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Liya Pi, Jianwen Liu, Anitha Shenoy, Paulette Robinson, Daniel Gibson, Gregory Schultz, Edward Scott; Connective Tissue Growth Factor Regulates Neovessel Formation via Targeting Structurally Conserved Cystine Knot Motifs in Multiple Angiogenic Regulators. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3003. doi: https://doi.org/.
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This study investigated the molecular mechanism of CTGF regulation of retinal angiogenesis by characterizing CTGF binding proteins.
Binding of CTGF to angiogenic growth factors was analyzed using immunoprecipitation assay and yeast two-hybrid cDNA analysis. Gene expression was assessed using transgenic mice carrying CTGF promoter-driven-GFP, immunohistochemistry, and confocol microscopy during retinal angiogenesis. Mice at postnatal day 7 (P7) were treated by 75% oxygen for 5 days to induce retinal retinopathy (OIR). A CTGF mutant carrying the first three modules was injected into the vitreous at P12 and P14 and retinal neovascularization was analyzed at P17 by retinal flat-mounting using Griffonia simplicifolia Isolectin B4 to stain blood vessels.
Key angiogenic regulators of the cystine knot superfamily including vascular endothelial growth factor (VEGF)-A, Slit3, platelet derived growth factor (PDGF)-B, and von Willebrand factor (vWF), bound with CTGF through its first three modules. The immunofluorescent staining showed that CTGF and Slit3 were co-localized during retinal angiogenesis in neonatal mice. CTGF functioned in concert with Slit3 to promote endothelial tube formation and downstream activation of Cdc42, whereas a truncation mutant that contained the first three modules of CTGF and retained the full ability to bind all the tested cystine knot motifs inhibited the angiogenic activities of CTGF and Slit3. Intravitreal injection of this CTGF fragment significantly inhibited retinal neovascularization at P17 in the OIR model.
These results demonstrate that CTGF plays key roles in regulating retinal neovessel formation by binding other key angiogenic regulators with cystine knot motifs. A CTGF fragment consisting of the three N-terminal modules that binds key angiogenic growth factors with cystine knot motifs inhibits OIR in mice and may provide a unique therapeutic strategy to reduce pathological angiogenesis in eye diseases.
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