title: Antisense Egr-1 RNA driven by the CMV promoter is an inhibitor of vascular smooth muscle cell proliferation and regrowth after injury.
abstract: Smooth muscle cell (SMC) migration and proliferation are key events in the pathogenesis of atherosclerotic and post-angioplasty restenotic lesions. Mechanical injury to the artery wall induces the SMC expression of the zinc finger transcription factor, early growth response factor-1 (Egr-1). Egr-1 in turn can bind and activate the promoters of many genes, whose products influence vascular repair. Here, a 127-bp cDNA fragment corresponding to the 5' region of murine Egr-1 mRNA was cloned into a CMV-driven expression vector, in the sense or antisense orientation. We demonstrate that antisense Egr-1 RNA inhibited rat vascular SMC proliferation, whereas the sense counterpart produced only a modest effect. By semi-quantitative reverse-transcription PCR, antisense Egr-1 RNA blocked serum-inducible Egr-1 mRNA expression. Western blot analysis demonstrated that antisense RNA overexpression inhibited Egr-1 protein synthesis, without affecting levels of the immediate early gene product, c-fos. Finally, antisense Egr-1 RNA overexpression inhibited SMC regrowth after mechanical injury in vitro. In contrast, sense Egr-1 RNA had no effect on SMC repair, Egr-1 mRNA expression or protein synthesis. Analysis of transfection efficiencies revealed that both CMV-driven constructs (sense and antisense) were taken up by the SMCs with equivalent efficiency. These findings provide the first demonstration of antisense RNA strategies targeting Egr-1 as inhibitors of Egr-1 and Egr-1-dependent cellular processes. The antisense RNA approach may be potentially useful in gene therapeutic efforts to control SMC growth in the injured artery wall.
authors.affiliation: Centre for Thrombosis and Vascular Research, School of Pathology, The University of New South Wales, Sydney NSW 2052, Australia.
authors.forename: Roger G
authors.initials: RG
authors.lastname: Fahmy
authors.forename: Levon M
authors.initials: LM
authors.lastname: Khachigian
mesh_terms: D000818:Animals; D002455:Cell Division; D002460:Cell Line; D003470:Culture Media; D003587:Cytomegalovirus / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics*; D051766:Early Growth Response Protein 1; D004705:Endocytosis; D017874:Immediate-Early Proteins; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000502:physiology; D020320:Oligoribonucleotides, Antisense / Q000494:pharmacology*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013314:Stress, Mechanical; D014157:Transcription Factors / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics*; D014945:Wound Healing
chemical_list: D003470:Culture Media; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486110:Egr1 protein, rat; D017874:Immediate-Early Proteins; D020320:Oligoribonucleotides, Antisense; D012333:RNA, Messenger; D014157:Transcription Factors