P177	3-Deazaadenosine interferes with Ras-signaling and prevents neointima formation.
1D.Sedding, 1M.Troebs, 1F.Reich, 1G.Walker, 2L.Fink, 4W.Haberbosch, 1H.Tillmanns, 3W.Rau, 2R.Bohle, 3A.Langheinrich
1Cardiology - Giessen University Clinic, Gießen, DE; 2Pathology - Giessen University Clinic, Gießen, DE; 3Radiology - Giessen University Clinic, Gießen, DE; 4Cardiology - Central Hospital Suhl, Suhl, DE.

Background: 3-Deazaadenosine (c3Ado) is a potent inhibitor of S-adenosylhomocysteine hydrolase which regulates cellular methyltransferase activity. We previously reported that c3Ado prevents atherosclerotic lesion formation by its anti-inflammatory properties. In the present study we sought to determine c3Ados effect on vascular smooth muscle cell (VSMC) function and neointima formation in vivo. Methods and Results: Human VSMC were exposed to 10% FCS in the absence or presence of different concentrations of c3Ado. c3Ado prevented VSMC proliferation (52 ± 31 % vs. 199 ± 29 % increase in cell number after 48 h; P<0.001) and migration (29 ± 2 vs. 74 ± 9 cells/ high power field; P<0.001) at 50 µM. c3Ado dose dependently increased the expression of the cyclin-dependend kinase inhibitors p21WAF1/Cip1, p27Kip1, decreased the expression of cyclin A and cyclin E and prevented retinoblastoma protein hyperphosphorylation. In conclusion with these findings, FACS analysis of propidium iodide stained cells indicated a cell cycle arrest in the G0/G1 phase. Importantly, c3Ado did not affect the number of viable (trypan blue exclusion) or apoptotic cells (TUNEL). c3Ado prevented FCS-induced Ras carboxyl methylation, membrane translocation and activity and reduced FCS-induced ERK 1/2 and Akt phosphorylation in a dose-dependent manner. Furthermore, rescuing signal transduction by overexpression of a constitutive active Ras mutant abrogated c3Ados effect on proliferation. For in vivo studies, the femoral artery of C57BL/6 mice (n=6/group) was dilated and mice were fed with a diet containing 150 µg c3Ado/day. c3Ado prevented dilation-induced Ras-activation and ERK 1/2 and Akt phosphorylation. At day 21, VSMC proliferation (7.9 ± 0.7 % vs. 10.8 ± 0.8 % PCNA pos. cells P<0.05) as well as the neointima/media ratio (0.7 ± 0.2 vs. 1.6 ± 0.4; P<0.05) were significantly reduced, without any changes in the number of apoptotic cells or in medial area. Conclusion: Our data indicate that c3Ado interferes with Ras carboxyl methylation and function and thereby with mitogenic activation of ERK 1/2 and Akt, preventing VSMC cell cycle entry and proliferation and neointima formation in vivo. Thus, therapeutic application of c3Ado may represent a novel approach to prevent vascular proliferative disease.

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