April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Collagen XVIII Derived Peptides Regulate Lymphangiogenesis
Author Affiliations & Notes
  • J.-H. Chang
    Department of Ophthalmology and Visual Sciences, Univ of Illinois at Chicago, Chicago, Illinois
  • K.-Y. Han
    Department of Ophthalmology and Visual Sciences, Univ of Illinois at Chicago, Chicago, Illinois
  • A. Sabri
    Department of Ophthalmology and Visual Sciences, Univ of Illinois at Chicago, Chicago, Illinois
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 4968. doi:
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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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Abstract

Purpose: : To determine the involvement of neostatin-7, a collagen XVIII-derived peptide, in corneal lymphangiogenesis in vitro and in vivo.

Methods: : 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.

Results: : 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.

Conclusions: : Neostatin-7 inhibits corneal lymphangiogenesis. This is mediated, in part, via competitive inhibition of VEGF receptor 3.

Keywords: neovascularization • cornea: basic science • cornea: epithelium 
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