Inhibition of endothelial cell adhesion molecule expression with antisense oligonucleotides.
In response to inflammatory stimuli, expression of a group of proteins that bind circulating leukocytes (endothelial-leukocyte adhesion molecules) are induced on the luminal surface of vascular endothelium. A series of phosphorothioate oligonucleotides 18 to 21 bases in length were designed and synthesized to hybridize selectively to the mRNA, which encodes three such endothelial-leukocyte adhesion molecules; human intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Antisense oligonucleotides were identified that selectively inhibited ICAM-1, VCAM-1, and E-selectin expression in HUVEC. Oligonucleotides that hybridized to the 3'-untranslated region of either ICAM-1, VCAM-1, or E-selectin mRNAs promoted a selective reduction in the respective mRNA levels. In contrast, oligonucleotides that hybridized to 5'-untranslated sequences did not significantly reduce target mRNA levels, although they did promote a reduction in protein expression. With the use of flow cytometry to measure cell surface expression, ICAM-1 and E-selectin were selectively inhibited by their respective antisense oligonucleotide. At low concentrations of oligonucleotides, only VCAM-1 antisense oligonucleotides inhibited VCAM-1 expression. However, at an oligonucleotide concentration of 50 nM or greater, phosphorothioate oligonucleotides not predicted to hybridize to VCAM-1 mRNA also reduced VCAM-1 expression. The sequence-independent inhibition of VCAM-1 expression by phosphorothioate oligonucleotides could be the result of a perturbation in the transcriptional regulation of the VCAM-1 gene. ICAM-1, VCAM-1, and E-selectin antisense oligonucleotides reduced adhesion of HL-60 cells to TNF-activated HUVEC. These data demonstrate that phosphorothioate oligonucleotides are capable of selectively inhibiting the expression of ICAM-1, VCAM-1, and E-selectin in HUVEC.
Bennett
C F
CF
Department of Molecular Pharmacology, ISIS Pharmaceuticals, Carlsbad, CA 92008.
Condon
T P
TP
Grimm
S
S
Chan
H
H
Chiang
M Y
MY
D001483:Base Sequence; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000235:genetics*; D002478:Cells, Cultured; D019040:E-Selectin; D004730:Endothelium, Vascular / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D018799:Intercellular Adhesion Molecule-1; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D019010:Vascular Cell Adhesion Molecule-1
D015815:Cell Adhesion Molecules; D019040:E-Selectin; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1