- Email: [email protected]
Vascular Progenitors
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Vascular Progenitors: From Biology to Treatment Aernout Luttun, Geert Carmeliet, and Peter Carmeliet*
The identification of endothelial progenitor cells (EPCs) in the adult human has forced us to reconsider how new blood vessels grow in physiological and pathological conditions in the adult human. An important question in angiogenesis research is to what extent these progenitors functionally contribute to revascularization of ischemic tissues and and to what extent they can be used for therapeutic angiogenic cell transplantation. Even more challenging is the concept that hematopoietic and other bone-marrow–derived stem cells might be recruited in the context of ischemia to induce neovessel formation. This review discusses some of the recent insights and outstanding questions on EPCs, both from a biological and therapeutic perspective. (Trends Cardiovasc Med 2002;12:88–96). © 2002, Elsevier Science Inc.
• Endothelial Progenitors in the Embryo Endothelial cells, lining the inside of blood vessels (endo-, inside), arise from mesodermal precursor cells, the angioblasts. These precursors have the capacAernout Luttun and Peter Carmeliet are from the Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium. Geert Carmeliet is from the Laboratory of Experimental Medicine and Endocrinology, Leuven, Belgium. * Address correspondence to: P. Carmeliet, Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, KU Leuven, Campus Gasthuisberg, Herestraat 49, B-3000, Leuven, Belgium. Tel: (132) 16-34.57.72; fax: (132) 16-34.59.90; e-mail: peter.carmeliet@med. kuleuven.ac.be. © 2002, Elsevier Science Inc. All rights reserved. 1050-1738/02/$-see front matter
ity to proliferate, migrate, and differentiate into endothelial cells but have not yet acquired characteristic mature endothelial markers. In vitro differentiation studies and studies in zebrafish have indicated that endothelial and blood cell (hematopoietic) precursors originate from a common ancestor, the hemangioblast (Figure 1). Hemangioblasts undergo their first critical steps of differentiation within the blood islands. Cells at the perimeter of the blood islands give rise to precursors for endothelial cells, whereas those in the center constitute hematopoietic precursors. 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. These include Ets-1, Fli1, Hex or Hhex , Vezf-1, Hox, a4-integrin, members of the GATA-family, basic helixloop-helix (bHLH) factors (like SCL/
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Tal-1), and the Id-proteins and hedgehog protein Indian Hedgehog (Carmeliet 2000b, Dyer et al. 2001). Studies in quail/chick chimeras have shown that the fibroblast growth factor-2 (FGF-2) mediates the induction of angioblasts from the mesoderm (Poole et al. 2001). In addition, embryonic angioblasts express the receptor-2 for vascular endothelial growth factor (VEGFR-2; flk-1) and divide in response to VEGF, a pleiotropic angiogenic factor in vitro. In addition, in vivo studies in the Xenopus embryo have documented a role for VEGF in mediating angioblast migration (Poole et al. 2001). Its role may, however, not relate to determining endothelial fate because endothelial cells still differentiate in embryos lacking VEGF. Inactivation of the VEGFR-2 gene in mice results in embryonic lethality, with lack of development of both hematopoietic and endothelial cell lineages, supporting the critical importance of this receptor at that developmental stage, although not defining the steps regulating differentiation into endothelial versus hematopoietic cell. However, because VEGFR-2 deficiency blocks differentiation of blood and vascular cells, other VEGFR-2 ligands may be essential for endothelial cell fate in vivo. Angioblast differentiation may be promoted by VEGF, FGF-2, and VEGFR-2, whereas VEGF receptor-1 (VEGFR-1; flt-1) has been determined to suppress hemangioblast commitment (Ferrara 2001). • Arterial and Venous Angioblasts Following commitment to the endothelial lineage, angioblasts assemble into a primitive vascular plexus of veins and arteries, a process called vasculogenesis. This primitive vasculature is subsequently refined into a functional network by angiogenesis (vascular sprouting from preexisting vessels, vascular fusion and intussusception) and by remodeling and “muscularization” (arteriogenesis) of newly formed vessels (Figure 2) (Carmeliet 2000b). Whether endothelial cells in veins and arteries have distinct precursors remains to be determined (Figure 1). The specification between vessels conducting blood from or to the heart (arteries and veins, respectively) occurs very early during embryonic development. Only recently have possible molecular players in this process been TCM Vol. 12, No. 2, 2002
suggested. The existence of molecular differences in arterial and venous endothelium before vessel formation implies that establishing the identity of arteries and veins is independent of blood flow and pressure. Genetic studies in the zebrafish have identified the bHLH transcription factor gridlock A as a possible candidate, with gridlock A favoring differentiation of prearterial at the expense of prevenous angioblasts (Wilkinson 2000). Notch-derived signals, which are often involved in cell fate determination via lateral specification and inductive signaling between distinct cell types, might also be implicated. Recent evidence indicates that certain Notch ligands such as DeltaC (Wilkinson 2000) are expressed in arterial endothelium and that defective Notch signaling caused vascular remodeling defects (Krebs et al. 2000). The hairy-related bHLH factor HeyL, an effector of Notch, is expressed in smooth muscle of all arteries, overlapping with that of Notch3, mutations of which underlie the cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy vascular (CADASIL) disorder (Leimeister et al. 2000). The TGF-b receptor activin receptor-like kinase 1 also appears to control early arterial differentiation (Urness et al. 2000). There is increasing evidence that members of the large ephrin family may also play a role (Roman and Weinstein 2000, Wilkinson 2000). Ephrins are ligands for their corresponding Eph receptors, comprising a family of at least 14 receptor tyrosine kinases. Ephrins activate their receptors when membrane bound. Ephrin A1 and ephrin A2 are angiogenic in different vascular beds. Ephrins B1 and B2 also induce vascular sprouting. During embryonic development, expression of ephrin B2 is restricted to endothelial cells in arteries at early stages and progressively extends to also marking arterial smooth muscle cells (SMCs) (Gale et al. 2001). In addition, ephrin B2 continues to selectively mark arteries in the adult with expression in endothelial cells, SMCs and pericytes (Gale et al. 2001, Shin et al. 2001). In contrast, its receptor, EphB4, is found primarily in veins. Gene inactivation of either ephrin B2 or of EphB4 results in normal vasculogenesis but abnormal angiogenesis, the latter with disrupted remodeling of both arteries and veins
into large and small branches. Complex ligand-receptor bidirectional signaling interactions via ephrins and ephrin receptors have been described, with responses dependent on a variety of factors, including phosphorylation, multimerization, and the presence of adaptor proteins such as Grb2, Grb10, and Nck. A novel cytoplasmic tyrosine kinase gene, bone marrow tyrosine kinase (Bmx), was identified in arterial endothelium (Ekman et al. 1997). Because its loss did not affect physiological growth of arteries in knockout mice, the function of Bmx needs to be further defined. We have found that selective loss of the VEGF164 isoform in mice impairs arterial development in the retina (unpublished observations in collaboration with P. D’Amore). Intriguingly, neuropilin-1, a VEGF164-isoform specific receptor, was also more abundantly expressed in arterioles (unpublished observations, Moyon et al. 2001). To what extent vascular growth factors selectively affect arterial or venous growth in the adult remains to be established. Future studies will be required to unravel the differentiation characteristics of veins, arteries, and capillaries. • Smooth Muscle Progenitors in the Embryo Endothelial channels are covered by multiple layers of SMCs in large vessels in proximal parts of the vasculature and by single pericytes around smaller distal vessels. These mural cells have a complex origin, depending on their location in the embryo (Gittenberger-de Groot et al. 1999) (Figure 1). The first SMCs around endothelial tubes in the embryo transdifferentiate from the endothelium (Gittenberger-de Groot et al. 1999). Endothelial cells also transform to smoothmuscle–like myofibroblasts in the prospective cardiac valves, a process involving signaling by transforming growth factor-b3 (TGF-b3) (Nakajima et al. 1997). TGF-b1, another family member, has been implicated in the differentiation of a mesenchymal stem cell to a progenitor that expresses platelet-derived growth factor receptor-b (PDGFR-b) (Hellstrom et al. 1999, Hirschi et al. 1998). By PDGF-BB, endothelial cells stimulate subsequent growth and differentiation of this precursor. Pericytes and SMCs of the coronary vessels are derived from a
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Figure 1. A common origin for the two types of blood-vessel cell. Endothelial and smooth muscle cells (SMCs) arise from separate types of precursor. Endothelial cells arise from precursors called angioblasts or hemangioblasts in the embryo, or from circulating endothelial progenitors in the adult. Angioblasts give rise to arterial and venous lineages. SMCs and pericytes, by contrast, can form from a variety of progenitors. These include mesenchymal stem cells, neural crest cells, and progenitors in the epicardium in the embryo. A new common vascular progenitor cell that gives rise to both types of blood-vessel cell has been recently identified. Whether this vascular progenitor and the hemangioblast both arise from a multipotent stem cell remains elusive. Vascular endothelial growth factor (VEGF) promotes the development of endothelial cells from this precursor. TGF-b1 has been involved in differentiation of mesenchymal stem cells to progenitors that express the receptor for PDGF-BB. The latter stimulates their development into SMCs and pericytes and is responsible for their recruitment around nascent vessels. Circulating endothelial progenitors have been identified postnatally, but the persistence of smooth muscle or common vascular progenitors in adult life remains elusive. Adapted from Carmeliet (2000a).
putative progenitor that infiltrates the heart from its external (epicardial) layers (Gittenberger-de Groot et al. 1999). Coronary vein SMCs are derived from the atrial myocardium, whereas those of the coronary arteries come from the epicardial layer (Dettman et al. 1998). Cardiac neural crest cells are the source of SMCs of the large thoracic blood vessels (Creazzo et al. 1998). Whereas the periendothelial cells in the dorsal vascular compartment of the brain have a mesodermal origin, vessels in the forebrain and the retinae derive their pericytes and SMCs from cephalic neural crest cells (Etchevers et al. 2001). Whether SMCs and pericytes in arteries, veins, and lymphatic vessels or even within the inner and medial layer of an artery arise from distinct progenitors remains unknown. • Common Vascular Progenitors in the Embryo Yamashita et al. (2000) discovered an embryonic common vascular progenitor that differentiates into endothelial and SMCs. The SMCs arising from these progenitors were not simply transdifferentiated endothelial cells expressing the atypical smooth muscle a-actin marker. Instead, they expressed an entire set of smooth muscle markers and surrounded endothelial channels in vivo. This vascular progenitor resembles the putative common precursor of the endothelial cells that line the inner surface of the heart (the endocardium) and their surrounding cardiac muscle fibers. Like these hemangioblasts, vascular progenitors express VEGFR-2 (Yamashita et al. 2000), raising the question of whether
both cell types arise from a multipotent stem cell. The vascular progenitors differentiated to endothelial cells in response to VEGF, whereas they developed into SMCs in response to PDGF-BB (Yamashita et al. 2000). It is possible that PDGF-BB is a determinant of smooth muscle cell fate, but PDGFRb-positive progenitors still develop in the absence of PDGF-BB in vivo (Hellstrom et al. 1999) and in vitro (Yamashita et al. 2000). Thus, PDGF-BB may favor the selection and growth of PDGFRb-expressing progenitors. Such a hypothesis is consistent with a model whereby PDGF-BB stimulates PDGFRb-expressing progenitors to migrate along preexisting endothelial channels and to divide during arterial enlargement (Hellstrom et al. 1999). A common vascular progenitor could contribute to the formation of naked endothelial capillaries (angiogenesis) and muscle-coated vessels (arteriogenesis). It remains to be determined whether these common precursors or smooth muscle cell progenitors persist during adult life and whether these cells contribute to postnatal vessel growth (see below) (Figure 1). • Circulating Endothelial Progenitors in the Adult Neovascularization during adult life has long been attributed to angiogenesis only. However, recent studies have revealed that EPCs also circulate postnatally in the peripheral blood and may be recruited and incorporated into sites of active neovascularization in ischemic hindlimbs, ischemic myocardium, injured corneas, cutaneous wounds, and
tumor vasculature (Asahara et al. 1999, Kalka et al. 2000a, Schatteman et al. 2000, Takahashi et al. 1999), a process termed postnatal vasculogenesis (Figures 1 and 3). In addition, EPCs have been shown to be involved in maintenance angiogenesis replacing lost endothelial cells and in reendothelialization of implants (Peichev et al. 2000 and references therein). EPCs were initially identified and isolated on the basis of their expression of VEGFR-2 and CD34, antigens shared by the angioblast and the hematopoietic progenitor (Asahara et al. 1997). These EPCs were subsequently shown to express VE-cadherin, a junctional molecule, and AC133, an orphan receptor that is specifically expressed on EPCs but whose expression is lost once they differentiate into more mature endothelial cells (Peichev et al. 2000). Their high proliferation rate distinguishes circulating marrow-derived EPCs in the adult from mature endothelial cells shed from the vessel wall (Lin et al. 2000). Thus far, a bipotential common vascular progenitor, giving rise to both endothelial and periendothelial cells, has not been documented in the adult (Figure 1). • Local Signals in the Bone Marrow Compartment Controlling EPC Mobilization The majority of circulating EPCs reside in the bone marrow in close association with hematopoietic stem cells and the bone marrow stroma that provides the optimal microenvironment for hematopoiesis (Figure 4). Several observations suggest that hematopoietic (stem)
Figure 2. Angioblasts in the embryo assemble in a primitive network (vasculogenesis) that expands and remodels (angiogenesis). Smooth muscle cells (SMCs), originating from SMC precursors, cover endothelial cells during vascular myogenesis and stabilize vessels during arteriogenesis. CL, collagen; EL, elastin; Fib, fibrillin. Adapted from Carmeliet (1999).
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Figure 3. Pathological vascular growth in the adult may occur via vasculogenesis (endothelial progenitor cell [EPC] mobilization), angiogenesis (sprouting) or arteriogenesis (collateral growth). Mf, macrophages; SMC, smooth muscle cells. Adapted from Carmeliet (1999).
Figure 4. Bone and bone marrow constitute a single organ (the “bone–bonemarrow organ”) in which marrow cells reside in close vicinity to bone cells. Within the marrow, hematopoietic stem cells (HSC) produce the angiogenic factor VEGF, and erythroblasts (EB) are a source of both VEGF and its homologue PlGF. These angiogenic factors can influence the bone marrow microenvironment in different ways. First, they can induce stroma cells (expressing neuropilin-1 [NP-1]) to produce hematopoietic growth factors like thrombopoietin (Tpo) and Flt3-L and induce bone marrow endothelial cells (BMEC) (expressing VEGFR-1 and VEGFR-2) to secrete GM-CSF. Second, they can act as survival and proliferation factors for HSCs and endothelial progenitor cells (EPC), which both express VEGFR-2. Third, they might influence EPC or HSC mobilization by regulating adhesion molecule levels like E-selectin on BMECs or by expanding the bone-marrow vasculature. In addition, different bone cells express VEGF-receptors. Osteoclasts (OC) express VEGFR-1 and VEGFR-2, and NP-1 is expressed on osteoblasts (OB). Expression of VEGFR-1 and VEGFR-2 on OB is a matter of debate. Whereas VEGF has been shown to be involved in bone formation by influencing OB differentiation and OC recruitment and survival, it remains to be determined whether OB and OC, which can produce VEGF, also influence the bone marrow microenvironment. Left: Cellular organization of the bone–bone-marrow organ. Upper right: Distinct bone marrow cell types producing and responding to VEGF/PlGF. Lower right: Interaction between the bone and bone marrow cells producing and responding to VEGF/PlGF. Dotted lines represent hypothetical interactions. The left panel was adapted with permission from Bianco et al. (1999).
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cells as well as stroma cells might contribute to the local proliferation and transmigration of EPCs across the bonemarrow/blood barrier. Angiogenic factors are produced in the bone marrow, and their receptors are expressed on different cell types. Hematopoietic stem cells secrete VEGF, which is significantly upregulated upon cytokine stimulation (Bautz et al. 2000). VEGF, in turn, induces the release of hematopoietic growth factors, such as granulocyte macrophagecolony stimulating factor (GM-CSF) by bone marrow endothelial cells (BMECs) (Bautz et al. 2000) and thrombopoietin and Flt3-L by stromal cells (Tordjman et al. 1999). Recently, erythroblasts were shown to produce VEGF and placental growth factor (PlGF) (Tordjman et al. 2001), a VEGF homologue that specifically modulates VEGF function during pathological conditions (Carmeliet et al. 2001). VEGF-R2 is expressed on EPCs and a subset of hematopoietic stem cells (Hattori et al. 2001, Peichev et al. 2000). Neuropilin-1, a receptor for both VEGF and PlGF, is expressed on stroma cells (Tordjman et al. 1999) (Figure 4). VEGF has been shown to stimulate growth of embryonic angioblasts (see above). In addition, VEGF and angiopoietin (Ang)1 exert a synergistic effect in promoting the survival of bone marrow–derived hematopoietic progenitor cells (Hattori et al. 2001). Moreover, these growth factors might increase recruitment of bone marrow cells by expansion of the bone marrow vasculature (Hattori et al. 2001). To allow selective migration of hematopoietic stem cells through the bone marrow/blood barrier, BMECs constitutively express adhesion molecules such as E-selectin and vascular adhesion molecule-1. Down- or upregulation of these adhesion molecules or their corresponding ligands may contribute to the regulation of transendothelial migration of hematopoietic stem cells, and analogous mechanisms might be involved in EPC transmigration. VEGF has been shown to induce expression of E-selectin on BMECs (Naiyer et al. 1999). In addition to the contribution of adhesion molecules, paracrine chemokines and proteinases also influence stem cell homing and mobilization. Virtually all CD341VEGFR-21 cells (a cell population that includes EPCs) express CXCR-4, a receptor for the chemokine stromal-derived factor (SDF)-1 and miTCM Vol. 12, No. 2, 2002
grate in response to SDF-1 (Peichev et al. 2000), a factor that is important for HSC homing into the bone marrow. Desensitization of CXCR-4 results in mobilization of hematopoietic progenitors from the bone marrow into the peripheral blood (Shen et al. 2001). Interleukin8 induces rapid release of matrix metalloproteinase (MMP)-9 concurrent with the mobilization of hematopoietic precursors, consistent with an MMPmediated release of these precursor cells from the extracellular matrix (Fibbe et al. 2000). • Possible Interaction Between Bone Cells and the Bone Marrow Compartment Bone and bone marrow together form a single organ (the “bone–bone-marrow organ”) in which marrow cells and bone lining cells (osteoblasts and osteoclasts) reside in close contact with each other (Figure 4) (Bianco et al. 1999). The cells within the marrow cavity form a continuous network in which stromal cells are interspersed with hematopoietic and endothelial progenitors (Figure 4). Stromal cells lining the endothelium of the marrow vasculature are related to pericytes and have the potential to differentiate into skeletal tissue components such as bone and cartilage as well as adipocytes. Although a physical continuity exists between bone and marrow, the interaction between the different cell types is only now being unraveled. VEGF might play an important role in cellular communication because many cells in the bone–bone-marrow organ are responsive to and/or produce this growth factor. VEGF is expressed by hypertrophic chondrocytes (Gerber et al. 1999), (differentiating) osteoblasts, and osteoclasts (Horner et al. 1999) and significantly regulates capillary invasion during bone development, thereby triggering cartilage remodeling and bone formation. The functions of VEGF in bone metabolism may extend beyond its angiogenic role. Indeed, VEGF receptors are not only present on endothelial cells but also on bone cells, suggesting an autocrine loop (Figure 4). Osteoclasts express both VEGFR-1 and VEGFR-2 (Nakagawa et al. 2000). Neuropilin-1 is expressed by osteoblasts and late hypertrophic chondrocytes during development in vivo (Colnot and Helms 2001,
Harper et al. 2001) as well as by stromal cells in vitro (Tordjman et al. 1999). The expression of VEGFR-1 and VEGFR-2 on osteoblasts is a matter of debate (Harper et al. 2001 and references therein) (Figure 4). VEGF is involved in osteoclast formation from hematopoietic stem cells and has overlapping acitivities with macrophage colony-stimulating factor in this process (Niida et al. 1999). In addition, VEGF stimulates survival and resorption activity of osteoclasts in vitro (Niida et al. 1999) and is involved in osteoclast recruitment into developing long bones (Engsig et al. 2000). In contrast to the well characterized role of VEGF in bone formation, possible effects of VEGFproducing bone cells on VEGF-responsive cells residing in the marrow (hematopoietic stem cells, EPCs, stromal cells) remain elusive (Figure 4). VEGF from bone cells may regulate vasculogenesis by acting on endothelial or hematopoietic precursors or by affecting the vasculature in the marrow space (see above). • Signals from Outside the Bone Marrow Controlling EPC Mobilization Which signals determine where and when EPCs initiate vasculogenesis is largely a mystery. A variety of growth factors have been implicated in EPC mobilization, including VEGF, FGF-2, GM-CSF, granulocyte colony stimulating factor (G-CSF), and angiopoietins (Kalka et al. 2000b, Kocher et al. 2001, Takahashi et al. 1999). Ischemia or vascular trauma may be a significant stimulus for mobilization of EPCs from the bone marrow, as evidenced by the increased recruitment of EPCs in corneal angiogenesis when hindlimb ischemia was also present in mice (Takahashi et al. 1999) as well as by the rapid EPC mobilization in patients with vascular trauma (Gill et al. 2001). Recently it was reported that elevation of VEGF plasma levels results in rapid recruitment of both hematopoietic stem cells and EPCs, whereas increased Ang-1 plasma levels caused a delayed mobilization of both precursor cell types (Hattori et al. 2001). The presence of tyrosine kinase receptors for both angiogenic factors on the recruited cells suggests a direct interaction with these growth factors.
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• EPCs and Bone Marrow Cells Create an Angiogenic Microenvironment Labeling of donor progenitors revealed that these cells incorporate into newly formed vessels after transplantation. In a recent study, Kocher et al. (2001) reported that transplantation of G-CSF– mobilized CD341 human cells (containing both EPCs and hematopoietic stem cells) stimulated vascularization both in myocardial infarcts and infarct borders. Intriguingly, endothelial cells of donor (human) origin were present in the core of the infarct, whereas endothelial cells from host (rat) origin contributed to new vessels in the infarct border. This suggests that bone-marrow–derived progenitors directly contribute to in situ vessel formation (vasculogenesis) in the core region and stimulate angiogenic sprouting from resident endothelium (angiogenesis) in the peri-infarct area, possibly by secretion of angiogenic growth factors. Hematopoietic stem cells are known to secrete VEGF, especially after stimulation with different cytokines including G-CSF (Bautz et al. 2000). PlGF might also be one of the trophic factors secreted by bone-marrow–derived (stem) cells as transplantation of wildtype bone marrow partially restored the impaired neovascularization of matrigel implants in PlGF-deficient mice (Carmeliet et al. 2001). Hematopoietic and endothelial stem cells not only share a common origin; the former can also stimulate the assembly of endothelial cells into nascent blood vessels in the embryo. Indeed, hematopoietic stem cells are present at sites of active vascular expansion. By producing Ang-1, these cells stimulated endothelial growth in the embryo (Takakura et al. 2000). Hematopoietic stem cells are also present in adult bone marrow and can be mobilized in response to angiogenic factors such as VEGF and Ang-1 (Hattori et al. 2001). Whether hematopoietic stem cells also contribute to postnatal angiogenesis remains to be seen. Bone marrow contains mesenchymal stem cells, which may act as feeder cells for EPCs or hematopoietic stem cells, which in turn might secrete angiogenic growth factors. Bone marrow–derived macrophages might also contribute to neovascularization by in situ transdifferentiation to endothelial-like cells, as recently reported by Schmeisser et al. (2001).
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• VEGF: More Than an Angiogenic Factor during Adult Life It has long been thought that the stimulating effects of VEGF on postnatal neovascularization could be fully attributed to its influence on mature endothelial cells present at the site of neovascularization. However, recent findings indicate that the impact of VEGF on neovascularization during adult life is the combined result of angiogenesis and vasculogenesis, the latter by controlling EPCs. Indeed, VEGF might be one of the key factors in EPC regulation and influence these cells at different levels on their way from the bone marrow to the site of neovascularization. In the bone– bone-marrow compartment, different cell types, including bone cells, secrete and are responsive to VEGF (Figure 4). The presence of this growth factor might affect EPC recruitment in different ways by exhibiting a chemoattractive effect, inducing EPC proliferation, influencing the bone marrow/blood barrier (through its effects on vascular permeability), expanding the bone marrow vasculature, or by modulating expression of adhesion molecules on BMECs. VEGF secreted outside this compartment (i.e., in the ischemic tissue) can mobilize EPCs as well as HSCs. These cells, in turn, can be an additional source of VEGF, thereby creating a local angiogenic environment. An intriguing question is whether VEGF analogues, such as PlGF, VEGFB, VEGF-C, and VEGF-D, equally affect EPC regulation.
• Adult Bone Marrow: A Source of EPCs and Other Stem Cells The concept that pluripotent stem cells, able to differentiate to most cell types, would only exist in the embryo has been challenged by recent studies that show that adult stem cells also differentiate to distinct cell types under the influence of the local environment (Blau et al. 2001). The adult bone marrow not only harbors hematopoietic stem cells but also precursor cells able to differentiate into muscle, liver, neuronal cells of the brain, and cells of the vessel wall (endothelial cells and SMCs). When bone marrow cells are cultured under specific conditions, progenitors of particular lineages can be enriched. For instance, peripheral blood mononuclear cells from adult
humans can be enriched for EPCs by supplementation of VEGF, FGF-2, insulinlike growth factor, and epidermal growth factor to the culture medium for 7–10 days. After local injection in vivo, these cells contributed to the formation of new vessels in the ischemic limb (Kalka et al. 2000a). Recent publications further indicated that, after heterotypic cardiac (Hillebrands et al. 2001, Saiura et al. 2001) or aortic (Hillebrands et al. 2001) transplantation in mice, most of the neointimal a-actin positive SMCs in the donor coronary arteries or aortas were from host origin, suggesting that these SMCs might at least in part be derived from bone marrow–derived smooth muscle progenitor cells. In support of this concept, transplantation of b-galactosidase–expressing bone marrow into irradiated aortic allograft recipient mice revealed that part of the neointimal SMC-like population consisted of marrow-derived cells (Shimizu et al. 2001). Orlic et al. (2001) demonstrated that intracardial delivery of Lin2c-Kit1 cells stimulated both new vessel growth and de novo generation of cardiomyocytes. In a similar study, CD342/lowc-Kit1Sca-11 cells differentiated into cardiomyocytes and endothelial cells in the ischemic myocardium (Jackson et al. 2001). Studies from our own laboratory demonstrated the essential role of bone marrow–derived cells in the revascularization of the ischemic myocardium as well as in infarct healing (Heymans et al. 1999). These studies revealed that the defective healing and revascularization response in u-PA– deficient mice could be rescued by transplantation of whole bone marrow from wild-type littermates. Although neutrophils and monocytes were involved, we cannot exclude that bone marrow–derived EPCs or other cells also contributed to infarct revascularization and healing. It remains to be determined whether unfractionated bone marrow might be better suited to regenerate new vessels, precisely because all cell types (endothelial and hematopoietic precursors, stromal cells) synergize in new vessel growth. • Bone Marrow and EPCs: Therapeutic Potential Hopes are raised that EPCs and bone marrow cells will be of therapeutic value in the future. Reports on the contribuTCM Vol. 12, No. 2, 2002
tion of these progenitors to neovascularization have been quite variable. After transplantation of human CD341 cells into nude rats, human cells were detected in 25% of newly formed vessels in the infarcted myocardium (Kocher et al. 2001). In hind limb ischemia, transplantation of human CD341 cells into nude mice resulted in incorporation of human cells in ,13% of the capillaries in the ischemic limbs (Asahara et al. 1997). After ex vivo expansion of human EPCs (by culture for 7–10 days in the presence of angiogenic growth factors), human cells were found in ,56% of the vessels of the ischemic limb (Kalka et al. 2000a). These percentages reflect the number of vessels containing at least one EPCderived endothelial cell per vessel but do not necessarily imply that the entire new vessel was derived from EPCs. However, the percentages mentioned above might be an underestimation because EPCs and/or other bone marrow cells may provide a local source of angiogenic factors that further enhance new vessel formation (see above). Importantly, the transplanted cells should contribute to the formation of functional vessels that restore blood flow to the ischemic region and improve cardiac or limb function. Injection of unfractionated bone marrow significantly improved blood flow and increased running time on a motor-driven treadmill in rats with hind limb ischemia (Ikenaga et al. 2001). In other studies, transplantation of either ex vivo expanded hEPCs (Kawamoto et al. 2001), freshly isolated CD341 cells (Kocher et al. 2001), or total bone marrow (Fuchs et al. 2001) resulted in increased vessel formation and improved cardiac function, indicating that the newly formed vessels were functional. Cell transplantation might complement current strategies of therapeutic angiogenesis, on the basis of the administration of recombinant growth factors or on gene transfer, for patients in whom resident endothelial cells fail to sufficiently respond to growth factor treatment. Because animal studies have demonstrated that atherosclerosis, diabetes, aging, etc. impair the angiogenic response, EPC-transplantation might expand the therapeutic armamentarium to improve perfusion of ischemic tissues by providing a new set of endothelial cells capable of better responding to angiogenic stimuli. The function of vascu-
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lar progenitors may also be impaired. For instance, in vitro studies revealed that insulin plays a significant role in EPC function and differentiation, suggesting that EPC function may be impaired in diabetics (Schatteman et al. 2000). Moreover, a recent study by Vasa et al. (2001) reported an inverse correlation between the number of cardiovascular risk factors (including diabetes and hypertension) and the number and migratory activity of EPCs, implying that cell transplantation might be an appropriate strategy for revascularization in patients with coronary artery disease. In addition, EPCs might be used as vectors to deliver angiogenic factors to the ischemic tissue, thereby providing an additional stimulus for neovascularization. A recent study in primates proves that CD341 cell-mediated gene delivery into sites of active angiogenesis is a feasible strategy (Gomez-Navarro et al. 2000).
References Asahara T, Masuda H, Takahashi T, et al.: 1999. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 85: 221–228. Asahara T, Murohara T, Sullivan A, et al.: 1997. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275:964–967. Bautz F, Rafii S, Kanz L, Mohle R: 2000. Expression and secretion of vascular endothelial growth factor-A by cytokine-stimulated hematopoietic progenitor cells: possible role in the hematopoietic microenvironment. Exp Hematol 28:700–706. Bianco P, Riminucci M, Kuznetsov S, Robey PG: 1999. Multipotential cells in the bone marrow stroma: regulation in the context of organ physiology. Crit Rev Eukaryot Gene Expr 9:159–173. Blau HM, Brazelton TR, Weimann JM: 2001. The evolving concept of a stem cell: entity or function? Cell 105:829–841. Carmeliet P: 1999. Basic concepts of (myocardial) angiogenesis: role of vascular endothelial growth factor and angiopoietin. Curr Interv Cardiol 1:322–335. Carmeliet P: 2000a. Developmental biology: one cell, two fates. Nature 408:43, 45. Carmeliet P: 2000b. Mechanisms of angiogenesis and arteriogenesis. Nat Med 6:389– 395. Carmeliet P, Moons L, Luttun A, et al.: 2001. Synergism between vascular endothelial
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zation of VEGFR2(1)AC133(1) endothelial precursor cells. Circ Res 88:167–174. Gittenberger-de Groot AC, DeRuiter MC, Bergwerff M, Poelmann RE: 1999. Smooth muscle cell origin and its relation to heterogeneity in development and disease. Arterioscler Thromb Vasc Biol 19:1589–1594. Gomez-Navarro J, Contreras JL, Arafat W, et al.: 2000. Genetically modified CD341 cells as cellular vehicles for gene delivery into areas of angiogenesis in a rhesus model. Gene Ther 7:43–52. Harper J, Gerstenfeld LC, Klagsbrun M: 2001. Neuropilin-1 expression in osteogenic cells: down-regulation during differentiation of osteoblasts into osteocytes. J Cell Biochem 81:82–92. Hattori K, Dias S, Heissig B, et al.: 2001. Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells. J Exp Med 193:1005–1014. Hellstrom M, Kal NM, Lindahl P, et al.: 1999. Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development 126:3047–3055. Heymans S, Luttun A, Nuyens D, et al.: 1999. Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. Nat Med 5:1135–1142. Hillebrands JL, Klatter FA, van Den Hurk BM, et al.: 2001. Origin of neointimal endothelium and alpha-actin-positive smooth muscle cells in transplant arteriosclerosis. J Clin Invest 107:1411–1422. Hirschi KK, Rohovsky SA, D’Amore PA: 1998. PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J Cell Biol 141:805–814. Horner A, Bishop NJ, Bord S, et al.: 1999. Immunolocalisation of vascular endothelial growth factor (VEGF) in human neonatal growth plate cartilage. J Anat 194:519–524. Ikenaga S, Hamano K, Nishida M, et al.: 2001. Autologous bone marrow implantation induced angiogenesis and improved deteriorated exercise capacity in a rat ischemic hindlimb model. J Surg Res 96: 277–283. Jackson KA, Majka SM, Wang H, et al.: 2001. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J Clin Invest 107:1395–1402. Kalka C, Masuda H, Takahashi T, et al.: 2000a. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA 97:3422–3427.
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Kalka C, Tehrani H, Laudenberg B, et al.: 2000b. VEGF gene transfer mobilizes endothelial progenitor cells in patients with inoperable coronary disease. Ann Thorac Surg 70:829–834. Kawamoto A, Gwon HC, Iwaguro H, et al.: 2001. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 103:634–637. Kocher AA, Schuster MD, Szabolcs MJ, et al.: 2001. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 7:430–436. Krebs LT, Xue Y, Norton CR, et al.: 2000. Notch signaling is essential for vascular morphogenesis in mice. Genes Dev 14: 1343–1352. Leimeister C, Schumacher N, Steidl C, Gessler M: 2000. Analysis of HeyL expression in wild-type and Notch pathway mutant mouse embryos. Mech Dev 98:175–178. Lin Y, Weisdorf DJ, Solovey A, Hebbel RP: 2000. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest 105:71–77. Moyon D, Pardanaud L, Yuan L, et al.: 2001. Plasticity of endothelial cells during arterialvenous differentiation in the avian embryo. Development 128:3359–3370. Naiyer AJ, Jo DY, Ahn J, et al.: 1999. Stromal derived factor-1-induced chemokinesis of cord blood CD34(1) cells (long-term cultureinitiating cells) through endothelial cells is mediated by E-selectin. Blood 94:4011–4019. Nakagawa M, Kaneda T, Arakawa T, et al.: 2000. Vascular endothelial growth factor (VEGF) directly enhances osteoclastic bone resorption and survival of mature osteoclasts. FEBS Lett 473:161–164. Nakajima Y, Mironov V, Yamagishi T, et al.: 1997. Expression of smooth muscle alphaactin in mesenchymal cells during formation of avian endocardial cushion tissue: a role for transforming growth factor beta3. Dev Dyn 209:296–309. Niida S, Kaku M, Amano H, et al.: 1999. Vascular endothelial growth factor can substitute for macrophage colony-stimulating factor in the support of osteoclastic bone resorption. J Exp Med 190:293–298. Orlic D, Kajstura J, Chimenti S, et al.: 2001. Bone marrow cells regenerate infarcted myocardium. Nature 410:701–705. Peichev M, Naiyer AJ, Pereira D, et al.: 2000. Expression of VEGFR-2 and AC133 by circulating human CD34(1) cells identifies a population of functional endothelial precursors. Blood 95:952–958. Poole TJ, Finkelstein EB, Cox CM: 2001. The role of FGF and VEGF in angioblast induction and migration during vascular development. Dev Dyn 220:1–17.
Roman BL, Weinstein BM: 2000. Building the vertebrate vasculature: research is going swimmingly. Bioessays 22:882–893. Saiura A, Sata M, Hirata Y, et al.: 2001. Circulating smooth muscle progenitor cells contribute to atherosclerosis. Nat Med 7:382–383. Schatteman GC, Hanlon HD, Jiao C, et al.: 2000. Blood-derived angioblasts accelerate blood-flow restoration in diabetic mice. J Clin Invest 106:571–578. Schmeisser A, Garlichs CD, Zhang H, et al.: 2001. Monocytes coexpress endothelial and macrophagocytotic lineage markers and form cord-like structures in matrigel under angiogenic conditions. Cardiovasc Res 49:639–680. Shen H, Cheng T, Olszak I, et al.: 2001. Cxcr-4 desensitization is associated with tissue localization of hemopoietic progenitor cells. J Immunol 166:5027–5033. Shimizu K, Sugiyama S, Aikawa M, et al.: 2001. Host bone-marrow cells are a source of donor intimal smooth-muscle-like cells in murine aortic transplant arteriopathy. Nat Med 7:738–741. Shin D, Garcia-Cardena G, Hayashi S, et al.: 2001. Expression of ephrinB2 identifies a stable genetic difference between arterial and venous vascular smooth muscle as well as endothelial cells, and marks subsets of microvessels at sites of adult neovascularization. Dev Biol 230:139–150. Takahashi T, Kalka C, Masuda H, et al.: 1999. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 5:434–438. Takakura N, Watanabe T, Suenobu S, et al.: 2000. A role for hematopoietic stem cells in promoting angiogenesis. Cell 102:199–209. Tordjman R, Delaire S, Plouet J, et al.: 2001. Erythroblasts are a source of angiogenic factors. Blood 97:1968–1974. Tordjman R, Ortega N, Coulombel L, et al.: 1999. Neuropilin-1 is expressed on bone marrow stromal cells: a novel interaction with hematopoietic cells? Blood 94:2301–2309. Urness LD, Sorensen LK, Li DY: 2000. Arteriovenous malformations in mice lacking activin receptor-like kinase-1. Nat Genet 26:328–331. Vasa M, Fichtlscherer S, Aicher A, et al.: 2001. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res 89:E1–E7. Wilkinson DG: 2000. Eph receptors and ephrins: regulators of guidance and assembly. Int Rev Cytol 196:177–244. Yamashita J, Itoh H, Hirashima M, et al.: 2000. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 408:92–96. PII S1050-1738(01)00152-9
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Vascular Progenitors: From Biology to Treatment Aernout Luttun, Geert Carmeliet, and Peter Carmeliet*
The identification of endothelial progenitor cells (EPCs) in the adult human has forced us to reconsider how new blood vessels grow in physiological and pathological conditions in the adult human. An important question in angiogenesis research is to what extent these progenitors functionally contribute to revascularization of ischemic tissues and and to what extent they can be used for therapeutic angiogenic cell transplantation. Even more challenging is the concept that hematopoietic and other bone-marrow–derived stem cells might be recruited in the context of ischemia to induce neovessel formation. This review discusses some of the recent insights and outstanding questions on EPCs, both from a biological and therapeutic perspective. (Trends Cardiovasc Med 2002;12:88–96). © 2002, Elsevier Science Inc.
• Endothelial Progenitors in the Embryo Endothelial cells, lining the inside of blood vessels (endo-, inside), arise from mesodermal precursor cells, the angioblasts. These precursors have the capacAernout Luttun and Peter Carmeliet are from the Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium. Geert Carmeliet is from the Laboratory of Experimental Medicine and Endocrinology, Leuven, Belgium. * Address correspondence to: P. Carmeliet, Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, KU Leuven, Campus Gasthuisberg, Herestraat 49, B-3000, Leuven, Belgium. Tel: (132) 16-34.57.72; fax: (132) 16-34.59.90; e-mail: peter.carmeliet@med. kuleuven.ac.be. © 2002, Elsevier Science Inc. All rights reserved. 1050-1738/02/$-see front matter
ity to proliferate, migrate, and differentiate into endothelial cells but have not yet acquired characteristic mature endothelial markers. In vitro differentiation studies and studies in zebrafish have indicated that endothelial and blood cell (hematopoietic) precursors originate from a common ancestor, the hemangioblast (Figure 1). Hemangioblasts undergo their first critical steps of differentiation within the blood islands. Cells at the perimeter of the blood islands give rise to precursors for endothelial cells, whereas those in the center constitute hematopoietic precursors. 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. These include Ets-1, Fli1, Hex or Hhex , Vezf-1, Hox, a4-integrin, members of the GATA-family, basic helixloop-helix (bHLH) factors (like SCL/
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Tal-1), and the Id-proteins and hedgehog protein Indian Hedgehog (Carmeliet 2000b, Dyer et al. 2001). Studies in quail/chick chimeras have shown that the fibroblast growth factor-2 (FGF-2) mediates the induction of angioblasts from the mesoderm (Poole et al. 2001). In addition, embryonic angioblasts express the receptor-2 for vascular endothelial growth factor (VEGFR-2; flk-1) and divide in response to VEGF, a pleiotropic angiogenic factor in vitro. In addition, in vivo studies in the Xenopus embryo have documented a role for VEGF in mediating angioblast migration (Poole et al. 2001). Its role may, however, not relate to determining endothelial fate because endothelial cells still differentiate in embryos lacking VEGF. Inactivation of the VEGFR-2 gene in mice results in embryonic lethality, with lack of development of both hematopoietic and endothelial cell lineages, supporting the critical importance of this receptor at that developmental stage, although not defining the steps regulating differentiation into endothelial versus hematopoietic cell. However, because VEGFR-2 deficiency blocks differentiation of blood and vascular cells, other VEGFR-2 ligands may be essential for endothelial cell fate in vivo. Angioblast differentiation may be promoted by VEGF, FGF-2, and VEGFR-2, whereas VEGF receptor-1 (VEGFR-1; flt-1) has been determined to suppress hemangioblast commitment (Ferrara 2001). • Arterial and Venous Angioblasts Following commitment to the endothelial lineage, angioblasts assemble into a primitive vascular plexus of veins and arteries, a process called vasculogenesis. This primitive vasculature is subsequently refined into a functional network by angiogenesis (vascular sprouting from preexisting vessels, vascular fusion and intussusception) and by remodeling and “muscularization” (arteriogenesis) of newly formed vessels (Figure 2) (Carmeliet 2000b). Whether endothelial cells in veins and arteries have distinct precursors remains to be determined (Figure 1). The specification between vessels conducting blood from or to the heart (arteries and veins, respectively) occurs very early during embryonic development. Only recently have possible molecular players in this process been TCM Vol. 12, No. 2, 2002
suggested. The existence of molecular differences in arterial and venous endothelium before vessel formation implies that establishing the identity of arteries and veins is independent of blood flow and pressure. Genetic studies in the zebrafish have identified the bHLH transcription factor gridlock A as a possible candidate, with gridlock A favoring differentiation of prearterial at the expense of prevenous angioblasts (Wilkinson 2000). Notch-derived signals, which are often involved in cell fate determination via lateral specification and inductive signaling between distinct cell types, might also be implicated. Recent evidence indicates that certain Notch ligands such as DeltaC (Wilkinson 2000) are expressed in arterial endothelium and that defective Notch signaling caused vascular remodeling defects (Krebs et al. 2000). The hairy-related bHLH factor HeyL, an effector of Notch, is expressed in smooth muscle of all arteries, overlapping with that of Notch3, mutations of which underlie the cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy vascular (CADASIL) disorder (Leimeister et al. 2000). The TGF-b receptor activin receptor-like kinase 1 also appears to control early arterial differentiation (Urness et al. 2000). There is increasing evidence that members of the large ephrin family may also play a role (Roman and Weinstein 2000, Wilkinson 2000). Ephrins are ligands for their corresponding Eph receptors, comprising a family of at least 14 receptor tyrosine kinases. Ephrins activate their receptors when membrane bound. Ephrin A1 and ephrin A2 are angiogenic in different vascular beds. Ephrins B1 and B2 also induce vascular sprouting. During embryonic development, expression of ephrin B2 is restricted to endothelial cells in arteries at early stages and progressively extends to also marking arterial smooth muscle cells (SMCs) (Gale et al. 2001). In addition, ephrin B2 continues to selectively mark arteries in the adult with expression in endothelial cells, SMCs and pericytes (Gale et al. 2001, Shin et al. 2001). In contrast, its receptor, EphB4, is found primarily in veins. Gene inactivation of either ephrin B2 or of EphB4 results in normal vasculogenesis but abnormal angiogenesis, the latter with disrupted remodeling of both arteries and veins
into large and small branches. Complex ligand-receptor bidirectional signaling interactions via ephrins and ephrin receptors have been described, with responses dependent on a variety of factors, including phosphorylation, multimerization, and the presence of adaptor proteins such as Grb2, Grb10, and Nck. A novel cytoplasmic tyrosine kinase gene, bone marrow tyrosine kinase (Bmx), was identified in arterial endothelium (Ekman et al. 1997). Because its loss did not affect physiological growth of arteries in knockout mice, the function of Bmx needs to be further defined. We have found that selective loss of the VEGF164 isoform in mice impairs arterial development in the retina (unpublished observations in collaboration with P. D’Amore). Intriguingly, neuropilin-1, a VEGF164-isoform specific receptor, was also more abundantly expressed in arterioles (unpublished observations, Moyon et al. 2001). To what extent vascular growth factors selectively affect arterial or venous growth in the adult remains to be established. Future studies will be required to unravel the differentiation characteristics of veins, arteries, and capillaries. • Smooth Muscle Progenitors in the Embryo Endothelial channels are covered by multiple layers of SMCs in large vessels in proximal parts of the vasculature and by single pericytes around smaller distal vessels. These mural cells have a complex origin, depending on their location in the embryo (Gittenberger-de Groot et al. 1999) (Figure 1). The first SMCs around endothelial tubes in the embryo transdifferentiate from the endothelium (Gittenberger-de Groot et al. 1999). Endothelial cells also transform to smoothmuscle–like myofibroblasts in the prospective cardiac valves, a process involving signaling by transforming growth factor-b3 (TGF-b3) (Nakajima et al. 1997). TGF-b1, another family member, has been implicated in the differentiation of a mesenchymal stem cell to a progenitor that expresses platelet-derived growth factor receptor-b (PDGFR-b) (Hellstrom et al. 1999, Hirschi et al. 1998). By PDGF-BB, endothelial cells stimulate subsequent growth and differentiation of this precursor. Pericytes and SMCs of the coronary vessels are derived from a
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Figure 1. A common origin for the two types of blood-vessel cell. Endothelial and smooth muscle cells (SMCs) arise from separate types of precursor. Endothelial cells arise from precursors called angioblasts or hemangioblasts in the embryo, or from circulating endothelial progenitors in the adult. Angioblasts give rise to arterial and venous lineages. SMCs and pericytes, by contrast, can form from a variety of progenitors. These include mesenchymal stem cells, neural crest cells, and progenitors in the epicardium in the embryo. A new common vascular progenitor cell that gives rise to both types of blood-vessel cell has been recently identified. Whether this vascular progenitor and the hemangioblast both arise from a multipotent stem cell remains elusive. Vascular endothelial growth factor (VEGF) promotes the development of endothelial cells from this precursor. TGF-b1 has been involved in differentiation of mesenchymal stem cells to progenitors that express the receptor for PDGF-BB. The latter stimulates their development into SMCs and pericytes and is responsible for their recruitment around nascent vessels. Circulating endothelial progenitors have been identified postnatally, but the persistence of smooth muscle or common vascular progenitors in adult life remains elusive. Adapted from Carmeliet (2000a).
putative progenitor that infiltrates the heart from its external (epicardial) layers (Gittenberger-de Groot et al. 1999). Coronary vein SMCs are derived from the atrial myocardium, whereas those of the coronary arteries come from the epicardial layer (Dettman et al. 1998). Cardiac neural crest cells are the source of SMCs of the large thoracic blood vessels (Creazzo et al. 1998). Whereas the periendothelial cells in the dorsal vascular compartment of the brain have a mesodermal origin, vessels in the forebrain and the retinae derive their pericytes and SMCs from cephalic neural crest cells (Etchevers et al. 2001). Whether SMCs and pericytes in arteries, veins, and lymphatic vessels or even within the inner and medial layer of an artery arise from distinct progenitors remains unknown. • Common Vascular Progenitors in the Embryo Yamashita et al. (2000) discovered an embryonic common vascular progenitor that differentiates into endothelial and SMCs. The SMCs arising from these progenitors were not simply transdifferentiated endothelial cells expressing the atypical smooth muscle a-actin marker. Instead, they expressed an entire set of smooth muscle markers and surrounded endothelial channels in vivo. This vascular progenitor resembles the putative common precursor of the endothelial cells that line the inner surface of the heart (the endocardium) and their surrounding cardiac muscle fibers. Like these hemangioblasts, vascular progenitors express VEGFR-2 (Yamashita et al. 2000), raising the question of whether
both cell types arise from a multipotent stem cell. The vascular progenitors differentiated to endothelial cells in response to VEGF, whereas they developed into SMCs in response to PDGF-BB (Yamashita et al. 2000). It is possible that PDGF-BB is a determinant of smooth muscle cell fate, but PDGFRb-positive progenitors still develop in the absence of PDGF-BB in vivo (Hellstrom et al. 1999) and in vitro (Yamashita et al. 2000). Thus, PDGF-BB may favor the selection and growth of PDGFRb-expressing progenitors. Such a hypothesis is consistent with a model whereby PDGF-BB stimulates PDGFRb-expressing progenitors to migrate along preexisting endothelial channels and to divide during arterial enlargement (Hellstrom et al. 1999). A common vascular progenitor could contribute to the formation of naked endothelial capillaries (angiogenesis) and muscle-coated vessels (arteriogenesis). It remains to be determined whether these common precursors or smooth muscle cell progenitors persist during adult life and whether these cells contribute to postnatal vessel growth (see below) (Figure 1). • Circulating Endothelial Progenitors in the Adult Neovascularization during adult life has long been attributed to angiogenesis only. However, recent studies have revealed that EPCs also circulate postnatally in the peripheral blood and may be recruited and incorporated into sites of active neovascularization in ischemic hindlimbs, ischemic myocardium, injured corneas, cutaneous wounds, and
tumor vasculature (Asahara et al. 1999, Kalka et al. 2000a, Schatteman et al. 2000, Takahashi et al. 1999), a process termed postnatal vasculogenesis (Figures 1 and 3). In addition, EPCs have been shown to be involved in maintenance angiogenesis replacing lost endothelial cells and in reendothelialization of implants (Peichev et al. 2000 and references therein). EPCs were initially identified and isolated on the basis of their expression of VEGFR-2 and CD34, antigens shared by the angioblast and the hematopoietic progenitor (Asahara et al. 1997). These EPCs were subsequently shown to express VE-cadherin, a junctional molecule, and AC133, an orphan receptor that is specifically expressed on EPCs but whose expression is lost once they differentiate into more mature endothelial cells (Peichev et al. 2000). Their high proliferation rate distinguishes circulating marrow-derived EPCs in the adult from mature endothelial cells shed from the vessel wall (Lin et al. 2000). Thus far, a bipotential common vascular progenitor, giving rise to both endothelial and periendothelial cells, has not been documented in the adult (Figure 1). • Local Signals in the Bone Marrow Compartment Controlling EPC Mobilization The majority of circulating EPCs reside in the bone marrow in close association with hematopoietic stem cells and the bone marrow stroma that provides the optimal microenvironment for hematopoiesis (Figure 4). Several observations suggest that hematopoietic (stem)
Figure 2. Angioblasts in the embryo assemble in a primitive network (vasculogenesis) that expands and remodels (angiogenesis). Smooth muscle cells (SMCs), originating from SMC precursors, cover endothelial cells during vascular myogenesis and stabilize vessels during arteriogenesis. CL, collagen; EL, elastin; Fib, fibrillin. Adapted from Carmeliet (1999).
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Figure 3. Pathological vascular growth in the adult may occur via vasculogenesis (endothelial progenitor cell [EPC] mobilization), angiogenesis (sprouting) or arteriogenesis (collateral growth). Mf, macrophages; SMC, smooth muscle cells. Adapted from Carmeliet (1999).
Figure 4. Bone and bone marrow constitute a single organ (the “bone–bonemarrow organ”) in which marrow cells reside in close vicinity to bone cells. Within the marrow, hematopoietic stem cells (HSC) produce the angiogenic factor VEGF, and erythroblasts (EB) are a source of both VEGF and its homologue PlGF. These angiogenic factors can influence the bone marrow microenvironment in different ways. First, they can induce stroma cells (expressing neuropilin-1 [NP-1]) to produce hematopoietic growth factors like thrombopoietin (Tpo) and Flt3-L and induce bone marrow endothelial cells (BMEC) (expressing VEGFR-1 and VEGFR-2) to secrete GM-CSF. Second, they can act as survival and proliferation factors for HSCs and endothelial progenitor cells (EPC), which both express VEGFR-2. Third, they might influence EPC or HSC mobilization by regulating adhesion molecule levels like E-selectin on BMECs or by expanding the bone-marrow vasculature. In addition, different bone cells express VEGF-receptors. Osteoclasts (OC) express VEGFR-1 and VEGFR-2, and NP-1 is expressed on osteoblasts (OB). Expression of VEGFR-1 and VEGFR-2 on OB is a matter of debate. Whereas VEGF has been shown to be involved in bone formation by influencing OB differentiation and OC recruitment and survival, it remains to be determined whether OB and OC, which can produce VEGF, also influence the bone marrow microenvironment. Left: Cellular organization of the bone–bone-marrow organ. Upper right: Distinct bone marrow cell types producing and responding to VEGF/PlGF. Lower right: Interaction between the bone and bone marrow cells producing and responding to VEGF/PlGF. Dotted lines represent hypothetical interactions. The left panel was adapted with permission from Bianco et al. (1999).
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cells as well as stroma cells might contribute to the local proliferation and transmigration of EPCs across the bonemarrow/blood barrier. Angiogenic factors are produced in the bone marrow, and their receptors are expressed on different cell types. Hematopoietic stem cells secrete VEGF, which is significantly upregulated upon cytokine stimulation (Bautz et al. 2000). VEGF, in turn, induces the release of hematopoietic growth factors, such as granulocyte macrophagecolony stimulating factor (GM-CSF) by bone marrow endothelial cells (BMECs) (Bautz et al. 2000) and thrombopoietin and Flt3-L by stromal cells (Tordjman et al. 1999). Recently, erythroblasts were shown to produce VEGF and placental growth factor (PlGF) (Tordjman et al. 2001), a VEGF homologue that specifically modulates VEGF function during pathological conditions (Carmeliet et al. 2001). VEGF-R2 is expressed on EPCs and a subset of hematopoietic stem cells (Hattori et al. 2001, Peichev et al. 2000). Neuropilin-1, a receptor for both VEGF and PlGF, is expressed on stroma cells (Tordjman et al. 1999) (Figure 4). VEGF has been shown to stimulate growth of embryonic angioblasts (see above). In addition, VEGF and angiopoietin (Ang)1 exert a synergistic effect in promoting the survival of bone marrow–derived hematopoietic progenitor cells (Hattori et al. 2001). Moreover, these growth factors might increase recruitment of bone marrow cells by expansion of the bone marrow vasculature (Hattori et al. 2001). To allow selective migration of hematopoietic stem cells through the bone marrow/blood barrier, BMECs constitutively express adhesion molecules such as E-selectin and vascular adhesion molecule-1. Down- or upregulation of these adhesion molecules or their corresponding ligands may contribute to the regulation of transendothelial migration of hematopoietic stem cells, and analogous mechanisms might be involved in EPC transmigration. VEGF has been shown to induce expression of E-selectin on BMECs (Naiyer et al. 1999). In addition to the contribution of adhesion molecules, paracrine chemokines and proteinases also influence stem cell homing and mobilization. Virtually all CD341VEGFR-21 cells (a cell population that includes EPCs) express CXCR-4, a receptor for the chemokine stromal-derived factor (SDF)-1 and miTCM Vol. 12, No. 2, 2002
grate in response to SDF-1 (Peichev et al. 2000), a factor that is important for HSC homing into the bone marrow. Desensitization of CXCR-4 results in mobilization of hematopoietic progenitors from the bone marrow into the peripheral blood (Shen et al. 2001). Interleukin8 induces rapid release of matrix metalloproteinase (MMP)-9 concurrent with the mobilization of hematopoietic precursors, consistent with an MMPmediated release of these precursor cells from the extracellular matrix (Fibbe et al. 2000). • Possible Interaction Between Bone Cells and the Bone Marrow Compartment Bone and bone marrow together form a single organ (the “bone–bone-marrow organ”) in which marrow cells and bone lining cells (osteoblasts and osteoclasts) reside in close contact with each other (Figure 4) (Bianco et al. 1999). The cells within the marrow cavity form a continuous network in which stromal cells are interspersed with hematopoietic and endothelial progenitors (Figure 4). Stromal cells lining the endothelium of the marrow vasculature are related to pericytes and have the potential to differentiate into skeletal tissue components such as bone and cartilage as well as adipocytes. Although a physical continuity exists between bone and marrow, the interaction between the different cell types is only now being unraveled. VEGF might play an important role in cellular communication because many cells in the bone–bone-marrow organ are responsive to and/or produce this growth factor. VEGF is expressed by hypertrophic chondrocytes (Gerber et al. 1999), (differentiating) osteoblasts, and osteoclasts (Horner et al. 1999) and significantly regulates capillary invasion during bone development, thereby triggering cartilage remodeling and bone formation. The functions of VEGF in bone metabolism may extend beyond its angiogenic role. Indeed, VEGF receptors are not only present on endothelial cells but also on bone cells, suggesting an autocrine loop (Figure 4). Osteoclasts express both VEGFR-1 and VEGFR-2 (Nakagawa et al. 2000). Neuropilin-1 is expressed by osteoblasts and late hypertrophic chondrocytes during development in vivo (Colnot and Helms 2001,
Harper et al. 2001) as well as by stromal cells in vitro (Tordjman et al. 1999). The expression of VEGFR-1 and VEGFR-2 on osteoblasts is a matter of debate (Harper et al. 2001 and references therein) (Figure 4). VEGF is involved in osteoclast formation from hematopoietic stem cells and has overlapping acitivities with macrophage colony-stimulating factor in this process (Niida et al. 1999). In addition, VEGF stimulates survival and resorption activity of osteoclasts in vitro (Niida et al. 1999) and is involved in osteoclast recruitment into developing long bones (Engsig et al. 2000). In contrast to the well characterized role of VEGF in bone formation, possible effects of VEGFproducing bone cells on VEGF-responsive cells residing in the marrow (hematopoietic stem cells, EPCs, stromal cells) remain elusive (Figure 4). VEGF from bone cells may regulate vasculogenesis by acting on endothelial or hematopoietic precursors or by affecting the vasculature in the marrow space (see above). • Signals from Outside the Bone Marrow Controlling EPC Mobilization Which signals determine where and when EPCs initiate vasculogenesis is largely a mystery. A variety of growth factors have been implicated in EPC mobilization, including VEGF, FGF-2, GM-CSF, granulocyte colony stimulating factor (G-CSF), and angiopoietins (Kalka et al. 2000b, Kocher et al. 2001, Takahashi et al. 1999). Ischemia or vascular trauma may be a significant stimulus for mobilization of EPCs from the bone marrow, as evidenced by the increased recruitment of EPCs in corneal angiogenesis when hindlimb ischemia was also present in mice (Takahashi et al. 1999) as well as by the rapid EPC mobilization in patients with vascular trauma (Gill et al. 2001). Recently it was reported that elevation of VEGF plasma levels results in rapid recruitment of both hematopoietic stem cells and EPCs, whereas increased Ang-1 plasma levels caused a delayed mobilization of both precursor cell types (Hattori et al. 2001). The presence of tyrosine kinase receptors for both angiogenic factors on the recruited cells suggests a direct interaction with these growth factors.
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• EPCs and Bone Marrow Cells Create an Angiogenic Microenvironment Labeling of donor progenitors revealed that these cells incorporate into newly formed vessels after transplantation. In a recent study, Kocher et al. (2001) reported that transplantation of G-CSF– mobilized CD341 human cells (containing both EPCs and hematopoietic stem cells) stimulated vascularization both in myocardial infarcts and infarct borders. Intriguingly, endothelial cells of donor (human) origin were present in the core of the infarct, whereas endothelial cells from host (rat) origin contributed to new vessels in the infarct border. This suggests that bone-marrow–derived progenitors directly contribute to in situ vessel formation (vasculogenesis) in the core region and stimulate angiogenic sprouting from resident endothelium (angiogenesis) in the peri-infarct area, possibly by secretion of angiogenic growth factors. Hematopoietic stem cells are known to secrete VEGF, especially after stimulation with different cytokines including G-CSF (Bautz et al. 2000). PlGF might also be one of the trophic factors secreted by bone-marrow–derived (stem) cells as transplantation of wildtype bone marrow partially restored the impaired neovascularization of matrigel implants in PlGF-deficient mice (Carmeliet et al. 2001). Hematopoietic and endothelial stem cells not only share a common origin; the former can also stimulate the assembly of endothelial cells into nascent blood vessels in the embryo. Indeed, hematopoietic stem cells are present at sites of active vascular expansion. By producing Ang-1, these cells stimulated endothelial growth in the embryo (Takakura et al. 2000). Hematopoietic stem cells are also present in adult bone marrow and can be mobilized in response to angiogenic factors such as VEGF and Ang-1 (Hattori et al. 2001). Whether hematopoietic stem cells also contribute to postnatal angiogenesis remains to be seen. Bone marrow contains mesenchymal stem cells, which may act as feeder cells for EPCs or hematopoietic stem cells, which in turn might secrete angiogenic growth factors. Bone marrow–derived macrophages might also contribute to neovascularization by in situ transdifferentiation to endothelial-like cells, as recently reported by Schmeisser et al. (2001).
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• VEGF: More Than an Angiogenic Factor during Adult Life It has long been thought that the stimulating effects of VEGF on postnatal neovascularization could be fully attributed to its influence on mature endothelial cells present at the site of neovascularization. However, recent findings indicate that the impact of VEGF on neovascularization during adult life is the combined result of angiogenesis and vasculogenesis, the latter by controlling EPCs. Indeed, VEGF might be one of the key factors in EPC regulation and influence these cells at different levels on their way from the bone marrow to the site of neovascularization. In the bone– bone-marrow compartment, different cell types, including bone cells, secrete and are responsive to VEGF (Figure 4). The presence of this growth factor might affect EPC recruitment in different ways by exhibiting a chemoattractive effect, inducing EPC proliferation, influencing the bone marrow/blood barrier (through its effects on vascular permeability), expanding the bone marrow vasculature, or by modulating expression of adhesion molecules on BMECs. VEGF secreted outside this compartment (i.e., in the ischemic tissue) can mobilize EPCs as well as HSCs. These cells, in turn, can be an additional source of VEGF, thereby creating a local angiogenic environment. An intriguing question is whether VEGF analogues, such as PlGF, VEGFB, VEGF-C, and VEGF-D, equally affect EPC regulation.
• Adult Bone Marrow: A Source of EPCs and Other Stem Cells The concept that pluripotent stem cells, able to differentiate to most cell types, would only exist in the embryo has been challenged by recent studies that show that adult stem cells also differentiate to distinct cell types under the influence of the local environment (Blau et al. 2001). The adult bone marrow not only harbors hematopoietic stem cells but also precursor cells able to differentiate into muscle, liver, neuronal cells of the brain, and cells of the vessel wall (endothelial cells and SMCs). When bone marrow cells are cultured under specific conditions, progenitors of particular lineages can be enriched. For instance, peripheral blood mononuclear cells from adult
humans can be enriched for EPCs by supplementation of VEGF, FGF-2, insulinlike growth factor, and epidermal growth factor to the culture medium for 7–10 days. After local injection in vivo, these cells contributed to the formation of new vessels in the ischemic limb (Kalka et al. 2000a). Recent publications further indicated that, after heterotypic cardiac (Hillebrands et al. 2001, Saiura et al. 2001) or aortic (Hillebrands et al. 2001) transplantation in mice, most of the neointimal a-actin positive SMCs in the donor coronary arteries or aortas were from host origin, suggesting that these SMCs might at least in part be derived from bone marrow–derived smooth muscle progenitor cells. In support of this concept, transplantation of b-galactosidase–expressing bone marrow into irradiated aortic allograft recipient mice revealed that part of the neointimal SMC-like population consisted of marrow-derived cells (Shimizu et al. 2001). Orlic et al. (2001) demonstrated that intracardial delivery of Lin2c-Kit1 cells stimulated both new vessel growth and de novo generation of cardiomyocytes. In a similar study, CD342/lowc-Kit1Sca-11 cells differentiated into cardiomyocytes and endothelial cells in the ischemic myocardium (Jackson et al. 2001). Studies from our own laboratory demonstrated the essential role of bone marrow–derived cells in the revascularization of the ischemic myocardium as well as in infarct healing (Heymans et al. 1999). These studies revealed that the defective healing and revascularization response in u-PA– deficient mice could be rescued by transplantation of whole bone marrow from wild-type littermates. Although neutrophils and monocytes were involved, we cannot exclude that bone marrow–derived EPCs or other cells also contributed to infarct revascularization and healing. It remains to be determined whether unfractionated bone marrow might be better suited to regenerate new vessels, precisely because all cell types (endothelial and hematopoietic precursors, stromal cells) synergize in new vessel growth. • Bone Marrow and EPCs: Therapeutic Potential Hopes are raised that EPCs and bone marrow cells will be of therapeutic value in the future. Reports on the contribuTCM Vol. 12, No. 2, 2002
tion of these progenitors to neovascularization have been quite variable. After transplantation of human CD341 cells into nude rats, human cells were detected in 25% of newly formed vessels in the infarcted myocardium (Kocher et al. 2001). In hind limb ischemia, transplantation of human CD341 cells into nude mice resulted in incorporation of human cells in ,13% of the capillaries in the ischemic limbs (Asahara et al. 1997). After ex vivo expansion of human EPCs (by culture for 7–10 days in the presence of angiogenic growth factors), human cells were found in ,56% of the vessels of the ischemic limb (Kalka et al. 2000a). These percentages reflect the number of vessels containing at least one EPCderived endothelial cell per vessel but do not necessarily imply that the entire new vessel was derived from EPCs. However, the percentages mentioned above might be an underestimation because EPCs and/or other bone marrow cells may provide a local source of angiogenic factors that further enhance new vessel formation (see above). Importantly, the transplanted cells should contribute to the formation of functional vessels that restore blood flow to the ischemic region and improve cardiac or limb function. Injection of unfractionated bone marrow significantly improved blood flow and increased running time on a motor-driven treadmill in rats with hind limb ischemia (Ikenaga et al. 2001). In other studies, transplantation of either ex vivo expanded hEPCs (Kawamoto et al. 2001), freshly isolated CD341 cells (Kocher et al. 2001), or total bone marrow (Fuchs et al. 2001) resulted in increased vessel formation and improved cardiac function, indicating that the newly formed vessels were functional. Cell transplantation might complement current strategies of therapeutic angiogenesis, on the basis of the administration of recombinant growth factors or on gene transfer, for patients in whom resident endothelial cells fail to sufficiently respond to growth factor treatment. Because animal studies have demonstrated that atherosclerosis, diabetes, aging, etc. impair the angiogenic response, EPC-transplantation might expand the therapeutic armamentarium to improve perfusion of ischemic tissues by providing a new set of endothelial cells capable of better responding to angiogenic stimuli. The function of vascu-
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lar progenitors may also be impaired. For instance, in vitro studies revealed that insulin plays a significant role in EPC function and differentiation, suggesting that EPC function may be impaired in diabetics (Schatteman et al. 2000). Moreover, a recent study by Vasa et al. (2001) reported an inverse correlation between the number of cardiovascular risk factors (including diabetes and hypertension) and the number and migratory activity of EPCs, implying that cell transplantation might be an appropriate strategy for revascularization in patients with coronary artery disease. In addition, EPCs might be used as vectors to deliver angiogenic factors to the ischemic tissue, thereby providing an additional stimulus for neovascularization. A recent study in primates proves that CD341 cell-mediated gene delivery into sites of active angiogenesis is a feasible strategy (Gomez-Navarro et al. 2000).
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