Suppressive effect of short-interfering RNA on hyperglycemia-induced expression of intercellular adhesion molecule-1 on cultured vascular endothelial cells.
OBJECTIVE
The pathology of diabetic retinopathy involves endothelial dysfunction, in which leukocyte adhesion to the vascular endothelium via intercellular adhesion molecule-1 (ICAM-1) may play a key role. Short-interfering RNAs (siRNAs) are unique modulators of gene expression in mammalian cells. The purpose of this study was to evaluate the enhanced effect of hyperglycemia and the attenuating effect of siRNAs on ICAM-1 expression in cultured endothelial cells.


METHODS
Human umbilical vein endothelial cells (HUVECs) were seeded onto 24-well culture plates. The following day, ICAM-1-specific siRNAs were transfected using Lipofectamine 2000. Glucose (15, 30, or 45 mM) or interleukin-1ss as a positive control was added to the medium to stimulate ICAM-1. After 48 hours, the HUVECs were collected to measure the ICAM-1 expression by enzyme-linked immunosolvent assay. Fluoresceinated siRNAs were transfected into HUVECs to confirm transfection of the siRNAs into HUVECs by fluorescein microscopy.


RESULTS
Glucose enhanced the ICAM-1 expression in a dose-dependent manner. ICAM-1 expression stimulated by hyperglycemia decreased significantly in the HUVECs transfected with corresponding siRNAs. Transfection of siRNAs was confirmed with enhanced fluorescence in HUVECs incubated with control siRNAs.


CONCLUSIONS
These results suggested that hyperglycemia stimulated protein expression of ICAM-1 and that siRNAs suppressed gene expression of ICAM-1 in HUVECs. The RNA-targeting approach using siRNAs may provide a novel therapeutic option for diabetic retinopathy.
Takase
Ayae
A
Department of Ophthalmology and Visual Science, Nagoya City University Graduate School of Medical Sciences, Mizuho-ku, Nagoya-shi, Aichi 467-8601, Japan.
Yasukawa
Tsutomu
T
Kato
Aki
A
Ogura
Yuichiro
Y
D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism*; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism*; D053583:Interleukin-1beta / Q000494:pharmacology; D008055:Lipids; D008856:Microscopy, Fluorescence; D034622:RNA Interference / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology
D053583:Interleukin-1beta; D008055:Lipids; C086724:Lipofectamine; D034741:RNA, Small Interfering; D018799:Intercellular Adhesion Molecule-1; D005947:Glucose