Purchase this article with an account.
J-H Chang, G-Y Chang, EE Gabison, N Fukai, DT Azar; Endostatin-Like Molecule Generated by Matrilysin Cleavage of Collagen Type XVIII Inhibits Vascular Endothelial Cell Proliferation . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1867.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose:We have previously reported the formation of 28 kDa proteolytic fragment, including the site for endostatin, based upon MMP-7 cleavage of the NCI domain in collagen XVIII (Lin et al., IOVS 2001). Our current goal is to compare the anti-angiogenic properties of this endostatin-spanning molecule to those of endostatin. Methods: Recombinant human NC1 fragments were purified according to Yamaguchi et al., (EMBO J. 1999). Purified NC1 fragments were cleaved with MMP-1, -2, -3, -7, -9, -12). Recombinant His-endostatin and endostatin-like molecules were purified from pET system. Bovine calf pulmonary artery endothelial (CPAE) cells were grown to confluence in DMEM with 10% fetal calf serum (FCS) and remained confluent for 48 hours. Cells were then trypsinized at 37oC for five minutes and subsequently harvested. A suspension of 2,000 cells in 200 ml of DMEM with 0.5% FCS was added to each well of a 96-well plate. The cells were incubated for 24 hours at 37oC before the medium was replaced with DMEM containing either 10% FCS, then treated with either mouse recombinant endostatin or 28 kDa fragment (10, 5, and 2.5 ml/ml). CPAE cells in 0.5% FCS and 10% FCS were used as controls. After 24 hours, cell density was determined using the CellTiter 96 assay (Promega). Statistical analyses were performed using ANOVA test and t-test, P<0.05 Results: Matrilysin (MMP-7) cleaved recombinant human NC-1 to generate a 28 kDA endostatin-like molecules. In control, MMP-1, -2, -3, -9 and -12 did not cleave the recombinant NC1 domain of collagen XVIII. MMP-7 cleavage occurred at the hinge domain of collagen XVIII which is different from that of cathepsin cleavage site. Affinity purified endostatin and endostatin-spanning fragments were confirmed by Western blotting analysis. In the functional assays, both His-endostatin and the endostatin-spanning 28 kDa fragment significantly inhibited the proliferation of the CPAE cells when compared to controls. Conclusion: The 28 kDa endostatin-spanning molecule generated by matrilysin cleavage of collagen XVIII has similar anti-angiogenic effect as endostatin in vitro.
This PDF is available to Subscribers Only