Circulating endothelial precursors: mystery, reality, and promise

Abstract

Postnatal physiological and pathophysiological processes such as wound healing and tumor growth require endothelial migration, proliferation, and stabilization, a process referred to as angiogenesis. Initial phases of angiogenesis involve the recruitment of endothelial cells to the site of injury or to the tumor vascular bed. Two possible sources of endothelialization are (a) migration and co-option (1, 2) of preexisting vascular wall endothelial cells and (b) recruitment of endothelial precursor cells from the circulation (Figure ​(Figure1).1). There is ample evidence for the first scenario. However, the existence of angioblast-like circulating endothelial precursor cell (CEP) in adult humans has only recently been suggested, and their role in supporting postnatal angiogenesis is under intensive investigation. Figure 1 Models of postnatal angiogenesis and vasculogenesis. Recruitment of endothelial cells from preexisting vessel wall or CEPs plays a critical role in the regulation of postnatal angiogenesis and vasculogenesis. Mobilized BM-derived CEPs with high proliferative ... CEP may reflect the phenotype of embryonic angioblasts, which are migratory endothelial cells with the capacity to circulate, proliferate, and differentiate into mature endothelial cells, but which have neither acquired characteristic markers of mature endothelium nor formed lumina (3, 4). Although there is abundant evidence of the existence of angioblasts during embryonic development, the isolation and characterization of angioblast-like CEPs from adult circulation has been hampered by the absence of specific endothelial markers and functional assays to distinguish angioblast-like CEPs from sloughed mature endothelial and hematopoietic cells. Both putative CEPs with angioblastic potential and vessel wall–derived mature endothelial cells may express similar endothelial-specific markers, including vascular endothelial growth factor receptor-2 (VEGFR2, KDR, Flk-1) (5), Tie-2, Tie-1 (6, 7), VE-cadherin (8), CD34, and E-selectin (Table ​(Table1).1). Therefore, it may be impractical to use these markers to distinguish between the 2 populations. Discrimination between CEPs and mature endothelial cells is further complicated by the fact that subsets of hematopoietic cells express markers similar to those of endothelial cells such as CD34, PECAM (CD31), Tie-1, Tie-2, von Willebrand factor (vWF), and VEGFR2 (9). Furthermore, certain hematopoietic cells are also able to incorporate acetylated LDL (AcLDL) and to bind lectins such as BS-1 and ulex europaeus, which are usually considered endothelial specific. Table 1 Phenotypic analysis of putative endothelial precursor cells Several studies have attempted to identify CEPs by performing bone marrow (BM) transplantation studies in which recipient and donor cells could be distinguished by endothelial-specific markers. One study (10) has suggested that BM-derived VEGFR2+ endothelial progenitor cells could be detected in the peripheral circulation and contribute to vasculogenesis. However, although 27% of freshly isolated CD34+ cells were VEGFR2+ as determined by flow cytometry, the authors of this study were not able to detect VEGFR2 mRNA message in the first 7 days of culture using RT-PCR, perhaps because the isolated cells were not, in fact, of endothelial origin. This concern arises because of the use of an antibody that was specific to the intracellular, rather than the extracellular, domain of VEGFR2. In subsequent studies, populations of cells enriched for endothelial progenitor cells were derived from murine peripheral blood cells or BM and labeled with DiI to track the mobilization and recruitment of these cells into ischemic limbs (11, 12). However, given that subsets of hematopoietic cells have the capacity to express CD31, Tie-2, and incorporate AcLDL, it is not clear whether the mobilized cells and the cells incorporated into the vessel wall were of primarily hematopoietic or endothelial origin. We have shown that allogeneic sex-mismatched BM transplantation results in the transfer of endothelial cells to recipient dogs (13). Replacement of the aorta of the recipient dogs months after transplantation with impervious Dacron grafts resulted in graft endothelialization arising exclusively from the transplanted BM. In humans, evidence for CEPs originates from patients implanted with a left ventricular assist device (LVAD). We have demonstrated colonization of the flow surface of the titanium housing of LVADs with CD34+ endothelial-like cells 6 months after the devices were removed (14). These studies suggest that transplanted CEPs residing in the BM have the capacity to be mobilized to the peripheral circulation and incorporate in the sites of active angiogenesis. However, because transplantation of whole BM results in transfer of both mature BM endothelium and putative angioblast-like CEPs, none of these studies have conclusively demonstrated the exis