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.