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ARBESU Y MIAR Anais (INSERM U1011 éq. 02 - Eric VAN BELLE)
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@mail :  arbesu.anais@gmail.com      tél. :  0032472401619

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Titre de la communication :
Characterization of human valvular interstitial cells isolated from normal and fibrocalcified aortic valves.
Auteurs (et leurs adresses) de la communication :
A. Arbesu Y Miar, A. Ung, R. Lorenzi, F. Juthier, A. Dupont, B. Staels, E. Van Belle, G. Chinetti-Gbaguidi, S. Susen, D. Corseaux Université Lille Nord de France, EA 2693, Faculté de Médecine, Pôle recherche, 1 place de Verdun, 59045 Lille, France
Résumé de la communication :
Purpose: Aortic Valve Stenosis (AVS) affects 2% to 6% of population over 65 years in industrialized countries. AVS involves Valve Interstitial Cells (VIC) proliferation and commitment to osteoblast-like cells. This prevalent cell type of thevalve is heterogeneous and presents five identifiable phenotypes: embryonic progenitor endothelial/mesenchymal cells, progenitor, quiescent, activated and osteoblastic VIC.To study the pathophysiology of AVS, their in vitro cultures are oftenused. Our purpose is to characterize VIC\'s phenotype from isolation from normal and fibrocalcified human aortic valves to different passages.

Methods: VIC were isolated by collagenase digestion from normal and fibrocalcific human tricuspid aortic valves (n=5 each). One cusp was conserved for immunohistological analyses. Characterization was assessed at different passages (P0, P3 to P5) by immunofluorescence and flow cytometry. We analyzed markers of progenitor cells (SSEA4 and ABCG2), fibroblasts (vimentin and HSP47), smooth muscle cells (SMC) (α-actin) and osteoblasts (OsteoBlast CaDHerin (OBCDH)). Viability and proliferation of VIC, in standard and starvation medium at 48h, were analyzed by blue trypan and Cell Titer MTS.

Results: Immunohistology showed the presence of all VIC phenotypes, independently of the valve naturebut in specific layer distribution. At the third passage, no statistical difference was found in progenitor markers (SSEA4: 68±10.2 % vs 47±14.5 % and ABCG2: 91±4.5 % vs 81.7±6.4 %; positive normal VIC percent vs pathological ones ± SEM). No difference was observed for fibroblast and SMC markers (vimentin: 73.7±34.5 % vs 84.7±17.8%; α-actin 5.7±5.4 % vs 29.1±27.9 %) except for HSP47 (21.9±3.7 % vs 46.8±4.7%, p=0.05). Osteoblast markers are more expressed by pathological VIC (OBCDH 0.6±0.5 %vs 3.1±0.9 %, p=0.05). However, there are changes in VIC subpopulations from cell isolation through culture passages (eg. 0.6 fold higher of SSEA4 positive pathological VIC between P0 and P3). Moreover, pathological VIC had lowerviability (89.6±7.9 % vs 76.5±5.3 %, p=0.02) but a higher proliferation index (1.4 fold normal VIC, p=0.02).

Conclusion: There is a fluctuation in the distribution of VIC subpopulations from the explanted valve through the progression of cell culture. Although all phenotypes persist through different passages, the prevalence of one or another depends on the nature of the aortic valve. These novel findings are of the utmost importance to analyze the responses of the mixed VIC population in vitro to understand their participation and the mechanisms of AVS.

ARBESU Y MIAR Anais (INSERM U1011 éq. 02 - Eric VAN BELLE)