This Article Abstract Full Text (PDF) Correction (v111,p1718) All Versions of this Article: 108/20/2511    most recent 01.CIR.0000096483.29777.50v1 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 Urbich, C. Articles by Dimmeler, S. Search for Related Content PubMed PubMed Citation Articles by Urbich, C. Articles by Dimmeler, S. Related Collections Angiogenesis Cell biology/structural biology Other Vascular biology

(Circulation. 2003;108:2511.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Relevance of Monocytic Features for Neovascularization Capacity of Circulating Endothelial Progenitor Cells

Carmen Urbich, PhD^*; Christopher Heeschen, MD^*; Alexandra Aicher, MD; Elisabeth Dernbach, MD; Andreas M. Zeiher, MD; Stefanie Dimmeler, PhD

From Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Germany.

Correspondence to Stefanie Dimmeler, PhD, Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. E-mail dimmeler{at}em.uni-frankfurt.de

Received April 15, 2003; de novo received July 3, 2003; revision received July 22, 2003; accepted July 22, 2003.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background— Transplantation of ex vivo expanded circulating endothelial progenitor cells (EPCs) from peripheral blood mononuclear cells improves the neovascularization after critical ischemia. However, the origin of the endothelial progenitor lineage and its characteristics have not yet been clearly defined. Therefore, we investigated whether the phenotype and functional capacity of EPCs to improve neovascularization depend on their monocytic origin.

Methods and Results— Monocytic CD14⁺ cells were isolated from mononuclear cells and incubated on fibronectin-coated dishes in endothelial medium in the presence of vascular endothelial growth factor. After 4 days of cultivation, adherent cells deriving from CD14⁺ or CD14^- mononuclear cells showed equal expression of endothelial marker proteins and capacity for clonal expansion as determined by measuring endothelial colony-forming units. In addition, transplanted EPCs (5x10⁵ cells) deriving from CD14⁺ or CD14^- cells were incorporated into vascular structures of nude mice after hind-limb ischemia and significantly improved neovascularization from 0.27±0.12 (no cells) to 0.66±0.12 and 0.65±0.17, respectively (P<0.001; laser Doppler-derived relative blood flow). In contrast, no functional improvement of neovascularization was detected when freshly isolated CD14⁺ mononuclear cells without ex vivo expansion were used (0.33±0.17). Moreover, macrophages or dendritic cells differentiated from isolated CD14⁺ cells were significantly less effective in improving neovascularization than EPCs cultivated from the same starting population (P<0.01).

Conclusions— These data demonstrate that EPCs can be generated from nonmonocytic CD14^- peripheral blood mononuclear cells and exhibit a unique functional activity to improve neovascularization after hind-limb ischemia.

Key Words: angiogenesis • endothelium • cells

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Improvement of neovascularization after critical ischemia is an important novel therapeutic strategy. Growth of new blood vessel in the adult occurs through arteriogenesis, angiogenesis or vasculogenesis.¹ Arteriogenesis is defined as the growth of collateral vessels, whereas angiogenesis refers to the growth of new capillaries by sprouting of preexisting vessels through migration and proliferation of mature endothelial cells (ECs). The concept of vasculogenesis was originally described by Sabin² and others,^3,4 who investigated the embryonic de novo blood vessel formation and assigned the term "angioblast" to the endothelial cell precursor. Now, the term "vasculogenesis" is also used to describe adult blood vessel formation, referring to the mobilization of bone marrow-derived endothelial stem cells, which home to sites of ischemia and contribute to new blood vessel formation.¹ The finding that vasculogenesis also contributes to blood vessel formation in the adult offers novel therapeutic strategies for the use of cultivated circulating endothelial progenitor cells (EPCs) or their precursors for cell therapy of tissue ischemia.^5–8

EPC populations can be grown from mononuclear cells (MNCs) or purified populations of CD34-positive or CD133-positive hematopoietic cells.^9,10 In addition, CD14-positive MNCs have been used as the starting population for cultivation of EPCs.¹¹ Cultivated EPCs grown from different starting populations, including peripheral blood MNCs, have been shown to express endothelial marker proteins such as von Willebrand factor (vWF), vascular endothelial growth factor (VEGF)-receptor 2 (KDR), VE-cadherin, CD146, and CD31.^7,9,12 Moreover, these cells were identified by their functional capacity to form EC colonies and enhanced eNOS expression after shear-stress exposure.^13–15 Various studies have demonstrated that MNC-derived EPCs improve neovascularization after critical ischemia in animal models of hind-limb ischemia and myocardial infarction.^7,16–18 Moreover, a recent clinical study suggests that intracoronary infusion of blood-derived EPCs can be used to improve coronary flow reserve and cardiac function in patients after acute myocardial infarction.⁸

Although there is convincing evidence for the improvement of neovascularization by EPC transplantation, the origin of the endothelial progenitor lineage and its characterization is not clear. Monocytic cells and the attraction of monocytic cells by monocyte chemoattractant protein-1 have been shown to enhance arteriogenesis (collateral growth).^19,20 In addition, a recent study suggests that MNC-derived EPCs have monocytic characteristics.²¹ Therefore, we investigated whether the neovascularization capacity of EPCs is dependent on their monocytic origin. Our results demonstrate that EPCs can be cultivated from purified populations of both CD14-positive and CD14-negative cells. EPCs derived from both subpopulations showed equal expression of endothelial marker proteins and functional activity for improving neovascularization in a hind-limb ischemia model. Of note, whereas EPCs cultivated from CD14-positive cells effectively improved neovascularization, infusion of freshly isolated CD14-positive or CD14-negative cells did not exhibit any therapeutic effect. These data suggest that EPCs can be differentiated ex vivo from nonmonocytic lineages out of peripheral blood.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
Human recombinant VEGF, macrophage-colony stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-4 were purchased from Peprotech EC Ltd.

Cell Culture
MNCs were isolated by density-gradient centrifugation with Ficoll from peripheral blood of healthy human volunteers as described by Dimmeler et al.¹² Immediately after isolation, total MNCs (8x10⁶ cells/mL medium; cell density, 2.5x10⁶ cells/cm²) were plated on culture dishes coated with human fibronectin (Sigma) and maintained in endothelial basal medium (EBM; CellSystems) supplemented with EGM SingleQuots, VEGF (100 ng/mL), and 20% FCS. CD14-positive monocytes were purified from MNCs by positive selection with anti-CD14 microbeads (Miltenyi Biotec) using a magnetic cell sorter device (Miltenyi Biotec). Purity assessed by fluorescence-activated cell sorting (FACS) analysis was >95%.

CD14-positive monocytes were incubated in RPMI with 10% FCS in the presence of M-CSF (50 ng/mL) to induce macrophage differentiation or GM-CSF (100 ng/mL) and interleukin-4 (50 ng/mL) to stimulate dendritic differentiation.

Dil-Ac-LDL-Lectin Staining
Cells were incubated with 2.4 µg/mL 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated LDL (Dil-Ac-LDL) (Harbor Bio-Products) in fresh medium for 1 hour. Cells were fixed in 2% paraformaldehyde and counterstained with FITC-labeled lectin from Ulex europaeus (Sigma).

Colony Assay
After 4 days of culture, adherent cells were washed twice with PBS and detached with EDTA. Freshly isolated total MNCs, CD14-positive, or CD14-negative cells were washed with PBS. Cells (each 5x10⁴) were seeded in methylcellulose plates (Methocult GF H4434, CellSystems) with 100 ng/mL human recombinant VEGF. Plates were studied under phase-contrast microscopy, and colonies were counted after 14 days of incubation by 2 independent investigators. Colonies that took up Dil-Ac-LDL and bound lectin were defined as colony-forming unit (CFU)-ECs.²²

FACS Analysis
Adherent cells were washed twice with PBS, detached with EDTA, washed in PBS, and incubated in PBS/1% BSA in the presence of the following antibodies. Staining of KDR (Reliatech), vWF (Oncogene), and CD105 (Neomarkers) was visualized with FITC-conjugated rabbit anti-mouse immunoglobulins (Dako) or swine anti-rabbit immunoglobulins (Dako). CD45 and CD14 (all BD Biosciences) were used directly FITC- or PE-conjugated.

Murine Model of Hind-Limb Ischemia
The therapeutic potential of different cell types was investigated in a murine model of hind-limb ischemia in 8- to 10-week-old female athymic NMRI nude mice (The Jackson Laboratory, Bar Harbor, ME). Briefly, the proximal femoral artery including the superficial and the deep branch as well as the distal saphenous artery were ligated with 6-0 silk suture. EPCs cultivated from MNCs, CD14-positive, or CD14-negative cells, freshly isolated CD14-positive or CD14-negative cells, and monocyte (CD14)-derived macrophages and dendritic cells were injected intravenously 24 hours after induction of hind-limb ischemia (n5; each 5x10⁵ cells/mouse).

Limb Perfusion
Two weeks after induction of ischemia, ischemic (right)/normal (left) limb blood flow ratio was measured by laser Doppler imager (MoorLDI-Mark 2, Moor Instruments). After 2 recordings of laser Doppler color images, the average perfusion of the ischemic and nonischemic limbs was calculated on the basis of colored histogram pixels.

Histological Evaluation
Capillary density was determined in 5-µm frozen sections of the adductor and semimembranous muscles. ECs were stained with CD146-FITC (Chemicon International). Capillary density was expressed as number of capillaries per myocyte. Injected human cells were identified by costaining for HLA class I-APC (BD Pharmingen) and CD146-FITC (Chemicon International).

Statistical Analysis
Results for continuous variables are expressed as mean±SEM. Comparisons between groups were analyzed by t test (2-sided) or ANOVA for experiments with more than 2 subgroups. Post hoc range tests and pairwise multiple comparisons were performed with the t test (2-sided) with Bonferroni adjustment. Probability values of P<0.05 were considered statistically significant. All analyses were performed with SPSS 11.5 software (SPSS Inc.).

	Results

Top Abstract Introduction Methods Results Discussion References

 
Characterization of EPCs Cultivated From CD14-Positive or CD14-Negative Cells
To investigate whether EPCs are derived from monocytic cells, we purified CD14-positive and CD14-negative cells from blood-derived MNCs by use of magnetic beads. The experimental setup is outlined in Figure 1A. The purity of the CD14-positive population was 95.4±0.7% (Figure 1B). CD14-negative cell populations contained <3.7±2.7% CD14-positive cells (Figure 1B). Equal numbers of CD14-positive (CD14⁺), CD14-negative (CD14^-), and total unfractionated MNCs were cultivated on fibronectin-coated dishes in EBM supplemented with human epidermal growth factor, bovine brain extract, gentamicin, hydrocortisone, VEGF, and 20% FCS to cultivate EPCs ex vivo as previously described.^7,9,12,23 After 4 days of cultivation, the supernatant including the suspending cells was removed, and the adherent cells were washed twice. Adherent cells grown from total MNCs, CD14-positive, and CD14-negative cells stained positive for Dil-Ac-LDL and lectin (Figures 1A and 2 A). Moreover, FACS or Western blot analysis revealed comparable expression of the endothelial marker proteins KDR, CD105, endothelial nitric oxide synthase (eNOS), and vWF (Figure 2B and data not shown). Further cultivation up to 7 days leads to an additional increase in KDR and eNOS expression (data not shown). Of note, all cells expressed the panleukocyte marker protein CD45 (Figure 2B). To exclude the possibility that contaminating CD14⁺ cells within the CD14^- fraction are the source of EPCs, we additionally purified the CD14-negative cells with anti-CD11b microbeads as a second monocytic marker protein. These CD14^-CD11b^- cells (CD14⁺ cells, <0.25±0.16%) also give rise to Dil-Ac-LDL/lectin-double-positive cells after 4 days in culture (Figure 2A), thereby excluding the possibility that adherent EPCs are deriving from contaminating monocytic cells after the first purification step.

View larger version (45K): [in this window] [in a new window]   Figure 1. Characterization of circulating EPCs. A, Experimental setup is shown as scheme. CD14-positive and CD14-negative cells were purified from blood-derived MNCs by use of magnetic beads. Equal numbers of CD14-positive, CD14-negative, and total unfractionated MNCs were cultivated on fibronectin-coated dishes in EBM with supplements of VEGF and 20% FCS. After 4 days of cultivation, supernatant including suspending cells was removed and cells were washed twice. Adherent cells were stained with Dil-Ac-LDL. CD14-positive monocytes were incubated in RPMI with 10% FCS in presence of M-CSF (50 ng/mL) to induce macrophage differentiation or GM-CSF (100 ng/mL) and interleukin-4 (50 ng/mL) to stimulate dendritic differentiation. After 4 days of cultivation, cells were stained with Dil-Ac-LDL. Representative confocal micrographs of adherently growing cells (EPCs, macrophages) or nonadherent DCs (cytospins) are shown. B, Purity of CD14-positive and CD14-negative populations was assessed by FACS analysis. Representative histograms out of 5 independent experiments are shown.

View larger version (44K): [in this window] [in a new window]   Figure 2. Expression of endothelial markers on EPCs. A, Total unfractionated MNCs, CD14-positive, CD14-negative, and CD14-negative/CD11b-negative cells were cultivated on fibronectin-coated dishes in EBM with supplements, VEGF, and 20% FCS. After 4 days of cultivation, adherent cells were stained with Dil-Ac-LDL and lectin. Representative micrographs are shown. B, Staining of KDR, CD105, vWF, and CD45 (bold lines) by FACS analysis is shown compared with isotype controls (thin lines); n=3.

To demonstrate that cultivated EPCs have a proliferative capacity and stem/progenitor cell characteristics, we performed endothelial colony assays (CFU-EC) (Figure 3, A–C). Similar numbers of CFU-ECs could be grown from EPCs originating from CD14-positive, CD14-negative, and total unfractionated MNCs (Figure 3C). In contrast, the number of granulocyte/monocyte-colony forming units (CFU-GM) was very low in all 3 experimental settings (<1/plate). In contrast, when directly incubating CD14-positive cells without previous culture, we detected predominantly CFU-GM colonies and only a minor proportion of CFU-EC (Figure 3B).

View larger version (42K): [in this window] [in a new window]   Figure 3. Colony-forming activity of circulating EPCs. A, Cells were seeded in methylcellulose with VEGF (100 ng/mL). A representative CFU-EC (left) and CFU-GM (right) is shown. B and C, Colonies per high-power field were quantified by light microscopy after 14 days of cultivation. Data are mean±SEM; n=8.

Improvement of Neovascularization by EPCs Growing From CD14-Positive or CD14-Negative MNCs
To assess the functional capacity of CD14-positive, CD14-negative, and MNC-derived EPCs, we used a hind-limb ischemia model. The severely impaired neovascularization of nude mice in the control group was almost completely rescued by intravenous infusion of human EPCs cultivated from CD14⁺, CD14^-, or total MNC as assessed by laser Doppler-monitored blood flow measurements (Figure 4A). Interestingly, infusion of purified CD14⁺ cells or CD14^- cells without previous culture did not result in a functional improvement of neovascularization (Figure 4A). Capillary density was significantly augmented by use of cultured CD14-positive, CD14-negative or MNC-derived EPCs but not by CD14⁺ cells without previous incubation (Figure 5B and data not shown). Importantly, integrated EPCs were detected in immunohistochemical sections of the limbs (Figure 5A). Again, the efficiency of incorporation was similar when EPCs from CD14⁺, CD14^-, or total MNCs were infused (Figure 5A and data not shown).

View larger version (42K): [in this window] [in a new window]   Figure 4. Neovascularization capacity of EPCs. A, EPCs derived from MNCs, CD14-positive, or CD14-negative cells or freshly isolated CD14-positive or CD14-negative cells (each 5x105 cells; n=5 or 6) were injected intravenously into a murine model of hind-limb ischemia. Representative laser Doppler images obtained 2 weeks after induction of hind-limb ischemia are shown. Perfusion signal is subdivided into 6 different intervals, each displayed as a separate color. Low or no perfusion is displayed as dark blue; highest perfusion interval is displayed as red. Quantitative results are presented as medians, with box plots representing 25th and 75th percentiles as boxes and 5th and 95th percentiles as whiskers. B, EPCs, macrophages (M), or dendritic cells (DC; each 5x105 cells; n=5 or 6) were injected intravenously into a murine model of hind-limb ischemia. Representative laser Doppler images obtained 2 weeks after induction of hind-limb ischemia are shown. Results are presented as medians, with box plots representing 25th and 75th percentiles as boxes and 5th and 95th percentiles as whiskers.

View larger version (33K): [in this window] [in a new window]   Figure 5. Immunohistochemistry of limb muscle sections. A, Immunohistochemistry of limb muscle sections from nude mice treated with human EPCs or macrophages (HLA-APC, red fluorescence). Human and murine ECs were detected by anti-CD146-FITC (green fluorescence). Double-positive cells appear in yellow. Representative confocal images are shown. Higher magnification is shown in lower panels. B, Capillary density is expressed as % of control. Results are shown as box plots representing median, 25th and 75th percentiles as boxes, and 5th and 95th percentiles as whiskers. *P<0.05 vs macrophages. C, Incorporation of EPCs was quantified, and results are shown as box plots representing median, 25th and 75th percentiles as boxes, and 5th and 95th percentiles as whiskers. *P<0.05 vs macrophages.

Finally, we investigated whether cells that can be differentiated ex vivo from CD14⁺ monocytes, such as macrophages or dendritic cells, are capable of rescuing impaired neovascularization in the hind-limb ischemia model. We therefore differentiated, ex vivo, blood-derived CD14⁺ monocytes to macrophages or dendritic cells as described in the Methods. However, infusion of dendritic cells did not improve neovascularization after ischemia (Figure 4B). Moreover, macrophages revealed a significantly lower capacity to improve neovascularization compared with EPCs and did not incorporate into the vascular structures (Figures 4B and 5A). Consistently, limb neovascularization assessed by capillary densitometry as well as the percentage of double-positive vessels was significantly increased in mice receiving EPCs compared with macrophages (Figure 5, B and C).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The data of the present study demonstrate that the origin of EPCs isolated from peripheral blood is not restricted to the monocytic lineage marker CD14. Thus, EPCs cultivated from purified CD14⁺, CD14^-, and total MNC fractions revealed identical expression of endothelial marker proteins, colony forming capacity, and functional activity as assessed by neovascularization improvement after hind-limb ischemia. Importantly, transplantation of freshly isolated monocytic CD14⁺ cells or CD14^- cells without cultivation did not rescue neovascularization after hind-limb ischemia, clearly demonstrating that expression of the monocytic lineage marker per se does not define a functionally active cell population that is capable of improving neovascularization. Obviously, different monocyte-derived cell lineages, including macrophages and dendritic cells, can be isolated and ex vivo expanded from blood-derived MNCs depending on the culture conditions used. However, whereas MNC-derived ex vivo-expanded EPCs potently improved neovascularization, macrophages or dendritic cells derived from the identical starting MNC population were significantly less effective in improving new blood vessel formation. These data suggest that EPCs generated from peripheral blood MNCs exhibit a unique functional activity. Of note, EPCs not only express endothelial markers but also stain positive for other markers, such as the panleukocyte marker protein CD45, after 4 days of cultivation. This observation may be rationalized with the progenitor cell phenotype of the ex vivo-expanded cells and may be essential for the functional activity of EPCs. Our data as well as various previous studies show that infusion of finally differentiated mature ECs does not improve neovascularization after ischemia^7,17,18 (data not shown). Thus, a completely differentiated endothelial phenotype is not suitable for cell transplantation, although these cells can form tube-like structures in angiogenesis assays in vitro. This might be explained by the lack of homing receptors expressed on more mature cells.

Previous studies suggested that monocytic cells may contribute to neovascularization via the release of proangiogenic growth factors.^19,24,25 A recent publication corroborated these earlier findings by demonstrating that cultivated MNCs, which do not express endothelial marker proteins but are of monocytic origin, release VEGF, hepatocyte growth factor (HGF), and G-CSF.²¹ In line with these findings, we could demonstrate that EPCs cultivated from different sources showed a marked expression of growth factors such as VEGF, HGF, and IGF-1 (C.U., unpublished data). Thus, EPCs may release a variety of growth factors that act in a paracrine manner and contribute to the profound angiogenic effect. However, EPCs also incorporated into the newly formed vessel structures and showed endothelial marker protein expression in vivo. These data suggest that EPCs do not act exclusively via releasing paracrine factors. Indeed, the infusion of macrophages, which are known to release growth factors^24–28 but were not incorporated into vessel-like structures, induced only a slight increase in neovascularization after ischemia. Further studies will be necessary to define potential factor(s) contributing to the proangiogenic capacity of different cell sources that may be used for therapeutic neovascularization. However, because macrophages were significantly less effective in promoting neovascularization after hind-limb ischemia, the capacity of EPCs to physically contribute to vessel-like structures may contribute significantly to their potent capacity to improve neovascularization.

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft (FOR 501: Di 600/4-1). We thank Melanie Näher, Andrea Knau, and Christiane Mildner-Rihm for excellent technical help.

	Footnotes

 
*Drs Urbich and Heeschen contributed equally to this work.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med. 2000; 6: 389–395.[CrossRef][Medline] [Order article via Infotrieve]
   
2. Sabin FR. Studies on the origin of blood vessels and of red blood corpuscles as seen in the living blastoderm of chicks during the second day of incubation. Carnegie Inst Washington Contrib Embryol. 1920; 9: 215–262.
   
3. His W. Lecithoblast und Angioblast der Wirbeltiere. Abhandl Math-Phys Ges Wiss. 1900; 26.
   
4. Stockard GR. The origin of blood and vascular endothelium in embryos without a circulation of blood and in the normal embryo. Am J Anat. 1915; 18: 227–237.[CrossRef]
   
5. Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res. 1999; 85: 221–228.[Abstract/Free Full Text]
   
6. Takahashi T, Kalka C, Masuda H, et al. 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]
   
7. Kalka C, Masuda H, Takahashi T, et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A. 2000; 97: 3422–3427.[Abstract/Free Full Text]
   
8. Assmus B, Schachinger V, Teupe C, et al. Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI). Circulation. 2002; 106: 3009–3017.[Abstract/Free Full Text]
   
9. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275: 964–967.[Abstract/Free Full Text]
   
10. Gehling UM, Ergun S, Schumacher U, et al. In vitro differentiation of endothelial cells from AC133-positive progenitor cells. Blood. 2000; 95: 3106–3112.[Abstract/Free Full Text]
    
11. Fernandez Pujol B, Lucibello FC, Gehling UM, et al. Endothelial-like cells derived from human CD14 positive monocytes. Differentiation. 2000; 65: 287–300.[CrossRef][Medline] [Order article via Infotrieve]
    
12. Dimmeler S, Aicher A, Vasa M, et al. HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway. J Clin Invest. 2001; 108: 391–397.[CrossRef][Medline] [Order article via Infotrieve]
    
13. Shi Q, Rafii S, Wu MH, et al. Evidence for circulating bone marrow-derived endothelial cells. Blood. 1998; 92: 362–367.[Abstract/Free Full Text]
    
14. Heissig B, Hattori K, Dias S, et al. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell. 2002; 109: 625–637.[CrossRef][Medline] [Order article via Infotrieve]
    
15. Vasa M, Fichtlscherer S, Aicher A, et al. 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]
    
16. Murohara T, Ikeda H, Duan J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest. 2000; 105: 1527–1536.[Medline] [Order article via Infotrieve]
    
17. Kocher AA, Schuster MD, Szabolcs MJ, et al. 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]
    
18. Kawamoto A, Gwon HC, Iwaguro H, et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation. 2001; 103: 634–637.[Abstract/Free Full Text]
    
19. Arras M, Ito WD, Scholz D, et al. Monocyte activation in angiogenesis and collateral growth in the rabbit hindlimb. J Clin Invest. 1998; 101: 40–50.[Medline] [Order article via Infotrieve]
    
20. van Royen N, Hoefer I, Buschmann I, et al. Effects of local MCP-1 protein therapy on the development of the collateral circulation and atherosclerosis in Watanabe hyperlipidemic rabbits. Cardiovasc Res. 2003; 57: 178–185.[Abstract/Free Full Text]
    
21. Rehman J, Li J, Orschell CM, et al. Peripheral blood "endothelial progenitor cells" are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation. 2003; 107: 1164–1169.[Abstract/Free Full Text]
    
22. Assmus B, Urbich C, Aicher A, et al. HMG-CoA reductase inhibitors reduce senescence and increase proliferation of endothelial progenitor cells via regulation of cell cycle regulatory genes. Circ Res. 2003; 92: 1049–1055.[Abstract/Free Full Text]
    
23. Vasa M, Fichtlscherer S, Adler K, et al. Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation. 2001; 103: 2885–2890.[Abstract/Free Full Text]
    
24. Leibovich SJ, Polverini PJ, Shepard HM, et al. Macrophage-induced angiogenesis is mediated by tumour necrosis factor-alpha. Nature. 1987; 329: 630–632.[CrossRef][Medline] [Order article via Infotrieve]
    
25. Berse B, Brown LF, Van de Water L et al. Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors. Mol Biol Cell. 1992; 3: 211–220.[Abstract]
    
26. Polverini PJ, Cotran PS, Gimbrone MA Jr, et al. Activated macrophages induce vascular proliferation. Nature. 1977; 269: 804–806.[CrossRef][Medline] [Order article via Infotrieve]
    
27. Wahl SM, Hunt DA, Wakefield LM, et al. Transforming growth factor type beta induces monocyte chemotaxis and growth factor production. Proc Natl Acad Sci U S A. 1987; 84: 5788–5792.[Abstract/Free Full Text]
    
28. Iijima K, Yoshikawa N, Connolly DT, et al. Human mesangial cells and peripheral blood mononuclear cells produce vascular permeability factor. Kidney Int. 1993; 44: 959–966.[Medline] [Order article via Infotrieve]
    

This article has been cited by other articles:

				G Coppolino, S Campo, D Bolignano, A Sturiale, M S Giacobbe, S Loddo, and M Buemi Effect of immunoglobulin treatment on endothelial progenitor cells in systemic lupus erythematosus Ann Rheum Dis, July 1, 2008; 67(7): 1047 - 1048. [Full Text] [PDF]

				A. Zampetaki, J. P. Kirton, and Q. Xu Vascular repair by endothelial progenitor cells Cardiovasc Res, June 1, 2008; 78(3): 413 - 421. [Abstract] [Full Text] [PDF]

				J.-S. Silvestre, Z. Mallat, A. Tedgui, and B. I. Levy Post-ischaemic neovascularization and inflammation Cardiovasc Res, May 1, 2008; 78(2): 242 - 249. [Abstract] [Full Text] [PDF]

				E. Chavakis, G. Carmona, C. Urbich, S. Gottig, R. Henschler, J. M. Penninger, A. M. Zeiher, T. Chavakis, and S. Dimmeler Phosphatidylinositol-3-Kinase-{gamma} Is Integral to Homing Functions of Progenitor Cells Circ. Res., April 25, 2008; 102(8): 942 - 949. [Abstract] [Full Text] [PDF]

				A. D. Bhatwadekar, J. V. Glenn, G. Li, T. M. Curtis, T. A. Gardiner, and A. W. Stitt Advanced Glycation of Fibronectin Impairs Vascular Repair by Endothelial Progenitor Cells: Implications for Vasodegeneration in Diabetic Retinopathy Invest. Ophthalmol. Vis. Sci., March 1, 2008; 49(3): 1232 - 1241. [Abstract] [Full Text] [PDF]

				G. Carmona, E. Chavakis, U. Koehl, A. M. Zeiher, and S. Dimmeler Activation of Epac stimulates integrin-dependent homing of progenitor cells Blood, March 1, 2008; 111(5): 2640 - 2646. [Abstract] [Full Text] [PDF]

				E. Shantsila, T. Watson, H.-F. Tse, and G. Y.H. Lip New insights on endothelial progenitor cell subpopulations and their angiogenic properties. J. Am. Coll. Cardiol., February 12, 2008; 51(6): 669 - 671. [Full Text] [PDF]

				M. L. Balestrieri, C. Schiano, F. Felice, A. Casamassimi, A. Balestrieri, L. Milone, L. Servillo, and C. Napoli Effect of Low Doses of Red Wine and Pure Resveratrol on Circulating Endothelial Progenitor Cells J. Biochem., February 1, 2008; 143(2): 179 - 186. [Abstract] [Full Text] [PDF]

				A. Nareika, Y.-B. Im, B. A Game, E. H Slate, J. J Sanders, S. D London, M. F Lopes-Virella, and Y. Huang High glucose enhances lipopolysaccharide-stimulated CD14 expression in U937 mononuclear cells by increasing nuclear factor {kappa}B and AP-1 activities J. Endocrinol., January 1, 2008; 196(1): 45 - 55. [Abstract] [Full Text] [PDF]

				D. Tang, J. Lu, J. P. Walterscheid, H.-H. Chen, D. A. Engler, T. Sawamura, P.-Y. Chang, H. J. Safi, C.-Y. Yang, and C.-H. Chen Electronegative LDL circulating in smokers impairs endothelial progenitor cell differentiation by inhibiting Akt phosphorylation via LOX-1 J. Lipid Res., January 1, 2008; 49(1): 33 - 47. [Abstract] [Full Text] [PDF]

				G. Grenier, A. Scime, F. Le Grand, A. Asakura, C. Perez-Iratxeta, M. A. Andrade-Navarro, P. A. Labosky, and M. A. Rudnicki Resident Endothelial Precursors in Muscle, Adipose, and Dermis Contribute to Postnatal Vasculogenesis Stem Cells, December 1, 2007; 25(12): 3101 - 3110. [Abstract] [Full Text] [PDF]

				W. Schaper Prevention of Tissue Death by Killer Cells?: The Role of the Immune System in Arteriogenesis Arterioscler. Thromb. Vasc. Biol., November 1, 2007; 27(11): 2273 - 2274. [Full Text] [PDF]

				J. Tongers and D. W. Losordo Frontiers in Nephrology: The Evolving Therapeutic Applications of Endothelial Progenitor Cells J. Am. Soc. Nephrol., November 1, 2007; 18(11): 2843 - 2852. [Abstract] [Full Text] [PDF]

				M. F. Denny, S. Thacker, H. Mehta, E. C. Somers, T. Dodick, F. J. Barrat, W. J. McCune, and M. J. Kaplan Interferon-{alpha} promotes abnormal vasculogenesis in lupus: a potential pathway for premature atherosclerosis Blood, October 15, 2007; 110(8): 2907 - 2915. [Abstract] [Full Text] [PDF]

				M. C. Deregibus, V. Cantaluppi, R. Calogero, M. Lo Iacono, C. Tetta, L. Biancone, S. Bruno, B. Bussolati, and G. Camussi Endothelial progenitor cell derived microvesicles activate an angiogenic program in endothelial cells by a horizontal transfer of mRNA Blood, October 1, 2007; 110(7): 2440 - 2448. [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]

				P. E Westerweel, R. K M A C Luijten, I. E Hoefer, H. A Koomans, R. H W M Derksen, and M. C Verhaar Haematopoietic and endothelial progenitor cells are deficient in quiescent systemic lupus erythematosus Ann Rheum Dis, July 1, 2007; 66(7): 865 - 870. [Abstract] [Full Text] [PDF]

				C. K. Kissel, R. Lehmann, B. Assmus, A. Aicher, J. Honold, U. Fischer-Rasokat, C. Heeschen, I. Spyridopoulos, S. Dimmeler, and A. M. Zeiher Selective Functional Exhaustion of Hematopoietic Progenitor Cells in the Bone Marrow of Patients With Postinfarction Heart Failure J. Am. Coll. Cardiol., June 19, 2007; 49(24): 2341 - 2349. [Abstract] [Full Text] [PDF]

				H. F. Langer, K. Daub, G. Braun, T. Schonberger, A. E. May, M. Schaller, G. M. Stein, K. Stellos, A. Bueltmann, D. Siegel-Axel, et al. Platelets Recruit Human Dendritic Cells Via Mac-1/JAM-C Interaction and Modulate Dendritic Cell Function In Vitro Arterioscler. Thromb. Vasc. Biol., June 1, 2007; 27(6): 1463 - 1470. [Abstract] [Full Text] [PDF]

				H.-K. Oh, J.-M. Ha, E. O, B. H. Lee, S. K. Lee, B.-S. Shim, Y.-K. Hong, and Y. A. Joe Tumor Angiogenesis Promoted by Ex vivo Differentiated Endothelial Progenitor Cells Is Effectively Inhibited by an Angiogenesis Inhibitor, TK1-2 Cancer Res., May 15, 2007; 67(10): 4851 - 4859. [Abstract] [Full Text] [PDF]

				S. Zbinden, L. C. Clavijo, B. Kantor, H. Morsli, G. A. Cortes, J. A. Andrews, G. J. Jang, M. S. Burnett, and S. E. Epstein Interanimal variability in preexisting collaterals is a major factor determining outcome in experimental angiogenesis trials Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1891 - H1897. [Abstract] [Full Text] [PDF]

				P. E. Westerweel, I. E. Hoefer, P. J. Blankestijn, P. de Bree, D. Groeneveld, O. van Oostrom, B. Braam, H. A. Koomans, and M. C. Verhaar End-stage renal disease causes an imbalance between endothelial and smooth muscle progenitor cells Am J Physiol Renal Physiol, April 1, 2007; 292(4): F1132 - F1140. [Abstract] [Full Text] [PDF]

				E. Chavakis, A. Hain, M. Vinci, G. Carmona, M. E. Bianchi, P. Vajkoczy, A. M. Zeiher, T. Chavakis, and S. Dimmeler High-Mobility Group Box 1 Activates Integrin-Dependent Homing of Endothelial Progenitor Cells Circ. Res., February 2, 2007; 100(2): 204 - 212. [Abstract] [Full Text] [PDF]

				M. Sahara, M. Sata, T. Morita, K. Nakamura, Y. Hirata, and R. Nagai Diverse Contribution of Bone Marrow Derived Cells to Vascular Remodeling Associated With Pulmonary Arterial Hypertension and Arterial Neointimal Formation Circulation, January 30, 2007; 115(4): 509 - 517. [Abstract] [Full Text] [PDF]

				A. Aicher, C. Heeschen, K.-i. Sasaki, C. Urbich, A. M. Zeiher, and S. Dimmeler Low-Energy Shock Wave for Enhancing Recruitment of Endothelial Progenitor Cells: A New Modality to Increase Efficacy of Cell Therapy in Chronic Hind Limb Ischemia Circulation, December 19, 2006; 114(25): 2823 - 2830. [Abstract] [Full Text] [PDF]

K. H. Wu, Y. L. Liu, B. Zhou, and Z. C. Han Cellular therapy and myocardial tissue engineering: the role of adult stem and progenitor cells Eur. J. Cardiothorac. Surg., November 1, 2006; 30(5): 770 - 781.