| Background and purpose Vascular disease are main life-threatening comeplex in world. Arterial interventional therapy such as balloon angioplasty and stenting is presently a potential strategy against coronary and cerebral artery disease. However, the efficacy of these techniques is limited by restenosis ( ie, the recurrence of the treated lesions, in 20% to 50% of procedures). Several lines of evidence indicate that this restenosis might be multifactorial and caused mainly by vascular remodeling, the migration of vascular smooth muscle cells (VSMCs) from the media into the intima, and their unregulated proliferation. Smooth muscle cell proliferation is a key factor in the pathogenesis of restenosis and antiproliferative molecular strategies is an inviting target. Although some pharmacological agents and devices have been shown to decrease the incidence of restenosis, the results are not sufficient and other novel therapies are required for the further decrease or elimination of restenosis. The possibility of transferring foreign DNA into arterial wall cells has brought new insights into the pathophysiology of arterial disease and holds considerable promise as a therapeutic tool. When transferred into vascular cells at specific site, the transgene ( ie the foreign DNA sequences) is expressed locally, resulting in prolonged and progressive production of the recombinant protein encoded by the transgene at that specific site. Prostacyclin (PGI2) an arachidonic acid metabolite, is produced in vessel walls and binds to PGI2 receptor, which induces the production of cAMP. PGI2 exerts its multiple effects mostly by the production of cAMP, such as vasodilation, anti-platelet aggregation, inhibition of smooth muscle proliferation. Prostacyclin synthase (PCS) is known to catalyze the conversion of prostaglandin H2 (PGH2) to prostacyclin. The cDNA for bovine PCS has been cloned from aorta endothelial cells and contain a 1500-bp open reading frame coding for a 500-amino acid polypeptide with an Mr of 56,628. Miyata et al accomplished the cDNA cloning of human PCS from aortic endothelial cells which encoded a 500-amino acid polypeptide. Successively, cDNA for rat and mouse PCS was cloned sharing 84% and 78% identity with that of human PCS, respectively. It was possible that PCS cDNA was transferred to the carotid artery after balloon injury and inhibited restenosis. Therefore, to test our hypothesis that an over expression of endogenous PGI2 may inhibit neointimal formation in injured artery, we constructed a plasmid expressing PGI2. In the present study, we examined the transfection of PCS gene resulted in over expression of PCS increased PGI2 synthsis and concomitant suppression of the growth of cultured VSMCS, furthermore, using a balloon-injured carotid artery rabbit model, we examined whether the local delivery of the PCS gene could prevent neointimal formation.Methods Plasmid DNA Preparation: Based on the obtained sequence of the rat PCS gene, we designed the primers (forward primer, 5'-CATGTTCTGGGCCGCT-3'; reverse primer, 5'-GGAACAACAGGAAGTCAGGAG-3'; underlined region indicates starting codon). Total cellular RNA was extracted from rat aorta by the modified method of Chomczinski and Sacchi, and subjected to reverse-transcription using a first-strand cDNA synthesis kit. In each tube, oligo(dT)12 to 18 primer was used as a primer for first-strand cDNA synthesis. PCR was performed for 40 cycles of denaturation at 95°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 1 minute with a final 10 minute extension period. The PCR product was electrophoresed on a 1.5% agarose gel, stained with ethidium bromide, and extracted. The 1.6-kb rat PCS cDNA containing the open reading frame sequence (nucleotides 7 to 1512) was cloned in the cytomegalovirus enhancer/promoter-directed vector. A plasmid carrying the LacZ gene substituted for the PCS gene was constructed as a control (pCMV-LacZ). The plasmids were purified with a plasmid purification kit according to the manufacturer's...
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