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Nirbhai Singh, Pooja D. Jani, Tushar Suthar, Shivan Amin, Balamurali K. Ambati; Flt-1 Intraceptor Induces the Unfolded Protein Response, Apoptotic Factors, and Regression of Murine Injury–Induced Corneal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2006;47(11):4787-4793. doi: 10.1167/iovs.06-0419.
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purpose. To determine whether Flt24K, a recombinant construct of domains 2 to 4 of VEGFR-1 (Flt) coupled with an endoplasmic reticulum retention signal (KDEL) can bind VEGFR-2 and induce apoptosis, unfolded protein response (UPR), and regression of injury-induced corneal neovascularization.
methods. Human microvascular endothelial cells were transfected with pCMV.Flt24K and subjected to hypoxia. Cell lysates underwent Western blot analysis with anti-XBP-1 antibody and RT-PCR for CHOP. Human malignant melanoma cells (which express VEGFR-2 but not Flt), were transfected with pCMV.Flt24K, and lysates underwent immunoprecipitation with anti-FLT antibody, and Western blot analysis for VEGF and VEGFR-2. Mouse corneas sustained injury induced by topical NaOH and mechanical scraping and were injected with pCMV.Flt24K 2 weeks later. Corneas were harvested 2 days later for Western blot analysis for XBP-1 and caspase-3 or 1 week later for quantification of neovascularization and TUNEL staining. Saline and empty pCMV vector were used in control experiments.
results. The mean percentage area of corneal neovascularization in mice 3 weeks after corneal injury and 1 week after intrastromal injection of empty pCMV vector or pCMV.Flt24K was 55.4% ± 2.7% vs. 19.3% ± 6.1%, respectively (P < 0.001). Flt24K was found to bind VEGFR-2 and upregulate activated XBP-1 and CHOP in vitro. In vivo, pCMV.Flt24K upregulated activated XBP-1 and caspase-3. Apoptosis was observed in corneal neovascular endothelium in corneas treated with pCMV.Flt24K but not in the control.
conclusions. The Flt24K intraceptor can bind VEGFR-2 within cells, induce the unfolded protein response in vitro and in vivo, elicit apoptosis of vascular endothelial cells in vivo, and induce regression of corneal neovascularization in vivo.
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