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Online Appendix for the following February 20 JACC article TITLE: Endothelial Progenitor Cells in Cardiovascular Disorders AUTHORS: Eduard Shantsila, MD, Haemostasis, Thrombosis & Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham, United Kingdom, Timothy Watson, MRCP, Haemostasis, Thrombosis & Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham, United Kingdom, Gregory Y.H. Lip, MD, FRCP, Haemostasis, Thrombosis & Vascular Biology Unit, University Department of Medicine, City Hospital, Birmingham, United Kingdom APPENDIX SEARCH STRATEGY We performed a comprehensive literature search using electronic bibliographic databases (i.e., MEDLINE, EMBASE, DARE, COCHRANE DATABASE), scanning reference lists from included articles and hand searching abstracts from national and international cardiovascular meetings. For the search, we used the term “endothelial progenitor cell(s).” Bibliographies of all selected articles and review articles were reviewed for other relevant articles. Finally, the supplements of major journals were hand searched to identify relevant abstracts that had not been published as peer-reviewed articles. Where necessary, study authors were contacted to obtain further data. ENDOTHELIAL PROGENITOR CELLS: PATHOPHYSIOLOGICAL CONSIDERATIONS—DEFINITION AND LINKS TO ANGIOGENESIS Endothelial Markers In 1997, Asahara et al. (1) first reported the isolation of endothelial precursor cells (EPCs) from peripheral blood because they had found that CD34+ hematopoietic progenitor cells can differentiate to cells with endothelial characteristics. The EPCs were defined as cells positive for both hematopoietic stem cell and endothelial cell markers, such as CD34 and vascular endothelial growth factor (VEGF) receptor-2, respectively. The latter VEGF receptor-2 is often referred to as kinase insert domain receptor (KDR). The putative CD34+ EPC is able to proliferate and differentiate to mature endothelial cells with expression of different endothelial markers (Figure) such as KDR (2,3), plateletendothelial cell adhesion molecule (CD31) (2,4), von Willebrand factor (2,3,5), VE-cadherin (2,3), caveolin-1 (4,6), and endothelial nitric oxide synthase (4,6). While in vitro, EPCs can form vascular-like structures (3,6), and in vivo, incorporate into neovessels at sites of tissue ischemia (3,5,7). Of note, CD34 antigen density is highest on early progenitors and decreases progressively as cells mature (8); however, CD34 is expressed not only on EPCs but on mature endothelial cells, albeit at a lower density (9). Therefore, an early hematopoietic stem cells marker, CD133, was adopted as an alternative additional marker to indicate a “true” EPC (10,12). FIGURE Potential Origin and Differentiation of Endothelial Progenitor Cells BONE MARROW Hematopoietic stem cells CD34+ CD133++ CD45+ Mesenchymal stem cells CD34- c-kit- Side population cells Myeloid Precursors CD14+ CD45+ Tissue-resident stem cells c-kit+ “Early” EPCs CD34++ CD133++ KDR+ Macrophages Monocytes “More mature” EPCs + + ++ CD14 + CD14+++ CD45+ CD45 + CD34 CD133 KDR BLOOD VESSEL Mature EPCs CD133- CD34low KDR+ CD31+ VE-Cadherin+ vWF+ Are these cells the same? The marker CD133 (also known as prominin or AC133) is a 120-kDa transmembrane polypeptide with an (as yet) unknown biological function. It is expressed on hematopoietic stem and progenitor cells from human bone marrow, fetal liver, and peripheral blood (11). As progenitors develop to more mature endothelium-like cells, CD133 is rapidly downregulated (10). The CD133+ cells are able to form both early and late outgrowing colonies in vitro (10). Thus, CD133 might provide a more reliable means of defining and tracking human angioblast-like EPCs and distinguishing these from mature endothelial or monocytic cells. The combination of CD34, CD133, and KDR is commonly used for this purpose, and this represents a rare subset of peripheral blood cells. Interestingly, the CD34−/133+ subpopulation of endothelial progenitors was recently found to be a possible precursor of classic CD34+/133+ EPCs, but to possess even more potent angioregenerative properties in vivo than on the latter (13). Hematopoietic Markers Hematopoietic cell markers such as CD34, CD133, and CD117 (c-kit) and monocytic/macrophagic cell markers such as CD11 and CD14 are expressed by early immature EPCs, and are subsequently lost in the more differentiated state (10,11). Paradoxically, expression of endothelial markers progressively increases with EPC maturation (3). Subtypes of these cells that do not express VE-cadherin and von Willebrand factor seem to be an early subpopulation of EPCs localized predominantly in the bone marrow or detected immediately after their migration into the systemic circulation (14). More mature circulating CD34-positive EPCs also present CD31, CD146, VE-cadherin, and endothelial nitric oxide synthase, as well as begin to express von Willebrand factor (10,11). Thus, the surface marker profile of EPCs seems to depend predominantly on their stage of differentiation (15). The current most detailed phenotypic description of circulating EPCs proposes the co-expression of several common hematopoietic and endothelial antigens: CD34, CD133, CD31, CD38, CD45, KDR, VE-cadherin, c-kit, and Flt-1 (16). Cultivation of peripheral blood mononuclear cells (MNCs) in medium favoring endothelial growth is another approach widely used for definition and quantitative analysis of EPCs. Adherent cells grow colonies and have been shown to possess endothelial characteristics, such as expression of von Willebrand factor and staining for Dil-acetylated low-density lipoprotein and Fluorescein isothiocyanate (FITC)-conjugated BS-lectin (17–20). Despite the relatively low numbers of CD34+ of circulating endothelial precursors in peripheral blood (100 to 500/ml), relatively large numbers of adherent cells are found during culture (approximately 100,000 from 1 mL blood). This raises some controversy with respect to the identification and the origin of isolated EPCs, as these cells seem to reflect a functional subpopulation within the blood MNCs that have the potential to differentiate into an endothelial phenotype in vivo (18,21–23,24). There are at least 2 morphologically and functionally distinct endothelial cell populations can be grown from circulating MNCs (2). The early spindle-like outgrowth cells possess a relatively low proliferative capacity and low ability to express mature endothelial proteins (3). These cells presumably represent cells of different lineage, which include a subset of CD14+/CD34− monocytic cells, which have the potential to differentiate (transdifferentiate) into endothelial-like cells under certain environmental condition in the presence of special growth factors (e.g., VEGF, fibroblast growth factor, and so on) (22). Late “outgrowth cells” show a high proliferative potential and originate predominantly from bone marrow donors and are considered as circulating angioblasts (2). It is important to appreciate that although monocyte-derived EPCs have a lower in vitro proliferation potential than hematopoietic stem cells or cord-blood-derived EPCs (25), the different progenitor types seem to have a similar ability to enhance neovascularization in experimental models (3,26,27). One may speculate that proliferation capacity is not the decisive factor and that the reduced proliferation of the monocyte-derived EPCs is likely to be attributable to increased release of growth factors, which may act in a paracrine manner to support angiogenesis and arteriogenesis (28). The expression of common markers by hematopoietic and endothelial progenitor cells in embryonic development and transdifferentiation potential of monocytes into cells with endothelial characteristics would suggest a possible common origin from a bone marrow precursor, perhaps a putative hemangioblast (29). Bone marrow also contains mesenchymal cells, which have been shown to differentiate into endothelial cells (30), improve vascularization in vivo (31,32), and contribute to tissue repair (33). Likewise, other bonemarrow-derived EPCs mesenchymal stem cells release a variety of angiogenic growth factors (34). In addition to bone-marrow-derived cells, other cell populations, such as fat tissue (35,36), cardiac tissue (37), neural stem cells (38), and fetal liver cells (39), can give rise to endothelial cells, suggesting that tissue-resident stem/progenitor cells can contribute to vascular growth in the adult. In summary, a universal single or complex EPC marker still remains to be identified, showing the heterogeneous nature of endothelial precursors. As a result, both different surface markers and culture properties have been used by research workers to define EPCs. ANGIOGENIC CYTOKINES AND CHEMOKINES: THE LINK TO EPCs EPCs have been linked to angiogenic cytokines and chemokines. For example, VEGF is one of the key angiogenic factors involved in the earliest stages of vasculogenesis and angiogenesis (40–42), and is the primary determinant of hemangioblast differentiation into angioblasts (EPCs) and hematopoietic stem cells (43). Asahara et al. first showed the effects of VEGF on adult endothelial progenitors, which included enhanced EPC mobilization from bone marrow, proliferation, differentiation, and incorporation of bone-marrow-derived EPCs into the neovasculature (44). This physiological pathway may be another target of cardiovascular risk factors, such as oxidized low-density lipoprotein; the latter inhibits VEGFinduced EPC differentiation (45). For patients with inoperable CAD (46) or critical limb ischemia (47), VEGF gene transfer results in mobilization of EPCs, which could potentially be involved in the success of such therapy (46). Interestingly, the dose of EPCs required to obtain clinical improvement in murine hind limb ischemia reduced by as much as 30-fold when VEGF gene transfer was simultaneously used (48). Furthermore, combination of VEGF gene therapy with cytokine (granulocyte colony-stimulating factor [G-CSF] and stem cell factor) administration led to enhanced neovascularization, bone marrow cell mobilization, and incorporation into the neovasculature, as well as improvements in cardiac performance, at least in animal models of cardiac ischemia (49). However, intramyocardial injection of VEGF-A plasmid followed by G-CSF in patients with severe chronic ischemic heart disease failed to induce angiogenesis, as well as to show any improvement in cardiac perfusion or function (50). This might be explained partly by the low VEGF-A165 plasmid dose chosen (probably for safety reasons) because no significant increase in CD34+ cells was observed. In another rodent study, VEGF gene transfer was associated with enhanced angiogenesis and mobilization of bone marrow cells and the recruitment of cultured MNCs to the sites of neovascularization (51). Both G-CSF and granulocyte-macrophage colony-stimulating factor, which are already widely used clinically in oncology and in transplant donors, were considered attractive candidates for stimulating arteriogenesis. Pre-clinical studies uniformly report positive results of their infusion alone (52) or in combination with bone marrow MNC transplantation (53) or cytokines to mobilize EPCs in different models of ischemia (54–56). However, 3 clinical trials of G-CSF and granulocyte-macrophage colony-stimulating factor treatment to stimulate neovasculogenesis were stopped early because of serious safety concerns (57–59). For example, the MAGIC cell-randomized clinical trial of intracoronary administration of peripheral blood leucocytes with subcutaneous infusion of G-CSF in patients after myocardial infarction was stopped because of a high rate of in-stent restenosis despite good periprocedural results and short-term safety (59). In the study by Zbinden et al. (57), an acute occlusion of a coronary artery developed in 2 of 7 patients with stable angina undergoing percutaneous coronary intervention within 12 days of treatment with granulocyte-macrophage colony-stimulating factor. In the study by Hill et al. (58), G-CSF increased the number of circulating CD34+/CD133+ progenitors in patients with severe CAD, but also increased Creactive protein levels and white cell counts. At 1 month after treatment, there was no improvement in either cardiac contractility or exercise duration, and a trend toward a greater number of ischemic segments was seen. Worryingly, 2 of 16 patients included in the study experienced myocardial infarction, which was fatal in 1 patient. Although in 1 study, treatment with G-CSF in patients with myocardial infarction subjected to primary PCI stenting with abciximab not only mediated mobilization of CD34+ progenitors, but significantly improved left ventricular function and geometry without signs of accelerated restenosis or other serious complications (60), it was proposed to restrict ongoing trials using G-CSF or granulocyte-macrophage colony-stimulating factor to patients with no other therapeutic options, such as those with refractory angina or critical limb ischemia (61). The mechanisms of the reported complications are unclear, but may relate to the proinflammatory and procoagulant effects of G-CSF (62), possibly leading to vascular smooth muscle cell proliferation. Indeed, recently G-CSF was shown to induce neointimal overgrowth in a rabbit iliac artery balloon injury model after bare-metal stenting, possibly as a result of concomitant mobilization of smooth muscle progenitors. Implantation of paclitaxel-eluting stents substantially reduced neointimal overgrowth, probably by preferential inhibition of proliferation of smooth muscle lineage cells rather than EPCs, and G-CSF treatment in this group significantly accelerated endothelial recovery (63). A further clinical trial by Zohlnhufer et al. (64) has failed to reignite interest in the potential of G-CSF to induce neoangiogenesis. In this study, a cohort of patients with acute myocardial infarction undergoing percutaneous coronary intervention within 12 h of symptom onset were randomized to a 5-day course of G-CSF or placebo. The CD34 cell counts were quantified by flow cytometry up to 1 week after randomization. Although CD34+ cells did increase in the G-CSF group, there was notably no difference in infarct size as assessed by technetium Tc99m sestamibi scintigraphy 4 to 6 months compared with baseline. Importantly though, the previously noted excess of restenosis did not feature strongly in this study. A positive impact on the EPC characteristics and potential proangiogenic properties has been shown in the experimental studies for a number of cytokines/chemokines such as stromal cellderived factor-1-alpha (65), stem cell factor (55), hepatocyte grow factor (66), transforming growth factor-beta1 (67), hemangiopoietin (68), and secretoneurin (69). For example, stromal cell-derived factor-1alpha was shown to play an important role in the mobilization of CD34+CXCR4+ progenitor cells during cardioplegia and cardiopulmonary bypass (70). Members of the platelet-derived growth factor (PDGF) family are involved in vascular physiology, and may be possible promoters of angiogenesis. Secreted by ECs, PDGF-BB plays an important role in the maturation of blood vessels via recruiting mural cells (71), as well as in the promotion of angiogenesis indirectly by producing VEGF (72). Also, PDGF-BB was shown to induce differentiation of bone marrow EPCs into smooth muscle cells (73). When administered alone, PDGF-BB reduces EC survival and causes destabilization of vessels as a result of impaired mural cell coverage. Both PDGF-BB and PDGF-AB show positive vascular effects only when coadministered with other cytokines or bone marrow cells (73–75). Recently, Li et al. (76) showed multiple proangiogenic properties of PDGF-CC, a novel member of PDGF family: mobilization, differentiation of EPCs and their homing to the sites of ischemia, migration of mature endothelial cells and microvessel sprouting, and release of VEGF. Moreover, PDGF-CC can induce differentiation of progenitors into endothelial cells and smooth-muscle cells, which represent another essential component of blood vessels (76). Given its multiple angiogenic actions, PDGF-CC is considered to be an attractive candidate for combination therapy with other growth factors (77). REFERENCES 1. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–7. 2. Lin Y, Weisdorf DJ, Solovey A, Hebbel RP. Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest 2000;105:71–7. 3. Hur J, Yoon CH, Kim HS, et al. Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis. Arterioscler Thromb Vasc Biol 2004;24:288–93. 4. Gulati R, Jevremovic D, Peterson TE, et al. Autologous culture-modified mononuclear cells confer vascular protection after arterial injury. Circulation 2003;108:1520–6. 5. Shi Q, Rafii S, Wu MH, et al. Evidence for circulating bone marrow-derived endothelial cells. Blood 1998;92:362–7. 6. Gulati R, Jevremovic D, Peterson TE, et al. Diverse origin and function of cells with endothelial phenotype obtained from adult human blood. Circ Res 2003;93:1023–5. 7. Zhang ZG, Zhang L, Jiang Q, Chopp M. Bone marrow–derived endothelial progenitor cells participate in cerebral neovascularization after focal cerebral ischemia in the adult mouse. Circ Res 2002;90:284–8. 8. Civin CI, Banquerigo ML, Strauss LC, Loken MR. Antigenic analysis of hematopoiesis. VI. Flow cytometric characterization of My-10 positive progenitor cells in normal human bone marrow. Exp Hematol 1987;15:10–7. 9. Fina L, Molgaard HV, Robertson D, et al. Expression of the CD34 gene in vascular endothelial cells. Blood 1990;75:2417–26. 10. Peichev M, Naiyer AJ, Pereira D, et al. Expression of VEGFR-2 and AC133 by circulating human CD34 (+) cells identifies a population of functional endothelial precursors. Blood 2000;95:952–8. 11. Yin AH, Miraglia S, Zanjani ED, et al. AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 1997;90:5002–12. 12. Miraglia S, Godfrey W, Yin AH, et al. A novel five-transmembrane hematopoietic stem cell antigen: isolation, characterization, and molecular cloning. Blood 1997;90:5013–21. 13. Friedrich EB, Walenta K, Scharlau J, Nickenig G, Werner N. CD34−/CD133+/VEGFR2+ endothelial progenitor cell subpopulation with potent vasoregenerative capacities. Circ Res 2006;98:e20–5. 14. Quirici N, Soligo D, Caneva L, Servida F, Bossolasco P, Deliliers GL. Differentiation and expansion of endothelial cells from human bone marrow CD133+ cells. Br J Haematol 2001;115:186–94. 15. Murohara T, Ikeda H, Duan J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J Clin Invest 2000;105:1527–36. 16. Burger PE, Coetzee S, McKeehan WL, et al. Fibroblast growth factor receptor-1 is expressed by endothelial progenitor cells. Blood 2002;100:3527–35. 17. Shintani S, Murohara T, Ikeda H, et al. Mobilization of endothelial progenitor cells in patients with acute myocardial infarction. Circulation 2001;103:2776–9. 18. Hill JM, Zalos G, Halcox JP, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003;348:593–600. 19. Vasa M, Fichtlscherer S, Adler K, et al. Increase in circulating endothelial progenitor cells by statin therapy in patients with stable coronary artery disease. Circulation 2001;103:2885– 90. 20. Vasa M, Fichtlscherer S, Aicher A, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res 2001;89:E1–7. 21. Fernandez Pujol B, Lucibello FC, Gehling UM, et al. Endothelial-like cells derived from human CD14 positive monocytes. Differentiation 2000;65:287–300. 22. Harraz M, Jiao C, Hanlon HD, Hartley RS, Schatteman GC. CD34(-) blood-derived human endothelial cell progenitors. Stem Cells 2001;19:304–12. 23. Schmeisser A, Garlichs CD, Zhang H, et al. Monocytes coexpress endothelial and macrophagocytic lineage markers and form cord-like structures in Matrigel under angiogenic conditions. Cardiovasc Res 2001;49:671–80. 24. Rookmaaker MB, Vergeer M, van Zonneveld AJ, Rabelink TJ, Verhaar MC. Endothelial progenitor cells: mainly derived from the monocyte/macrophage containing CD34- but also partly derived from the hematopoietic stem cell containing CD34+ mononuclear population. Circulation 2003;108:150e. 25. Ingram DA, Mead LE, Tanaka H, et al. Identification of a novel hierarchy of endothelial progenitor cells utilizing human peripheral and umbilical cord blood. Blood 2004;104:2752– 60. 26. Urbich C, Heeschen C, Aicher A, Dernbach E, Zeiher AM, Dimmeler S. Relevance of monocytic features for neovascularization capacity of circulating endothelial progenitor cells. Circulation 2003;108:2511–6. 27. Kalka C, Masuda H, Takahashi T, et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci U S A 2000;97:3422– 7. 28. Urbich C, Dimmeler S. Endothelial progenitor cells: functional characterization. Trends Cardiovasc Med 2004;14:318–22. 29. Nishikawa SI. A complex linkage in the developmental pathway of endothelial and hematopoietic cells. Curr Opin Cell Biol 2001;13:673–8. 30. Oswald J, Boxberger S, Jorgensen B, et al. Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells 2004;22:377–84. 31. Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 2002;109:337–46. 32. Al-Khaldi A, Eliopoulos N, Martineau D, Lejeune L, Lachapelle K, Galipeau J. Postnatal bone marrow stromal cells elicit a potent VEGF-dependent neoangiogenic response in vivo. Gene Ther 2003;10:621–9. 33. Kuznetsov SA, Mankani MH, Gronthos S, Satomura K, Bianco P, Robey PG. Circulating skeletal stem cells. J Cell Biol 2001;153:1133–40. 34. Kinnaird T, Stabile E, Burnett MS, et al. Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation 2004;109:1543–9. 35. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7:211–28. 36. Planat-Benard V, Silvestre JS, Cousin B, et al. Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation 2004;109:656–63. 37. Beltrami AP, Barlucchi L, Torella D, et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114:763–76. 38. Wurmser AE, Nakashima K, Summers RG, et al. Cell fusion-independent differentiation of neural stem cells to the endothelial lineage. Nature 2004;430:350–6. 39. Dan YY, Riehle KJ, Lazaro C, et al. Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages. Proc Natl Acad Sci U S A 2006;103:9912–7. 40. Shalaby F, Rossant J, Yamaguchi TP, et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 1995;376:62–6. 41. Carmeliet P, Ferreira V, Breier G, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996;380:435–9. 42. Coultas L, Chawengsaksophak K, Rossant J. Endothelial cells and VEGF in vascular development. Nature 2005;438:937–45. 43. Wilting J, Brand-Saberi B, Kurz H, Christ B. Development of the embryonic vascular system. Cell Mol Biol Res 1995;41:219–32. 44. Asahara T, Takahashi T, Masuda H, et al. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J 1999;18:3964–72. 45. Imanishi T, Hano T, Matsuo Y, Nishio I. Oxidized low-density lipoprotein inhibits vascular endothelial growth factor-induced endothelial progenitor cell differentiation. Clin Exp Pharmacol Physiol 2003;30:665–70. 46. Kalka C, Tehrani H, Laudenberg B, et al. VEGF gene transfer mobilizes endothelial progenitor cells in patients with inoperable coronary disease. Ann Thorac Surg 2000;70:829– 34. 47. Kalka C, Masuda H, Takahashi T, et al. Vascular endothelial growth factor (165) gene transfer augments circulating endothelial progenitor cells in human subjects. Circ Res 2000;86:1198–202. 48. Iwaguro H, Yamaguchi J, Kalka C, et al. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration. Circulation 2002;105:732–8. 49. Kawamoto A, Murayama T, Kusano K, et al. Synergistic effect of bone marrow mobilization and vascular endothelial growth factor-2 gene therapy in myocardial ischemia. Circulation 2004;110:1398–405. 50. Ripa RS, Wang Y, Jorgensen E, Johnsen HE, Hesse B, Kastrup J. Intramyocardial injection of vascular endothelial growth factor-A165 plasmid followed by granulocyte-colony stimulating factor to induce angiogenesis in patients with severe chronic ischaemic heart disease. Eur Heart J 2006;27:1785–92. 51. Zentilin L, Tafuro S, Zacchigna S, et al. Bone marrow mononuclear cells are recruited to the sites of VEGF-induced neovascularization but are not incorporated into the newly formed vessels. Blood 2006;107:3546–54. 52. Cho HJ, Kim HS, Lee MM, et al. Mobilized endothelial progenitor cells by granulocytemacrophage colony-stimulating factor accelerate reendothelialization and reduce vascular inflammation after intravascular radiation. Circulation 2003;108:2918–25. 53. Takagi Y, Omura T, Yoshiyama M, et al. Granulocyte-colony stimulating factor augments neovascularization induced by bone marrow transplantation in rat hindlimb ischemia. J Pharmacol Sci 2005;99:45–51. 54. Minamino K, Adachi Y, Okigaki M, et al. Macrophage colony-stimulating factor (MCSF), as well as granulocyte colony-stimulating factor (G-CSF), accelerates neovascularization. Stem Cells 2005;23:347–54. 55. Sesti C, Hale SL, Lutzko C, Kloner RA. Granulocyte colony-stimulating factor and stem cell factor improve contractile reserve of the infarcted left ventricle independent of restoring muscle mass. J Am Coll Cardiol 2005;46:1662–9. 56. Woo YJ, Grand TJ, Berry MF, et al. Stromal cell-derived factor and granulocytemonocyte colony-stimulating factor form a combined neovasculogenic therapy for ischemic cardiomyopathy. J Thorac Cardiovasc Surg 2005;130:321–9. 57. Zbinden S, Zbinden R, Meier P, Windecker S, Seiler C. Safety and efficacy of subcutaneous-only granulocyte-macrophage colony stimulating factor for collateral growth promotion in patients with coronary artery disease. J Am Coll Cardiol 2005;46:1636–42. 58. Hill JM, Syed MA, Arai AE, et al. Outcomes and risks of granulocyte colony-stimulating factor in patients with coronary artery disease. J Am Coll Cardiol 2005;46:1643–8. 59. Kang HJ, Kim HS, Zhang SY, et al. Effects of intracoronary infusion of peripheral blood stem-cells mobilised with granulocyte-colony stimulating factor on left ventricular systolic function and restenosis after coronary stenting in myocardial infarction: the MAGIC cell randomized clinical trial. Lancet 2004;363:751– 6. 60. Ince H, Petzsch M, Kleine HD, et al. Preservation from left ventricular remodeling by front-integrated revascularization and stem cell liberation in evolving acute myocardial infarction by use of granulocyte-colony-stimulating factor (FIRSTLINE-AMI). Circulation 2005;112:3097–106. 61. Wilson RF, Henry TD. Granulocyte colony-stimulating factor and granulocytemacrophage colony-stimulating factor: double-edged swords. J Am Coll Cardiol 2005;46:1649–50. 62. Anderlini P, Przepiorka D, Champlin R, Korbling M. Biologic and clinical effects of granulocyte colony-stimulating factor in normal individuals. Blood 1996;88:2819–25. 63. Cho HJ, Kim TY, Cho HJ, et al. The effect of stem cell mobilization by granulocyte- colony stimulating factor on neointimal hyperplasia and endothelial healing after vascular injury with bare-metal versus paclitaxel-eluting stents. J Am Coll Cardiol 2006;48:366–74. 64. Zohlnhofer D, Ott I, Mehilli J, et al., REVIVAL-2 Investigators. Stem cell mobilization by granulocyte colony-stimulating factor in patients with acute myocardial infarction: a randomized controlled trial. JAMA 2006;295:1003–10. 65. Hiasa K, Ishibashi M, Ohtani K, et al. Gene transfer of stromal cell-derived factor-1alpha enhances ischemic vasculogenesis and angiogenesis via vascular endothelial growth factor/endothelial nitric oxide synthase-related pathway: next-generation chemokine therapy for therapeutic neovascularization. Circulation 2004;109:2454–61. 66. Shirakawa Y, Sawa Y, Takewa Y, et al. Gene transfection with human hepatocyte growth factor complementary DNA plasmids attenuates cardiac remodeling after acute myocardial infarction in goat hearts implanted with ventricular assist devices. J Thorac Cardiovasc Surg 2005;130:624–32. <3>67. Sales VL, Engelmayr GC Jr., Mettler BA, Johnson JA Jr., Sacks MS, Mayer JE Jr. Transforming growth factor-beta1 modulates extracellular matrix production, proliferation, and apoptosis of endothelial progenitor cells in tissue-engineering scaffolds. Circulation 2006;114 Suppl:I193–9.</3> 68. Liu YJ, Lu SH, Xu B, et al. Hemangiopoietin, a novel human growth factor for the primitive cells of both hematopoietic and endothelial cell lineages. Blood 2004;103:4449–56. 69. Kirchmair R, Egger M, Walter DH, et al. Secretoneurin, an angiogenic neuropeptide, induces postnatal vasculogenesis. Circulation 2004;110:1121–7. 70. Mieno S, Ramlawi B, Boodhwani M, et al. Role of stromal-derived factor-1{alpha} in the induction of circulating CD34+CXCR4+ progenitor cells after cardiac surgery. Circulation 2006;114 Suppl:186–92. 71. Hellstrom M, Kalen M, Lindahl P, Abramsson A, Betsholtz C. Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development 1999;126:3047–55. 72. Sato N, Beitz JG, Kato J, et al. Platelet-derived growth factor indirectly stimulates angiogenesis in vitro. Am J Pathol 1993;142:1119–30. 73. Miyata T, Iizasa H, Sai Y, Fujii J, Terasaki T, Nakashima E. Platelet-derived growth factor-BB (PDGF-BB) induces differentiation of bone marrow endothelial progenitor cellderived cell line TR-BME2 into mural cells, and changes the phenotype. J Cell Physiol 2005;204:948–55. 74. Hao X, Mansson-Broberg A, Gustafsson T, et al. Angiogenic effects of dual gene transfer of bFGF and PDGF-BB after myocardial infarction. Biochem Biophys Res Commun 2004;315:1058–63. 75. Richardson TP, Peters MC, Ennett AB, Mooney DJ. Polymeric system for dual growth factor delivery. Nat Biotechnol 2001;19:1029–34. 76. Li X, Tjwa M, Moons L, et al. Revascularization of ischemic tissues by PDGF-CC via effects on endothelial cells and their progenitors. J Clin Invest 2005;115:118–27. BONE MARROW 77. Dimmeler S. Platelet-derived growth factor CC:a clinically useful angiogenic factor at last? N Engl J Med 2005;352:1815–6.