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K. Nishijima, D. Krilleke, G.S. Robinson, A.P. Adamis, Y.–S. Ng, D.T. Shima; A Functional Heparin–Binding Domain is Required for Vegf164–Induced Retinal Leukostasis . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5502.
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© ARVO (1962-2015); The Authors (2016-present)
The heparin–binding domain (HBD) in the C–terminus of VEGF164 confers VEGF isoform–specific biological activities in vascular development and disease. We have previously shown that VEGF164 has increased potency as a pro–inflammatory factor and subsequently, a more fundamental role in ocular neovascular disease models. The aim of this study was to evaluate the biological effect of VEGF164 HBD functional mutants on leukocyte recruitment in retinal vasculature.
Wild–type and non–heparin–binding VEGF164 HBD mutant proteins were produced using the yeast P. Pastoris protein production system. The biological activity of the VEGF164 HBD mutants was assessed using the rat aorta ring angiogenesis assay. Leukocyte recruitment in retinal microvasculature was determined by acridine orange digital fluorography and FITC–coupled ConA perfusion following intravitreous injection of wild–type and mutant VEGF164.
Alanine substitutions in basic residues R13, R14, and R49 of the heparin–binding domain resulted in VEGF164 mutants that cannot bind heparin and more importantly lose the heightened ability to induce leukocyte recruitment in retina as compared to VEGF120. However, the HBD mutants can induce angiogenesis in ex vivo cultures of rat aorta rings, comparable with wild type controls. PlGF–2, which binds to VEGF receptor–1, heparin, and neuropilin–1 (Np–1), could also recruit leukocytes as much as wild–type VEGF164, whereas PlGF–1 that can only bind VEGFR–1 could not. Furthermore, VEGF–E binding to VEGF receptor–2 did not induce leukostasis in retina. In addition, intravitreous injection of purified heparin–binding domain protein inhibited VEGF164–induced leukocyte recruitment to retinal vasculature in a dose dependent manner.
These data suggested that residues R13, R14, and R49 of the heparin–binding domain govern VEGF164–induced retinal leukostasis, but are not required for VEGF164–induced angiogenesis. Furthermore, VEGF164 binding to VEGF receptor–1, in combination with heparin and/or Np–1, mediates potent retinal leukostasis.
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