EXPERIMENTAL CELL RESEARCH 237, 223–230 (1997) ARTICLE NO. EX973792 Hyaluronan Oligosaccharides Induce Tube Formation of a Brain Endothelial Cell Line in Vitro Mehdi Rahmanian,* Ha ˚ kan Pertoft,* Shigeru Kanda,† Rolf Christofferson,‡ , § Lena Claesson-Welsh,* and Paraskevi Heldin* ,1 *Department of Medical and Physiological Chemistry, Box 575, †Ludwig Institute for Cancer Research, Box 595, ‡Department of Human Anatomy, Uppsala University, Biomedical Center, S-751 23 Uppsala, Sweden; and §Department of Pediatric Surgery, The Children’s Academic Hospital, Uppsala, Sweden compounds are implicated in the regulation of angio- In remodeling tissues the formation of new blood genesis such as vascular endothelial cell growth factor vessels is an essential process which is regulated by (VEGF) and fibroblast growth factors (FGF-1, FGF-2), different factors. During such processes an increase as well as proteoglycans and glycosaminoglycans [for in hyaluronan synthesis and turnover is often seen and reviews see 4, 5]. recent observations have suggested that hyaluronan Hyaluronan is a high-molecular-weight glucosami- can both promote and inhibit neovascularization de- noglycan located in the extracellular matrix and com- pending on its molecular mass. In this work we show posed of repeating disaccharide units of D-glucuronic that a brain capillary endothelial cell line forms tubes acid and N-acetyl-D-glucosamine. Its synthesis is regu- in a collagen gel after stimulation by hyaluronan oligo- lated by platelet-derived growth factor (PDGF), trans- saccharides. Ultrastructural examination of the tubes forming growth factor-b (TGF-b), FGF-2, and phorbol by transmission electron microscopy revealed that the esters [6, 7]. Hyaluronan is ubiquitously distributed in cord-like outgrowths consisted of 2 – 10 tightly packed body tissues and has been shown to mediate several cells containing a continuous lumen. The tube forma- biological functions such as cell proliferation, differen- tion in response to hyaluronan oligosaccharide was tiation, and tumor metastasis through its interactions not mediated by activation of receptors for fibroblast with specific hyaluronan-binding proteins, hyaladher- growth factor. The endothelial cell line, which does not ins [for a review see 8]. Furthermore, both in vivo and synthesize hyaluronan, exhibited specific hyaluronan- in vitro studies have revealed that regulation of the binding sites, with about 3000 hyaluronan molecules rate of hyaluronan synthesis and the rate of its catabo- (M r 3.85 1 10 6 ) bound per cell at saturation and K d was 0.05 1 10 09 M. Furthermore, the cell line was stained lism, through the action of hyaluronan synthases and with mAb IVd4 that recognizes a novel class of hyalur- hyaluronidases, respectively, play important roles dur- onan-binding proteins and mAb IM7 which recognizes ing angiogenesis [9, for a review see Ref. 4]. In vitro CD44 molecules.  1997 Academic Press studies have demonstrated that high-molecular-weight hyaluronan is antiangiogenic, whereas hyaluronan oli- gosaccharides of specific size, 3 to 10 disaccharide INTRODUCTION units, stimulate proliferation and migration of endo- thelial cells [9, 10]. Angiogenesis, i.e, the formation of new blood vessels, A brain capillary endothelial cell line was recently occurs during the embryonal development, as well as established from H-2K b -tsA58 transgenic mice [11]. during wound healing, tumor growth, and metastasis The cell line is stable in culture (ú150 passages) and [1]. A series of sequential events are involved in angio- expresses common endothelial markers, e.g., factor genesis including local degradation of endothelial base- VIII-related antigen, binding sites for Bandeiraea sim- ment membrane by the action of proteases, migration plicifolia lectin, and binding sites for Ulex Europaeus of endothelial cells and formation of sprouts, prolifera- Agglutinin-1 lectin and has a specific uptake of ace- tion of endothelial cells and formation of a lumen which tylated LDL. In vitro studies demonstrated that the gives a functional vessel [for reviews see 2, 3]. Several established cell line can form capillary-like tubes in three-dimensional collagen gels in response to stimula- tion by FGF-1 and FGF-2 [11]. 1 To whom correspondence and reprint requests should be ad- The aim of the present investigation was to investi- dressed. Fax:46 18 4714975. E-mail: Paraskevi.Heldin@medkem. uu.se. gate the effect of hyaluronan oligosaccharides on the 223 0014-4827/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.