SEARCH

SEARCH BY CITATION

Keywords:

  • Stem/progenitor cell;
  • Cell therapy;
  • Ischemia;
  • Angiogenesis;
  • Regeneration

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IDENTIFICATION OF EPCs
  5. ROLE OF EPCs IN POSTNATAL NEOVASCULARIZATION
  6. EPC-BASED THERAPEUTIC ANGIOGENESIS
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES

Endothelial progenitor cells (EPCs) have been isolated and shown to be effective in animal models of ischemia, and many groups involved in clinical trials have demonstrated that EPC therapy is safe and feasible for the treatment of critical limb ischemia and cardiovascular diseases. However, many issues in the field of EPC biology, especially in regards to the proper and unambiguous molecular characterization of these cells still remain unresolved, hampering not only basic research but also the effective therapeutic use and widespread application of these cells. In this review, we introduce the recent concept of EPC identification in terms of hematopoietic and nonhematopoietic EPCs along with the development of EPC biology research. Furthermore, we define the role of circulating EPCs in postnatal neovascularization to illustrate the future direction of EPC therapeutic applications. Next, we review on-going medical applications of EPC for cardiovascular and peripheral vascular diseases, introduce the practical example of therapeutic application of EPCs to patients with ischemic disease, and discuss about the feedback of clinical researches. STEM CELLS 2011;29:1650–1655


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IDENTIFICATION OF EPCs
  5. ROLE OF EPCs IN POSTNATAL NEOVASCULARIZATION
  6. EPC-BASED THERAPEUTIC ANGIOGENESIS
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES

Endothelial progenitor cells (EPCs) were first isolated from adult peripheral blood (PB) in 1997 [1] and were shown to derive from bone marrow (BM) and to incorporate into foci of physiological or pathological neovascularization [2, 3]. Historically, a postnatal neovascularization was originally recognized to be constituted by the mechanism of “angiogenesis,” operated by in situ proliferation and migration of pre-existing endothelial cells (ECs) [4]. The finding that EPCs can home to sites of neovascularization and differentiate into ECs in situ is consistent with “vasculogenesis,” a critical paradigm not only well described for embryonic neovascularization [5] but also proposed recently for the adult organism in which a reservoir of progenitor cells contributes to postnatal neovascular formation [6]. The discovery of EPCs has therefore drastically changed our understanding of adult blood vessel formation. Furthermore, we and other groups notice the valuable capability to translate potential of EPC to improve the clinical applicability in the fight against cardiovascular diseases.

The following review will highlight the potential value of EPCs for therapeutic vasculogenesis in ischemic diseases, focusing particularly on “circulating EPCs” in terms of regenerative biology and medicine.

IDENTIFICATION OF EPCs

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IDENTIFICATION OF EPCs
  5. ROLE OF EPCs IN POSTNATAL NEOVASCULARIZATION
  6. EPC-BASED THERAPEUTIC ANGIOGENESIS
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES

Circulating EPCs can be subdivided into two main categories, hematopoietic lineage EPCs and nonhematopoietic lineage EPCs (Fig. 1). The hematopoietic EPCs originate from BM and represent a provasculogenic subpopulation of hematopoietic stem cells (HSCs) [7–9]. The hematopoietic EPCs can enter circulation on stimulation as cellular components of blood, compromising a possibly heterogeneous cell population, represented by, for example, colony forming EPCs, non-colony forming “differentiating” EPCs, myeloid EPCs, or angiogenic cells. The nonhematopoietic EPCs are not HSC-derived cells, which can be isolated from blood or tissue samples via the help of successive culture and distinguished by their rather obvious EC (-like) phenotype [10] or differentiation capability into EC (-like) phenotype [11]. The origin of nonhematopoietic EPCs remains to be clarified, but they are generally thought to be derived from nonhematopoietic tissue-prone lineage stem cells or organ blood vessels but not likely from HSC [12].

thumbnail image

Figure 1. Putative EPC kinetics. Abbreviations: BM, bone marrow; EC, endothelial cell; EOCs, endothelial outgrowth cells; EPCs, endothelial progenitor cells; HSC, hematopoietic stem cell.

Download figure to PowerPoint

Hematopoietic EPCS

Taking embryological and hematological experimental evidence suggests that HSCs and angioblasts (EPCs) are derived from a common precursor (a putative hemangioblast or hemogenic EC) [5, 8, 13], the origin of circulating EPCs can very well be speculated to be related to HSCs. In this regard, the development of characterization and identification of EPCs are tightly linked to and associated with the methods and markers already applied in the hematopoietic field. EPCs and HSCs can both be isolated using antibodies against various cell surface markers, including membrane receptors like CD34, CD133, Flk-1/KDR, CXCR4, CD105 (Endoglin) for human samples and receptors like c-Kit, Sca-1, and CD34 in combination with Flk-1 (vascular endothelial growth factor receptor (VEGFR2) in case of mouse samples (referring to well-established review [14]). Nevertheless, the identification of a unique combination of receptors specific and selective for primary EPCs, enabling an unambiguous distinction between EPCs and HSCs is still missing. The introduction of a definitive assay system capable of clearly distinguishing between EPCs and HSCs, thus enabling the identification of the long sought precise primary EPC phenotype, is highly anticipated.

A novel EPC colony forming assay (EPC-CFA) system [15–18] has been developed recently to improve the above-mentioned limitations of existing assays. This is reconsidering and uniting several conceptual opinions about EPCs and enabling a differential hierarchic view on circulating EPCs (Fig. 1). The EPC-CFA of progenitor-enriched populations, such as c-Kit+/Sca-1+/lineage negative cells in mouse [16–18] and CD34+ or CD133+ cells in human [15], identify two kinds of cell colony type each derived from a single cell, small EPC colony and large EPC colony, respectively. Because of the observed in vitro and in vivo characteristics of colony types, small EPCs are classed to represent “primitive EPCs,” a highly immature and proliferative population of cells, compared to large EPCs which are to represent “definitive EPCs,” cells prone to differentiate and promote vasculogenesis.

In contrast to “colony forming EPCs,” “non-colony forming EPCs” such as adhesive endothelial lineage (-like) cells derived from PB or BM mononuclear cells with endothelial growth factor supplemented media are deduced to represent further differentiating EPC phenotypes [15, 19-21]. These overall reproducible and standardized assay systems were used for the characterization of a wide range of EPCs, ranging from “cultured EPCs” [19, 21-24], “EC-like cells” [14], “early EPCs” [24–26] to so-called “circulating angiogenic cells” [27, 28], which generally do not form colonies under conventional endothelial differentiation conditions. These EPCs are potentially vasculogenic to create new blood vessels by themselves and/or angiogenic to support new blood vessel formations.

Several groups described endothelial contribution of hematopoietic cells, noting specifically that myelo-monocytic cells are capable of vascular integration [29–31]. Myeloid lineage cells may therefore function as differentiating EPCs, giving rise to EC-like cells in vitro and possibly even in vivo, contributing to neovascularization in ischemia or tissue damage.

The advantages of these hematopoietic EPCs are convenience for clinical application in terms of a medical regulatory feasibility of sampling from blood cells by antibody targeting isolation and potent effectiveness through vasculogenic and angiogenic mechanisms by primary cells.

Nonhematopoietic EPCs

The main member of this group of EPCs, which shall be discussed here very briefly, is the so-called endothelial outgrowth cell (EOC). EOCs are the products of an endothelial colony formation assay system developed and reported by Yoder's group and others [32, 33].

This carefully conceived culture assay system contributes significantly to the development of EPC biology via the introduction of a differential hierarchy system for adhesive EPCs. EOC is a more distinct endothelial lineage phonotype than hematopoietic EPC and easily forms tube-like structures in culture. As the primary origin and character of EOCs is still under debate, these cells cannot be placed into the existing hematopoietic category, albeit still incomplete map of EPC biology, as likely derivatives of organ blood vessel or nonhematopoietic BM cells (Fig. 1). EOC is also a candidate for cell therapy for neovascularization [34, 35].

The other source of nonhematopoietic EPC was found by Aicher et al. [11]. They have investigated the contribution of circulating cells from BM and non-BM sources to the vasculature by using a parabiosis model with or without reverse BM transplantation and disclosed the evidence for the mobilization of tissue resident c-Kit+/CD45 progenitor cells, such as from liver and small intestine. Although the experimental condition is not patho-physiologically compatible with natural bodies, this indicates that non-BM-derived EPCs populate on some level such as 10%.

Recently, another member of nonhematopoietic cells for EPCs has been described. Wojakowski et al. identified BM-derived cells that expressed a number of ESC-specific transcripts and which were mobilized into PB in patients following acute myocardial infarction (AMI). These cells, termed very small embryonic-like stem cells based on their size (7–8 μm), may potentially represent a cell type for use to enhance neovascularization by functioning as EPC in ischemic diseases [36, 37].

These nonhematopoietic EPCs are all vasculogenic and angiogenic for new blood vessel formations but not yet practiced clinically meantime by cumbersome medical regulations to be developed ex vivo. The establishment of medical grade culture methodology and practical culture efficiency are eagerly anticipated.

ROLE OF EPCs IN POSTNATAL NEOVASCULARIZATION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IDENTIFICATION OF EPCs
  5. ROLE OF EPCs IN POSTNATAL NEOVASCULARIZATION
  6. EPC-BASED THERAPEUTIC ANGIOGENESIS
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES

Direct EPC Contribution to Neovascularization

In the context of EPC biology, vasculogenesis covers the de novo formation of blood vessel via in situ migration, proliferation, differentiation, and/or incorporation of BM-derived EPCs into regenerating vasculature [3]. The incorporation of BM-derived EPCs into foci of physiological and pathological neovascularization has been demonstrated in various animal models, albeit remaining a still controversial topic in the field of EPC biology with several contradicting reports being published so far. Nevertheless, one well-established model, allowing the detection of BM-derived EPCs, uses transplantation of BM cells from transgenic mice in which LacZ is expressed under the regulation of an EC lineage-specific promoter, such as Flk-1 or Tie-2 (Flk-1/LacZ/BMT, Tie-2/LacZ/BMT) into wild-type control mice, followed by their use as a base for several different ischemic injury models. Using such a model, it has been shown that BM-derived Flk-1- and/or Tie-2-expressing endothelial lineage cells can localize to vascular structures during tumor growth [3, 38], wound healing [39], skeletal [3] and cardiac ischemia [40, 41], corneal neovascularization [42], and endometrial remodeling following hormone-induced ovulation [3, 38]. Regardless of the origin of EPCs, they undoubtedly play a significant role contributing to neovascularization via vasculogenesis in ischemic tissues.

Indirect EPC Contribution to Neovascularization

Albeit the well-established model of EPC action during neovascularization, that is the “direct participation/integration into the forming neovasculature of ischemic organs via vasculogenesis,” EPCs migrating to distressed tissues and organs urgently requiring vascular regeneration do not always participate in the formation of the neovasculature but rather “stay out” residing in the interstitial tissue [43, 44]. These tissue-bound “resting EPCs” produce a variety of proangiogenic cytokines and growth factors, promoting proliferation and migration of pre-existing ECs, activating angiogenesis, and contributing “indirectly” to vascular regeneration and the re-establishment of tissue homeostasis. EPCs, thus do not only work via the activation and support of vasculogenesis but may also be major players involved in the activation and mediation of angiogenesis [4], the process of new vessel formation, via in situ proliferation and migration of pre-existing ECs.

In fact, regardful observations of the preclinical studies remind us the evidence of enhanced intrinsic recipient angiogenesis by extrinsic factors derived from transplanted EPCs in myocardial ischemia models [43, 45, 46].

This paracrine aspect of EPC activity reflecting its indirect contribution to neovascularization was confirmed by several reports, demonstrating the presence of various cytokines and other secreting proangiogenic factors in EPCs such as VEGF, hepatic growth factor (HGF), Ang-1, stroma derived factor (SDF)-1α, insulin-like growth factor (IGF)-1, and endothelial nitric oxide synthase (eNOS)/inducible nitric oxide synthase (iNOS) [40, 47, 48].

Therefore, EPCs can mediate tissue-protective effects and contribute to neovascularization in ischemic tissues via production of “indirect working supportive” factors.

EPC-BASED THERAPEUTIC ANGIOGENESIS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IDENTIFICATION OF EPCs
  5. ROLE OF EPCs IN POSTNATAL NEOVASCULARIZATION
  6. EPC-BASED THERAPEUTIC ANGIOGENESIS
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES

Since EPCs were first described more than a decade ago, we and other groups focused especially on the regenerative potential of these progenitor cells trying to unravel and understand their unique properties and characteristics with the ultimate goal to translate this knowledge and to improve the clinical applicability/efficacy of these cells in the fight against cardiovascular diseases [6, 45, 49, 50]. The transplantation of blood/BM-derived vasculogenic progenitor cells, of EPCs, opened unprecedented opportunities for the treatment of ischemic diseases, which thus far was bound to and limited by the classic paradigm of angiogenesis.

Although there have been a lot of therapeutic approaches using EPC biology, including total mononuclear cell transplantations and mobilization therapies using cytokines, we focus to describe, in the context of this review, only cellular therapies using enriched EPC populations.

EPC Transplantation in Animal Models

It was shown that therapeutic approaches using culture-expanded EPCs could successfully promote neovascularization and regeneration of ischemic tissues, even when administered as “sole therapy,” that is, in the absence of other supportive pro-angiogenic growth factors. Such a “supply-side” version of therapeutic neovascularization was first reported for the i.v. transplantation of human PB-derived cultured EPCs into immunodeficient mice with hind limb ischemia [22]. These experimental findings proved that exogenously administered EPCs could restore impaired neovascularization in a murine ischemic hind limb model. A similar study in which human culture-expanded EPCs were transplanted in a nude rat myocardial ischemia model demonstrated that transplanted EPCs recruited to ischemic myocardium and were able to differentiate into ECs in sites of neovascularization. These findings were consistent with the observed preservation of left ventricular (LV) function and a reduction in infraction size [51, 52]. Another study in which human cord blood-derived EPCs were transplanted in a nude rat hind limb ischemia model also demonstrated similar findings with enhanced neovascularization in ischemic tissues [23].

Recently, several groups have explored the therapeutic potential of CD34+ cells as a possible EPC-enriched fraction. As described above, a clear distinction between HSCs and EPCs with the currently available methodology in many cases is not possible; nonetheless is the use of the “hematopoietic” cell surface marker CD34, for the isolation/enrichment of EPCs a widely used approach in the field of EPC biology. Schatteman et al. [53] transplanted freshly isolated human CD34+ cells into diabetic nude mice with hind limb ischemia and showed significant blood flow recovery in ischemic limbs. Kocher et al. [49] infused freshly isolated human CD34+ cells into a nude rat model of myocardial ischemia and observed preservation of LV function and inhibition of cardiac apoptosis. Dose-dependent contribution of CD34+ cells to LV functional recovery and neovascularization in ischemic myocardium has also been demonstrated by Iwasaki et al. [54].

EPC Transplantation in Clinical Trials

Numerous clinical trials are now on going and trying to elucidate the therapeutic effects of EPCs seen in animal models on ischemic diseases, using cell populations which are all believed to consist of or enriched by EPCs [55] (Table 1; Fig. 2).

thumbnail image

Figure 2. Development of EPC therapy. Abbreviations: BM, bone marrow; EPCs, endothelial progenitor cells; GCSF, granulocyte colony stimulating factor; MNCs, mononuclear cells; PB, peripheral blood.

Download figure to PowerPoint

Table 1. Clinical trials for ischemic diseases with EPCs
inline image

One example for such a clinical study using EPCs is our reported phase I/II clinical trial regarding the intramuscular transplantation of autologous and G-colony stimulating factor (CSF)-mobilized CD34+ cells in patients with intractable critical limb ischemia (CLI) [63]. The first-in-man trial was conducted as a prospective, multicenter, single-blind, and dose-escalation study since 2002 in our institute. G-CSF was used to efficiently mobilize BM-EPCs into the PB, and the mobilized CD34+ cells were isolated as an EPC-enriched fraction. In all subjects, primary endpoint of efficacy score at week 12 was positive indicating improvement of lower limb ischemia after cell therapy. In addition, both subjective and objective parameters of lower limb ischemia such as toe brachial pressure index, transcutaneous partial oxygen pressure, total walking distance, pain-free walking distance, Wang-Baker's pain rating scale, and ulcer size improved significantly after the transplantation of CD34+ cells. Because this was not a controlled randomized study, the possibility of a placebo effect after CD34+ cell transplantation needs to be evaluated in a large-scale trial in the future. As for the evaluation of safety issues, neither death nor life-threatening adverse events were observed in this study, and no severe adverse events except for transient and expected mild to moderate ones could be observed as a result of the performed cell therapeutic approach. These outcomes suggest the safety and feasibility of this cell-based therapy in patients with CLI.

Feedbacks from Clinical EPC Transplantation

Our animal studies as well as the results of other groups suggest that heterologous EPC transplantation requires systemic injections of 0.5–2.0 × 104 human EPCs per gram body weight of the recipient animal to achieve a satisfactory improvement of hind limb ischemia [3, 22, 51, 54, 65]. In general, cultured EPCs obtained from healthy human volunteers yield 5.0 × 106 cells per 100 milliliter of PB on day 7. Based on these data in human, a blood volume of as much as 12 liters will be necessary to obtain a sufficient number of EPCs for the treatment of patients with critical hind limb ischemia. Therefore, the background factors in clinical patients such as aging [66], diabetes [21, 67], hypercholesterolemia [21], hypertension [21, 68], and smoking [69, 70] that may reduce the number and biological activity of circulating/BM EPCs represent possible major limitations for the success of primary EPC transplantations. In reality, most of the patients who are going to undergo EPC therapy for ischemic diseases have background diseases as described above. Considering autologous EPC therapy, certain technical improvements that may help to overcome the shortcomings of EPCs should include: (1) local delivery of EPCs, (2) endogenous EPC mobilization, that is, cytokine/growth factor supplementation to promote BM-derived EPC mobilization [3, 20], (3) enrichment procedures, that is, leukapheresis or BM aspiration, (4) enhancement of EPC functions by gene transduction, or (5) culture expansion of EPCs from self-renewable primitive stem/progenitor cells isolated from BM or other sources.

Strategies that will recover potential EPC dysfunction and improve the bioactivity of these cells for the successful treatment of ischemic disorders should be considered, especially, in light of the current findings implicating that EPC function and mobilization may be impaired in certain diseases. For years, several groups have reported the expansion of EPCs for therapeutic application [51, 71, 72]. Recently, we have also succeeded to improve EPC culture to expand freshly isolated G-CSF mobilized PB-derived human CD34+ cells. The strategy to use qualified culture system might compensate the current disadvantages of applying dysfunctional EPCs for autologous cell transplantation therapy in ischemic diseases by increasing the quantity and quality of the applied EPCs (Fig. 2).

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IDENTIFICATION OF EPCs
  5. ROLE OF EPCs IN POSTNATAL NEOVASCULARIZATION
  6. EPC-BASED THERAPEUTIC ANGIOGENESIS
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES

Accumulating evidence suggests that BM-derived EPCs have the potential to promote postnatal vasculogenesis in adults, thus opening the way for possible clinical applications and the targeted cellular therapy of cardiovascular diseases. For a successful therapeutic EPC-based approach, the isolation and preparation of an optimal quality and quantity of EPCs is essential to remedy certain unresolved issues in the field.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IDENTIFICATION OF EPCs
  5. ROLE OF EPCs IN POSTNATAL NEOVASCULARIZATION
  6. EPC-BASED THERAPEUTIC ANGIOGENESIS
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES

We thank Sachie Ota and Noriko Kakuta for their secretarial assistance. This work was supported by grants from the Riken Center for Developmental Biology Collaborative Research Fund, Kobe (08001475).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. IDENTIFICATION OF EPCs
  5. ROLE OF EPCs IN POSTNATAL NEOVASCULARIZATION
  6. EPC-BASED THERAPEUTIC ANGIOGENESIS
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  • 1
    Asahara T, Murohara T, Sullivan A et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997; 275: 9647.
  • 2
    Shi Q, Rafii S, Wu MH et al. Evidence for circulating bone marrow-derived endothelial cells. Blood 1998; 92: 362367.
  • 3
    Asahara T, Masuda H, Takahashi T et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999; 85: 221228.
  • 4
    Folkman J, Shing Y. Angiogenesis. J Biol Chem 1992; 267: 1093110934.
  • 5
    Risau W, Sariola H, Zerwes HG et al. Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies. Development 1988; 102: 471478.
  • 6
    Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest 1999; 103: 12311236.
  • 7
    Bailey AS, Jiang S, Afentoulis M et al. Transplanted adult hematopoietic stems cells differentiate into functional endothelial cells. Blood 2004; 103: 1319.
  • 8
    Pelosi E, Valtieri M, Coppola S et al. Identification of the hemangioblast in postnatal life. Blood 2002; 100: 32033208.
  • 9
    Grant MB, May WS, Caballero S et al. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med 2002; 8: 607612.
  • 10
    Ingram DA, Caplice NM, Yoder MC. Unresolved questions, changing definitions, and novel paradigms for defining endothelial progenitor cells. Blood 2005; 106: 15251531.
  • 11
    Aicher A, Rentsch M, Sasaki K et al. Nonbone marrow-derived circulating progenitor cells contribute to postnatal neovascularization following tissue ischemia. Circ Res 2007; 100: 581589.
  • 12
    Timmermans F, Van Hauwermeiren F, De Smedt M et al. Endothelial outgrowth cells are not derived from CD133+ cells or CD45+ hematopoietic precursors. Arterioscler Thromb Vasc Biol 2007; 27: 15721579.
  • 13
    Flamme I, Risau W. Induction of vasculogenesis and hematopoiesis in vitro. Development 1992; 116: 435439.
  • 14
    Timmermans F, Plum J, Yoder MC et al. Endothelial progenitor cells: Identity defined? J Cell Mol Med 2009; 13: 87102.
  • 15
    Masuda H, Alev C, Akimaru H et al. Methodological development of a clonogenic assay to determine endothelial progenitor cell potential. Circ Res 2011; 109: 2037.
  • 16
    Kwon SM, Eguchi M, Wada M et al. Specific Jagged-1 signal from bone marrow microenvironment is required for endothelial progenitor cell development for neovascularization. Circulation 2008; 118: 157165.
  • 17
    Tanaka R, Wada M, Kwon SM et al. The effects of flap ischemia on normal and diabetic progenitor cell function. Plast Reconstr Surg 2008; 121: 19291942.
  • 18
    Kamei N, Kwon SM, Alev C et al. Lnk deletion reinforces the function of bone marrow progenitors in promoting neovascularization and astrogliosis following spinal cord injury. Stem Cells 2010; 28: 365375.
  • 19
    Dimmeler S, Aicher A, Vasa M et al. HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway. J Clin Invest 2001; 108: 391397.
  • 20
    Takahashi T, Kalka C, Masuda H et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 1999; 5: 434438.
  • 21
    Vasa M, Fichtlscherer S, Aicher A et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res 2001; 89: E1E7.
  • 22
    Kalka C, Masuda H, Takahashi T et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA 2000; 97: 34223427.
  • 23
    Murohara T, Ikeda H, Duan J et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest 2000; 105: 15271536.
  • 24
    Sharpe EE,3rd, Teleron AA, Li B et al. The origin and in vivo significance of murine and human culture-expanded endothelial progenitor cells. Am J Pathol 2006; 168: 17101721.
  • 25
    Gulati R, Jevremovic D, Peterson TE et al. Diverse origin and function of cells with endothelial phenotype obtained from adult human blood. Circ Res 2003; 93: 10231025.
  • 26
    Hur J, Yang HM, Yoon CH et al. Identification of a novel role of T cells in postnatal vasculogenesis: Characterization of endothelial progenitor cell colonies. Circulation 2007; 116: 16711682.
  • 27
    Rehman J, Li J, Orschell CM et al. Peripheral blood “endothelial progenitor cells” are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation 2003; 107: 11641169.
  • 28
    Shepherd RM, Capoccia BJ, Devine SM et al. Angiogenic cells can be rapidly mobilized and efficiently harvested from the blood following treatment with AMD3100. Blood 2006; 108: 36623667.
  • 29
    Bailey AS, Willenbring H, Jiang S et al. Myeloid lineage progenitors give rise to vascular endothelium. Proc Natl Acad Sci USA 2006; 103: 1315613161.
  • 30
    Romagnani P, Annunziato F, Liotta F et al. CD14+CD34low cells with stem cell phenotypic and functional features are the major source of circulating endothelial progenitors. Circ Res 2005; 97: 314322.
  • 31
    Moldovan NI. Role of monocytes and macrophages in adult angiogenesis: A light at the tunnel's end. J Hematother Stem Cell Res 2002; 11: 179194.
  • 32
    Lin Y, Weisdorf DJ, Solovey A et al. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest 2000; 105: 7177.
  • 33
    Ingram DA, Mead LE, Tanaka H et al. Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood 2004; 104: 27522760.
  • 34
    Hur J, Yoon CH, Kim HS et al. Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis. Arterioscler Thromb Vasc Biol 2004; 24: 288293.
  • 35
    Sieveking DP, Buckle A, Celermajer DS et al. Strikingly different angiogenic properties of endothelial progenitor cell subpopulations: Insights from a novel human angiogenesis assay. J Am Coll Cardiol 2008; 51: 660668.
  • 36
    Wojakowski W, Kucia M, Kazmierski M et al. Circulating progenitor cells in stable coronary heart disease and acute coronary syndromes: Relevant reparatory mechanism? Heart 2008; 94: 2733.
  • 37
    Losordo DW, Kishore R. A big promise from the very small identification of circulating embryonic stem-like pluripotent cells in patients with acute myocardial infarction. J Am Coll Cardiol 2009; 53: 1012.
  • 38
    Masuda H, Kalka C, Takahashi T et al. Estrogen-mediated endothelial progenitor cell biology and kinetics for physiological postnatal vasculogenesis. Circ Res 2007; 101: 598606.
  • 39
    Bauer SM, Goldstein LJ, Bauer RJ et al. The bone marrow-derived endothelial progenitor cell response is impaired in delayed wound healing from ischemia. J Vasc Surg 2006; 43: 134141.
  • 40
    Ii M, Nishimura H, Iwakura A et al. Endothelial progenitor cells are rapidly recruited to myocardium and mediate protective effect of ischemic preconditioning via “imported” nitric oxide synthase activity. Circulation 2005; 111: 11141120.
  • 41
    Iwakura A, Shastry S, Luedemann C et al. Estradiol enhances recovery after myocardial infarction by augmenting incorporation of bone marrow-derived endothelial progenitor cells into sites of ischemia-induced neovascularization via endothelial nitric oxide synthase-mediated activation of matrix metalloproteinase-9. Circulation 2006; 113: 16051614.
  • 42
    Murayama T, Tepper OM, Silver M et al. Determination of bone marrow-derived endothelial progenitor cell significance in angiogenic growth factor-induced neovascularization in vivo. Exp Hematol 2002; 30: 96772.
  • 43
    Urbich C, Aicher A, Heeschen C et al. Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells. J Mol Cell Cardiol 2005; 39: 733742.
  • 44
    Dai Y, Ashraf M, Zuo S et al. Mobilized bone marrow progenitor cells serve as donors of cytoprotective genes for cardiac repair. J Mol Cell Cardiol 2008; 44: 607617.
  • 45
    Kawamoto A, Asahara T, Losordo DW. Transplantation of endothelial progenitor cells for therapeutic neovascularization. Cardiovasc Radiat Med 2002; 3: 221225.
  • 46
    Wu Y, Ip JE, Huang J et al. Essential role of ICAM-1/CD18 in mediating EPC recruitment, angiogenesis, and repair to the infarcted myocardium. Circ Res 2006; 99: 315322.
  • 47
    Jujo K, Ii M, Losordo DW. Endothelial progenitor cells in neovascularization of infarcted myocardium. J Mol Cell Cardiol 2008; 45: 530544.
  • 48
    Miyamoto Y, Suyama T, Yashita T et al. Bone marrow subpopulations contain distinct types of endothelial progenitor cells and angiogenic cytokine-producing cells. J Mol Cell Cardiol 2007; 43: 627635.
  • 49
    Kocher AA, Schuster MD, Szabolcs MJ et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001; 7: 430436.
  • 50
    Aicher A, Brenner W, Zuhayra M et al. Assessment of the tissue distribution of transplanted human endothelial progenitor cells by radioactive labeling. Circulation 2003; 107: 21342139.
  • 51
    Kawamoto A, Gwon HC, Iwaguro H et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 2001; 103: 634637.
  • 52
    Kawamoto A, Iwasaki H, Kusano K et al. CD34-positive cells exhibit increased potency and safety for therapeutic neovascularization after myocardial infarction compared with total mononuclear cells. Circulation 2006; 114: 21632169.
  • 53
    Schatteman GC, Hanlon HD, Jiao C et al. Blood-derived angioblasts accelerate blood-flow restoration in diabetic mice. J Clin Invest 2000; 106: 571578.
  • 54
    Iwasaki H, Kawamoto A, Ishikawa M et al. Dose-dependent contribution of CD34-positive cell transplantation to concurrent vasculogenesis and cardiomyogenesis for functional regenerative recovery after myocardial infarction. Circulation 2006; 113: 13111325.
  • 55
    Burt RK, Testori A, Oyama Y et al. Autologous peripheral blood CD133+ cell implantation for limb salvage in patients with critical limb ischemia. Bone Marrow Transplant 2010; 45: 1111116.
  • 56
    Kawamoto A, Katayama M, Handa N et al. Intramuscular transplantation of G-CSF-mobilized CD34(+) cells in patients with critical limb ischemia: a phase I/IIa, multicenter, single-blinded, dose-escalation clinical trial. Stem Cells 2009; 27: 28572864.
  • 56
    Schachinger V, Assmus B, Britten MB et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction: Final one-year results of the TOPCARE-AMI Trial. J Am Coll Cardiol 2004; 44: 16901699.
  • 57
    Bartunek J, Vanderheyden M, Vandekerckhove B et al. Intracoronary injection of CD133-positive enriched bone marrow progenitor cells promotes cardiac recovery after recent myocardial infarction: Feasibility and safety. Circulation 2005; 112( 9 suppl): I178I183.
  • 58
    Li ZQ, Zhang M, Jing YZ et al. The clinical study of autologous peripheral blood stem cell transplantation by intracoronary infusion in patients with acute myocardial infarction (AMI). Int J Cardiol 2007; 115: 5256.
  • 59
    Stamm C, Westphal B, Kleine HD et al. Autologous bone-marrow stem-cell transplantation for myocardial regeneration. Lancet 2003; 361: 4546.
  • 60
    Stamm C, Kleine HD, Choi YH et al. Intramyocardial delivery of CD133+ bone marrow cells and coronary artery bypass grafting for chronic ischemic heart disease: Safety and efficacy studies. J Thorac Cardiovasc Surg 2007; 133: 717725.
  • 61
    Boyle AJ, Whitbourn R, Schlicht S et al. Intra-coronary high-dose CD34+ stem cells in patients with chronic ischemic heart disease: A 12-month follow-up. Int J Cardiol 2006; 109: 2127.
  • 62
    Losordo DW, Schatz RA, White CJ et al. Intramyocardial transplantation of autologous CD34+ stem cells for intractable angina: A phase I/IIa double-blind, randomized controlled trial. Circulation 2007; 115: 31653172.
  • 64
    Kuroda R, Matsumoto T, Miwa M et al. Local Transplantation of G-CSF-mobilized CD34+ cells in a patient with tibial nonunion: A case report. Cell Transplant 2010 [Epub ahead of print].
  • 65
    Hung HS, Shyu WC, Tsai CH et al. Transplantation of endothelial progenitor cells as therapeutics for cardiovascular diseases. Cell Transplant 2009; 18: 10031012.
  • 66
    Heiss C, Keymel S, Niesler U et al. Impaired progenitor cell activity in age-related endothelial dysfunction. J Am Coll Cardiol 2005; 45: 14411448.
  • 67
    Ii M, Takenaka H, Asai J et al. Endothelial progenitor thrombospondin-1 mediates diabetes-induced delay in reendothelialization following arterial injury. Circ Res 2006; 98: 697704.
  • 68
    Imanishi T, Moriwaki C, Hano T et al. Endothelial progenitor cell senescence is accelerated in both experimental hypertensive rats and patients with essential hypertension. J Hypertens 2005; 23: 18311837.
  • 69
    Kondo T, Hayashi M, Takeshita K et al. Smoking cessation rapidly increases circulating progenitor cells in peripheral blood in chronic smokers. Arterioscler Thromb Vasc Biol 2004; 24: 14421447.
  • 70
    Michaud SE, Dussault S, Haddad P et al. Circulating endothelial progenitor cells from healthy smokers exhibit impaired functional activities. Atherosclerosis 2006; 187: 423432.
  • 71
    Pesce M, Orlandi A, Iachininoto MG et al. Myoendothelial differentiation of human umbilical cord blood-derived stem cells in ischemic limb tissues. Circ Res 2003; 93: e51e62.
  • 72
    Ott I, Keller U, Knoedler M et al. Endothelial-like cells expanded from CD34+ blood cells improve left ventricular function after experimental myocardial infarction. FASEB J 2005; 19: 992994.