Heparan–Sulfate–ligand interaction in corneal angiogenesis.

Abstract

Abstract: : Purpose: Inhibiting pathologic neovascularizations represents an important therapeutic modality for a variety of diseases, including corneal neovascularisations. Of the multiple endogenous factors controlling angiogenesis heparan sulfate–like glycosaminoglycan (HSGAG) play a major role. They act as a co–receptor for of growth factors e.g. bFGF, VEGF as well as factors of the coagulation cascade such as antithrombin III. It is, depending on its fine chemical structure, capable of rendering these growth factors active or inactive. This chemical diversity arises from chain modifications, introduced by a series of enzymes like the N–acetylase/N–sulfotransferase (NST), glucosaminyl 3–O–sulfotransferase (3–OST), the iduronosyl 2–O–sulfotransferase (2–OST) and the glucosaminyl–6–O–sulfotransferase (6–OST). Herein, we study the structure, angiostatic ability and localization of HLGAG in–vitro and in–vivo in corneal neovascularisations. Methods: Heparinase III (hep III) was expressed and purified. The antiproliferative effect of Heparinase III treatment on bovine capillary endothelial cells was tested. The angiostatic ability of hep III was tested in a corneal pocket assay. Further changes in the expression levels of the HSGAG modifying enzymes NST, 2–OST, 3–OST and 6–OST in corneal neovascularisationes, were measured in normal and vascularised mouse corneas. Co–staining for heparan sulfate (HS) and its ligand AT III in vascularised mouse corneas, was performed to test the hypothesis, that HS might play a role in suppressing the coagulation cascade in these vessel. Results: At 100 nM, hep III inhibited endothelial cell proliferation by up to 74. In the corneal pocket assay 2 pmol of hep III, resulted in a 61% reduction of bFGF induced corneal neovascularization. In vascularised mouse corneas an increase in the expression of HSGAG modifying enzymes was seen. NST increased by 33±8%, 2–OST by 101±9% and 6–OST by 130±6%. 3–OST expression was increased by 203±4%. HS and AT III were found both to co–localize to the vascular endothelial cells of new formed vessels. Conclusions: Herein we demonstrate that through enzymatic digestion of cell surface HLGAG inhibition of eondothelial cell proliferation and angiogenesis is achieved. We also found an increase in HSGAG modifying enzymes supporting the notion of a relevance of the chemical diversity of the sugar side chains in the development of neovascularisations.

Keywords: cornea: basic science • proteoglycans/glycosaminoglycans