Endothelial Progenitor Cells: Unexpected Disclosures

Editorials See related article, pages 314 –322

Endothelial Progenitor Cells Unexpected Disclosures Annarosa Leri, Jan Kajstura

T

he formation of coronary collateral vessels constitutes a compensatory adaptation of the heart to ischemia. This vascular response increases blood supply to the myocardium reducing the area of hypoxic damage and, ultimately, infarct size. The recruitment of collateral vessels within the coronary circulation is favored by multiple episodes of ischemia, which provide the stimuli necessary for the growth of vascular structures and increased blood flow. The degree of collateralization significantly influences the prognosis of an ischemic episode, which is more severe with diabetes, metabolic syndrome, or in the elderly, the patient population mostly affected by coronary artery disease.1 Originally, angiogenesis, ie, sprouting of vessels from the preexisting circulation, was considered the exclusive mechanism of vascular remodeling and growth. The identification of circulating endothelial progenitor cells (EPCs) has modified this view.2 Migration and homing of EPCs to ischemic regions leads to de novo formation of vascular structures. In analogy with vessel development in the embryo, this process has been called vasculogenesis. Also, circulating EPCs participate in reendothelization of damaged vessels, together with resident vascular progenitor cells.3 EPCs represent a heterogeneous cell population of multiple origins and distinct phenotypes, which are able to give rise to functionally competent endothelial cells.4 In the hierarchy established in the hematopoietic system, progenitors identify cells with lower differentiation potential than stem cells (SCs). However, EPCs possess degrees of stemness, which include self-renewal, clonogenicity, and differentiation capacity.4 EPCs represent various subsets of progenitor cells that have distinct phenotypes but share the ability to differentiate into mature endothelial cells. During embryogenesis, hematopoietic cells and endothelial cells develop temporally and spatially in close association. Blood cells are surrounded by layers of endothelial cells suggesting a joint origin of these lineages from a common precursor, the embryonic hemangioblast.4 The adult hemangioblast resides in the bone marrow (BM), and hematopoietic stem cells (HSCs) are considered the major source of circulating EPCs. Moreover, EPCs can derive from cells of the The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Cardiovascular Research Institute, Department of Medicine, New York Medical College, Valhalla. Correspondence to Dr Annarosa Leri, Cardiovascular Research Institute, Vosburgh Pavilion, New York Medical College, Valhalla, NY 10595. E-mail [email protected] (Circ Res. 2005;97:299-301.) © 2005 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/01.RES.0000179774.56498.d2

myeloid/monocytic lineage and from BM mesenchymal stromal cells. The recognition of vascular progenitors in the vessel wall, adipose tissue, brain, and heart suggests that EPCs are released from different sites into the peripheral circulation.4,5 In the absence of tissue-related markers, it is impossible to establish the relative contribution of different organs to the circulating pool of EPCs. It cannot be excluded, however, that EPCs are stored in the bone marrow and, when the need arises, take residence in distant organs (Figure 1). A specific phenotype of EPCs has not yet emerged. In spite of the identification of tissue-resident immature endothelial cells, the prevailing view remains that HSCs are the ancestors of EPCs. And the presence of EPCs in organs other than the bone marrow has been interpreted as the result of trafficking of cells from the BM through the bloodstream or preservation of HSC-derived embryonic remnants.4,5 Accordingly, markers of EPCs have been searched for among HSC epitopes. The historic antigen of EPCs, CD34, was identified in 1997 and this work promoted the study of circulating angioblasts.2 Subsequently, other markers were detected including CD133, an immature HSC epitope, and CD14, an indicator of monocyte lineage. The coexpression of the VEGF receptor-2, flk1, with CD34 and CD14 is a fundamental attribute of EPCs.4,5 In this issue of Circulation Research, Romagnani and collaborators6 report on a novel population of human peripheral blood mononuclear cells (PBMCs). PBMCs were identified as a subset with a uniform, bright presence of CD14 and flk1 accompanied by low expression of CD34. This epitope was undetectable by FACS and apparent only with a highly sensitive technique. This approach consisted of a combination of immunofluorescence and immunomagnetism. Liposomes were loaded with fluorescein and magnetic beads, whereas CD34 antibodies were conjugated to the liposomes. By this technique, staining intensity increased 100- to 1000-fold, whereas background fluorescence was unchanged.6 Because of magnetic labeling, cells with low-level antigens were isolated by magnetic sorting, and CD14POSCD34LOW cells were identified in the peripheral blood and BM. Contrary to expectation, these cells were clonogenic and multipotent generating, in addition to mature endothelial cells, osteoblasts, adipocytes, and neural cells. Growth and differentiation of CD14POSCD34LOW cells required the presence of VEGF. Thus, CD14POSCD34LOW cells possess the criteria of SCs and give rise to cells from different germ layers comprising the mesoderm and ectoderm. The level of enrichment of CD14POSCD34LOW cells, 95%, indicates that commitment to adipocytes, osteoblasts and neural cells occurred by transdifferentiation. The possibility that fusion between CD14POSCD34LOW cells and CD14NEGCD34NEG cells took place is unlikely. CD14NEGCD34NEG cells constituted only

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Figure 1. EPCs originate from several organs and are involved in re-endothelization of injured vessels and in the formation of new vessels in ischemic areas.

5% of the population, and their fusion with the stem cell pool would have not been followed by a significant level of replication of the hybrid cells. This would have led to a small fraction of differentiated cells, slightly higher than 5%. Conversely, the number of endothelial cells, osteoblasts, adipocytes, and neural cells was several-fold higher, supporting the notion of plasticity of CD14POSCD34LOW cells. CD14POS cells have been shown to be pluripotent SCs capable of generating blood, epithelial, endothelial, neuronal, and liver cells.7 In contrast to CD14POSCD34LOW cells,6 this cell expresses high levels of CD34, a membrane-bound sialomucin, indicative of stemness and pluripotency.8 Whether the presence of CD34 determines the function of this SC cannot be determined.7 Equally limited is our understanding of the properties of CD14, which up to now has been considered a receptor of low-density lipoproteins.8 Whether this epitope needs to be included in the identification and purification of human HSCs remains to be established. A novel observation in the study by Romagnani and colleagues is the documentation that CD14POSCD34LOW cells express Bmi-1, Nanog, and Oct4.6 These transcription factors are implicated in the self-renewing and pluripotent nature of SCs. Bmi-1 is required for postnatal preservation of SCs in the central and peripheral nervous system and in the BM.9 Several organs are sustained throughout the lifespan of the organism by a small number of SCs. SC growth is regulated by sophisticated molecular pathways that promote asymmetric division and prevent premature senescence and apoptosis preserving the SC pool.1 Bmi-1, a member of the Polycomb family of proteins, is essential for self-renewal of adult SCs via repression of genes associated with replicative senescence, and activation of genes involved in cell proliferation. The target downstream genes of Bmi-1 include p16INK4a, p19ARF, and telomerase; p16INK4a and p19ARF are markers of cellular senescence whereas telomerase function protects telomere integrity and

cell growth.1 By this mechanism, Bmi-1 extends SC lifespan without inducing immortalization. The cell pool identified here may be less sensitive to the effects of aging than other subsets of EPCs, and thus would have great clinical relevance. Nanog and Oct4, together with Sox2, are transcription factors critical for the maintenance of the pluripotent phenotype of embryonic SCs. These molecules are strictly correlated; Oct4 in combination with Sox2 is the upstream regulatory element of Nanog.10 Oct4 was considered restricted to the embryonic life of SCs. However, adult peripheral blood and BMCs possess these genes and their products. Oct4 has recently been documented in adult SCs of self-renewing organs. Oct4-positive cells are scattered in the basal layer of the epidermis, and in the breast, pancreas, liver, gut, and mesenchymal compartment of the BM.10 Oct4 confers to CD14POSCD34LOW cells the status of undifferentiated cells with high proliferative capacity and pluripotent property. The rigorous documentation of the SC phenotype of circulating CD14POSCD34LOW cells has led to the conclusion that this cell pool represents the major source of EPCs and endothelial cells.6 However, the lack of in vivo assessment of the functional behavior of these cells after ischemic injury precludes a definitive answer. CD14POSCD34LOW SCs may be more efficient and powerful in regenerating dead myocardium than other more committed BMCs (Figure 2). If this property were to be present, CD14POSCD34LOW cells could become a new form of cell therapy for the infarcted heart. Currently, the critical question is whether nonresident primitive cells are preferable to cardiac stem cells (CSCs) or represent a second option. Like the CD14POSCD34LOW cells, the c-kitPOS or Sca-1POS CSCs are self-renewing, clonogenic and multipotent, and programmed to form cardiomyocytes and coronary vessels.11–14 However, difficulties exist in the acquisition and growth of adult CSCs. Conversely, CD14POSCD34LOW cells are easily obtained and expanded,

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Acknowledgments This work was supported by the National Institutes of Health.

References

Figure 2. Bone marrow cells injected in proximity of an acute myocardial infarct generate numerous coronary arterioles (␣-smooth muscle actin, red) over a period of 1 month. SM indicates surviving myocardium (␣-sarcomeric actin, white).

providing a potential source of effective cells for cardiac repair and vascular growth. Nevertheless, CSCs can be activated and mobilized locally by growth factors representing, theoretically, the optimal cell for myocardial regeneration and possible restitutio ad integrum of the damaged organ. Romagnani’s study has important biological implications. It refutes the paradigm that adult SCs cannot change their original identity and commit to cell lineages beyond their own tissue boundaries. CD14POSCD34LOW cells generate not only endothelial cells, adipocytes, and osteoblasts but also glial and neuronal cells. The controversy concerning SC plasticity required major efforts on the part of the scientific community to understand whether SCs actually control their fate. Results supporting this view have been contrasted with the possibility of cell fusion as an alternative mechanism to SC plasticity. Debate and controversy are critical for scientific progress because discoveries offer unexpected disclosures regarding the biology of the organism and, ultimately, our sense of identity.

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myocardial regeneration

Endothelial Progenitor Cells: Unexpected Disclosures Annarosa Leri and Jan Kajstura Circ Res. 2005;97:299-301 doi: 10.1161/01.RES.0000179774.56498.d2 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2005 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571

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