P335	Inhibitors of histone deacetylation suppress the activation of the MAPK signaling and block the proliferation of human cSMC.
1T.Gierse, 2K.Peters, 1H.Kneer, 1J.Leng, 1H.Eschert, 3F.Jesacher, 3P.Loidl, 4M.Jung, 1A.Skaletz-Rorowski
1Leibniz-Inst. for Arteriosclerosis Research and Dept. of Cardiology and Angiology, Univ. of Münster, Münster, DE; 2Inst. of Pathology, Johannes Gutenberg Univ., Mainz, DE; 3Dept. of Molecular Biology, Univ. of Innsbruck, Innsbruck, AT; 4Inst. of Pharmaceutical Sciences, Univ. of Freiburg, Freiburg, DE.

The pharmacological modulation of gene expression is gaining increasing interest but only a few possible mechanisms of drug action have been evaluated for their therapeutical potential concerning atherosclerosis and restenosis. Both processes are characterised by uncontrolled proliferation of coronary smooth muscle cells (cSMC) which depends on the reversible acetylation of histone proteins by histone acetyltransferases or deacetylases (HDAC). We investigated the effect of the histone deacetylase inhibitor (HDAI) trichostatin A (TSA) and 37 new synthetic analogues, derived from the general structure of HDAI, on the proliferation of cSMC in vitro and we analyzed the molecular link between HDAC inhibition and control of proliferation of human cSMC in order to explore the therapeutic potential of HDAI in proliferative cardiovascular disorders. Using available antibodies for HDAC 1-7 we showed that all of these isoforms are expressed in human cSMC. Furthermore, all of the HDAI tested inhibit the deacetylase activity as shown by an in vitro assay. 20 compounds of the synthetic analogues inhibit the proliferation of bovine and human cSMC in a dose-dependent manner (0.05µM-50µM). Eight of the HDAI block the cSMC proliferation up to 90% in a concentration range between 0.05µM and 5µM. Two compounds induce a 50% inhibition of human cSMC proliferation at 0.1µM HDAI treatment. To understand these antiproliferative mechanisms, we studied the action of TSA and its analogue MD85 on cell cycle progression in human cSMC. HDAI induce a concentration-dependent reversible growth arrest in G1 phase, an inhibition of Retinoblastoma protein (pRb) phosphorylation, decreased cyclin D1 and A protein levels and CDK-inhibitory protein (CDKI) p21Waf1 upregulation. HDAI treatment resulted in both an inhibition of p42 mitogen activated protein kinase (MAPK) phosphorylation and a decrease of the MAPK-dependent c-myc expression inducing a downregulation of cyclin D1 and A protein levels and pRb hypophosphorylation, while upregulation of p21Waf1 is p42 MAPK (and p53) independent. Taken together, the ability of HDAI to disrupt cell cycle progression makes them interesting candidates for vascular proliferative disorders.

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