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J.-H. Chang, T. Kojima, T. Onguchi; Anti-Lymphangiogenic Effect of Collagen XVIII and Neostatin. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3301. doi: https://doi.org/.
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To determine the anti-lymphangiogenic effect of collagen XVIII and its MMP-7-derived proteolytic product, neostatin-7.
Recombinant GST- neostatin-7 and GST proteins were purified from E. coli (BL21DE3) transformed with pGEX or pGEX-neostatin-7. Purified proteins were dialyzed against PBS. Corneal micropocket assay was performed. Hydron pellets containing 80 ng of human bFGF with either 500ng of GST or GST-neostatin-7 were implanted into corneal pockets. The eyes were then examined and photographed on day 7 post-pellet implantation by slit lamp microscopy. The neovascular area was calculated using a NIH ImageJ program. Corneas were whole mount immunostained with anti-LYVE-1 and CD31 antibodies and analyzed by confocal microscopy. VEGFR3 proteins were incubated with GST or GST-neostain-7 in vitro and pulldown using glutathione bead. In vitro association of neostatin-7 and VEGFR3 were determined by Western blot analysis using anti-VEGFR3 antibody.
Enhanced bFGF-induced corneal lymphangiogenesis in MMP-7 knockout mice. Micropellet implantation of recombinant GST-neostatin-7 with bFGF resulted in a significant reduction in bFGF-induced corneal neovascularization (1.5mm2+0.3mm2) and lymphangiogenesis (40,900pixels + 3,600pixels) when compared to GST treated corneal NV (2.6mm2+0.5mm2) and lymphangiogenesis (17,000pixels+3,300pixels). Western blot analysis showed neostatin-7 binds to VEGFR-3 (flt4) in vitro. Enhanced corneal lymphangiogenesis and VEGF-C expression were revealed in collagen XVIII knockout mice using a keratectomy model when compared to wild type mice.
These results provide supportive evidence of a possible anti-lymphangiogenic effect of neostatin-7 on corneal
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