A possible fourth angiogenesis inhibitor in bovine cartilage has been purified not from cartilage but from the culture fluid of bovine chondrocytes grown in the laboratory. This inhibitor, which has been named chondrocyte-derived inhibitor (ChDI), is a protein that has a molecular mass of approximately 36,000. It has been reported that ChDI and CDI/TIMP-1 have similar antiangiogenic activities, Reviewed in [17,34] but the relationship between these proteins is unclear because amino acid sequence information for ChDI is not available. Thus, whether CDI/TIMP-1 is a breakdown product of ChDI or whether ChDI is truly the fourth angiogenesis inhibitor identified in bovine cartilage is unknown.
As indicated previously, shark cartilage, like bovine cartilage, contains more than one type of angiogenesis inhibitor. One shark cartilage inhibitor, named U-995, reportedly contains two small proteins, one with a molecular mass of approximately 14,000 and the other with a molecular mass of approximately 10,000. Both proteins have shown antiangiogenic activity when tested individually. The exact relationship between these two proteins and their relationship to the larger bovine angiogenesis inhibitors are not known because amino acid sequence information for U-995 is not available. U-995 has been reported to inhibit endothelial cell proliferation, endothelial cell migration, matrix metalloproteinase activity in vitro, and the formation of new blood vessels in the chorioallantoic membrane of chicken embryos. It does not appear to inhibit the proliferation of other types of normal cells or of cancer cells in vitro. Intraperitoneal but not oral administration of U-995 has been shown to inhibit the growth of mouse sarcoma-180 tumors implanted subcutaneously on the backs of mice and the formation of lung metastases of mouse B16-F10 melanoma cells injected into the tail veins of mice.
The second angiogenesis inhibitor identified in shark cartilage appears to have been studied independently by three groups of investigators.[2,27,36] This inhibitor, which was named SCF2 by one of the groups, is a proteoglycan that has a molecular mass of about 10,000. Proteoglycans are combinations of glycosaminoglycans and protein. Reviewed in  The principal glycosaminoglycan in SCF2 is keratan sulfate. SCF2 has been shown to block endothelial cell proliferation in vitro,[2,27,36] the formation of new blood vessels in the chorioallantoic membrane of chicken embryos,[2,27] and tumor-induced angiogenesis in the corneas of rabbits.[2,27]
Other studies have demonstrated that AE-941/Neovastat, the previously mentioned aqueous extract of shark cartilage, has antiangiogenic activity,[13,28,29] Reviewed in [35,37,38,39,40] but the molecular basis for this activity has not been defined. Therefore, whether AE-941/Neovastat contains U-995 and/or SCF2 or some other angiogenesis inhibitor is not known. It has been reported that AE-941/Neovastat inhibits endothelial cell proliferation and matrix metalloproteinase activity in vitro and the formation of new blood vessels in the chorioallantoic membrane of chicken embryos.[13,28,32] In addition, AE-941/Neovastat has been shown to induce endothelial cell apoptosis by activating caspases, enzymes important in the promotion and regulation of apoptosis. Reviewed in [35,39] It also appears to inhibit the action of vascular endothelial growth factor, thus interfering with the communication between tumor cells and nearby blood vessels. Reviewed in [35,38,39] AE-941/Neovastat may also inhibit angiogenesis through promotion of tissue plasminogen activator (tPA) activity. Neovastat stimulates tPA expression in endothelial cells through an increase in the transcription of the tPA gene. This transcriptional activation is associated with activation of c-Jun N-terminal kinase (JNK) and nuclear factor-kappa B (NF-kappa B) signaling pathways to an extent similar to tumor necrosis factor-alpha (TNF-alpha). Furthermore, AE-941/Neovastat has been reported to inhibit the growth of DA3 mammary adenocarcinoma cells and the metastasis of Lewis lung carcinoma cells in vivo in mice. Reviewed in [5,35,42] In the Lewis lung carcinoma experiments, AE-941/Neovastat enhanced the antimetastatic effect of the chemotherapy drug cisplatin. Reviewed in [5,35,42] All the aspects of preclinical development have been reviewed.