P245	Gene regulation in coronary atherosclerotic plaques from patients with stable angina pectoris and acute coronary syndromes via nuclear remodeling.
2N.Marziliano, 1M.L.Rossi, 3P.Merlini, 2E.Arbustini, 1P.Presbitero, 4D.Ardissino
1Istituto Clinico Humanitas, Cardiologia, Milan- IT, JM; 2IRCCS Policlinco San Matteo, Transplant Area, Pavia-IT, JM; 3Opsedale Cà Granda Niguarda, Cardiologia, Milan-IT, DE; 4Ospedale Maggiore di Parma, Parma-IT, DE.

Background. Proliferation of vascular smooth muscle cell (VSMC) and differential expression of inflammatory and apoptotic markers have been demonstrated to be among one of the most important mechanisms in the atherosclerosis progression. Over-expression of inflammatory and pro-apoptotic genes from the VSMC may be the initial triggering for both plaque rupture and thrombotic response. Moreover, Tissue Factor (F3) has been found more abundant in acute coronary syndromes (ACS) patients with respect to those experiencing stable angina (SA) thus suggesting that its content may determine different thrombotic response to plaque rupture in human coronary arteries. Our aim was: a) to collect those VSMC from atherosclerotic plaques b) establish long term primary cell cultures c) evaluating whether there is a phenotypic commitment of tissue factor expressing cells within the atheroma. Methods and Results. We have isolated VSMC from 15 patients with SA and 15 with ACS after dissection under light microscopy. VSMC grown out, were confirmed by their typical “hill and valley” morphology and by α-actin immunofluorescent staining up to the 25th plating passages. Whole gene expression profiling was done at each passage via microarray analysis. Different cellular commitment in the ACS and SA VSMC was evaluated by applying small interfering RNA (siRNA) to revert the ACS pattern into SA and viceversa: this reversibility of ACS and SA VSMC was possible up the 15 passage, while only SA to ACS transitions were allowed after this time-point. In situ RT-PCR followed by FISH analysis with partial chromosome painting on VSMC was done for F3 (Ch 1p22-p21) and its inhibitor WISP3 (Ch 6q22-q23). Conclusions. We have demonstrated that the cell differentiation between SA and ACS VSMC is kept in primary cell cultures up to 25 plating passages. F3 from ACS VSMCs was 68 times more expressed than in SA plaques (p<0.01). FISH analysis revealed two distinct mutually exclusive arm domains for both Ch 1 and 6 being the two clustered together in ACS and apart in the SA plaques. Thus we demonstrated: a) a cell-specific orchestration of gene activity -that uses also nuclear remodeling of chromosomal domains- is present in SA and ACS plaques and b) the cell system is a suitable model for further studies on the pathophysiology of unstable angina and myocardial infarction.

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