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J.-H. Chang, K.-Y. Han, A. Sabri; Collagen XVIII Derived Peptides Regulate Lymphangiogenesis. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4968.
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To determine the involvement of neostatin-7, a collagen XVIII-derived peptide, in corneal lymphangiogenesis in vitro and in vivo.
In vitro assays: Neostatin-7 and nine ~27-mer Neostatin-7 peptides (peptides 1-9) were synthesized. The binding of neostatin-7 and its peptides to recombinant VEGFR3 were determined by western blot analysis and Surface plasmon resonance (biacore T100) assays. Lymphatic endothelial cells were divided into 4 groups: Group 1, untreated LECs; Group 2, VEGF-C stimulated LECs; Group 3, neostatin-7 treated LECs; and Group 4, VEGF-C stimulated LECs in the presence of neostatin-7. LECs were lysed and equal amount of proteins were subjected to 2-D gel analysis. Differentially expressed proteins from these four groups were subjected to Mass spectrometric analysis. In vivo assays: 1.5-mm mid-stromal keratectomy wounds were performed in WT and collagen XVIII knockout mice and the corneas were dissected 7 days following wounding. Corneal micropocket assays were performed and pellets containing 80 ng of human bFGF with either 500 ng of GST or GST-neostatin-7 were implanted into the corneal pockets. The eyes were then examined and photographed on day 7. Whole mount immunohistochemical staining was used to assay corneal lymphangiogenesis.
Using biacore T100 binding assays, neostatin-7 peptide 9 showed preferential binding to recombinant VEGF receptor-3 in vitro. Several differentially-expressed molecules were detected by Mass spectrometric analysis in the four LEC subgroups. These included PI-3 kinase and other signal transduction molecules. Enhanced corneal lymphangiogenesis and VEGF-C expression in collagen XVIII knockout mice were observed following corneal keratectomy wounding. Corneal micropellet implantation of neostatin-7 reduced bFGF-induced corneal lymphangiogenesis.
Neostatin-7 inhibits corneal lymphangiogenesis. This is mediated, in part, via competitive inhibition of VEGF receptor 3.
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