Hyaluronan Fragments Induce Endothelial Cell Differentiation in a CD44- and CXCL1/GRO1-dependent Manner

Yoshinori Takahashi,Lingli Li,Masaru Kamiryo,Trias Asteriou,Aristidis Moustakas,Hidetoshi Yamashita,Paraskevi Heldin

doi:10.1074/jbc.m411913200

Yoshinori Takahashi, Lingli Li + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m411913200

Copy DOI

Abstract

Hyaluronan is a glycosaminoglycan of the extracellular matrix. In tumors and during chronic inflammatory diseases, hyaluronan is degraded to smaller fragments, which are known to stimulate endothelial cell differentiation. In this study, we have compared the molecular mechanisms through which hyaluronan dodecasaccharides (HA12), and the known angiogenic factor, fibroblast growth factor 2 (FGF-2), induce capillary endothelial cell sprouting in a three-dimensional collagen gel. The gene expression profiles of unstimulated and HA12- or FGF-2-stimulated endothelial cells were compared using a microarray analysis approach. The data revealed that both FGF-2 and HA12 promoted endothelial cell morphogenesis in a process depending on the expression of ornithine decarboxylase (Odc) and ornithine decarboxylase antizyme inhibitor (Oazi) genes. Among the genes selectively up-regulated in response to HA12 was the chemokine CXCL1/GRO1 gene. The notion that the induction of CXCL1/GRO1 is of importance for HA12-induced endothelial cell sprouting was supported by the fact that morphogenesis was inhibited by antibodies specifically neutralizing the CXCL1/GRO1 protein product. HA12-stimulated endothelial cell differentiation was exerted via binding to CD44 since it was inhibited by antibodies blocking CD44 function. Our data show that hyaluronan fragments and FGF-2 affect endothelial cell morphogenesis by the induction of overlapping but also by distinct sets of genes.

Highlights

Hyaluronan is a glycosaminoglycan of the extracellular matrix
Since considerable changes in the levels and molecular mass of hyaluronan molecules have been detected in tissues during the progression of cancer, wounding, and inflammation, we have studied how hyaluronan fragments affect endothelial cell differentiation at the molecular level
We showed that defined hyaluronan fragments of six disaccharides induced endothelial sprouting through up-regulation of the mouse chemokine CXC chemokine ligand 1 gene (CXCL1)/GRO1 since blocking antibodies completely suppressed the HA12induced capillary morphogenesis (Fig. 4)

Summary

EXPERIMENTAL PROCEDURES

Antibodies and Reagents—Monoclonal anti-mouse CXC chemokine antibody (rat IgG2A, Clone 124014) was purchased from R&D Systems. Endothelial cells were cultured for 48 h on fibronectin-gelatin coated 75-cm bottles in Ham’s F-12 medium containing 0.25% BSA. In the experiments in which antibodies were used, the Ham’s F-12 medium containing 0.25% BSA was premixed with each one of the monoclonal antibodies against CXCL1/ GRO1 or CD44, as well as the isotope controls rat IgG2A and rat IgG1, respectively, and added to the cultures for 30 min before stimulation of the cells with HA12 or FGF-2. CDNA Microarray Analysis—Total RNA was prepared from mouse endothelial cells cultured in three-dimensional collagen gels after different time periods (1, 4, and 12 h) of incubation in the absence or presence of HA12 (100 ␮g/ml) or FGF-2 (5 ng/ml).

RESULTS

GenBankTM accession number bp

DISCUSSION