Effects of VEGFR-1, VEGFR-2, and IGF-IR hammerhead ribozymes on glucose-mediated tight junction expression in cultured human retinal endothelial cells.
OBJECTIVE
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.
Spoerri
Polyxenie E
PE
Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, FL, USA. spoerri_PE01@hotmail.com
Afzal
Aqeela
A
Li Calzi
Sergio
S
Shaw
Lynn C
LC
Cai
Jun
J
Pan
Hao
H
Boulton
Michael
M
Grant
Maria B
MB
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D042783:Endothelial Cells; D005947:Glucose / Q000008:administration & dosage / Q000494:pharmacology*; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D062793:Occludin; D016337:RNA, Catalytic / Q000235:genetics / Q000494:pharmacology*; D012333:RNA, Messenger / Q000187:drug effects; D017526:Receptor, IGF Type 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D040262:Receptors, Vascular Endothelial Growth Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012171:Retinal Vessels / Q000166:cytology / Q000378:metabolism*; D019108:Tight Junctions / Q000187:drug effects*; D014162:Transfection; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme; D017526:Receptor, IGF Type 1; D040262:Receptors, Vascular Endothelial Growth Factor; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D005947:Glucose