Oligomeric collagen XVIII‐derived endostatin requires cell surface heparan sulfate in order to induce morphological changes in endothelial and epithelial cell lines in vitro

Abstract

Introduction Monomeric endostatin (HEM) has been extensively investigated as an endogenous anti‐angiogenic molecule. In contrast, oligomeric endostatin (HED) induces a promigratory phenotype in endothelial cells in vitro that can be inhibited by an EM in a putative negative feedback loop (Kuo et al. 2001). Endostatin is heparin‐binding but the role of cell‐surface glycosaminoglycans (GAG) in regulating the activity of HEM/HED is unclear.Methods and results Bovine aortic endothelial (BAE) cells plated onto a Matrigel substratum spontaneously form capillary‐like tubules. Addition of 50 nm HED, 16 h after plating induces the migration of BAE cells away from these tubules. This pro‐migratory action can be inhibited by coincubation with HEM. We have demonstrated that exogenous GAGs can also inhibit the actions of HED. This property is size dependent, with heparin‐derived oligosaccharides containing more than 20 monosaccharide units being optimal inhibitors. HED (50 nm) was also shown to induce marked morphological changes in the Chinese hamster ovary (CHO) epithelial cell line plated in Matrigel, i.e. cell elongation, lamellipodia formation and intercellular bridging. This novel observation allowed us to address the role of cell surface GAGs in HED function using a panel of CHO mutants with defined defects in GAG biosynthesis. CHO 745 cells, which lack all cell surface sulfated GAGs, fail to respond to HED stimulation, while CHO pgsD cells, which specifically lack cell surface heparan sulfate (HS), displayed an attenuated phenotype on HED stimulation with less cell elongation/intercellular bridging. Notably, CHO pgsF17 cells, which lack 2‐O‐sulfation of HS, respond normally to HED indicating that HS 2‐O‐sulfate groups are not necessary for the bioactivity of HED.To investigate the endostatin–GAG interaction further, zero‐length cross‐linking was utilized. This indicated that multiple HEM subunits can simultaneously bind to a single oligosaccharide chain with up to 3 HEM molecules cross‐linking to a heparin dp22. HED cross‐linking experiments indicate that both endostatin subunits in this molecule may bind to the same oligosaccharide chain. These results suggest that one potential anti‐angiogenic mechanism of HEM is to block the GAG‐binding sites for HED.Discussion Cell surface GAGs are absolutely necessary for the bioactivity of HED. HS is required for a wild‐type response, although 2‐O‐sulfation is not essential. The ability to use a panel of GAG‐mutant CHO cells provides a well‐understood system in which to investigate the role of HS in HEM : HED regulation.