ABSTRACT
To investigate antisense peptide nucleic acid (PNA) as a gene therapy for the arterial proliferative diseases, the authors designed and examined the effects of an antisense PNA targeting platelet-derived growth factor (PDGF) A-chain on expression of PDGF A-chain and growth of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats. A 15-mer antisense PNA complementary to the initiation codon of rat and human PDGF A-chain mRNA was synthesized and purified by high-performance liquid chromatography. Gel-shift assay and biomolecular interaction analysis (BIAcore) revealed that the antisense PNA bound weakly to the target RNA, whereas it bound strongly to the target DNA. Fluorescein-isothiocyanate-labeled antisense PNA to PDGF A-chain was taken up slowly and maintained in VSMCs for a prolonged period of time. Antisense PNA inhibited expression of PDGF A-chain mRNA and protein as well as DNA synthesis in VSMCs in a dose-independent manner. Inhibition of DNA synthesis by the antisense PNA was greater than that by the antisense DNA at a low concentration (0.5 micromol/L). These results suggest that antisense PNA to PDGF A-chain will be used as a gene therapy for vascular proliferative diseases such as hypertensive vascular diseases, restenosis of coronary arteries after angioplasty, and atherosclerosis.
Subject(s)
Muscle, Smooth, Vascular/drug effects , Oligonucleotides, Antisense/pharmacology , Peptide Nucleic Acids/pharmacology , Platelet-Derived Growth Factor/drug effects , Animals , Humans , Muscle, Smooth, Vascular/growth & development , Oligonucleotides, Antisense/chemical synthesis , Peptide Nucleic Acids/chemical synthesis , Rats , Rats, Inbred SHR , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Bisphosphonates have been reported to exhibit antiarteriosclerotic and anticalcification effects. We investigated the effect of a bisphosphonate, etidronate, on growth and phenotype of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR). Etidronate (10 microM) significantly decreased DNA synthesis evaluated by [3H]thymidine incorporation in VSMC cultured without serum, and 1 microM etidronate significantly inhibited DNA synthesis in the presence of 10% calf serum. Etidronate (10 microM) significantly inhibited VSMC proliferation after 72h incubation. Etidronate (100 microM) significantly increased the expression of SM22alpha mRNA and protein in VSMC, while 10 microM etidronate significantly decreased the expression of matrix Gla mRNA. These findings indicate that etidronate inhibits the exaggerated growth of VSMC from SHR, while altering their phenotype from synthetic to contractile one. These effects of etidronate may account for its antiarteriosclerotic action.
Subject(s)
Etidronic Acid/pharmacology , Gene Expression Regulation/drug effects , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/growth & development , Phenotype , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Gene Expression Regulation/physiology , Muscle, Smooth, Vascular/metabolism , RNA, Messenger/biosynthesis , Rats , Rats, Inbred SHR , Rats, Inbred WKYABSTRACT
Troglitazone, a thiazolizidinedione, has recently been reported to possess anti-arteriosclerotic properties. To evaluate mechanisms underlying the anti-arteriosclerotic effects of troglitazone, we examined the effect of troglitazone on growth, expression of growth factors, and insulin signaling in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) which produce angiotensin II (Ang II) in a homogeneous culture. Troglitazone inhibited basal and serum-stimulated DNA synthesis and inhibited increases in the number of VSMC from SHR and normotensive Wistar-Kyoto (WKY) rats. Its inhibition was greater in VSMC from SHR. Troglitazone abolished DNA synthesis in response to Ang II in VSMC from both rat strains and markedly inhibited DNA synthesis in response to epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-AA in VSMC from SHR. Troglitazone did not alter the expression of transforming growth factor (TGF)-beta1, PDGF A-chain, or basic fibroblast growth factor (bFGF) mRNAs in VSMC from WKY rats, but it markedly decreased expression of these growth factor mRNAs in VSMC from SHR. Troglitazone markedly decreased basal and Ang II-stimulated expression of extracellular signal-regulated kinase proteins in VSMC from both rat strains. Troglitazone abolished Ang II-induced suppression of phosphatidilinositol 3-kinase (PI3-kinase) activity, insulin receptor substrate-1 (IRS-1) associated tyrosine phosphorylation, and IRS-1 associated p85 levels in VSMC from WKY rats. Basal PI3-kinase activity, tyrosine phosphorylation of IRS-1, and IRS-1 associated p85 levels were lower in VSMC from SHR than in cells from WKY rats. Troglitazone significantly increased PI3-kinase activity, IRS-1 associated tyrosine phosphorylation, and IRS-1 associated p85 levels in VSMC from SHR. These results indicate that troglitazone produce its anti-arteriosclerotic effects through suppression of the action of growth-promoting factors including Ang II, and that troglitazone inhibits Ang II-induced suppression of insulin signaling in VSMC from SHR, suggesting that tissue Ang II may lead to insulin resistance and to arteriosclerosis in hypertension. Troglitazone may be useful in the treatment of insulin resistance as well as of hypertensive vascular diseases.
