Differential effects of Heparitinase I and Heparitinase III on endothelial tube formation in vitro

Abstract

Heparan sulfate proteoglycans (HSPGs) play vital roles in many steps of angiogenesis under physiological and pathological conditions. HSPGs on endothelial cell surfaces act as co-receptors for a variety of pro-angiogenic growth factors such as FGF and VEGF and anti-angiogenic factors such as endostatin. However, the fine structural requirements of these binding interactions are dependent on the sulfation patterns of HSPGs. Previous studies have shown that Heparitinases, heparin lyases isolated from Flavobacterium heparinum, can cleave heparan sulfate chains. These enzymes have been shown to reduce tumor-derived neovascularization in vivo in mice. However, the results from these experiments could not conclusively pinpoint the origin of the HS fragments. Thus, in this study we utilized an in vitro assay to assess the differential effects of Heparitinase I (Hep I) and Heparitinase III (Hep III) on endothelial tube formation. Hep III was found to be a more potent inhibitor of tube formation than Hep I. In conclusion, differential cleavage of endothelial cell surface bound HS can affect the extent of inhibition of tube formation.

Figure 1

Site of action of a) Hep I and b) Hep III. Hep I fragments NA domains so that only NS domains are left intact. Hep III fragments NS domains and leaves NA domains intact.

Figure 2

Hep III and Hep I were added at equal activities onto BLMVEC on GFR matrigel. a) Control without enzymes b) –Control with 20μM Sulforaphane c) Hep I 30 mU d) Hep I 100 mU e) Hep III 30 mU f) Hep III 100 mU. Results are from three independent experiments performed in triplicate.