1Stem Cell Translational Research, Kobe Institute of Biomedical Research and Innovation/RIKEN Center of Developmental Biology, Chuo-ku, Kobe 650-0047; and 2Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan
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ABSTRACT |
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cell transplantation; ischemia; neovascularization; stem cell
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STEM CELL BIOLOGY FOR REGENERATION |
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Whereas most cells in adult organs are composed of differentiated cells, which express a variety of specific phenotypic genes adapted to each organ's environment, quiescent stem or progenitor cells are maintained locally or in the systemic circulation and are activated by environmental stimuli for physiological and pathological tissue regeneration. In the past decade researchers have defined the stem or progenitor cells from various tissues, including bone marrow, peripheral blood, brain, liver, and reproductive organs, in both adult animals and humans (Fig. 1).
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EMBRYONIC EPCs |
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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 showed that fibroblast growth factor-2 (FGF-2) mediates the induction of EPCs from the mesoderm (48). These embryonic EPCs express Flk-1, the type 2 receptor for vascular endothelial growth factor (VEGFR-2), and respond to a pleiotropic angiogenic factor, VEGF, for proliferation and migration. Deletion of the Flk-1 gene in mice results in embryonic lethality, lacking both hematopoietic and endothelial lineage development, supporting the critical importance of Flk-1 at that developmental stage, although the steps regulating differentiation into endothelial vs. hematopoietic cells had not yet been defined at the time of those studies. The Flk-1-expressing mesodermal cell has also been defined as an embryonic common vascular progenitor that differentiates into endothelial and smooth muscle cells (69). The vascular progenitors differentiated to ECs in response to VEGF, whereas they developed into smooth muscle cells in response to platelet-derived growth factor (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.
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POSTNATAL EPCs |
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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 (BMT) 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 (10) or growth factor-induced neovascularization (45).
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IDENTIFICATION OF EPCs AND THEIR PRECURSORS |
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Lin et al. (39) cultivated peripheral MNCs from patients receiving gender-mismatched BMT and studied their growth in vitro. In that study, they 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. (23) investigated a chronic myelogenous leukemia model and disclosed that bone marrow-derived EPCs contribute to postnatal neovascularization in humans (23). Reyes et al. (50) recently isolated multipotent adult progenitor cells (MAPCs) from bone marrow MNCs, which differentiated into EPCs. These findings strongly proposed MAPCs as the origin of EPCs (49). These studies therefore provide evidence to support the presence of bone marrow-derived EPCs that take part in neovascularization (Fig. 2).
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EPC KINETICS FOR REGENERATION |
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Previous 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 constitute a natural response to tissue ischemia (26). The former murine BMT model presented the direct evidence of enhanced bone marrow-derived EPC incorporation into foci of corneal neovascularization after the development of hindlimb ischemia. Light microscopic examination of corneas excised 6 days after micropocket injury and concurrent surgery to establish hindlimb ischemia demonstrated a statistically significant increase in cells expressing -galactosidase in the corneas of mice with, versus those without, an ischemic limb (63). This finding indicates that circulating EPCs are mobilized endogenously in response to tissue ischemia, after which they may be incorporated into neovascular foci to promote tissue repair. This was confirmed by clinical findings of EPC mobilization in patients with coronary artery bypass grafting, burns (22), and acute myocardial infarction (60).
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 effective means to augment the resident population of ECs that is competent to respond to administered angiogenic cytokines. Such a program might thereby address the issue of endothelial dysfunction or depletion that may compromise strategies of therapeutic neovascularization in older, diabetic, and/or hypercholesterolemic animals and patients. Granulocyte macrophage colony-stimulating factor (GM-CSF), which stimulates hematopoietic progenitor cells and myeloid lineage cells as well as nonhematopoietic cells including bone marrow stromal cells and ECs, has been shown to exert a potent stimulatory effect on EPC kinetics: mobilization from bone marrow, incorporation into sites of neovascularization, and proliferation and differentiation in culture (63). Such cytokine-induced EPC mobilization could enhance neovascularization of severely ischemic tissues as well as de novo corneal vascularization (63).
The mechanisms whereby these EPCs are mobilized to the peripheral circulation are in the early stage of definition. Among other growth factors, VEGF is the most critical factor for vasculogenesis and angiogenesis (6, 15, 57). Recently collected data indicate that VEGF is an important factor for the mobilization of EPCs from bone marrow, as well. Our studies performed first in mice (2) and subsequently in patients undergoing VEGF gene transfer for critical limb ischemia (32) and myocardial ischemia (32) established that a previously unappreciated mechanism by which VEGF contributes to neovascularization is via mobilization of bone marrow-derived EPCs. The similar EPC kinetics modulation has been observed in response to other hematopoietic stimulators, such as granulocyte colony-stimulating factor (G-CSF) (20), angiopoietin-1 (24), and stroma-derived factor-1 (SDF-1) (47).
This therapeutic strategy of EPC mobilization has recently been implicated not only by natural hematopoietic or angiogenic stimulants but also by recombinant pharmaceuticals. The statins inhibit the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which catalyzes the synthesis of mevalonate, a rate-limiting step in cholesterol biosynthesis. The statins rapidly activate Akt signaling in ECs, thereby stimulating EC bioactivity in vitro and enhancing angiogenesis in vivo (37). Recently, we (41) and Dimmeler and colleagues (12, 67) 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. With regard to its pharmacological safety and 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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THERAPEUTIC POTENTIALS OF EPC TRANSPLANTATION |
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We therefore considered a novel strategy of EPC transplantation to provide a source of robust ECs that might supplement fully differentiated ECs thought to migrate and proliferate from preexisting blood vessels according to the classic paradigm of angiogenesis developed by Folkman (19). Our studies indicated that ex vivo cell therapy, consisting of culture-expanded EPC transplantation, successfully promotes neovascularization of ischemic tissues, even when administered as "sole therapy," i.e., in the absence of angiogenic growth factors. Such a "supply side" version of therapeutic neovascularization in which the substrate (ECs as EPCs) rather than the ligand comprises the therapeutic agent was first demonstrated in the hindlimb ischemia model of immunodeficient mouse, using donor cells from human volunteers (32). These findings provided novel evidence that exogenously administered EPCs augment naturally impaired neovascularization in an animal model of experimentally induced critical limb ischemia. Not only did heterologous cell transplantation improve neovascularization and blood flow recovery, but important biological consequences, notably limb necrosis and autoamputation, were reduced by 50% compared with mice receiving differentiated ECs or control mice receiving media in which harvested cells were expanded ex vivo before transplantation. A similar strategy applied in a model of myocardial ischemia in the nude rat demonstrated that transplanted human EPCs incorporated into rat myocardial neovascularization, differentiated into mature ECs in ischemic myocardium, enhanced neovascularization, preserved left ventricular (LV) function, and inhibited myocardial fibrosis (34).
Recently, Shatteman et al. (56) conducted local injection of freshly isolated human CD34+ MNCs into diabetic nude mice with hindlimb ischemia and showed an increase in the restoration of limb flow. Kocher et al. (28) attempted intravenous infusion of freshly isolated (not cultured) human CD34+ MNCs (EPC-enriched fraction) into nude rats with myocardial ischemia. This strategy resulted in preservation of LV function associated with inhibition of cardiomyocyte apoptosis. These experimental findings obtained using immunodeficient animals suggest that both cultured and freshly isolated human EPCs have therapeutic potential in peripheral and coronary artery diseases.
Induction of angiogenic diseases, such as diabetic retinopathy and malignant tumors, is a possible deleterious effect of EPC transplantation. Such harmful events should be carefully monitored in future clinical trials, although no adverse effects have ever been reported in previous basic and preclinical studies.
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IMPACT OF CLINICAL PHENOTYPE ON EPCs |
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Endogenous cytokine expression, however, is not the only factor contributing to impaired neovascularization. Older, diabetic, and hypercholesterolemic animals, like human subjects (7, 8, 13, 21, 30, 42, 62, 65), also exhibit evidence of age-related endothelial dysfunction. Although endothelial dysfunction does not necessarily preclude a favorable response to cytokine replacement therapy, indexes of limb perfusion fail to reach ultimate levels recorded in wild-type animals, reflecting limitations imposed by a less responsive EC substrate (9, 54, 55, 66).
It is then conceivable that unfavorable clinical situations (such as aging) might be associated with dysfunctional EPCs, defective vasculogenesis, and, thus, impaired neovascularization. Indeed, preliminary results from our laboratory indicated that replacement of native bone marrow (including its compartment of progenitor cells) of young mice with bone marrow transplanted from old animals leads to a marked reduction in neovascularization following corneal micropocket injury, compared with young mice transplanted with young bone marrow (53). These studies thus established evidence of an age-dependent impairment in vasculogenesis (as well as angiogenesis) and the origin of progenitor cells as a critical parameter influencing neovascularization. Moreover, analysis of clinical data in older patients at our institution disclosed a significant reduction in the number of circulating EPC both at baseline and after VEGF165 gene transfer (31); specifically, the number of circulating EPCs of younger patients with critical limb ischemia was five times more than the number in older individuals. Impaired EPC mobilization and/or activity in response to VEGF may thus contribute to the age-dependent defect in postnatal neovascularization. Recently, Tepper et al. (64) reported that proliferation and tube formation of EPCs was impaired in patients with type 2 diabetes compared with normal subjects.
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GENE THERAPY OF EPCs |
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Our recent findings provide the first evidence that exogenously administered, gene-modified EPCs augment naturally impaired neovascularization in an animal model of experimentally induced limb ischemia (29). Transplantation of heterologous EPCs transduced with adenovirus encoding VEGF not only improved neovascularization and blood flow recovery but also had meaningful biological consequences: limb necrosis and autoamputation were reduced by 63.7% compared with controls. The dose of EPCs used in the current in vivo experiments was subtherapeutic; i.e., this dose of EPCs was 30 times less than that required in previous experiments to improve the rate of limb salvage above that seen in untreated controls. Adenoviral VEGF gene transfer of EPC, however, accomplished a therapeutic effect, as evidenced by a functional outcome, despite a subtherapeutic dose of EPCs. Thus VEGF gene transfer of EPC constitutes one option to address the limited number of EPCs that can be isolated from peripheral blood prior to ex vivo expansion and subsequent autologous readministration.
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EPCs IN OTHER FIELDS |
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EPCs also have been investigated in the cerebrovascular field. Embolization of the middle cerebral artery in Tie-2/LacZ/BMT mice disclosed that the formation of new blood vessels in the adult brain after stroke involves vasculogenesis/EPCs (70). Similar data were reported by investigators using gender-mismatched wild-type mice transplanted with bone marrow from green fluorescent protein-transgenic mice (26). However, whether autologous EPC transplantation would augment cerebral revascularization has yet to be examined.
To date, the role of EPCs in tumor angiogenesis has been demonstrated by several groups. Davidoff et al. (11) showed that bone marrow-derived EPCs contribute to tumor neovasculature and that bone marrow cells transduced with an antiangiogenic gene can restrict tumor growth in mice. Lyden et al. (43) recently demonstrated the critical role of bone marrow-derived EPCs in tumor neovascularization. Id-mutant (Id1+/Id3/) mice are angiogenic defective and tumor-resistant double-mutant mice in which implanted tumors rapidly regress in association with poor development of tumor neovessels. BMT from wild-type mice, not from Id-mutant mice, restored the tumor neovascularization and growth in Id-mutant mice (43). These data demonstrate that EPCs are not only important but also critical to tumor neovascularization. Although it is not known whether local administration of exogenous EPCs may augment tumor neovascularization, this issue should be carefully considered for clinical application of EPC cell therapy to treat cardiovascular diseases.
In conclusion, EPCs isolated from adult species, which have characteristics similar to those of embryonic angioblasts, have the capacity to proliferate, migrate, and differentiate into endothelial lineage cells but have not yet acquired mature endothelial markers. EPCs are mobilized from bone marrow into the circulation and then home to the site of neovascularization in response to physiological and pathological stimuli, thereby contributing to postnatal neovascularization. Since animal experiments on EPC transplantation proved the therapeutic potential of the cell-based strategy, the application of EPCs for regenerative medicine has been watched with keen interest. The clinical impact of EPC regenerative properties will be evaluated in a phase I-II trial being started at our institution.
However, a number of issues remain to be addressed in this research field. Some of the future perspectives are as follows: 1) identification of a specific marker for EPC with which other lineage cells do not share; 2) evaluation of EPC transdifferentiation in vitro and in physiological, pathological, and iatrogenic regeneration of tissues and organs; 3) methodological optimization of EPC purification, expansion, gene transfer, and administration to improve the efficacy of EPC transplantation; and 4) comparison of the therapeutic impact between purified EPCs and total bone marrow MNCs.
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FOOTNOTES |
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