Stem Cell Migration - Ingenta Connect

Current Stem Cell Research & Therapy, 2007, 2, 89-103

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Stem Cell Migration: A Quintessential Stepping Stone to Successful Therapy Corinna Weidt*,1 , Bernd Niggemann2 , Benjamin Kasenda2 , Theodore L. Drell3 , Kurt S. Zänker2 and Thomas Dittmar *,2 1Department

of Medicine, Hematology and Oncology, University Hospital Muenster, Albert-Schweitzer-Strasse 33, D-48129 Muenster, Germany 2Institute 3i3

of Immunology, Witten/ Herdecke University, Stockumer Str. 10, D-58448 Witten, Germany

Research SKM Oncology, Taunusstr. 9, D-65183 Wiesbaden, Germany Abstract: Migration is an innate and fundamental cellular function that enables hematopoietic stem cells (HSCs) and endothelial progenitors (EPCs) to leave the bone marrow, relocate to distant tissue, and to return to the bone marrow. An increasing number of studies demonstrate the widening scope of the therapeutic potential of both HSCs and endothelial cells. Therapeutic success however not only relies upon their ability to repair damaged tissue, but is also fundamentally dependent on the migration to these areas. Extensive in vivo and in vitro research efforts have shown that the most significant effects seen on HSC migration are initiated by the chemokine SDF-1α. In this review we will elucidate the many cellular and systemic factors of HSC and EPC cell migration and their modi operandi.

Keywords: Hematopoietic stem cell migration, homing, mobilization. INTRODUCTION Hematopoietic stem cells are of increasing interest due to an accumulating evidence of their therapeutic potential. This self-renewal and trans-differentiation capacity is however worthless, if these cells loose their capacity to migrate to regions of interest. Because transplantation protocols utilize intravenous injections, diseases that require hematopoietic stem cell transplantation would fail to rescue lethally irradiated recipients if their homing potential was impaired. Moreover, current research is investigating whether cells of both hematopoietic and angiogenic potential, such as hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs), may be recruited in both ischemic and malignant tissues. This, on the one hand, will provide important information to aid the therapeutic reconstitution of damaged tissue, and on the other, prevent angiogenesis in malignant disease. One of the most prominent chemokines that initiate stem cell migration is the stromal cell-derived factor-1α (SDF1α). It provides a gradient toward which stem cells migrate, and in which they are retained in the bone marrow microenvironment. In this review we will report about the cellular and molecular pathways that regulate in the migration of HSCs and EPCs to and from the bone marrow, ischemic and malignant tissue. STROMAL CELL-DERIVED FACTOR-1 Chemokines are a group of small peptides that by definition initiate the migration of effector cells. To date *Address correspondence to these authors at the 1 Department of Medicine, Hematology and Oncology, University Hospital Muenster, Albert-Schweitzer-Strasse 33, D-48129 Muenster, Germany; Tel: +49 251 8356225; Fax: +49 251 8356709; E-mail: [email protected] 2 Institute of Immunology, Witten/ Herdecke University, Stockumer Str. 10, D-58448 Witten, Germany; Tel: +49 2302 926165; Fax: +49 2302 926158; E-mail: [email protected] 1574-888X/07 $50.00+.00

more than 50 chemokines and 18 chemokine receptors have been identified. Chemokines bind to several receptors as well as several receptors binding to several ligands [1, 2]. SDF-1α is a member of the CXC chemokine family, and has been shown to be essential for chemotaxis of both progenitor and mature blood cells, and is involved in Blymphopoiesis and myelopoiesis. It binds to the CXCR4 receptor, also known as CD184, which belongs to the class of G-protein coupled receptors, whereby chemokine binding will initiate the migration of lymphocytes [3], hematopoietic stem cells [4, 5], and tumor cells [6-8]. A recent study of Balabanian and colleagues indicated that SDF-1α also binds to and signals through the orphan receptor RDC1 in Tlymphocytes [9]. SDF-1α was isolated originally from a murine bonemarrow stromal cell line, and was reported to function as a pre-B-cell growth factor [10]. At present three SDF-1α isoforms, SDF-1α, SDF-1β, and SDF-1γ , are known. SDF1α and SDF-1β are encoded by a single gene, and are generated by alternative splicing [11]. SDF-1β (93 amino acids) has an additional 4 amino acids at the C-terminal end, whereby SDF-1α is made up of 89 amino acids. Sequence alignment analyses have revealed that SDF-1α is highly conserved between species. For instance, murine SDF-1α and SDF-1β sequences are more than 92% identical to those of human origin [11]. The SDF-1 gene was mapped to chromosome 10q, in contrast to other members of the intercrine family, which are localized on chromosome 4q and 17q. It has thus been hypothesized that SDF-1α may have important functions distinct from those of the other members of the intercrine family [11]. The third known isoform, SDF-1γ , was originally isolated from the rat [12]. Compared to mice and humans in which SDF-1α/β is expressed ubiquitously [11], Gleichmann and colleagues found that neurons and Schwann cells are the main regions of both SDF-1β and SDF-1γ mRNA expression in the rat [12]. SDF-1β is the © 2007 Bentham Science Publishers Ltd.

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predominant isoform in embryonic and early postnatal rat nerve tissue, whereas SDF-1γ is expressed at higher levels in adulthood. Regulation of SDF-1 gene expression may be induced by the transcription factor hypoxia-inducible factor-1 (HIF-1) in endothelial cells. This results in selective in vivo expression of SDF in direct proportion to reduced oxygen tension [13]. Further characterization of the SDF promoter region has shown that the proximal promoter has 6 putative Sp1binding motifs [14]. Promotor regulation was demonstrated in three tested cell lines by using phorbol myristate acetate, ionomycin, gamma irradiation, and interferon gamma. In bone marrow, SDF-1α availability has been shown to be regulated by the uptake of SDF-1α via CXCR4 expressing cells, which then translocate the chemokine into the bone marrow. This transporter function is characteristic of both endothelial and stromal cells [15]. CXCR4 The SDF-1 receptor CXCR4 was independently described by several authors, thus explaining the various alternative names, such as neuropeptide Y (NPY) Y3 receptor [16], D2S201E [17], leukocyte-derived seventransmembrane domain receptor (LESTR) [18], or fusin [19]. All studies revealed a single open reading frame (ORF) of 352 amino acids that shared features common to many other 7-transmembrane G protein-coupled receptors, such as the human interleukin-8 (IL-8) receptor (36 – 37% homology), and bovine NPY receptor (92 to 93% homology). Bleul et al. and Oberlin et al. reported that SDF-1α is a ligand for this receptor, which they referred to as CXCR4 [20, 21]. Both groups showed that binding of SDF-1α to its’ receptor CXCR4 blocked the infection by lymphocytetropic HIV-1 strains in vitro. Similar results were obtained by Feng et al., showing that blocking of the CXCR4 receptor using anti-CXCR4 antibodies strongly inhibited HIV-1 infection of normal human CD4+ target cells [19]. Moreover, transient expression of the CXCR4 gene allowed non-human cells co-expressing recombinant CD4 to undergo Env-CD4-mediated cell fusion and productive HIV-1 infection, revealing that CXCR4 acts as a cofactor for HIV-1 infection in CD4 + T cells. CXCR4 is expressed in a variety of tissues, including brain, endothelial and epithelial cells, and tissues of hematopoietic origin [17]. Studies performed with CXCR4 knock-out mice revealed that CXCR4 is required for several physiological processes during late embryonic and early postnatal stages, as well as in the adult. CXCR4-deficient mice exhibited hematopoietic and cardiac defects [22], or died perinatally with defects in both the hematopoietic and nervous system [23]. Tachibana et al. showed that CXCR4 is essential for vascularization of the gastrointestinal tract [24]. Similar observations were made with SDF-1α deficient mice, highlighting the importance of the SDF-1α/CXCR4 signaling. Additionally, a reduced B-lymphopoiesis and myelopoiesis were observed in the fetal liver, whereby bone marrow myelopoiesis was completely ablated in these mice. Both groups showed that fetal cerebellar development was markedly different compared to wild type animals. For

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instance, numerous proliferating granule cells abnormally invaded within the cerebellar anlage. The hypothesis that the SDF-1α/CXCR4 axis is involved in neuronal cell migration and patterning in the central nervous system was recently substantiated by the finding that CXCR4 mRNA is expressed at sites of neuronal and progenitor cell migration in the hippocampus at late embryonic and early postnatal stages [25]. The morphology of the hippocampal dentate gyrus is dramatically altered in CXCR4 knock-out mice due to an underlying defect in the stream of post mitotic cells and secondary dentate progenitor cells that migrate toward and from the dentate gyrus. The lack of CXCR4 signaling led to reduced migratory activity of neuronal progenitor cells, whereby neurons appeared to differentiate prematurely before reaching their target [25]. Mice deficient in CXCR4 showed a massive loss of spinal cord motoneurons and dorsal root ganglion neurons. This led to a reduced innervation of the developing mouse fore and hind limbs. Increased death of motoneurons in CXCR4deficient animals seems to result from impaired limb myogenesis and a subsequent loss of muscle-derived neurotrophic support [26]. CXCR4 AND SDF-1 The interaction between CXCR4 and its’ ligand SDF-1α plays a pivotal role in regulating the retention, migration, and mobilization of HSCs during steady state homeostasis and injury [27]. Administration of anti-CXCR4 antibodies prevented the engraftment of murine bone marrow by human SCID repopulating stem cells [28], whereas cytokine mediated CXCR4 up-regulation led to increased SDF-1α mediated migration, in vivo homing, and repopulation of HSCs [28-30]. Similarly, CXCR4 over-expression by human cord blood and mobilized peripheral blood CD34+ cells by lentiviral gene transfer resulted in an increased proliferation, SDF-1α mediated migration, and bone marrow engraftment of these cells [31, 32]. The expression of CXCR4 on HSCs is very dynamic and known to be regulated by both cytokines and chemokines. Exposure of cord blood derived and mobilized peripheral blood derived CD34+ HSCs to stem cell factor (SCF; also named c-kit-ligand) or a combination of SCF and IL6 led to a CXCR4 up-regulation, concomitant with increased motility of SCID repopulating cells (SRC) [28]. Similar results were achieved with hepatocyte growth factor (HGF), alone or in combination with SCF [29]. Sorted CD34+ CXCR4 - HSCs express intracellular CXCR4, which can be up-regulated and expressed on the cell surface in response to stimulation with 5 cytokines (SCF, Flt3-ligand, IL6, IL-3, and G-CSF), thereby converting these cells into definitive SRC with high CXCR4 expression levels (both intracellular and cell surface) and SDF-1α induced migration [33]. In contrast, our findings showed that incubation of CD133 HSCs from cord blood with Flt3-ligand alone or in combination with IL6 for 5 days resulted in a CXCR4 down-regulation in both populations (unpublished observations). However, despite a reduced CXCR4 expression, CD133 cord blood HSCs incubated with Flt3ligand and IL6 responded to SDF-1α with an increased locomotory activity and an altered migration pattern. This is

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in contrast to cells solely incubated with Flt3-ligand, where only a slight increase in the migratory activity was detected [34]. Thus, cell-surface level expression of the SDF-1α receptor alone does not necessarily correlate with elevated migration or successful engraftment. On this note, a recent publication by Bertheboud and colleagues demonstrated that the regulators of G protein signaling (RGS) are responsible for negative regulation of G protein coupled receptors and regulate the activation of SDF1α signaling. The authors were able to show that an RGS16 over-expression in megakaryocytes inhibited SDF-1αinduced migration, mitogen activated protein kinase and protein kinase B activation [35]. MOBILIZATION CELLS

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Mobilization/recruitment and the homing of HSCs are mirror processes that are regulated by the interplay of cytokines, chemokines and proteases [36]. In brief, the process of HSC mobilization is characterized by both a loss of cell to cell contacts due to down-regulation and degradation of cell adhesion molecules, and a desensitization of the SDF-1α/CXCR4 axis. In contrast, up-regulation of cell adhesion molecules and activation of the SDF1α/CXCR4 axis is essential for stem cell homing, as we shall shortly see. Mobilization of HSCs from bone marrow is achieved by the application of cytokines, such as granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophagecolony-stimulating factor (GM-CSF) [37, 38], Flt-3 ligand [39-41], SCF [42, 43], and vascular endothelial growth factor (VEGF) [44], as well as several chemokines such as IL-8 [45-48], growth regulated oncogene-β (GROβ) [49], and macrophage inflammatory protein-2 (MIP-2) [50]. The aforementioned cytokines require 5 to 6 days for a peak level response to be reached; chemokines however induce mobilization within the time span of 30 minutes to a few hours. G-CSF G-CSF induced mobilization is a multi-step process that depends on both the disruption of cell-cell contacts by cleavage of cell adhesion molecules, and the CXCR4/SDF1α axis by inactivation of the SDF-1α receptor. Levesque and colleagues demonstrated that following GCSF application, active neutrophil granulocyte proteases, e.g. such as elastase and cathepsin G, accumulate within the bone marrow [51, 52]. An increase in the concentration of these proteases coincided with a down-regulation of the SCF receptor (CD117)[51], as well as a sharp reduction of the VCAM-1/CD106 expression in the bone marrow [52]. Conjointly, accumulation of neutrophil granulocyte secreted proteases within the bone marrow coincides with the cleavage of the N-terminus of CXCR4 on HSCs resident in the bone marrow and mobilized into the peripheral blood [53]. Recent findings indicate that CD26/Dipetidylpeptidase IV (DPPIV) plays an important role in G-CSF mediated HSC mobilization [54-57]. CD26/DPPIV is a membrane-

bound extracellular peptidase expressed on a subset of CD34 positive HSCs. It inactivates SDF-1α by cleaving it at position-2 proline [56]. Inhibition of CD26/DPPIV activity enhanced the migratory response of HSC to SDF-1α [54], thus pointing toward a regulatory role of CD26/DPPIV in SDF-1α mediated chemotaxis. Treatment of mice with CD26 inhibitors during G-CSF induced mobilization resulted in a reduced number of progenitor cells in the periphery, compared to the G-CSF regimen alone [54]. Similar results were obtained in CD26 (-/-) mice treated with G-CSF. Due to the absence of CD26/DPPIV activity, the number of HSCs in the peripheral blood was significantly lessened, compared to the corresponding number of HSCs detected in G-CSF treated wild-type mice [55]. It nevertheless remains to be determined whether CD26/DPPIV activity is directly triggered by G-CSF. Furthermore, the same group was able to show that endogenous CD26 expression on donor cells negatively regulates homing and engraftment [57]. FLT3-LIGAND Flt3 is a type III tyrosine kinase receptor expressed on primitive HSCs. Its ligand, aptly called Flt3-ligand, induces HSC mobilization in mice and acts synergistically with GCSF or GM-CSF. In a recent study by Fukuda and colleagues, Flt3-ligand was shown to enhance SDF-1α induced migration in the short term. Long term exposure to Flt3-ligand however caused a down-regulation of CXCR4 [39]. These findings suggest that Flt3-ligand plays an extrinsic regulatory role in the migration of HSCs that may be relevant in both homing to the bone marrow as well as mobilization. IL-8 In contrast to G-CSF-induced accumulation of elastase and cathepsin G in the bone marrow, IL-8 induced mobilization has been proposed to be dependent upon matrix metalloprotease-9 (MMP-9) activity. MMPs belong to the group of gelatinases/type IV collagenases and have been found to be of relevance to HSC migration and mobilization. The MMP family comprises 16 members [58] and are produced as latent pro-enzymes which are proteolytically processed to attain their activated status. The gelatinases predominantly degrade denatured collagens (gelatin) and also degrade collagen types IV, V, VII and X, as well as fibronectin and laminin (for a review see [59]). MMP-9 has been implicated in HSC mobilization in both mice and monkeys [45, 60-62]. Studies on rhesus monkeys showed that a single intravenous injection of IL-8 resulted in a dramatic increase in the plasma levels of MMP9, followed by a 10- to 100-fold increase of HSCs in the peripheral blood within 30 minutes after injection [60]. Pretreatment of the animals with a specific MMP-9 antibody blocked the IL-8 induced HSC mobilization. The mobilization of cells via the IL-8 and MMP pathway is not well understood. In a study by Pruijt and colleagues [63], the authors showed that the IL-8 induced mobilization was restored in neutropenic mice by injection with polymorphonuclear cells and non-purified mononuclear cells.

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Furthermore, IL-8 induced mobilization was not impaired in MMP-9 deficient mice, demonstrating that MMP-9 is dispensable for mobilization.

caused the release of soluble SCF and permitted the transfer of HSCs from a quiescent to a proliferative niche [66], thus implying an active role of SDF-1α in HSC mobilization (Fig. ( 1)).

GRO

Mobilization via an increase in plasma SDF-1α levels has furthermore been demonstrated with both fucan sulfate and dextransulfate [68]. The authors were able to show that a single injection of these sulfated glycans bind to SDF-1α, and thereby inhibit SDF-1/heparin binding, and likewise cause an increase in plasma concentrations. Homologously MMP-9 deficient mice did not prevent mobilization, indicating that MMP-9 it is not essential for this mechanism.

CXCR2 mediated mobilization is initiated via the chemokine CXCL2, also known as GROβ, and has been shown to be mediated via neutrophil derived proteases [49]. Mobilization induced by GROβ/CXCL2 was associated with elevated plasma and bone marrow levels of MMP-9, whereas mobilization and the associated increase in MMP-9 levels did not take place in neutrophil granulocyte-depleted mice [49]. Interestingly, the authors reported that GROβ/ CXCL2 acted synergistically with G-CSF. HSC mobilization by both G-CSF and GROβ/CXCL2 correlated with a synergistic rise in the plasma levels of MMP-9 and a simultaneous increase in peripheral blood neutrophil granulocytes. In contrast, marrow levels of neutrophil granulocytes and neutrophil granulocyte-derived proteases were decreased. Synergistic mobilization was absent in MMP-9-deficient or neutrophil granulocyte-depleted mice and completely blocked in animals pretreated with antiMMP-9 [49]. SDF-1 The chemokine SDF-1α is to date one of the best described and widely confirmed chemoattractants for HSCs [4, 5]; several studies indicated that this chemokine is pivotal in HSC mobilization. Aiuti and colleagues reported that the migration capacity of G-CSF mobilized HSCs from peripheral blood in response to SDF-1α was lower than of HSCs from bone marrow, and suggested that an altered response to SDF-1α may be associated with HSC mobilization [5]. Furthermore, exposure of adult mice to the SDF-1β analog methionin-SDF-1β (Met-SDF-1β) resulted in HSC mobilization with a more than 30-fold increase compared to the PBS control [64]. Over-expression of SDF1α in the peripheral circulation of SCID mice via intravenous injection of an adenoviral vector expressing SDF-1α (AsSDF1) has also been shown to result in HSC mobilization [65, 66]. Although these studies implicate a direct role of SDF-1α in HSC mobilization, the reported effects are predominantly attributed to an impairment of the CXCR4/SDF-1α axis or a change in the SDF-1α concentration in peripheral blood and bone marrow. For example, the decreased migration rate of mobilized peripheral blood HSCs to SDF-1α [5] is attributed to a desensitization of the CXCR4/SDF-1α axis due to SDF-1α cleavage, mediated by G-CSF induced activation of neutrophil granulocyte-derived elastase [67]. Similarly, administration of Met-SDF, which is resistant to cleavage causes a prolonged CXCR4 desensitization, which again impairs CXCR4/SDF-1α signaling [64]. In contrast to the desensitization of the CXCR4/SDF-1α axis, mobilization of HSCs by adenovirus-driven SDF-1α was attributed to a change in the SDF-1α gradient between bone marrow and peripheral blood [66]. However, the same group reported that SDF-1α over-expression resulted in an activation of MMP-9 induced in bone marrow cells, which

OTHER MOBILIZING FACTORS More recent data has shown that more factors other than the above mentioned proteases and cytokines are involved in the mobilization process. For example, the calcium sensing receptor is expressed on HSCs, a method by which cells are able to respond to extracellular calcium concentrations. It serves to retain HSCs close to the endosteal surface [69]. The activation of noradrenergic neurons has also been shown to decrease the production of SDF-1α. Ablation of adrenergic neurotransmission indicated that norepinephrine signaling controls G-CSF induced osteoblast suppression, SDF down-regulation and HSC mobilization [70]. The regulation of stem cell release and expansion has not only been shown to be regulated by osteoblasts, but by osteoclasts as well. Zhang and colleagues found that the number of osteoblastic cells correlates with the number of HSCs, whereby long term HSCs were found to be attached to these cells, and bone morphogenic protein indirectly controlled the number of stem cells [71]. Recently the role of osteoclasts in stem cell homeostasis has been investigated by Kollet and colleagues. The mobilization of HSCs associated increase in osteoclast number led the authors to investigate a possible relationship between stem cell release from the endosteal niche [72]. Stimulation of the osteoclasts was then shown to cause a release of stem cells into the circulation in a CXCR4 and MMP-9 dependent manner. The cytokine RANKL caused an altered expression of the proteases MMP-9 and cathepsin K, which degrades membrane bound SCF, and also caused a decrease in osteopontin. In a clinical setting it is of continual concern to increase the mobilization efficacy of HSCs for therapeutic purposes. Concurrently several strategies for the mobilization of stem cells are being tested in the clinical setting. King and colleagues have shown that a truncated form of the CXC chemokine GROβ (SB-251353) alone or in combination with G-CSF mobilize hematopoietic stem cells with long term repopulating ability [73]. Likewise a combination of the CXCR4 antagonist AMD3100 and G-CSF to improve the efficacy of stem cell mobilization is being tested [74, 75]. AMD3100 is a bicyclam antagonist of the SDF-1α receptor CXCR4 and has been shown to induce rapid mobilization of CD34+ hematopoietic cells in mice, dogs and humans [74]. Recent findings by Broxmeyer and colleagues show that AMD3100 synergizes with G-CSF and leads to the mobilization of long-term repopulating (LTR)

Stem Cell Migration


Fig. (1). Stem cell homing and mobilization to and from the bone marrow endosteum. HSC mobilization and homing are mirror processes that strongly depend on the activation state of cell-cell contacts and the SDF-1α/ CXCR4 axis. HSC mobilization from bone marrow mediated by cytokines, e.g. G-CSF or chemokines such as IL-8 is caused by proteases (MMPs, elastase, and cathepsin G) that degrades adhesion molecules, membrane bound c-kit, and SDF-1α and its receptor CXCR4. In contrast, up-regulation of cell adhesion molecules and activation of the SDF-1α/ CXCR4 axis is essential for stem cell homing. The blue coloring represents the SDF-1α gradient in the bone marrow.

cells [76]. Moreover lymphoma and myeloma cells do not appear to be mobilized by this drug [75]. AMD3100 is currently being tested in phase I-III clinical trials. HOMING OF HEMATOPOIETIC STEM CELLS Homing, as defined by Lapidot et al., is a descriptive term used for the crossing of circulating HSCs across the blood/bone endothelial barrier into the bone marrow compartment within a fairly short time span of hours to days. Successful homing is measured by the successful reconstitution of hematopoiesis [77]. HSC homing is a multi-step process requiring an interplay of adhesion molecules, cytokines and chemokines, and extracellular matrix degrading proteases. This resembles the process of leukocyte/ lymphocyte extravasation [78], as

well as trans-endothelial migration of tumor cells during the hematogenous metastasis of cancer [79]. Extravasating cells (1) adhere to the vascular endothelium, (2) transmigrate across the endothelial lining and the underlying basement membrane, and (3) migrate into the surrounding tissue. Although prefatory work on molecular pathways guiding extravasation of HSCs has provided some insight, the mechanisms explaining this complex process remain nebulous [80]. Studies investigating the mechanisms of homing were first published in the early 90s. These provided a first indication that galactose and mannose specific lectins and their receptors were important for successful bone marrow engraftment [81]. As in the case of lymphocytes/ leucocytes, the rolling phase of extravasation is mediated by E- and Pselectins [82, 83]. Subsequent firm adhesion is mediated through intercellular adhesion molecule-1 (ICAM-

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1)/leukocyte function-associated antigen-1 (LFA-1), vascular adhesion molecule-1 (VCAM-1)/very late antigen-4 (VLA-4) ligand pairs, and junctional adhesion molecules, such as platelet endothelial cell adhesion molecule (PECAM) [83]. The importance of VLA-4 (α 4β1 integrin) and VCAM interactions have been demonstrated by the use of a blocking anti-α 4-antibody; results showed that HSC homing was inhibited by more than 90% [80, 84]. Similar results were achieved with hypomorphic VCAM-1 mice with domain 4 deletion (D4D) and low expression of VCAM-1. Experiments with the latter showed that homing was virtually abrogated when the animals were treated with antiVCAM-1 antibody and were given anti-CD11a-treated cells [80]. However, β2-integrins and selectins may be used as a fall-back if the dominant VLA-4/ VCAM-1 (α 4β1/ VCAM1) interaction is compromised [80]. Furthermore α4 integrins have been shown to selectively mediate the homing of cells to the bone marrow, but not to the spleen (as was demonstrated in transplantation studies using α4 (-/-) cells) [85].

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Central to the regulation of this process is SDF-1α, which has been shown to trigger the firm adhesion to LFA-1 to ICAM, as well as the VLA-4 to VCAM-1 under shear flow conditions [86]. Studies by Peled and colleagues indicated that the SDF-1α induced polarization and extravasation of CD34+ /CXCR4 + HSCs through the extracellular matrix underlining the endothelium is dependent upon both VLA-4 and VLA-5 [87]. A myriad of publications have shown that polarization per se is a prerequisite for migration [88], as has been shown in leucocytes as well as in tumor cells. SDF-1α causes a relocalization of CXCR4 to the leading edge of transfected KG1a cells upon contact with HUVECS. CXCR4 has furthermore been shown to be co-localized with lipid rafts, and is found at cell to cell interaction sites when in contact with the endothelial cell surface [89] (Fig. (2)). Polarization was also induced when cells stimulated with SDF-1α were brought together with immobilized hyaluronic acid (HA). CD44 was found at the leading edge of pseudopodia and is involved in adhesion and immobilization of cells [90]. Pretreatment of these cells with blocking anti-CD44 antibodies

Fig. (2). Migrating HSCs show a polarized phenotype. A confocal laser scanning microscope picture of a CD34 cord blood derived progenitor cell embedded in a 3D collagen lattice. Anti βtubulin (green) and phalloidin (red) staining reveals the polarization of the migrating cell. The fibers of the collagen lattice appear in blue.

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prevented these morphologic alterations implying that CD44-HA interaction is a prerequisite for polarization. Pilarski and colleagues found that the HA receptor RHAMM on CD34 cells leads to an increase in motility, whereby CD44 leads to an increase in adhesion. CD44-HA has furthermore been shown to play a role in the lodging of HSCs in the spleen [90, 91] and bone marrow [90-92]. However, studies performed on CD44 deficient mice showed that these animals have no hematopoietic abnormalities and that CD44-/- HSCs are capable of homing to the bone marrow of normal or CD44-/- animals [93]. Inconsistencies in these results may be readily explained by the expression of various CD44 isoforms [94]. A recent study has shown that HSCs express a novel glycoform of CD44 named HCELL (hematopoietic cell E-/L-selectin ligand) and most likely plays a pivotal role in HSC trafficking to the bone marrow [95]. Cytokines trigger the adhesion of HSCs during homing. Several studies have shown that various cytokines such as GM-CSF, IL-3, and SCF temporarily increase the adhesiveness of HSCs by activating the β1-integrins VLA-4 and VLA-5. Cytokines (Flt3-ligand, SCF, IL-3, IL6, HGF) up-regulate CXCR4 expression on HSCs in vitro and in vivo [29, 86, 96], thereby enhancing the intracellular signals generated through the SDF-1α/ CXCR4 axis [97]. Our investigations and those published by Fukuda et al. have shown that prolonged exposure of CD133 + or CD34+ HSCs from cord blood to Flt3-ligand leads to a down-regulation of CXCR4 expression [34, 39]. Interestingly, short-term (2 day) exposure of CD34+ HSCs with Flt3-ligand inhibited the SDF-1α mediated phosphorylation of MAPKp42/p44, CREB, and Akt, and impaired migration toward CXCL12 [39], whereas long-term (5 day) Flt3-ligand incubation of CD133+ HSCs responded to SDF-1α with an increased migration rate, despite reduced CXCR4 expression [34]. Moreover, similar results were observed for cord-blood derived CD133+ HSCs cultured in Flt3-ligand and IL6. Prolonged incubation time led to a decrease in CXCR4 expression levels, whereas the migratory response to SDF1α was enhanced. These cells displayed an altered migration pattern, as well as a recruitment of non-moving cells. The mechanisms responsible for the altered migration pattern of cells cultured in IL6 remain to be elucidated. Some research has however shown that the differentiation of HSCs is regulated by IL6 [98-101]. It may thus be speculated that these phenotypical changes could be attributed to an altered gene expression profile. Transendothelial migration of cells presupposes the degradation of the basement membrane, which requires the production of matrix-degrading enzymes, especially those capable of degrading type IV collagen such as MMPs [58]. Both MMP-2 and MMP-9 are known to be expressed by all cell types that migrate through the endothelial cell layer including leukocytes [78, 102-105], tumor cells [58, 106114], and HSCs [27, 29, 115, 116]. Their expression and secretion are stimulated by various chemokines and cytokines, as discussed in the previous section. Two recent studies by Zheng and colleagues showed that CD34+ HSC from cord blood exhibited significantly lower expression levels of CD49e, CD49f, CXCR4 as well as MMP-2 and MMP-9, compared to their counterparts from peripheral blood and bone marrow, which may be a reason for delayed


hematopoietic reconstitution after umbilical cord blood transplantation [117, 118]. However, upon incubation with recombinant human SCF, these cells gained increased expression of the homing related molecules, including CXCR4 and MMP-2/-9, and showed an increased ex vivo transmigratory and in vivo homing potential [117, 118]. SDF-1 /CXCR4 MIGRATION

SIGNAL

TRANSDUCTION

AND

Cell migration is an essential component of both successful mobilization and homing. Its’ initiation, as discussed in the previous sections, is controlled by a multitude of extracellular parameters. These include extrinsic initiation, such as the extracellular matrix, signaling via chemokine receptors and receptor tyrosine kinases [119]. These pathways ultimately lead to an activation of actin associated proteins, reorganization of the actin cytoskeleton, as well as the activation of focal adhesion kinases. The SDF-1 receptor CXCR4 belongs to the group of 7transmembrane receptors, also known as serpentine receptors, and are linked to G proteins [19, 20], as described in previous sections. SDF-1α induced chemotaxis in HSCs may be inhibited by the addition of pertussis toxin, indicating that it is associated with a Gα i-protein subtype [5, 120, 121]. In the case of both T lymphocytes and CD34 positive cells, the PI3-kinase, phospholipase C-γ (PLC-γ )/ protein kinase C (PKC) cascade and MAPKp42/44 (ERK-1/2) are activated upon SDF-1α binding [122, 123]. Studies on human T cell lines indicated that SDF-1α triggers CXCR4 dimerization and activates the JAK/STAT pathway, which suggests gene regulation [124]. Similarly, Ganju and colleagues have reported that SDF-1α treatment led to increased NF-κB activity in nuclear extracts of CXCR4 transfectants, indicating that changes of the gene expression level can be initiated via two independent signal transduction pathways down-stream of the SDF-1α/ CXCR4 axis [122]. In contrast, Lee and colleagues have demonstrated that NF-κB is activated after addition of SDF1α [97]. The same group has however demonstrated that MAPK p42/44 (ERK-1/2), ribosomal S6 kinase (p90RSK) and Akt are synergistically activated by SDF-1α in combination with GM-CSF, SCF, TPO, or Flt3-ligand, and that this correlated with an enhanced survival of cord blood derived CD34+ HSCs [97]. The actin cytoskeleton is one of the central mechanical components responsible for the motility of cells, and its’ analysis is an effective method of determining a migratory phenotype. Actin polymerization in migration is induced by the PI3-kinase and the PLC-γ / PKC cascade in a variety of cell types [119, 125-127], including HSCs. So far several groups have convincingly demonstrated that SDF-1α induces actin polymerization [5, 39] as well as tyrosine phosphorylation of several components of focal adhesion complexes such as paxillin, the related adhesion focal tyrosine kinase (RAFTK/ pyk2), p130cas , Crk-II, and Crk-L [122, 123]. Inhibition of PI3-kinase signaling using wortmannin partially inhibited the SDF-1α induced migration and tyrosine phosphorylation of paxillin [122], further underpinning the role PI3-kinase in cell migration. In a recent study by Petit and co-workers, SDF-1α induced cell migration was shown to activate an atypical

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PKC subtype known as PKC-ζ [128]. Activation of this subtype is not dependent upon calcium or diacylglycerole (DAG) [129, 130]. Furthermore cell polarization, adhesion to bone marrow stromal cells and chemotaxis were all shown to be PKC-ζ-dependent. PKC-ζ phosphorylation, translocation to the plasma membrane, and kinase activity, was furthermore shown to be dependent upon PI3-kinase activity, Pyk-2 and MAPKp42/44 (ERK-1/2). MMP-9 was also found to be activated after PKC-ζ phosphorylation [128]. Moreover, in vivo studies showed that engraftment, but not homing, of human CD34+ HSCs was also dependent upon PKC-ζ activity [128]. Cancelas and colleagues were recently able to demonstrate that a post engraftment deletion of the Rho GTPases Rac1 and Rac2 led to a massive mobilization of HSCs from the bone marrow. Rac1(-/-) mutants were not able to rescue hematopoiesis after transplantation, but a deletion of Rac1 did not prevent steady state hematopoiesis. The authors were thus able to demonstrate that Rac proteins regulate HSC motility in both homing and mobilization [131]. The examination of HSC migration however should not only be limited to an analysis of signal transduction pathways, but should also include a study of the migratory phenotype. For the general study of migration Maheshweri and Lauffenburger proposed that cell population migration should be deconstructed in terms of a mathematical model, comprising of cell population parameters such as random motility, chemotaxis/haptotaxis, and chemotaxis/haptotaxis coefficients [132]. They recognized that individual cell paths can be analyzed in a model of individual cell parameters which include translocation, speed, and directional persistence in time, which in turn depend upon membrane extension and retraction, cell to substratum adhesion, cell contractile force and front vs. rear symmetry. Commonplace migration analyses are carried out in a 2D environment, which regrettably offer but an indication of the situation in vivo, and are thus insufficient for the study of the aforementioned parameters. In the benchmark work by Friedl and colleagues, leukocytes were introduced into a 3D environment, in which the migratory parameters such as speed, directionality, persistence, trajectory, and percentage locomoting cells were analyzed and determined for CD4 and CD8 lymphocytes [133]. Comparative experiments using lymphocytes and tumor cells demonstrated that these parameters were determined, and seen to be characteristic for different cell types [134]. Video microscopic observations concomitantly present the opportunity to further scrutinize the relationship between morphology and migration, and have successfully been implemented in the case of tumor cells and cord blood derived CD133 + cells [34, 134]. In a recent comparative study of both 2D and 3D migration [135], the authors were able to determine that the 2D model predictions were qualitatively similar to the 3D situation, but that new effects such as matrix sterics and mechanics arose in the 3D situation. In our work we analyzed various migratory parameters in a 3D collagen matrix. These parameters included the time active within the period of the assay, the velocity of the cells (speed without pauses), the overall speed (with pauses), the pause length, pause frequency, and the overall distance migrated. Upon stimulation with SDF-1α and following extensive single

Weidt et al.

cell analysis, the migratory phenotype showed a recruitment of non-moving cells, an increase in speed and frequency of pauses, but a decrease in pause duration. This resulted in an increase in the distance migrated. The study of cell migration should be considered on both a molecular and cell population level, for both are integral components of homing and mobilization. The understanding of these parameters becomes particularly relevant in a clinical setting where engraftment and mobilization rates vary greatly from patient to patient. Both isolated HSCs as well as whole bone marrow are commonly used to restore hematopoiesis in hematopoietic maladies, whereby bone marrow transplantation is preferred in clinical transplantation. Bone marrow transplantation is currently the only therapeutic approach for several lethal malignant and non-malignant disorders. The use of cord blood as a source of progenitor cells was quickly established as an alternative. To date over 50 banks worldwide serve as a source for cord blood, and over 2000 patients have received transplantation [136]. Progenitors are also being targeted as potential vectors for gene therapy to invoke immune tolerance with the aim of facilitating allogeneic or xenogeneic organ transplants. Despite the fact that cord blood transplantation strategies have meanwhile become routine in clinical practice, engraftment is still very variable [137]. Intensive studies in the recent years indicate that the ability of hematopoietic stem cells to engraft is dependent on cell their cycle status. Studies on murine HSCs that have been exposed to a cytokine cocktail revealed that the engraftment phenotype showed marked fluctuations over 24h intervals, whereby least effective responses were recorded with cells in late S and early G2 phase [138]. Enhanced long-term donor-derived multilineage reconstitution of the peripheral blood was observed in recipients of G0/G1-phase HSCs from fetal liver compared to recipients of S/G 2/Mphase cells [139]. Similar results were observed for human cord blood derived CD34+ HSCs that were stimulated to proliferate [139]. HSCs that had entered the S/G2/M-phase lost engraftment potential and did not reenter the G0-phase. No differences in the expression level of VLA-4, VLA-5, and CXCR4 were detectable in cultured G0/G1-phase and S/G 2/M-phase cord blood CD34+ HSCs indicating that an unknown cell cycle-associated mechanism selectively prevents proliferating HSCs to successfully engraft [139]. Yamaguchi and colleagues have however demonstrated that CD34+ HSCs from various sources (bone marrow, peripheral blood, and apheresis peripheral blood samples), and in different stages of the cell cycle (G0/G1-phase versus S/G 2/M-phase), exhibited altered adhesive characteristics and VLA-4 expression [140]. In a comparative study by Ramirez and colleagues, CD34 cells were expanded for six days. The authors showed that the adhesion molecules VLA-4, VLA-5, MAC-1 (α Mβ2 integrin) were up-regulated, but that the adhesion to fibronectin was significantly decreased [141]. Different results are probably due to different culture conditions and times used: Ramirez cultured the cells for six days in IL-3, IL6, and SCF or alternatively in IL-11, SCF, and Flt3-ligand [141]. Glimm and colleagues used a five day culture with Flt3-ligand, SCF, IL-3, IL6, and G-CSF [139], and Yamaguchi on the other hand used a 4 day culture with IL-3 and SCF [140].

Stem Cell Migration

The cycling of long term culture initiating cells and primitive human colony forming cells is stopped upon addition of SDF-1α to the culture media of CD34 HSCs [142, 143]. The recovery rate of transplanted NOD/SCID mice increased two fold under this regimen [144]. Thus, blocking the cell cycle of HSCs suggests new strategies to improve the engraftment activity of ex vivo expanded HSCs. SDF-1 IN INFLAMMATORY CONDITIONS The role that SDF-1α plays in inflammatory processes has long remained unclear. SDF-1α is ubiquitously expressed in various tissues [11]; it was thus suggested to play a role in immune surveillance and in the basal extravasation of lymphocytes, rather than in inflammation [120]. A study by Gonzalo and colleagues delivered the first indication that SDF-1α is involved in inflammatory processes. They demonstrated that SDF-1α is a critical inflammatory component in allergic airway disease [145]. Administration of neutralizing antibodies to CXCR4 in a mouse model of allergic airway disease resulted in reduced eosinophilia (broncho-alveolar lavage fluid and interstitium) by half, which was accompanied by a significant decrease in airway hyper-responsiveness. A later study revealed that the influence of SDF-1α in the inflammatory component of allergic airway disease is more passive than active [146]. Exposure of human airway epithelial cells with proinflammatory cytokines resulted in CXCR4 up-regulation, thus enabling the cells to respond more efficiently to constitutively expressed SDF-1α. The treatment of an asthma mouse model with the CXCR4 antagonist AMD3100 furthermore significantly reduced airway hyper-reactivity, peribronchial eosinophilia and the overall inflammatory response [18]. The study of bacterial neurological infections have shown that SDF-1α participates in the recruitment on dendritic cells in the cerebrospinal fluid [147, 148]. Patients suffering from noninflammatory neurological diseases, as well as inflammatory neurological diseases, were seen to have elevated SDF-1α in the cerebrospinal fluid. The application of blocking antibodies led to a decrease in the recruitment of both mature and immature dendritic cells [148]. For rheumatoid arthritis it has been reported that SDF-1α plays an important role in CD4+ memory T cell accumulation in the rheumatoid arthritis synovium [149, 150]. CXCR4 expression on CD4+ T cells was enhanced by IL-15 [150] and/or TGF-β [149], whereas SDF-1α expression of the synovium and on synovial endothelial cells was increased by CD40 stimulation [150], and led to a strong integrin mediated adhesion of synovial fluid T cells to fibronectin and ICAM1 [149]. Recent findings indicate that SDF-1α is modulated by inflammatory cytokines and proteases. Gene profiling analysis of endothelial cells showed that the inflammatory cytokines TNF-α and IFN-γ inhibit the expression of proangiogenic molecules including SDF-1α [151]. Similar results were reported by Fedyk et al., showing that secretion of TNF-α and IL-1α from activated macrophages inhibited the expression of SDF-1α by human fibroblasts in vitro [152]. The biological activity of SDF-1α is not only impaired by inflammatory cytokine-mediated down-


regulation of the gene, but also by degradation of the chemokine by proteases, such as elastase [153], MMPs [154], and CD26/Dipeptidylpeptidase IV (DPPIV) [56]. Modulation of SDF-1α function by inflammatory cytokines and/or proteases plays a role in various biological processes. Salvucci et al. showed that down-regulation of endothelial cell-derived SDF-1α by TNF-α and IFN-γ correlated with a disruption of extracellular matrix-dependent endothelial cell tube formation, an in vitro morphogenic process that recapitulates critical steps in angiogenesis [151]. Degradation of endothelial-bound SDF-1α by elastase released by transmigrating neutrophils may prove to be involved in the regulation of leukocyte trafficking to sites of inflammation [153]. Pre-treatment of endothelial cells with purified neutrophil elastase resulted in an inhibition of T lymphocyte transmigration. Elastase selectively cleaves the aminoterminus of endothelial-bound SDF-1α, which is required for chemotactic activity. The emerging inactivated cytokine is immobilized on the endothelial cell, thereby preventing T lymphocyte transmigration. In contrast, within the bone marrow elastase plays a major role in dislodging HSCs from bone marrow stroma by degrading VLA-4, VCAM-1, CXCR4, and SDF-1α [51-53]. Due to desensitization of the CXCR4/SDF-1α axis concomitant with a loss of cell-cell contacts, CD34+ HSCs are successfully released from the bone marrow (see above). HOMING OF HSCs TO ORGANS In this section we would briefly like to discuss the role of SDF-1α and HSCs in tissue regeneration. The first work that suggested that HSCs were homed to damaged tissue by SDF-1α was published by Kollet and co-workers [29]. Previous findings that the level of HSC engraftment into the liver is amplified during liver injury or viral inflammation prompted the authors to investigate the role that SDF-1α plays in this process. Both irradiation and inflammation led to elevated SDF-1α expression in liver bile and duct epithelial cells. Conjointly, hepatic injury induced MMP-9 activity, leading to both increased CXCR4 expression and SDF-1α mediated recruitment of hematopoietic progenitors to the liver. Recruited HSCs were found localized in clusters surrounding the bile ducts, in close proximity to SDF-1α expressing epithelial cells, and had differentiated into albumin-producing cells [29]. In a recent publication by Jin and colleagues, the authors were able to show that nonendothelial CXCR4 + , VEGFR1+ progenitors constitute a major determinant of revascularization in ischemia [155]. A series of cytokines such as SCF, TPO, erythropoietin (EPO), and GM-CSF induced the release of SDF-1α from platelets. It has been shown that stress signals, such as tissue injury and/or inflammation, cause up-regulation of SDF-1α in endothelial cells, thereby recruiting HSCs. This has been demonstrated in tissues such as heart [156, 157], kidney [157], and brain [158]. Ceradini et al. found that the recruitment of CXCR4-positive progenitor cells to regenerating tissues is mediated by the hypoxic gradient, namely via HIF-1 induced expression of SDF-1α in endothelial cells [13]. The up-regulation of SDF-1α in ischemic tissue is directly proportional to reduced oxygen tension, and was correlated with increased adhesion,

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migration, and homing of circulating HSCs to ischemic tissue. MALIGNANT DISEASE, SDF-1 AND HSCs Tumor tissue is very heterogeneous; it includes not only tumor cells but also fibroblasts, lymphocytes and macrophages. Tumor associated macrophages (TAMs) for example, have been suggested to sustain microenvironmental conditions in malignant tissue [159]. TAMs are attracted to tumor tissue via hypoxia and they themselves initiate angiogenesis [160]. They likewise secrete proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), IL-1, IL6 and also chemokines such as monocyte chemoattractant protein-1, -2, -3 (MCP-1, -2, -3) [161, 162]. Several publications have also shown that bone marrow derived cells contribute to malignant tissue. Using the angiogenic deficient Id-mutant mouse model, Lyden and colleagues were able to show that both wild type and VEGF mobilized bone marrow was able to restore tumor angiogenesis. By targeting both VEGFR1 and VEGFR2 complete ablation of tumor growth was achieved [163]. Other studies have shown that CD34-positive cells of hematopoietic nature with endothelial morphology were found in tumor tissue [164-166]. So called tie2, CD11b expressing monocytes were likewise found in tumor tissue [167]. In this study the authors were able to show that a knockout of tie2 expressing monocytes completely prevented human glioma neovascularization in the mouse brain and brought about substantial tumor regression. But the role that bone marrow derived cells may assume in a malignant environment is not yet fully explored. The aforementioned studies, as well as many others, suggest that the bone marrow derived cells may contribute to malignant tissue. If one however combines both the thought that stem cells recruit to malignant tissues with that of the tumor as an instructing milieu [168-171] not only in the sense that they contribute to neovascularization, but to the development of new malignant tissue, current paradigms of stem cell therapy should be (re)considered carefully. Stem cells recruited to tumor tissue may on the one hand remain dormant in this environment, but may also boost cancer progression. In a hypothetical process of transdifferentiation or fusion, signals from the malignant environment may cause these stem cells to both retain stem cell characteristics and attain a variety of common features of cancer cells. In an important work by Müller et al., the authors were able to demonstrate that breast cancer metastasis to the regional lymph nodes, bone marrow, liver and lung is directed by SDF-1α. The target organs were shown to predominantly express SDF-1α, and breast cancer cells themselves expressed CXCR4 [6]. The application of a neutralizing SDF-1α specific antibody effectively impaired metastasis formation in regional lymph nodes and lung. The pivotal role that SDF-1α plays has furthermore been shown for bone marrow metastasis formation in neuroblastoma [172], and in metastasis formation in prostate cancer [8, 173]. In melanoma, SDF-1α has been shown to promote tumor cell invasion across basement membranes via stimulation of MT1-MMP activity [174]. Moreover, SDF1α triggered the activation of the GTPases RhoA, Rac1, and

Weidt et al.

Cdc42 in the highly metastatic BLM melanoma cell line [174]. Furthermore, CXCR4 expression on melanoma cells was notably augmented by transforming growth factor-β1 (TGF-β1), a matrigel component, whereas anti-TGF-β1 antibodies inhibited increases in CXCR4 expression and melanoma cell invasion toward SDF-1α. It is well known that tumor tissue is hypoxic, and that under these conditions the transcription factor HIF regulates gene expression and allows tumor cells to adapt to hypoxic conditions [175, 176]. Recently the expression of SDF-1α and CXCR4 have been suggested to be regulated by HIF [13, 177]. Taken together these findings suggest that SDF-1α signaling is involved in metastasis formation. ENDOTHELIAL PROGENITOR CELL MOBILIZATION Bone marrow derived endothelial progenitors have become the focus of intense investigation in the past decade. First indications of the existence of a bone marrow derived endothelial precursor population were discovered by Asahara and colleagues [178]. They found that up to 20% of the CD34 positive population of peripheral blood was also VEGFR2 positive. They have been phenotypically characterized by antigens that to date have concomitantly been associated with hematopoietic stem and progenitor cells. These encompass CD133, CD34, c-kit, VEGRR2, CD144 (VE-Cadherin), and Sca-1 [179]. An ever increasing number of studies have provided multiple indications that endothelial precursors contribute to neo-angiogenesis in wound healing, post myocardial ischemia, cerebral ischemia, limb ischemia and tumor growth. The elucidation of mechanisms that enable the mobilization of endothelial progenitor cells (EPCs) will provide a new target for therapeutical strategies in the treatment of cardiovascular diseases and malignancies. The mobilization of these cells out of the bone marrow may be induced by G-CSF [180], GM-CSF [181], SDF-1α [182], VEGF [182, 183], angiopoietin-1 [182, 183], erythropoietin (EPO) [184, 185], estrogen [186], physical activity [187] and two members of the platelet-derived growth factor (PDGF) family, PDGF-AA and PDGF–CC [188]. In a study by Bergers and colleagues, MMP-9 was shown to render normal pancreatic islets angiogenic via VEGF. MMPs made membrane bound cytokines such as VEGF-A biologically available [189], and invoked an angiogenic switch in carcinogenesis. Furthermore, osteoclast migration may be initiated by VEGF [190], these are cells which now have been shown to regulate the release of HSCs via MMP-9 and CXCR4. Kopp and colleagues have furthermore been able to show that marrow suppression (or release of VEGF-A or placental growth factor) result in an increase in MMP-9, as well as soluble SCF within the bone marrow [179]. The increase of soluble kit ligand promotes stem cell cycling and motility. Another very interesting study published by Grunewald et al., this year [191] analyzed the role of VEGF in the recruitment of circulating mononuclear myeloid cells from the bone marrow. These cells were held in close proximity to angiogenic vessels by SDF-1α, the expression of which

Stem Cell Migration

was induced by VEGF in perivascular myofibroblasts. They enhanced the in situ proliferation of endothelial cells, and not the recruitment of cells from the bone marrow. In regard to metastasis the mobilization of VEGFR1 positive cells from the bone marrow cells have been suggested to provide the soil for metastases [192]. In light of this data, one can conclude that this population provides an ideal target for anti-angiogenic therapy, but must in light of the Kaplan study be considered carefully so as not to provide additional soil for metastases. Even though several factors that stimulate EPC mobilization are known, the cellular and molecular mechanisms remain to be elucidated. This also applies of EPC homing, which is still poorly understood. Recently, β2-integrins have been reported to be play a role in EPC homing by showing in a murine model of hind limb ischemia. Preactivation of β2-integrins expressed on EPCs by activating antibodies augments the EPC-induced neovascularization in vivo [193]. Elucidation of these mechanisms will provide a valuable means to regulate the processes involving EPCs such as ischemia and neoangiogenesis. This is still a challenge in light of technical difficulties with EPC numbers as well as the still open discussion of EPC antigen profile.

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[6] [7] [8] [9] [10] [11] [12]

CONCLUSION In this review, we have elucidated the current understanding of the role of migration in HSC and EPC function. We have furthermore discussed the myriad of factors, and their complex cellular and systemic regulation of HSC migration. The most prominent known regulators of migration include MMPs and elastases, c-kit, and SCF which govern the expression of SDF-1α and its receptor CXCR4. This however begs the question: How does this knowledge help nurture the current (often insufficient) therapies for various illnesses? Current therapies rely on the harvesting of HSCs from the bone marrow, isolating the clinically relevant sub-population and re-administering via intravenous injection. These therapies unfortunately do not always deliver the desired clinical benefit. In these processes, migration plays a pivotal role which is central in both the mobilization of cells from the bone marrow, and subsequent engraftment in the recipient. An understanding of migration and its initiation, regulation, and mechanisms may thus provide a promising approach to enhance and enrich current therapeutic strategies involving HSC and EPC engraftment for a range of critical illnesses.

[13]

ACKNOWLEDGEMENTS

[22]

We would like to apologize to all those authors whose invaluable work was not mentioned in the above. This work was supported by the Fritz-Bender-Foundation, Munich, Germany.

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Received: June 28, 2006

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Accepted: August 17, 2006