This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 26/8/1760    most recent 01.ATV.0000229243.49320.c9v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Loomans, C.J.M. Articles by Staal, F.J.T. Search for Related Content PubMed PubMed Citation Articles by Loomans, C.J.M. Articles by Staal, F.J.T.

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:1760.)
© 2006 American Heart Association, Inc.

Vascular Biology

Angiogenic Murine Endothelial Progenitor Cells Are Derived From a Myeloid Bone Marrow Fraction and Can Be Identified by Endothelial NO Synthase Expression

C.J.M. Loomans; H. Wan; R. de Crom; R. van Haperen; H.C. de Boer; P.J.M. Leenen; H.A. Drexhage; T.J. Rabelink; A.J. van Zonneveld; F.J.T. Staal

From the Departments of Immunology (C.J.M.L., H.W., P.J.M.L., H.A.D., F.J.T.S.), Cell Biology and Genetics (R.d.C., R.v.H.), and Vascular Surgery (R.d.), Erasmus Medical Center, Rotterdam, the Netherlands; and Department of Nephrology (C.J.M.L., H.C.d.B., T.J.R., A.J.v.Z.), University Medical Center, Leiden, the Netherlands.

Correspondence to Dr Frank J.T. Staal, PhD, Department of Immunology, Erasmus MC, Dr Molewaterplein 50, 3015GE Rotterdam, the Netherlands. E-mail f.staal{at}erasmusmc.nl

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Objective— Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization and are therefore of great interest for autologous cell therapies to treat ischemic vascular disease. However, the origin and functional properties of these EPCs are still in debate.

Methods and Results— Here, ex vivo expanded murine EPCs were characterized in terms of phenotype, lineage potential, differentiation from bone marrow (BM) precursors, and their functional properties using endothelial NO synthase (eNOS)–green fluorescent protein transgenic mice. Despite high phenotypic overlap with macrophages and dendritic cells, EPCs displayed unique eNOS expression, endothelial lineage potential in colony assays, and angiogenic characteristics, but also immunologic properties such as interleukin-12p70 production and low levels of T-cell stimulation. The majority of EPCs developed from an immature, CD31⁺Ly6C⁺ myeloid progenitor fraction in the BM. Addition of myeloid growth factors such as macrophage–colony-stimulating factor (M-CSF) and granulocyte/macrophage (GM)-CSF stimulated the expansion of spleen-derived EPCs but not BM-derived EPCs.

Conclusion— The close relationship between EPCs and other myeloid lineages may add to the complexity of using them in cell therapy. Our mouse model could be a highly useful tool to characterize EPCs functionally and phenotypically, to explore the origin and optimize the isolation of EPC fractions for therapeutic neovascularization.

The lineage relationship of EPCs and other blood cells has remained elusive. Using eNOS–GFP transgenic mice, we show that EPCs share phenotypic and functional characteristics with myeloid cells and develop from myeloid precursors in the bone marrow. However, they have unique angiogenic function, providing a rationale for therapeutic neovascularization applications.

Key Words: endothelial progenitor cells • myeloid cells • neovascularization • lineage differentiation • eNOS

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human peripheral blood (PB) contains bone marrow (BM)–derived progenitor cells with angiogenic properties.^1–3 These cells have the potential to differentiate toward endothelial cells (ECs) and are therefore named endothelial progenitor cells (EPCs). Transplantation of EPCs has been shown to be effective in animal models for re-endothelialization^4,5 and adult neovascularization^6,7 as well as in human patient studies aimed to enhance myocardial regeneration after acute myocardial infarction.⁷ Although EPCs are used in clinical trials, the exact phenotypic and lineage/differentiation parameters of ex vivo–expanded EPCs are poorly defined, and it is not clear which cell populations will be most effective in repair studies. EPCs can be derived from CD34+ as well as CD34– or CD34^low cells and can be isolated and expanded ex vivo using BM aspirates and PB CD14+ mononuclear cell fractions.^8–12 In many studies, EPCs are characterized by their adhesive spindle-like morphology, staining with the EC-binding lectin Ulex europaeus agglutinin (Ulex), and the capacity to endocytose acetylated low-density lipoprotein (acLDL).^2,11 Although this may generally suffice for EPC studies dealing with EPCs obtained from healthy animal models or humans, the different culture conditions and sources used may lead to a large heterogeneity and functionally suboptimal EPC populations.^13,14 It has even been suggested that transplantation of certain cell fractions may contribute to adverse side effects.¹⁵ Clinical studies demonstrated that in patients experiencing diabetes and hypertension, the number of circulating EPCs is severely decreased, and the cells are dysfunctional.^16–19 This altered phenotype of EPCs could contribute to and might even endow the progression of the pathogenesis of ischemic vascular disease in these patients.

It has been shown that cells from the myeloid lineage (eg, EPCs) show a wide phenotypic overlap²⁰ and, as we demonstrate here, that Ulex and the uptake of acLDL, among other often used endothelial markers, are not specific for EPCs. Therefore, discrimination is difficult between EPCs and other myeloid cells such as dendritic cells (DCs) and macrophages (Mphs), which are also in close contact with the vascular system.^21,22 Myeloid progenitor cells exhibit a very high plasticity, and under different circumstances, a precursor cell can be skewed toward alternative differentiation directions.^14,23–26

To better characterize the nature of the angiogenic myeloid cell (EPCs) compared with other myeloid cells and mature ECs, we first performed a detailed comparative phenotypic and functional analysis of cells stimulated to differentiate into EPCs, DCs, or Mphs starting from the same progenitor cell populations. Second, we used a transgenic mouse model expressing endothelial NO synthase (eNOS) fused to green fluorescent protein (GFP).²⁷ The expression of the transgene is driven by the native human eNOS promoter and the transgenic mice show an endothelium-specific expression pattern in many different organs. Therefore, this transgenic mouse model is expected to precisely distinguish cells from the EC lineage from other myeloid cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
For detailed methods, please see the online Materials and Methods, available at http://atvb.ahajournals.org.

Animals
C57BL/6J and FVB wild-type mice 6 to 22 weeks of age were used. eNOS-GFP transgenic mice with C57BL/6J as well as FVB background were generated and bred as described previously.²⁷

Isolation and Differentiation of Murine EPCs, DCs, and Mphs
Single-cell BM suspensions were prepared by flushing femora and tibiae. BM isolates were used to culture EPCs, Mphs, and DCs for 7 days using optimal culture conditions to differentiate the cells. For activation, EPCs, Mphs, and DCs were incubated overnight with lipopolysaccharide (LPS) at day 6, and overnight culture supernatants were collected and frozen for cytokine measurements.

Antibodies and Conjugates for Cell Sorting, Flow Cytometric, and Immunohistochemical Analysis
Antibodies against ER-MP12 (CD31), ER-MP20 (Ly-6C),²⁸ F4/80, mouse endothelial cell antigen (MECA)-20 MECA-32, CD11c, major histocompatibility complex (MHC) class II, CD14, kinase insert domain receptor (KDR), Sca-1, c-kit, CD34, vascular endothelial–cadherin, and Flt-1 antigens were used to phenotype and characterize cells. Flowcytometric analyses were done with proper isotype controls for the antibodies. For lectin staining, cells were stained with rhodamine-labeled Bandeiraea Simplicifolia lectin and Ulex europaeus agglutinin-1. The uptake of DiI-labeled acLDL cells was measured by flowcytometry.

Cell Sorting
Before sorting of cells, labeled cell suspensions were filtered more than a 30-µm pore size sieve to avoid clogging of the nozzle. The purity of the sorted cell suspensions exceeded 95%.

Cytokine Detection
Interleukin-10 (IL-10) and IL-6 ELISA kit (Biosource) and IL-12p40 and IL-12p70 ELISA kits (R & D Systems) were used according to manufacturer protocol.

Mixed Leukocyte Reaction Assay
Allogeneic mixed leukocyte reactions (MLRs) were performed to evaluate the ability of the various cells to stimulate a T-cell response.

In Vitro Angiogenesis Assay
Conditioned media (16 hours; serum-free medium) were obtained from 6-day EPC cultures and applied on an in vitro angiogenesis assay using human umbilical vein ECs (P3) as tube forming cells. After 14 hours, tube formation was measured and quantified.

The ability of labeled EPCs, DCs, and Mphs to incorporate or participate in the formation of vessel-like structures was tested using the same in vitro angiogenesis assay kit.

Endocytosis Assay
Uptake of dextran–fluorescein isothiocyanate was done at 37°C for 30 minutes, and cells were washed and then measured by fluorescence-activated cell sorter.

Real-Time Quantitative Polymerase Chain Reaction
Quantitative analyses of mRNA levels of eNOS were measured using iCylcer polymerase chain reaction technology. GAPDH and actin were used as normalization genes.

EPC Colony Formation
An established colony forming unit–endothelial cell assay was used to assess the property of EPCs to proliferate and to differentiate to ECs.

Statistical Analysis
Results are expressed as mean±SD. P values of P<0.05 were considered statistically significant (Student t test).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
For supplemental Figures I through IV, please see the online supplement, available at http://atvb.ahajournals.org.

Morphological and Phenotypic Comparison of EPCs, DCs, and Mphs Derived From BM
Because of the high phenotypic overlap of EPCs with other cells of the myeloid lineage, it is important to define the criteria that characterize EPCs in more detail. To that end, we first investigated morphological and functional differences between EPCs, DCs, and Mph cultures obtained from mouse BM. In Figure 1A (top), the distinct morphology of the different cells at day 7 is shown. EPCs showed typical spindle-shaped morphology, DCs displayed long-extended dendrites or veils, and Mphs were more rounded up and attaching. EPCs were capable of binding Ulex and taking up acLDL to the same extent as Mphs. DCs stained for Ulex but hardly took up acLDL particles. Therefore, Ulex staining combined with the uptake of acLDL are not appropriate markers restricted to EPCs.

View larger version (62K): [in this window] [in a new window]   Figure 1. Morphological and phenotypic characterization of murine EPCs compared with DCs and Mphs. A, Phase-contrast microscopic morphological appearance (top) and flow cytometric analyses of the ability of the different cells to bind the lectin Ulex and to take up DiI-labeled acLDL particles (bottom). The analyses were plotted and nonstained cells served as negative controls (see quadrilles). B, Representative flow cytometric analyses of different lineage specific antigens. The thick green lines represent the DCs, the thin dark line the EPCs, and the pink-dashed line, the Mphs. Cells were cultured for 7 days starting with total BM under optimized culture conditions as described in the Methods.

Next, we determined the expression of surface markers to further characterize EPCs (CD31, MECA-20, MECA-32, BS-1 lectin, Flt-1, c-kit, Sca-1, KDR, VE-cadherin, and CD14), DCs (CD11c and MHCII), and Mphs (F4/80, CD11b; Figure 1B; supplemental Figure I). EPCs displayed a higher expression of MECA-20, CD14 and CD31, in comparison to Mphs and DCs (Figure 1B). EPCs and Mphs showed a lower expression level of CD11c and MHCII when compared with DCs. The Flt-1 receptor is highly upregulated in total population of the EPCs but also on a small population of DCs (supplemental Figure I). MECA-32 antibody showed expression on a very small subset of the EPCs and no expression on Mphs and DCs, whereas MECA-20 (also reported as EC specific^30,31) does show a higher expression on the EPC fraction when compared with DCs an Mphs. Thus, a unique marker specifically defining EPCs was lacking. At best, EPCs could be characterized and distinguished from DCs and Mphs as spindle-shaped cells that were CD31^hi, MECA-20^hi, Flt^hi, and F4/80^lo.

Functional Comparison of EPCs, DCs, and Mphs Derived From BM
Conditioned medium (CM) of EPCs, Mphs, and DCs was tested for supporting formation of tube-like structures in an in vitro angiogenesis assay. Although CM of DCs and Mphs hardly showed any induction of tube-like structures, EPC CM significantly augmented the formation of tube-like structures (supplemental Figure IIA). Second, using confocal microscopy, we compared the 3 different cell types for their ability to incorporate into or to participate in the formation of tube-like structures. Although DCs, Mphs, and EPCs all attached to the protrusions of the EC, only EPCs were able to specifically adhere to and line up in tube-like structures (supplemental Figure IIB, arrows). Thus, only EPCs and not DCs or Mphs display genuine proangiogenic properties by both factor production and participation in tube formation.

Next, we addressed functional properties specific for Mphs and DCs. Mphs endocytose to clear the body of pathogens, whereas DCs mainly use their endocytic properties to present antigens to T lymphocytes. Mphs displayed a high endocytic capacity (supplemental Figure IIC), but EPCs also showed an almost similar capacity to take up the dextran molecules. DCs barely showed endocytic capabilities above control values. A typical feature of DCs is antigen presentation to, and cytokine activation of, naïve T lymphocytes, for instance, in an MLR. Because expected, mature/activated DCs were able to trigger T-cell proliferation. EPCs activated by LPS could do this as well but to a lesser extent (supplemental Figure IID). Unstimulated EPCs hardly induced T-cell proliferation (data not shown).

To evaluate the cytokine profile of EPCs compared with that of DCs and Mphs, we measured IL-6, IL-10, IL-12p70, and IL-12p40 in CM of nonstimulated and LPS-stimulated cells. Although DCs and Mphs were capable of producing all 4 cytokines, EPCs secreted detectable levels of IL-12p70 and IL-12p40 only. IL-12p70 was produced by the EPCs to a similar level as Mphs and DCs, and LPS stimulation of the EPCs strongly enhanced this IL-12p70 production (supplemental Figure IIE). IL-12p40 was produced by EPCs, although to a lower extent than by DCs and LPS-stimulated Mphs. IL-12p70 has been shown to be an active subunit of IL-12, which can regulate T cell–mediated immune responses by promoting Th1 development. It is striking that IL-12p70 is the predominant IL-12 subtype produced by EPCs.

Concluding, only EPCs have the capacity to induce in vitro angiogenesis, yet they share with DCs and Mphs the capability to endocytose and are also able to act, to some extent, as activated protein C with IL-12–producing capacity.

Tracking EPC Differentiation by Using the Endothelial-Specific Marker eNOS Coupled to GFP
Because there was a considerable phenotypic and also some functional overlap between the EPCs, DCs, and Mphs, we aimed to specifically track BM-derived cells differentiating toward the endothelial lineage (EPCs). Therefore, a transgenic mouse model was used in which the mice show an endothelium-specific GFP expression pattern.²⁷ When BM of eNOS-GFP transgenic C57BL/6J mice was harvested (day 0), a small population of cells (0.05% of total cells) expressed GFP in the transgenic mice, which is not present in control BM isolates (day 0) of wild-type mice (Figure 2A). At day 7 of culture under EPC culture conditions, 15% (n=6; representative experiment shown) of the attached cells were GFP+ in the transgenic EPCs. There is a high autofluorescent background of cells in the EPC cultures at day 7 in both FL1 and FL2 channels, which is seen in transgenic BM cultures as well as wild-type BM cultures.

View larger version (52K): [in this window] [in a new window]   Figure 2. EPCs from eNOS-GFP transgenic mice show a specific expression of GFP. A, Expression of eNOS was measured in total BM of eNOS-GFP C57BL/6J transgenic mice and compared with wild-type controls at day 0 and in day 7 EPC cultures using flow cytometry and GFP as a fluorescent marker. The FL2 channel shows autofluorescent staining. GFP+ and GFP– cells from day 7 EPC cultures were then sorted. Clear spindle-shaped morphology typical for EPCs was observed in the GFP+ fraction by phase-contrast microscopy. B, DCs and Mphs were cultured for 7 days under their specific growth conditions and measured for eNOS-GFP expression. DCs and Mphs were stained with antibodies against typical mouse DCs (CD11c) and Mphs (F4/80) antigens. Only BM cells differentiated under EPC conditions showed a high eNOS-GFP expression, whereas Mphs and DC cultures displayed no or hardly any GFP expression in combination with lineage specific antigens. C, BM cells cultured in an CFU-EC assay give rise to GFP+ colonies after 3 days of culturing, demonstrating the specificity of the eNOS marker for the EC lineage.

When EPC cultures were flow-sorted at day 7 and the GFP– and GFP+ populations were replated separately at the same concentrations, only the GFP+ fraction (by definition, expressing eNOS; supplemental Figure IV) cells displayed the typical EPC morphology of spindle-shaped cells. The GFP– population hardly reattached, indicating that these did not represent EPCs.

To ensure that the GFP reporter specifically tracks ECs and EPCs, BM cells of the transgenic mice were cultured with either granulocyte/macrophage (GM)-CSF to differentiate them to DCs or with M-CSF for Mph differentiation (Figure 2B). In the Mph culture, no GFP+ cells were present, and >90% of the culture was F4/80^hi. In the DC culture, only a very small percentage (3%) of cells was found to express GFP at a low level. However, these GFP+ cells did not express CD11c, suggesting that these few GFP+ cells were not DCs.

To assess the property of BM-derived EPCs to differentiate and proliferate in an in vitro colony assay and to exclude the possibility of a minute fraction of mature ECs growing out in our cultures, we performed an established CFU-EC assay. BM of transgenic mice was plated on fibronectin-coated dishes for 48 hours, and nonattaching cells were then replated and assessed for colony outgrowth (GFP+ colonies). There were hardly any cells attached to the plates after 2 days, and these few cells did not survive or proliferate in the next 3 days (data not shown). However, the nonattached fraction did form GFP+ colonies as shown in Figure 2C. This observation was extended when we sorted out the very small GFP+ population, presumably corresponding to a minute fraction of mature ECs in BM. When cultured under EPC culture conditions, the GFP+ population did not survive and did not expand (data not shown), whereas the GFP– population proliferated significantly and differentiated into eNOS+ GFP+ cells.

We conclude that using this mouse model EPC differentiation can be tracked, allowing identification and separation of true EPCs from cells not committed to the endothelial lineage.

EPC Differentiation Varies Between Different Mouse Strains
To further explore commitment of BM-derived cells toward the endothelial lineage, GFP expression was followed in time up to 7 days. Because there could be differences between mouse strains, we studied the kinetics of EPC differentiation in 2 different genetic backgrounds: C57BL/6J and FVB eNOS-GFP transgenic mice. At day 0, there was no significant difference in the already very low number of GFP+. At day 1, the attached cells were GFP– (Figure 3A); however, at day 4, eNOS-GFP+ cells appeared in both strains that expanded further in time. At day 4, a trend of higher numbers of GFP+ cells was observed in the FVB background mice, but this was not statistically significant. At day 7, >4-fold more GFP+ cells were observed for FVB over the C57BL/6J strain (FVB mice 65% ± 11 GFP+ cells [n=6] versus C57BL/6J 15±7.5 [n=6]; *P<0.01; Figure 3B). These data indicate that eNOS-expressing EPCs can be derived from BM of both strains tested but more readily from FVB mice.

View larger version (36K): [in this window] [in a new window]   Figure 3. Kinetics of EPC differentiation varies between different mouse strains. A, BM of FVB– (FVB tg) and C57BL/6J-transgenic mice (BL6 tg) was cultured to generate EPCs, and the expression of eNOS-GFP was measured in time by flow cytometry for 7 days. The FL2 channel shows autofluorescence. B, The percentage of GFP+ cells of the attached cells in culture was measured by flow cytometry and plotted (n=6 for both groups FVB tg and BL6 tg). At day 7, BM from BL6 tg mice showed fewer eNOS-positive EPCs than BM from FVB tg; *P<0.01, BL6 tg vs FVB tg.

Ex Vivo–Expanded EPCs From BM Are Mainly Derived From a Specific Myeloid Precursor Fraction
Next, we investigated which subfraction of the BM contains progenitors for EPCs. Based on a 2-color flow cytometry analysis with ER-MP12 (anti-CD31) and ER-MP20 (anti-Ly-6C), total BM cells can be separated in 6 phenotypically and functionally distinct subsets.²⁹ We previously showed that 3 of these subsets contain myeloid progenitor cells^28,32,33 that can give rise to Mphs and DCs. Here, based on CD31/Ly-6C profiles, all 6 subsets were flow-sorted from total BM of both eNOS-GFP transgenic mice and cultured under EPC conditions (Figure 4).

View larger version (71K): [in this window] [in a new window]   Figure 4. EPCs are mainly derived from a specific myeloid CD31+/Ly-6C+ precursor fraction of the BM. A, Fresh BM (day 0) of C57BL/6J (BL6 tg) and FVB transgenic mice (FVB tg) was stained with CD31 and Ly-6C antibodies, revealing different myeloid fractions of the BM. B, Several fractions P1/2 (P1 and P2 together), P4, and P6 were sorted by flow cytometry, and the fractions were cultured separately. After 7 days, the cells were isolated and measured for eNOS-GFP expression by flow cytometry. Cells sorted from the P4 (CD31+/Ly-6C+) subpopulation differentiated most efficiently into EPCs. FVB tg mice revealed higher numbers and more efficient EPC differentiation when compared with BL6 tg mice.

In 4 of 4 sort experiments, GFP+ EPCs appeared in the cultures derived from the CD31⁺/Ly-6C⁺ (P4) subset. As expected, significantly fewer GFP+ cells appeared in the culture of the eNOS-GFP C57BL/6J background compared with the eNOS-GFP FVB. We demonstrated previously that nearly 80% of this CD31⁺/Ly-6C⁺ (P4) cell fraction is comprised of myeloid progenitor cells, indicating that the majority of EPCs are derived from these cells. In 2 of 4 experiments, we observed a few GFP+ cells in the CD31^lo/Ly-6C^hi (P6) subset but only in the FVB background. In 1 of 4 sorting experiments, a very small fraction of GFP+ cells was also seen in the CD31^dim/hi/Ly-6C^lo (P1/2) subfraction. Because this fraction contains lymphoid progenitor cells and hematopoietic stem cells, it could be that it takes longer to induce EPC differentiation from this fraction or that the necessary factors are missing in the in vitro culture system used here. In conclusion, the main source of EPCs from BM is the CD31⁺/Ly-6C⁺ (P4) subset, whereas DCs and Mphs can also be differentiated from the P1/2 and P6 fraction.

Additional phenotyping of the CD31⁺/Ly-6C⁺ (P4) subpopulation compared with cultured EPCs and mature ECs (bEnd3 cells) showed that c-kit was markedly present in the BM P4 fraction but very minor in the EPC cultures and absent in the mature EC cultures (supplemental Figure IIIB). A subpopulation of 11% of this P4 fraction showed Sca-1 expression, whereas Sca-1 expression seemed to be highly present on EPCs as well as mature ECs. The observation that Sca-1 is expressed on ECs and even a possible function of expression of Sca-1 on ECs has been proposed previously by Luna et al.³⁴ Vascular endothelial growth factor-1 (Flt-1) is highly expressed on eNOS+ cells, whereas KDR is not yet detectable. VE-cadherin is positive on a small subset of cells and has been confirmed by immunohistochemical staining (data not shown). CD31 is upregulated in EPC fraction and showed an even higher expression on mature ECs (supplemental Figure IIIC). Myeloid markers such as CD11b were downregulated on GFP+ EPCs and even further on mature ECs, especially when compared with Mphs and DCs. BS-1 lectin staining of the total population of both EPCs and mature ECs was confirmed by flowcytometric as well as immunohistochemical analyses. We conclude that with the notable exception of Sca-1, EPCs express higher levels of progenitor/stem cell markers than mature ECs and begin to express EC-specific markers while downregulating classical myeloid markers, consistent with a further narrowing of differentiation potential toward the EC lineage.

Spleen-Derived EPCs Can Be Expanded Using Myeloid-Specific Growth Factors
The therapeutic potential of EPCs has elicited a number of studies that demonstrated that myeloid growth factors can stimulate recruitment, differentiation, or outgrowth of EPCs and may have favorable effects on their function.^35–37 Therefore, the effects of GM-CSF and M-CSF on EPC differentiation from BM were determined. Addition of these myeloid growth factors to the cultures lowered the numbers of EPCs (GFP+ cells) derived from the BM precursors (Figure 5A). Other sources than BM have been used to derive human EPCs and murine EPCs. Human EPCs can be cultured from CD14+ mononuclear cell fractions isolated from PB mononuclear cells (PBMNCs)^10,11 or from CD34+ progenitor cells isolated from G-CSF–mobilized PB stem cells,³⁷ umbilical cord blood,¹³ or BM.³⁸ Murine EPCs have been cultured from BM and spleen. The mononuclear cell fraction of the spleen is often used as a homologue of PBMNCs from mice because it is described as a reservoir of PB stem/progenitor cells.³⁹ Spleen-derived murine EPCs have similar functional (angiogenic) and phenotypic characteristics as BM-derived EPCs (data not shown), but they show a lower proliferation capacity. Using the same culture conditions as described above for the generation of BM-derived EPCs, spleen-derived cultures yielded 10- to 50-fold lower numbers of GFP+ EPCs (Figure 5B). Addition of myeloid growth factors to spleen-derived cultures showed an increase in the number of EPCs. Thus, addition of myeloid growth factors as GM-CSF and M-CSF could be useful for expanding PB- or spleen-derived EPCs ex vivo but not for BM derived EPCs.

View larger version (12K): [in this window] [in a new window]   Figure 5. Addition of specific growth factors to EPC cultures has different effects depending on the source of the EPCs. EPCs were cultured using either BM (A) or spleen mononuclear cells (B). The cells were cultured under standard optimized EPC conditions (medium) or with addition of M-CSF (10 ng/mL), GM-CSF (20 ng/mL), or a combination of both. These cytokines were either refreshed every 2 days (black bars), or they were only added once at day 0 (gray bars). After 7 days, cells in culture were counted, and the total number of GFP+ cells per well was determined by fluorescence-activated cell sorter. A 2- to 3-fold increase in the number of GFP+ EPCs was observed by addition of growth factors in cultures from spleen-derived cells but not with BM cells. Refreshing the growth factors every 2 days or addition only once at day 0 showed a similar proliferation/differentiation pattern. A representative experiment is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we characterized the ex vivo commitment of BM precursors toward ECs in terms of phenotype, lineage potential, differentiation from BM precursors, and angiogenic properties. To address these issues in detail and to have an endothelium-specific marker, we made use of eNOS-GFP transgenic mice. This well-characterized system²⁷ allows a careful appreciation of the relationship between myeloid and endothelial lineages. Our report emphasizes the high phenotypic overlap and close relationship of EPCs, DCs, and Mphs. Consequently, frequently used markers for EPCs, such as the uptake of acLDL and binding of Ulex, are relatively unspecific because these are also markers for Mphs. Despite this high phenotypic overlap of EPCs, DCs, and Mphs, the capacity of EPCs to support angiogenesis is a unique feature of EPCs when compared with DCs and Mphs. Although we could demonstrate a potent angiogenic capacity in the CM of EPCs, we observed that only a small fraction of the EPCs did incorporate (as do mature ECs) in tubes. The majority of the EPCs appear to function as pericytes and localize around the tubes and under the junctions but do not form an integral part of it. Other investigators also found that attaching cells derived from BM or PBMNCs under culture conditions with vascular endothelial growth factor did not differentiate into ECs but stimulated angiogenesis in other ways.^11,40 Therefore, the term EPCs might not be an adequate definition of the total cell culture because not all cells might become true ECs under the conditions used. Although we generally refer to these attaching cells with angiogenic capacity as EPCs, after the consensus in the field, the term angiogenic myeloid cells may be more appropriate. Nevertheless, the cells referred to as EPCs are different from mature ECs, as demonstrated in the CFU-EC assays and by phenotypic analysis. EPCs also express higher levels of stem cell markers but lower levels of eNOS, although they are clearly positive for this marker.

We showed that there is a strain difference between FVB and C57BL/6J mice in their capacity to generate EPCs from BM precursors. FVB mice are less susceptible for atherosclerosis,⁴¹ and this might possibly indicate a role for the plasticity of BM precursors to differentiate toward ECs. As a corollary, we conclude that C57BL/6J mice might not be the best strain to choose for studying short-term cultured murine EPCs.

A number of studies indicated that myeloid growth factors such as GM-CSF can be used to augment neovascularization in animal models and in patients.⁴² In this study, only for spleen-derived EPCs, the number of eNOS-GFP+ EPCs increased. In BM, addition of M-CSF and GM-CSF to the culture resulted in a decreased number of EPCs, probably because of extensive expansion of myeloid progenitors that are driven into another differentiation lineage than ECs, such as DCs and Mphs.

It is becoming increasingly apparent that cells of the myeloid lineage display a high plasticity, and that some of these seemingly "lineage-committed" myeloid cells can, under specific growth conditions, differentiate into cells of another lineage with distinct functional properties.^20,25,43 For instance, in the presence of inflammatory cytokines, the normal differentiation of monocytes into Mphs can be skewed to yield DCs.²⁶ Another example is the differentiation of myeloid cells into cells of the mesenchymal lineages.⁴⁴ Likewise, several reports have described the myeloid character of ECs.^12,20,45 Cultures of adhered mononuclear cells¹² or DCs^46,47 grown under stringent angiogenic differentiation conditions have been shown to differentiate into endothelial like cells. We argue that this large degree of plasticity among cells of the myeloid lineage and the close phenotypic overlap between many of these different myeloid lineages (including cells that stimulate angiogenesis) caution the use of these cells in clinical cell transplantation protocols aimed to augment neovascularization in peripheral or cardiac ischemia. In particular, when early outgrowth EPCs are derived from patients subject to chronic systemic inflammation, transplanted cells might have suboptimal angiogenic properties or even induce an unwanted immunologic response.

In the present study, we observed that LPS-stimulated EPCs cultures have the capacity, although to a low extent, to induce T-cell proliferation in an MLR.

Using short cultured cell sorting experiments, we here show that the best and almost exclusive source for murine EPCs are the myeloid progenitors in the BM. This myeloid character of EPCs is in line with a recent study from Dimmeler et al showing that CD34^lowCD14+ cells in PB are a major source of EPCs.¹⁰ Translating our results to the human situation suggest that further purification of human CD34+ cells to include only CD33+ (immature myeloid marker)/CD34+ myeloid progenitors, but exclude contaminating cells that may yield unwanted side effects, could be of clinical relevance. Further experiments have to determine whether human myeloid progenitor cells from BM or cord blood provide a superior source of EPCs.

	Acknowledgments

 
We thank Dr M. Versnel for her contributions to work discussions. We also thank Drs A. Nigg and G. van Cappellen for their help with the confocal microscopy images.

This work was supported in part by the Netherlands Heart Foundation (grants NHS 2000B019 and 2002B157).

Disclosures

None.

	Footnotes

 
Original received January 13, 2006; final version accepted May 10, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Urbich C, Dimmeler S. Endothelial progenitor cells: characterization and role in vascular biology. Circ Res. 2004; 95: 343–353.[Abstract/Free Full Text]
2. 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: 964–967.[Abstract/Free Full Text]
3. Crosby JR, Kaminski WE, Schatteman G, Martin PJ, Raines EW, Seifert RA, Bowen-Pope DF. Endothelial cells of hematopoietic origin make a significant contribution to adult blood vessel formation. Circ Res. 2000; 87: 728–730.[Abstract/Free Full Text]
4. Walter DH, Rittig K, Bahlmann FH, Kirchmair R, Silver M, Murayama T, Nishimura H, Losordo DW, Asahara T, Isner JM. Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation. 2002; 105: 3017–3024.[Abstract/Free Full Text]
5. Werner N, Junk S, Laufs U, Link A, Walenta K, Bohm M, Nickenig G. Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury. Circ Res. 2003; 93: e17–e24.[Abstract/Free Full Text]
6. Kawamoto A, Asahara T, Losordo OW. Transplantation of endothelial progenitor cells for therapeutic neovascularization. Cardiovasc Radiat Med. 2002; 3: 221–225.[CrossRef][Medline] [Order article via Infotrieve]
7. Assmus B, Schachinger V, Teupe C, Britten M, Lehmann R, Dobert N, Grunwald F, Aicher A, Urbich C, Martin H, Hoelzer D, Dimmeler S, Zeiher AM. Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI). Circulation. 2002; 106: 3009–3017.[Abstract/Free Full Text]
8. Schatteman GC, Awad O. In vivo and in vitro properties of CD34+ and CD14+ endothelial cell precursors. Adv Exp Med Biol. 2003; 522: 9–16.[Medline] [Order article via Infotrieve]
9. Rookmaaker MB, Vergeer M, van Zonneveld AJ, Rabelink TJ, Verhaar MC. Endothelial progenitor cells: mainly derived from the monocyte/macrophage-containing CD34-mononuclear cell population and only in part from the hematopoietic stem cell-containing CD34+ mononuclear cell population. Circulation. 2003; 108: e150.[Free Full Text]
10. Romagnani P, Annunziato F, Liotta F, Lazzeri E, Mazzinghi B, Frosali F, Cosmi L, Maggi L, Lasagni L, Scheffold A, Kruger M, Dimmeler S, Marra F, Gensini G, Maggi E, Romagnani S. CD14+CD34low cells with stem cell phenotypic and functional features are the major source of circulating endothelial progenitors. Circ Res. 2005; 97: 314–322.[Abstract/Free Full Text]
11. Rehman J, Li J, Orschell CM, March KL. Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation. 2003; 107: 1164–1169.[Abstract/Free Full Text]
12. Fernandez Pujol B, Lucibello FC, Gehling UM, Lindemann K, Weidner N, Zuzarte ML, Adamkiewicz J, Elsasser HP, Muller R, Havemann K. Endothelial-like cells derived from human CD14 positive monocytes. Differentiation. 2000; 65: 287–300.[CrossRef][Medline] [Order article via Infotrieve]
13. Murohara T, Ikeda H, Duan J, Shintani S, Sasaki K, Eguchi H, Onitsuka I, Matsui K, Imaizumi T. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest. 2000; 105: 1527–1536.[Medline] [Order article via Infotrieve]
14. Urbich C, Heeschen C, Aicher A, Dernbach E, Zeiher AM, Dimmeler S. Relevance of monocytic features for neovascularization capacity of circulating endothelial progenitor cells. Circulation. 2003; 108: 2511–2516.[Abstract/Free Full Text]
15. Silvestre JS, Gojova A, Brun V, Potteaux S, Esposito B, Duriez M, Clergue M, Le Ricousse-Roussanne S, Barateau V, Merval R, Groux H, Tobelem G, Levy B, Tedgui A, Mallat Z. Transplantation of bone marrow-derived mononuclear cells in ischemic apolipoprotein E-knockout mice accelerates atherosclerosis without altering plaque composition. Circulation. 2003; 108: 2839–2842.[Abstract/Free Full Text]
16. Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Dimmeler S. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res. 2001; 89: E1–E7.[Medline] [Order article via Infotrieve]
17. Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, Finkel T. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 2003; 348: 593–600.[Abstract/Free Full Text]
18. Loomans CJ, de Koning EJ, Staal FJ, Rookmaaker MB, Verseyden C, de Boer HC, Verhaar MC, Braam B, Rabelink TJ, van Zonneveld AJ. Endothelial progenitor cell dysfunction: a novel concept in the pathogenesis of vascular complications of type 1 diabetes. Diabetes. 2004; 53: 195–199.[Abstract/Free Full Text]
19. Tepper OM, Galiano RD, Capla JM, Kalka C, Gagne PJ, Jacobowitz GR, Levine JP, Gurtner GC. Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures. Circulation. 2002; 106: 2781–2786.[Abstract/Free Full Text]
20. Schmeisser A, Graffy C, Daniel WG, Strasser RH. Phenotypic overlap between monocytes and vascular endothelial cells. Adv Exp Med Biol. 2003; 522: 59–74.[Medline] [Order article via Infotrieve]
21. Schmeisser A, Strasser RH. Phenotypic overlap between hematopoietic cells with suggested angioblastic potential and vascular endothelial cells. J Hematother Stem Cell Res. 2002; 11: 69–79.[CrossRef][Medline] [Order article via Infotrieve]
22. Schatteman GC, Awad O. Hemangioblasts, angioblasts, and adult endothelial cell progenitors. Anat Rec A Discov Mol Cell Evol Biol. 2004; 276: 13–21.[CrossRef][Medline] [Order article via Infotrieve]
23. Planat-Benard V, Silvestre JS, Cousin B, Andre M, Nibbelink M, Tamarat R, Clergue M, Manneville C, Saillan-Barreau C, Duriez M, Tedgui A, Levy B, Penicaud L, Casteilla L. Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation. 2004; 109: 656–663.[Abstract/Free Full Text]
24. Choi K. The hemangioblast: a common progenitor of hematopoietic and endothelial cells. J Hematother Stem Cell Res. 2002; 11: 91–101.[CrossRef][Medline] [Order article via Infotrieve]
25. Chomarat P, Dantin C, Bennett L, Banchereau J, Palucka AK. TNF skews monocyte differentiation from macrophages to dendritic cells. J Immunol. 2003; 171: 2262–2269.[Abstract/Free Full Text]
26. Abuljadayel IS. Induction of stem cell-like plasticity in mononuclear cells derived from immobilized adult human peripheral blood. Curr Med Res Opin. 2003; 19: 355–375.[CrossRef][Medline] [Order article via Infotrieve]
27. van Haperen R, Cheng C, Mees BM, van Deel E, de Waard M, van Damme LC, van Gent T, van Aken T, Krams R, Duncker DJ, de Crom R. Functional expression of endothelial nitric oxide synthase fused to green fluorescent protein in transgenic mice. Am J Pathol. 2003; 163: 1677–1686.[Abstract/Free Full Text]
28. de Bruijn MF, Slieker WA, van der Loo JC, Voerman JS, van Ewijk W, Leenen PJ. Distinct mouse bone marrow macrophage precursors identified by differential expression of ER-MP12 and ER-MP20 antigens. Eur J Immunol. 1994; 24: 2279–2284.[Medline] [Order article via Infotrieve]
29. van der Loo JC, Slieker WA, Kieboom D, Ploemacher RE. Identification of hematopoietic stem cell subsets on the basis of their primitiveness using antibody ER-MP12. Blood. 1995; 85: 952–962.[Abstract/Free Full Text]
30. Murray JC, Smith KA, Lauk S. Vascular markers for murine tumors. Radiother Oncol. 1989; 16: 221–234.[CrossRef][Medline] [Order article via Infotrieve]
31. Duijvestijn AM, Kerkhove M, Bargatze RF, Butcher EC. Lymphoid tissue- and inflammation-specific endothelial cell differentiation defined by monoclonal antibodies. J Immunol. 1987; 138: 713–719.[Abstract]
32. Nikolic T, de Bruijn MF, Lutz MB, Leenen PJ. Developmental stages of myeloid dendritic cells in mouse bone marrow. Int Immunol. 2003; 15: 515–524.[Abstract/Free Full Text]
33. McCormack JM, Leenen PJ, Walker WS. Macrophage progenitors from mouse bone marrow and spleen differ in their expression of the Ly-6C differentiation antigen. J Immunol. 1993; 151: 6389–6398.[Abstract]
34. Luna G, Paez J, Cardier JE. Expression of the hematopoietic stem cell antigen Sca-1 (LY-6A/E) in liver sinusoidal endothelial cells: possible function of Sca-1 in endothelial cells. Stem Cells Dev. 2004; 13: 528–535.[CrossRef][Medline] [Order article via Infotrieve]
35. Woo YJ, Grand TJ, Berry MF, Atluri P, Moise MA, Hsu VM, Cohen J, Fisher O, Burdick J, Taylor M, Zentko S, Liao G, Smith M, Kolakowski S, Jayasankar V, Gardner TJ, Sweeney HL. Stromal cell-derived factor and granulocyte-monocyte colony-stimulating factor form a combined neovasculogenic therapy for ischemic cardiomyopathy. J Thorac Cardiovasc Surg. 2005; 130: 321–329.[Abstract/Free Full Text]
36. Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, Magner M, Isner JM, Asahara T. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med. 1999; 5: 434–438.[CrossRef][Medline] [Order article via Infotrieve]
37. Kocher AA, Schuster MD, Szabolcs MJ, Takuma S, Burkhoff D, Wang J, Homma S, Edwards NM, Itescu S. Neovascularization of ischemic myocardium by human bone-marrow- derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med. 2001; 7: 430–436.[CrossRef][Medline] [Order article via Infotrieve]
38. Quirici N, Soligo D, Caneva L, Servida F, Bossolasco P, Deliliers GL. Differentiation and expansion of endothelial cells from human bone marrow CD133(+) cells. Br J Haematol. 2001; 115: 186–194.[CrossRef][Medline] [Order article via Infotrieve]
39. Heeschen C, Aicher A, Lehmann R, Fichtlscherer S, Vasa M, Urbich C, Mildner-Rihm C, Martin H, Zeiher AM, Dimmeler S. Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization. Blood. 2003; 102: 1340–1346.[Abstract/Free Full Text]
40. Ziegelhoeffer T, Fernandez B, Kostin S, Heil M, Voswinckel R, Helisch A, Schaper W. Bone marrow-derived cells do not incorporate into the adult growing vasculature. Circ Res. 2004; 94: 230–238.[Abstract/Free Full Text]
41. Wang X, Paigen B. Comparative genetics of atherosclerosis and restenosis: exploration with mouse models. Arterioscler Thromb Vasc Biol. 2002; 22: 884–886.[Free Full Text]
42. Seiler C, Pohl T, Wustmann K, Hutter D, Nicolet PA, Windecker S, Eberli FR, Meier B. Promotion of collateral growth by granulocyte-macrophage colony-stimulating factor in patients with coronary artery disease: a randomized, double-blind, placebo-controlled study. Circulation. 2001; 104: 2012–2017.[Abstract/Free Full Text]
43. Harraz M, Jiao C, Hanlon HD, Hartley RS, Schatteman GC. CD34- blood-derived human endothelial cell progenitors. Stem Cells. 2001; 19: 304–312.[Abstract/Free Full Text]
44. Kuwana M, Okazaki Y, Kodama H, Izumi K, Yasuoka H, Ogawa Y, Kawakami Y, Ikeda Y. Human circulating CD14+ monocytes as a source of progenitors that exhibit mesenchymal cell differentiation. J Leukoc Biol. 2003; 74: 833–845.[Abstract/Free Full Text]
45. Nishimura H, Asahara T. Bone marrow-derived endothelial progenitor cells for neovascular formation. EXS. 2005: 147–154.
46. Fernandez Pujol B, Lucibello FC, Zuzarte M, Lutjens P, Muller R, Havemann K. Dendritic cells derived from peripheral monocytes express endothelial markers and in the presence of angiogenic growth factors differentiate into endothelial-like cells. Eur J Cell Biol. 2001; 80: 99–110.[CrossRef][Medline] [Order article via Infotrieve]
47. Moldenhauer A, Nociari M, Lam G, Salama A, Rafii S, Moore MA. Tumor necrosis factor alpha-stimulated endothelium: an inducer of dendritic cell development from hematopoietic progenitors and myeloid leukemic cells. Stem Cells. 2004; 22: 144–157.[Abstract/Free Full Text]

This article has been cited by other articles:

				K. K. Hirschi, D. A. Ingram, and M. C. Yoder Assessing Identity, Phenotype, and Fate of Endothelial Progenitor Cells Arterioscler. Thromb. Vasc. Biol., September 1, 2008; 28(9): 1584 - 1595. [Full Text] [PDF]

				T. Sugiura, T. Kondo, Y. Kureishi-Bando, Y. Numaguchi, O. Yoshida, Y. Dohi, G. Kimura, R. Ueda, T. J. Rabelink, and T. Murohara Nifedipine Improves Endothelial Function: Role of Endothelial Progenitor Cells Hypertension, September 1, 2008; 52(3): 491 - 498. [Abstract] [Full Text] [PDF]

				D. P. Sieveking, A. Buckle, D. S. Celermajer, and M. K.C. Ng Strikingly different angiogenic properties of endothelial progenitor cell subpopulations: insights from a novel human angiogenesis assay. J. Am. Coll. Cardiol., February 12, 2008; 51(6): 660 - 668. [Abstract] [Full Text] [PDF]

				M. B. Rookmaaker, M. C. Verhaar, H. C. de Boer, R. Goldschmeding, J. A. Joles, H. A. Koomans, H.-J. Grone, and T. J. Rabelink Met-RANTES reduces endothelial progenitor cell homing to activated (glomerular) endothelium in vitro and in vivo Am J Physiol Renal Physiol, August 1, 2007; 293(2): F624 - F630. [Abstract] [Full Text] [PDF]

				S. A. Sorrentino, F. H. Bahlmann, C. Besler, M. Muller, S. Schulz, N. Kirchhoff, C. Doerries, T. Horvath, A. Limbourg, F. Limbourg, et al. Oxidant Stress Impairs In Vivo Reendothelialization Capacity of Endothelial Progenitor Cells From Patients With Type 2 Diabetes Mellitus: Restoration by the Peroxisome Proliferator-Activated Receptor-{gamma} Agonist Rosiglitazone Circulation, July 10, 2007; 116(2): 163 - 173. [Abstract] [Full Text] [PDF]

				J. E. Deanfield, J. P. Halcox, and T. J. Rabelink Endothelial Function and Dysfunction: Testing and Clinical Relevance Circulation, March 13, 2007; 115(10): 1285 - 1295. [Full Text] [PDF]

				G. C. Schatteman, M. Dunnwald, and C. Jiao Biology of bone marrow-derived endothelial cell precursors Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H1 - H18. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 26/8/1760    most recent 01.ATV.0000229243.49320.c9v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Loomans, C.J.M. Articles by Staal, F.J.T. Search for Related Content PubMed PubMed Citation Articles by Loomans, C.J.M. Articles by Staal, F.J.T.