Short Report Endostatin’s heparan sulfate-binding site is essential for inhibition of angiogenesis and enhances in situ binding to capillary-like structures in bone explants Sabine Gaetzner a , Martine M.L. Deckers b,1 , Sonja Stahl a , Clemens Lfwik b , Bjorn R. Olsen c , Ute Felbor a, * a Department of Human Genetics, University of Wu ¨rzburg, Biozentrum, Am Hubland, D-97074 Wu ¨rzburg, Germany b Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands c Department of Cell Biology, Harvard Medical School, Boston, MA, USA Received 19 August 2004; received in revised form 11 October 2004; accepted 12 October 2004 Abstract The functional role of endostatinTs affinity for heparan sulfates was addressed using an ex vivo bone angiogenesis model. Capillary-like sprouts showed prominent expression of collagen XVIII/endostatin. Outgrowth of endothelial cells was not altered in the absence of collagen XVIII but inhibited by the addition of recombinant endostatin. Mutant non-heparan sulfate binding endostatin and the collagen XV endostatin homologue were ineffective. The ability of mutant endostatin to bind to capillary structures was reduced when compared to endostatin. Endostatin-XV completely failed to bind to endothelial cells. Our data indicate that endostatinTs angiostatic function is heparan sulfate- dependent, and that in situ-binding of endostatin to endothelial cells is increased by heparan sulfates. D 2004 Elsevier B.V./International Society of Matrix Biology. All rights reserved. Keywords: Endostatin; Collagen XVIII; Collagen XV; Heparan sulfate; Bone angiogenesis 1. Introduction Endostatin, a 20-kDa proteolytic fragment from the C- terminal domain of collagen XVIII, is a naturally occurring inhibitor of angiogenesis and tumour growth whose affinity for heparin facilitated purification (O’Reilly et al., 1997). Replacing arginines 158 and 270 with alanines by in vitro mutagenesis resulted in loss of heparin and heparan sulfate binding associated with loss of anti-angiogenic activity in FGF-2-stimulated chick chorioallantoic membrane angio- genesis (Sasaki et al., 1999). The C-terminal domain of collagen XV (endostatin-XV) is highly homologous to endostatin but contains a four-residue deletion around arginine 158 and lacks heparin binding. While endostatin- XV did not affect endothelial cell proliferation (Ramchan- dran et al., 1999), it reduced angiogenesis in the chick chorioallantoic membrane (Sasaki et al., 2000). Further conflicting reports exist regarding the importance of heparin/heparan sulfate affinity for endostatin activity and binding. Four studies report loss of inhibitory activity of mutant endostatin R158/270A, whereas two studies dem- onstrate biological activity of non-heparin binding endo- statin in various assay systems (Table 1). Binding of heparin mutant endostatin to the endothelial cells used in the respective assay was analyzed in only two studies with contradictory results. Javaherian et al. (2002) did not find binding of human Fc-endostatin R27/139A (which corre- sponds to R158/270A in the non-collagenous domain of 0945-053X/$ - see front matter D 2004 Elsevier B.V./International Society of Matrix Biology. All rights reserved. doi:10.1016/j.matbio.2004.10.001 Abbreviations: VEGF, vascular endothelial growth factor; AP, alkaline phosphatase; ES, endostatin; AP-mES, alkaline phosphatase fused to the N- terminus of murine endostatin. * Corresponding author. Tel.: +49 931 888 4097; fax: +49 931 888 4058. E-mail address: felbor@biozentrum.uni-wuerzburg.de (U. Felbor). 1 Current affiliation: Department of Cellular Biochemistry, The Nether- lands Cancer Institute, Amsterdam, The Netherlands. Matrix Biology 23 (2005) 557 – 561 www.elsevier.com/locate/matbio