May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Flt-1 Intraceptor Induces the Unfolded Protein Response and Regress Injury-Induced Corneal Neovascularization in vitro and in vivo
Author Affiliations & Notes
  • N. Singh
    Medical College of Georgia, Augusta, Georgia
    Ophthalmology,
  • C. Jenkins
    Medical College of Georgia, Augusta, Georgia
    Ophthalmology,
  • P. Jani
    Medical College of Georgia, Augusta, Georgia
    Ophthalmology,
  • R. Kaur
    Medical College of Georgia, Augusta, Georgia
    Institute of Molecular Medicine and Genetics,
  • J. Ambati
    Ophthalmology & Visual Sciences, University of Kentucky, Lexington, Kentucky
  • B. K. Ambati
    Medical College of Georgia, Augusta, Georgia
    Ophthalmology,
  • Footnotes
    Commercial Relationships N. Singh, None; C. Jenkins, None; P. Jani, None; R. Kaur, None; J. Ambati, None; B.K. Ambati, None.
  • Footnotes
    Support Knights-Templar Eye Foundation and ARVO-Fight For Sight Fellowship
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 1698. doi:https://doi.org/
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    • Get Citation

      N. Singh, C. Jenkins, P. Jani, R. Kaur, J. Ambati, B. K. Ambati; Flt-1 Intraceptor Induces the Unfolded Protein Response and Regress Injury-Induced Corneal Neovascularization in vitro and in vivo. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1698. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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 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.

Conclusions:: The Flt24K intraceptor can bind VEGFR-2 within cells, induce the unfolded protein response in vitro and in vivo, and induce regression of corneal neovascularization in vivo.

Keywords: cornea: basic science • neovascularization • wound healing 
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