8 research outputs found

    Characterization of Carotid Smooth Muscle Cells during Phenotypic Transition

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    Vascular smooth muscle cells (VSMCs) are central players in carotid atherosclerosis plaque development. Although the precise mechanisms involved in plaque destabilization are not completely understood, it is known that VSMC proliferation and migration participate in plaque stabilization. In this study, we analyzed expression patterns of genes involved in carotid atherosclerosis development (e.g., transcription factors of regulation of SMC genes) of VSMCs located inside or outside the plaque lesion that may give clues about changes in phenotypic plasticity during atherosclerosis. VSMCs were isolated from 39 carotid plaques extracted from symptomatic and asymptomatic patients by endarterectomy. Specific biomarker expression, related with VSMC phenotype, was analyzed by qPCR, western immunoblot, and confocal microscopy. MYH11, CNN1, SRF, MKL2, and CALD1 were significantly underexpressed in VSMCs from plaques compared with VSMCs from a macroscopically intact (MIT) region, while SPP1, KLF4, MAPLC3B, CD68, and LGALS3 were found significantly upregulated in plaque VSMCs versus MIT VSMCs. The gene expression pattern of arterial VSMCs from a healthy donor treated with 7-ketocholesterol showed high similarity with the expression pattern of carotid plaque VSMCs. Our results indicate that VSMCs isolated from plaque show a typical SMC dedifferentiated phenotype with macrophage-like features compared with VSMCs isolated from a MIT region of the carotid artery. Additionally, MYH11, KLF5, and SPP1 expression patterns were found to be associated with symptomatology of human carotid atherosclerosis.This work was supported by Spanish Institute for Health Carlos III, RETICS program (Grant Number RD16/0019/0007) and by Basque Government, Education Department, Consolidated Groups program (Grant Number IT512-10)

    Autophagic Marker <i>MAP1LC3B</i> Expression Levels Are Associated with Carotid Atherosclerosis Symptomatology

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    <div><p>Objectives</p><p>The mechanism by which atheroma plaque becomes unstable is not completely understood to date but analysis of differentially expressed genes in stable versus unstable plaques may provide clues. This will be crucial toward disclosing the mechanistic basis of plaque instability, and may help to identify prognostic biomarkers for ischaemic events. The objective of our study was to identify differences in expression levels of 59 selected genes between symptomatic patients (unstable plaques) and asymptomatic patients (stable plaques).</p><p>Methods</p><p>80 carotid plaques obtained by carotid endarterectomy and classified as symptomatic (>70% stenosis) or asymptomatic (>80% stenosis) were used in this study. The expression levels of 59 genes were quantified by qPCR on RNA extracted from the carotid plaques obtained by endarterectomy and analyzed by means of various bioinformatic tools.</p><p>Results</p><p>Several genes associated with autophagy pathways displayed differential expression levels between asymptomatic and symptomatic (i.e. <i>MAP1LC3B</i>, <i>RAB24</i>, <i>EVA1A</i>). In particular, mRNA levels of <i>MAP1LC3B</i>, an autophagic marker, showed a 5−fold decrease in symptomatic samples, which was confirmed in protein blots. Immune system−related factors and endoplasmic reticulum-associated markers (i.e. <i>ERP27</i>, <i>ITPR1</i>, <i>ERO1LB, TIMP1, IL12B</i>) emerged as differently expressed genes between asymptomatic and symptomatic patients.</p><p>Conclusions</p><p>Carotid atherosclerotic plaques in which <i>MAP1LC3B</i> is underexpressed would not be able to benefit from <i>MAP1LC3B</i>−associated autophagy. This may lead to accumulation of dead cells at lesion site with subsequent plaque destabilization leading to cerebrovascular events. Identified biomarkers and network interactions may represent novel targets for development of treatments against plaque destabilization and thus for the prevention of cerebrovascular events.</p></div

    Validation of selected markers in an extended sample set.<sup>a</sup>

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    <p>The statistical significance was analyzed with the non-parametrical statistical test Mann-Whitney U test (* P≤0.05 and **** P≤0.0001). FC; +, overexpressed and –, underexpressed).</p>a<p>qPCR data analysis was performed in the combined set of the first (80 samples) and second cohorts (32 samples).</p><p>Validation of selected markers in an extended sample set.<sup><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115176#nt104" target="_blank">a</a></sup></p

    Ranking of gene expression markers according to highest fold change in asymptomatic (A) compared with symptomatic (S) samples quantified by Real Time RT−PCR.

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    <p>The statistical significance was analyzed with the non-parametrical statistical test Mann-Whitney U test (* P≤0.05 and ** P≤0.0001).</p><p>Ranking of gene expression markers according to highest fold change in asymptomatic (A) compared with symptomatic (S) samples quantified by Real Time RT−PCR.</p

    Correlation networks.

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    <p>The correlation has been computed as the normalised conditional mutual information. Only correlations above 0.7 are shown in this figure and with a significance of P<0.001.</p

    Differences in MAP1LC3B protein expression between symptomatic and asymptomatic.

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    <p>(A) MAP1LC3B and GAPDH carotid atheroma plaque protein levels were analyzed by Western blot and signal was detected on a ChemiDoc (XRS) detection system (BioRad). The blot shows the results from 5 asymptomatic and 4 symptomatic samples. (B) Densitometric analysis of western blot of LC3-II relative to GAPDH. *P = 0.015 symptomatic vs asymptomatic (Mann-Whitney U test).</p
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