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This study investigated the functional interplay between vascular endothelial growth factor (VEGF) and metalloproteinases (MMPs) in ovarian carcinomas. Levels of MMP9 (pro and activated form) and proMMP2 in ascites correlated with VEGF and with the ascitic volume in nude mice bearing human ovarian carcinoma xenografts (HOC22 and HOC8). The MMP inhibitor batimastat (BB-94) reduced VEGF release and ascitic fluid formation. Exogenous, activated MMP9, and, to a lesser extent, MMP2, increased VEGF release by SKOV3 and OVCAR3 ovarian carcinoma cells. The effect was dose and time dependent and inhibited by BB-94. MMP9-released VEGF was biologically active, because it induced endothelial cell motility, and its activity was prevented by the VEGF inhibitor SU5416. Our results indicate that MMPs, mainly MMP9, play a role in the release of biologically active VEGF and consequently in the formation of ascites.

The tubulin-binding agent ZD6126 is a novel vascular-targeting agent in clinical development for the treatment of solid tumors. In vivo, ZD6126 is rapidly converted into N-acetylcolchinol (ZD6126 phenol). In this study, we have explored the antivascular property of N-acetylcolchinol in vitro and ZD6126 in vivo. In cell culture, N-acetylcolchinol induced rapid changes in the morphology of human umbilical vein and lung microvessel endothelial cells. Within 40 min, the compound induced endothelial cell contraction, destabilization of the tubulin cytoskeleton, induction of actin stress fibers, and membrane blebbing. These effects occurred at noncytotoxic concentrations and were rapidly reversed on removal of the drug. Nonconfluent endothelial cells were more sensitive than confluent, quiescent cells. Among different cell types, endothelial cells were the most sensitive to the induction of morphological changes, whereas smooth muscle cells were not affected. In vitro, N-acetylcolchinol rapidly disrupted a network of newly formed cords. In vivo, ZD6126 caused shut down of newly formed vessels in the Matrigel plug assay, shortly after injection. This study indicates that rapid alteration of endothelial cell morphology may be responsible for the loss of tumor blood vessel integrity, vessel shut down, and extensive tumor necrosis induced by ZD6126 in experimental tumor models.

Polyoma virus middle T-transformed murine endothelioma cell lines provide a useful model for studying vascular lesions such as hemangiomas, hemangiosarcomas, and Kaposi's sarcoma and tumor-associated angiogenesis. In vivo they produce fast-growing, hemorrhaging, cavernous blood-filled hemangiomas, mainly formed by recruited host endothelial cells, suggesting an angiogenesis-like process underlying the lesion. The molecular mechanism(s) responsible for the recruitment of host endothelial cells by endothelioma cells has not yet been identified. We found that five different cultured endothelioma cell lines produced a soluble factor, named endothelioma-derived motility factor (EDMF) that stimulates chemotaxis (motility induced by a gradient of soluble attractant), haptotaxis (motility in response to substrate-bound attractant), and chemoinvasion (migration through a layer of reconstituted basement membrane, Matrigel) of normal human, bovine, and murine endothelial cells. The inhibitory effect of actinomycin D and of enzymatic treatment on its activity proved that EDMF is a protein. EDMF binds to heparin, since its activity was inhibited by heparin, and it was retained on a heparin-Sepharose column. Its molecular weight, as assessed by Sephacryl S-200 gel filtration, ranges from 40,000-65,000. Although in many aspects EDMF is similar to vascular permeability factor-vascular endothelial growth factor, this was not detected in endothelioma cell supernatants, as assessed by enzyme-linked immunosorbent assay, thus indicating that EDMF might be related to, but is not identical with, vascular permeability factor. Our findings support the notion that recruitment of host endothelial cells by endothelioma cells in vivo might be mediated by a still unidentified, soluble factor that stimulates and directs endothelial cell migration.

Stable attachment of cancer cells to the endothelium is a key step in the formation of metastasis. In this study, we have investigated the possibility that interaction between laminin and its Mr 67,000 high-affinity receptor (67 LR) could play a major role in this process. Scatchard analysis of laminin-binding studies showed that bovine aortic endothelial cells exhibit 46,000 high-affinity receptors that mediate, at least in part, the attachment of highly invasive melanoma cells. This endothelial cell-melanoma cell interaction was significantly inhibited by soluble laminin and by anti-laminin antibodies. Peptide G, an eicosapeptide derived from the complementary DNA sequence of the 67 LR precursor (IPCNNKGAHSVGLMWWMLAR) that specifically binds to laminin and presumably contains the active ligand-binding site of the receptor, specifically prevented attachment of the melanoma cells to both the bovine aortic endothelial cell monolayer and human umbilical vein endothelium. Thus, peptide G may selectively interfere with the metastatic cascade by inhibiting tumor cell attachment to endothelium via the laminin-67 LR pathway and is a potential new antimetastatic agent.

Polyclonal antisera were made against biochemically purified laminin receptor protein as well as against synthetic peptides deduced from a complementary DNA clone corresponding to the COOH-terminal end of the laminin receptor (U.M. Wewer et al., Proc. Natl. Acad. Sci. USA, 83: 7137-7141, 1986). These antisera were used to study the potential role of laminin receptor in laminin-mediated attachment and haptotactic migration of human A2058 melanoma cells. The anti-laminin receptor antisera reacted with the surface of suspended, nonpermeabilized melanoma and carcinoma cells. The anti-laminin receptor antisera blocked the surface interaction of A2058 cells with endogenous laminin, resulting in the inhibition of laminin-mediated cell attachment. The A2058 melanoma cells migrated toward a gradient of solid phase laminin or fibronectin (haptotaxis). Anti-laminin antiserum abolished haptotaxis on laminin but not on fibronectin. Synthetic peptide GRGDS corresponding to the fibronectin cell-binding domain inhibited haptotaxis on fibronectin but not on laminin. Both types of anti-laminin receptor antisera inhibited haptotaxis on laminin but not on fibronectin. Using immunohistochemistry, invading human carcinoma cells in vivo exhibited a marked cytoplasmic immunoreactivity for the receptor antigen. Together these findings indicate a specific role for the laminin receptor in laminin-mediated migration and that the ligand binding of the laminin receptor is encompassed in the COOH-terminal end of the protein.