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 mole cules) are induced on the luminal surface of vascular endothelium. A s eries of phosphorothioate oligonucleotides 18 to 21 bases in length we re designed and synthesized to hybridize selectively to the mRNA, whic h encodes three such endothelial-leukocyte adhesion molecules; human i ntercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion mole cule-1 (VCAM-1), and E-selectin. Antisense oligonucleotides were ident ified that selectively inhibited ICAM-1, VCAM-1, and E-selectin expres sion in HUVEC. Oligonucleotides that hybridized to the 3'-untranslated region of either ICAM-1, VCAM-1, or E-selectin mRNAs promoted a selec tive reduction in the respective mRNA levels. In contrast, oligonucleo tides that hybridized to 5'-untranslated sequences did not significant ly reduce target mRNA levels, although they did promote a reduction in protein expression. With the use of flow cytometry to measure cell su rface expression, ICAM-1 and E-selectin were selectively inhibited by their respective antisense oligonucleotide. At low concentrations of o ligonucleotides, only VCAM-1 antisense oligonucleotides inhibited VCAM -1 expression. However, at an oligonucleotide concentration of 50 nM o r greater, phosphorothioate oligonucleotides not predicted to hybridiz e to VCAM-1 mRNA also reduced VCAM-1 expression. The sequence-independ ent inhibition of VCAM-1 expression by phosphorothioate oligonucleotid es could be the result of a perturbation in the transcriptional regula tion of the VCAM-1 gene. ICAM-1, VCAM-1, and E-selectin antisense olig onucleotides reduced adhesion of HL-60 cells to TNF-activated HUVEC. T hese data demonstrate that phosphorothioate oligonucleotides are capab le of selectively inhibiting the expression of ICAM-1, VCAM-1, and E-s electin in HUVEC.