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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].

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

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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).

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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.

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Table 1. Clinical trials for ischemic diseases with EPCs
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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