Angiostatin was one of the first tumor-generated angiogenesis inhibitors to be identified. The mechanisms of action appears to be highly specific, as angiostatin inhibits migration and induces apoptosis specifically in endothelial cells in vitro and interferes with blood vessel formation in vivo. Here we report the identification of angiomotin, a novel protein mediating angiostatin inhibition of migration and tube formation of endothelial cells. We used the yeast two-hybrid system to identify angiostatin-binding peptides in a human placental cDNA library. This screening procedure has been widely used to identify protein–protein interactions such as proteins that interact with the cytoplasmic parts of receptors. However, it has also been shown to be a useful tool to analyze the interactions of extracellular ligands and their receptors (Zhu and Kahn 1997
). The sequence encoding the angiostatin-binding domain of angiomotin was identified in three independent clones after screening a placental cDNA library with the K1–4 domains of angiostatin. The amino acid sequence was rich in alanine (40%) and proline (20%). The proline/alanine rich sequence included 4 (PXXP) motifs which may be potential binding sites for Src homology 3 domains which may be found in a diverse group of signal-transducing molecules (Sudol 1998
). The angiomotin amino acid sequence did not appear to contain any of the signal sequences that are normally found in membrane receptors or secreted proteins. This is in analogy with other plasminogen- or angiostatin-binding proteins such as alpha enolase, annexin II, or the more recently identified angiostatin-binding protein, ATP synthase (Miles et al. 1991
; Das et al. 1994
; Hajjar et al. 1996
). These proteins lack the endoplasmic reticulum targeting peptide but are still localized to and are able to bind plasminogen or angiostatin on the endothelial cell surface.
The expression of angiomotin in the endothelium was verified by immunohistochemical staining of placental vessels. In the human term placenta, positive staining was detected in larger vessels as well as in capillaries. In addition, positive staining could also be detected in cytotrophoblasts, a cell type characterized by its ability to invade the endometrium and form a transitional circulatory system during early embryogenesis. Interestingly, these cells have been shown to express other “endothelial cell–specific” proteins such as the vascular endothelial growth factor (VEGF)-receptor 1 and tyrosine kinase with immunoglobin and EGF homology domains (TIE)-2 (Ahmed et al. 1995
; Dunk et al. 2000
). We also analyzed the expression pattern of angiomotin in Kaposis sarcoma lesion, a tumor with endothelial origin. Positive staining could be detected in vessels infiltrating the tumor. Interestingly, dermal capillaries in adjacent normal tissue in the same section were negative, thus indicating that there is a differential expression of angiomotin in vessels of normal and pathological tissues.
The endothelial cell migration assay has proven to be a consistent predictor of inhibition of neovascularization in vivo. Indeed, angiostatin, endostatin, and thrombospondin all inhibit endothelial cell migration in vitro (Good et al. 1990
; Ji et al. 1998
; Yamaguchi et al. 1999
). Thrombospondin is probably the best characterized angiogenesis inhibitor with regard to signaling pathways. It binds the CD36 receptor and activates p38 mitogen-activated protein kinase (MAPK) via the src-related kinase, c-fyn (Jimenez et al. 2000
). In contrast, angiostatin-induced inhibition of angiogenesis is not dependent on either CD36 or c-fyn, as was recently shown in the mouse cornea angiogenesis assay (Jimenez et al. 2000
Our findings indicate a direct functional role for angiomotin in endothelial cell migration, as angiomotin is located in areas of actin reorganization and cells expressing angiomotin migrate significantly faster than control cells. Treatment of angiomotin-transfected cells with angiostatin efficiently inhibited basal as well as bFGF-stimulated migration of these cells, indicating that there is a direct functional link between angiomotin and angiostatin. Furthermore, we also showed that angiostatin reduced the total tube length generated by angiomotin-transfected cells in the matrigel assay by 90%. However, endothelial cells that are plated on Matrigel rapidly migrate to form cell aggregates. Endothelial tubes are then extended between these aggregates of cells. Angiostatin-treated MAE-angiomotin cells remained primarily as single cells on the matrigel and did not form aggregates. This observation suggests that angiostatin interferes with the early processes of tube formation involving cell motility.
The angiomotin sequence provides little information into exactly how it may be involved in mediating angiostatin inhibition of in vitro angiogenesis. The lack of a signal peptide and transmembrane domain argues that angiomotin does not act as a typical membrane receptor. The NH2-terminal coil–coil domain as well as the proline rich sequences in the angiostatin-binding domain suggest that angiomotin forms protein complexes. Considering the stimulatory effect of angiomotin on endothelial cell migration, we speculate that angiostatin antagonizes angiomotin function by inhibiting the formation of complexes with other proteins.