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Therapeutic Vasculogenesis Using Human Cord Blood-Derived Endothelial Progenitors
BRIEF REVIEWS Therapeutic Vasculogenesis Using Human Cord Blood-Derived Endothelial Progenitors Toyoaki Murohara*
Peripheral blood of adult species contains endothelial progenitor cells (EPCs) that participate in neovascularization, consistent with postnatal vasculogenesis. Abundant EPCs can be isolated from a relatively small volume of human umbilical cord blood, and that cultureexpanded EPCs participate in endothelial network formation in vitro. Transplanted EPCs incorporated into sites of active neovascularization and formed capillaries among preserved skeletal myocytes in the ischemic hindlimb of athymic nude rats in vivo. Furthermore, transplantation of EPC quantitatively and effectively augmented neovascularization in response to hindlimb ischemia. Thus, human umbilical cord blood seems to be a novel source for EPCs, and the transplantation of cord blood-derived EPCs may become a useful strategy to modulate postnatal neovascularization. (Trends Cardiovasc Med 2001;11:303– 307). © 2001, Elsevier Science Inc.
Therapeutic angiogenesis is an important survival mechanism that preserves the integrity of tissues subjected to ischemia (Isner and Asahara 1999, Isner et al. 1996). Supplemental administration of angiogenic cytokines such as vascular endothelial growth factor (VEGF) and
Toyoaki Murohara is from The Cardiovascular Research Institute and Department of Internal Medicine III, Kurume University School of Medicine Kurume, Fukuoka, Japan. * Address correspondence to: Toyoaki Murohara, MD, PhD, The Cardiovascular Research Institute and Department of Internal Medicine III, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. Tel.: (181) 942-35-3311 (ext. 3801); fax: (181) 942-31-7707; e-mail: [email protected]. © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter
TCM Vol. 11, No. 8, 2001
basic fibroblast growth factor (bFGF)— in the forms of either recombinant protein or gene—have been shown to augment collateral development when endogenous angiogenesis is insufficient. Thus, therapeutic angiogenesis constitutes a novel option for the treatment of ischemic cardiovascular diseases. Recent advances in vascular biology and developmental biology have led to a new paradigm of therapeutic neovascularization: cell-mediated vascular regeneration. This concept was rapidly introduced after the discovery of endothelial progenitor cells (EPCs) in circulating adult human peripheral blood (Asahara et al. 1997). Transplantation of either culture-expanded EPCs or adult stem cells isolated from bone marrow have been recently shown to effectively enhance angiogenesis in ischemic tissues (Kalka et al. 2000,
Shintani et al. 2001). Currently, EPCs have been identified in adult peripheral blood, bone marrow, and human umbilical cord blood (Asahara et al. 1999a, Murohara et al. 2000, Nieda et al. 1997). This review summarizes recent advances in therapeutic angiogenesis using cell transplantation method, and discusses potential utility of EPCs found in human umbilical cord blood. • Vasculogenesis and Angiogenesis The development of vascular tissues may be considered in several different contexts. Vasculogenesis and angiogenesis are the two major processes responsible for the development of new blood vessels (Isner and Asahara 1999). Vasculogenesis refers to the in situ formation of blood vessels from EPCs or angioblasts (Risau 1995). It begins with the formation of cell clusters or blood islands. Growth and fusion of multiple blood islands in the embryo ultimately give rise to the capillary network structure (Risau and Flamme 1995); after the onset of blood circulation, this network differentiates into an arteriovenous vascular system. EPCs are located at the periphery of the blood islands, while hematopoietic stem cells (HSCs) are located in the center of the blood islands. EPCs give rise to vascular ECs, whereas HSCs develop into mature blood cells. In addition to this spatial association, HSCs and EPCs share several cell surface antigens, such as Flk-1, Tie-2, and CD34. These progenitor cells are considered to derive from a common precursor, putatively termed hemangioblast (Flamme and Risau 1992, Weiss and Orkin 1996). In contrast, angiogenesis is a process mediated through the sprouting of new capillaries from pre-existing mature ECs. Angiogenesis has been suggested to begin with the “activation” of ECs within a parent vessel, followed by disruption of the basement membrane, and subsequent migration of ECs into the interstitial
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space, possibly in response to an ischemic stimulus (D’Amore and Thompson 1987, Folkman 1995). Subsequent EC proliferation, pericyte recruitment, and production of a basement membrane complete the angiogenesis process. Thus, the proliferative and migratory activities of ECs constitute the basal mechanistic of angiogenesis. Until recently, vasculogenesis was considered to be restricted to the embryo, and neovascular formation in adult species was thought to be the consequence of angiogenesis alone. • Postnatal Vasculogenesis Although vasculogenesis has been considered to be restricted to embryos (Risau et al. 1988), we recently discovered that the peripheral blood of adult species contains EPCs that are presumably derived from CD34-positive mononuclear blood cells (MNCCD341) (Asahara et al. 1997). In vitro, these cells differentiate into mature ECs. In animal models of tissue ischemia, transplanted heterologous, homologous, and/or autologous EPCs incorporate into sites of active angiogenesis (Asahara et al. 1997). These findings suggest that naturally circulating EPCs or exogenously transplanted EPCs may contribute to neovascularization in adults, consistent with “postnatal vasculogenesis.” This paradigm may have some implications regarding the enhancement of collateral vessel growth and angiogenesis in ischemic tissues (therapeutic angiogenesis) as well as the delivery of anti- or proangiogenic agents to sites of pathologic or utilitarian angiogenesis, respectively. In fact, transplantation of culture-expanded EPCs has been shown to effectively augment angiogenesis and collateralization in ischemic tissues in several animal models (Kalka et al. 2000, Kawamoto et al. 2001, Murohara et al. 2000). EPCs identified in peripheral blood have been shown to derive from bone marrow in response to ischemic stimuli in adult animals (Asahara et al. 1999a, Takahashi et al. 1999). We and other investigators recently identified EPCs in human umbilical cord blood, a previously unknown source for EPCs. • Human Umbilical Cord Blood Is a Rich Source of Hematopoietic Stem Cells Human umbilical cord blood has been shown to contain a large number of he-
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matopoietic colony-forming cells or stem cells (Gluckman et al. 1989, Nakahata and Ogawa 1982). In fact, transfusion of human cord blood in severe combined immunodeficiency (SCID) mice demonstrated repopulation of the bone marrow with colonogenic progenitors, which support development of erythroid, myoloid, and B- and T-lymphocyte lineages. In contrast to HSCs isolated from adult bone marrow, cord blood progenitors have distinctive proliferative characteristics, including the capacity to form a greater number of colonies, a higher cellcycle rate and self-renewal potential, and longer telomeres (Mayani and Lansdorp 1994, Vaziri et al. 1994). When used for stem cell transplantation to reconstitute hematopoiesis (HSC transplantation), all of these properties should favor the growth of the cord blood progenitors compared to adult bone marrow-derived progenitors. • Isolation of EPCs from Human Umbilical Cord Blood Because HSCs and EPCs are believed to derive from a common precursor cell (i.e., hemangioblast) and because cord blood contains a large amount of HSCs, cord blood would be a novel source for isolating EPCs. Cell surface molecules such as CD34, KDR, Tie-2 and VE-cadherin are expressed by ECs at an early stage of differentiation (Millauer et al. 1993, Nishikawa et al. 1998, Sato et al. 1995, Yamaguchi et al. 1993). Similarly, HSCs express CD34, KDR and Tie-2 on their surface (Katoh et al. 1995, Yano et al. 1997). However, as they differentiate into mature blood cells HSCs lose CD34 (Civin et al. 1984). Importantly, Rafii et al. (1995) showed colonization of the flow surface of left ventricular assist devices with CD34-positive ECs, and Shi et al. (1998) showed that transplanted bone marrow-derived CD34-positive cells participated in the endothelialization of impervious Dacron grafts in vivo. Therefore, we consider that CD34 antigen is an appropriate marker for isolation of EPCs from human umbilical cord blood (Murohara et al. 2000). Flow cytometric analysis revealed that cord blood contained a 10-fold excess of MNCCD341 compared to adult peripheral blood. When cord blood MNCs were isolated and cultured on fibronectincoated culture plates, numerous cell
clusters appeared within 48 h, and spindleshaped and attached (AT) cells sprouted from the edge of those clusters. Cell clusters and AT cells formed linear cord-like structures (Figure 1a). Mature ECs differentiated and sprouted from these structures, and eventually formed cobblestone-like EC monolayers (Figure 1b and c). The morphological structure of AT cells resembled that of EPCs derived from adult peripheral blood (Asahara et al. 1997). AT cells stained positive for von Willebrand factor (vWF), CD31 (PECAM-1), and KDR, also took up DiI-acLDL, which are the characteristic features of ECs. AT cells were positive for endothelial nitric oxide synthase (eNOS), and released NO in response to VEGF (50 ng/mL) as assessed by DAF2DA, an NO-specific fluorescent indicator. Thus, AT cells might functionally respond to VEGF via its receptor tyrosine kinase KDR followed by activation of eNOS (Murohara et al. 1998). These findings suggest that AT cells express multiple endothelial-lineage markers and functions (Murohara et al. 2000). To examine whether cord blood-derived AT cells participate in the formation of an angiogenesis-like network in vitro, AT cells were expanded and collected on day 7 of culture, fluorescence-labeled and co-cultured with unlabeled human umbilical vein endothelial cells (HUVECs) on a three-dimensional matrix gel. Within 3 days, incorporation of labeled AT cells to the networks was confirmed (Figure 1d). Thus, AT cells actively formed endothelial networks together with mature ECs. Because cord blood-derived AT cells expressed multiple endothelial lineage markers and functions, and because AT cells participated in angiogenesis, we considered AT cells as constituting a major population of EPCs. Interestingly, a greater number of EPCs differentiated from cord blood MNCs than from the same number of adult peripheral blood MNCs. These findings were consistent with the data obtained by flow cytometric analysis showing that cord blood contained a 10-fold excess of MNCCD341 compared with adult peripheral blood. Thus, cord blood MNCs seem to be a robust source of EPC compared to the same amount of peripheral blood MNCs. However, the total number of EPCs derived from peripheral blood is not necessarily less than that of EPCs derived from cord blood just because TCM Vol. 11, No. 8, 2001
Figure 1. Differentiation of EPCs from human cord blood MNCs. (a) When cord blood MNCs were cultured on fibronectin, cell clusters appeared, and spindle-shaped and attaching (AT) cells sprouted. (b) Clusters and AT cells formed linear cord-like structures, and then mature ECs differentiated from clusters. (c) These cells formed EC monolayer with typical cobblestone appearance. (d) Fluorescence-labeled EPCs formed network structure with unlabeled human umbilical vein endothelial cells (HUVECs) cultured on three-dimensional matrix gel.
the volume of umbilical cord blood is limited. Kalka et al. (1999) recently reported that EPCs derived from human umbilical cord blood showed faster differentiation than EPCs derived from adult peripheral blood. These data support the finding that EPCs derived from cord blood have a greater proliferative activity than those derived from adult peripheral blood, presumably because of their higher cell cycle rate and longer tolomere activity (Mayani and Lansdorp 1994, Vaziri et al. 1994). These properties of cord blood-derived EPCs may contribute to therapeutic vasculogenesis more effectively. Crisa et al. (1999) recently reported that transplanted cord blood-derived progenitors sustained not only T-lymphocyte reconstitution but also induced EPCdependent neovascularization in the implanted human thymus within the kidney of severe combined immunodeficient (SCID) mice. These data also support the notion that cord blood can be a source for EPC isolation. • Therapeutic Vasculogenesis Using Human Umbilical Cord Blood-Derived EPCs We examined whether transplanted human cord blood-derived EPCs participated in postnatal neovascularization in vivo in immunodeficient animals. EPCs were isolated on day 7 of culture and then fluorescence labeled. Unilateral hindlimb ischemia was surgically induced in nude rats, and 3 days after surgery animals were injected with fluorescence labeled cord blood-derived EPCs (3 3 105 cells/ TCM Vol. 11, No. 8, 2001
animal) in the ischemic thigh skeletal muscles. On day 14 after limb ischemia, frozen tissue sections were prepared from the ischemic tissues. Fluorescence microscopy revealed that numerous labeled EPCs had been incorporated and had arranged into EC capillary-like structures among the preserved skeletal myocytes in the ischemic limbs. Moreover, transplanted EPCs often formed tubular structures with a round lumen. When adjacent sections were stained for alkaline phosphatase to identify viable ECs (Ziada et al. 1984), capillary ECs were detected at exactly the same locations where fluorescence-positive implanted EPCs were identified. Thus, transplanted EPCs had survived, and had been incorporated into capillary-like network structures in the ischemic hindlimb in vivo. Finally, we examined whether in vivo transplantation of EPCs would quantitatively augment neovascularization in the ischemic limb in nude rats. Unilateral limb ischemia was surgically induced, and human umbilical cord blood-derived EPCs isolated on day 7 of culture were directly transplanted into the ischemic thigh muscles (3 3 105 EPCs/rat). Serial laser Doppler blood flow analyses revealed significantly augmented ratios of the ischemic/normal hindlimb blood flow in the EPC-transplanted group compared to saline-treated control animals on days 7, 14, and 21. Moreover, on day 14, immunohistochemical analysis for vWF expression and histochemical staining for alkaline phosphatase in the ischemic tissues revealed a significant increase in capillary density after transplantation of
EPCs compared with the controls. No significant difference in capillary density was observed between the two groups in the contralateral nonischemic limb. Thus, transplantation of human cord bloodderived EPCs is a novel strategy to enhance tissue neovascularization in adult animals (Murohara et al. 2000), a notion consistent with “therapeutic vasculogenesis” (Isner and Asahara 1999). Our results are consistent with a recent study by Kalka et al. (2000), who showed that transplantation of adult peripheral bloodderived EPCs significantly augmented ischemia-induced neovascularization in the hindlimb and promoted limb salvage in nude mice. • Limitations of the Clinical Utilization of Human Umbilical Cord Blood-Derived EPCs for Therapeutic Angiogenesis At present, human cord blood-derived EPCs may be difficult to use for therapeutic vasculogenesis because cord blood transplantation is currently only allogenic. Transplantation of cord blood EPCs for therapeutic angiogenesis into patients with normal bone marrow function would induce immunological graft-versus-host diseases (GVHDs), and thus, implanted cells would be rejected after transplantation by the host’s immune defense mechanisms. In the next decade, however, cryopreserved autologous cord blood cells might be used as a source of HSCs to reconstitute the hematopoietic system in patients with marrow degenerative diseases or after myeloablative chemother-
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apy. Similarly, cryo-preserved autologous cord blood-derived EPCs for therapeutic vasculogenesis may become available in future. Several recent studies showed that transplantation of culture expanded EPCs enhanced neovascularization in ischemic tissues. However, the number of EPCs required for therapeutic angiogenesis is still unknown. Similarly, we do not know the yields of EPCs from cord blood-, peripheral blood-, or bone marrow-MNCs. Nevertheless, one could estimate the yields of EPCs based on the mean percentages of CD34-positive MNCs among total MNCs (i.e., approximately 3, 2, and 0.2% of total MNCs in bone marrow, cord blood, and adult peripheral blood). Thus, the estimated yields of EPCs from bone marrow, cord blood, and adult peripheral blood would be 15:10:1 from the same number of MNCs. • Isolation of EPCs From an Alternative Source: Utilization of Bone Marrow Stem Cells for Therapeutic Vasculogenesis Because at present it is difficult to use autologous cord blood-derived EPCs clinically, other source(s) for EPCs should be explored. Currently, autologous EPCs can be isolated and expanded from adult human peripheral blood for therapeutic angiogenesis (Kalka et al. 2000). However, the number of EPCs obtained from peripheral blood may also be limited. To overcome this issue, the use of genetically modified EPCs that are transfected with adenovirus vector encoding VEGF gene, is being considered (Asahara et al. 1999b). This strategy seems to be promising in animal experiments, but its clinical application needs further careful evaluation because a recent study showed that a powerful expression of VEGF transgene has been shown to induce angioma formation in experimental animal models (Carmeliet 2000, Lee et al. 2000). Rather, simple combined administration of culture expanded EPCs together with naked plasmid vectors containing VEGF or VEGF-2 genes will be a safer and a more feasible clinical strategy. As an alternative source, autologous bone marrow cells are receiving great attention (Kamihata et al. 2001, Shintani et al. 2001, Tomita et al. 1999). Bone marrow contains a large number of HSCs, EPCs and other stem cells for stroma
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tissues (Prockop 1997, Asahara et al. 1999a, Shi et al. 1998). In fact, implantation of autologous bone marrow cells has been shown to effectively augment angiogenesis in ischemic tissues (Kamihata et al. 2001, Shintani et al. 2001, Ueno et al. 2001). The greatest advantage of using bone marrow-derived EPCs is that one can use the patient’s own EPCs (autologous cell implantation). So that there will be no immunological adverse reactions. Secondly, one can obtain a significant number of CD34-positive MNCs from bone marrow. The safety and efficacy of autologous implantation of bone marrow-derived MNCs (i.e., stem cellrich fraction) for therapeutic angiogenesis are currently under investigation in patients with critical limb ischemia in a multi-center clinical trial in Japan (TACT study; Therapeutic Angiogenesis by Cell Transplantation study) (Tateishi et al. 2001). The initial results of this clinical study will be reported in the near future.
References Asahara T, Murohara T, Sullivan A, et al.: 1997. Isolation of putative progenitor endothelial cells for angiogenesis. Science 275:965–967. Asahara T, Masuda H, Takahashi T, et al.: 1999a. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 85:221– 228. Asahara T, Takahashi T, Masuda H, et al.: 1999b. Gene therapy of endothelial progenitor cell for vascular development in severe ischemic disease [abst]. Circulation 100:I-481. Carmeliet P. 2000. VEGF gene therapy: stimulating angiogenesis or angioma-genesis? Nat Med 6:1102–1103. Civin CI, Strauss LC, Brovall C, et al.: 1984. Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a. J Immunol 133:157–165. Crisa L, Cirulli V, Smith KA, et al.: 1999. Human cord blood progenitors sustain thymic T-cell development and a novel form of angiogenesis. Blood 94:3928–3940. D’Amore PA, Thompson RW: 1987. Mechanisms of angiogenesis. Annu Rev Physiol 49:453–464. Flamme I, Risau W: 1992. Induction of vasculogenesis and hematopoiesis in vitro. Development 116:435–439.
Folkman J: 1995. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature Med 1:27–31. Gluckman E, Broxmeyer HA, Auerbach AD, et al.: 1989. Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 321:1174–1178. Isner JM, Asahara T: 1999. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest 103:1231–1236. Isner JM, Pieczek A, Schainfeld R, et al.: 1996. Clinical evidence of angiogenesis following arterial gene transfer of phVEGF165. Lancet 348:370–374. Kalka C, Iwaguro H, Masuda H, et al.: 1999. Generation of differentiated endothelial cells from mononuclear cells of human umbilical cord blood. Circulation 100(Suppl I):I-749. Kalka C, Masuda H, Takahashi T, et al.: 2000. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA 97:3422–3427. Kamihata K, Matsubara H, Nishiue T, et al.: 2001. Implantation of autologous bone marrow cells into ischemic myocardium enhances collateral perfusion and regional function via side-supply of angioblasts, angiogenic ligands and cytokines. Circulation 104:1046–1052. Katoh O, Tauchi H, Kawaishi K, et al.: 1995. Expression of the vascular endothelial growth factor (VEGF) receptor gene, KDR, in hematopoietic cells and inhibitory effect of VEGF on apoptotic cell death caused by ionizing radiation. Cancer Res 55:5687– 5692. 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. Lee RJ, Springer ML, Blanco-Bose WE, et al.: 2000. VEGF gene delivery to myocardium. Deleterious effects of unregulated expression. Circulation 102:898–901. Mayani H, Lansdorp PM: 1994. Thy-1 expression is linked to functional properties of primitive hematopoietic progenitor cells from human umbilical cord blood. Blood 83:2410–2417. Millauer B, Wizigmann-Voos S, Schnurch H, et al.: 1993. High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell 72:835–846. Murohara T, Horowitz JR, Silver M, et al.: 1998. Vascular endothelial growth factor/ vascular permeability factor enhances vascular permeability via nitric oxide and prostacyclin. Circulation 97:99–107. Murohara T, Ikeda H, Duan J, et al.: 2000. Transplanted cord blood-derived endothelial
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precursor cells augment postnatal neovascularization. J Clin Invest 105:1527–1536. Nakahata T, Ogawa M: 1982. Hemopoietic colony-forming cells in umbilical cord blood with extensive capability to generate mono- and multipotential hemopoietic progenitors. J Clin Invest 70:1324–1328.
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Nieda M, Nicol A, Denning-Kendall P, et al.: 1997. Endothelial cell precursors are normal components of human umbilical cord blood. Br J Haematol 98:775–777.
Weiss M, Orkin SH. 1996. In vitro differentiation of murine embryonic stem cells: new approaches to old problems. J Clin Invest 97:591–595.
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Nishikawa SI, Nishikawa S, Hirashima M, et al.: 1998. Progressive lineage analysis by cell sorting and culture identifies FLK11VEcadherin1 cells at a diverging point of endothelial and hemopoietic lineages. Development 125:1747–1757.
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Prockop DJ: 1997. Marrow stromal cells as stem cells for non-hematopoietic tissues. Science 276:71–74. Rafii S, Oz MC, Seldomridge JA, et al.: 1995. Characterization of hematopoietic cells arising on the textured surface of left ventricular assist devices. Ann Thorac Surg 60:1627–1632. Risau W, Sariola H, Zerwes HG, et al.: 1988. Vasculogenesis and angiogenesis in embryonic stem cell-derived embryoid bodies. Development 102:471–478. Risau W: 1995. Differentiation of endothelium. FASEB J 9:926–933. Risau W, Flamme I: 1995. Vasculogenesis. Ann Rev Cell Dev Biol 11:73–91. Sato TN, Tozawa Y, Deutsch U, et al.: 1995. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 376:70–74. Shi Q, Rafii S, Wu MH, et al.: 1998. Evidence for circulating bone marrow-derived endothelial cells. Blood 92:362–367. Shintani S, Murohara T, Ikeda H, et al.: 2001. Augmentation of postnatal neovascularization with autologous bone marrow transplantation. Circulation 103:897–903. 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. Tateishi E, Masaki H, Matsubara H, et al.: 2001. Clinically feasible therapeutic angiogenesis using autologous implantation of bone marrow-derived mononuclear cells to ischemic limbs [abst]. Jpn Circ J 65(Suppl 1-A):181. Tomita S, Li RK, Weisel RD, et al.: 1999. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation 100:II247–II256. Ueno T, Coussement P, Murohara T, et al.: 2001. Therapeutic angiogenesis by bone marrowderived cell transplantation in pigs with coronary constrictor-induced chronic myocar-
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TCM
Tissue Factor in the Heart Multiple Roles in Hemostasis, Thrombosis, and Inflammation Thomas Luther and Nigel Mackman*
Tissue factor (TF) is the primary cellular initiator of the coagulation protease cascade. It plays an essential role in hemostasis to limit hemorrhage in the event of vascular injury. Indeed, loss of TF is incompatible with life. Recent studies have suggested that TF also plays non-hemostatic roles in blood vessel development, tumor angiogenesis, tumor metastasis, cell migration, and inflammation. This review discusses the roles of TF in hemostasis, thrombosis, and inflammation in the heart as well as other potential roles of TF in the maintenance of cardiac muscle. (Trends Cardiovasc Med 2001;11:307–312). © 2001, Elsevier Science Inc.
Tissue factor (TF) is a transmembrane glycoprotein that binds plasma FVII/FVIIa (Bach 1988, Edgington et al. 1991, Nemerson 1988). Proteolytic cleavage of the substrates FIX and FX (Figure 1) initiates the coagulation protease cascade and leads to thrombin generation, fibrin deposition, and platelet activation (Bach 1988, Edgington et al. 1991, Nemerson Thomas Luther is from Universitatsklinikum Carl Gustav Carus der Technischen Universitat, Dresden, Germany. Nigel Mackman is from Scripps Research Institute, La Jolla, California. * Address correspondence to: Nigel Mackman, Ph.D., Departments of Immunology and Vascular Biology, C-204, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Tel.: (11) 858-784-8594; fax: (11) 858-784-8480. © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter
1988). TF is constitutively expressed at extravascular sites and plays an essential role in hemostasis by limiting hemorrhage in the event of vascular injury (Drake et al. 1989). High levels of TF antigen and activity are detected in atherosclerotic lesions, particularly in advanced lesions. When plaques rupture, TF exposure to blood initiates thrombus formation. Readers are referred to a recent review on this subject (Tremoli et al. 1999); the role of TF in atherosclerosis is not discussed in the current review. • TF Expression in Cardiac Muscle Analysis of TF mRNA levels in various mouse and rabbit tissues by northern blotting revealed constitutive expression in the heart and no detectable expression in skeletal muscle (Hartzell et
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Peripheral blood of adult species contains endothelial progenitor cells (EPCs) that participate in neovascularization, consistent with postnatal vasculogenesis. Abundant EPCs can be isolated from a relatively small volume of human umbilical cord blood, and that cultureexpanded EPCs participate in endothelial network formation in vitro. Transplanted EPCs incorporated into sites of active neovascularization and formed capillaries among preserved skeletal myocytes in the ischemic hindlimb of athymic nude rats in vivo. Furthermore, transplantation of EPC quantitatively and effectively augmented neovascularization in response to hindlimb ischemia. Thus, human umbilical cord blood seems to be a novel source for EPCs, and the transplantation of cord blood-derived EPCs may become a useful strategy to modulate postnatal neovascularization. (Trends Cardiovasc Med 2001;11:303– 307). © 2001, Elsevier Science Inc.
Therapeutic angiogenesis is an important survival mechanism that preserves the integrity of tissues subjected to ischemia (Isner and Asahara 1999, Isner et al. 1996). Supplemental administration of angiogenic cytokines such as vascular endothelial growth factor (VEGF) and
Toyoaki Murohara is from The Cardiovascular Research Institute and Department of Internal Medicine III, Kurume University School of Medicine Kurume, Fukuoka, Japan. * Address correspondence to: Toyoaki Murohara, MD, PhD, The Cardiovascular Research Institute and Department of Internal Medicine III, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. Tel.: (181) 942-35-3311 (ext. 3801); fax: (181) 942-31-7707; e-mail: [email protected]. © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter
TCM Vol. 11, No. 8, 2001
basic fibroblast growth factor (bFGF)— in the forms of either recombinant protein or gene—have been shown to augment collateral development when endogenous angiogenesis is insufficient. Thus, therapeutic angiogenesis constitutes a novel option for the treatment of ischemic cardiovascular diseases. Recent advances in vascular biology and developmental biology have led to a new paradigm of therapeutic neovascularization: cell-mediated vascular regeneration. This concept was rapidly introduced after the discovery of endothelial progenitor cells (EPCs) in circulating adult human peripheral blood (Asahara et al. 1997). Transplantation of either culture-expanded EPCs or adult stem cells isolated from bone marrow have been recently shown to effectively enhance angiogenesis in ischemic tissues (Kalka et al. 2000,
Shintani et al. 2001). Currently, EPCs have been identified in adult peripheral blood, bone marrow, and human umbilical cord blood (Asahara et al. 1999a, Murohara et al. 2000, Nieda et al. 1997). This review summarizes recent advances in therapeutic angiogenesis using cell transplantation method, and discusses potential utility of EPCs found in human umbilical cord blood. • Vasculogenesis and Angiogenesis The development of vascular tissues may be considered in several different contexts. Vasculogenesis and angiogenesis are the two major processes responsible for the development of new blood vessels (Isner and Asahara 1999). Vasculogenesis refers to the in situ formation of blood vessels from EPCs or angioblasts (Risau 1995). It begins with the formation of cell clusters or blood islands. Growth and fusion of multiple blood islands in the embryo ultimately give rise to the capillary network structure (Risau and Flamme 1995); after the onset of blood circulation, this network differentiates into an arteriovenous vascular system. EPCs are located at the periphery of the blood islands, while hematopoietic stem cells (HSCs) are located in the center of the blood islands. EPCs give rise to vascular ECs, whereas HSCs develop into mature blood cells. In addition to this spatial association, HSCs and EPCs share several cell surface antigens, such as Flk-1, Tie-2, and CD34. These progenitor cells are considered to derive from a common precursor, putatively termed hemangioblast (Flamme and Risau 1992, Weiss and Orkin 1996). In contrast, angiogenesis is a process mediated through the sprouting of new capillaries from pre-existing mature ECs. Angiogenesis has been suggested to begin with the “activation” of ECs within a parent vessel, followed by disruption of the basement membrane, and subsequent migration of ECs into the interstitial
303
space, possibly in response to an ischemic stimulus (D’Amore and Thompson 1987, Folkman 1995). Subsequent EC proliferation, pericyte recruitment, and production of a basement membrane complete the angiogenesis process. Thus, the proliferative and migratory activities of ECs constitute the basal mechanistic of angiogenesis. Until recently, vasculogenesis was considered to be restricted to the embryo, and neovascular formation in adult species was thought to be the consequence of angiogenesis alone. • Postnatal Vasculogenesis Although vasculogenesis has been considered to be restricted to embryos (Risau et al. 1988), we recently discovered that the peripheral blood of adult species contains EPCs that are presumably derived from CD34-positive mononuclear blood cells (MNCCD341) (Asahara et al. 1997). In vitro, these cells differentiate into mature ECs. In animal models of tissue ischemia, transplanted heterologous, homologous, and/or autologous EPCs incorporate into sites of active angiogenesis (Asahara et al. 1997). These findings suggest that naturally circulating EPCs or exogenously transplanted EPCs may contribute to neovascularization in adults, consistent with “postnatal vasculogenesis.” This paradigm may have some implications regarding the enhancement of collateral vessel growth and angiogenesis in ischemic tissues (therapeutic angiogenesis) as well as the delivery of anti- or proangiogenic agents to sites of pathologic or utilitarian angiogenesis, respectively. In fact, transplantation of culture-expanded EPCs has been shown to effectively augment angiogenesis and collateralization in ischemic tissues in several animal models (Kalka et al. 2000, Kawamoto et al. 2001, Murohara et al. 2000). EPCs identified in peripheral blood have been shown to derive from bone marrow in response to ischemic stimuli in adult animals (Asahara et al. 1999a, Takahashi et al. 1999). We and other investigators recently identified EPCs in human umbilical cord blood, a previously unknown source for EPCs. • Human Umbilical Cord Blood Is a Rich Source of Hematopoietic Stem Cells Human umbilical cord blood has been shown to contain a large number of he-
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matopoietic colony-forming cells or stem cells (Gluckman et al. 1989, Nakahata and Ogawa 1982). In fact, transfusion of human cord blood in severe combined immunodeficiency (SCID) mice demonstrated repopulation of the bone marrow with colonogenic progenitors, which support development of erythroid, myoloid, and B- and T-lymphocyte lineages. In contrast to HSCs isolated from adult bone marrow, cord blood progenitors have distinctive proliferative characteristics, including the capacity to form a greater number of colonies, a higher cellcycle rate and self-renewal potential, and longer telomeres (Mayani and Lansdorp 1994, Vaziri et al. 1994). When used for stem cell transplantation to reconstitute hematopoiesis (HSC transplantation), all of these properties should favor the growth of the cord blood progenitors compared to adult bone marrow-derived progenitors. • Isolation of EPCs from Human Umbilical Cord Blood Because HSCs and EPCs are believed to derive from a common precursor cell (i.e., hemangioblast) and because cord blood contains a large amount of HSCs, cord blood would be a novel source for isolating EPCs. Cell surface molecules such as CD34, KDR, Tie-2 and VE-cadherin are expressed by ECs at an early stage of differentiation (Millauer et al. 1993, Nishikawa et al. 1998, Sato et al. 1995, Yamaguchi et al. 1993). Similarly, HSCs express CD34, KDR and Tie-2 on their surface (Katoh et al. 1995, Yano et al. 1997). However, as they differentiate into mature blood cells HSCs lose CD34 (Civin et al. 1984). Importantly, Rafii et al. (1995) showed colonization of the flow surface of left ventricular assist devices with CD34-positive ECs, and Shi et al. (1998) showed that transplanted bone marrow-derived CD34-positive cells participated in the endothelialization of impervious Dacron grafts in vivo. Therefore, we consider that CD34 antigen is an appropriate marker for isolation of EPCs from human umbilical cord blood (Murohara et al. 2000). Flow cytometric analysis revealed that cord blood contained a 10-fold excess of MNCCD341 compared to adult peripheral blood. When cord blood MNCs were isolated and cultured on fibronectincoated culture plates, numerous cell
clusters appeared within 48 h, and spindleshaped and attached (AT) cells sprouted from the edge of those clusters. Cell clusters and AT cells formed linear cord-like structures (Figure 1a). Mature ECs differentiated and sprouted from these structures, and eventually formed cobblestone-like EC monolayers (Figure 1b and c). The morphological structure of AT cells resembled that of EPCs derived from adult peripheral blood (Asahara et al. 1997). AT cells stained positive for von Willebrand factor (vWF), CD31 (PECAM-1), and KDR, also took up DiI-acLDL, which are the characteristic features of ECs. AT cells were positive for endothelial nitric oxide synthase (eNOS), and released NO in response to VEGF (50 ng/mL) as assessed by DAF2DA, an NO-specific fluorescent indicator. Thus, AT cells might functionally respond to VEGF via its receptor tyrosine kinase KDR followed by activation of eNOS (Murohara et al. 1998). These findings suggest that AT cells express multiple endothelial-lineage markers and functions (Murohara et al. 2000). To examine whether cord blood-derived AT cells participate in the formation of an angiogenesis-like network in vitro, AT cells were expanded and collected on day 7 of culture, fluorescence-labeled and co-cultured with unlabeled human umbilical vein endothelial cells (HUVECs) on a three-dimensional matrix gel. Within 3 days, incorporation of labeled AT cells to the networks was confirmed (Figure 1d). Thus, AT cells actively formed endothelial networks together with mature ECs. Because cord blood-derived AT cells expressed multiple endothelial lineage markers and functions, and because AT cells participated in angiogenesis, we considered AT cells as constituting a major population of EPCs. Interestingly, a greater number of EPCs differentiated from cord blood MNCs than from the same number of adult peripheral blood MNCs. These findings were consistent with the data obtained by flow cytometric analysis showing that cord blood contained a 10-fold excess of MNCCD341 compared with adult peripheral blood. Thus, cord blood MNCs seem to be a robust source of EPC compared to the same amount of peripheral blood MNCs. However, the total number of EPCs derived from peripheral blood is not necessarily less than that of EPCs derived from cord blood just because TCM Vol. 11, No. 8, 2001
Figure 1. Differentiation of EPCs from human cord blood MNCs. (a) When cord blood MNCs were cultured on fibronectin, cell clusters appeared, and spindle-shaped and attaching (AT) cells sprouted. (b) Clusters and AT cells formed linear cord-like structures, and then mature ECs differentiated from clusters. (c) These cells formed EC monolayer with typical cobblestone appearance. (d) Fluorescence-labeled EPCs formed network structure with unlabeled human umbilical vein endothelial cells (HUVECs) cultured on three-dimensional matrix gel.
the volume of umbilical cord blood is limited. Kalka et al. (1999) recently reported that EPCs derived from human umbilical cord blood showed faster differentiation than EPCs derived from adult peripheral blood. These data support the finding that EPCs derived from cord blood have a greater proliferative activity than those derived from adult peripheral blood, presumably because of their higher cell cycle rate and longer tolomere activity (Mayani and Lansdorp 1994, Vaziri et al. 1994). These properties of cord blood-derived EPCs may contribute to therapeutic vasculogenesis more effectively. Crisa et al. (1999) recently reported that transplanted cord blood-derived progenitors sustained not only T-lymphocyte reconstitution but also induced EPCdependent neovascularization in the implanted human thymus within the kidney of severe combined immunodeficient (SCID) mice. These data also support the notion that cord blood can be a source for EPC isolation. • Therapeutic Vasculogenesis Using Human Umbilical Cord Blood-Derived EPCs We examined whether transplanted human cord blood-derived EPCs participated in postnatal neovascularization in vivo in immunodeficient animals. EPCs were isolated on day 7 of culture and then fluorescence labeled. Unilateral hindlimb ischemia was surgically induced in nude rats, and 3 days after surgery animals were injected with fluorescence labeled cord blood-derived EPCs (3 3 105 cells/ TCM Vol. 11, No. 8, 2001
animal) in the ischemic thigh skeletal muscles. On day 14 after limb ischemia, frozen tissue sections were prepared from the ischemic tissues. Fluorescence microscopy revealed that numerous labeled EPCs had been incorporated and had arranged into EC capillary-like structures among the preserved skeletal myocytes in the ischemic limbs. Moreover, transplanted EPCs often formed tubular structures with a round lumen. When adjacent sections were stained for alkaline phosphatase to identify viable ECs (Ziada et al. 1984), capillary ECs were detected at exactly the same locations where fluorescence-positive implanted EPCs were identified. Thus, transplanted EPCs had survived, and had been incorporated into capillary-like network structures in the ischemic hindlimb in vivo. Finally, we examined whether in vivo transplantation of EPCs would quantitatively augment neovascularization in the ischemic limb in nude rats. Unilateral limb ischemia was surgically induced, and human umbilical cord blood-derived EPCs isolated on day 7 of culture were directly transplanted into the ischemic thigh muscles (3 3 105 EPCs/rat). Serial laser Doppler blood flow analyses revealed significantly augmented ratios of the ischemic/normal hindlimb blood flow in the EPC-transplanted group compared to saline-treated control animals on days 7, 14, and 21. Moreover, on day 14, immunohistochemical analysis for vWF expression and histochemical staining for alkaline phosphatase in the ischemic tissues revealed a significant increase in capillary density after transplantation of
EPCs compared with the controls. No significant difference in capillary density was observed between the two groups in the contralateral nonischemic limb. Thus, transplantation of human cord bloodderived EPCs is a novel strategy to enhance tissue neovascularization in adult animals (Murohara et al. 2000), a notion consistent with “therapeutic vasculogenesis” (Isner and Asahara 1999). Our results are consistent with a recent study by Kalka et al. (2000), who showed that transplantation of adult peripheral bloodderived EPCs significantly augmented ischemia-induced neovascularization in the hindlimb and promoted limb salvage in nude mice. • Limitations of the Clinical Utilization of Human Umbilical Cord Blood-Derived EPCs for Therapeutic Angiogenesis At present, human cord blood-derived EPCs may be difficult to use for therapeutic vasculogenesis because cord blood transplantation is currently only allogenic. Transplantation of cord blood EPCs for therapeutic angiogenesis into patients with normal bone marrow function would induce immunological graft-versus-host diseases (GVHDs), and thus, implanted cells would be rejected after transplantation by the host’s immune defense mechanisms. In the next decade, however, cryopreserved autologous cord blood cells might be used as a source of HSCs to reconstitute the hematopoietic system in patients with marrow degenerative diseases or after myeloablative chemother-
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apy. Similarly, cryo-preserved autologous cord blood-derived EPCs for therapeutic vasculogenesis may become available in future. Several recent studies showed that transplantation of culture expanded EPCs enhanced neovascularization in ischemic tissues. However, the number of EPCs required for therapeutic angiogenesis is still unknown. Similarly, we do not know the yields of EPCs from cord blood-, peripheral blood-, or bone marrow-MNCs. Nevertheless, one could estimate the yields of EPCs based on the mean percentages of CD34-positive MNCs among total MNCs (i.e., approximately 3, 2, and 0.2% of total MNCs in bone marrow, cord blood, and adult peripheral blood). Thus, the estimated yields of EPCs from bone marrow, cord blood, and adult peripheral blood would be 15:10:1 from the same number of MNCs. • Isolation of EPCs From an Alternative Source: Utilization of Bone Marrow Stem Cells for Therapeutic Vasculogenesis Because at present it is difficult to use autologous cord blood-derived EPCs clinically, other source(s) for EPCs should be explored. Currently, autologous EPCs can be isolated and expanded from adult human peripheral blood for therapeutic angiogenesis (Kalka et al. 2000). However, the number of EPCs obtained from peripheral blood may also be limited. To overcome this issue, the use of genetically modified EPCs that are transfected with adenovirus vector encoding VEGF gene, is being considered (Asahara et al. 1999b). This strategy seems to be promising in animal experiments, but its clinical application needs further careful evaluation because a recent study showed that a powerful expression of VEGF transgene has been shown to induce angioma formation in experimental animal models (Carmeliet 2000, Lee et al. 2000). Rather, simple combined administration of culture expanded EPCs together with naked plasmid vectors containing VEGF or VEGF-2 genes will be a safer and a more feasible clinical strategy. As an alternative source, autologous bone marrow cells are receiving great attention (Kamihata et al. 2001, Shintani et al. 2001, Tomita et al. 1999). Bone marrow contains a large number of HSCs, EPCs and other stem cells for stroma
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tissues (Prockop 1997, Asahara et al. 1999a, Shi et al. 1998). In fact, implantation of autologous bone marrow cells has been shown to effectively augment angiogenesis in ischemic tissues (Kamihata et al. 2001, Shintani et al. 2001, Ueno et al. 2001). The greatest advantage of using bone marrow-derived EPCs is that one can use the patient’s own EPCs (autologous cell implantation). So that there will be no immunological adverse reactions. Secondly, one can obtain a significant number of CD34-positive MNCs from bone marrow. The safety and efficacy of autologous implantation of bone marrow-derived MNCs (i.e., stem cellrich fraction) for therapeutic angiogenesis are currently under investigation in patients with critical limb ischemia in a multi-center clinical trial in Japan (TACT study; Therapeutic Angiogenesis by Cell Transplantation study) (Tateishi et al. 2001). The initial results of this clinical study will be reported in the near future.
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Tissue Factor in the Heart Multiple Roles in Hemostasis, Thrombosis, and Inflammation Thomas Luther and Nigel Mackman*
Tissue factor (TF) is the primary cellular initiator of the coagulation protease cascade. It plays an essential role in hemostasis to limit hemorrhage in the event of vascular injury. Indeed, loss of TF is incompatible with life. Recent studies have suggested that TF also plays non-hemostatic roles in blood vessel development, tumor angiogenesis, tumor metastasis, cell migration, and inflammation. This review discusses the roles of TF in hemostasis, thrombosis, and inflammation in the heart as well as other potential roles of TF in the maintenance of cardiac muscle. (Trends Cardiovasc Med 2001;11:307–312). © 2001, Elsevier Science Inc.
Tissue factor (TF) is a transmembrane glycoprotein that binds plasma FVII/FVIIa (Bach 1988, Edgington et al. 1991, Nemerson 1988). Proteolytic cleavage of the substrates FIX and FX (Figure 1) initiates the coagulation protease cascade and leads to thrombin generation, fibrin deposition, and platelet activation (Bach 1988, Edgington et al. 1991, Nemerson Thomas Luther is from Universitatsklinikum Carl Gustav Carus der Technischen Universitat, Dresden, Germany. Nigel Mackman is from Scripps Research Institute, La Jolla, California. * Address correspondence to: Nigel Mackman, Ph.D., Departments of Immunology and Vascular Biology, C-204, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. Tel.: (11) 858-784-8594; fax: (11) 858-784-8480. © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter
1988). TF is constitutively expressed at extravascular sites and plays an essential role in hemostasis by limiting hemorrhage in the event of vascular injury (Drake et al. 1989). High levels of TF antigen and activity are detected in atherosclerotic lesions, particularly in advanced lesions. When plaques rupture, TF exposure to blood initiates thrombus formation. Readers are referred to a recent review on this subject (Tremoli et al. 1999); the role of TF in atherosclerosis is not discussed in the current review. • TF Expression in Cardiac Muscle Analysis of TF mRNA levels in various mouse and rabbit tissues by northern blotting revealed constitutive expression in the heart and no detectable expression in skeletal muscle (Hartzell et
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