Influence of sustained mechanical stress on Egr-1 mRNA expression in cultured human endothelial cells

Restenosis after initially successful balloon angioplasty of coronary arter y stenosis remains a major problem in clinical cardiology. Previous studies have identified pathogenetic factors which trigger cell proliferation and vascular remodeling ultimately leading to restenosis. Since there is eviden ce that endothelial cells adjacent to the angioplasty wound area synthesize factors which may initiate this process, we investigated the effects of me chanical stimulation on endothelial gene expression in vitro and focussed o n the influence of sustained mechanical stress on expression of immediate e arly genes which have previously been shown to be induced in the vascular w all in vivo. Primary cultured human umbilical vein endothelial cells (HUVEC ) and the human endothelial cell line EA.hy 926 were plated on collagen-coa ted silicone membranes and subjected to constant longitudinal stress of app roximately 20% for 10 min to 6 h. Total RNA was isolated and the expression of the immediate early genes c-Fos and Egr-1 was studied by Northern blot analysis. We found a rapid upregulation c-Fos and Egr-1 mRNA which started at 10 min and reached its maxima at 30 min. HUVEC lost most of their stretc h response after the third passage whereas immediate early gene expression was constantly in EA.hy 926 cells. Using specific inhibitors we investigate d the contribution of several signal transduction pathways to stretch-activ ated Egr-1 mRNA expression. We found significant suppression of stretch-ind uced Egr-1 mRNA expression by protein kinase C (PKC) inhibition (p < 0.05) and by calcium depletion (EA.hy926, p < 0.05; HUVEC, p = 0.063). No effect on stretch-activated Egr-1 mRNA expression was detected by inhibition of pr otein kinase A, blockade of stretch-activated cation channels or inhibition of microtubule synthesis. We conclude that sustained mechanical strain ind uces Egr-1 mRNA expression by PKC- and calcium-dependent mechanisms.