG, Kong D, Zhang L, Mann MJ, Pratt RE, Mulligan RC, Dzau VJ. Isolation and transplantation of autologous circulating endothelial cells into denuded vessels and prosthetic grafts: implications for cell-based vascular therapy. Circulation. 2003;108(21):2710–5.

    Article  PubMed  Google Scholar 

  5. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997;275(5302):964–7.

    Article  CAS  PubMed  Google Scholar 

  6. Sieveking DP, Ng MK. Cell therapies for therapeutic angiogenesis: back to the bench. Vasc Med. 2009;14(2):153–66.

    Article  PubMed  Google Scholar 

  7. Dixit P, Katare R. Challenges in identifying the best source of stem cells for cardiac regeneration therapy. Stem Cell Res Ther. 2015;6:26.

    Article  PubMed Central  PubMed  Google Scholar 

  8. Pearson JD. Endothelial progenitor cells—hype or hope? J Thromb Haemost. 2009;7(2):255–62.

    Article  CAS  PubMed  Google Scholar 

  9. Fadini GP, Losordo D, Dimmeler S. Critical reevaluation of endothelial progenitor cell phenotypes for therapeutic and diagnostic use. Circ Res. 2012;110(4):624–37.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Tura O, Skinner EM, Barclay GR, Samuel K, Gallagher RC, Brittan M, Hadoke PW, Newby DE, Turner ML, Mills NL. Late outgrowth endothelial cells resemble mature endothelial cells and are not derived from bone marrow. Stem Cells. 2013;31(2):338–48.

    Article  CAS  PubMed  Google Scholar 

  11. Daub K, Langer H, Seizer P, Stellos K, May AE, Goyal P, Bigalke B, Schonberger T, Geisler T, Siegel-Axel D, et al. Platelets induce differentiation of human CD34 + progenitor cells into foam cells and endothelial cells. FASEB J. 2006;20(14):2559–61.

    Article  CAS  PubMed  Google Scholar 

  12. de Boer HC, Verseyden C, Ulfman LH, Zwaginga JJ, Bot I, Biessen EA, Rabelink TJ, van Zonneveld AJ. Fibrin and activated platelets cooperatively guide stem cells to a vascular injury and promote differentiation towards an endothelial cell phenotype. Arterioscler Thromb Vasc Biol. 2006;26(7):1653–9.

    Article  PubMed  Google Scholar 

  13. Lev EI, Estrov Z, Aboulfatova K, Harris D, Granada JF, Alviar C, Kleiman NS, Dong JF. Potential role of activated platelets in homing of human endothelial progenitor cells to subendothelial matrix. Thromb Haemost. 2006;96(4):498–504.

    CAS  PubMed  Google Scholar 

  14. Massberg S, Konrad I, Schurzinger K, Lorenz M, Schneider S, Zohlnhoefer D, Hoppe K, Schiemann M, Kennerknecht E, Sauer S, et al. Platelets secrete stromal cell-derived factor 1 alpha and recruit bone marrow-derived progenitor cells to arterial thrombi invivo. J Exp Med. 2006;203(5):1221–33.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Stellos K, Langer H, Daub K, Schoenberger T, Gauss A, Geisler T, Bigalke B, Mueller I, Schumm M, Schaefer I, et al. Platelet-derived stromal cell-derived factor-1 regulates adhesion and promotes differentiation of human CD34 + cells to endothelial progenitor cells. Circulation. 2008;117(2):206–15.

    Article  CAS  PubMed  Google Scholar 

  16. Feng W, Madajka M, Kerr BA, Mahabeleshwar GH, Whiteheart SW, Byzova TV. A novel role for platelet secretion in angiogenesis: mediating bone marrow-derived cell mobilization and homing. Blood. 2011;117(14):3893–902.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Raz O, Lev DL, Battler A, Lev EI. Pathways mediating the interaction between endothelial progenitor cells (EPCs) and platelets. PLoS ONE. 2014;9(6):e95156.

    Article  PubMed Central  PubMed  Google Scholar 

  18. Abou-Saleh H, Yacoub D, Theoret JF, Gillis MA, Neagoe PE, Labarthe B, Theroux P, Sirois MG, Tabrizian M, Thorin E, et al. Endothelial progenitor cells bind and inhibit platelet function and thrombus formation. Circulation. 2009;120(22):2230–9.

    Article  PubMed Central  PubMed  Google Scholar 

  19. Bou Khzam L, Hachem A, Zaid Y, Boulahya R, Mourad W, Merhi Y. Soluble CD40 ligand impairs the anti-platelet function of peripheral blood angiogenic outgrowth cells via increased production of reactive oxygen species. Thromb Haemost. 2013;109(5):940–7.

    Article  CAS  PubMed  Google Scholar 

  20. Shirota T, He H, Yasui H, Matsuda T. Human endothelial progenitor cell-seeded hybrid graft: proliferative and antithrombogenic potentials in vitro and fabrication processing. Tissue Eng. 2003;9(1):127–36.

    Article  CAS  PubMed  Google Scholar 

  21. Li XQ, Meng QY, Wu HR. Effects of bone marrow-derived endothelial progenitor cell transplantation on vein microenvironment in a rat model of chronic thrombosis. Chin Med J. 2007;120(24):2245–9.

    CAS  PubMed  Google Scholar 

  22. Alexandru N, Popov D, Dragan E, Andrei E, Georgescu A. Circulating endothelial progenitor cell and platelet microparticle impact on platelet activation in hypertension associated with hypercholesterolemia. PLoS ONE. 2013;8(1):e52058.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Bou Khzam L, Boulahya R, Abou-Saleh H, Hachem A, Zaid Y, Merhi Y. Soluble CD40 ligand stimulates the pro-angiogenic function of peripheral blood angiogenic outgrowth cells via increased release of matrix metalloproteinase-9. PLoS ONE. 2013;8(12):e84289.

    Article  PubMed Central  PubMed  Google Scholar 

  24. Aoki J, Serruys PW, van Beusekom H, Ong AT, McFadden EP, Sianos G, van der Giessen WJ, Regar E, de Feyter PJ, Davis HR, et al. Endothelial progenitor cell capture by stents coated with antibody against CD34: the HEALING-FIM (healthy endothelial accelerated lining inhibits neointimal growth-first in man) Registry. J Am Coll Cardiol. 2005;45(10):1574–9.

    Article  CAS  PubMed  Google Scholar 

  25. Kaushal S, Amiel GE, Guleserian KJ, Shapira OM, Perry T, Sutherland FW, Rabkin E, Moran AM, Schoen FJ, Atala A, et al. Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo. Nat Med. 2001;7(9):1035–40.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Shirota T, Yasui H, Shimokawa H, Matsuda T. Fabrication of endothelial progenitor cell (EPC)-seeded intravascular stent devices and in vitro endothelialization on hybrid vascular tissue. Biomaterials. 2003;24(13):2295–302.

    Article  CAS  PubMed  Google Scholar 

  27. Ranjan AK, Kumar U, Hardikar AA, Poddar P, Nair PD, Hardikar AA. Human blood vessel-derived endothelial progenitors for endothelialization of small diameter vascular prosthesis. PLoS ONE. 2009;4(11):e7718.

    Article  PubMed Central  PubMed  Google Scholar 

  28. Abou-Saleh H, Hachem A, Yacoub D, Gillis MA, Merhi Y. Endothelial progenitor cells inhibit platelet function in a P-selectin-dependent manner. J Transl Med. 2015;13:142.

    Article  PubMed Central  PubMed  Google Scholar 

  29. Ingram DA, Mead LE, Tanaka H, Meade V, Fenoglio A, Mortell K, Pollok K, Ferkowicz MJ, Gilley D, Yoder MC. Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood. 2004;104(9):2752–60.

    Article  CAS  PubMed  Google Scholar 

  30. Prater DN, Case J, Ingram DA, Yoder MC. Working hypothesis to redefine endothelial progenitor cells. Leukemia. 2007;21(6):1141–9.

    Article  CAS  PubMed  Google Scholar 

  31. Gulati R, Jevremovic D, Peterson TE, Chatterjee S, Shah V, Vile RG, Simari RD. Diverse origin and function of cells with endothelial phenotype obtained from adult human blood. Circ Res. 2003;93(11):1023–5.

    Article  CAS  PubMed  Google Scholar 

  32. Yoder MC. Endothelial progenitor cell: a blood cell by many other names may serve similar functions. J Mol Med. 2013;91(3):285–95.

    Article  PubMed Central  PubMed  Google Scholar 

  33. Resch T, Pircher A, Kahler CM, Pratschke J, Hilbe W. Endothelial progenitor cells: current issues on characterization and challenging clinical applications. Stem Cell Rev. 2012;8(3):926–39.

    Article  CAS  PubMed  Google Scholar 

  34. Yang N, Li D, Jiao P, Chen B, Yao S, Sang H, Yang M, Han J, Zhang Y, Qin S. The characteristics of endothelial progenitor cells derived from mononuclear cells of rat bone marrow in different culture conditions. Cytotechnology. 2011;63(3):217–26.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Lin RZ, Moreno-Luna R, Li D, Jaminet SC, Greene AK, Melero-Martin JM. Human endothelial colony-forming cells serve as trophic mediators for mesenchymal stem cell engraftment via paracrine signaling. Proc Natl Acad Sci. 2014;111(28):10137–42.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Mead LE, Prater D, Yoder MC, Ingram DA. Isolation and characterization of endothelial progenitor cells from human blood. Curr Protoc Stem Cell Biol. 2008;Chapter 2:Unit 2C 1.

  37. Zhang Y, Ingram DA, Murphy MP, Saadatzadeh MR, Mead LE, Prater DN, Rehman J. Release of proinflammatory mediators and expression of proinflammatory adhesion molecules by endothelial progenitor cells. Am J Physiol Heart Circ Physiol. 2009;296(5):H1675–82.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Stroncek JD, Grant BS, Brown MA, Povsic TJ, Truskey GA, Reichert WM. Comparison of endothelial cell phenotypic markers of late-outgrowth endothelial progenitor cells isolated from patients with coronary artery disease and healthy volunteers. Tissue Eng Part A. 2009;15(11):3473–86.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Yamamoto K, Takahashi T, Asahara T, Ohura N, Sokabe T, Kamiya A, Ando J. Proliferation, differentiation, and tube formation by endothelial progenitor cells in response to shear stress. J Appl Physiol. 2003;95(5):2081–8.

    Article  PubMed  Google Scholar 

  40. Yoon CH, Hur J, Park KW, Kim JH, Lee CS, Oh IY, Kim TY, Cho HJ, Kang HJ, Chae IH, et al. Synergistic neovascularization by mixed transplantation of early endothelial progenitor cells and late outgrowth endothelial cells: the role of angiogenic cytokines and matrix metalloproteinases. Circulation. 2005;112(11):1618–27.

    Article  PubMed  Google Scholar 

  41. Krenning G, van der Strate BW, Schipper M, van Seijen XJ, Fernandes BC, van Luyn MJ, Harmsen MC. CD34 + cells augment endothelial cell differentiation of CD14 + endothelial progenitor cells in vitro. J Cell Mol Med. 2009;13(8B):2521–33.

    Article  CAS  PubMed  Google Scholar 

  42. Cheng CC, Chang SJ, Chueh YN, Huang TS, Huang PH, Cheng SM, Tsai TN, Chen JW, Wang HW. Distinct angiogenesis roles and surface markers of early and late endothelial progenitor cells revealed by functional group analyses. BMC Genom. 2013;14:182.

    Article  CAS  Google Scholar 

  43. Langer HF, May AE, Vestweber D, De Boer HC, Hatzopoulos AK, Gawaz M. Platelet-induced differentiation of endothelial progenitor cells. Semin Thromb Hemost. 2007;33(2):136–44.

    Article  CAS  PubMed  Google Scholar 

  44. Mitchell JA, Ali F, Bailey L, Moreno L, Harrington LS. Role of nitric oxide and prostacyclin as vasoactive hormones released by the endothelium. Exp Physiol. 2008;93(1):141–7.

    Article  CAS  PubMed  Google Scholar 

Download references

Authors’ contributions

LBK designed the study, performed the experiments, acquired and interpreted the data, and drafted the manuscript. OB contributed to EPC isolation and western blot experiments. RB participated in platelet isolation and aggregation, and provided technical support. AH participated in platelet isolation and flow cytometry experiments and revised the manuscript. HAS participated in manuscript preparation and revision, provided technical support, and drafted the manuscript. YM conceived and designed the study, evaluated data, and drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by grants from the Canadian Institute for Health Research and the Heart and Stroke Foundation of Canada. We thank Mr. Louis Villeneuve for his technical assistance with confocal microscopy imaging and Dr. Daniel Yacoub for his help in editing the manuscript.

Competing interests

The authors declare that they have no competing interests.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Yahye Merhi.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bou Khzam, L., Bouchereau, O., Boulahya, R. et al. Early outgrowth cells versus endothelial colony forming cells functions in platelet aggregation. J Transl Med 13, 353 (2015). https://doi.org/10.1186/s12967-015-0723-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12967-015-0723-6

Keywords