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Activating mutations of anaplastic lymphoma kinase ( ALK ) have been identified as important players in neuroblastoma development. Our goal was to evaluate the significance of overall ALK activation in neuroblastoma. Expression of phosphorylated ALK, ALK, and its putative ligands, pleiotrophin and midkine, was screened in 289 neuroblastomas and 56 paired normal tissues. ALK was expressed in 99% of tumors and phosphorylated in 48% of cases. Pleiotrophin and midkine were expressed in 58% and 79% of tumors, respectively. ALK activation was significantly higher in tumors than in paired normal tissues, together with ALK and midkine expression. ALK activation was largely independent of mutations and correlated with midkine expression in tumors. ALK activation in tumors was associated with favorable features, including a younger age at diagnosis, hyperdiploidy, and detection by mass screening. Antitumor activity of the ALK inhibitor TAE684 was evaluated in wild-type or mutated ALK neuroblastoma cell lines and xenografts. TAE684 was cytotoxic in vitro in all cell lines, especially those harboring an ALK mutation. TAE684 efficiently inhibited ALK phosphorylation in vivo in both F1174I and R1275Q xenografts but demonstrated antitumor activity only against the R1275Q xenograft. In conclusion, ALK activation occurs frequently during neuroblastoma oncogenesis, mainly through mutation-independent mechanisms. However, ALK activation is not associated with a poor outcome and is not always a driver of cell proliferation and/or survival in neuroblastoma.

Contains fulltext : 108052.pdf (Publisher’s version ) (Open Access) Successful use of stem cell-based therapeutic products is conditioned by transplantation of optimized cells in permissive microenvironment. Mesenchymal stem cell (MSC) fates are tightly regulated by humoral factors, cellular interactions and extracellular matrix (ECM) components, such as glycosaminoglycans (GAG), which are complex polysaccharides with structural heterogeneity. During osteogenesis, a temporally controlled expression of particular GAG species is required to interact with specific growth promoting and differentiating factors to regulate their biological activities. As a comparative tool to study natural GAG, we used structurally and functionally related synthetic GAG mimetics. One of these compounds [OTR(4120)] was previously shown to stimulate bone repair in rat models. Here, we demonstrate that structurally distinct GAG mimetics stimulate differentially clonogenicity, proliferation, migration and osteogenic phenotype of MSC in vitro, according to their specific chemical signature, underlying the role of sulfate and acetyl groups in specific interactions with heparin binding factors (HBF). These effects are dependent on FGF-2 interactions since they are inhibited by a FGF receptor 1 signaling pathway blocker. These data suggest that the in vivo [OTR(4120)] bone regenerative effect could be due to its ability to induce MSC migration and osteogenic differentiation. To conclude, we provide evidences showing that GAG mimetics may have great interest for bone regeneration therapy and represent an alternative to exogenous growth factor treatments to optimize potential therapeutic properties of MSC. 01 maart 2012

Item does not contain fulltext Supraspinatus tendon overuse injuries lead to significant pain and disability in athletes and workers. Despite the prevalence and high social cost of these injuries, the early pathological events are not well known. We analyzed the potential relation between glycosaminoglycan (GAG) composition and phenotypic cellular alteration using a rat model of rotator cuff overuse. Total sulfated GAGs increased after 4 weeks of overuse and remained elevated up to 16 weeks. GAG accumulation was preceded by up-regulation of decorin, versican, and aggrecan proteoglycans (PGs) mRNAs and proteins and biglycan PG mRNA after 2 weeks. At 2 weeks, collagen 1 transcript decreased whereas mRNAs for collagen 2, collagen 3, collagen 6, and the transcription factor Sox9 were increased. Protein levels of heparin affine regulatory peptide (HARP)/pleiotrophin, a cytokine known to regulate developmental chondrocyte formation, were enhanced especially at 4 weeks, without up-regulation of HARP/pleiotrophin mRNA. Further results suggest that the increased GAGs present in early lesions may sequester HARP/pleiotrophin, which could contribute to a loss of tenocyte's phenotype. All these modifications are characteristic of a shift towards the chondrocyte phenotype. Identification of these early changes in the extra-cellular matrix may help to prevent the progression of the pathology to more disabling, degenerative alterations. 01 januari 2012

Recent studies have implicated the involvement of cell surface forms of nucleolin in tumor growth. In this study, we investigated whether a synthetic ligand of cell-surface nucleolin known as N6L could exert antitumor activity. We found that N6L inhibits the anchorage-dependent and independent growth of tumor cell lines and that it also hampers angiogenesis. Additionally, we found that N6L is a proapoptotic molecule that increases Annexin V staining and caspase-3/7 activity in vitro and DNA fragmentation in vivo. Through affinity isolation experiments and mass-spectrometry analysis, we also identified nucleophosmin as a new N6L target. Notably, in mouse xenograft models, N6L administration inhibited human tumor growth. Biodistribution studies carried out in tumor-bearing mice indicated that following administration N6L rapidly localizes to tumor tissue, consistent with its observed antitumor effects. Our findings define N6L as a novel anticancer drug candidate warranting further investigation. Cancer Res; 71(9); 3296–305. ©2011 AACR.

Pleiotrophin (PTN) is a heparin-binding growth factor that plays a significant role in tumor growth and angiogenesis. We have previously shown that in order for PTN to induce migration of endothelial cells, binding to both ανβ3 integrin and its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is required. In the present study we show that a synthetic peptide corresponding to the last 25 amino acids of the C-terminal region of PTN (PTN112–136) inhibited angiogenesis in the in vivo chicken embryo chorioallantoic membrane (CAM) assay and PTN-induced migration and tube formation of human endothelial cells in vitro. PTN112–136 inhibited binding of PTN to ανβ3 integrin, and as shown by surface plasmon resonance (SPR) measurements, specifically interacted with the specificity loop of the extracellular domain of β3. Moreover, it abolished PTN-induced FAK Y397 phosphorylation, similarly to the effect of a neutralizing ανβ3-selective antibody. PTN112–136 did not affect binding of PTN to RPTPβ/ζ in endothelial cells and induced β3 Y773 phosphorylation and ERK1/2 activation to a similar extent with PTN. This effect was inhibited by down-regulation of RPTPβ/ζ by siRNA or by c-src inhibition, suggesting that PTN112–136 may interact with RPTPβ/ζ. NMR spectroscopy studies showed that PTN112–136 was characterized by conformational flexibility and absence of any element of secondary structure at room temperature, although the biologically active peptide segment 123–132 may adopt a defined structure at lower temperature. Collectively, our data suggest that although PTN112–136 induces some of the signaling pathways triggered by PTN, it inhibits PTN-induced angiogenic activities through inhibition of PTN binding to ανβ3 integrin. J. Cell. Biochem. 112: 1532–1543, 2011. © 2011 Wiley-Liss, Inc.

There is considerable evidence showing that the neurodegenerative processes that lead to sporadic Parkinson's disease (PD) begin many years before the appearance of the characteristic motor symptoms and that impairments in olfactory, cognitive and motor functions are associated with time-dependent disruption of dopaminergic neurotransmission in different brain areas. Midkine is a 13-kDa retinoic acid-induced heparin-binding growth factor involved in many biological processes in the central nervous system such as cell migration, neurogenesis and tissue repair. The abnormal midkine expression may be associated with neurochemical dysfunction in the dopaminergic system and cognitive impairments in rodents. Here, we employed adult midkine knockout mice (Mdk(-/-)) to further investigate the relevance of midkine in dopaminergic neurotransmission and in olfactory, cognitive and motor functions. Mdk(/-) mice displayed pronounced impairments in their olfactory discrimination ability and short-term social recognition memory with no gross motor alterations. Moreover, the genetic deletion of midkine decreased the expression of the enzyme tyrosine hydroxylase in the substantia nigra reducing partially the levels of dopamine and its metabolites in the olfactory bulb and striatum of mice. These findings indicate that the genetic deletion of midkine causes a partial loss of dopaminergic neurons and depletion of dopamine, resulting in olfactory and memory deficits with no major motor impairments. Therefore, Mdk(-/-) mice may represent a promising animal model for the study of the early stages of PD and for testing new therapeutic strategies to restore sensorial and cognitive processes in PD.

The aim of this study was to investigate the development of an oral dextran derivative OTR4120. Pharmacokinetics (PK) parameters of OTR4120 were determined after treatment with intravenous injection (i.v.) at dose 5 mg/kg, intraperitoneal injection (i.p.) at dose 50 mg/kg or oral administration at dose 70 mg/kg. To study distribution at dose 70 mg/kg after oral administration, OTR4120 was given by gavage to mice. In ex vivo experiments, SDS-PAGE showed that plasma of mice treated orally at dose 70 mg/kg induced the formation of covalently linked complexes between antithrombin III and thrombin. OTR4120 were absorbed and metabolized following oral administration. OTR4120 given i.v., i.p. and oral had relatively small volume of distribution 0.95 L/kg and 4.68 L/kg respectively, plasma clearance was 45, 520 and 514 ml/h per kg after i.v., i.p. or oral administration respectively. Short elimination half-life was 80 min after i.p. administration and 383 min after oral administration. OTR4120 was distributed in the spleen and kidney and accumulated there over a long period, whereas the OTR4120 levels in liver were negligible a 24 hours after oral administration. Food do not change the oral bioavailability of OTR4120 in mice, AUC, C(max) and T(max) of OTR4120 were not significantly different when mice received the oral dose with food compared with under fasting conditions. This work presents another therapeutic agents administration way using dextran delivery system.

Contraction and energy metabolism are functions of skeletal muscles co-regulated by still largely unknown signals. To help elucidating these interconnecting pathways, we are developing new cellular models that will allow to control the switch from a neonatal to an adult slow-oxidative or fast-glycolytic phenotype of myofibers, during in vitro differentiation. Thus, our purpose was to direct the differentiation of the newly characterized WTt clone, from a mixed towards either fast or slow phenotype, by modifying amounts of two transcription factors respectively involved in control of glycolytic and oxidative energy metabolism, namely HIF-1alpha and PPARdelta. Our data support the idea that HIF-1alpha protein stabilization would favor expression of fast phenotypic markers, accompanied or not by a decreased expression of slow markers, depending on treatment conditions. Conversely, PPARdelta over-expression appears to enhance the slow-oxidative phenotype of WTt myotubes. Furthermore, we have observed that expression of PGC-1alpha, a coregulator of PPAR, is also modified in this cell line upon conditions that stabilize HIF-1alpha protein. This observation points to the existence of a regulatory link between pathways controlled by the two transcription factors HIF-1alpha and PPARdelta. Therefore, these cells should be useful to analyze the balance between oxidative and glycolytic energy production as a function of phenotypic transitions occurring during myogenic maturation. The newly characterized murine WTt clone will be a good tool to investigate molecular mechanisms implicating HIF-1alpha and PPARdelta in the coordinated metabolic and contractile regulations involved in myogenesis.

Pleiotrophin (PTN) is a polypeptide that belongs to a family of heparin-binding growth factors; it displays mitogenic activity for a wide variety of cells. In a previous study, we reported that PTN induces the stimulation of expression of inflammatory cytokines, including tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta and IL-6, in quiescent human peripheral blood mononuclear cells (PBMCs) through B-lymphocyte binding. These results emphasize the importance of PTN in the regulation of inflammatory processes. Moreover, using in vitro infection of PBMCs or using PBMCs from AIDS patients, we showed that PTN was sufficient to induce human immunodeficiency virus type 1 (HIV-1) replication. Moreover, neutralization of TNF-alpha, IL-1beta and IL-6 suppressed HIV replication in PTN-stimulated PBMCs. As these cytokines are potent upregulators of virus expression, these results should prove useful in investigating the role of PTN as a host factor in the regulation of pathological disorders in HIV-1 infection. Identification of this host factor could be important for understanding HIV disease and designating therapeutic approaches.

Heparan sulfate proteoglycans are key regulators of complex molecular networks due to the interaction of their sugar chains with a large number of partner proteins, which in humans number more than 200 (Ori, A., Wilkinson, M. C., and Fernig, D. G. (2008) The heparanome and regulation of cell function: structures, functions and challenges. Front. Biosci. 13, 4309–4338). We developed a method to selectively label residues involved in heparin binding that matches the requirements for medium/high throughput applications called the “Protect and Label” strategy. This is based on the protection against chemical modification given by heparin/heparan sulfate to the residues located in the heparin-binding site. Thus, analysis of fibroblast growth factor-2 bound to heparin and incubated with N-hydroxysuccinimide acetate showed that lysines involved in the sugar binding are protected against chemical modification. Moreover following release from heparin, the protected lysine side chains may be specifically labeled with N-hydroxysuccinimide biotin. After protein digestion, the biotinylated peptides were readily isolated and identified by MALDI-Q-TOF mass spectrometry. The analysis of labeled peptides obtained from three well characterized heparin-binding proteins with very different heparin-binding sites, fibroblast growth factor-2, platelet factor-4, and pleiotrophin demonstrates the success of this new approach, which thus provides a rapid and reliable procedure to identify heparin-binding sites.

International audience; We have recently reported that the CXC-chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 induces proliferation, migration, and invasion of the Huh7 human hepatoma cells through its G-protein-coupled receptor CXCR4 and that glycosaminoglycans (GAGs) are involved in these events. Here, we demonstrate by surface plasmon resonance that the chemokine binds to GAG mimetics obtained by grafting carboxylate, sulfate or acetate groups onto a dextran backbone. We also demonstrate that chemically modified dextrans inhibit SDF-1/CXCL12-mediated in vitro chemotaxis and anchorage-independent cell growth in a dose-dependent manner. The binding of GAG mimetics to the chemokine and their effects in modulating the SDF-1/CXCL12 biological activities are mainly related to the presence of sulfate groups. Furthermore, the mRNA expression of enzymes involved in heparan sulfate biosynthesis, such as exostosin-1 and -2 or N-deacetylase N-sulfotransferases remained unchanged, but heparanase mRNA and protein expressions in Huh7 cells were decreased upon GAG mimetic treatment. Moreover, decreasing heparanase-1 mRNA levels by RNA interference significantly reduced SDF-1/CXCL12-induced extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation. Therefore, we suggest that GAG mimetic effects on SDF-1/CXCL12-mediated hepatoma cell chemotaxis may rely on decreased heparanase expression, which impairs SDF-1/CXCL12's signaling. Altogether, these data suggest that GAG mimetics may compete with cellular heparan sulfate chains for the binding to SDF-1/CXCL12 and may affect heparanase expression, leading to reduced SDF-1/CXCL12 mediated in vitro chemotaxis and growth of hepatoma cells.

Functionalized iron oxide magnetic nanoparticles (MNP) are an innovative tool for cancer detection and treatment. In this study, a cell-surface nucleolin antagonist, N6L, was used as targeting ligand for MNP. This N6L, which exhibits antitumor activities, specifically targets bind tumor cells by binding to cell-surface nucleolin and glycosaminoglycans. N6L was covalently conjugated to dimercaptosuccinic acid coated magnetic nanoparticles (MNP-N6L). Using immunoprecipitation, gene invalidation and enzymatic degradation of glycosaminoglycans, we showed that MNP-N6L targets human breast cancer MDA-MB 231 cells through interaction with nucleolin and sulfated glycosaminoglycans. In vivo biodistribution studies were carried out in MDA-MB 231 tumor-bearing mice, using iron detection assay by spectrometric analysis and Prussian blue staining. Whereas both non-functionalized and functionalized nanoparticles were found in liver and spleen, only MNP-N6L was found in the tumor. Our findings indicate that MNP-N6L is a promising targeting system for theranostic applications in cancer detection and treatment.

Introduction Tumor cells can effectively be killed by heat, e.g. by using magnetic hyperthermia. The main challenge in the field, however, is the generation of therapeutic temperatures selectively in the whole tumor region. We aimed to improve magnetic hyperthermia of breast cancer by using innovative nanoparticles which display a high heating potential and are functionalized with a cell internalization and a chemotherapeutic agent to increase cell death. Methods The superparamagnetic iron oxide nanoparticles (MF66) were electrostatically functionalized with either Nucant multivalent pseudopeptide (N6L; MF66-N6L), doxorubicin (DOX; MF66-DOX) or both (MF66-N6LDOX). Their cytotoxic potential was assessed in a breast adenocarcinoma cell line MDA-MB-231. Therapeutic efficacy was analyzed on subcutaneous MDA-MB-231 tumor bearing female athymic nude mice. Results All nanoparticle variants showed an excellent heating potential around 500 W/g Fe in the alternating magnetic field (AMF, conditions: H = 15.4 kA/m, f = 435 kHz). We could show a gradual inter- and intracellular release of the ligands, and nanoparticle uptake in cells was increased by the N6L functionalization. MF66-DOX and MF66-N6LDOX in combination with hyperthermia were more cytotoxic to breast cancer cells than the respective free ligands. We observed a substantial tumor growth inhibition (to 40% of the initial tumor volume, complete tumor regression in many cases) after intratumoral injection of the nanoparticles in vivo. The proliferative activity of the remaining tumor tissue was distinctly reduced. Conclusion The therapeutic effects of breast cancer magnetic hyperthermia could be strongly enhanced by the combination of MF66 functionalized with N6L and DOX and magnetic hyperthermia. Our approach combines two ways of tumor cell killing (magnetic hyperthermia and chemotherapy) and represents a straightforward strategy for translation into the clinical practice when injecting nanoparticles intratumorally. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0576-1) contains supplementary material, which is available to authorized users.

Conventional cancer chemotherapies are often limited by their non-targeted nature and their inadequate delivery to the tumor affecting the normal tissues and leading to toxic adverse effects. In order to improve the anticancer efficacy and safety of these drugs, as well as the diagnosis capacity of imaging agents, nanoparticles drug delivery system has been developed combining ligands enabling tumor targeting at a cellular level and drug carrier capacity. Nucleolin (NCL) is a multifunctional protein that could shuttle from nucleolus to nucleoplasm, cytoplasm and cell surface. This ribonucleo protein over expressed at the cell surface of cancer cells is involved in many cancer processes supporting tumorigenesis such as cell proliferation and apoptosis. Additionally, NCL expression is enhanced in angiogenic vessels, enabling multi-targeting strategies toward the tumor microenvironment. In this context, several compounds targeting NCL, such as the aptamer AS1411, the peptide F3 and the multivalent pseudopeptide N6L, have been developed and investigated for cancer therapy. Due to their cancer cell targeting capacities, these compounds have been evaluated to mediate highly specific and effective nanoparticles for drug delivery to the tumor. In this report, we present a review of literature focusing on drug-loaded nanoparticles conjugated with these nucleolin ligands, strikingly emphasizing the success of such a strategy.

Pleiotrophin (PTN) is a heparin-binding growth factor involved in nerve regeneration after peripheral nerve injury. After crush injury, PTN is found in distal nerve segments in several non-neural cell types, including Schwann cells, macrophages, and endothelial cells, but not in axons. To further clarify the role for PTN in nerve regeneration, we investigated the effects of PTN applied to lesioned peripheral nerve in vivo. PTN in a dose of 1 mg/kg impaired muscle reinnervation. Thus, gastrocnemius muscle failed to recover its contractile properties as assessed by in situ maximal isometric tetanic force. PTN also decreased non-neural cell densities and delayed macrophage recruitment in the distal crushed nerve. These results are discussed in the light of recent evidence that PTN is a multifunctional polypeptide.

In a previous study, we showed that in situ injection of glycosaminoglycan mimetics called RGTAs (ReGeneraTing Agents) enhanced neovascularization after skeletal muscular ischemia (Desgranges, P., Barbaud, C., Caruelle, J. P., Barritault, D., and Gautron, J. (1999) FASEB J. 13, 761-766). In the present study, we showed that the RGTA OTR4120 modulated angiogenesis in the chicken embryo chorioallantoic membrane assay, in a dose-dependent manner. We therefore investigated the effect of OTR4120 on one of the most specific angiogenesis-regulating heparin-binding growth factors, vascular endothelial growth factor 165 (VEGF165). OTR4120 showed high affinity binding to VEGF165 (Kd = 2.2 nm), as compared with heparin (Kd = 15 nm), and potentiated the affinity of VEGF165 for VEGF receptor-1 and -2 and for neuropilin-1. In vitro, OTR4120 potentiated VEGF165-induced proliferation and migration of human umbilical vein endothelial cells. In the in vivo Matrigel plug angiogenesis assay, OTR4120 in a concentration as low as 3 ng/ml caused a 6-fold increase in VEGF165-induced angiogenesis. Immunohistochemical staining showed a larger number of well differentiated VEGFR-2-expressing-cells in Matrigel sections of OTR4120-treated plug than in control sections. These findings indicate that OTR4120 enhances the VEGF165-induced angiogenesis and therefore may hold promise for treating disorders characterized by deficient angiogenesis.

To better understand the particular vulnerability of mesencephalic dopaminergic neurons to toxins or gene mutations causing parkinsonism, we have taken advantage of a primary cell culture system in which these neurons die selectively. Antimitotic agents, such as cytosine arabinoside or cAMP, prevent the death of the neurons by arresting astrocyte proliferation. To identify factors implicated in either the death of the dopaminergic neurons or in the neuroprotective effect of cAMP, we constructed cDNA libraries enriched by subtractive hybridization and suppressive PCR in transcripts that are preferentially expressed in either control or cAMP-treated cultures. Differentially expressed transcripts were identified by hybridization of the enriched cDNAs with a commercially available cDNA expression array. The proteoglycan receptors syndecan-3 and the receptor protein tyrosine phosphatase zeta/beta were found among the transcripts preferentially expressed under control conditions, and their ligand, the cytokine pleiotrophin, was highly represented in the cDNA libraries for both conditions. Since pleiotrophin is expressed during embryonic and perinatal neural development and following lesions in the adult brain, we investigated its role in our cell culture model. Pleiotrophin was not responsible for the death of dopaminergic neurons under control conditions, or for their survival in cAMP-treated cultures. It was, however, implicated in the initial and cAMP-dependent enhancement of the differentiation of the dopaminergic neurons in our cultures. In addition, our experiments have provided evidence for a cAMP-dependent regulatory pathway leading to protease activation, and the identification of pleiotrophin as a target of this pathway.

Heparin affin regulatory peptide (HARP) is a growth factor displaying high affinity for heparin. It is present in the extracellular matrix of many tissues, interacting with heparan sulfate and dermatan/chondroitin sulfate glycosaminoglycans. We have previously shown that HARP is implicated in the control of angiogenesis and its effects are mimicked, at least in part, by synthetic peptides that correspond to its N and C termini. In the present work, we show that HARP is cleaved by plasmin, leading to the production of five peptides that correspond to distinct domains of the molecule. Heparin, heparan sulfate and dermatan sulfate, at various HARP to glycosaminoglycan ratios, partially protect HARP from plasmin degradation. The molecules with higher affinity to HARP are the more protective, heparin being the most efficient. The peptides that are produced from cleavage of HARP by plasmin, affect in vivo and in vitro angiogenesis and modulate the angiogenic activity of vascular endothelial growth factor on human umbilical vein endothelial cells. Similar results were obtained in vitro with recombinant HARP peptides, identical to the peptides generated after treatment of HARP with plasmin. These results suggest that different regions of HARP may induce or inhibit angiogenesis.

Heparin affin regulatory peptide (HARP) is an heparin-binding molecule involved in the regulation of cell proliferation and differentiation. Here, we report that HARP inhibited the biological activity induced by the 165-amino-acid form of vascular endothelial growth factor (VEGF165) on human umbilical vein endothelial cells. Endothelial-cell proliferation induced by VEGF165 showed about 50% inhibition in the presence of HARP in a concentration of 3 nM. In similar range of concentrations, HARP blocked tube formation induced by VEGF165 in three-dimensional angiogenesis assay. In vivo studies showed that HARP inhibited the VEGF165-induced Matrigel trade mark infiltration of endothelial cells. We then investigated the mechanisms of this inhibition and shown that HARP inhibited the binding of 125I-VEGF165 to the VEGF receptors of endothelial cells. Additional studies using VEGF soluble receptors indicated that binding of 125I-VEGF165 to kinase insert domain-containing receptor and neuropilin receptor was inhibited by HARP, but conversely the binding of 125I-VEGF165 to fms-like tyrosine kinase I receptor was unaffected. A competitive affinity-binding assay demonstrated that HARP interacted directly with VEGF165 with a dissociation coefficient of 1.38 nM. Binding assay using deletion mutants of HARP revealed that the thrombospondin type-1 repeats domains were involved in this interaction. These data demonstrate for the first time that the angiogenic factor HARP can also negatively regulates the angiogenic activity of VEGF165.

Heparin affin regulatory peptide (HARP) and midkine (MK) are growth factors, expressed in carcinomas, neuroblastomas and gliomas. In this study, we measured the levels of HARP and MK in plasma samples from 77 cancer patients. The patients had advanced tumors with loco-regional (n=18) or metastatic (n=49) diseases and 10 patients have their diseases limited to the primary site. HARP and MK plasma concentrations were significantly higher in all of these different subgroups of cancer patients (P

Heparin affin regulatory peptide (HARP) is a heparin binding growth factor that belongs to a family of molecule whose biological function in myogenesis has been suspected without formal demonstration. In the present study, we investigated the expression and the distribution of HARP and its mRNA during soleus muscle regeneration using a crushed-induced regeneration model and also during differentiation of muscle satellite cells in primary cultures. We show that HARP mRNA and protein expression are increased during the regeneration process with a peak at day 5 after muscle crushing when new myotubes are formed. In situ hybridization and immunohistochemical studies showed that activated myoblasts expressed HARP at day two after crushing. Five days after muscle lesion, HARP is localised in newly formed myotubes as well as in prefused activated myoblasts. In regenerated myofibers, 15 days after crushing, expression of HARP was reduced. In vitro experiments using primary cultures of rat satellite cells indicated that HARP expression level increased during the differentiation process and peaked on fusion of myoblasts into myotubes. This is the first study demonstrating the presence of HARP in fusing myogenic cells suggests that this growth factor could play a function in myogenic differentiation.

Background Receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is a chondroitin sulphate (CS) transmembrane protein tyrosine phosphatase and is a receptor for pleiotrophin (PTN). RPTPβ/ζ interacts with ανβ3 on the cell surface and upon binding of PTN leads to c-Src dephosphorylation at Tyr530, β3 Tyr773 phosphorylation, cell surface nucleolin (NCL) localization and stimulation of cell migration. c-Src-mediated β3 Tyr773 phosphorylation is also observed after vascular endothelial growth factor 165 (VEGF165) stimulation of endothelial cells and is essential for VEGF receptor type 2 (VEGFR2) - ανβ3 integrin association and subsequent signaling. In the present work, we studied whether RPTPβ/ζ mediates angiogenic actions of VEGF. Methods Human umbilical vein endothelial, human glioma U87MG and stably transfected Chinese hamster ovary cells expressing different β3 subunits were used. Protein-protein interactions were studied by a combination of immunoprecipitation/Western blot, immunofluorescence and proximity ligation assays, properly quantified as needed. RPTPβ/ζ expression was down-regulated using small interference RNA technology. Migration assays were performed in 24-well microchemotaxis chambers, using uncoated polycarbonate membranes with 8 μm pores. Results RPTPβ/ζ mediates VEGF165-induced c-Src-dependent β3 Tyr773 phosphorylation, which is required for VEGFR2-ανβ3 interaction and the downstream activation of phosphatidylinositol 3-kinase (PI3K) and cell surface NCL localization. RPTPβ/ζ directly interacts with VEGF165, and this interaction is not affected by bevacizumab, while it is interrupted by both CS-E and PTN. Down-regulation of RPTPβ/ζ by siRNA or administration of exogenous CS-E abolishes VEGF165-induced endothelial cell migration, while PTN inhibits the migratory effect of VEGF165 to the levels of its own effect. Conclusions These data identify RPTPβ/ζ as a cell membrane binding partner for VEGF that regulates angiogenic functions of endothelial cells and suggest that it warrants further validation as a potential target for development of additive or alternative anti-VEGF therapies. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0287-3) contains supplementary material, which is available to authorized users.

Angiogenesis is the physiological process involving the growth of new blood vessels from pre-existing vessels. In normal or pathological angiogenesis, angiogenic growth factors activate cognate receptors on endothelial cells. Fibroblast growth factor-2 (FGF-2) and heparin affin regulatory peptide (HARP) are two heparin-binding growth factors and were described for their pro-angiogenic properties on human umbilical endothelial cells (HUVEC). We now show that HARP acts as a mediator of FGF-2's stimulatory effects, since it is able to inhibit the proliferation and migration of HUVEC induced by FGF-2. We demonstrate by ELISA and optical biosensor binding assay that HARP and FGF-2 interact through direct binding. We have adapted a previously developed structural proteomics method for the identification of residues involved in protein–protein interactions. Application of this method showed that two sequences in HARP were involved in binding FGF-2. One was in the C-thrombospondin type 1 repeat (C-TSR-1) domain and the other in the C-terminal domain of HARP. The identification of these regions as mediating the binding of FGF-2 was confirmed by ELISA using synthetic peptides, which are as well mediators of FGF-2-induced proliferation, migration and tubes formation on HUVEC in vitro . These results imply that besides a regulation of the proliferation, migration and angiogenesis of HUVEC by direct interaction of FGF-2 with its receptors, an alternative pathway exists involving its binding to growth factors such as HARP.

Pleiotrophin (PTN) is a growth factor present in the extracellular matrix of the growth plate during bone development and in the callus during bone healing. Bone healing is a complicated process that recapitulates endochondral bone development and involves many cell types. Among those cells, mesenchymal stromal cells (MSC) are able to differentiate toward chondrogenic and osteoblastic lineages. We aimed to determine PTN effects on differentiation properties of human bone marrow stromal cells (hBMSC) under chondrogenic induction using histological analysis and quantitative reverse transcription polymerase chain reaction. PTN dramatically potentiated chondrogenic differentiation as indicated by a strong increase of collagen 2 protein, and cartilage-related gene expression. Moreover, PTN increased transcription of hypertrophic chondrocyte markers such as MMP13, collagen 10 and alkaline phosphatase and enhanced calcification and the content of collagen 10 protein. These effects are dependent on PTN receptors signaling and PI3 K pathway activation. These data suggest a new role of PTN in bone regeneration as an inducer of hypertrophy during chondrogenic differentiation of hBMSC.

International audience

Heparin affin regulatory peptide (HARP) is a 18-kDa heparin-binding polypeptide that is highly expressed in developing tissues and in several primary human tumors. It seems to play a key role in cellular growth and differentiation. In vitro, HARP displays mitogenic, angiogenic, and neurite outgrowth activities. It is a secreted protein that is organized in two beta-sheet domains, each domain containing a cluster of basic residues. To assess determinants involved in the biological activities of HARP, C-terminally truncated proteins were produced in Chinese hamster ovary-K1 cells and tested for their mitogenic, tumor formation in nude mice and neurite outgrowth activities. Our data clearly indicate that the residues 111-136 of the lysine-rich C-terminal domain are involved in the mitogenic and tumor formation activities of HARP. Correlatively, no signal transduction was detected using the corresponding mutant, suggesting the absence of HARP binding to its high affinity receptor. However, this C-terminal domain of HARP is not involved in the neurite outgrowth activity. We also demonstrate that HARP signal peptide cleavage could led to two maturated forms that are both but differentially mitogenic.

BACKGROUND Heparin affin regulatory peptide (HARP) composes, together with midkine (MK), a new family of heparin-binding growth/differentiation factors. Recently, HARP was incriminated in cancer progression, as an angiogenic factor and as a tumor growth factor. In this study, we analyzed the possible involvement of HARP in human prostate cancer (Pca). METHODS The localization of HARP protein and its mRNAs in normal prostate (n = 5), benign prostate hyperplasia (BPH) (n = 7), and prostate cancer (Pca) (n = 9) was analyzed by immunohistochemistry and in situ hybridization. The mitogenic activity of this growth factor for prostate epithelial cells was determined with a thymidine incorporation assay. HARP cDNA was transfected into normal prostate epithelial (PNT-1A) cells, and their growth was evaluated by soft-agar growth assay. RESULTS We found HARP protein associated with epithelial cells in PCa but not in normal prostate or BPH, while the corresponding mRNAs were located in the stromal compartment. Furthermore, HARP is mitogenic for PNT-1A, LNCaP, and DU-145 cells. Overexpression of the human HARP in PNT-1A transfected cells induced both anchorage-independent growth and growth at low serum concentrations. CONCLUSIONS Our results suggest that HARP may act in a paracrine manner from mesenchymal to tumoral epithelial cells, and may play a role in the molecular mechanisms that regulate prostate tumor cell growth. Prostate 38:126–136, 1999. © 1999 Wiley-Liss, Inc.

In human breast cancer MCF-7 and MCF-7ras cells, we demonstrated that whereas insulin had a mitogenic effect on both cell lines, fibroblast growth factor-2 (FGF-2) had opposite effects, stimulating MCF-7 and inhibiting MCF-7ras cell proliferation. The inhibitory signal induced by FGF-2 was related to sustained mitogen-activated protein kinase (MAPK) activation in MCF-7ras cells, while transient MAPK activation was associated with MCF-7 cell proliferation. FGF-2 was further used in combination with insulin or cAMP. In MCF-7 cells, insulin and cAMP reversed the mitogenic effect of FGF-2. In MCF-7ras cells, insulin did not modify the inhibitory effect of FGF-2, but cAMP markedly enhanced it. These effects were also associated with an increased level and duration of MAPK activation. PD98056 abolished the effect of FGF-2 on DNA synthesis in both cell lines, demonstrating that the dual effect of FGF-2 on cell proliferation is dependent on the activity of the extracellular-signal-regulated kinase 1 and 2 (ERK1/2) signalling pathway.

Tissue engineering approaches to stimulate bone formation currently combine bioactive scaffolds with osteocompetent human mesenchymal stem cells (hMSC). Moreover, osteogenic and angiogenic factors are required to promote differentiation and survival of hMSC through improved vascularization through the damaged extracellular matrix (ECM). Glycosaminoglycans (GAGs) are ECM compounds acting as modulators of heparin-binding protein activities during bone development and regenerative processes. GAG mimetics have been proposed as ECM stabilizers and were previously described for their positive effects on bone formation and angiogenesis after local treatment. Here, we developed a strategy associating the GAG mimetic [OTR4120] with bone substitutes to optimize stem cell-based therapeutic products. We showed that [OTR4120] was able to potentiate proliferation, migration, and osteogenic differentiation of hMSC in vitro. Its link to tricalcium phosphate/hydroxyapatite scaffolds improved their colonization by hMSC. Surprisingly, when these combinations were tested in an ectopic model of bone formation in immunodeficient mice, the GAG mimetics inhibit bone formation induced by hMSC and promoted an osteoclastic activity. Moreover, the inflammatory response was modulated, and the peri-implant vascularization stimulated. All together, these findings further support the ability of GAG mimetics to organize the local ECM to coordinate the host response toward the implanted biomaterial, and to inhibit the abnormal bone formation process on a subcutaneous ectopic site.

The multifunctional protein nucleolin (NCL) is overexpressed on the surface of activated endothelial and tumor cells and mediates the stimulatory actions of several angiogenic growth factors, such as pleiotrophin (PTN). Because α(v)β(3) integrin is also required for PTN-induced cell migration, the aim of the present work was to study the interplay between NCL and α(v)β(3) by using biochemical, immunofluorescence, and proximity ligation assays in cells with genetically altered expression of the studied molecules. Interestingly, cell surface NCL localization was detected only in cells expressing α(v)β(3) and depended on the phosphorylation of β(3) at Tyr(773) through receptor protein-tyrosine phosphatase β/ζ (RPTPβ/ζ) and c-Src activation. Downstream of α(v)β(3,) PI3K activity mediated this phenomenon and cell surface NCL was found to interact with both α(v)β(3) and RPTPβ/ζ. Positive correlation of cell surface NCL and α(v)β(3) expression was also observed in human glioblastoma tissue arrays, and inhibition of cell migration by cell surface NCL antagonists was observed only in cells expressing α(v)β(3). Collectively, these data suggest that both expression and β(3) integrin phosphorylation at Tyr(773) determine the cell surface localization of NCL downstream of the RPTPβ/ζ/c-Src signaling cascade and can be used as a biomarker for the use of cell surface NCL antagonists as anticancer agents.

HARP (heparin affin regultory peptide) is an 18 kDa heparin binding protein, also known as HB-GAM or pleiotrophin (PTN) which has been primarily isolated from brain and uterus, and displays neurite outgrowth, angiogenic and mitogenic activities. Previously, we have expressed the human cDNA encoding human HARP in NIH 3T3 cells. Purified recombinant HARP displayed mitogenic activity for endothelial cells. Its NH2-terminal sequence indicates that the HARP molecule possesses a three amino acid extension from the signal peptide more than the NH2-terminal described. For HB-GAM or PTN, these three amino acids may be essential for the stability and the mitogenic activity of this growth factor. In an attempt to further study the mode of action of this growth factor, we have investigated the mitogenic effect of HARP on various cell types. In contrast to FGF-2, HARP failed to induce stimulation of DNA synthesis on a CCL39 cell line. However, we found that in quiescent bovine epithelial lens (BEL) cells, the stimulation of DNA synthesis induced by HARP is dose-dependent (EC50: 2.5 ng/ml) and maximal stimulation is as potent as that induced by FGF-2 (EC50: 25 pg/ml). Interestingly, when BEL cells were allowed to quiesce in the presence of serum, the stimulation induced by HARP is considerably less potent. In this highly responsive cell system, heparin could potentiate the mitogenic activity of HARP at very low doses (0.1-1 mg/ml) and inhibit this activity at concentrations of 10 mg/ml. In contrast to its protective effect on FGF-1 and -2, heparin was unable to preserve HARP from tryptic and chymotryptic degradations. © 1995 Wiley-Liss, Inc.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor, mostly resistant to the standard treatments. NCL is overexpressed in cancers and its inhibition impairs tumor growth. Herein we described, that NCL was overexpressed in human specimens of PDAC, and low NCL staining patients had increased overall survival. Previously, we described a family of multivalent pseudopeptides binding to NCL and inhibiting tumour growth. Here, NCL antagonist N6L, strongly impaired tumor growth, liver metastasis formation and angiogenesis in an orthothopic mouse model of PDAC. N6L inhibited both human and mouse tumor cell proliferation and invasion. Proteome analysis of endothelial cell secreted proteins showed that NCL inhibition decreased Ang-2 levels and switched a pro-angiogenic signature. Importantly, Ang-2 levels were decreased in plasma of N6L-treated PDAC mice. The analysis of tumor vasculature revealed a strong increase of pericyte coverage and vessel perfusion in parallel to an inhibition of tumor hypoxia. As consequence of N6L-induced tumor vessel normalization, pre-treatment with N6L efficiently improved chemotherapeutic drug delivery and increased the anti-tumor properties of gemcitabine in PDAC mice. In conclusion, NCL inhibition is a new anti-tumor therapeutic strategy that dually blocks tumor progression and normalizes tumor vessels improving the delivery and efficacy of chemotherapeutic drugs in PDAC cancers. Moreover, we identified Ang-2 as a potential target and suitable response biomarker for N6L treatment in PDAC. Citation Format: Maud-Emmanuelle Gilles, Federica Maione, Mélissande Cossutta, Gilles Carpentier, Laure Caruana, Silvia Di Maria, Damien Destouches, Ksenya Shchors, Christopher Prochasson, Anne Couvelard, José Courty, Enrico Giraudo, Ilaria Cascone. NCL targeting impairs the progression of pancreatic ductal adenocarcinoma and promotes tumor vessel normalization through Ang-2 inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3366.

Recently, we have found that the skin secretions of the Amazonian tree frog Phyllomedusa bicolor contains molecules with antitumor and angiostatic activities and identified one of them as the antimicrobial peptide dermaseptin (Drs) B2. In the present study we further explored the in vitro and in vivo antitumor activity of this molecule and investigated its mechanism of action. We showed that Drs B2 inhibits the proliferation and colony formation of various human tumor cell types, and the proliferation and capillary formation of endothelial cells in vitro. Furthermore, Drs B2 inhibited tumor growth of the human prostate adenocarcinoma cell line PC3 in a xenograft model in vivo. Research on the mechanism of action of Drs B2 on tumor PC3 cells demonstrated a rapid increasing amount of cytosolic lactate dehydrogenase, no activation of caspase-3, and no changes in mitochondrial membrane potential. Confocal microscopy analysis revealed that Drs B2 can interact with the tumor cell surface, aggregate and penetrate the cells. These data together indicate that Drs B2 does not act by apoptosis but possibly by necrosis. In conclusion, Drs B2 could be considered as an interesting and promising pharmacological and therapeutic leader molecule for the treatment of cancer.

International audience; The discovery of new molecules with potential antitumor activity continues to be of great importance in cancer research. In this respect, natural antimicrobial peptides isolated from various animal species including humans and amphibians have been found to be of particular interest. Here, we report the presence of two anti-proliferative peptides active against cancer cells in the skin secretions of the South American tree frog, Phyllomedusa bicolor. The crude skin exudate was fractioned by size exclusion gel followed by reverse-phase HPLC chromatography. After these two purification steps, we identified two fractions that exhibited anti-proliferative activity. Sequence analysis indicated that this activity was due to two antimicrobial alpha-helical cationic peptides of the dermaseptin family (dermaseptins B2 and B3). This result was confirmed using synthetic dermaseptins. When tested in vitro, synthetic B2 and B3 dermaseptins inhibited the proliferation of the human prostatic adenocarcinoma PC-3 cell line by more than 90%, with an EC(50) of around 2-3 mu M. No effect was observed on the growth of the NIH-3T3 non-tumor mouse cell line with Drs B2, whereas a slight inhibiting effect was observed with Drs B3 at high dose. In addition, the two fractions obtained after size exclusion chromatography also inhibited PC-3 cell colony formation in soft agar. Interestingly, inhibition of the proliferation and differentiation of activated adult bovine aortic endothelial cells was observed in cells treated with these two fractions. Dermaseptins B2 and B3 could, therefore, represent interesting new pharmacological molecules with antitumor and angiostatic properties for the development of a new class of anticancer drugs.

Nucleolin (NCL) is a nucleolar protein regulating ribogenesis and cell cycle progression, and is overexpressed in tumor cells. Shuttling to the cell surface of tumor cells and tumor vessels NCL is a marker of tumor tissues and a target for cancer therapy. Recently, we developed a family of nucleolin antagonist pseudopeptides (NucANT). The N6L peptide, strongly inhibits human tumor growth by inducing apoptosis of tumor cells (1), and is currently in clinical trial for cancer. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human malignancies, and an explanation of the failure of treatments is that chemotherapies are poorly delivered to the tumor because of a deficient and pathologic vasculature. We investigated the possibility that N6L could dually target tumor cells and tumor vasculature and be a promising new option for the treatment of PDAC. NCL targeting by N6L inhibited pancreatic tumor cell proliferation and pancreatic tumor cell motility similarly to other tumor cell types (1). Concerning endothelial cells (EC), N6L inhibited EC proliferation, motility, adhesion and tubulogenesis. We screened proteins involved in vascular stability and showed that N6L treatment or NCL depletion regulated the secretion of Ang-2. This N6L effect was specific of NCL targeting because NCL depletion rescued the inhibition of Ang-2 secretion induced by N6L. We investigated the effect of N6L on the growth and metastasis of a PDAC orthotopic mouse model that was deficient in vasculature such as the human tumors and NCL was highly expressed in tumor ductal epithelial cells. We showed that N6L strongly inhibited the tumor growth by inhibiting NCL expression, tumor cell proliferation, and increasing tumor cell apoptosis. Coherently with the in vitro data, N6L decreased Ang-2 plasma concentration of PDAC mice. By characterizing the PDAC tumor vasculature after treatment, we demonstrated that N6L induced both vessel pruning and normalization of tumor vasculature by improving pericyte coverage and tumor blood vessel perfusion. Moreover, N6L-induced tumor vessel normalization was accompanied by an improved efficiency of chemotherapeutic drugs delivery to cancer tissues and an inhibition of liver metastasis incidence. We conclude that NCL inhibition by N6L normalizes pancreatic tumor vasculature and suggest Ang-2 as a target and a biomarker of N6L response. N6L-induced PDAC vessel normalization promotes drug delivery and contributes to decrease metastasis formation. 1. Destouches D, et al. (2011) A simple approach to cancer therapy afforded by multivalent pseudopeptides that target cell-surface nucleoproteins. Cancer Res 71(9):3296-3305. Citation Format: Maud-Emmanuelle Gilles, Federica Maione, Gilles Carpentier, damien Destouches, José Courty, Enrico Giraudo, Ilaria Cascone. Nucleolin antagonist peptide N6L, normalizes tumor vasculature by decreasing Ang-2 secretion and inhibits pancreatic ductal adenocarcinoma growth and metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A02.

We have previously described the purification of a heparin binding growth factor from adult bovine brain named heparin affin regulatory peptide (HARP), which was identical to an uterus derived growth factor named pleiotrophin and to a developmentally regulated neurite promoting factor named heparin-binding growth associated molecule. However, for yet unclear reasons, the mitogenic activity of this purified polypeptide following isolation from animal tissue extracts is a subject of controversy, due to conflicting and irreproducible data when produced by recombinant DNA technologies in E. coli or insect cells. The purified protein was inactive in mitogenic assays but the natural molecule was active in assay of neurite outgrowth. In order to clarify these conflicting results and to obtain a recombinant protein free from other contaminating heparin-binding growth factors, we have cloned human cDNA encoding human HARP, engineered its expression in NIH 3T3 cells and characterised the resulting recombinant polypeptide. Purified recombinant HARP displayed mitogenic activity for capillary endothelial cells with half-maximal stimulation at approximately 1 ng/ml (55 pM) and induced angiogenesis in an in vitro model. Interestingly, while the NH2 terminal sequence of tissue purified HARP was NH2-GKKEKPEKK, the NH2 terminal sequence of the biologically active recombinant protein was NH2-AEAGKKEKPEKK, corresponding to a three amino acid extended form.

Pleiotrophin, also known as heparin affin regulatory peptide (HARP), is a growth factor expressed in various tissues and cell lines. In this work, HARP was tested for its capacity to modulate the anticoagulant activity of heparin and heparan sulphate mimetics (OTR4120). We used both in vitro and in vivo assays. HARP was found to be differently effective for neutralization of the anticoagulant activity of the mimetic heparan sulphate (OTR4120) and heparin in purified system and human plasma. HARP was shown to compete with both antithrombin and thrombin for binding to heparin and to OTR4120, respectively. In the presence of OTR4120, the V(max) was constant and the calculated maximum velocity was 1.56 U/min; the thrombin Km value (0.011 nM) was affected by HARP concentrations. The Km (HARP) value was 0.085 nM, which is consistent with high affinity of HARP to OTR4120. Under the same conditions, initial velocity patterns for antithrombin-heparin were determined in the presence or in the absence of HARP. The antithrombin value Km (0.022 nM) was affected by HARP (0.077 nM). HARP exhibits efficacy equivalent to or greater than protamine. Interestingly, intraperitoneally administered HARP decreased the anticoagulant activity of heparin and of OTR4120 in mice. Taken together, these data provide the first evidence for a physiological role of HARP in the modulation of anticoagulant activity of heparin and heparin-like material.

We previously demonstrated, using the glioblastoma cell line U87MG as an experimental model, that the adenoviral mediated overexpression of the truncated protein HARPΔ111-136 inhibits the proliferation of these cells in vitro as well as tumor growth and angiogenesis in vivo. This study focused on identifying the underlying mechanisms for the observed antitumoral effect. The present study demonstrated that HARPΔ111-136 induced the ATF4/ATF3/CHOP cascade resulting in a strong expression of the proapoptotic protein CHOP, leading to tumor cell apoptosis as demonstrated by PARP cleavage and FACS analysis. siRNA-mediated CHOP gene silencing abolished Ad-HARPΔ111-136 induced apoptosis. Moreover, Ad-HARPΔ111-136 increased the expression of the death receptor DR5 and enhanced U87MG cells sensitivity in vitro to TRAIL a DR5 ligand with subsequent activation of caspase 8. Infection of U87MG cells with Ad-HARPΔ111-136 also enhanced radiation-induced apoptosis. In vivo, the combination of Ad-HARPΔ111-136 and radiation therapy resulted in a striking inhibition (92%) of the growth of U87MG xenografts, resulting from the potent effect on tumor angiogenesis and tumor cell apoptosis as determined by TUNEL analysis. Taken together, our results indicated that the inhibitor HARPΔ111-136 sensitized U87MG cells to apoptosis.

Background Pleiotrophin, also known as HARP (Heparin Affin Regulatory Peptide) is a growth factor expressed in various tissues and cell lines. Pleiotrophin participates in multiple biological actions including the induction of cellular proliferation, migration and angiogenesis, and is involved in carcinogenesis. Recently, we identified and characterized several pleiotrophin proteolytic fragments with biological activities similar or opposite to that of pleiotrophin. Here, we investigated the biological actions of P(122-131), a synthetic peptide corresponding to the carboxy terminal region of this growth factor. Results Our results show that P(122-131) inhibits in vitro adhesion, anchorage-independent proliferation, and migration of DU145 and LNCaP cells, which express pleiotrophin and its receptor RPTPβ/ζ. In addition, P(122-131) inhibits angiogenesis in vivo, as determined by the chicken embryo CAM assay. Investigation of the transduction mechanisms revealed that P(122-131) reduces the phosphorylation levels of Src, Pten, Fak, and Erk1/2. Finally, P(122-131) not only interacts with RPTPβ/ζ, but also interferes with other pleiotrophin receptors, as demonstrated by selective knockdown of pleiotrophin or RPTPβ/ζ expression with the RNAi technology. Conclusions In conclusion, our results demonstrate that P(122-131) inhibits biological activities that are related to the induction of a transformed phenotype in PCa cells, by interacing with RPTPβ/ζ and interfering with other pleiotrophin receptors. Cumulatively, these results indicate that P(122-131) may be a potential anticancer agent, and they warrant further study of this peptide.

International audience; Glycosaminoglycans (GAG) are sulfated polysaccharides that play an important role in regulating cell functions. GAG mimetics called RGTAs (for ReGeneraTing Agents) have been shown to stimulate tissue repair. In particular they accelerate myogenesis, in part via their heparin-mimetic property towards growth factors. RGTAs also increase activity of calcium-dependent intracellular protease suggesting an effect on calcium cellular homeostasis. This effect was presently investigated on myoblasts in vitro using one member of the RGTA family molecule named OTR4120. We have shown that OTR4120 or heparin induced transient increases of intracellular calcium concentration ([Ca(2+)]i) in pre-fusing myoblasts from both mouse SolD7 cell line and rat skeletal muscle satellite cells grown in primary culture by mobilising sarcoplasmic reticulum store. This [Ca(2+)]i was not mediated by ryanodine receptors but instead resulted from stimulation of the Inositol-3 phosphate-phospholipase C activation pathway. OTR4120-induced calcium transient was not mediated through an ATP, nor a tyrosine kinase, nor an acetylcholine receptor but principally through serotonin 5-HT2A receptor. This original finding shows that the GAG mimetic can induce calcium signal through serotonin receptors and the IP3 pathway may be relevant to its ability to favour myoblast differentiation. It supports a novel and unexpected function of GAGs in the regulation of calcium homeostasis.

At the mouse neuromuscular junction (NMJ), there are two distinct cholines-terases (ChE): acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Until now, it has been difficult to determine the precise localization of BChE at the NMJ. In this study, we use a modification of Koelle's method to stain AChE and BChE activity. This method does not interfere with fluorescent co-staining, which allows precise co-localization of ChE and other synaptic molecules at the NMJ. We demonstrate that AChE and BChE exhibit different localization patterns at the mouse NMJ. AChE activity is present both in the primary cleft and in the secondary folds, whereas BChE activity appears to be almost absent in the primary cleft and to be concentrated in subsynaptic folds. The same localization for BChE is observed in the AChE-knockout (KO) mouse NMJ. Collagenase treatment removed AChE from the primary cleft, but not from secondary folds in the wild-type mouse, whereas in the AChE-KO mouse, BChE remains in the secondary folds. After peripheral nerve injury and regeneration, BChE localization is not modified in either normal or KO mice. In conclusion, specific localization of BChE in the secondary folds of the NMJ suggests that this enzyme is not a strict surrogate of AChE and that the two enzymes have two different roles. (J Histochem Cytochem 58:1075–1082, 2010)

International audience; The anionic polymerization of a spiro monomer containing both an ester‐activated cyclopropane moiety and a 1,4,7,10,13‐pentaoxacyclohexadecane‐14,16‐dione crown ether bislactone unexpectedly yielded a linear alternating poly(ether‐ester) via the ring‐opening polymerization of the crown ether cycle. Ion conductivity measurements using black lipid membranes as model systems showed that oligomers of this structure are able to permeabilize bilipidic membranes, with single‐ion channel behaviors being observed. Biological assays on fibroblast cells indicated a significant cytotoxicity, probably related to the above permeabilization mechanism.

International audience; A new form of high affinity fibroblast growth factor receptor has been purified from adult bovine brain membranes. Purification was performed by chromatography on DEAE-Trisacryl and wheat germ agglutinin-agarose followed by FGF-2 affinity chromatography. Affinity labeling of purified fractions with 125I-FGF-2 showed after cross-linking a 170-kDa complex, suggesting the existence of a 150-kDa FGF receptor. No cross-reactivity with anti-FGF receptor 1 (FGFR-1 or flg) or with anti-receptor 2 (FGFR-2 or bek) antibodies could be detected with this partially purified receptor. Heparitinase treatment of the partially purified FGF receptor abolished the formation of the ligand receptor complex. The complex was restored in the presence of heparin in a dose dependent fashion, supporting the idea that heparin-like molecules are needed for proper binding. Further purification of the receptor was achieved by heparin-Sepharose affinity chromatography and yielded a purification of over 320,000-fold. The purified receptor fraction was radiolabeled and loaded on RPLC C4 column. Eluted fractions were analysed by SDS-PAGE. A major 150-kDa band was detected. These data show for the first time a new form of FGF receptor isolated from bovine brain membranes. This purified receptor displays affinity for heparin and was therefore named heparin binding FGF receptor (HB-FGFR). It remains unclear whether the receptor is a proteo-heparin sulfate or whether heparans are strongly associated and therefore are copurified. Large scale preparations are in progress for core protein structure studies.

Heparan sulfate mimetic polymers promotes tissue repair when injected locally in doses of 1-2mg/kg by various routes. These biopolymers, have been extensively studied for their diverse biological activities. However, there is no detailed report investigating the toxicity of OTR4120. In this study, the acute and subchronic (30 days) toxicity of varying levels of OTR4120 was investigated in mice after intraperitoneal administration. The results showed that no significant toxicological changes were observed when 50mg/kg body weight per day OTR4120 was administered to mice. But when the dose was increased to 60 and 70 mg/kg body weight per day, the clotting time was significantly prolonged. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activities were reduced female and male at dose 70 mg/kg body weight per day. These blood biochemistry data suggest that OTR4120 have a hepatoprotective effect. Based on these results, it can be concluded that the no adverse effect level of OTR4120 is 50 mg/kg body weight per day.

Glioblastoma is the most common primary brain tumor in human adults. Since existing treatments are not effective enough, novel therapeutic targets must be sought. The heparin-binding growth factor, heparin affin regulatory peptide (HARP), also known as pleiotrophin (PTN), could potentially represent such a target. We have previously shown that a mutant protein, HARPDelta111-136, which lacks HARP's C-terminal 26 amino acids, acts as a dominant negative HARP effector by heterodimerizing with the wild-type growth factor. The aim of our study was to evaluate the potential inhibitory activity of HARPDelta111-136 on the U87 MG human glioblastoma cell line. By overexpressing the truncated form of HARP in stably established clones of U87 MG cells, we observed an inhibition of proliferation under both anchorage-dependent and anchorage-independent conditions. We confirmed these results in an in vivo subcutaneous tumor xenograft model. In addition, we found that HARPDelta111-136 inhibited cell proliferation in a paracrine manner. Analysis of key cellular pathways revealed a decrease of cell adhesion in U87 MG cells that overexpressed the mutant protein, which could explain this inhibitory effect. A replication-defective adenovirus model that encoded HARPDelta111-136 supported a putative antiproliferative role for the truncated protein in vitro and in vivo. Interestingly, HARPDelta111-136 was also able to abolish angiogenic activity in HUVEC proliferation and in a Matrigel plug assay. These results demonstrate that considering its antiproliferative and angiostatic effects, HARPDelta111-136 could be of great interest when used in conjunction with standard treatments.

Pleiotrophin (PTN) is a 136-amino acid secreted heparin-binding protein that is considered as a rate-limiting growth and an angiogenic factor in the onset, invasion, and metastatic process of many tumors. Its mitogenic and tumorigenic activities are mediated by the COOH-terminal residues 111 to 136 of PTN, allowing it to bind to cell surface tyrosine kinase-linked receptors. We investigated a new strategy consisting in evaluating the antitumor effect of a truncated PTN, lacking the COOH-terminal 111 to 136 portion of the molecule (PTNΔ111-136), which may act as a dominant-negative effector for its mitogenic, angiogenic, and tumorigenic activities by heterodimerizing with the wild-type protein. In vitro studies showed that PTNΔ111-136 selectively inhibited a PTN-dependent MDA-MB-231 breast tumor and endothelial cell proliferation and that, in MDA-MB-231 cells expressing PTNΔ111-136, the vascular endothelial growth factor-A and hypoxia-inducible factor-1α mRNA levels were significantly decreased by 59% and 71%, respectively, compared with levels in wild-type cells. In vivo, intramuscular electrotransfer of a plasmid encoding a secretable form of PTNΔ111-136 was shown to inhibit MDA-MB-231 tumor growth by 81%. This antitumor effect was associated with the detection of the PTNΔ111-136 molecule in the muscle and tumor extracts, the suppression of neovascularization within the tumors, and a decline in the Ki-67 proliferative index. Because PTN is rarely found in normal tissue, our data show that targeted PTN may represent an attractive and new therapeutic approach to the fight against cancer. [Mol Cancer Ther 2008;7(9):2817–27]

Objective Glycosaminoglycans (GAG) are major components of bone marrow extracellular matrix because they have the property to interact with cells and growth factors in hematopoietic niches. In this study, we investigated the effect of two different chemically defined GAG mimetics on mobilization of hematopoietic stem and progenitor cells (HSPCs) in mice peripheral blood. Materials and Methods Mobilization was achieved by intraperitoneal injection of GAG mimetics. Mobilized cells were characterized phenotypically by reverse transcription polymerase chain reaction and fluorescence-activated cell sorting analysis and functionally by colony-forming cell, cobblestone area−forming cell and long-term culture-initiating cell assays in vitro. Radioprotection assays were performed to confirm the functionality of primitive hematopoietic cells in vivo. Involvement of stromal-derived factor−1 (SDF-1) and matrix metalloproteinase-9 (MMP-9) were investigated. Results GAG mimetics treatment induces hyperleukocytosis and mobilization of HSPC. They synergize with the effects of granulocyte colony-stimulating factor or AMD3100 on hematopoietic progenitors mobilization. Reconstitution of lethally irradiated recipient mice with peripheral blood mononuclear cells from GAG mimetic-treated donor mice improves engraftment and survival. BiAcore studies indicate that the mimetics interact directly with SDF-1. In addition, GAG mimetics-induced mobilization is associated with increased levels of pro- and active MMP-9 from bone marrow cells and increased level of SDF-1 in peripheral blood. Finally, mobilization is partially inhibited by co-injection with anti−SDF-1 antibody. Conclusion This study demonstrates that GAG mimetics induce efficient mobilization of HSPCs, associated with an activation of pro−MMP-9 and a modification in the SDF-1 concentration gradient between bone marrow and peripheral blood. We suggest that structural features of GAGs can modify the nature of mobilized cells.

The incidence of ischemic cardiac diseases increases with age and elderly subjects are more vulnerable to myocardial infarction. Endurance exercise (e.g. treadmill training) provides cardioprotection against an ischemia and reperfusion (IR) event in adult heart but such a potential beneficial effect of regular exercise has never been evaluated during aging. Therefore, this study investigated the effects of moderate running training on post-ischemic recovery of contractile function and coronary perfusion in senescent myocardium. Isolated hearts of sedentary (24 mo-sedentary; n=10) and trained senescent (24 mo-trained; n=11; moderate running: 1h/day, 5 days/week for 12 weeks) rats were submitted to 45min low-flow ischemia (15% of initial coronary flow (CF)) followed by 30min reperfusion. Active tension (AT) and CF were recorded at baseline and after 1, 3, 5, 10, 15, 20, 25 and 30min of reperfusion. Left ventricular protein carbonylation, and both heat-shock-protein 70 (HSP70) and endothelial nitric oxide synthase (eNOS) contents were determined by Oxyblotting and Western blotting, respectively. Regular physical exercise improves impairment of functional post-ischemic recovery (AT and CF) of aged hearts during reperfusion and this cardioprotection is associated to limited protein oxidation and increased HSP70 and eNOS myocardial contents.

Pleiotrophin (PTN) is a polypeptide that belongs to a family of heparin-binding growth factor, which displays mitogenic activity for a wide variety of cells. Since PTN induces the proliferation of immune cells the mechanism of action was investigated. In the present study, we show for the first time that PTN induces the expression of inflammatory cytokines including TNF-alpha, IL-1beta and IL-6 in quiescent human peripheral blood mononuclear cells (PBMC). These results emphasize the importance of PTN in the regulation of inflammatory processes. Elucidation of the mechanisms by which a host factor such PTN regulates cytokines production will significantly advance our understanding of endothelium-immunity interactions.