In their initial report of endothelial progenitor cells Asahara and colleagues showed that following systemic injection, endothelial progenitors incorporate into sites of vessel growth and repair in rodent hind limb ischemia models. Other investigators have since repeated this finding. Several experimental designs have been used to document this process, although each design has possible pitfalls. The supply of endothelial progenitors has varied between experiments. Both undifferentiated early progenitor cells and cells that have been differentiated in vitro to a more endothelial-like phenotype have been delivered systemically and directly into areas of vessel growth. Additionally, bone
Table I. Characteristics of Endothelial Progenitor Preparations. Hematopoietic cells of different types and from different sources have been shown to express endothelial characteristics when cultured with pro-angiogenic cytokines. Starting cell populations and sources are shown in addition to culture additives; vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor I (IGF-I), and epidermal growth factor (EGF). The expression of a set of endothelial and myeloid markers is indicated for each study. Endothelial-like cells derived from hematopoietic progenitors and monocytes are grouped separately.
Starting cell population
Cells with Progenitor Markers
Total human PB mononuclear cells
CD34+ human periperal blood
CD34+ human bone marrow, fetal liver, cord blood, peripheral blood
VEGF, bFGF, IGF-1, EGF, hydrocortisone, heparin, FBS
Bovine brain extract FBS VEGF, bFGF, IGF-1
CD133+ GCSF mobilized human FBS, VEGF, SCGF, peripheral blood
CD133+ human bone marrow followed by selection with UEA-1 binding CD34+/CD133+/VEGFR-2+ human peripheral blood, fetal liver, and cord blood hydrocortisone FBS, bFGF, heparin
FBS, bFGF, heparin
CD34+, CD31+, VE-Cad+, Flk-1+, Tie2+, UEA-i, vWF+, CDia-, CD14-
VE-Cad, vWF, P1H12, UEA-i, CD1G5, KDR, WP bodies, CD14-, CD45-, CD34-
AcLDL, VE-Cad, CD13, CD31
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Quirici et al. (2001). Br. J. Haematol. 115, 186-194
Peichev et al. (2000). Blood 95, 952-958
Unselected human peripheral blood mononuclear cells
CD14+ human peripheral blood monocytes
Human CD34+mobilized peripheral blood
Human CD34- peripheral blood monocytes
CD14+ human peripheral blood
FBS, insulin, VEGF, bFGF
FBS, bovine brain extract
VEGF, bFGF, EGF, IGF-1, hydrocortisone, heparin, FBS
AcLDL, UEA-1, CD45+, CD14+, CD11b+, CD11c+, CD31, low VE-Cad, low CD34
VWF, VE-Cad, ecNOS, CD68
VE-Cad, ecNOS, vWF
EcNOS, vWF, VE-Cad, Tie-2, MUC18, CD1G5, CD1a, CD45, acLDL
Ac-LDL, vWF, VE-Cad, CD1G5, CD36, Flt-1, Flk-1, CD1a-, CD83-, CD68, HLA-DR
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Nakul-Aquaronne et al. (2003).
Cardiovasc Res 57, 816-823
Harraz et al. (2001). Stem Cells 19, 304-312
Fernandez Pujol et al. (2000).
Differentiation 65, 287-300
marrow transplant studies have been performed to document the incorporation of bone marrow-derived cells into a growing endothelium. The methods of marking the endothelial progenitors have also differed and include dye labeling, green fluorescent protein (GFP) expression, use of transgenic donor animals expressing GFP or b-galactosidase, and identification of the Y chromosome in sex-mismatched donor/recipient pairs. Some criticisms of these studies have been the possibility of dye uptake by other native cells and the inherent difficulty in demonstrating that a cell is functionally incorporated into the vasculature as opposed to spatially located in the vasculature. Despite these criticisms, the ability of several groups to derive essentially the same conclusions using varied experimental methodologies make the data compelling.
Although the potential for circulating cells to incorporate into new vasculature appears to be established, there contin ues to be debate on the relative importance of angiogenesis versus vasculogenesis in the adult. An effort has been made to quantitate the relative amount of endothelium derived from circulating cells versus mature local endothelial cells using rodent bone marrow transplant models. Several groups have used this strategy and found wide ranges of donor versus recipient contribution to new vascular endothelium. In different experimental situations, the donor contribution ranges from 0 to 95 percent (see Table II). The comparison of these experiments is complicated by the use of different models of adult vascular formation ranging from tumors to limb ischemia and by other factors such as differences in the extent of bone marrow engraftment and accuracy with which transplanted cells are differentiated from host cells. Several studies in human transplant patients have also been published. Host contributions to endothelium in donor hearts ranging from 0 to -25 percent have been
Table II. Contribution of Bone Marrow-Derived Cells to Neovasculature in Mouse Transplant Models. The contribution of bone marrow-derived cells in various models of neovascular growth in the adult mouse has been determined using murine transplant models. The wide range of bone marrow-derived cells illustrates possible differences in the contributions of angiogenesis versus vasculogenesis with different experimental conditions.
Model of vascular growth
Percentage of bone marrow-derived cells
Regenerative lung growth
Ischemic cardiac injury
B6RV2 lymphoma xenograft
Glioma xenograft rare
Cells present at infarct border 0
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demonstrated, and recipient-derived endothelial cells have been reported in transplanted kidneys and livers. Transplanted bone marrow has also been found to contribute to endothelium in recipients. There are wide variations in the reported relative contribution of circulating cells to new vascular endothelium even among different patients in the same study. This variation in transplant patients as well as rodent bone marrow transplant models suggests that many factors influence the interplay between angiogenesis and vasculo-genesis and also illustrates the complexity and variability of these experiments.
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