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

Nirbhai Singhb, Shivan Amin, Elizabeth Richter, Saadia Rashid, Vincent Scoglietti, Pooja D. Jani, Jin Wang, Rajwinder Kaur, Jayakrishna Ambati, Zheng Dong, Balamurali K. Ambati

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)1647-1652
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Volume46
Issue number5
DOIs
StatePublished - 2005
Externally publishedYes

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Corneal Neovascularization
Vascular Endothelial Growth Factor A
Leukocytes
Plasmids
Epithelial Cells
Endoplasmic Reticulum
Vascular Endothelial Growth Factor Receptor
Cornea
Up-Regulation
Enzyme-Linked Immunosorbent Assay
Hypoxia
In Vitro Techniques
Wounds and Injuries
Transfection

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Flt-1 intraceptors inhibit hypoxia-induced VEGF expression in vitro and corneal neovascularization in vivo. / Singhb, Nirbhai; Amin, Shivan; Richter, Elizabeth; Rashid, Saadia; Scoglietti, Vincent; Jani, Pooja D.; Wang, Jin; Kaur, Rajwinder; Ambati, Jayakrishna; Dong, Zheng; Ambati, Balamurali K.

In: Investigative Ophthalmology and Visual Science, Vol. 46, No. 5, 2005, p. 1647-1652.

Research output: Contribution to journalArticle

Singhb, N, Amin, S, Richter, E, Rashid, S, Scoglietti, V, Jani, PD, Wang, J, Kaur, R, Ambati, J, Dong, Z & Ambati, BK 2005, 'Flt-1 intraceptors inhibit hypoxia-induced VEGF expression in vitro and corneal neovascularization in vivo', Investigative Ophthalmology and Visual Science, vol. 46, no. 5, pp. 1647-1652. https://doi.org/10.1167/iovs.04-1172
Singhb, Nirbhai ; Amin, Shivan ; Richter, Elizabeth ; Rashid, Saadia ; Scoglietti, Vincent ; Jani, Pooja D. ; Wang, Jin ; Kaur, Rajwinder ; Ambati, Jayakrishna ; Dong, Zheng ; Ambati, Balamurali K. / Flt-1 intraceptors inhibit hypoxia-induced VEGF expression in vitro and corneal neovascularization in vivo. In: Investigative Ophthalmology and Visual Science. 2005 ; Vol. 46, No. 5. pp. 1647-1652.
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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.",
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T1 - Flt-1 intraceptors inhibit hypoxia-induced VEGF expression in vitro and corneal neovascularization in vivo

AU - Singhb, Nirbhai

AU - Amin, Shivan

AU - Richter, Elizabeth

AU - Rashid, Saadia

AU - Scoglietti, Vincent

AU - Jani, Pooja D.

AU - Wang, Jin

AU - Kaur, Rajwinder

AU - Ambati, Jayakrishna

AU - Dong, Zheng

AU - Ambati, Balamurali K.

PY - 2005

Y1 - 2005

N2 - 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.

AB - 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.

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U2 - 10.1167/iovs.04-1172

DO - 10.1167/iovs.04-1172

M3 - Article

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EP - 1652

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

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