Published online 31 March 2009
Haematologica, Vol 94, Issue 5, 670-678 doi:10.3324/haematol.13427
Copyright © 2009 by Ferrata Storti Foundation
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Hemostasis

Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

Maartje van den Biggelaar1,2, Eveline A.M. Bouwens1,2, Neeltje A. Kootstra3,4, Robert P. Hebbel5, Jan Voorberg1,3, Koen Mertens1,2

1 Department of Plasma Proteins, Sanquin Research, Amsterdam, The Netherlands
2 Department of Pharmaceutics, Utrecht I nstitute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands
3 Landsteiner Laboratory of Academic Medical Centre and Sanquin, University of Amsterdam, Amsterdam, The Netherlands
4 Department of Experimental Immunology, Center for Infectious Disease and Immunity, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
5 Department of Medicine, Vascular Biology Center and Division of Hematology-Oncology-Transplantation, University of Minnesota Medical School, Minneapolis, MI, USA

Correspondence: Jan Voorberg, Department of Plasma Proteins, Sanquin Research, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands. E-mail:j.voorberg{at}sanquin.nl

Background: Gene therapy provides an attractive alternative for protein replacement therapy in hemophilia A patients. Recent studies have shown the potential benefit of directing factor (F)VIII gene delivery to cells that also express its natural carrier protein von Willebrand factor (VWF). In this study, we explored the feasibility of blood outgrowth endothelial cells as a cellular FVIII delivery device with particular reference to long-term production levels, intracellular storage in Weibel-Palade bodies and agonist-induced regulated secretion.

Design and Methods: Human blood outgrowth endothelial cells were isolated from peripheral blood collected from healthy donors, transduced at passage 5 using a lentiviral vector encoding human B-domain deleted FVIII-GFP and characterized by flow cytometry and confocal microscopy.

Results: Blood outgrowth endothelial cells displayed typical endothelial morphology and expressed the endothelial-specific marker VWF. Following transduction with a lentivirus encoding FVIII-GFP, 80% of transduced blood outgrowth endothelial cells expressed FVIII-GFP. Levels of FVIII-GFP positive cells declined slowly upon prolonged culturing. Transduced blood outgrowth endothelial cells expressed 1.6±1.0 pmol/1x106 cells/24h FVIII. Morphological analysis demonstrated that FVIII-GFP was stored in Weibel-Palade bodies together with VWF and P-selectin. FVIII levels were only slightly increased following agonist-induced stimulation, whereas a 6- to 8-fold increase of VWF levels was observed. Subcellular fractionation revealed that 15–22% of FVIII antigen was present within the dense fraction containing Weibel-Palade bodies.

Conclusions: We conclude that blood outgrowth endothelial cells, by virtue of their ability to store a significant portion of synthesized FVIII-GFP in Weibel-Palade bodies, provide an attractive cellular on-demand delivery device for gene therapy of hemophilia A.

Key words: factor VIII, von Willebrand factor, endothelial progenitor cells, Weibel-Palade bodies, gene therapy.