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Characterization of human vascular smooth muscle cells from atherosclerotic and non-atherosclerotic carotid artery

Guastella, Giuseppe (2013) Characterization of human vascular smooth muscle cells from atherosclerotic and non-atherosclerotic carotid artery. PhD Thesis , Università degli Studi di Ferrara.

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    Background-Vascular smooth muscle cells (SMC), which are a major component of the atherosclerotic lesions, have an important role in initiation and progression of the atherosclerotic process. VSMCs migrate from the media into the intima layer of the arterial wall where they switch from a contractile to a synthetic phenotype, causing intimal thickening and plaque progression. Aim- The major aims of this study were 1)to reproducibly isolate and thoroughly characterize human arterial SMC subpopulations in order to evaluate their role in atheroma formation and their phenotypic changes associated to the atherosclerotic process; 2)to analyze the expression profiles of SMC subpopulations, in particular to validate signals stemming from a previous microarray study and 3) to investigate these signals both at RNA and protein level, on cultured SMCs cells and on carotid tissues directly. Methods-The following methods were used: 1)primary cell cultures of SMCs from normal (NP) and diseased (DP) portions of human carotid artery by tissue explantation; 2) quantitative real-time PCR (qPCR) on cultured VSMCs; 3)immunofluorescence (IF) on cultured VSMCs and carotid specimens 4) immunohistochemistry (IHC) on carotid specimens. Results- This study shows that large and small SMC subpopulations with different phenotypes can be isolated form CEA. Characterization of both subpopulations indicates that small SMCs exhibit "atheroma-prone" phenotype, characterized by high proliferation and low level of differentiation. We show that small SMCs, overexpressing CaM, proliferate more actively than large SMCs. Coculture of large SMCs with plaque-derived macrophages or conditioned medium or plaque induced a transition to the small phenotype with increased CaM expression. The CaM inhibitor W-7 decreased the proliferation of small SMCs and partially prevented the large to small phenotypic transition. Analysis of the expression profiles in SMC subpopulations and in carotid tissue specimens showed differences, which point to NOV (which belongs to CCN family) and ENPP2 (the enzyme that catalyzes the production of LPA) as potential components of the athero-prone phenotype. Additional studies in a larger number of samples, both at RNA and protein levels in cells and in tissue are needed to confirm data obtained. Moreover experiments addressing functional implications would required to corroborate the suggested association of markers, like CaM, NOV and ENPP2, with the atherosclerotic process.

    Item Type:Thesis (PhD Thesis)
    Date:25 March 2013
    Tutor:Marchetti, Giovanna
    Coordinator:Barbujani, Guido
    Institution:Università degli Studi di Ferrara
    Divisions:Dipartimento > Scienze della vita e biotecnologie
    Subjects:Area 05 - Scienze biologiche > BIO/10 Biochimica
    Uncontrolled Keywords:Cellule muscolari lisce vascolari, aterosclerosi, calmodulina, NOV(CCN3), ENPP2, livelli di espressione di RNA, vascular smooth muscle cells, atherosclerosis, calmodulin, expression levels
    Identification Number:10.5072//799
    Deposited on:17 Jun 2015 13:56


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