alexa Flt-1 intraceptors inhibit hypoxia-induced VEGF expression in vitro and corneal neovascularization in vivo.
Ophthalmology

Ophthalmology

Journal of Clinical & Experimental Ophthalmology

Author(s): Singh N, Amin S, Richter E, Rashid S, Scoglietti V,

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Abstract PURPOSE: To determine whether subunits of VEGF receptor-1 coupled with an endoplasmic reticulum retention signal can block hypoxia-induced upregulation of VEGF secretion in corneal epithelial cells and block murine corneal angiogenesis induced by corneal injury. METHODS: Human corneal epithelial cells, transfected with plasmids encoding Flt23K or Flt24K (the VEGF-binding domains of the Flt-1 receptor coupled with the endoplasmic reticulum retention peptide KDEL), were subjected 2 days after transfection to 5\% hypoxia for 24 hours. Supernatant was sampled at 24 hours and assayed for VEGF by ELISA. For in vivo models, mouse corneas underwent intrastromal injections of plasmids encoding Flt23K or Flt24K, and 2 days later, sustained injury induced by topical NaOH and mechanical scraping. Corneas were assessed 2 days later for VEGF ELISA and leukocyte counting or 1 week later for quantification of neovascularization. RESULTS: Hypoxia induced VEGF by human corneal epithelial cells was sequestered by both Flt23K and Flt24K; Flt-1 23K suppressed VEGF secretion as well. Intrastromal delivery of plasmid Flt23K suppressed VEGF by 40.4\% (P = 0.009), leukocytes by 49.4\% (P < 0.001), and neovascularization by 66.8\% (P = 0.001). Flt24K suppressed VEGF expression by 30.8\% (P = 0.042), leukocytes by 25.8\% (P < 0.001), and neovascularization by 49.5\% (P = 0.015). CONCLUSIONS: Flt-1 intraceptors, which are endoplasmic reticulum retention signal-coupled VEGF receptors, significantly suppress hypoxia-induced VEGF secretion by corneal epithelial cells in vitro. In vivo, delivery of naked plasmids expressing these intraceptors inhibits injury-induced upregulation of VEGF, leukocyte infiltration, and corneal neovascularization. This article was published in Invest Ophthalmol Vis Sci and referenced in Journal of Clinical & Experimental Ophthalmology

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