Embryonic stem cell research has opened a novel door for vascular biology, as for any medical field, to elucidate the history of vascular development. Embryonic endothelial progenitor cells, so-called angioblasts, for blood vessel development arise from migrating mesodermal cells. EPCs have the capacity to proliferate, migrate, and differentiate into endothelial lineage cells, but have not yet acquired characteristic mature endothelial markers. Available evidence suggests that hematopoietic stem cells (HSCs) and EPCs (3,4) are derived from a common precursor (hemangioblast) (5-7). Growth and fusion of multiple blood islands in the yolk sac of the embryo ultimately give rise to the yolk sac capillary network (8); after the onset of blood circulation, this network differentiates into an arteriovenous vascular system (4). The integral relationship between the elements that circulate in the vascular system (the blood cells) and the cells principally responsible for the vessels themselves (the ECs) is implied by the composition of the embryonic blood islands. The cells destined to generate hemato-poietic cells are situated in the center of the blood island and are termed HSCs. EPCs are located at the periphery of the blood islands.
The key molecular players determining the fate of the hemangioblast are not fully elucidated. However, several factors have been identified that may play a role in this early event. Studies in quail/chick chimeras have shown that the fibroblast growth factor 2 (FGF-2) mediates the induction of EPCs from the mesoderm (9). These embryonic EPCs express flk-1, the receptor 2 for vascular endothelial growth factor (VEGFR-2), and respond to a pleio-tropic angiogenic factor, vascular endothelial growth factor (VEGF), for proliferation and migration. Deletion of the flk-1 gene in mice results in embryonic lethality because of a lack of both hematopoietic and endothelial lineage development, supporting the critical importance of flk-1 at that developmental stage, although not defining the steps regulating differentiation into endothelial vs hematopoietic cells.
Mesodermal cells expressing flk-1 have also been defined as an embryonic common vascular progenitor that differentiates into endothelial and SMCs (10). The vascular progenitors differentiated to ECs in response to VEGF, whereas they developed into SMCs in response to PDGF-BB. It remains to be determined whether embryonic EPCs or vascular progenitor
cells persist with an equivalent capability during adult life and whether these cells contribute to postnatal vessel growth (see below).
3. adult endothelial progenitor cells 3.1. Identification of Adult Endothelial Progenitor Cells
The identification of putative HSCs in peripheral blood and bone marrow and the demonstration of sustained hematopoietic reconstitution with these HSC transplants have constituted inferential evidence for HSCs in adult tissues (11-14). The related descendents (EPCs) have been isolated along with HSCs in hematopoietic organs. Flk-1 and CD34 antigens were used to detect putative EPCs from the mononuclear cell (MNC) fraction of peripheral blood (15). This is supported by the former findings that embryonic HSCs and EPCs share certain antigenic determinants, including Flk-1, Tie-2, c-Kit, Sca-1, CD133, and CD34. These progenitor cells have consequently been considered derived from a common precursor, putatively termed a hemangioblast (5-7) (Fig. 1).
In vitro, these cells differentiated into endothelial lineage cells and, in animal models of ischemia, heterologous, homologous, and autologous EPCs were shown to incorporate into sites of active neovascularization. This finding was followed by diverse identifications of EPCs using equivalent or different methodologies (16-20). EPCs were subsequently shown to express VE-cadherin, a junctional molecule, and AC133, an orphan receptor specifically expressed on EPCs, but with expression that is lost once they differentiate into more mature ECs (19). Their high proliferation rate distinguishes circulating marrow-derived EPCs in the adult from mature ECs shed from the vessel wall (18). Thus far, a bipotential common vascular progenitor, giving rise to both ECs and SMCs, has not been documented in the adult.
These findings have raised important questions regarding fundamental concepts of blood vessel growth and development in adult subjects. Does the differentiation of EPCs in situ (vasculogenesis) play an important role in adult neovascularization, and would impairments in this process lead to clinical diseases? There is now a strong body of evidence suggesting that vasculogenesis does in fact make a significant contribution to postnatal neovascularization. Recent studies with animal bone marrow transplantation models in which bone marrow (donor)-derived EPCs could be distinguished have shown that the contribution of EPCs to neovessel formation may range from 5 to 25% in response to granulation tissue formation (21) or growth factor-induced neovascularization (22).
3.2. Diverse Identifications of Human EPCs and Their Precursors
Since the initial report of EPCs (15), a number of groups have set out to define this cell population better. Because EPCs and HSCs share many surface markers and no simple definition of EPCs exists, various methods of EPC isolation have been reported (15-20,23-33) (Table 1). The term EPC may therefore encompass a group of cells that exist in a variety of stages, ranging from hemangioblasts to fully differentiated ECs. Although the true differentiation lineage of EPCs and their putative precursors remains to be determined, there is overwhelming evidence in vivo that a population of EPCs exists in humans.
Lin et al. (18) cultivated peripheral MNCs from patients receiving gender-mismatched bone marrow transplantation and studied their growth in vitro. This study identified a population of bone marrow (donor)-derived ECs with high proliferative potential (late outgrowth); these bone marrow cells likely represent EPCs. Gunsilius et al. (17) investigated a chronic myelogenous leukemia model and disclosed that bone marrow-derived EPCs
Methods of Endothelial Progenitor Cell Isolation
26 17 16 24
CB PB BM
MNCs Nonadherent CD34+ cells
BM, Hydroxyurea/ MNCs G-CSF-PB
CD133+ cells CD14+ cells
CD34+ cells Negative cells for CD3, CD7, CD19, CD34, CD45RA, CD56, and IgE MNCs CD34- cells CD133+ cells
CD45- and acid, glycophorin A- dexamethasone, ascorbate, VEGF on FN cells
On type I collagen, 1 mo (outgrowth) bFGF, VEGF, heparin on collagen, 2 wk
VEGF, bFGF, IGF-1, EGF, ascorbate on FN, 7-10 d BBE, VEGF, SCGF on FN, 10 d
Hydrocortisone, SCGF, VEGF on
FN, 2 wk VEGF, bFGF, EGF, IGF-1, hydrocortisone, heparin, ascorbate on FN, 1 wk BBE, heparin on FN, 7 d
VEGF, bFGF, IGF-1 on FN, 3 wk, then UEA-1 selection Insulin, transferrin, selenium, linoleic acLDL, vWF, CD144, KDR, CD36 vWF, CD144
acLDL, UEA-1, KDR, CD144, CD31
CD31, factor VIII, vWF, UEA-1, acLDL
CD31, CD144, KDR, Tie-2, UEA-1, vWF, Weibel-Palade body vWF, CD144, CD105, acLDL, CD36, flt-1, KDR, Weibel-Palade body
CD31, CD144, KDR, eNOS, vWF, acLDL Tie-2, acLDL vWF, CD144, eNOS
KDR, CD31, CD144 Tie-2, eNOS, CD144 vWF, CD105, KDR, CD31, CD144
Abbr: acLDL, acetylated low-density lipoprotein; BBE, bovine brain extract; BM, bone marrow; CB, cord blood; CM, conditioned media; eNOS, endothelial nitric oxide synthase; FN, fibronectin; G-CSF-PB, granulocyte colony-stimulating factor-mobilized PB; IgE, immunoglobulin E; MNCs, mononuclear cells; PB, peripheral blood; SCGF, stem cell growth factor.
contribute to postnatal neovascularization in humans. Multipotent adult progenitor cells (MAPCs) were isolated from bone marrow MNCs (34), differentiated into EPCs, and were proposed as the origin of EPCs (31). These studies therefore provided evidence to support the presence of bone marrow-derived EPCs that take part in neovascularization.
3.3. Kinetics of Endothelial Progenitor Cells in the Adult Body
Given the result of common antigenicity, bone marrow has been considered the origin of EPCs as HSCs in adult. The bone marrow transplantation experiments have demonstrated the incorporation of bone marrow-derived EPCs into foci of physiological and pathological neovascularization (35). Wild-type mice were lethally irradiated and transplanted with bone marrow harvested from transgenic mice, in which constitutive LacZ expression is regulated by an EC-specific promoter, Flk-1 or Tie-2. The tissues in growing tumor, healing wound, ischemic skeletal, and cardiac muscles and cornea micropocket surgery have shown localization of Flk-1 or Tie-2 expressing endothelial lineage cells derived from bone marrow in blood vessels and stroma around vasculatures. The similar incorporation was observed in physiological neovascularization in uterus endometrial formation following induced ovulation and estrogen administration (35).
Investigators have shown that wound trauma causes mobilization of hematopoietic cells, including pluripotent stem or progenitor cells in spleen, bone marrow, and peripheral blood. Consistent with EPC/HSC common ancestry, recent data from our laboratory have shown that mobilization of bone marrow-derived EPCs constitutes a natural response to tissue ischemia (36). The murine bone marrow transplantation model presented direct evidence of enhanced bone marrow-derived EPC incorporation into foci of corneal neovascularization following the development of hind limb ischemia. Light microscopic examination of corneas excised 6 d after micropocket injury and concurrent surgery to establish hind limb ischemia demonstrated a statistically significant increase in cells expressing P-galactosidase in the corneas of mice with an ischemic limb vs those without one (36). The finding indicates that circulating EPCs are mobilized endogenously in response to tissue ischemia, following which they may be incorporated into neovascular foci to promote tissue repair. This was confirmed by clinical findings of EPC mobilization in patients of coronary artery bypass grafting, burns (37), and acute myocardial infarction (38).
Having demonstrated the potential for endogenous mobilization of bone marrow-derived EPCs, we considered that iatrogenic expansion and mobilization of this putative EC precursor population might represent an effec-
tive means to augment the resident population of ECs competent to respond to administered angiogenic cytokines. Such a program might thereby address the issue of endothelial dysfunction or depletion, which may compromise strategies of therapeutic neovascularization in older, diabetic, or hypercho-lesterolemic animals and patients. Granulocyte macrophage colony-stimulating factor (GM-CSF), which stimulates hematopoietic progenitor cells, myeloid lineage cells, and nonhematopoietic cells, including bone marrow stromal cells and ECs, has been shown to exert a potent stimulatory effect on EPC kinetics (36). Such cytokine-induced EPC mobilization could enhance neovascularization of severely ischemic tissues as well as de novo corneal vascularization (36) (Fig. 2).
The mechanisms by which these EPCs are mobilized to the peripheral circulation are in their early definitions. Among other growth factors, VEGF is the most critical factor for vasculogenesis and angiogenesis (39-41). Recent data indicate that VEGF is an important factor for the kinetics of EPC as well. Our studies, performed first in mice (42) and subsequently in patients undergoing VEGF gene transfer for critical limb ischemia (43) and myocardial ischemia (44), established that a previously unappreciated mechanism by which VEGF contributes to neovascularization is via mobilization of bone marrow-derived EPCs. Similar EPC kinetics modulation has been observed in response to other hematopoietic stimulators, such as granulocyte colony-stimulating factor (G-CSF) (16), angiopoietin 1 (45) and stroma-derived factor 1 (SDF-1) (19).
This therapeutic strategy of EPC mobilization has been implicated not only by natural hematopoietic or angiogenic stimulants, but also by recombinant pharmaceuticals. Statins, the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, inhibit the activity of HMG-CoA reductase, which catalyzes the synthesis of mevalonate, a rate-limiting step in cholesterol biosynthesis. The statins rapidly activate Akt signaling in ECs, and this stimulates EC bioactivity in vitro and enhances angiogenesis in vivo (46). Our group and Dimmeler et al. demonstrated a novel function for HMG-CoA reductase inhibitors that contributes to postnatal neovascularization by augmented mobilization of bone marrow-derived EPCs through stimulation of the Akt signaling pathway (47-49). Regarding its pharmacological safety and the effectiveness on hypercholesterolemia, one of the risk factors for atherogenesis, the statin might be a potent medication against atherosclerotic vascular diseases.
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