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Open Access Research

Pleiotrophin expression and role in physiological angiogenesis in vivo: potential involvement of nucleolin

Marina Koutsioumpa1, Georgia Drosou1, Constantinos Mikelis15, Katerina Theochari1, Dionussios Vourtsis1, Panagiotis Katsoris2, Efstathia Giannopoulou16, Jose Courty3, Christos Petrou4, Vassiliki Magafa4, Paul Cordopatis4 and Evangelia Papadimitriou1*

  • * Corresponding author: Evangelia Papadimitriou epapad@upatras.gr

  • † Equal contributors

Author Affiliations

1 Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, Patras GR 26504, Greece

2 Division of Genetics, Cell & Developmental Biology, Department of Biology, University of Patras, Patras, Greece

3 Laboratoire CRRET, Université Paris Est Créteil, Val de Marne, 61 avenue du Général de Gaulle, 94010 Créteil, Cedex, France

4 Laboratory of Pharmacognocy and Chemistry of Natural Products, Department of Pharmacy, University of Patras, Athens, Greece

5 Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Building 30, Room 203, Bethesda, MD 20892-4340, USA

6 Clinical Oncology Laboratory, Division of Oncology, Department of Medicine, University Hospital of Patras, Patras Medical School, 26504 Rio, Greece

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Vascular Cell 2012, 4:4  doi:10.1186/2045-824X-4-4

Published: 16 March 2012

Abstract

Background

Pleiotrophin (PTN) is a heparin-binding growth factor with significant role(s) in tumour growth and angiogenesis. Although implication of endogenous PTN has been studied in several in vivo models of tumour angiogenesis, its role in physiological angiogenesis has not been addressed. In the present work, we studied expression and functional significance of endogenous PTN during angiogenesis in the chicken embryo chorioallantoic membrane (CAM).

Methods

Using molecular, cellular and biochemical assays, we studied the expression pattern of PTN in CAM and human endothelial cells and its possible interaction with nucleolin (NCL). CAM cells were transfected with a pCDNA3.1 vector, empty (PC) or containing full length cDNA for PTN in antisense orientation (AS-PTN). Angiogenesis was estimated by measuring total vessel length. In vitro, human endothelial cells migration was studied by using a transwell assay, and down-regulation of NCL was performed by using a proper siRNA.

Results

Endogenous PTN mRNA and protein levels, as well as protein levels of its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) were maximal at early stages, when CAM angiogenesis is active. Application of AS-PTN onto CAM at days of active angiogenesis was not toxic to the tissue and led to dose-dependent decreased expression of endogenous PTN, ERK1/2 activity and angiogenesis. Interestingly, endogenous PTN was also immunolocalized at the endothelial cell nucleus, possibly through interaction with NCL, a protein that has a significant role in the nuclear translocation of many proteins. Down-regulation of NCL by siRNA in human endothelial cells significantly decreased nuclear PTN, verifying this hypothesis. Moreover, it led to abolishment of PTN-induced endothelial cell migration, suggesting, for the first time, that PTN-NCL interaction has a functional significance.

Conclusions

Expression of endogenous PTN correlates with and seems to be involved in angiogenesis of the chicken embryo CAM. Our data suggest that NCL may have a role, increasing the number of growth factors whose angiogenic/tumorigenic activities are mediated by NCL.

Keywords:
angiogenesis; endothelial cells; migration; nucleolin; pleiotrophin; receptor protein tyrosine phosphatase