Effects of VEGFR-1, VEGFR-2, and IGF-IR hammerhead ribozymes on glucose-mediated tight junction expression in cultured human retinal endothelial cells

Polyxenie E. Spoerri, Aqeela Afzal, Sergio Li Calzi, Lynn C. Shaw, Jun Cai, Hao Pan, Michael Boulton, Maria B. Grant

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Purpose: To evaluate whether transfection of human retinal endothelial cells (HRECs) with plasmids expressing ribozymes designed to specifically cleave the mRNA and reduce expression of either vascular endothelial growth factor (VEGF) receptor-1 (VEGFR-1), or VEGF receptor-2 (VEGFR-2), or insulin-like growth factor-I receptor (IGF-IR) modulates occludin expression in high glucose-treated cells. Methods: Hammerhead ribozymes that specifically cleave the human VEGFR-1, VEGFR-2, and IGF-IR mRNAs were developed and tested in vitro to determine ribozyme kinetics and cleavage specificity. HRECs grown in normal (5.5 mM) and high (25 mM) glucose medium were transfected with plasmids expressing VEGFR-1, VEGFR-2, or IGF-IR hammerhead ribozymes. VEGF and IGF-I levels were measured in conditioned medium of HREC exposed to high glucose conditions, and the effect of varying glucose concentration on VEGFR-1 and VEGFR-2 phosphorylation was examined. The amount of the tight junction protein occludin was determined by western analysis, and the protein was localized by immunohistochemistry. Results: Exposure of HRECs to high glucose resulted in increased VEGF and IGF-I expression as well as VEGFR-2 but not VEGFR-1 phosphorylation. Immunocytochemistry and western analysis revealed that HRECs exposed to high glucose had reduced occludin staining and protein expression, respectively. Transfection of HRECs exposed to high glucose with either VEGFR-1, VEGFR-2, or IGF-IR hammerhead ribozymes prevented the downregulation of occludin protein expression. Conclusions: Our studies support that activation of VEGFR-1, VEGFR-2, and IGF-IR by high glucose contributes to disruption of tight junctions by decreasing occludin expression and may be important in the pathogenesis of blood-retinal barrier dysfunction in diabetic retinopathy.

Original languageEnglish (US)
Pages (from-to)32-42
Number of pages11
JournalMolecular Vision
Volume12
StatePublished - Jan 12 2006
Externally publishedYes

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IGF Type 1 Receptor
Vascular Endothelial Growth Factor Receptor
Tight Junctions
Endothelial Cells
Glucose
Occludin
Catalytic RNA
Vascular Endothelial Growth Factor A
Transfection
hammerhead ribozyme
Plasmids
Immunohistochemistry
Phosphorylation
Blood-Retinal Barrier
Tight Junction Proteins
Vascular Endothelial Growth Factor Receptor-1
Vascular Endothelial Growth Factor Receptor-2
Messenger RNA
Proteins
Diabetic Retinopathy

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Spoerri, P. E., Afzal, A., Calzi, S. L., Shaw, L. C., Cai, J., Pan, H., ... Grant, M. B. (2006). Effects of VEGFR-1, VEGFR-2, and IGF-IR hammerhead ribozymes on glucose-mediated tight junction expression in cultured human retinal endothelial cells. Molecular Vision, 12, 32-42.

Effects of VEGFR-1, VEGFR-2, and IGF-IR hammerhead ribozymes on glucose-mediated tight junction expression in cultured human retinal endothelial cells. / Spoerri, Polyxenie E.; Afzal, Aqeela; Calzi, Sergio Li; Shaw, Lynn C.; Cai, Jun; Pan, Hao; Boulton, Michael; Grant, Maria B.

In: Molecular Vision, Vol. 12, 12.01.2006, p. 32-42.

Research output: Contribution to journalArticle

Spoerri, PE, Afzal, A, Calzi, SL, Shaw, LC, Cai, J, Pan, H, Boulton, M & Grant, MB 2006, 'Effects of VEGFR-1, VEGFR-2, and IGF-IR hammerhead ribozymes on glucose-mediated tight junction expression in cultured human retinal endothelial cells', Molecular Vision, vol. 12, pp. 32-42.
Spoerri, Polyxenie E. ; Afzal, Aqeela ; Calzi, Sergio Li ; Shaw, Lynn C. ; Cai, Jun ; Pan, Hao ; Boulton, Michael ; Grant, Maria B. / Effects of VEGFR-1, VEGFR-2, and IGF-IR hammerhead ribozymes on glucose-mediated tight junction expression in cultured human retinal endothelial cells. In: Molecular Vision. 2006 ; Vol. 12. pp. 32-42.
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abstract = "Purpose: To evaluate whether transfection of human retinal endothelial cells (HRECs) with plasmids expressing ribozymes designed to specifically cleave the mRNA and reduce expression of either vascular endothelial growth factor (VEGF) receptor-1 (VEGFR-1), or VEGF receptor-2 (VEGFR-2), or insulin-like growth factor-I receptor (IGF-IR) modulates occludin expression in high glucose-treated cells. Methods: Hammerhead ribozymes that specifically cleave the human VEGFR-1, VEGFR-2, and IGF-IR mRNAs were developed and tested in vitro to determine ribozyme kinetics and cleavage specificity. HRECs grown in normal (5.5 mM) and high (25 mM) glucose medium were transfected with plasmids expressing VEGFR-1, VEGFR-2, or IGF-IR hammerhead ribozymes. VEGF and IGF-I levels were measured in conditioned medium of HREC exposed to high glucose conditions, and the effect of varying glucose concentration on VEGFR-1 and VEGFR-2 phosphorylation was examined. The amount of the tight junction protein occludin was determined by western analysis, and the protein was localized by immunohistochemistry. Results: Exposure of HRECs to high glucose resulted in increased VEGF and IGF-I expression as well as VEGFR-2 but not VEGFR-1 phosphorylation. Immunocytochemistry and western analysis revealed that HRECs exposed to high glucose had reduced occludin staining and protein expression, respectively. Transfection of HRECs exposed to high glucose with either VEGFR-1, VEGFR-2, or IGF-IR hammerhead ribozymes prevented the downregulation of occludin protein expression. Conclusions: Our studies support that activation of VEGFR-1, VEGFR-2, and IGF-IR by high glucose contributes to disruption of tight junctions by decreasing occludin expression and may be important in the pathogenesis of blood-retinal barrier dysfunction in diabetic retinopathy.",
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T1 - Effects of VEGFR-1, VEGFR-2, and IGF-IR hammerhead ribozymes on glucose-mediated tight junction expression in cultured human retinal endothelial cells

AU - Spoerri, Polyxenie E.

AU - Afzal, Aqeela

AU - Calzi, Sergio Li

AU - Shaw, Lynn C.

AU - Cai, Jun

AU - Pan, Hao

AU - Boulton, Michael

AU - Grant, Maria B.

PY - 2006/1/12

Y1 - 2006/1/12

N2 - Purpose: To evaluate whether transfection of human retinal endothelial cells (HRECs) with plasmids expressing ribozymes designed to specifically cleave the mRNA and reduce expression of either vascular endothelial growth factor (VEGF) receptor-1 (VEGFR-1), or VEGF receptor-2 (VEGFR-2), or insulin-like growth factor-I receptor (IGF-IR) modulates occludin expression in high glucose-treated cells. Methods: Hammerhead ribozymes that specifically cleave the human VEGFR-1, VEGFR-2, and IGF-IR mRNAs were developed and tested in vitro to determine ribozyme kinetics and cleavage specificity. HRECs grown in normal (5.5 mM) and high (25 mM) glucose medium were transfected with plasmids expressing VEGFR-1, VEGFR-2, or IGF-IR hammerhead ribozymes. VEGF and IGF-I levels were measured in conditioned medium of HREC exposed to high glucose conditions, and the effect of varying glucose concentration on VEGFR-1 and VEGFR-2 phosphorylation was examined. The amount of the tight junction protein occludin was determined by western analysis, and the protein was localized by immunohistochemistry. Results: Exposure of HRECs to high glucose resulted in increased VEGF and IGF-I expression as well as VEGFR-2 but not VEGFR-1 phosphorylation. Immunocytochemistry and western analysis revealed that HRECs exposed to high glucose had reduced occludin staining and protein expression, respectively. Transfection of HRECs exposed to high glucose with either VEGFR-1, VEGFR-2, or IGF-IR hammerhead ribozymes prevented the downregulation of occludin protein expression. Conclusions: Our studies support that activation of VEGFR-1, VEGFR-2, and IGF-IR by high glucose contributes to disruption of tight junctions by decreasing occludin expression and may be important in the pathogenesis of blood-retinal barrier dysfunction in diabetic retinopathy.

AB - Purpose: To evaluate whether transfection of human retinal endothelial cells (HRECs) with plasmids expressing ribozymes designed to specifically cleave the mRNA and reduce expression of either vascular endothelial growth factor (VEGF) receptor-1 (VEGFR-1), or VEGF receptor-2 (VEGFR-2), or insulin-like growth factor-I receptor (IGF-IR) modulates occludin expression in high glucose-treated cells. Methods: Hammerhead ribozymes that specifically cleave the human VEGFR-1, VEGFR-2, and IGF-IR mRNAs were developed and tested in vitro to determine ribozyme kinetics and cleavage specificity. HRECs grown in normal (5.5 mM) and high (25 mM) glucose medium were transfected with plasmids expressing VEGFR-1, VEGFR-2, or IGF-IR hammerhead ribozymes. VEGF and IGF-I levels were measured in conditioned medium of HREC exposed to high glucose conditions, and the effect of varying glucose concentration on VEGFR-1 and VEGFR-2 phosphorylation was examined. The amount of the tight junction protein occludin was determined by western analysis, and the protein was localized by immunohistochemistry. Results: Exposure of HRECs to high glucose resulted in increased VEGF and IGF-I expression as well as VEGFR-2 but not VEGFR-1 phosphorylation. Immunocytochemistry and western analysis revealed that HRECs exposed to high glucose had reduced occludin staining and protein expression, respectively. Transfection of HRECs exposed to high glucose with either VEGFR-1, VEGFR-2, or IGF-IR hammerhead ribozymes prevented the downregulation of occludin protein expression. Conclusions: Our studies support that activation of VEGFR-1, VEGFR-2, and IGF-IR by high glucose contributes to disruption of tight junctions by decreasing occludin expression and may be important in the pathogenesis of blood-retinal barrier dysfunction in diabetic retinopathy.

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