Growth-related responses in arterial smooth muscle cells are arrested by thrombin receptor antisense sequences

The capacity of antisense sequences to the thrombin receptor to selectively inhibit thrombin receptor expression and limit mitogenic responses in vascular wall cells was investigated in vitro. Eight phosphorothioate oligodeoxynucleotides based on the sequences of the rat thrombin receptor (including sense, antisense, scrambled, and missense controls) were synthesized, characterized, and purified by high performance liquid chromatography. The antisense oligodeoxynucleotide (ODN 4) inhibitory effect was sequence-specific and both time-and concentration-dependent. A reduction in serum or alpha-thrombin-induced smooth muscle cell (SMC) proliferation was noted as early as 3 days at 30 microM (82%; 6.17 +/- 1.01 versus 34.08 +/- 3.89 x 10(4) cells/well; p < 0.05) and at a dose as low as 15 microM after 4 days in culture (19%; p < 0.05). Nonspecific effects were enhanced after prolonged exposure of SMC to the antisense oligodeoxynucleotide (> or = 6 days). A reduction of inositol phosphate generation greater than 50% (p < 0.05) was detected after exposure of SMC to antisense but not to sense or scrambled nucleotide sequences. This was observed after stimulation with both thrombin and SFFLRN (thrombin receptor peptide agonist). Northern blot analysis and enzyme-linked immunosorbent assays revealed 50 and 22% decreases, respectively, in thrombin receptor mRNA and protein (cell surface) levels in antisense oligonucleotide-treated (72 h) SMC as compared to untreated cells, suggesting that thrombin receptor down-regulation occurred at the pretranslational level. Thus, thrombin receptor-specific antisense sequences inhibit growth-related effects both of serum and thrombin on smooth muscle cells, potentially providing a new strategy for selective inhibition of receptor-mediated arterial injury responses.