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Pleiotrophin (PTN) has a moderate stimulatory effect on endothelial cell migration through ανβ3 integrin, while it decreases the stimulatory effect of vascular endothelial growth factor A (VEGFA) and inhibits cell migration in the absence of ανβ3 through unknown mechanism(s). In the present work, by using a multitude of experimental approaches, we show that PTN binds to VEGF receptor type 2 (VEGFR2) with a KD of 11.6 nM. Molecular dynamics approach suggests that PTN binds to the same VEGFR2 region with VEGFA through its N-terminal domain. PTN inhibits phosphorylation of VEGFR2 at Tyr1175 and still stimulates endothelial cell migration in the presence of a selective VEGFR2 tyrosine kinase inhibitor. VEGFR2 downregulation by siRNA or an anti-VEGFR2 antibody that binds to the ligand-binding VEGFR2 domain also induce endothelial cell migration, which is abolished by a function-blocking antibody against ανβ3 or the peptide PTN112−136 that binds ανβ3 and inhibits PTN binding. In cells that do not express ανβ3, PTN decreases both VEGFR2 Tyr1175 phosphorylation and cell migration in a VEGFR2-dependent manner. Collectively, our data identify VEGFR2 as a novel PTN receptor involved in the regulation of cell migration by PTN and contribute to the elucidation of the mechanism of activation of endothelial cell migration through the interplay between VEGFR2 and ανβ3.

The secreted growth factor pleiotrophin (PTN) is expressed in all species and is evolutionarily highly conserved, suggesting that it plays a significant role in the regulation of important processes. The observation that it is highly expressed at early stages during development and in embryonic progenitor cells highlights a potentially important contribution to development. There is ample evidence of the role of PTN in the development of the nervous system and hematopoiesis, some, albeit inconclusive, evidence of its role in the skeletomuscular system, and limited evidence of its role in the development of other organs. Studies on its role in the cardiovascular system and angiogenesis suggest that PTN has a significant regulatory effect by acting on endothelial cells, while its role in the functions of smooth or cardiac muscle cells has not been studied. This review highlights what is known to date regarding the role of PTN in the development of various organs and in angiogenesis. Wherever possible, evidence on the crosstalk between the receptors that mediate PTN’s functions is also quoted, highlighting the complex regulatory pathways that affect development and angiogenesis.

Η πλειοτροπίνη (pleiotrophin, PTN) είναι ένας αυξητικός παράγοντας που φαίνεται να παίζει σημαντικό ρόλο στην ανάπτυξη του καρκίνου του προστάτη και την αγγειογένεση, ενώ έχει αναφερθεί ότι ίσως παίζει ρόλο στην ανάπτυξη και αναδιαμόρφωση των οστών. Η ΡΤΝ ασκεί τις δράσεις της μέσω του υποδοχέα με δράση φωσφατάσης τυροσίνης β/ζ (PTPRZ1), της ιντεγκρίνης ανβ3, και της μεμβρανικής νουκλεολίνης. Η ΡΤΝ επηρεάζει, επίσης τις δράσεις του αυξητικού παράγοντα του αγγειακού ενδοθηλίου Α (vascular endothelial growth factor A, VEGF-A), ο οποίος εκφράζεται και από τους προ-οστεοβλάστες κι έχει αυτοκρινείς και παρακρινείς δράσεις, επάγοντας τον πολλαπλασιασμό, τη μετανάστευση και τη διαφοροποίηση των οστεοβλαστών, και διεγείροντας την αγγειογένεση στα οστά. Η έκφραση του VEGF-Α είναι αυξημένη σε περιπτώσεις καρκίνου του προστάτη και σχετίζεται με την οστεοβλαστικού τύπου μετάσταση των όγκων. Πρόσφατες μελέτες της ερευνητικής μας ομάδας έδειξαν ότι ΡΤΝ και VEGF-A ανταγωνίζονται για δέσμευση στον PTPRZ1 και ο PTPRZ1 φαίνεται να εμπλέκεται σε σημαντικές αγγειογενετικές δράσεις του VEGF-A, όπως είναι η μετανάστευση των ενδοθηλιακών κυττάρων. Με βάση τα παραπάνω, στην παρούσα διατριβή μελετήθηκε ο ρόλος της ΡΤΝ στον πολλαπλασιασμό, τη μετανάστευση και τη διαφοροποίηση οστεοβλαστών, καθώς και στην επιβίωση και την ικανότητα προσκόλλησης των καρκινικών κυττάρων προστάτη σε οστεοβλάστες. Επιπλέον, διερευνήθηκε η αλληλεπίδραση της ΡΤΝ με τον VEGFR2 σε ενδοθηλιακά κύτταρα και οστεοβλάστες και μελετήθηκε η λειτουργική σημασία της αλληλεπίδρασης αυτής. Βρέθηκε ότι η ΡΤΝ εκφράζεται στους οστεοβλάστες (χρησιμοποιήθηκαν οι κυτταρικές σειρές Saos-2, MG-63 και MC3T3-E1) και εντοπίζεται στη μεμβράνη, στο κυτταρόπλασμα και στον πυρήνα τους, παρόμοια με τον εντοπισμό της στα ενδοθηλιακά κύτταρα. Η ΡΤΝ προκαλεί μια μικρή μείωση του πολλαπλασιασμού των οστεοβλαστών, η οποία είναι στατιστικά σημαντική μόνο στους προ-οστεοβλάστες MC3T3-E1, και μειώνει στατιστικά σημαντικά τη μετανάστευση όλων των κυτταρικών σειρών οστεοβλαστών που μελετήθηκαν, ενώ επάγει τη διαφοροποίηση των προ-οστεοβλαστών MC3T3-E1 σε ώριμους οστεοβλάστες. Στα καρκινικά κύτταρα προστάτη PC3, μείωση της έκφρασης της ΡΤΝ οδηγεί σε επαγωγή της απόπτωσης και μείωση της προσκόλλησης των καρκινικών κυττάρων στους οστεοβλάστες. Στην προσπάθεια διαλεύκανσης των υποδοχέων της ΡΤΝ που εμπλέκονται σε αυτήν την προσκόλληση, βρέθηκε με τη χρήση πολλών διαφορετικών πειραματικών προσεγγίσεων ότι η ΡΤΝ αλληλεπιδρά εκλεκτικά και με υψηλή χημική συγγένεια με τον VEGFR2 σε οστεοβλάστες και σε ενδοθηλιακά κύτταρα. Η PΤΝ φαίνεται να δεσμεύεται μέσω της αμινοτελικής της περιοχής σε θέση του VEGFR2 που επηρεάζει τη δέσμευση του VEGF-A, και μειώνει τη φωσφορυλίωση του VEGFR2 που προκαλεί ο VEGF-A165. H δράση αυτή εξηγεί την ανασταλτική δράση της ΡΤΝ στη μετανάστευση των οστεοβλαστών, αλλά όχι την επαγωγική δράση στα ενδοθηλιακά κύτταρα, που διαμεσολαβείται από την ιντεγκρίνη ανβ3. Βρέθηκε, ότι μονοκλωνικό αντίσωμα που δεσμεύεται στην εξωκυτταρική περιοχή του VEGFR2 ή αποσιώπηση του VEGFR2 με siRNA οδηγούν σε επαγωγή της κυτταρικής μετανάστευσης, η οποία αναστέλλεται από αντίσωμα που αναστέλλει την ιντεγκρίνη ανβ3 ή από πεπτίδιο της ΡΤΝ που αντιστοιχεί στο καρβοξυτελικό της άκρο και επίσης αναστέλλει την ιντεγκρίνη ανβ3. Τέλος, με τη χρήση πεπτιδίων που αντιστοιχούν σε διαφορετικές περιοχές του μορίου της ΡΤΝ και αναστέλλουν την αλληλεπίδρασή της με διαφορετικούς υποδοχείς, καθώς και με φαρμακολογικούς αναστολείς των διαφορετικών υποδοχέων της ΡΤΝ, βρέθηκε ότι η μεμβρανική νουκλεολίνη είναι ο υποδοχέας που εμπλέκεται σε σημαντικό βαθμό στην αλληλεπίδραση των καρκινικών κυττάρων προστάτη με οστεοβλάστες και ταυτοποιήθηκαν πεπτίδια που θα μπορούσαν να μελετηθούν και αξιοποιηθούν περαιτέρω ως αναστολείς της αγγειογένεσης και/ή της μετάστασης καρκινικών κυττάρων προστάτη στα οστά.

Pleiotrophin (PTN) is a growth factor that appears to play an important role in prostate cancer growth and angiogenesis. We have previously shown that decreased PTN expression in human prostate cancer PC3 cells leads to decreased adhesion of prostate cancer cells to osteoblasts, suggesting that PTN mediates this interaction. In the current work, using peptides that correspond to different regions of the PTN protein, we identified that a domain responsible for the adhesion of prostate cancer cells to osteoblasts corresponds to amino acids 16-24 of the mature PTN protein. Given that a synthetic PTN

Pleiotrophin (PTN) has a moderate stimulatory effect on endothelial cell migration through α

Pleiotrophin (PTN) stimulates endothelial cell migration through binding to receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) and ανβ3 integrin. Screening for proteins that interact with RPTPβ/ζ and potentially regulate PTN signaling, through mass spectrometry analysis, identified cyclin-dependent kinase 5 (CDK5) activator p35 among the proteins displaying high sequence coverage. Interaction of p35 with the serine/threonine kinase CDK5 leads to CDK5 activation, known to be implicated in cell migration. Protein immunoprecipitation and proximity ligation assays verified p35-RPTPβ/ζ interaction and revealed the molecular association of CDK5 and RPTPβ/ζ. In endothelial cells, PTN activates CDK5 in an RPTPβ/ζ- and phosphoinositide 3-kinase (PI3K)-dependent manner. On the other hand, c-Src, ανβ3 and ERK1/2 do not mediate the PTN-induced CDK5 activation. Pharmacological and genetic inhibition of CDK5 abolished PTN-induced endothelial cell migration, suggesting that CDK5 mediates PTN stimulatory effect. A new pyrrolo[2,3-α]carbazole derivative previously identified as a CDK1 inhibitor, was found to suppress CDK5 activity and eliminate PTN stimulatory effect on cell migration, warranting its further evaluation as a new CDK5 inhibitor. Collectively, our data reveal that CDK5 is activated by PTN, in an RPTPβ/ζ-dependent manner, regulates PTN-induced cell migration and is an attractive target for the inhibition of PTN pro-angiogenic properties.

The natural product artemisinin and derivatives thereof are currently considered as the drugs of choice for the treatment of malaria. At the same time, a significant number of such drugs have also shown interesting anticancer activity. In the context of the present research work, artemisinin was structurally modified and anchored to naturally occurring polyamines to afford new artemisinin dimeric conjugates whose potential anticancer activity was evaluated. All artemisinin conjugates tested were more effective than artemisinin itself in decreasing the number of MCF7 breast cancer cells. The effect required conjugation and was not due to the artemisinin analogue or the polyamine, alone or in combination. To elucidate potential mechanism of action, we used the most effective conjugates 6, 7, 9 and 12 and found that they decreased expression and secretion of the angiogenic growth factor pleiotrophin by the cancer cells themselves, and inhibited angiogenesis in vivo and endothelial cell growth in vitro. These data suggest that the new artemisinin dimers are good candidates for the development of effective anticancer agents.

Bone has an enormous capacity for growth, regeneration, and remodelling, largely due to induction of osteoblasts that are recruited to the site of bone formation. Although the pathways involved have not been fully elucidated, it is well accepted that the immediate environment of the cells is likely to play a role via cell–matrix interactions, mediated by several growth factors. Formation of new blood vessels is also significant and interdependent to bone formation, suggesting that enhancement of angiogenesis could be beneficial during the process of bone repair. Pleiotrophin (PTN), also called osteoblast-specific factor 1, is a heparin-binding angiogenic growth factor, with a well-defined and significant role in both physiological and pathological angiogenesis. In this review we summarise the existing evidence on the role of PTN in bone repair.

Retinoids constitute a family of organic compounds that are being used for the treatment of various diseases, ranging from acne vulgaris to acute promyelocytic leukemia. Their use however is limited due to serious adverse effects and there is a great need for analogues with better safety profile. In the present work, the effect of N(1),N(12)-bis(all-trans-retinoyl)spermine (RASP), a conjugate of all-trans-retinoic acid (atRA) with spermine, on angiogenesis in vivo and viability of human endothelial and prostate cancer cells in vitro were studied. Both atRA and RASP dose-dependently inhibited angiogenesis in the chicken embryo chorioallantoic membrane model. RASP was more effective and could be used in a wider dose range due to lower toxicity compared with atRA. Both retinoids decreased the number of human umbilical vein endothelial and prostate cancer LNCaP and PC3 cells in a concentration-dependent manner. RASP was more effective and potent compared with atRA, spermine, their combination, or conjugates of spermine with other acidic retinoids and/or psoralens in prostate cancer cells. The inhibitory effect of both atRA and RASP seems to be related to an increase of the tumour repressing gene retinoic acid receptor beta mRNA, was mediated by retinoic acid receptor alpha, and was proportional to endogenous retinoic acid receptor beta expression. These data suggest that RASP is more effective than atRA in decreasing angiogenesis and prostate cancer cell growth and identify retinoic acid receptor alpha as the receptor through which it causes retinoic acid receptor beta up-regulation and decrease of prostate cancer cell growth.

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.

Colloidal clusters of magnetic iron oxide nanocrystals (MIONs), particularly in the condensed pattern (co-CNCs), have emerged as new superstructures to improve further the performance of MIONs in applications pertaining to magnetic manipulation (drug delivery) and magnetic resonance imaging (MRI). Exploitation of the advantages they represent and their establishment in the area of nanomedicine demands a particular set of assets. The present work describes the development and evaluation of MION-based co-CNCs featuring for the first time such assets: High magnetization, as well as magnetic content and moment, high relaxivities (r2 = 400 and r2* = 905 s(-1) mMFe(-1)) and intrinsic loss power (2.3 nH m(2) kgFe(-1)) are combined with unprecedented colloidal stability and structural integrity, stealth and drug-loading properties. The reported nanoconstructs are endowed with additional important features such as cost-effective synthesis and storage, prolonged self-life and biocompatibility. It is finally showcased with in vivo multispectral optoacoustic tomography how these properties culminate in a system suitable for targeting breast cancer and for forceful in vivo manipulation with low magnetic field gradients.

Mechanical loading of the spine is a major causative factor of degenerative changes and causes molecular and structural changes in the intervertebral disc (IVD) and the vertebrae end plate (EP). Pleiotrophin (PTN) is a growth factor with a putative role in bone remodeling through its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ). The present study investigates the effects of strain on PTN and RPTPβ/ζ protein expression in vivo. Tails of eight weeks old Sprague-Dawley rats were subjected to mechanical loading using a mini Ilizarov external apparatus. Rat tails untreated (control) or after 0 degrees of compression and 10°, 30° and 50° of angulation (groups 0, I, II and III respectively) were studied. PTN and RPTPβ/ζ expression were evaluated using immunohistochemistry and Western blot analysis. In the control group, PTN was mostly expressed by the EP hypertrophic chondrocytes. In groups 0 to II, PTN expression was increased in the chondrocytes of hypertrophic and proliferating zones, as well as in osteocytes and osteoblast-like cells of the ossification zone. In group III, only limited PTN expression was observed in osteocytes. RPTPβ/ζ expression was increased mainly in group 0, but also in group I, in all types of cells. Low intensity RPTPβ/ζ immunostaining was observed in groups II and III. Collectively, PTN and RPTPβ/ζ are expressed in spinal deformities caused by mechanical loading, and their expression depends on the type and severity of the applied strain.

Pleiotrophin (PTN) is a heparin-binding growth factor that induces cell migration through binding to its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) and integrin alpha v beta 3 (ανβ3). In the present work, we studied the effect of PTN on the generation of reactive oxygen species (ROS) in human endothelial cells and the involvement of ROS in PTN-induced cell migration. Exogenous PTN significantly increased ROS levels in a concentration and time-dependent manner in both human endothelial and prostate cancer cells, while knockdown of endogenous PTN expression in prostate cancer cells significantly down-regulated ROS production. Suppression of RPTPβ/ζ through genetic and pharmacological approaches, or inhibition of c-src kinase activity abolished PTN-induced ROS generation. A synthetic peptide that blocks PTN–ανβ3 interaction abolished PTN-induced ROS generation, suggesting that ανβ3 is also involved. The latter was confirmed in CHO cells that do not express β3 or over-express wild-type β3 or mutant β3Y773F/Y785F. PTN increased ROS generation in cells expressing wild-type β3 but not in cells not expressing or expressing mutant β3. Phosphoinositide 3-kinase (PI3K) or Erk1/2 inhibition suppressed PTN-induced ROS production, suggesting that ROS production lays down-stream of PI3K or Erk1/2 activation by PTN. Finally, ROS scavenging and xanthine oxidase inhibition completely abolished both PTN-induced ROS generation and cell migration, while NADPH oxidase inhibition had no effect. Collectively, these data suggest that xanthine oxidase-mediated ROS production is required for PTN-induced cell migration through the cell membrane functional complex of ανβ3 and RPTPβ/ζ and activation of c-src, PI3K and ERK1/2 kinases.

Objectives The aim of this study was to examine whether asymmetric loading influences macrophage elastase (MMP12) expression in different parts of a rat tail intervertebral disc and growth plate and if MMP12 expression is correlated with the severity of the deformity. Methods A wedge deformity between the ninth and tenth tail vertebrae was produced with an Ilizarov-type mini external fixator in 45 female Wistar rats, matched for their age and weight. Three groups were created according to the degree of deformity (10°, 30° and 50°). A total of 30 discs and vertebrae were evaluated immunohistochemically for immunolocalisation of MMP12 expression, and 15 discs were analysed by western blot and zymography in order to detect pro- and active MMP12. Results No MMP12 expression was detected in the nucleus pulposus. Expression of MMP12 in the annulus progressively increased from group I to groups II and III, mainly at the concave side. Many growth plate chondrocytes expressed MMP12 in the control group, less in group I and rare in groups II and III. Changes in cell phenotype and reduction of cell number were observed, together with disorganisation of matrix microstructure similar to disc degeneration. ProMMP12 was detected at the area of 54 kDa and active MMP12 at 22 kDa. Conclusions Expression of MMP12 after application of asymmetric loading in a rat tail increased in the intervertebral disc but decreased in the growth plate and correlated with the degree of the deformity and the side of the wedged disc. Cite this article: Bone Joint Res 2014;3:273–9.

Pleiotrophin (PTN) is a heparin-binding growth factor with diverse functions related to tumor growth, angiogenesis, and metastasis. Pleiotrophin seems to have a significant role in prostate cancer cell growth and to mediate the stimulatory actions of other factors that affect prostate cancer cell functions. However, all studies carried out up to date are in vitro, using different types of human prostate cancer cell lines. The aim of the present work was to study the role of endogenous PTN in human prostate cancer growth in vivo. For this purpose, human prostate cancer PC3 cells were stably transfected with a plasmid vector, bearing the antisense PTN sequence, in order to inhibit PTN expression (AS-PC3). Migration, apoptosis, and adhesion on osteoblastic cells were measured in vitro. In vivo, PC3 cells were s.c. injected into male NOD/SCID mice, and tumor growth, survival rates, angiogenesis, apoptosis, and the number of metastasis were estimated. Pleiotrophin depletion resulted in a decreased migration capability of AS-PC3 cells compared with the corresponding mock-transfected or the non-transfected PC3 cells, as well as increased apoptosis and decreased adhesiveness to osteoblastic cells in vitro. In prostate cancer NOD/SCID mouse xenografts, PTN depletion significantly suppressed tumor growth and angiogenesis and induced apoptosis of cancer cells. In addition, PTN depletion decreased the number of metastases, providing a survival benefit for the animals bearing AS-PC3 xenografts. Our data suggest that PTN is implicated in human prostate cancer growth in vivo and could be considered a potential target for the development of new therapeutic approaches for prostate cancer.

Pleiotrophin (PTN) is a heparin-binding growth factor with diverse biological activities, the most studied of these being those related to the nervous system, tumor growth and angiogenesis. Although interest in the involvement of PTN in tumor growth is increasing, many questions remain unanswered, particularly concerning the receptors and the signaling pathways involved. In this review, we briefly introduce PTN, and summarize data on its involvement in tumor growth and angiogenesis, and on what is known to date concerning the receptors and pathways involved.