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ORIGINAL RESEARCH ARTICLE

Heparanase Upregulation in High Glucose-Treated Endothelial Cells Is Prevented by Insulin and Heparin

Juying Han^*, Adrienne E. Woytowich^*, Anil K. Mandal and Linda M. Hiebert^*,1

^* Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5B4; and Department of Medicine, University of Florida, Jacksonville, Florida 32086

To whom requests for reprints should be addressed at ¹ Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan, S7N 5B4 Canada. E-mail: linda.hiebert{at}usask.ca

Heparan sulfate proteoglycans on the endothelial cell surface and extracellular matrix play an important role in vascular homeostasis. Previous studies have shown that the quantity of heparan sulfate is reduced in kidney and other organs in diabetes. The objectives of this study were to determine if heparanase is induced by high glucose in endothelial cells and if heparin and/or insulin or basic fibroblast growth factor (bFGF) affect this upregulation. Cultured porcine aortic endothelial cells in M199 medium were treated with high glucose (30 mM) and/or bFGF (1 or 10 ng/ml) or high glucose plus insulin (1 U/ml) and/or heparin (0.5 µg/ml) for 7 days. To help define the mechanism of endothelial damage, cells were also exposed to H₂O₂ (0.1 mM) for 1 day or mannitol (30 mM) for 7 days. Heparanase mRNA was detected by reverse transcription polymerase chain reaction. Heparanase activity was measured by incubating cell lysates with [³⁵S]labeled extracellular matrix of bovine corneal endothelial cells and analyzing released radioactive products by gel filtration and ß-scintillation. Heparanase mRNA was found in high-glucose– and H₂O₂-treated cells; however, it was not found in control cells, mannitol- or high glucose plus insulin– and/or heparin-treated cells, or fresh porcine tissue. Heparanase activity was only found in high-glucose– and H₂O₂-treated cells. As well, bFGF did not prevent heparanase mRNA upregulation by high glucose. From these observations, we concluded that heparanase upregulation by high glucose is prevented by insulin and/or heparin but not bFGF. Reactive oxygen species, but not changes in osmolarity, may be involved in the upregulation of heparanase.

Key Words: endothelial cells • high glucose • heparanase • heparin • insulin