Pleiotrophin (PTN) is a secreted growth factor that induces neurite outgrowth and is mitogenic for fibroblasts, epithelial, and endothelial cells. During tumor growth PTN can serve as an angiogenic factor and drive tumor invasion and metastasis. To identify a receptor for PTN, we panned a phage display human cDNA library against immobilized PTN protein as a bait. From this we isolated a phage insert that was homologous to an amino acid sequence stretch in the extracellular domain (ECD) of the orphan receptor tyrosine kinase anaplastic lymphoma kinase (ALK). In parallel with PTN, ALK is highly expressed during perinatal development of the nervous system and down-modulated in the adult. Animal studies demonstrated that PTN can serve as a ratelimiting angiogenic factor during tumor growth, invasion, and metastasis (8 -12). Clinical studies showed elevated serum levels and tumor expression of PTN in samples from patients with colon, stomach, pancreatic, and breast cancer (5, 13). Furthermore, PTN has been implicated in neonatal brain development as well as in neurodegenerative disorders (reviewed in Ref. 14).Obviously, understanding of PTN-mediated signal transduction as well as identification of a receptor for PTN would enhance studies on the biology and pathology of this growth factor family. Our previous studies have shown that the activation of mitogen-activated protein kinase and PI 3-kinase pathways is required for mitogenic activity of PTN, and we had found that the adaptor molecule Shc participated in signal transduction (15). Based on the work of different laboratories in various cell types, it was hypothesized that proteins of 170 -220 kDa that are tyrosine-phosphorylated in response to PTN could be part of the receptor complex (15-17). More recently, several cell membrane-located proteins were shown to bind PTN at low affinity and serve as potential coreceptors or modulators of signal transduction (18 -21), but none of these molecules carried the hallmarks of a signal transducing receptor predicted from the earlier work.To identify a receptor for PTN, we rationalized that panning of a phage display cDNA library against immobilized PTN as a bait would allow us to isolate phage containing a ligand binding fragment of the receptor on their surface. Because of the high levels of expression of PTN during the perinatal development of the nervous system, we hypothesized that fetal brain would most likely also express a PTN receptor. We therefore panned a human fetal brain cDNA phage display library over several rounds against purified PTN that had been tested for biological activity (15). From this we isolated a phage insert homologous to an amino acid sequence stretch in the ECD of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK), a recently described orphan receptor with an apparent molecular mass of

Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2-Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2-Fc-stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy.

Anaplastic lymphoma kinase (ALK) is a novel neuronal orphan receptor tyrosine kinase that is essentially and transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. To determine whether ALK could play a role in neuronal differentiation, we established a model system that allowed us to mimic the normal activation of this receptor. We expressed, in PC12 cells, a chimeric protein in which the extracellular domain of the receptor was replaced by the mouse IgG 2b Fc domain. The Fc domain induced the dimerization and oligomerization of the chimeric protein leading to receptor phosphorylation and activation, thus mimicking the effect of ligand binding, whereas the wild type ALK remained as a monomeric nonphosphorylated protein. Expression of the chimera, but not that of the wild type ALK or of a kinase inactive form of the chimera, induced the differentiation of PC12 cells. Analysis of the signaling pathways involved in this process pointed to an essential role of the mitogen-activated protein kinase cascade. These results are consistent with a role for ALK in neuronal differentiation.The common structural features of a receptor tyrosine kinase (RTK) 1 include an extracellular ligand binding region, a hydrophobic membrane-spanning segment, and a cytoplasmic domain that carries the catalytic function. Following ligand binding, the RTK dimerizes and autophosphorylates (1). The activated RTK initiates signal transduction cascades through binding of SH2 domain-containing proteins to specific receptor phosphotyrosine residues (2). RTKs can regulate a wide variety of cellular processes involved in cell division, differentiation, survival, and motility. A number of RTKs play essential roles during the development of the nervous system by contributing to neuronal differentiation, survival, and function (reviewed in Ref.3). Most of these receptors have specific or shared ligands called neurotrophic factors that have been identified (reviewed in Ref. 3). However, for some of them, named orphan receptors, their ligands are still unknown (4 -6).Anaplastic lymphoma kinase (ALK), a novel orphan neuronal receptor, was originally identified as a member of the insulin receptor subfamily of receptor tyrosine kinases that acquires transforming capability when truncated and fused in the t(2;5) chromosomal rearrangement associated with the non-Hodgkin lymphoma (7). This translocation produces a fusion gene that encodes a soluble chimeric transforming protein comprised of the N-terminal portion of the phosphoprotein nucleophosmin (NPM), a highly conserved RNA-binding nucleolar protein, linked to the cytoplasmic portion of ALK (7). The NPM-ALK fusion protein was localized within both the cytoplasm and the nucleoplasm and also within the nucleoli of t(2;5)-translocation-positive lymphoma cells (8). However, whereas the NPM sequence is essential for the transforming activity (9), the nuclear localization, occurring via the shuttling activity of NPM (10...

Elevated serum pleiotrophin levels can indicate the presence of tumors expressing this factor. Monitoring serum levels of pleiotrophin may prove useful in determining the pharmacologic efficacy of cytotoxic or anti-pleiotrophin therapy.

Abstract-Cell-based therapy is a promising approach designed to enhance neovascularization and function of ischemic tissues. Interaction between endothelial and smooth muscle cells regulates vessels development and remodeling and is required for the formation of a mature and functional vascular network. Therefore, we assessed whether coadministration of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs) can increase the efficiency of cell therapy. Unilateral hindlimb ischemia was surgically induced in athymic nude mice treated with or without intravenous injection of EPCs (0.5ϫ10 6 ), SMPCs (0.5ϫ10 6 ) and EPCsϩSMPCs (0.25ϫ10 6 ϩ0.25ϫ10 6 ). Vessel density and foot perfusion were increased in mice treated with EPCsϩSMPCs compared to animals receiving EPCs alone or SMPCs alone (PϽ0.001). In addition, capillary and arteriolar densities were enhanced in EPCϩSMPC-treated mice compared to SMPC and EPC groups (PϽ0.01). We next examined the role of Ang-1/Tie2 signaling in the beneficial effect of EPC and SMPC coadministration. Small interfering RNA directed against Ang-1-producing SMPCs or Tie2-expressing EPCs blocked vascular network formation in Matrigel coculture assays, reduced the rate of incorporated EPCs within vascular structure, and abrogated the efficiency of cell therapy. Production of Ang-1 by SMPCs activates Tie2-expressing EPCs, resulting in increase of EPC survival and formation of a stable vascular network. Subsequently, the efficiency of EPC-and SMPC-based cotherapy is markedly increased. Therefore, coadministration of different types of vascular progenitor cells may constitute a novel therapeutic strategy for improving the treatment of ischemic diseases.

Pleiotrophin (PTN) is a developmentally regulated protein which exhibits neurite-outgrowth, mitogenic, and angiogenic properties. It has also been shown to be involved in tumor growth and metastasis. Here we used primary BEL (bovine epithelial lens) cells to investigate the signal transduction pathways involved in the mitogenic activity of recombinant PTN. PTN was purified from conditioned media of SW-13 cells transfected with the human PTN cDNA. We show that inhibitors of tyrosine kinase, mitogen-activated protein kinase, or phosphoinositide (PI) 3-kinase inhibit DNA synthesis stimulated by PTN. Analysis of tyrosine-phosphorylated proteins following PTN stimulation showed phosphorylation of two novel 190-and 215-kDa proteins in addition to SHC, ERK1, and ERK2. A mobility shift of phosphorylated ERK1 and ERK2 was detected with a panERK antibody confirming the phosphorylation of the two ERKs. Furthermore, in vitro immunocomplex kinase assay with Akt1, a natural substrate of PI 3-kinase, showed an activation of the kinase following PTN stimulation and a reversal by the PI 3-kinase inhibitor wortmannin. We conclude that the mitogenic activity of PTN is dependent on tyrosine kinase activation and utilizes the mitogen-activated protein kinase and the PI 3-kinase pathways to transduce a mitogenic signal. Pleiotrophin (PTN)1 (1) is an 18-kDa secreted protein belonging to a new family of heparin-binding factors which includes midkine (2) and the chicken retinoic-inducible heparin-binding protein (3). Both, PTN and midkine are developmentally regulated proteins which exibit neurite outgrowth, angiogenic and mitogenic properties (reviewed in Ref. 4), and which enhance plasminogen activator activity (5). PTN and midkine share 50% of sequence homology, but they have no homology with the fibroblast growth factor family of heparin-binding growth factors which display several overlapping biological properties (reviewed in Ref. 6).The mitogenic activity of PTN is a subject of controversy as conflicting results were reported by different laboratories. On one hand, PTN purified from tissues or expressed in bacteria and in insect cells is devoid of mitogenic activity but still promotes neurite outgrowth (7-9); however, Courty et al. (10) reported mitogenic activity after purification of PTN from bovine brain. On the other hand when expressed in eukaryotic cells, PTN is capable of stimulating thymidine incorporation and cell growth in different cell systems including fibroblasts (11), epithelial cells (11,12), and endothelial cells (11, 13). The failure of purifying mitogenically active PTN in the former systems listed above may be explained by either loss of mitogenic activity during the purification or erroneous folding of the protein when highly expressed. This latter explanation would be in agreement with the findings of Laaroubi et al. (13) who demonstrated that PTN expressed in NIH3T3 cells is produced in two forms distinct in their structure and properties. These two forms can be separated in two peaks on a Mono S column;...

Pleiotrophin (PTN) is a developmentally regulated protein that has been shown to be involved in tumor growth and metastasis presumably by activating tumor angiogenesis. To clarify the potential angiogenic activity of PTN and to analyze the signaling pathways involved in this process, we used an in vitro model of Human Umbilical Vein Endothelial Cells (HUVEC). We show that PTN was mitogenic toward a variety of endothelial cells including HUVEC, stimulated HUVEC migration across a reconstituted basement membrane and induced the formation of capillary-like structures by HUVEC grown as 3D-cultures in Matrigel or collagen. The signaling pathways triggered following endothelial cell stimulation by PTN were studied by using pharmacological inhibitors of the Phosphoinositide-3 kinase (PI3K) and endothelial Nitric Oxide Synthase (eNOS), two enzymes that have been shown to be crucial in the angiogenic response to Vascular Endothelial Growth Factor (VEGF). Whereas wortmannin (a PI3K inhibitor) and L-NAME (an eNOS inhibitor) dramatically reduced HUVEC growth induced by VEGF, only the former inhibitor reduced the growth induced by PTN and to a lesser extent that stimulated by basic Fibroblast Growth Factor. Thus, our results indicate that PTN induces angiogenesis and utilizes PI3K- but not eNOS-dependent pathways for its angiogenic activity.

Pleiotrophin is a heparin-binding growth factor involved in the differentiation and proliferation of neuronal tissue during embryogenesis, and also secreted by melanoma and breast carcinoma cells. Pleiotrophin exhibits mitogenic and angiogenic properties and has been shown to influence the vascular supply, expansion and metastasis of tumour cells. Our aim was to study the serum and plasma concentrations of pleiotrophin and the classical angiogenic growth factor vascular endothelial growth factor. Using a specific ELISA-test we studied patients with small cell lung cancer (n=63), and patients with non-small cell lung cancer (n=22) in comparison to healthy control subjects (n=41). In most of the lung cancer patients (81%), we found serum levels of pleiotrophin above those of control subjects (P50.001). Of the 63 small cell lung cancer patients in the study pleiotrophin serum levels were elevated in 55 cases (87%) and in 14 cases (63%) of the 22 non-small cell lung cancer patients. Pleiotrophin mean serum concentrations were 10.8-fold higher in the tumour patient group as compared to the control group (P50.001). Furthermore, pleiotrophin serum levels correlated positively with the stage of disease and inversely with the response to therapy. Plasma vascular endothelial growth factor concentrations were elevated in only in 28.6% of small cell lung cancer and 45.5% of non-small cell lung cancer patients by an average of 2.3-fold. Quite strikingly, there was no apparent correlation between the plasma vascular endothelial growth factor concentration and the stage of disease. Our study suggests that pleiotrophin may be an early indicator of lung cancer and might be of use in monitoring the efficacy of therapy, which needs to be confirmed by larger studies.