Generation of biologically active endostatin fragments from human collagen XVIII by distinct matrix metalloproteases.

Endostatin, a potent inhibitor of endothelial cell proliferation, migration, angiogenesis and tumor growth, is proteolytically cleaved from the C-terminal noncollagenous NC1 domain of type XVIII collagen. We investigated the endostatin formation from human collagen XVIII by several MMPs in vitro. The generation of endostatin fragments differing in molecular size (24-30 kDa) and in N-terminal sequences was identified in the cases of MMP-3, -7, -9, -13 and -20. The cleavage sites were located in the protease-sensitive hinge region between the trimerization and endostatin domains of NC1. MMP-1, -2, -8 and -12 did not show any significant activity against the C-terminus of collagen XVIII. The anti-proliferative effect of the 20-kDa endostatin, three longer endostatin-containing fragments generated in vitro by distinct MMPs and the entire NC1 domain, on bFGF-stimulated human umbilical vein endothelial cells was established. The anti-migratory potential of some of these fragments was also studied. In addition, production of endostatin fragments between 24-30 kDa by human hepatoblastoma cells was shown to be due to MMP action on type XVIII collagen. Our results indicate that certain, especially cancer-related, MMP family members can generate biologically active endostatin-containing polypeptides from collagen XVIII and thus, by releasing endostatin fragments, may participate in the inhibition of endothelial cell proliferation, migration and angiogenesis.

Differential expression of laminin isoforms in ovarian epithelial carcinomas suggesting different origin and providing tools for differential diagnosis.

Immunohistochemistry was used to study the distribution of laminin (Ln) chains, collagen types IV (alpha 1/2), VII, and XVIII and Lutheran antigen (Lu) in 36 frozen ovarian carcinoma samples. Surface epithelial basement membrane (BM) of the normal ovary showed immunoreactivity for Ln alpha1, alpha3-alpha5, beta1-3, gamma1, and gamma2 chains and type IV and XVIII collagens. Chains of Ln-5 (alpha3beta3gamma2) and Ln-10 (alpha5beta1gamma1) as well as type IV and XVIII collagens were found in most tumor BMs, but Ln alpha2 chain and type VII collagen were detected only in few tumors. Contrary to serous tumors, BMs of mucinous carcinomas showed Ln alpha4 chain, but not Ln alpha1 and beta2 chains. Ln alpha1 chain was found in most endometrioid carcinomas, whereas chains of Ln-5 were only moderately detectable in comparison with serous and mucinous carcinomas. In the normal ovary, Lu immunoreactivity was confined to basal aspect in the ovarian epithelial cells, but in tumor specimens Lu immunostainings showed variable polarized and nonpolarized patterns. The results suggest that the three types of ovarian carcinoma have distinct differences in their Ln distribution and can be grouped based on their expression pattern. This suggests that they may have histogenetically different precursors and may help to distinguish these tumors from each other.

Endostatin inhibits human tongue carcinoma cell invasion and intravasation and blocks the activation of matrix metalloprotease-2, -9, and -13.

Endostatin, a 20-kDa collagen XVIII fragment, inhibits angiogenesis and tumor growth in vivo, but the mechanisms are still unclear. Matrix metalloproteases (MMPs), a family of extracellular and membrane-associated endopeptidases, collectively digest almost all extracellular matrix and basement membrane components, and thus play an important role in tumor progression. We studied the effects of recombinant human endostatin on human MMP-2, -9, -8, and -13. We found that endostatin inhibited the activation and catalytic activity of pro-MMP-9 and -13 as well as recombinant pro-MMP-2. It prevented the fragmentation of pro-MMP-2 that was associated with reduction of catalytic activity. Endostatin had no effect on MMP-8 as shown by collagenase activity assays. An in vitro migration assay and an in vivo chicken chorioallantoic membrane intravasation assay with the human tongue squamous cell carcinoma cell line HSC-3 revealed the biphasic nature of endostatin; low endostatin concentrations inhibited intravasation and migration of these cells in a dose-dependent manner, but at increased concentrations, the inhibitory effect was far less efficient. The results show that endostatin blocks the activation and activities of certain tumor-associated pro-MMPs, such as pro-MMP-2, -9, and -13, which may explain, at least in part, the antitumor effect of endostatin. Our results also suggest that endostatin inhibits tumor progression by directly affecting the tumor cells and not just acting via endothelial cells and blockage of angiogenesis.