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

E. L. Chaikof, R. Caban, C. N. Yan, Rao Gadiparthi, M. S. Runge

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

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 α-thrombin-induced smooth muscle cell (SMC) proliferation was noted as early as 3 days at 30 μM (82%; 6.17 ± 1.01 versus 34.08 ± 3.89 x 104 cells/well; p < 0.05) and at a dose as low as 15 μM after 4 days in culture (19%; p < 0.05). Nonspecific effects were enhanced after prolonged exposure of SMC to the antisense oligodeoxynucleotide (≥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.

Original languageEnglish (US)
Pages (from-to)7431-7436
Number of pages6
JournalJournal of Biological Chemistry
Volume270
Issue number13
DOIs
StatePublished - Jan 1 1995
Externally publishedYes

Fingerprint

Thrombin Receptors
Smooth Muscle Myocytes
Muscle
Cells
Growth
Oligodeoxyribonucleotides
Thrombin
Immunosorbents
Inositol Phosphates
Antisense Oligonucleotides
Cell proliferation
High performance liquid chromatography
Serum
Northern Blotting
Cell Wall
Blood Vessels
Rats
Assays
Membrane Proteins
Down-Regulation

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Growth-related responses in arterial smooth muscle cells are arrested by thrombin receptor antisense sequences. / Chaikof, E. L.; Caban, R.; Yan, C. N.; Gadiparthi, Rao; Runge, M. S.

In: Journal of Biological Chemistry, Vol. 270, No. 13, 01.01.1995, p. 7431-7436.

Research output: Contribution to journalArticle

@article{0c2ae2a7f3f0468cbdab62632a93c039,
title = "Growth-related responses in arterial smooth muscle cells are arrested by thrombin receptor antisense sequences",
abstract = "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 α-thrombin-induced smooth muscle cell (SMC) proliferation was noted as early as 3 days at 30 μM (82{\%}; 6.17 ± 1.01 versus 34.08 ± 3.89 x 104 cells/well; p < 0.05) and at a dose as low as 15 μM after 4 days in culture (19{\%}; p < 0.05). Nonspecific effects were enhanced after prolonged exposure of SMC to the antisense oligodeoxynucleotide (≥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.",
author = "Chaikof, {E. L.} and R. Caban and Yan, {C. N.} and Rao Gadiparthi and Runge, {M. S.}",
year = "1995",
month = "1",
day = "1",
doi = "10.1074/jbc.270.13.7431",
language = "English (US)",
volume = "270",
pages = "7431--7436",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "13",

}

TY - JOUR

T1 - Growth-related responses in arterial smooth muscle cells are arrested by thrombin receptor antisense sequences

AU - Chaikof, E. L.

AU - Caban, R.

AU - Yan, C. N.

AU - Gadiparthi, Rao

AU - Runge, M. S.

PY - 1995/1/1

Y1 - 1995/1/1

N2 - 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 α-thrombin-induced smooth muscle cell (SMC) proliferation was noted as early as 3 days at 30 μM (82%; 6.17 ± 1.01 versus 34.08 ± 3.89 x 104 cells/well; p < 0.05) and at a dose as low as 15 μM after 4 days in culture (19%; p < 0.05). Nonspecific effects were enhanced after prolonged exposure of SMC to the antisense oligodeoxynucleotide (≥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.

AB - 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 α-thrombin-induced smooth muscle cell (SMC) proliferation was noted as early as 3 days at 30 μM (82%; 6.17 ± 1.01 versus 34.08 ± 3.89 x 104 cells/well; p < 0.05) and at a dose as low as 15 μM after 4 days in culture (19%; p < 0.05). Nonspecific effects were enhanced after prolonged exposure of SMC to the antisense oligodeoxynucleotide (≥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.

UR - http://www.scopus.com/inward/record.url?scp=0028946244&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028946244&partnerID=8YFLogxK

U2 - 10.1074/jbc.270.13.7431

DO - 10.1074/jbc.270.13.7431

M3 - Article

VL - 270

SP - 7431

EP - 7436

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 13

ER -