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Journal of Molecular and Cellular Cardiology 45 (2008) 514 522 www.elsevier.com/locate/yjmcc

Review article

Homing and engraftment of progenitor cells: A prerequisite for cell therapy

Emmanouil Chavakis, Carmen Urbich, Stefanie Dimmeler
Molecular Cardiology, Department of Internal Medicine III, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany Received 12 October 2007; received in revised form 17 December 2007; accepted 1 January 2008 Available online 18 January 2008

Abstract Cell therapy is a promising therapeutic option for treating patients with ischemic diseases. The efficiency of cell therapy to augment recovery after ischemia depends on the sufficient recruitment of applied cells to the target tissue. Using in vivo imaging techniques the extent of homing was shown to be rather low in most experimental and clinical studies. The elucidation of the molecular mechanisms of homing of different progenitor cell subpopulation to sites of injury is essential for the development of new specific therapeutic strategies, in order to improve the efficacy of cell-based therapies. Homing to sites of active neovascularization is a complex process depending on a timely and spatially orchestrated interplay between chemokines (e.g. SDF-1), chemokine receptors, intracellular signaling, adhesion molecules (selectins and integrins) and proteases. The review will focus on the mechanisms underlying homing of adult bone marrow-derived hematopoietic cells, mesenchymal stem cells, and vasculogenic circulating cells and discuss strategies how to optimize cell engraftment. 2008 Elsevier Inc. All rights reserved.
Keywords: Progenitor cells; Cell therapy; Homing; Chemokines; Integrins; Proteases; Neovascularization

Contents Extent of cell homing in experimental and clinical studies . . . . . . . . . . . . . . . . . . . Mechanism of homing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Role of chemo-/cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Role of adhesion molecules for EPC homing . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Role of selectins for rolling . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Role of integrins for adhesion, transendothelial migration and migration of EPC 2.3. Role of proteases for EPC homing and differentiation . . . . . . . . . . . . . . . . . . 3. Strategies to augment homing and engraftment . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Pre-activation of cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Pretreatment of the target tissue to augment cell homing and integration . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515 515 516 517 517 517 518 519 520 520 521

Cell therapy is a promising option to improve neovascularization and to enhance recovery of left ventricular function after acute myocardial infarction. Various different types of stem or progenitor cells have been successfully used in experimental models including various subsets of adult bone marrow-derived
Corresponding author. Tel.: +49 69 6301 5158 or 6667; fax: +49 69 6301 7113 or 6374. E-mail address: Dimmeler@em.uni-frankfurt.de (S. Dimmeler). 0022-2828/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2008.01.004

cells, tissue-resident stem cells (cardiac stem cells) and embryonic stem cells (for review see [1]). Some of these experimental studies have been moved to the clinic where bone marrowderived cells and endothelial progenitor cells (EPC) have been tested in patients with acute and chronic ischemic disease. So far, most studies used bone marrow mononuclear cells (BMC) preparation for the treatment of acute myocardial infarction overall demonstrating that intracoronary infusion of BMC is safe and feasible and improves ejection fraction [2]. The results of the

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REPAIR-AMI trial, the largest phase II/III trial, even suggested that BMC might improve event free survival [3]. However, when studies investigated the homing and the long term engraftment of cells, only a low percentage of cells were detectable in animal models as well as in clinical trials. Therefore, the understanding of homing mechanisms might be crucial for enhancing cell engraftment particularly when cells are infused via the vascular route. This review will focus on the mechanisms underlying homing of adult bone marrow-derived and vasculogenic cells (including pro-angiogenic cells and EPC). 1. Extent of cell homing in experimental and clinical studies In order to determine progenitor cell trafficking and in vivo distribution, life imaging is essential. Several non-invasive imaging modalities can be used to assess the biodistribution of the applied cells over time to track homing of the cells to the target tissue but also to visualize the up-take to other organs and tissues where cells potentially may exhibit unwanted sideeffects. Non-invasive imaging has been performed by direct labeling of cells with radionucleotides for single-photon emission computed tomography (SPECT) imaging, by using positron emission tomography (PET) tracers such as F18-FDG or by labeling of cells with iron particles for magnetic resonance imaging (MRI) (for review see [4,5]). After intravenous infusion of radioactively labeled circulating blood-derived ex vivo cultured endothelial progenitor cells in rats after myocardial infarction, some radioactivity was detected in the heart [6]. However, the amount of radioactivity detected in the ischemic heart was about 2% after 24 to 96h of infusion indicating that only a minor percentage of cells homed to the heart [6]. Similar findings were reported after infusion of uncultured Indiumlabeled peripheral blood mononuclear cells [7] or purified CD34+ cells [8]. The majority of the radioactivity after intravenously infusion of labeled bone marrow- or peripheral blood-derived cells was detected in the spleen and the liver [6,7]. Although infusion of the cells in the left ventricular cavity or direct intramuscular application increased the extent of homing, a maximal value of around 10% was achieved [6,7]. The extent of homing was similar in clinical studies where several groups labeled BMC, CD34+ or CD133+ cells and infused the cells in the coronary artery of patients with acute or chronic myocardial infarction [911]. In the first hours after infusion 2.6 to 11% of radioactivity was detected in the heart thereafter declining to 1 to 7%. Hofmann et al infused labeled BMC or CD34+ cell with the PET-tracer F18-FDG in three patients with acute myocardial infarction [12]. Purified CD34+ cells were shown to more efficiently home to the heart (1439%) [12]. Additional studies addressed the engraftment of mesenchymal stem cells (MSC). In contrast to hematopoietic or endothelial progenitor cell populations, the size of MSC is higher and these cells (at least the ones being ex vivo expanded) are not well adapted to circulate in the blood, and, therefore, MSC have initially been injected intramuscularly in the experimental studies [13,14]. Injected MSC have been detected up to 21days by MRT after labeling with iron particles in large animal models [13,14]. However, the body distribution after intravenous injection of

allogeneic MSC surprisingly documented a redistribution of the infused cells from initial lung homing to non target organs (liver, kidney, spleen) but also to the infarcted heart [15]. The targeted cardiac localization, however, was only detectable by SPECT/CT but not by MRI indicating that the concentration of incorporated cells in the heart is below the detection level of MRI [15]. In summary, the up-take of cells appears rather low independent on the used cell type or the route of delivery. Although one study by Hoffmann reports a significantly higher incorporation when using CD34+ cells, different studies with CD133+ cells did not confirm such a high up-take of purified hematopoietic stem cells. Since an overlapping set of cells (approximately 30%) express CD34 and CD133 further studies have to address putative differences between homing of purified cell populations. Clearly, the clinical trials assessing the biodistribution of the cells by non-invasive imaging cannot give insights into the morphological integration and the cell fate after prolonged time and may be confounded by either false positive (e.g. labeled cells are dying and are taken up by macrophages) or false negative effects (e.g. labeling is released by the cells or is diluted by proliferation). To get more detailed insights in the cell fate, immunohistochemistry would be required, which is unfortunately not useful in patients treated with autologous cells. 2. Mechanism of homing Homing and engraftment is a prerequisite for all cell types to exhibit any type of activity in the target tissue particularly when cells are infused via the vascular route. While the homing of leukocytes to sites of inflammation is well studied [1618], the mechanisms of progenitor cell homing to sites of ischemia or injury are poorly understood. During inflammation, the recruitment of inflammatory cells requires a coordinated sequence of multistep adhesive and signaling events including selectinmediated rolling, leukocyte activation by chemokines leading to activation of integrins, integrin-mediated firm adhesion on endothelial cell monolayers, diapedesis through the endothelial cell monolayers and finally the migration/invasion in the extracellular matrix involving integrin-dependent processes and matrix degrading proteases [1618]. Homing mechanisms of endothelial progenitor cells to sites of tumor neovascularization and to sites of ischemia appears to share at least some common features with the homing of leukocytes to sites of inflammation (Fig. 1). Embryonic EPC arrested within tumor microvessels, extravasated into the interstitium and incorporated into neovessels suggesting that adhesion and transendothelial migration are involved in the recruitment of endothelial progenitor cells to sites of tumor angiogenesis [19]. Furthermore, recent studies support the idea that EPC and progenitor cells utilize adhesion molecules for homing to sites of neovascularization similar to the adhesion molecules engaged by leukocytes for recruitment to sites of inflammation. In the following chapters we will focus on the role of chemokines, adhesion molecules and proteases for the homing of bone marrow-derived progenitor cells to ischemic tissues.

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Fig. 1. Multistep process of progenitor cell homing and engraftment. Recruitment and incorporation of progenitor cells into ischemic or injured tissue requires a coordinated multistep process including adhesion to the endothelium, transendothelial migration, chemotaxis, matrix degradation and invasion and in situ differentiation. The factors which are proposed to regulate the distinct steps are indicated.

antibodies against the IL-8 receptors, CXCR1 or CXCR2, reduced CD34+ cell-mediated improvement of neovascularization, establishing a role for CXC-chemokines (IL-8/Gro-) for homing and neovascularization improvement by CD34+ cells. In addition, blocking CXCR2 inhibited the incorporation of human EPCs expressing CXCR2 at sites of arterial injury [30]. Furthermore, ischemia-induced VEGF acts as a chemoattractant to EPC [31]. Interestingly, VEGF is sufficient to induce the organ recruitment of bone-marrow-derived circulating myeloid cells and their perivascular localization via induction of SDF-1 expression by perivascular myofibroblasts, suggesting that different cytokines may cooperate during homing of bone marrow cells [32]. In addition, invaded immune competent cells within the ischemic tissue may release further chemokines, such as MCP-1 or interleukins that can attract circulating progenitor cells [33]. Beside stimulating migration, MCP-1 and VEGF are capable of inducing the transendothelial migration of human ex vivo expanded myeloid EPC derived from peripheral blood in a 2-integrin-dependent manner in vitro [34]. In
Table 1 Summary of factors involved in homing of progenitor cells (Selection) Molecules Cell type Model Hind limb ischemia Reference [21,2426, 73,74]

2.1. Role of chemo-/cytokines Activation by chemokines is an important step during recruitment of a reasonable number of progenitor cells to the ischemic tissue. The factors that attract circulating pro-angiogenic cells and EPC to the ischemic tissues may be similar to those regulating hematopoietic stem cell engraftment to the bone marrow (Table 1). Indeed, SDF-1 has been shown to stimulate not only hematopoietic stem cell engraftment but also the recruitment of progenitor cells to the ischemic tissue [20]. The expression of SDF-1 is up-regulated during ischemia [21,22]. Inhibition of the SDF-1/CXCR4 axis partially blocks the homing of progenitor/ stem cells to the ischemic myocardium [23]. Likewise, inhibition of CXCR4 by neutralizing anti-CXCR4-antibodies significantly reduced SDF-1-induced adhesion of EPC to mature endothelial cell monolayers, the migration of EPC in vitro [21] and the in vivo homing of myeloid EPC to the ischemic limb in the model of hind limb ischemia [24]. Moreover, overexpression of SDF-1 enhanced stem cell homing and incorporation into ischemic tissues [25,26] clearly supporting that SDF-1 play a crucial role for recruitment of circulating or intravenously infused cells. Recent studies determined the involvement of additional other chemokines (Table 1). CXC-chemokines IL-8/Gro- and its cellular receptors CXCR2 and CXCR1 contribute to homing of intravenous infused CD34+ progenitor cells to the ischemic myocardium [27]. IL-8 is an inflammatory chemokine, which is able to stimulate angiogenesis [28]. Myocardial infarction induces an increase of the expression of cardiac IL-8/Gro- mRNA and increased serum concentrations of IL-8/Gro- were associated with the number of CD133+ cells [27,29]. CD34+/ CD117bright progenitor cells demonstrated a chemotactic response to IL-8 in vitro [27]. Moreover, local injection of IL8 in the non-ischemic myocardium increased the recruitment of CD34+ cells [27]. Neutralizing anti-IL-8/Gro--antibodies or

Chemokine/receptor SDF-1/CXCR4 PB-derived EPC BMC; CD34+ cells BM-derived c-kit+ cells MSC a IL-8/CXCR2 or CD34+ cells CXCR1 HMGB-1 Cardiac Stem Cells (c-kit+) PB-derived myeloid EPC VEGF PB-derived myeloid EPC BM-derived cells (RBCC) MCP-1 BM-derived CD34-/CD14+ cells Integrins 2-Integrin

Myocardial infarction Myocardial infarction Hind limb ischemia Hind limb ischemia VEGF-overexpression Arterial injury

[27] [36,37,72]

[32,75]

[33]

PB-derived myeloid EPC VEGF-R2+/lin- cells Sca-1+/lin- cells BM-derived EPC 41 integrin b BM-derived progenitor cells 1 integrin Mesenchymal stem cells a E- and P-selectin Embryonic EPC BM-derived EPC (outgrowth) PB-derived EPC
a

Hind limb ischemia Myocardial infarction

[34,44]

Tumor model Myocardial infarction Tumor (Intravital microscopy)

[38] [76] [19,41,40]

Hind limb ischemia

Cultivated mesenchymal stem cells (MSC) exhibit a low or absent expression of CXCR4 and are preferentially using 1-integrins other than the 41-integrin for homing. However, freshly isolated MSC, hypoxia prestimulated MSC or specific subsets of MSC were shown to express functionally active CXCR4. b The role of 41 appears more complex. Systemic anti-4 antibodies also induced mobilization and improved homing of EPC.

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accordance with these data, Spring et al. recently demonstrated the expression of the chemokine receptors CCR2 and CCR5 in EPC and the expression of CC chemokines in tumor vessels [35]. In the same study, the inhibition of chemokine receptor signaling by PTX significantly reduced the incorporation of EPC in tumor vessels [35] supporting the involvement of Gprotein-coupled chemokine receptors in the homing process. In conclusion, these data suggest that chemokines like IL-8, SDF-1 and probably others are involved in the trafficking of circulating pro-angiogenic cells and EPC from the bloodstream to ischemic tissues (Table 1). Beside classical chemokines, other factors, which could be present in the ischemic myocardium, may also influence the recruitment of EPC. E.g., high mobility group box-1 (HMGB-1) is a nuclear protein, which is released extracellularly upon activation of cells by inflammatory cytokines and during cell necrosis and acts as a chemoattractant for inflammatory cells, stem cells and EPC in vitro and in vivo [36,37]. Since necrosis and inflammation are hallmarks of ischemic and tumor tissues, it is conceivable, that HMGB-1 may be involved in the homing of EPC. 2.2. Role of adhesion molecules for EPC homing 2.2.1. Role of selectins for rolling The initial step of leukocytes homing involves the rolling of the leukocytes on the endothelium [28]. Circulating leukocytes must come in brief low affinity contacts (rolling) with the endothelial cell monolayer in order to get activated by cytokines and to arrest in the subsequent step on endothelial cells. In circulating leukocytes rolling on the endothelial cell monolayer is predominantly mediated by selectins and selectin ligands [28]. The selectin family of adhesion molecules consists of three related molecules. L-selectin is constitutively expressed in most leukocytes. Expression of E-selectin expression is restricted to endothelial cells activated by inflammatory cytokines, while P-selectin is expressed on both endothelial cells and platelets. P-selectin expression on platelets and endothelial cells can rapidly be induced on the surface of endothelial cells and platelets by exocytosis. Selectins bind sialyl-Lewis-X-like carbohydrate ligands presented by sialomucin-like surface molecules such as P-selectin-glycoprotein ligand-1 (PSGL-1). Interestingly, using intravital microscopy in a model of tumor angiogenesis no rolling of embryonic EPC was observed [19]. However, in the same study in vivo blocking experiments provided evidence that E- and P-selectin and P-selectin glycoprotein ligand 1 are involved in the initial cell arrest of embryonic EPC [19]. Additionally, in another study using also intravital microscopy, it was reported that murine bone marrow-derived progenitor cells (Sca-1+/Lin cells) display rolling on the endothelium of the tumor vasculature before firm adhesion [38]. Moreover, human hematopoietic progenitor CD34+ cells show efficient rolling on P-selectin, E-selectin, and the CD44 ligand hyaluronic acid under physiological shear flow in vitro [39]. The importance of E-selectin for recruitment was recently supported by studies in E-selectin deficient mice [40,41]. E-selectin deficient mice showed an impaired homing and recovery after ischemia. Surprisingly, the phenotype was reversed by the

soluble form of E-selectin (sE-selectin) indicating that the shed E-selectin is regulating the recruitment of bone marrow-derived EPC [40]. Indeed, sE-selectin increased the adhesion and migration of progenitor cells and stimulated the expression of the adhesion molecules ICAM-1 and VCAM-1 on endothelial cells thereby providing at least two distinct signals to increase homing of progenitor cells. Additional recent data provide evidence that activation of P-selectin may augment EPC-mediated neovascularization improvement [42]. The expression of the Pselectin ligand, PSGL-1, was increased by the activation of the ephrin receptor EphB4, and the siRNA mediated inhibition of PSGL-1 impaired the pro-angiogenic and adhesive effects of EPCs [42]. 2.2.2. Role of integrins for adhesion, transendothelial migration and migration of EPC The process of rolling is reversible. Many leukocytes that roll in vivo will not stop, but dissociate from the vessel surface and reenter the bloodstream. To stop rolling the low affinity rolling interactions must be replaced by high affinity adhesion. Thus, the subsequent step of leukocyte homing consists of the integrin activation by chemokines and the integrin-dependent arrest/firm adhesion of the leukocytes on the endothelium. Integrins are glycosylated heterodimeric proteins expressed on the cell surface mediating the adhesion of cells to extracellular matrix proteins (cellmatrix-adhesion) and to other cells (cellcell-adhesion) [43]. Integrins consist of non-covalent bound - and -subunits [43]. For the homing of leukocytes to sites of inflammation cell cell interactions of the leukocytes with the endothelium of the vessels mediated by integrins are mandatory. Fig. 2 summarizes the integrins and their respective counter ligands involved in homing of progenitor cells. Human adult peripheral blood-derived myeloid EPC, murine adult bone marrow-derived VEGFR2 +/ Lin cells, and bone marrow-derived hematopoietic Sca-1+/Lin progenitor cells express 2-integrins [34,44]. In vitro adhesion studies revealed that 2-integrins mediate the adhesion of human myeloid EPC to mature endothelial cell monolayers, while the 41-integrin was not involved in this process [34]. In addition, 2-integrins play an essential role for the homing of murine bone marrow Sca-1+/Lin hematopoietic progenitor cells and murine VEGF-R2+/Lin bone marrow EPC to ischemic tissues and for the neovascularization capacity of these cells in vivo [34]. Additional studies demonstrated the role of 2-integrins for the homing and neovascularization capacity of EPC in a murine model of myocardial infarction [34,44]. Since the 2-integrindeficiency led only to a partial inhibition of the homing to sites of ischemia and of the neovascularization capacity of progenitor cells [34], it is conceivable that other integrins may also be involved in these processes. The contribution of the 41 integrin is more complex. Regarding hematopoietic progenitor CD34+ cells, it was demonstrated that SDF-1 expressed on vascular endothelium is crucial for translating rolling adhesion of CD34+ progenitor cells into firm adhesion by increasing the adhesivity of the integrins 41 and LFA-1 to their respective endothelial ligands, VCAM-1 and ICAM-1 [39]. Interestingly, in a recent study the blockade of the 41-integrin did not inhibit homing of bone marrow-derived

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Fig. 2. Integrins and their counter ligands involved in hematopoietic and endothelial progenitor cell homing. Summary of integrins expressed on progenitor cells and their respective counter ligands expressed on endothelium or in the matrix involved in progenitor cell homing.

EPC to ischemic tissues but increased mobilization of progenitor cells from the bone marrow and enhanced the progenitor cellmediated neovascularization in the context of ischemia [45]. Nevertheless, in a tumor model the inhibition of 41-integrin significantly blocked adhesion and homing of bone marrow progenitor cells to sites of active tumor neovascularization as assessed by intravital microscopy [38]. A conceivable explanation for this discrepancy is that different integrins may play distinct context-specific roles (ischemic versus tumor neovascularization) for homing of progenitor cells. Interestingly, murine bone marrow Lin progenitor cells adhere to mature endothelial cell monolayers via both the 2-integrins and the 41-integrin (unpublished data) and human high proliferative potential-endothelial progenitor cells use 2-integrins (LFA-1) and the 41integrin for homing to ischemic limbs [46] suggesting that progenitor cells from different origin engage different mechanisms for adhesion to mature endothelial cells. Less is known for the transendothelial migration of EPC. In vivo studies demonstrated that embryonic EPC and murine bone marrow progenitor cells are able to extravasate [19,38]. However, it is not clear, whether EPC follow a paracellular and/or transcellular route during diapedesis. In vitro, 2-integrins appear to contribute to chemokine-induced transendothelial migration of EPC [34,46]. Beside integrins, PECAM-1 and CD99 were shown to be involved in the transendothelial migration of leukocytes [47]. Interestingly, CD99 was shown to be involved in transmigration and homing of CD34+ hematopoietic progenitor cells [48]. However, the role of PECAM-1 and CD99 in diapedesis and in in vivo homing of EPC to ischemic tissues has not been elucidated so far. 2.3. Role of proteases for EPC homing and differentiation Proteases are well established to be involved in angiogenesis, in particular in migration of endothelial cells. Meanwhile growing evidence suggests that pericellular proteases play also

an important role in vasculogenesis (Fig. 3). Thus, the mobilization, recruitment and invasion of stem- and progenitor cells during vasculogenesis involve proteolytic activity. Proteases comprise a large family of matrix metalloproteinases (MMPs), the related a disintegrin and metalloprotease domain proteins (ADAMs) and the catalytic classes of serine, aspartic as well as cysteine proteases exhibiting endo- or exopeptidase activities or a combination of both. Extracellular proteases may affect neovascularization by degradation of the extracellular matrix and cell surface receptors and activation, liberation and modification of angiogenic growth factors (Fig. 3). MMPs as well as ADAMs are well established to contribute to angiogenesis (for review see [49]), however, for vasculogenesis only a few studies assessed the role of MMPs. As shown by Rafiis group, the mobilization of stem- and progenitor cells requires MMP-9-mediated release of kit-ligand [50]. Thus, BM ablation induces the upregulation of MMP-9 within the BM microenvironment. MMP-9 releases soluble kit-ligand (sKitL), which augments mobility of EPC and HSC and mobilization to the peripheral circulation [50]. Moreover, circulating VEGF induced MMP-9 expression in the BM leading to the release of sKitL. In accordance, the reduction of MMP-9 activity may underlie the defective mobilization described in eNOS/ mice [51] suggesting a specific role of MMP-9 for mobilization. However, using positron emission tomography for in vivo realtime trafficking of EPC revealed that recruitment of EPC to the tumor tissue was only slightly reduced by treatment with the MMP inhibitor MMI270 [52]. Moreover, MMP inhibitors did not affect EPC invasion [53], leading to the speculation that MMPs may be specifically involved in EPC mobilization rather than homing to the target tissue. This is supported by the finding that homing of MMP-9-deficient bone marrow mononuclear cells into ischemic hind limbs is not impaired [53]. However, it is also conceivable that different subsets of progenitor cells may use a different set of proteases for either mobilization and/or homing. Thus, recruited bone marrow-derived myeloid VEGF-

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Fig. 3. Role of proteases in vasculogenesis and progenitor cell homing. Summary of proteases involved in different steps of vasculogenesis including the mobilization from the bone marrow, survival during circulation, chemotaxis, adhesion to the endothelium, transmigration, migration and invasion into the ischemic tissue, differentiation to endothelial cells and release of paracrine factors.

R1+ cells release MMP-9, which in turn may facilitate homing of other progenitor cells [32]. In contrast, MMPs may also limit engraftment by degrading chemokines or cytokines necessary for mediating cell recruitment. Thus, MMP-2 was shown to cleave and inactivate SDF-1 and its receptor CXCR4 [54], a ligandreceptor system which is essential for stem- and progenitor cells recruitment in ischemic tissues [21,24]. Moreover, VEGF is a target of MMP-3 and MMP-9 [55] indicating that MMPs may play a double-edged role in vasculogenesis. In vitro studies and experiments using knockout mice revealed an important role of the lysosomal cysteine protease cathepsin L for vasculogenesis. Myeloid EPC and bone marrowderived Sca-1+/lin cells showed a high expression and activity of cathepsin L. The improvement of neovascularization after hind limb ischemia was significantly impaired in cathepsin L/ mice and infused cathepsin L/ bone marrow mononuclear cells failed to home to sites of ischemia and to augment neovascularization. Cell engraftment was specifically dependent on cathepsin L since cathepsin D/ or MMP-9/ progenitor cells did not show an impaired phenotype. Mechanistically, cathepsin L was necessary for EPC invasion and proteolytic matrix degrading activity of EPC [53]. Finally, the serine proteases urokinase-type plasminogen activator (u-PA) proteolytically activates plasminogen into

plasmin. Tissue-type plasminogen activator (t-PA) cleaves plasminogen in the presence of fibrin during fibrinolysis of the blood. Both u-PA and t-PA are inhibited by plasminogen activator inhibitors (PAIs). u-PA as well as its receptor u-PAR are well established to be involved in cell migration and invasion (for review see [56]). However, a recent study has shown that outgrowing EPC displayed high levels of u-PA and u-PAR [57]. Moreover, inhibition of u-PA by blocking antibodies reduced proliferation, migration and tube forming activity of outgrowing EPC suggesting that the u-PA/u-PAR-dependent proteolysis not only contributes to angiogenesis but also to vasculogenesis. In summary, the matrix degrading activity of extracellular proteases is necessary for tissue invasion, however, additionally may regulate cytokines, receptors and ligands in ischemic tissue. In addition, intracellular proteolysis plays a key role in controlling signaling pathways involved in cell migration and survival, which need to be further explored in progenitor cell homing. 3. Strategies to augment homing and engraftment In principle two strategies might be used to augment homing of cells: pre-treat the cells to activate incorporation or to pre-treat the target tissue in order to provide the cytokines and chemoattractant factors to stimulate progenitor cell incorporation. The currently

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experimentally used strategies are based on the basic discoveries outlined in detail above and are summarized in Fig. 4. 3.1. Pre-activation of cells Most of the studies used overexpression systems to increase the expression of anti-apoptotic genes to promote survival of injected or infused cells (for review see [58]). Other studies used molecules to increase adhesion and migration of the cells. HMGCoA reductase inhibitors, statins, were first reported to increase the number of EPC in patients and in experimental models [59,60]. In addition to the systemic effects, statins directly augmented the migratory capacity of EPC in vitro and prestimulation of EPC with statins increased their neovascularization-promoting effect. The mechanisms by which statins affect the in vitro functions of EPC are not entirely clear but may involve the activation of the PI3-kinase and Akt and subsequent activation of the endothelial nitric oxide synthase (eNOS) [60,61]. NO plays a crucial role in progenitor cell function and the expression of eNOS is required for bone marrow-derived mobilization of EPC [51,62,63]. This might be particularly important in disease states, which are associated with reduced levels of bioavailable NO such as diabetes, coronary artery disease or heart failure [64]. eNOS is required for the basal, VEGF, and SDF-1 induced migration of EPC or bone marrow-derived progenitor cells in vitro and eNOS/ progenitor cells showed a reduced homing capacity in ischemia models [51,65]. The underlying mechanisms mediating the NO effects e.g. on chemokines or integrin expression or signaling needs to be further defined. The nitric oxide system was activated by compounds, which transcriptionally enhance eNOS expression in hematopoietic and endothelial progenitor cells [65]. The pretreatment of cells from patients with heart failure with these eNOS enhancing drugs improved the functional activity and partially rescued the impairment of the cell function [65]. The diabetic impairment of NO-mediated progenitor cell functions leading to

wound healing defects were rescued by hyperoxia and SDF-1 application [64]. Other strategies may include the use of sE-selectin to increase the homing activity of EPC [40] or the stimulation of the EphB4 receptor by Fc-ephrinB2 [42]. Interestingly, ex vivo pre-activation of 2-integrins in EPC by an activating antibody increased the homing and the neovascularization capacity of human EPC in a hind limb ischemia model [34]. In line with these results, prestimulation with HMGB-1 was shown to stimulate integrin activity in EPC and improve their homing to ischemic tissues, suggesting that ex vivo integrin activation in EPC is an effective strategy to improve homing and neovascularization capacity of EPC [37]. Kinases might be additional attractive targets. Thus, overexpression of Akt, GSK and ILK increased the function of delivered cells probably also because of an increased survival of the infused cells [66,67]. Of particular benefit in patient-derived cells might be the blockade of kinases, which are up-regulated during disease such as the MAP-kinase p38. Pharmacological inhibitors of p38 indeed improved peripheral blood-derived cells isolated from patients with diabetes and cardiovascular diseases [68]. 3.2. Pretreatment of the target tissue to augment cell homing and integration The enhancement of targeted recruitment might be useful to augment homing and incorporation of cells particularly in patients with chronic ischemia or heart failure. Here, the expression of VEGF, SDF-1 and CXCR4 was significantly suppressed even below the levels detected in controls suggesting a lack of recruitment signals in these patients [69]. Therefore, injection of cytokines may be useful to attract progenitor cells in the absence of necrosis or acute ischemia. Indeed, local injection of SDF-1 in ischemic hind limbs increased the recruitment of intravenously infused myeloid EPC [26]. SDF-1 stimulates the CXCR4 receptor, which is expressed on EPC and BMC, and, thereby, acts as a chemotactic and pro-migratory factor [21]. Moreover, an activation of the tissue by low energy shock wave application stimulated the expression of SDF-1 and VEGF within the target tissue and promoted homing of intravenously infused EPC [70]. Changing the environment may also be an attractive approach for increasing the efficiency of intramuscular injected cells. Local myocardial delivery of insulin-like growth factor 1 (IGF-1) with biotinylated peptide nanofibers improved the effect of cell therapy with intramuscularly injected neonatal rat cardiomyocytes after myocardial infarction [71]. Additionally, the local administration of high mobility group box protein1 (HMGB-1) enhanced the regeneration of infarcted myocardium by endogenous cardiac stem cells [72]. In summary, cell-based therapy provided promising results despite a rather low recruitment of cells to the target tissue. The low engraftment may not only limit regeneration in its pure sense by providing new contractile muscle tissue or blood vessels, but also reduces the paracrine activity, which may significantly contribute to early repair processes after acute ischemia. Based on ample basic science studies, molecular strategies are experimentally established to augment poor engraftment and survival. In addition to molecular strategies, cell delivery tools

Fig. 4. Putative strategies to improve homing and engraftment of cells to the target tissue. To increase homing and engraftment of cells to the target tissue either the cells could be pre-treated (e.g. modified by overexpression of genes or by incubation with small molecules) or the target tissue could be pre-activated (e.g. induction of cytokines and chemokines).

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may help to target cells for example by injecting cells selectively in the border zone where oxygen blood flow is maintained to improve the survival of the cells. The functional integration, however, will remain a challenge particularly if cardiac myocyte replacement should be achieved. References

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