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. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: Curr Opin Organ Transplant. 2010 Feb;15(1):68–72. doi: 10.1097/MOT.0b013e32833454b5

Endothelial Colony Forming Cell role in neoangiogenesis and tissue repair

Paul J Critser 1,2, Mervin C Yoder 1,2,3
PMCID: PMC2880951  NIHMSID: NIHMS180966  PMID: 19898235

Abstract

Purpose of review

Patients suffering from vascular disease often have impaired angiogenic ability contributing to impaired tissue repair. One potential therapy is to deliver cells that can aid in angiogenesis. This review will discuss the ability of endothelial progenitor cells (EPC), which have been reported to contribute to neoangiogenesis in both physiological and pathological conditions, to contribute to neoangiogenesis in tissue repair.

Recent findings

In recent years, various reports have described conflicting roles for EPC in vessel formation. Currently there are three different assays for outgrowth of EPC all resulting in the isolation of different cell populations. This confusion is partially due to limited functional characterization of putative EPC populations. One population, ECFC, have been shown to possess all the characteristics of a true endothelial progenitor.

Summary

The review overviews the role of putative EPC populations in angiogenesis and tissue repair. While all EPC populations have been shown to play a role in angiogenesis, only ECFC have demonstrated the ability to form de novo blood vessels in vivo. Additionally ECFC have been shown to play a role in neovascularization in several pre-clinical rodent models suggesting the may be an excellent cell source for treatment of patients with diminished vascular function.

Keywords: endothelial progenitor cell, endothelial colony forming cell, angiogenesis, wound healing

Introduction

Circulating endothelial progenitor cells (EPCs) have been studied as a cell source that contributes to neovascularization via postnatal vasculogenesis1. EPCs are reported to naturally home and integrate into sites of physiological vessel formation in vivo and incorporate into the vasculature of tumors, ischemic skeletal and cardiac muscle, and ulcers2,3. Further, several authors have demonstrated a relationship between the frequency of circulating EPCs and cardiovascular disease risk4,5.

Putative endothelial progenitor cell populations

Asahara et al. first described EPCs based on surface antigen expression, morphology and ability to incorporate into vessels1. Additional reports by many investigators characterizing EPCs have focuses on cell morphology and surface antigen expression1,4,6-8. Often these reports have lacked detailed characterization of cellular function and lineage of origin resulting in the term EPC encompassing different cell populations including cells of myeloid or endothelial origin9,10. Not surprisingly these putative EPC populations have demonstrated a mixed ability to contribute to the formation of blood vessels9-12. Currently, three methods for isolation and identification of putative EPCs from human mononuclear cells (MNCs) are in use13.

CFU-Hill

The first method, originally described by Asahara et al.1, has been modified4,7 in an attempt to remove contaminating mature endothelial cells and can now be performed using a commercially available kit (Endocult, StemCell Technologies). Low density MNCs plated on fibronectin coated tissue culture surfaces form adherent colonies after 5 to 9 days. These colonies are referred to as colony forming unit-Hill (CFU-Hill). CFU-Hill cells have been shown to express the cell surface antigens CD31, CD105, CD144, CD146, vWF, and KDR (VEGF-R2) which are certainly consistent with an endothelial cell phenotype, though not at all specific for this putative progenitor. CFU-Hill cells also display the ability to ingest acetylated low density lipoprotein (AcLDL), which is a behavior commonly ascribed to endothelial cells, but also to macrophages. CFU-Hill cells exhibit a low level of proliferative potential, express several monocytes/macrophage markers including CD14, CD45, and CD115, ingest bacteria, and display non-specific esterase activity1,9,10,14. In addition these colonies appear to be comprised of hematopoietic progenitor cells and T lymphocytes10,15 and several studies have shown that these cells arise from hematopoietic origins using several different human stem cell clonal blood disorders10,16. Thus, while CFU-Hill cells are involved in stimulation and regulation of angiogenesis11,17,18, the current evidence suggests that they are hematopoietic cells which may never become long-term intimal endothelial cells in vivo10,19.

Circulating angiogenic cells

Another method to identify putative EPC, involves the culture of peripheral blood MNCs on a fibronectin coated surface under “endothelial” differentiation conditions for four days using specific tissue culture medium and growth factors. Subsequently, non adherent cells are washed away and adherent cells with angiogenic potential (circulating angiogenic cells: CAC) remain6,14. CACs have been shown to express the endothelial cell surface antigens CD31, CD144, vWF, and KDR, bind Ulex Europaeus lectin6,14,20, and have been shown to uptake AcLDL6. CACs have also been shown to be enriched for monocyte/macrophages using a variety of cell surface antigens and functional assays9,12,14 and, as with the CFU-Hill cells, these cells never display all of the properties of an EPC19. In addition, recent evidence indicates that this particular method of CAC isolation and culture is complicated by the presence of numerous platelets that co-fractionate with the MNCs21. The contaminating platelets become attached to any adherent MNCs in the culture and platelet membrane proteins are transferred to the adherent cells (that do not express the mRNA for the various detectable surface proteins). Many of these platelet-derived surface antigens are those also expressed by endothelial cells (CD31, Ulex Europaeus lectin binding, and various integrins) and convey angiogenic properties to adherent MNCs in the culture, thus, erroneously qualifying these adherent cells as EPC. Thus, this method of putative EPC isolation can no longer be considered reliable (unless one can prove that no contaminating platelets or platelet proteins are present).

Endothelial colony forming cells

A third method of culture yields isolation and identification of endothelial colony forming cells (ECFCs)22, also termed blood outgrowth endothelial cells (BOECs)11,23. Umbilical cord blood derived circulating MNCs plated on a collagen I coated surface form adherent colonies with a cobblestone morphology that appear between day 7 and 1422. ECFCs express the cell surface antigens CD31, CD105, CD144, CD146, vWF, and KDR and uptake AcLDL10,22. ECFCs do not express the hematopoietic or monocytes/macrophage cell surface antigens CD14, CD45, or CD11510. Whether isolated from cord or adult peripheral blood, ECFC display clonal proliferative potential and relatively high levels of telomerase22.

EPC role angiogenesis

To further differentiate the putative EPC populations it is necessary to evaluate their functional capabilities. An endothelial progenitor cell should be able to give rise to endothelial progeny in a clonal fashion, form endothelial tubes with lumens in vitro, and form de novo vessels in vivo19. Ingram et al. developed a single cell assay to interrogate the clonogenic and proliferative potential of EPCs. While ECFCs exhibited a hierarchy of progenitor cells which vary in their proliferative potential and can be replated to form secondary colonies22, CFU-Hill cells do not form secondary colonies when replated10. CFU-Hill cells and ECFCs exhibit different tube formation potential when plated on Matrigel. While CFU-Hill cells incorporate into HUVEC formed tubes11, they have not demonstrated an ability to form tubes when plated alone11. However, ECFC form tubes when plated alone10 and integrate into HUVEC formed tubes on Matrigel11.

The Matrigel tube formation assay has often been used to test cell populations for the ability to function as endothelial cells. However this assay is unable to serve as a specific discriminator of endothelial cell behavior because Matrigel induces cord formation from several non-endothelial cells including fibroblasts24, baby murine kidney cells25, aortic smooth muscle cells, murine leydig cells26, and CD14+ monocytes seeded in Matrigel27. Further, the cord structures formed in Matrigel do not usually contain lumens suggesting a different mechanism of tube formation than angiogenesis or vasculogenesis24.

EPC contribution to neoangiogenesis

CACs, CFU-Hill cells, and ECFCs have been shown to contribute to neovascularization in a hind limb ischemia nude mouse model14,5. CACs increased blood vessel perfusion and capillary density in an athymic nude mouse hind limb ischemia model14. Histological examination revealed that human CACs homed to the area of new vessel formation and the authors claimed that the CACs were incorporated into “neovascular foci”14. Further, Hur et al. demonstrated improved perfusion and capillary density by either CFU-Hill cells or ECFCs in the hind limb ischemia nude mouse model compared with injection of a mature endothelial cells or media alone. While no significant difference in perfusion and capillary density was found between CFU-Hill cell and ECFC populations in this model11, Yoon et al. showed that injection of CFU-Hill cells and ECFCs together into a hind limb ischemia nude mouse model resulted in synergistic neovascularization compared to injection of either cell type alone18.

EPC ability to form de novo functional blood vessels

Human peripheral blood and umbilical cord blood ECFCs, but not CFU-Hill cells, form de novo functional blood vessels when seeded into a collagen fibronectin matrix and implanted in vivo10. This assay implants ECFCs, which have been seeded into a rat tail collagen fibronectin matrix in vitro, and then briefly cultured at 37 °C to permit polymerization, subcutaneously into the flank of an immunocompromised mouse, as described by Schechner et al.28. After two to four weeks cell seeded matrices are harvested and then investigated for the presence of proteins indicative of human blood vessel formation. ECFC seeded matrices contained functional human vessels determined by the presence of mouse anti human CD31 labeled endothelial lumens filled with circulating mouse red blood cells (Fig 1). However, CFU-Hill cells failed to demonstrate the ability to form functional vessels, though the cells did survive in the implanted matrix. This demonstrates that CFU-Hill cells and CACs are of hematopoietic origin and are unable to form de novo vessels in vivo. However, these cell populations may still contribute to neoangiogenesis and in the regulation of normal or abnormal angiogenesis17.

Figure 1. ECFCs but not CFU-Hill display the ability to form functional vessels in vivo.

Figure 1

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(A) Photomicrographs (original magnification, 20X) showing collagen fibronectin grafts with surrounding murine tissue after 28 days in NOD/SCID mice. Left and middle panels show consecutive sections of ECFC grafts stained with anti-murine CD31 (mCD31) and anti-human CD31 (hCD31). Right panel shows CFU-Hill seeded graft with anti hCD31 staining. (B) Photomicrographs (original magnification, 100X) showing hCD31 stained ECFC and CFU-Hill (far right) grafts after 28 days in vivo. ECFC for hCD31 positive vessels that contain murine RBCs (arrows) indicating that these vessels had inosculated with the host vasculature. CFU-Hill were unable to form functional vessels. Adapted from Yoder et al.10.

Additional investigators have also demonstrated the vasculogenic potential of ECFCs29-32. Melero-Martin et al.29 demonstrated that ECFCs seeded in Matrigel formed de novo blood vessels when implanted into mice. The authors noted that their vasculogenic potential decreased with passage number suggesting that the cells were becoming differentiated. The decreased vasculogenic potential could be overcome by increasing the seeding density29. Additionally, Au et al.30 used a mouse cranial window model to test the vasculogenic ability of adult peripheral blood and umbilical cord blood derived ECFCs. Adult ECFCs formed unstable vessels that regressed in several weeks when implanted alone or with 10T1/2 mouse embryonic fibroblast cells. Umbilical cord blood derived ECFC transiently formed vessels when implanted alone, but were stable for four months when co implanted with 10T1/2 cells30.

Human mesenchymal stem and progenitor cells can stabilize human endothelial derived vessels in vivo. Mesenchymal progenitor cells (MPCs) from adult peripheral blood or umbilical cord blood were shown to aid in ECFC in vivo vessel formation in a matrigel matrix32. MPC served as a perivascular cell surrounding ECFC derived vessels32. Bone marrow derived human mesenchymal stem cells (MSCs) also demonstrated the ability to stabilize HUVEC derived in vivo vessels in a collagen firbronectin matrix31. MSC derived perivascular cells stabilized vessels were shown to persist for greater than 130 days. Further, the HUVEC-MSC composite vessels were shown to constrict in response to the vasoconstrictive agent endothelin-131. Other MSC-derived populations such as adipose stromal cells (ASC) also provide perivascular support that promotes in vivo vessel formation upon implantation of cord blood ECFC with ASC in immunodeficient mice33.These studies suggest that MSCs could be used to improve the efficacy of potential ECFC based therapies.

EPC contribution to tissue repair

The ability of ECFCs to participate in neoangiogenesis gives them potential for treatment of impaired wound healing in patents with diminished angiogenic capabilities.

Kung et al. seeded acellular human cadaveric skin with keratinocytes and adult peripheral blood ECFCs. After in vitro culture the human skin substitute was transplanted onto immunocompromised mice, and within two weeks had formed functional human endothelial cell vessels which anastomosed with the host circulation34. Similarly, Shepherd et al.35 seeded tissue engineered human skin substitutes with keratinocytes and either umbilical cord blood derived ECFCs, adult peripheral blood derived ECFCs or HUVECs, transplanted them onto immunocompromised mice, and demonstrated the formation of human endothelial cell vessels within the skin substitute. Skin substitutes seeded with umbilical cord blood derived ECFCs exhibited a greater human vessel density than either adult blood derived ECFCs or HUVECs. Host cells also contributed to the vascularization of the implanted skin substitute. While the host angiogenic response could be diminished by the use of rapamycin, the extent of human derived vessels in the skin substitute was not affected. The authors state that the ability of implanted endothelial cells to form a vascular network when the host’s angiogenic response is inhibited suggests this strategy could be useful in treating patients with impaired wound healing35. These and other reports suggest that ECFCs represent an excellent cell source for vascular engineering strategies30.

Conclusion

Currently the term EPC encompasses several different cell populations with each population playing different roles in neoangiogenesis. The reliance on cell surface marker and morphology in the absence of functional tests to characterize EPCs has greatly contributed to this confusion. While all of the EPC populations have been shown to contribute in the promotion or regulation of angiogenesis, only ECFC display all the characteristic of an endothelial progenitor19. While there have been limited studies directly interrogating the potential of ECFCs in tissue repair and regeneration, ECFCs have been shown to contribute to vessel formation when implanted subcutaneously in a collagen matrix10,30 and other matrices29, in models of ischemia11,18, and in human skin substitutes. While there are no reports of the use of ECFCs in human clinical trials, the excellent results with pre-clinical rodent studies provides some hope for patients who suffer from impaired vascular function.

Acknowledgments

This work was supported by the Riley Children’s Foundation, Indianapolis, Indiana and the National Institues of Heath Grant F30-HL096350-01.

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