© 2001 by European Society of Cardiology
Copyright © 2001, European Society of Cardiology
Monocytes coexpress endothelial and macrophagocytic lineage markers and form cord-like structures in Matrigel® under angiogenic conditions
aDepartment of Cardiology, Technical University of Dresden, Heart Center Dresden, Fetscherstr. 76, D-01307 Dresden, Germany
bDivision of Molecular Cardiology, Department of Cardiology, University Erlangen-Nuernberg, Oestliche Stadtmauerstr. 29, D-91054 Erlangen, Germany
* Corresponding author. Tel.: +49-351-450-1704; fax: +49-351-450-1702 alexanderschmeis{at}t-online.de
Received 5 June 2000; accepted 23 October 2000
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
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Objectives: It has been shown that circulating human non-adherent CD34+ cells coexpressing vascular endothelial growth factor (VEGF)-R2 and AC133 have the capacity to differentiate into adherent mature endothelial cells. However, prior studies have demonstrated that a much bigger subset of primary adherent mononuclear cells can also form endothelial progenitor cells (EPC). To determine the origin of the latter cell population we tested the hypothesis: do monocytes as a firmly adherent and plastic cell type have the potential to differentiate into an endothelial phenotype. Methods: CD34–/CD14+ monocytes were isolated from human peripheral blood by adherence separation and magnetic bead selection (purity <90%) and cultured on fibronectin-coated plastic dishes (medium containing VEGF 10 ng/ml, basic fibroblast growth factor (bFGF) 2 ng/ml, insulin like growth factor (IGF-1) 1 ng/ml, 20% fetal calf serum). Results: After 2 weeks of culture, using fluorescence activated cell analysis we observed a new expression of the endothelial markers von Willebrand factor (vWf), VE-cadherin (VE) and ec-NOS in 45.2, 12.4 and 9.8% of the cells, respectively. The proportion of cells expressing these markers further increased after 4 weeks (94.2, 89.7 and 58.8% of these cells, respectively). The proportion of CD45 expressing cells remained unchanged during this period. However, after 14 days the specific macrophage antigen CD68 was newly expressed in 62% of the analysed cells with a further increase to 90% after 28 days of culture. In three-dimensional gel (Matrigel®) the formation of cord- and tubular-like structures was observed. Conclusion: The present data indicate that under angiogenic stimulation macrophages develop an endothelial phenotype with expression of specific surface markers and even form cord- and tubular-like structures in vitro suggesting that this cell population may be recruited for vasculogenesis.
KEYWORDS bFGF, basic fibroblast growth factor; EC, endothelial cells; EPC, endothelial progenitor cells; FACS, fluorescence activated cell analysis; FCS, fetal calf serum; Flk-1, fetal liver kinase-1; Flt-1, fms-like tyrosine kinase-1; HSC, hematopoietic stem cells; HUVEC, human umbilical vein endothelial cells; IGF-1, insulin like growth factor; Mo, monocytes; ox-LDL, oxidized low density lipoprotein; VE, VE-cadherin; VEGF, vascular endothelial growth factor; vWf, von Willebrand factor
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Neovascularization involves the recruitment of proliferating endothelial cells to the site of wound healing or tumor growth. Two possible sources of endothelialization are endothelial migration and sprouting from local residing endothelial cells, or recruitment of circulating endothelial progenitor cells (EPC) [1,2]. The first scenario is in accordance with the classic paradigm of angiogenesis. However, the existence of circulating EPC in adult humans as a characteristic feature of postnatal vasculogenesis has only recently been suggested and is under intensive scrutiny [2,3].
During embryogenesis, it has been reported that a single progenitor cell, the hemangioblast, can give rise to both the hematopoietic and vascular systems because targeted disruption of the gene encoding the vascular endothelial growth factor receptor-2 (VEGFR-2, also called fetal liver kinase-1 (Flk-1) and KDR in mice and humans) resulted in defects in both hematopoietic and endothelial/angioblastic differentiation [4]. For a long time vasculogenesis has been considered to be restricted to embryogenesis [5]. More recently there have been observations that vasculogenesis may also play a physiological role in adults. Asahara et al. [2] isolated endothelial progenitor cells from adult human peripheral blood using magnetic bead selection of CD34+ hematopoietic cells. In vitro, the majority of the primary adherent cells differentiate into spindle-shaped cells within 7–10 days of culture on fibronectin and express markers of the endothelial cell lineage, such as von Willebrand factor (vWf), E-selectin, PECAM (CD31), Flk-1, Tie-2, and ec-NOS and incorporate acetylated low-density lipoprotein. Kalka et al. [6] used the primary adherence on fibronectin to isolate EPCs from human total peripheral blood mononuclear cells and also demonstrated the appearance of cells with an endothelial phenotype at a very high frequency after 7–10 days of culture. In animal models of ischemia and tumor growth both demonstrated the contribution of EPCs to active neovascularization [2,6–8]. Shi et al. [9] and Nieda et al. [10], using CD34+ cells at a much higher purity (<93%) than Asahara et al. [2] (15.7±3.3%), observed adherent endothelial colonies only after 15–20 days and 5–6 weeks. In addition, Shi et al. [9] were able to show in accordance with embryonic vasculogenesis an absolute requirement for VEGF in endothelial cell colony formation from EPC. However the importance of the specific VEGFR-1 and -2, and also the true origin of EPCs within the very heterogeneous population of CD34+ hematopoietic progenitor and stem cells, remained unclear.
In a recent paper by Peichev et al. [3] it was demonstrated that human circulating CD34+ cells coexpressing the endothelial but also hemangioblastic marker VEGFR-2 and a early hematopoietic stem cell antigen AC133, constitute functional endothelial precursors with the capacity to differentiate into adherent mature endothelial cells (AC133–/VEGFR-2+) and to contribute to reendothelialization of left ventricular assist devices in human. Interestingly, the frequency of CD34+ cells coexpressing VEGFR-2 and AC133 in human peripheral blood was only 0.4±0.2% of the total CD34+ population (0.002% of total mononuclear cells). A further distinctive feature of these freshly isolated cells was their lack of capacity to adhere to extracellular matrix at the time of isolation. In contrast to Asahara et al. [2] and Kalka et al. [6], formation of mature endothelium from EPCs required at least a 2-week period.
Therefore, it may be suggested that Asahara et al. [2] and Kalka et al. [6] described a different cell population with endothelial cell quality. In the mononuclear cell fraction of peripheral human blood only monocytes have a high capacity to adhere to extracellular matrix at the time of isolation. They also have a multilineage potential of differentiation in response to different types of cytokine stimulation [11]. Consequently, the hypothesis of the present study was that human monocytes might contain a population of cells with the potential to differentiate in an endothelial phenotype under angiogenic stimulation.
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2 Methods |
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2.1 Reagents and antibodies
Monocyte and CD34 Progenitor Cell Isolation Kit were purchased from Miltenyi Biotec, Germany, and Ficoll-Paque Plus from Pharmacia Biotechnology, Germany. Monoclonal antibody (mAb) to human VE-cadherin (VE), von Willebrand factor (vWf), fms-like tyrosine kinase-1(Flt-1) and FITC-conjugated F(ab')2 fragments were purchased from Santa Cruz Biotechnology, USA, mAb to human Flk-1 (extracellular domain of KDR) from Sigma, Germany, mAb to human CD68 from Dako, monoclonal mouse anti-human macrophage Ab HAM56 from Biomeda, USA, mAb to CD34, CD31, CD41a, CD45 (leucocyte common-all isoforms) and CD14 from PharMingen, USA, CD64 from Dianova, Germany, anti-ec-NOS from Calbiochem, USA, fluorescein and phycoerythrin conjugated (Fc-specific) anti-mouse antibodies from PharMingen, and mouse-, goat- and rabbit serum from Sigma, Germany. VEGF, basic fibroblast growth factor (bFGF) and IGF were purchased from Biomol, Germany, Medium 199, fetal calf serum, penicillin and streptomycin were from Biochrom, Germany, and fibronectin, collagen, gelatine and Matrigel® Basement Membrane Matrix were from Becton Dickinson, USA. PCR primers were synthesized by Pharmacia, Germany. Dispase II, TRIzol reagent, Taq DNA polymerase, AMV reverse transcriptase and dNTP mix were purchased from Gibco BRL, Germany.
2.2 Cell lines and cell culture
Human monocytes (Mo) were obtained from peripheral blood, and CD34+ hematopoietic stem cells from leucapherisate of normal volunteers. The mononuclear cells were isolated by gradient centrifugation with Ficoll-Paque. Monocytes and CD34+ hematopoietic stem cells (HSC) were isolated using an Isolation Kit from Miltenyi Biotec. Briefly, in monocytes, 108 mononuclear cells were labeled after adherence separation on non-coated plastic dishes with 200 µl Hapten-Antibody Cocktail (containing CD3, CD7, CD19, CD34, CD45RA, CD56, and anti-IgE) and incubated in a volume of 500 µl PBS supplemented with 10% autologous serum, 0.5% bovine serum albumin and 2 mM EDTA for 15 min at 6°C. The cells were washed with buffer and resuspended in 600 µl PBS buffer. Then 200 µl MACS Anti-Hapten MicroBeads were added to the cell suspension and incubated at 6°C for 15 min. The cell suspension was separated by LS+/VS+ column and the monocyte fraction was passed through the column. In CD34+ hematopoietic stem cells there was positive selection with the same procedure but with CD34+ antibody-coated magnetic beads. Thus, it was necessary to reelute the target fraction with PBS. Monocyte and non-monocyte populations were seeded separately (100 000 cells/cm2) onto fibronectin-, collagen- or gelatine-coated six-well plastic dishes and cultured in 20% fetal bovine serum in Medium 199 containing VEGF 10 ng/ml, bFGF 2 ng/ml and insulin like growth factor (IGF-1) 1 ng/ml. After 24 h non-adherent cells were removed from the monocyte fraction.
2.3 Cell proliferation
Cell proliferation was measured on days 1, 7 and 20 with fluorescence activated cell analysis (FACS) of KI67 expression and with a cell proliferation kit (MTT, measurement of metabolic activity using the tetrazolium salts; BD Bioscience) according to the manufacturer's instructions .
2.4 Immunostaining
Macrophages were identified additionally by immunostaining for HAM56, a monoclonal mouse anti-human macrophage, according to the manufacturer's instructions.
2.5 Flow cytometry analysis
The differentiated cells were detached by 1 mM EDTA. After two washes in PBS, aliquots containing 105 cells were incubated at 4°C for 1 h with 100 µl of 5% blocking goat, mouse or rabbit serum to block Fc receptor binding by detecting Ab. After two additional washes in PBS, aliquots containing 105 cells were incubated at 4°C for 1 h with 100 µl saturating concentration of mAb to CD34, vWF, VE-cadherin, ec-NOS, CD14, CD31 (PECAM), CD45, CD64, CD41a, CD68, Flk-1 and Flt-1, respectively, or isotype-matched control. After two wash steps in PBS, the cells were incubated at 4°C for an additional hour with 100 µl FITC or PE-conjugated F(ab')2 fragments of mAb to mouse or rabbit IgG diluted 1:50 in 2% BSA/PBS, washed twice, and resuspended in PBS. Quantitative fluorescence analysis was performed using a FACS Calibur flow cytometer and CellQuest software program (Becton Dickinson, USA). Histograms of cell number versus fluorescence intensity were recorded for at least 5000 cells per sample.
2.6 RT-PCR
Monocytes were grown for different periods in 50-ml culture flasks and were rinsed twice with PBS. Total RNA of monocytes was isolated by using TRIzol reagent. Then 1 µg of total RNA was reversibly transcribed to cDNA in a reaction condition of 25 mM Tris–HCl (pH 8.3), 5 mM MgCl2, 50 mM KCl, 2 mM DTT, 1 mM dNTP each, 40 µg/ml primer dT15 and 200 U/ml AMV reverse transcriptase in a final volume of 25 µl and incubated for 40 min at 42°C. Reverse transcription was terminated by heating at 95°C for 5 min, and 5% of the cDNA was used as template for PCR. These reactions were performed in 10 mM Tris–HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.2 mM each dNTP, 0.5 mM each primer, and 1.25 U of Taq polymerase in a final reaction volume of 50 µl.
2.7 Primer
The primers used were as follows: vWf: sense, 5'-ATG TTG TGG GAG ATG TTT GC; antisense, 5'-GCA GAT AAG AGC TCA GCC TT; VE-cadherin: sense, 5'-CCC ACC GGC AAA AGA GAG ATT GG; antisense, 5'-CTG GGT TTC CTT CAG GAA GTG GT; ec-NOS: sense, 5'-GTT CGC TTC GAC GTG CT; antisense, 5'-TCC CCA TTC CCA ATG TG; Flk-1: sense, 5'-CTG GCA TGG TCT TCT GTG AAG CA; antisense, 5'-AAT ACC AGT CGA TGT GAT GCG GT; GAPDH: sense, 5'-TGA AGG TCG GAG TCA ACG GAT TTG; antisense, 5'-CAT GTG GGC CAT GAG GTC CAC CAC.
The primers are predicted to amplify 656-, 1036-, 836-, 819- and 983-bp DNA fragments, respectively. The PCR reactions were done at 94°C for 45 s, 60°C for 45 s, and 72°C for 1 min for 30 cycles. The PCR products were analyzed on a 1% agarose gel and stained by ethidium bromide. Afterwards, the sequences of the PCR products were analyzed by ALF express DNA Sequencer and Fragment Analysis System from Pharmacy Biotechnology.
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3 Results |
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CD34–/CD14+ monocytes were isolated from human peripheral blood by adherence separation and negative (CD34, CD3, CD7, CD19, CD45RA, CD56) magnetic bead selection. FACS analysis documented that 92±3% of selected cells compared with <0.8% of the remaining cells expressed CD14. In FACS analysis, the isolated CD14+ cells had no expression for CD34 or Flk-1. The remaining CD14 depleted cells, CD34+ hematopoietic stem cells after positive selection and human umbilical vein endothelial cells (HUVECs) were used as controls. The different mononuclear cells were cultured separately on fibronectin-coated plastic dishes in Medium 199 (20% fetal calf serum, FCS) containing the angiogenic growth factors VEGF (10 ng/ml), bFGF (2 ng/ml) and IGF-1 (1 ng/ml). On days 1, 7 and 20 there was no significant staining for KI67 in FACS analysis of CD14+ and CD34+ sorted cells. Additionally, in the MTT test there was no indication for proliferation. In contrast, there was a significant decrease of absorbance in the spectrophotometric assay from 0.7±0.2 on day 1 to 0.4±0.1 on day 20 (P<0.05) in CD14+ monocytes.
After 4–7 days in culture, the majority of CD14+ monocytes were attached and the morphology of these attached cells changed significantly. The cells gradually developed spindle shape (Fig. 1a). These cells appeared either as single cells or as clusters comprising round cells centrally and sprouts of spindle-shaped cells at the periphery. The same cell behavior was shown by Asahara et al. [2] in CD34-derived EPC, resembling blood island-like cell clusters [5]. On non-coated dishes the cells only rarely changed their morphology to the spindle-like form (data not shown). Additionally, in three-dimensional gel (Matrigel® Basement Membrane Matrix, 1:2 dilution with PBS, 150 µl/cm2 of growth surface) the formation of cord- and tubular-like structures from CD14+ monocytes was observed in 62% of the experiments. Such lines resembling the first stages of vasculogenesis appeared as early as 4 days in culture and were observed up to 10–20 days. After that time the structures became disorganized. There was great variability in line appearance, from multiple thin strands to thicker cord- and tubular-like like structures. Complex lines consisted of multiple rows of elongated cells in contact with each other (Fig. 1b,c). Cord formations usually continued to increase in complexity for up to 15–20 days.
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In the next step we looked for monocytic expression of specific endothelial cell markers under growth factor stimulation. FACS analysis and RT-PCR showed new expression of vWf, VE-cadherin and ec-NOS during the in vitro differentiation process after 2 and 4 weeks in culture (Figs. 2a, and 3). This switch in expression of cell surface markers occurred without cell proliferation indicating that the existing cells changed their characteristics.
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On the first day, no expression of the EC markers, VE-cadherin and ec-NOS, was observed. For vWf the FACS analysis showed a positive expression in only 22.3±4% of cells. After 2 weeks of culture, vWf, VE and ec-NOS were expressed in 45.2±5, 12.4±2 and 9.8±3% of the cells, respectively. After 4 weeks, 94.2±13, 89.7±15, and 58.8±10% of these cells were vWf, VE and ec-NOS positive, respectively (Fig. 2a). Because of positive staining for vWf in 22.3±4% of the freshly isolated cells, we assumed a contamination of monocytes with activated platelets, possibly induced by the procedure of cell isolation. In fact a high positive signal of the specific platelet marker CD41a was detected in the gated monocyte fraction immediately after the isolation of the cells. However, after 10 days of culture no CD41a was detected in the same cell population (data not shown). CD34 as a marker for hematopoietic stem cells but also for postnatal EPC [2] and activated vascular endothelial cells (EC) in adults [12], was not detected during the observation time (data not shown). It has been proposed that in particular VEGF and its receptor VEGFR-2 (Flk-1) are critical for inducing the emergence of the endothelial cell lineage in embryonic vasculogenesis [4,13,14]. However, during the differentiation of endothelial-like monocytic cells no fluorescence staining or detection of corresponding mRNA for Flk-1 could be detected (Fig. 2b, and Fig. 3). As expected for freshly isolated monocytes we found expression for VEGFR-1 (Flt-1) (62±9% of analysed cells). After 28 days in culture 56±7% of the cells expressed Flt-1 (Fig. 2b). Likewise, 96±13% of isolated monocytes expressed CD31 (PECAM) on the 1st day (data not shown). For this reason we did not follow the expression of PECAM as an endothelial cell marker (data not shown). We next investigated the expression of monocyte/macrophage lineage markers. In the course of 4 weeks, FACS analysis showed a drop in CD14 and CD64 expressing cells from 91.2±5 and 76.2±12% to 12.3±4 and 40.1±13%, respectively (Fig. 2c). Until 6 weeks of culture nearly all attached cells stained positive for the common leucocyte antigen (LCA) CD45, yet with decreasing fluorescence intensity (from 277.12±32.3 to 35.5±6.3). Furthermore, the mAb we used reacts with the 180-, 190-, 205- and 220-kDa isoforms of the LCA, but not with the CD45RO isoform. It might be noted that the expression of CD45RO in endothelial cells has been shown only under long-term stimulation with IL-1 [15].
FACS analysis of the human homologue for macrosialin, CD68, a member of the lysosomal-associated membrane protein family with a macrophage-specific mucin-like extracellular domain, showed that in contrast to day 1, after 14 and 28 days in culture 8±7, 62±13 and 90±10% of analysed monocytes express CD68, respectively (Fig. 2d). Moreover, after 28 days in culture 
90% of adherent cells stained positive for HAM56, an anti-human macrophage antibody (data not shown).
The non-monocyte, lymphocyte rich fraction after MACS was used as control in the present experiment: attached cells were observed only sporadically, and there was no significant staining for the investigated EC markers in FACS and the corresponding mRNA in RT-PCR.
HUVECs were used as positive controls as they constitutively express vWf, VE-cadherin, ec-NOS and Flk-1, both at the protein and mRNA level (Fig. 2a, and Fig. 3). In contrast, HUVECs in culture stained negative for CD14, CD64, and CD45 (Fig. 2c).
As an additional positive control for EPC [2,9] we used CD34+ hematopoietic stem cells under the identical culture conditions. After isolation from healthy human leucapherisate by means of magnetic beads coated with antibody to CD34 (purity 92±3%), the cells were seeded on fibronectin-coated dishes (100 000 cells/cm2). After 15–20 days in culture, only 1–5% of seeded cells showed adherence and approximately half of these adherent cells formed the spindle shape which has also been described by Asahara et al. [2] and Peichev et al. [3]. After 20 days in culture, 29±7 and 45±15% of adherent cells expressed CD34 and CD14 in FACS analysis, respectively (data not shown). After an additional 8 days of culture the cells showed expression of vWf, VE-cadherin and ec-NOS comparable to that of monocyte-derived endothelial-like cells (Fig. 4). There was only low expression for CD34 and Flk-1 antigen but under preservation of CD45 expression.
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4 Discussion |
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The salient finding of the present study is that monocytes (CD34–/CD14+) or a subfraction of monocytes have the potential to coexpress very specific endothelial markers and mono-/macrophagocytic antigens under growth factor stimulation in vitro. This is indicated by the facts that (i) after 4 weeks of in vitro culture, a significant part of the primary adherent monocytes express the endothelial markers vWf, VE-cadherin and ec-NOS, (ii) lineage markers of the monocyte cell population CD14 were downregulated completely, and CD64 was reduced by 50%, however, under persistence of the leucocyte common antigen (LCA) CD45, (iii) after this time over 90% of the cells stained positive for the macrophage receptor of oxidized low density lipoprotein (ox-LDL) CD68 and HAM56 and (iv) in three-dimensional gel-matrix (Matrigel®) the same cells formed cord- and tubular-like structures resembling the first stages of vasculogenesis.
The remaining lymphocytic-rich fraction in the magnetic field was recovered as negative control. After 14 days of culture, neither FACS nor RT-PCR analysis could show positive expression of those specific endothelial cell lineage markers.
It has been proposed that in embryogenesis hematopoietic and endothelial cells are derived from a very early stem cell, the hemangioblast [4]. In addition, recent data suggested the possible existence of a bone marrow-derived precursor endothelial cell in postembryonal life [2,3]. It has been demonstrated in vitro that the small subset of initially non-adherent CD34+ cells coexpressing VEGFR-2 and AC133 have the capacity to differentiate into mature adherent endothelial cells in the presence of the angiogenic growth factors bFGF, IGF-1 and VEGF. On the other hand, Asahara and Kalka et al. [2,6] isolated and cultured primary adherent endothelial progenitor cells from the mononuclear cell fraction of human peripheral blood with high frequency, but of at present unknown origin. However, in vivo data demonstrated that the different endothelial cell precursors have the potential to incorporate into sites of active angiogenesis and endothelialization, consistent with vasculogenesis, a paradigm otherwise restricted to embryogenesis [1–3,7,9].
Since the cell shape of isolated and cultured (CD34–/CD14+) mono-/macrophages and the surface expression of endothelial markers were very similar to EPCs as described by Asahara or Kalka et al. [2,6] we suggest for both cell types an identical cell source, i.e. CD14+ monocytes. Even the data of Kalka et al. [6] when analysed in detail support this suggestion. They showed that after 7 days of ex vivo expansion of suggested primary adherent EPCs, 90.7±1.8% of these cells express the marker CD14, which is characteristic for monocytes. In our experiments 45±14% of CD34-derived endothelial-like cells also stained positive for CD14 after 20 days in culture. On the other hand, Peichev et al. [3] demonstrated that endothelial cells at different stages, VEGFR-2/CD34/AC133-positive primary non-adherent endothelial precursors, mature endothelial cells and IL-1β activated endothelial cells, did not show any expression for CD14 [3]. However, neither Asahara et al. nor Kalka et al. [2,6] could demonstrate positive staining for CD68 on their isolated EPCs. It might be suggested that CD68 is a predominantly intracellular protein. Without a preactivation of monocytes/macrophages no or only low expression of CD68 on the cell surface may be detectable [16]. In contrast to Asahara et al. [2], CD31 (i.e. PECAM-1) was not used as an endothelial cell marker in this study, because it is constitutively expressed on the surface of circulating platelets, monocytes, neutrophils and selected T-cell subsets [17].
These data suggest that even at a later stage of cell differentiation, as described by Peichev et al. [3] for functional endothelial precursors, vasculogenesis may become stimulated in peripheral blood of adult humans. The present data, however, showed a persistence of LCA (CD45) expression and an increasing expression for the specific macrophage markers (CD68, HAM56). These data support the notion that the described cell type in this study is not identical to mature endothelium but remains a leucocyte.
The following controls were provided to demonstrate that a fraction of the CD34–/CD14+ monocytes without contaminating CD34+ hematopoietic stem cells or vascular endothelial cells do differentiate into an endothelial phenotype. After magnetic bead selection alone or in combination with adherence separation, no positive fluorescence for CD34 and Flk-1 within the monocytic cell population could be detected. In addition, both the present study with CD34+ hematopoietic cells and data by Peichev et al. [3] demonstrate no significant adherence of functional endothelial precursors within the first days of culture. Since the monocytes were also isolated in combination with adherence separation and CD14+ monocytes inhibit hematopoietic stem cell proliferation [18], the colony formation of contaminated CD34+ stem cells or VEGFR-2/AC133/CD34-positive functional endothelial precursors was very unlikely. Additionally, Lindner et al. [19] have suggested that mononuclear cells may secrete substances inhibitory or toxic to human endothelial cell colony development. Moreover, in the present study no indication for proliferation in cell culture on days 1, 7 and 20 could be detected suggesting a change of phenotype of the adherent cells.
The remaining lymphocytic-rich fraction in the magnetic field, recovered as negative control, could not show positive expression of endothelial cell lineage markers after 14 days of culture.
Circulating vascular endothelial cells have been detected only in acute myocardial infarction and unstable angina but not in healthy controls [20]. So, the experiments indicate that the monocytes obtained from healthy donors were not contaminated by circulating vascular endothelial cells.
The main limitation of our study is that the basic mechanisms underlying the differentiation of monocytes into an endothelial phenotype continue to be unclear. Several growth factors, in particular VEGF and its receptor VEGFR-2 (Flk-1), have been shown to be critical for inducing the emergence of the endothelial cell lineage in embryonic vasculogenesis [4,13,14]. Eichmann et al. [14] showed that early mesodermal Flk-1+ cells give rise to hematopoietic cell colonies and that the ligand VEGF supports the growth of endothelial colonies in chicken embryo. However, the monocyte is an example of a non-endothelial cell type that is positive for VEGFR-1 (Flt-1) but negative for the two other receptors of VEGF, VEGFR-2 and -3. This receptor is active in monocyte chemotaxis as shown by migration studies using VEGFR-1 specific ligands [21]. Furthermore it has been reported that Flt-1 is essential for the organization of embryonic vasculature, but not for embryonic endothelial cell proliferation and differentiation [22]. On the other hand, Flt-1 gene is a direct target of Egr-1, an transcription factor primarily induced on macrophage differentiation [23]. According to expectation, but in contrast to Asahara or Kalka et al. [2,6], the monocytic cell population in this study showed only the expression of Flt-1. Therefore, future studies need to address the influence of bFGF and related receptors known to be important in vasculogenesis [1] as well as the interaction of matrix integrins with monocytes in the observed angiogenic differentiation process.
In conclusion, the present data indicate that under angiogenic stimulation macrophages develop an endothelial phenotype with the expression of specific surface markers and even form cord- and tubular-like structures in vitro, suggesting that this leucocyte cell population may be recruited for vasculogenesis. Since the cord-like structures were observed in a relatively high percentage and with similar formations in many experiments it can be concluded that in combination with the formation of VE-cadherin as a cell contact signal, the structures might represent the preformation of vascular lines. Recently, these data have been supported by in vivo findings in transgenic mice overexpressing MCP-1 [24]. These mice suffer thrombotic occlusive arteriolar vasculopathy that results in ischemia and heart failure. The invading macrophages drill an extensive network of periodic acid-Schiff-positive tunnels within the myocardium. The tunnels and macrophages stain positive for mouse macrophage elastase, but they are not lined by endothelium and many tunnels seem to contain erythrocytes. This study also suggests another mechanism in addition to the secretion of angiogenic factors by which macrophages may participate in the elaboration of new blood vessels. In addition a role for monocyte/macrophages in the vascularization and growth of tumors is supported by a number of lines of evidence. For instance, macrophages are known to infiltrate murine and human tumors and mice depleted of monocytes show a strong reduction of tumor vascularization in implanted syngenic fibrosarcomas [25].
In total, the present data are not in contrast to those of Peichev et al. [3] or Shi et al. [9] since these authors actually investigated cell lines that are related still closer to conventional stem cells. However, they support the data by Asahara and Kalka et al. [2,6] indicating that cells may become involved in the process of vasculogenesis later in the myelomonocytic differentiation
The significance of the above phenomena in angiogenesis is quite speculative but if it held true that only monocytes/macrophages become involved in the process of capillarization not only by expression of angiogenic growth factors but also by converting into a cell type at least similar to endothelial phenotype with the potential to form vascular channels, this may open up a new window for diagnostic and therapeutic management in patients with ischemic diseases.
Time for primary review 37 days.
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Acknowledgements |
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This work was supported in part by grants to A.S., C.D.G. and W.G.D. from the IZKF and ELAN-Project of FAU Erlangen-Nuernberg. Heike Kloos (laboratory technician) and Christoph Requadt are acknowledged for technical assistance.
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Notes |
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1 These authors have contributed equally to this work.
* Presented in part at the 72nd Scientific Session of the American Heart Association, Atlanta, GA, November 7–10, 1999, and published in abstract form [Circulation 100 (1999) I-405]. During the review process of this manuscript similar findings where published by Fernandez Pujol et al. [Differentiation 65 (2000) 287–300].
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