Inhibition of angiogenesis by HC·HA, a complex of hyaluronan and the heavy chain of inter-α-inhibitor, purified from human amniotic membrane.

Purpose: To determine whether antiangiogenic action of the amniotic membrane (AM) can be mediated by HC·HA, a covalent complex of hyaluronan (HA) and the heavy chain (HC) of inter-α-inhibitor, purified from AM soluble extract.
Methods: HC·HA action on viability, proliferation, attachment, death, migration, and differentiation of human umbilical vein endothelial cells (HUVECs) and neovascularization in chicken chorioallantoic membrane (CAM) was examined by MTT assay, BrdU labeling, cell proliferation assay, cell death detection ELISA, transwell assay, tube formation assay, and CAM assay.
Results: HC·HA suppressed HUVEC viability more significantly than HA and AM stromal extract, and such suppression was not mediated by CD44. HC·HA also caused HUVECs to become small and rounded, with a decrease in spreading and filamentous actin. Without promoting cell detachment or death, HC·HA dose dependently inhibited proliferation (IC(50), 2.3 μg/mL) and was 100-fold more potent than HA. Migration triggered by VEGF and tube formation was also significantly inhibited by HC·HA. Purified HC·HA did not contain PEDF and TSP-1 but did contain IGFBP-1 and platelet factor 4 while significantly suppressing neovascularization in CAM.
Conclusions: The antiangiogenic activity of HC·HA might explain why AM is developmentally avascular and how AM might exert an antiangiogenic action when transplanted to the ocular surface, and it might indicate a potential therapeutic effect of HC·HA in diseases manifesting pathogenic angiogenesis. Roles of IGFBP-1 and platelet factor 4 in HC·HA antiangiogenic action warrant further investigation.