Angiogenesis involves the activation of vascular endothelium or endothelial progenitor cells through a series of cellular events including survival, migration, proliferation, and tube formation. Kobayashi et al.
33 noted that the conditioned medium of cultured amniotic epithelial and mesenchymal cells contains antiangiogenic activity, though the source of this activity remains obscure. The finding that ASE, which did not contain epithelial or basement membrane components, suppressed HUVEC viability (
Fig. 1) was in agreement with previous findings by Jiang et al.
11 PEDF, a potent antiangiogenic factor,
26 was predominantly found in the AM basement membrane.
27 Transcripts and proteins of tissue inhibitors of metalloproteinase and TSP-1—i.e., potential antiangiogenic factors—are expressed by both human amniotic epithelial and mesenchymal cells.
28 However, the roles of these three proteins in delivering antiangiogenic action in the AM cannot be ascertained because their protein levels are negligible in the medium after the incubation of human AM.
34 Our Western blot analysis showed that PEDF was present in the AM extract but not in the purified HC·HA complex (
Fig, 2A), but TSP-1 was present in neither AM extract nor purified HC·HA (not shown). These results from Western blot analysis were further confirmed by the data of human angiogenesis arrays (
Fig. 2B). Collectively, we conclude that the antiangiogenic action of the HC·HA complex, exerted by reducing HUVEC viability, proliferation, migration, and differentiation, is more potent than that of ASE and is not contributed by PEDF and TSP-1. Herein, our protein arrays showed that IGFBP-1, PF4, or both were selectively bound to the HC·HA complex (
Figs. 8B,
8C). Previously, IGFBP-1
35,36 has been shown to suppress cell growth and PF4
37 –39 has been shown to mediate antiangiogenic action. Thus, further studies are needed to determine whether the antiangiogenic actions of these two proteins are promoted by their binding to the HC·HA complex.