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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) 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.

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.