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  • 1. Endothelial Progenitor Cells : Characterization and Role in Vascular Biology Carmen Urbich and Stefanie Dimmeler Circulation Research 2004, 95:343-353 doi: 10.1161/01.RES.0000137877.89448.78Circulation Research is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TX 72514 Copyright © 2004 American Heart Association. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/95/4/343 Subscriptions: Information about subscribing to Circulation Research is online at http://circres.ahajournals.org//subscriptions/ Permissions: Permissions & Rights Desk, Lippincott Williams & Wilkins, a division of Wolters Kluwer Health, 351 West Camden Street, Baltimore, MD 21202-2436. Phone: 410-528-4050. Fax: 410-528-8550. E-mail: journalpermissions@lww.com Reprints: Information about reprints can be found online at http://www.lww.com/reprints Downloaded from http://circres.ahajournals.org/ at UNIV DE CONCEPCION on April 11, 2012
  • 2. Reviews This Review is part of a thematic series on Angiogenesis, which includes the following articles: Endothelial Progenitor Cells: Characterization and Role in Vascular Biology Bone Marrow–Derived Cells for Enhancing Collateral Development: Mechanisms, Animal Data, and Initial Clinical Experiences Arteriogenesis Innate Immunity and Angiogenesis Syndecans Growth Factors and Blood Vessels: Differentiation and Maturation Ralph Kelly, Guest Editor Endothelial Progenitor Cells Characterization and Role in Vascular Biology Carmen Urbich, Stefanie DimmelerAbstract—Infusion of different hematopoietic stem cell populations and ex vivo expanded endothelial progenitor cells augments neovascularization of tissue after ischemia and contributes to reendothelialization after endothelial injury, thereby, providing a novel therapeutic option. However, controversy exists with respect to the identification and the origin of endothelial progenitor cells. Overall, there is consensus that endothelial progenitor cells can derive from the bone marrow and that CD133/VEGFR2 cells represent a population with endothelial progenitor capacity. However, increasing evidence suggests that there are additional bone marrow– derived cell populations (eg, myeloid cells, “side population” cells, and mesenchymal cells) and non-bone marrow– derived cells, which also can give rise to endothelial cells. The characterization of the different progenitor cell populations and their functional properties are discussed. Mobilization and endothelial progenitor cell–mediated neovascularization is critically regulated. Stimulatory (eg, statins and exercise) or inhibitory factors (risk factors for coronary artery disease) modulate progenitor cell levels and, thereby, affect the vascular repair capacity. Moreover, recruitment and incorporation of endothelial progenitor cells requires a coordinated sequence of multistep adhesive and signaling events including adhesion and migration (eg, by integrins), chemoattraction (eg, by SDF-1/CXCR4), and finally the differentiation to endothelial cells. This review summarizes the mechanisms regulating endothelial progenitor cell–mediated neovascularization and reendothelialization. (Circ Res. 2004;95:343-353.) Key Words: progenitor cells Ⅲ neovascularization Ⅲ vasculogenesis Ⅲ angiogenesis Ⅲ endothelial cellsD ifferentiation of mesodermal cells to angioblasts and subsequent endothelial differentiation was believed toexclusively occur in embryonic development. This dogma mia. Rafii’s group in 19982 also reported the existence of “circulating bone marrow– derived endothelial progenitor cells” (CEPCs) in the adult. Again, a subset of CD34ϩwas overturned in 1997, when Asahara and colleagues1 hematopoietic stem cells was shown to differentiate to thepublished that purified CD34ϩ hematopoietic progenitor cells endothelial lineage and express endothelial marker proteinsfrom adults can differentiate ex vivo to an endothelial such as vWF and incorporated Dil-Ac-LDL. Most convinc-phenotype. These cells were named “endothelial progenitor ingly, bone marrow–transplanted genetically tagged cellscells” (EPCs), showed expression of various endothelial were covering implanted Dacron grafts.2 These pioneeringmarkers, and incorporated into neovessels at sites of ische- studies suggested the presence of circulating hemangioblasts Original received March 8, 2004; revision received May 27, 2004; accepted May 28, 2004. From Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Frankfurt, Germany. Correspondence to Stefanie Dimmeler, PhD, Molecular Cardiology, Dept of Internal Medicine IV, University of Frankfurt, Theodor-Stern-Kai 7, 60590Frankfurt, Germany. E-mail Dimmeler@em.uni-frankfurt.de © 2004 American Heart Association, Inc. Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000137877.89448.78 343 Downloaded from http://circres.ahajournals.org/ at UNIV DE CONCEPCION on April 11, 2012
  • 3. 344 Circulation Research August 20, 2004in the adult. According to the initial discovery, EPCs or endothelial lineage. Interestingly, lineage tracking showedCEPCs were defined as cells positive for both hematopoi- that myeloid cells are the hematopoietic stem cell– derivedetic stem cell markers such as CD34 and an endothelial intermediates, which contribute to muscle regeneration,10marker protein as VEGFR2. Because CD34 is not exclu- suggesting that myeloid intermediates may be part of thesively expressed on hematopoietic stem cells but, albeit at repair capacity after injury. Moreover, a subset of humana lower level, also on mature endothelial cells, further peripheral blood monocytes acts as pluripotent stem cells.11studies used the more immature hematopoietic stem cell Of note, a specific problem arises when cells are ex vivomarker CD1333 and demonstrated that purified CD133ϩ expanded and cultured, because the culture conditions (cul-cells can differentiate to endothelial cells in vitro.4 CD133, ture supplements such as FCS and cytokines, plastic) rapidlyalso known as prominin or AC133, is a highly conserved changes the phenotype of the cells. For example, supplemen-antigen with unknown biological activity, which is ex- tation of the medium with statins increased the number ofpressed on hematopoietic stem cells but is absent on endothelial cell colonies isolated from mononuclear cells.12mature endothelial cells and monocytic cells (see review).5 Moreover, continuous cultivation was shown to increaseThus, CD133ϩVEGFR2ϩ cells more likely reflect imma- endothelial marker protein expression.13 This may explainture progenitor cells, whereas CD34ϩVEGFR2ϩ may also why different groups may obtain cells with different surfacerepresent shedded cells of the vessel wall. At present, it is factor profile and functional activity although similar proto-unclear whether CD133 only represents a surface marker cols were used for cultivation.9,14 –16 Moreover, the interactionor has a functional activity involved in regulation of of cells within a heterogeneous mixture of cells such as theneovascularization. mononuclear cells from the blood may impact the yield and Overall, controversy exists with respect to the identifica- the functional activity of the cultivated cells.17tion and the origin of endothelial progenitor cells, which are Generally, several studies suggested that other cell popu-isolated from peripheral blood mononuclear cells by cultiva- lations beside hematopoietic stem cells also can give rise totion in medium favoring endothelial differentiation. In pe- endothelial cells (Figure 1). Thus, non-bone marrow– derivedripheral blood mononuclear cells, several possible sources for cells have been shown to replace the endothelial cells inendothelial cells may exist: (1) the rare number of hemato- grafts.18 In addition, adult bone marrow– derived stem/pro-poietic stem cells, (2) myeloid cells, which may differentiate genitor cells such as the side population cells and multipotentto endothelial cells under the cultivation selection pressure, adult progenitor cells, which are distinct from hematopoietic(3) other circulating progenitor cells (eg, “side population” stem cells, have also been shown to differentiate to thecells), and (4) circulating mature endothelial cells, which are endothelial lineage.19,20 Recently, tissue-resident stem cellsshed off the vessel wall6 and adhere to the culture dishes. First have been isolated from the heart, which are capable toevidence that there is more than one endothelial progeny differentiate to the endothelial lineage.21 These data supportwithin the circulating blood was provided by Hebbel and the notion that it will be difficult to define the “true”colleagues, who showed that morphological and functional endothelial progenitor cells. Overall, the field is reminiscentdistinct endothelial cell populations can be grown out of to immunology, where T-cells initially were defined as oneperipheral blood mononuclear cells.7 They stratified the cell population. However, the functional characterization (eg,different circulating endothelial cells according to their cytokine release and response to stimuli) helped to identifygrowth characteristics and morphological appearance as novel T-cell subpopulations with distinct functions and ca-“spindle-like cells,” which have a low proliferative capacity, pacities. Hopefully, better profiling of distinct cell popula-and outgrowing cells. Because the outgrowing cells showed a tions and fate mapping studies will help to identify markers,high proliferative potential and originated predominantly which distinguish the circulating endothelial precursor withinfrom the bone marrow donors, they were considered as circu- the blood and bone marrow/non-bone marrow– derived endo-lating angioblasts.7 The authors speculated that the spindle-like thelial cells.cells may likely represent mature endothelial cells, which areshed off the vessel wall. However, this hypothesis is difficult to Role of EPCs in Neovascularizationtest and has not yet been proven thus far. Improvement of neovascularization is a therapeutic option to Experimentally, preplating may be a way to reduce the rescue tissue from critical ischemia.22 The finding that boneheterogeneity of the cultivated EPCs, because this excludes marrow– derived cells can home to sites of ischemia andrapidly adhering cells such as differentiated monocytic or express endothelial marker proteins has challenged the use ofpossible mature endothelial cells.2 However, these protocols isolated hematopoietic stem cells or EPCs for therapeuticdo not eliminate myeloid and nonhematopoietic progenitor vasculogenesis. Infusion of various distinct cell types eithercells, which may contribute to the ex vivo cultivated cells. isolated from the bone marrow or by ex vivo cultivation wasThere is increasing evidence that myeloid cells can give rise shown to augment capillary density and neovascularization ofto endothelial cells as well. Specifically, CD14ϩ/CD34Ϫ ischemic tissue (Table 1 and Figure 2). In animal models ofmyeloid cells can coexpress endothelial markers and form myocardial infarction, the injection of ex vivo expandedtube-like structures ex vivo.8 Additionally, ex vivo expansion EPCs or stem and progenitor cells significantly improvedof purified CD14ϩ mononuclear cells yielded cells with an blood flow and cardiac function and reduced left ventricularendothelial characteristic, which incorporated in newly scarring.23,24 Similarly, infusion of ex vivo expanded EPCsformed blood vessels in vivo.9 These data would suggest that deriving from peripheral blood mononuclear cells in nudemyeloid cells can differentiate (or transdifferentiate) to the mice or rats improved the neovascularization in hind limb Downloaded from http://circres.ahajournals.org/ at UNIV DE CONCEPCION on April 11, 2012
  • 4. Urbich and Dimmeler Endothelial Progenitor Cells and Vascular Biology 345 Figure 1. Origin and differentiation of endothelial progenitor cells. Scheme depicts the potential origin and differenti- ation of endothelial progenitor cells from hematopoietic stem cells and nonhema- topoietic cells.ischemia models.9,15,23,25 Correspondingly, initial pilot trials uncultivated CD34Ϫ cells. Remarkably, terminally differenti-indicate that bone marrow– derived or circulating blood– ated mature endothelial cells (HMVECs, GEAECs, andderived progenitor cells are useful for therapeutically improv- SVECs) did not improve neovascularization15,24,33 suggestinging blood supply of ischemic tissue.26,27 Autologous implan- that a not-yet-defined functional characteristic (eg, chemo-tation of bone marrow mononuclear cells in patients with kine or integrin receptors mediating homing) is essential forischemic limbs significantly augmented ankle-brachial index EPC-mediated augmentation of blood flow after ischemia.and reduced rest pain.26 In addition, transplantation of ex vivo The functional capacity of EPCs to augment blood flowexpanded endothelial progenitor cells significantly improved further does not appear to be solely attributable to a mono-coronary flow reserve and left ventricular function in patients cytic phenotype. Ex vivo cultivated EPCs from CD14ϩwith acute myocardial infarction.27 mononuclear cells or CD14Ϫ mononuclear cell starting pop- Besides models of peripheral ischemia (hind limb ische- ulation improved neovascularization to a similar extent,mia), the angiogenic potential of EPCs was also investigated whereas the same number of freshly isolated mononuclearin animal models of tumor angiogenesis. Thereby, the inhi- cells taken from the starting culture did not.9 Interestingly,bition of VEGF-responsive bone marrow– derived endothelial these experimental data are supported by first clinical trialsand hematopoietic precursor cells blocks tumor angiogenesis showing that freshly isolated mononuclear cells are not welland growth.28 The use of various different models, cell suited to improve neovascularization in patients with periph-numbers, and species limits the comparability of the effi- eral vascular diseases.26 However, monocytic cells may playciency of distinct cell populations. However, the overall a crucial role in collateral growth (arteriogenesis). Thus, thefunctional improvement appear similar, when isolated human attraction of monocytic cells by monocyte chemoattractantCD34ϩ, CD133ϩ, EPC, MAPC, or murine Sca-1ϩ cells were protein-1 (MCP-1) enhanced arteriogenesis.34 Moreover, de-used.4,9,15,20,23,25,29 –32 Likewise, early spindle-like cells and pletion of the monocytes reduced PlGF-induced arteriogen-late outgrowing EPCs showed comparable in vivo vasculo- esis.35 A therapeutic benefit of monocyte infusion on arterio-genic capacity.33 These results suggest that the functional genesis was demonstrated under conditions of monocyteactivity of the cells to augment neovascularization is rather deficiency induced by chemical depletion.36 These data sug-independent of the type of (endothelial) progenitor cell used. gest that monocytic cells are necessary for arteriogenesis andHowever, the CD34Ϫ fractions of freshly isolated bone possibly neovascularization. For therapeutic application, themarrow– or blood-derived mononuclear cells showed a re- local enhancement of monocyte recruitment might be betterduced incorporation and functional activity.24,29 These data suited than systemic infusion of monocytic cells, which onlyindicate that the number of cells capable to augment neovas- leads to a relatively minor increase in the number of circu-cularization is low in this crude fraction of freshly isolated lating monocytes. Downloaded from http://circres.ahajournals.org/ at UNIV DE CONCEPCION on April 11, 2012
  • 5. 346 Circulation Research August 20, 2004TABLE 1. Neovascularization Induced by Injection of Progenitor Cells: Experimental and Clinical StudiesCells Surface Markers Improvement Models Incorporation RateExperimental studies Freshly isolated cells CD34ϩ cells CD34ϩ/flk-1ϩ, CD45ϩ 1 Incorporation1 13.4 Ϯ5.7% (mouse) or 9.7 Ϯ4.5% (rabbit) Dil-Ac-LDL-EPC in CD31ϩ capillaries1 Tie-2ϩ, Dil-Ac-LDLϩ 29 Hind limb ischemia29 Frequently detected (not quantified)29 CD117bright/GATAϪ2/VEGFR2/Tie-2/AC133 24 Myocardial infarction24 20–25% of total myocardial capillary vasculature24 Sca-1ϩ BM-MNCs Sca-1ϩ 30 Hind limb ischemia30 Detected (not quantified) PBMCs T and B lymphocytes and monocytes-depleted Hind limb ischemia30 MNCs30 Ex vivo expanded cells Ex vivo expanded EPC Dil-Ac-LDLϩ/lectinϩ VEGFR2ϩ, VE-cadherinϩ, Hind limb ischemia15,31 2.1 Ϯ0.4 EPCs into vessels in a CD31ϩ, CD14ϩ, CD34ϩ 15,23 Myocardial infarction23 ϫ10 field15 241 Ϯ25 cells/mm2 (day 3) 355 Ϯ30 cells/mm2 (day 7)31 Dil-Ac-LDLϩ, NOϩ, VEGFR2ϩ, VE-cadherinϩ, Hind limb ischemia25 Frequently detected (not CD31ϩ, vWFϩ, CD45Ϫ 25 quantified)25 CD31ϩ, vWFϩ, Dil-Ac-LDLϩ, VEGFR2ϩ, Tie-2ϩ 53 Vascular graft survival, Neovessel remodeling53 80% of graft lumen at day 1553 Dil-Ac-LDLϩ/lectinϩ Hind limb ischemia9 19.8 Ϯ8% CD146ϩ/HLA-DRϩ cell VEGFR2ϩ, CD105ϩ, vWFϩ, CD45ϩ 9 containing vessels9 Early EPC: Dil-Ac-LDLϩ/lectinϩ VEGFR2ϩ, CD31ϩ, Matrigel capillaries16 ND Tie-2ϩ, VE-cadherinϪ, eNOSϪ, CD14ϩ 16 Outgrowing ECs: exhibited a greater capacity for Outgrowing ECs: VEGFR2ϩ, CD31ϩ, Tie-2ϩ, capillary morphogenesis in in vitro and in vivo VE-cadherinϩ, eNOSϩ, CD14Ϫ 16 matrigel models Early EPC: weak VEGFR1, eNOS, vWF, In vitro: late EPC showed better incorporation and Detected (not quantified)33 VE-cadherin, VEGFR2, spindle shape33 tube formation. Early EPC: higher release of Late EPC: strong VE-cadherin, VEGFR1, VEGFR2, growth factors. In vivo: comparable vasculogenic eNOS, vWF, cobblestone morphology33 potential of early and late EPC (limb perfusion, capillary density) MAPC-derived ECs Co-purified MAPC: CD34Ϫ, VE-cadherinϪ, tumor growth/angiogenesis20 MAPC-derived ECs20 AC133ϩ, Flk-1ϩ 20 35% tumor angiogenesis, 30–45% Angioblast: CD34ϩ, VE-cadherinϩ, AC133Ϫ, ϩ 20 wound healing angiogenesis, Flk-1 undifferentiated MAPCs: 12%Clinical studies BMC and monocytes (TACT-trial) CD34ϩ/Dil-Ac-LDLϩ/lectinϩ Intramuscular injection in patients with peripheral ND ischemic disease; improved blood flow26 CPC and BMC (TOPCARE-AMI) CPC: Dil-Ac-LDLϩ/lectinϩ, VEGFR2ϩ, CD31ϩ, Intracoronary infusion in patients with AMI; ND vWFϩ, CD105ϩ; BMC: CD34ϩ/CD45ϩ, increase in coronary flow reserve27 CD34ϩ/CD133ϩ, CD34ϩ/VEGFR2ϩMechanisms by Which EPC derived cells adjacent to vessels, which do not expressImprove Neovascularization endothelial marker proteins.41,45 A reasonable explanationAlthough the role of EPCs in neovascularization has been might be that the model of ischemia (eg, intensity of injury orconvincingly shown by several groups, the question remains: ischemia)46 significantly influences the incorporation rate. Ahow do EPCs improve neovascularization? minor ischemia might not as profoundly induce a mobiliza- Bone marrow transplantation of genetically modified cells tion of bone marrow– derived endothelial progenitor cells(rosa-26, GFP, lacZ) was used to assess the incorporation of and, thus, may lead to a lower percentage of incorporation ofbone marrow-derived EPC into tissues. The basal incorpora- bone marrow– derived progenitor cells. The efficiency oftion rate of progenitor cells without tissue injury is extremely engraftment may additionally differ between distinct progen-low.37 In ischemic tissue, the incorporation rate of genetically itor subpopulations (pure hematopoietic stem cells versuslabeled bone marrow– derived cells, which coexpress endo- complete bone marrow cells). Indeed, therapeutic applicationthelial marker proteins, differs from 0% to 90% incorpora- of cells by intravenous infusion of ex vivo purified bonetion.19,28,37– 41 Likewise, the extent of incorporation of bone marrow mononuclear cells or expanded endothelial progeni-marrow– derived cells in cerebral vessels after stroke varies in tor cells led to a higher incorporation rate (Ϸ7% to 20%the literature.42– 44 Whereas two studies reported positive incorporation rate) as compared with the endogenously mo-vessels with an average of 34% endothelial marker express- bilized bone marrow– engrafted cells (Ϸ2%).9,47ing bone marrow– derived cells,42,43 other groups could not However, the number of incorporated cells with an endo-detect endothelial marker expressing cells.44 High amounts thelial phenotype into ischemic tissues is generally quite low.(Ͼ50%) were predominantly detected in models of tumor How can such a small number of cells increase neovascular-angiogenesis.28,40 Some studies only detected bone marrow– ization? A possible explanation might be that the efficiency Downloaded from http://circres.ahajournals.org/ at UNIV DE CONCEPCION on April 11, 2012
  • 6. Urbich and Dimmeler Endothelial Progenitor Cells and Vascular Biology 347 Figure 2. Role of EPCs in vascular biol- ogy. Injection of EPCs significantly improve reendothelialization and neovas- cularization after injury.of neovascularization may not solely be attributable to the be rapidly covered by bone marrow– derived cells derivingincorporation of EPCs in newly formed vessels, but may also from CD34ϩ hematopoietic stem cells in a dog model.2 Inbe influenced by the release of proangiogenic factors in a humans, the surface of ventricular assist devices was coveredparacrine manner. Indeed, the deletion of Tie-2–positive bone by even more immature CD133-positive hematopoietic stemmarrow– derived cells through activation of a suicide gene cells, which concomitantly express the VEGF-receptor 2.3blocked tumor angiogenesis, although these cells are not Additionally, Walter and coworkers demonstrated that circu-integrated into the tumor vessels but are detected adjacent to lating endothelial precursor cells can home to denuded partsthe vessel.41 Thus, EPCs may act similar to monocytes/mac- of the artery after balloon injury.51 Bone marrow transplan-rophages, which can increase arteriogenesis by providing tation experiments revealed that bone marrow– derived cellscytokines and growth factors. Indeed, EPCs cultivated from can contribute to reendothelialization of grafts and denudeddifferent sources showed a marked expression of growth arteries.51–53 However, in a model of transplant arteriosclero-factors such as VEGF, HGF, and IGF-1 (C.U., unpublished sis, bone marrow– derived cells appear to contribute only to adata, 2004). Moreover, adherent monocytic cells, which were minor extent to endothelial regeneration by circulating cells.18cultivated under similar conditions, but do not express endo- These data again indicate that there might be at least twothelial marker proteins, also release VEGF, HGF, and distinct populations of circulating cells that principally areG-CSF.14 The release of growth factors in turn may influence capable to contribute to reendothelialization, namely mobi-the classical process of angiogenesis, namely the proliferation lized cells from bone marrow and non-bone marrow– derivedand migration as well as survival of mature endothelial cells. The latter ones may arise from circulating progenitorcells.48 However, EPCs additionally incorporated into the cells released by non-bone marrow sources (eg, tissue resi-newly formed vessel structures and showed endothelial dent stem cells) or represent vessel wall– derived endothelialmarker protein expression in vivo. In contrast, infusion of cells.18,51–53macrophages, which are known to release growth fac- A rapid regeneration of the endothelial monolayer maytors,49,50 but were not incorporated into vessel-like struc- prevent restenosis development by endothelial synthesis oftures, induced only a slight increase in neovascularization antiproliferative mediators such as nitric oxide. Indeed, en-after ischemia, indicating— but not proving—that the ca- hanced incorporation of ␤-galactosidase–positive, bone mar-pacity of EPCs to physically contribute to vessel-like row– derived cells was associated with an accelerated reen-structures may contribute to their potent capacity to dothelialization and reduction of restenosis.51,52 Similarimprove neovascularization.9 Further studies will have to results were reported by Griese et al, who demonstrated thatbe designed to elucidate the contribution of physical infused peripheral blood monocyte– derived EPC home toincorporation, paracrine effects and possible effects on bioprosthetic grafts and to balloon-injured carotid arteries, thevessel remodeling and facilitating vessel branching to latter being associated with a significant reduction in neoin-EPC-mediated improvement of neovascularization. tima deposition.54 Likewise, infusion of bone marrow– de- rived CD34Ϫ/CD14ϩ mononuclear cells, which are not rep- EPCs and Endothelial Regeneration resenting the population of the “classical hemangioblast,”In the past, the regeneration of injured endothelium has been contributed to endothelial regeneration.13 The regeneratedattributed to the migration and proliferation of neighboring endothelium was functionally active as shown by the releaseendothelial cells. More recent studies, however, indicate that of NO,13 which is supposed to be one of the major vasculo-additional repair mechanisms may exist to replace denuded or protective mechanisms. Consistently, neointima developmentinjured arteries. Thus, implanted Dacron grafts were shown to was significantly reduced after cell infusion.13 Whereas the Downloaded from http://circres.ahajournals.org/ at UNIV DE CONCEPCION on April 11, 2012
  • 7. 348 Circulation Research August 20, 2004regeneration of the endothelium by EPCs protects lesion apy with plasmids encoding for VEGF demonstrated anformation, bone marrow– derived stem/progenitor cells may augmentation of EPC levels in humans.71 Additional factorsalso contribute to plaque angiogenesis, thereby potentially inducing mobilization of progenitor cells from the bonefacilitating plaque instability.55 However, in a recent study, marrow have been initially discovered in hematology tono influence of bone marrow cell infusion on plaque compo- harvest hematopoietic stem cells from the peripheral bloodsition was detected in nonischemic mice.56 An increase in for bone marrow transplantation. For instance, granulocyte-plaque size was only detected in the presence of ischemia, colony stimulating factor (G-CSF), a cytokine, which issuggesting that ischemia-induced release of growth factors typically used for mobilization of CD34ϩ cells in patients,predominantly accounts for this effect.56 also increased the levels of circulating endothelial progenitor Overall, these studies implicate that regardless of the origin cells. A related cytokine, the granulocyte monocyte-colonyof circulating endothelial progenitor cells, this pool of circu- stimulating factor (GM-CSF), augments EPC levels.30 More-lating endothelial cells may exert an important function as an over, erythropoietin (EPO), which stimulates proliferationendogenous repair mechanism to maintain the integrity of the and maturation of erythroid precursors, also increased periph-endothelial monolayer by replacing denuded parts of the eral blood endothelial progenitor cells in mice72 and in men.73artery (Figure 2). One can speculate that these cells may also The correlation between EPO serum levels and the number ofregenerate the low grade endothelial damage by ongoing CD34ϩ or CD133ϩ hematopoietic stem cells in the boneinduction of endothelial cell apoptosis induced by risk factors marrow in patients with ischemic coronary artery diseasefor coronary artery disease (see review).57 The maintenance further supports an important role of endogenous EPO levelsof the endothelial monolayer may prevent thrombotic com- as a physiologic determinant of EPC mobilization.72 Atplications and atherosclerotic lesion development. Although present, it is not clear which of the mobilizing factors mostthis concept has not yet been proven, several hints from potently elevates the levels of EPCs. SDF-1 and VEGF165recently presented data are supportive. Thus, transplantation showed similar effects and rapidly mobilize hematopoieticof ApoEϪ/Ϫ mice with wild-type bone marrow reduced stem cells and circulating endothelial precursor cells inatherosclerotic lesion formation.58 Moreover, various risk animal models, whereas angiopoietin-1 induced a delayedfactors for coronary artery disease, such as diabetes, hyper- and less pronounced mobilization of endothelial and hema-cholesterolemia, hypertension, and smoking, affect the num- topoietic progenitors.74,75 Whereas a similar increase in whiteber and functional activity of EPCs in healthy volunteers59 blood cell counts was achieved by G-CSF application, endo-and in patients with coronary artery disease.60 Likewise, thelial colonies (CFU-EC) were significantly lower in G-diabetic mice and patients were characterized by reduced CSF– compared with VEGF- or SDF-1–treated mice. Offunctional activity of EPCs.61– 63 In addition, factors that note, these data should be interpreted with caution, becausereduce cardiovascular risk such as statins38,51,52,64 or exer- the responsiveness toward cytokines may vary between dif-cise65 elevate EPC levels, which contribute to enhanced ferent mice strains and side-by-side comparisons in humansendothelial repair. The balance of atheroprotective and are lacking. Moreover, the extent of increasing neutrophil andproatherosclerotic factors, thus, may influence EPC levels lymphocyte levels, which may provoke proinflammatoryand subsequently reendothelialization capacity. responses, has to be considered for a potential therapeutic application. Mobilization of EPCs First evidence for potential pharmacological modulation ofBecause EPCs contribute to reendothelialization and neovas- systemic EPC levels by atheroprotective drugs came fromcularization, increasing the number of these cells may be an studies using HMG-CoA reductase inhibitors (statins). Statinsattractive therapeutic tool. The mobilization of stem cells in were shown to increase the number and the functional activitythe bone marrow is determined by the local microenviron- of EPCs in vitro,38,76 in mice,38,76 and in patients with stablement, the so-called “stem cell niche,” which consists of coronary artery disease.64 The increase in EPC numbers wasfibroblasts, osteoblasts, and endothelial cells (see review).66 associated with increased bone marrow– derived cells afterBasically, mobilizing cytokines hamper the interactions be- balloon injury and accelerated endothelial regeneration.51,52tween stem cells and stromal cells, which finally allow stem Although statins were shown to increase the number of stemcells to leave the bone marrow via transendothelial migration. cells within the bone marrow, the mechanism for enhancingThereby, activation of proteinases such as elastase, cathepsin EPC numbers and function may additionally include anG, and matrix metalloproteinases (MMPs) cleave adhesive increase in proliferation, mobilization, and prevention of EPCbonds on stromal cells, which interact with integrins on senescence and apoptosis.12,38,76 Interestingly, recent studieshematopoietic stem cells. MMP-9 was additionally shown to additionally demonstrated that estrogen increased the levelscleave the membrane-bound Kit ligand (mKitL) and induces of circulating EPCs.77,78 Moreover, exercise augmented EPCthe release of soluble Kit ligand (KitL; also known as stem levels in mice and in men.65 The molecular signaling path-cell factor, SCF).67 Physiologically, ischemia is believed to be ways have not been identified thus far. However, severalthe predominant signal to induce mobilization of EPCs from studies indicate that the activation of the PI3K/Akt pathway,the bone marrow. Ischemia thereby is believed to upregulate which has first been shown to be activated in matureVEGF or SDF-1,68,69 which in turn are released to the endothelial cells by statins,79 may also play an important rolecirculation and induce mobilization of progenitor cells from in statin-induced increase in EPC levels.12,76 Likewise,the bone marrow via a MMP-9 – dependent mecha- VEGF, EPO, estrogen, and exercise are well known tonism.30,46,67,70 Furthermore, clinical studies using gene ther- augment the PI3K/Akt-pathway. Thus, these factors may Downloaded from http://circres.ahajournals.org/ at UNIV DE CONCEPCION on April 11, 2012
  • 8. Urbich and Dimmeler Endothelial Progenitor Cells and Vascular Biology 349 Figure 3. Mechanism of EPC homing and differentiation. Recruitment and incorporation of EPCs into ischemic tis- sue requires a coordinated multistep process including mobilization, chemoat- traction, adhesion, transmigration, migra- tion, tissue invasion, and in situ differen- tiation. Factors that are proposed to regulate the distinct steps are indicated.share some common signaling pathways. Given that recent cells activated by cytokines and ischemia and the transmigra-data showed that eNOS is essential for mobilization of bone tion of the progenitor cells through the endothelial cellmarrow– derived stem and progenitor cells,47 one may spec- monolayer.80 Integrins are known to mediate the adhesion ofulate that these stimuli may increase progenitor cell mobili- various cells including hematopoietic stem cells and leuko-zation by PI3K/Akt-dependent activation of the NO-synthase cytes to extracellular matrix proteins and to endothelialwithin the bone marrow stromal cells. Indeed, exercise and cells.81– 83 Integrins capable of mediating cell-cell interactionsVEGF-stimulated EPC mobilization was blunted in eNOSϪ/Ϫ are the ␤2-integrins and the ␣4␤1-integrin. ␤1-Integrins aremice.47,65 expressed by various cell types including endothelial cells and hematopoietic cells, whereas ␤2-integrins are found Mechanism of Homing and Differentiation preferentially on hematopoietic cells.84 Because adhesion toAlthough the improvement of adult neovascularization is endothelial cells and transmigration events are involved in thecurrently under intensive investigations, the mechanism of in vivo homing of stem cells to tissues with active angiogen-homing and differentiation of endothelial progenitor cells is esis,80 integrins such as the ␤2-integrins and the ␣4␤1-integrinpoorly understood. In a previous study assessing in vivo may be involved in the homing of progenitor cells to ischemichoming of embryonic endothelial progenitor cells derived tissues. Consistent with the high expression of ␤2-integrins onfrom cord blood, the circulating cells arrested within tumor hematopoietic stem/progenitor cells, ␤2-integrins mediate ad-microvessels, extravasated into the interstitium, and incorpo- hesion and transmigration of hematopoietic stem/progenitorrated into neovessels, suggesting that adhesion and transmi- cells.85,86 ␤2-Integrins (CD18/CD11) are expressed on periph-gration are involved in the recruitment of endothelial progen- eral blood-derived EPCs and are required for EPC-adhesionitor cells to sites of tumor angiogenesis.80 Thus, it is to endothelial cells and transendothelial migration in vitroconceivable that ex vivo expanded adult EPCs and hemato-poietic stem/progenitor cells may engage similar pathways (S.D., personal communication, 2004). Moreover, hemato-for recruitment to sites of ischemia and incorporation in poietic stem cells (Sca-1ϩ/linϪ) lacking ␤2-integrins showednewly forming vessels. Recruitment and incorporation of reduced homing and a lower capacity to improve neovascu-EPCs requires a coordinated sequence of multistep adhesive larization after ischemia (S.D., personal communication,and signaling events including chemoattraction, adhesion, 2004). Interestingly, the homing of inflammatory cells duringand transmigration, and finally the differentiation to endothe- pneumonia or myocardial ischemia in ␤2-integrin– deficientlial cells (Figure 3). mice is mediated by the ␣4␤1-integrin87,88 suggesting that deficiency of ␤2-integrins can in part be compensated by theAdhesion and Transendothelial Migration ␣4␤1-integrin. Moreover, conditional deletion of the ␣4-The initial step of homing of progenitor cells to ischemic integrin selectively inhibited the homing of hematopoietictissue involves adhesion of progenitor cells to endothelial stem/progenitor cells to the bone marrow but not to the Downloaded from http://circres.ahajournals.org/ at UNIV DE CONCEPCION on April 11, 2012
  • 9. 350 Circulation Research August 20, 2004spleen,89 suggesting that the homing of progenitor cells to TABLE 2. Unresolved Questionsdifferent tissues is dependent on distinct adhesion molecules. How to define an endothelial progenitor cell?Furthermore, in vitro studies showed that MCP-1 stimulated Origin of endothelial progenitor cells?adhesion of bone marrow– derived CD34Ϫ/CD14ϩ monocytes Definition of subpopulations with different functional capacities?to the endothelium was blocked by anti–␤1-integrin antibod-ies.13 Interestingly, in this study, adhesion of CD34Ϫ/CD14ϩ Signals for EPC homing and differentiation in vivo?monocytes isolated from the peripheral blood to endothelial Optimization of ex vivo culture conditions to enhance the benefit of cellcells was less affected by MCP-1 and was not blocked by therapy?anti–␤1-integrin antibodies.13 Finally, the initial cell arrest of Influence of the severity of vascular damage on the contribution of EPCsembryonic progenitor cell homing during tumor angiogenesis to regeneration?was suggested to be mediated by E- and P-selectin and Mechanisms of action?P-selectin glycoprotein ligand-1.80 Yet, it is important to Transdifferentiation capacity of different progenitor cells?underscore that this study was performed with embryonic Importance of paracrine effects?endothelial progenitor cells. It is conceivable that differentcell types may use distinct mechanisms for homing to sites of interleukins, which can attract circulating progenitor cells.13angiogenesis. Whereas several studies shed some light on the mechanisms Cell-cell contacts and transmigration events might be less regulating attraction of EPCs to ischemic tissue, less is knownimportant for the reendothelialization of denuded arteries (incontrast to homing of progenitor cells to ischemic tissues). with respect to migration and tissue invasion. One mayWith respect to endothelial progenitor cells, studies investi- speculate that proteases such as cathepsins or metallopro-gated the contribution of integrins to reendothelialization, teases may mediate the tissue invasion of EPCs.which is mainly driven by adhesion to extracellular matrix Differentiationproteins. Adhesion of EPCs to denuded vessels appears to be Finally, maturation of EPCs to a functional endothelial cellmediated by vitronectin-receptors (␣v␤3- and ␣v␤5-integrins). may be important for functional integration in vessels. TheThus, inhibition of ␣v␤3- and ␣v␤5-integrins with cyclic RGD genetic cascades that regulate differentiation in the adultpeptides blocked reendothelialization of denuded arteries in system are largely unknown; however, several studies deter-vivo, suggesting that ␣v␤3- and ␣v␤5-integrins are involved in mined the differentiation of the common mesodermal precur-the reendothelialization of injured carotid arteries.51 How- sor, the hemangioblasts, during embryonic development.ever, other integrins such as the ␤1-integrins may also mediate Clearly, VEGF and its receptors play a crucial role foradhesion of progenitor cells to extracellular matrix proteinsduring reendothelialization of denuded arteries.13 stimulating endothelial differentiation in the embryonic de- velopment.96 –98 Likewise, VEGF induces differentiation ofChemotaxis, Migration, and Invasion endothelial cells in ex vivo culture assays using a variety ofGiven the low numbers of circulating progenitor cells, che- adult progenitor populations (CD34ϩ,1 CD133ϩ,4 peripheralmoattraction may be of utmost importance to allow for blood mononuclear cells).15,76 Studies with embryonic stemrecruitment of reasonable numbers of progenitor cells to the cells further revealed that the temporal regulation of Ho-ischemic or injured tissue. Various studies examined the meobox (Hox) genes might play an important role. Thus, thefactors influencing hematopoietic stem cell engraftment to orphan Hox gene termed Hex (also named Prh) is required forthe bone marrow. These factors include chemokines such as differentiation of the hemangioblast into the definitive hema-SDF-1,90,91 lipid mediators (sphingosine-1-phosphate),92 as topoietic progenitors and also affected endothelial differenti-well as factors released by heterologous cells.93 The factors ation.99 Additionally, the serine/threonine kinase Pim-1 wasattracting circulating EPCs to the ischemic tissue may be recently discovered as a VEGF-responsive gene, which con-similar. Indeed, SDF-1 has been proven to stimulate recruit- tributes to endothelial differentiation out of embryonic stemment of progenitor cells to the ischemic tissue.31 SDF-1 cells.100protein levels were increased during the first days afterinduction of myocardial infarction.94 Moreover, overexpres- Conclusionsion of SDF-1 augmented stem cell homing and incorporation Taken together, infusion of different hematopoietic stem cellinto ischemic tissues.31,94 Interestingly, hematopoietic stem populations and ex vivo expanded EPCs augmented neovas-cells were shown to be exquisitely sensitive to SDF-1 and did cularization of tissue after ischemia, thereby providing anot react to G-CSF or other chemokines (eg, IL-8 and novel therapeutic option. However, a variety of unresolvedRANTES).91 Moreover, VEGF levels are increased during questions remain to be answered (Table 2). The crucialischemia and capable to act as a chemoattractive factor to question is how to define an endothelial progenitor cell?EPCs.68,70,71 Interestingly, the migratory capacity of EPCs or Overall, there is consensus that endothelial progenitor cellsbone marrow cells toward VEGF and SDF-1, respectively, can derive from the bone marrow and that CD133/VEGFR2determined the functional improvement of patients after stem cells represent a population with endothelial progenitor ca-cell therapy.95 Beside genes, which are directly upregulated pacity. However, increasing evidence suggest that there areby hypoxia, the invasion of immune competent cells to the additional bone marrow– derived cell populations (eg, my-ischemic tissue may further augment the levels of various eloid cells) within the blood, which also can give rise tochemokines within the ischemic tissue, such as MCP-1 or endothelial cells. Moreover, non-bone marrow– derived cells Downloaded from http://circres.ahajournals.org/ at UNIV DE CONCEPCION on April 11, 2012
  • 10. Urbich and Dimmeler Endothelial Progenitor Cells and Vascular Biology 351with endothelial characteristic were isolated from the periph- genitor cells via regulation of cell cycle regulatory genes. Circ Res.eral blood. This might represent shed mature endothelial cells 2003;92:1049 –1055 13. Fujiyama S, Amano K, Uehira K, Yoshida M, Nishiwaki Y, Nozawa Y,or other endothelial cells deriving from other progenitor cell Jin D, Takai S, Miyazaki M, Egashira K, Imada T, Iwasaka T,populations. Clearly, one functional assay to define endothe- Matsubara H. Bone marrow monocyte lineage cells adhere on injuredlial progenitor cells independent of their progeny is the endothelium in a monocyte chemoattractant protein-1-dependent manner and accelerate reendothelialization as endothelial progenitordemonstration of clonal expansion activity. Possibly, func- cells. Circ Res. 2003;93:980 –989.tional assays will gain additional increasing importance, 14. Rehman J, Li J, Orschell CM, March KL. Peripheral blood “endothelialbecause recent studies suggest that endothelial progenitor progenitor cells” are derived from monocyte/macrophages and secretecells have a favorable survival and a better response toward angiogenic growth factors. Circulation. 2003;107:1164 –1169. 15. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearneyangiogenic growth factors compared with mature endothelial M, Li T, Isner JM, Asahara T. Transplantation of ex vivo expandedcells.101 From a therapeutic point of view, these functional endothelial progenitor cells for therapeutic neovascularization. Procactivities might be more important than the source of the Natl Acad Sci U S A. 2000;97:3422–3427. 16. Gulati R, Jevremovic D, Peterson TE, Chatterjee S, Shah V, Vile RG,progenitor cell. Another open question is which mechanism Simari RD. Diverse origin and function of cells with endothelial phe-underlies the improvement of neovascularization by infused notype obtained from adult human blood. Circ Res. 2003;93:1023–1025.EPCs? Likely, paracrine effects contribute in addition to the 17. Rookmaaker MB, Vergeer M, van Zonneveld AJ, Rabelink TJ, Verhaarphysical incorporation of EPC into newly formed capillaries. MC. Endothelial progenitor cells: mainly derived from the monocyte/ macrophage-containing CD34- mononuclear cell population and only inThe influence of the incorporation of a rather small number of part from the hematopoietic stem cell-containing CD34ϩ mononuclearcirculating cells on remodeling and vessel maturation has to cell population. Circulation. 2003;108:e150.be further elucidated. 18. Hillebrands JL, Klatter FA, van Dijk WD, Rozing J. Bone marrow does not contribute substantially to endothelial-cell replacement in transplant arteriosclerosis. Nat Med. 2002;8:194 –195. Acknowledgments 19. Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ, Majesky MW,This study is supported by the DFG (FOR 501: Di 600/6-1). We Entman ML, Michael LH, Hirschi KK, Goodell MA. 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