Unique homologous siRNA blocks hypoxia-induced VEGF upregulation in human corneal cells and inhibits and regresses murine corneal neovascularization

Nirbhai Singh, Eric Higgins, Shivan Amin, Pooja Jani, Elizabeth Richter, Anup Patel, Rajwinder Kaur, Jin Wang, Jayakrishna Ambati, Zheng Dong, Balamurali K. Ambati

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

PURPOSE: To determine whether RNA interference (RNAi) could block hypoxia-induced upregulation of vascular endothelial growth factor (VEGF) in human corneal epithelial cells in vitro and inhibit and regress injury-induced murine corneal neovascularization in vivo. METHODS: siRNA selected on the basis of target sequence homology between mouse and human VEGF was placed into expression cassettes and transfected into human corneal epithelial cells. Hypoxia-induced VEGF synthesis was assayed. Also, the effect of a plasmid capable of directing the expression of an siRNA against VEGF when injected into mouse corneas 8 hours before alkali-mechanical trauma was studied. Leukocyte count, VEGF protein levels, and degree of neovascularization in corneas were compared with that of a control siRNA plasmid. Plasmids were injected 1 week after injury to assess the ability of RNAi to regress corneal neovascularization. RESULTS: Hypoxia-induced VEGF mRNA synthesis and protein secretion by human corneal epithelial cells was efficiently suppressed by an siRNA targeted against a sequence uniquely identical for the mouse and human VEGF genes. Intrastromal delivery of a plasmid expressing this siRNA before murine corneal injury suppressed corneal VEGF by 55.7% versus control (P = 0.014), leukocyte infiltration by 69.5% (P < 0.001), and neovascularization 1 week after injury by 72.3% (P = 0.001). At the regression time point, treated corneas had 72.8% less neovascularization (P < 0.001). CONCLUSIONS: RNAi significantly suppresses expression of VEGF induced by hypoxia in human corneal epithelial cells in vitro. In vivo, intrastromal delivery of a plasmid expressing siRNA against VEGF suppresses injury-induced VEGF expression, leukocyte infiltration, and angiogenesis and was able to regress corneal neovascularization.

Original languageEnglish (US)
Pages (from-to)65-72
Number of pages8
JournalCornea
Volume26
Issue number1
DOIs
StatePublished - Jan 1 2007

Fingerprint

Corneal Neovascularization
Vascular Endothelial Growth Factor A
Small Interfering RNA
Up-Regulation
Plasmids
RNA Interference
Epithelial Cells
Cornea
Wounds and Injuries
Leukocytes
Hypoxia
Alkalies
Sequence Homology
Leukocyte Count
Proteins
Messenger RNA

Keywords

  • Angiogenesis
  • Inhibition
  • Neovascularization
  • RNA interference
  • Vascular endothelial growth factor
  • siRNA

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Unique homologous siRNA blocks hypoxia-induced VEGF upregulation in human corneal cells and inhibits and regresses murine corneal neovascularization. / Singh, Nirbhai; Higgins, Eric; Amin, Shivan; Jani, Pooja; Richter, Elizabeth; Patel, Anup; Kaur, Rajwinder; Wang, Jin; Ambati, Jayakrishna; Dong, Zheng; Ambati, Balamurali K.

In: Cornea, Vol. 26, No. 1, 01.01.2007, p. 65-72.

Research output: Contribution to journalArticle

Singh, N, Higgins, E, Amin, S, Jani, P, Richter, E, Patel, A, Kaur, R, Wang, J, Ambati, J, Dong, Z & Ambati, BK 2007, 'Unique homologous siRNA blocks hypoxia-induced VEGF upregulation in human corneal cells and inhibits and regresses murine corneal neovascularization', Cornea, vol. 26, no. 1, pp. 65-72. https://doi.org/10.1097/ICO.0b013e31802b4201
Singh, Nirbhai ; Higgins, Eric ; Amin, Shivan ; Jani, Pooja ; Richter, Elizabeth ; Patel, Anup ; Kaur, Rajwinder ; Wang, Jin ; Ambati, Jayakrishna ; Dong, Zheng ; Ambati, Balamurali K. / Unique homologous siRNA blocks hypoxia-induced VEGF upregulation in human corneal cells and inhibits and regresses murine corneal neovascularization. In: Cornea. 2007 ; Vol. 26, No. 1. pp. 65-72.
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T1 - Unique homologous siRNA blocks hypoxia-induced VEGF upregulation in human corneal cells and inhibits and regresses murine corneal neovascularization

AU - Singh, Nirbhai

AU - Higgins, Eric

AU - Amin, Shivan

AU - Jani, Pooja

AU - Richter, Elizabeth

AU - Patel, Anup

AU - Kaur, Rajwinder

AU - Wang, Jin

AU - Ambati, Jayakrishna

AU - Dong, Zheng

AU - Ambati, Balamurali K.

PY - 2007/1/1

Y1 - 2007/1/1

N2 - PURPOSE: To determine whether RNA interference (RNAi) could block hypoxia-induced upregulation of vascular endothelial growth factor (VEGF) in human corneal epithelial cells in vitro and inhibit and regress injury-induced murine corneal neovascularization in vivo. METHODS: siRNA selected on the basis of target sequence homology between mouse and human VEGF was placed into expression cassettes and transfected into human corneal epithelial cells. Hypoxia-induced VEGF synthesis was assayed. Also, the effect of a plasmid capable of directing the expression of an siRNA against VEGF when injected into mouse corneas 8 hours before alkali-mechanical trauma was studied. Leukocyte count, VEGF protein levels, and degree of neovascularization in corneas were compared with that of a control siRNA plasmid. Plasmids were injected 1 week after injury to assess the ability of RNAi to regress corneal neovascularization. RESULTS: Hypoxia-induced VEGF mRNA synthesis and protein secretion by human corneal epithelial cells was efficiently suppressed by an siRNA targeted against a sequence uniquely identical for the mouse and human VEGF genes. Intrastromal delivery of a plasmid expressing this siRNA before murine corneal injury suppressed corneal VEGF by 55.7% versus control (P = 0.014), leukocyte infiltration by 69.5% (P < 0.001), and neovascularization 1 week after injury by 72.3% (P = 0.001). At the regression time point, treated corneas had 72.8% less neovascularization (P < 0.001). CONCLUSIONS: RNAi significantly suppresses expression of VEGF induced by hypoxia in human corneal epithelial cells in vitro. In vivo, intrastromal delivery of a plasmid expressing siRNA against VEGF suppresses injury-induced VEGF expression, leukocyte infiltration, and angiogenesis and was able to regress corneal neovascularization.

AB - PURPOSE: To determine whether RNA interference (RNAi) could block hypoxia-induced upregulation of vascular endothelial growth factor (VEGF) in human corneal epithelial cells in vitro and inhibit and regress injury-induced murine corneal neovascularization in vivo. METHODS: siRNA selected on the basis of target sequence homology between mouse and human VEGF was placed into expression cassettes and transfected into human corneal epithelial cells. Hypoxia-induced VEGF synthesis was assayed. Also, the effect of a plasmid capable of directing the expression of an siRNA against VEGF when injected into mouse corneas 8 hours before alkali-mechanical trauma was studied. Leukocyte count, VEGF protein levels, and degree of neovascularization in corneas were compared with that of a control siRNA plasmid. Plasmids were injected 1 week after injury to assess the ability of RNAi to regress corneal neovascularization. RESULTS: Hypoxia-induced VEGF mRNA synthesis and protein secretion by human corneal epithelial cells was efficiently suppressed by an siRNA targeted against a sequence uniquely identical for the mouse and human VEGF genes. Intrastromal delivery of a plasmid expressing this siRNA before murine corneal injury suppressed corneal VEGF by 55.7% versus control (P = 0.014), leukocyte infiltration by 69.5% (P < 0.001), and neovascularization 1 week after injury by 72.3% (P = 0.001). At the regression time point, treated corneas had 72.8% less neovascularization (P < 0.001). CONCLUSIONS: RNAi significantly suppresses expression of VEGF induced by hypoxia in human corneal epithelial cells in vitro. In vivo, intrastromal delivery of a plasmid expressing siRNA against VEGF suppresses injury-induced VEGF expression, leukocyte infiltration, and angiogenesis and was able to regress corneal neovascularization.

KW - Angiogenesis

KW - Inhibition

KW - Neovascularization

KW - RNA interference

KW - Vascular endothelial growth factor

KW - siRNA

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