Submitted June 8, 2007
Accepted February 13, 2008
Angiogenic endothelium shows lactadherin-dependent phagocytosis of aged erythrocytes and apoptotic cells
Marcel H.A.M. Fens, Enrico Mastrobattista, Anko M. de Graaff, Frits M. Flesch, Anton Ultee, Jan T. Rasmussen, Grietje Molema, Gert Storm, and Raymond M. Schiffelers*
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Utrecht, Netherlands
Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands
Department of Pathobiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands
Protein Chemistry Laboratory, Department of Molecular Biology, Aarhus University, Aarhus, Denmark
Department of Pathology and Laboratory Medicine, Medical Biology Section, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
* Corresponding author; email: r.m.schiffelers{at}uu.nl.
Angiogenic endothelium plays a crucial role in tumor growth. During angiogenesis, complex alterations in the microenvironment occur. In response, the endothelium undergoes phenotypic changes, for example overexpression of 
v-integrins. Here, we show that the overexpression of v-integrins on angiogenic endothelial cells is engaged in phagocytic actions involving binding ('tethering') and uptake ('tickling') of lactadherin (also termed MFG-E8)-opsonized particles. Phosphatidylserine (PS)-exposing multilamellar vesicles, 'aged' erythrocytes and apoptotic melanoma cells incubated with lactadherin were all phagocytosed by angiogenic endothelial cells in vitro. Furthermore, we demonstrated lactadherin expression in and around tumor blood vessels making opsonisation in situ plausible. By engineering the surface of erythrocytes with covalently coupled cyclic Arg-Gly-Asp (RGD) peptides -mimicking lactadherin opsonization-, we could induce phagocytosis by angiogenic endothelial cells both in vitro and in vivo. In vitro this was confirmed by cytochalasin D pre-incubation. When RGD-erythrocytes were administered intravenously in tumor bearing mice, blood vessel congestion followed by tumor core necrosis was seen. Moreover, RGD-erythrocytes could delay tumor growth in a murine melanoma model, possibly through induction of tumor infarctions. These results reveal that angiogenic endothelial cells have phagocytic properties for lactadherin-opsonised large particles and apoptotic cells. Implications of our findings for diagnostic and therapy of angiogenesis-driven diseases are discussed.
