proteomic-profiling-of-macrophages-by-2d-electrophoresis

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

University Lille Nord de France

Macrophages are believed to play a crucial role in atherogenesis and atherosclerotic plaque progression, mainly through their role in the accumulation of large amounts of cholesteryl ester and foam cell formation after the uptake into the arterial intima of oxidized LDL (oxLDL) particles known to be proatherogenic. The aim of this study was to use a differential proteomic approach to identify the response of human monocyte-derived macrophages after treatment with oxLDL for 24 h. Mass spectrometry analysis (MALDI-TOF) of 2D-DIGE gels made it possible to identify 9 intracellular and 3 secreted proteins that were up-regulated, 11 intracellular and 1 secreted proteins that were down-regulated, and 2 secreted proteins that were induced. This methodological approach not only confirmed the differential expression levels of proteins known to be regulated by oxLDL in macrophages, such as catalase and pyruvate kinase, but also identified oxLDL modulation of other proteins for the first time, including heat shock proteins (HSP) and Actin cytoskeletal proteins. Semiquantitative Western blot confirmed their role. The HSPs identified included heat shock cognate 71 kDa protein (Hsc70), 75 kDa glucose-regulated protein (GRP75), heat shock 70 kDa protein (Hsp70), and 60 kDa (Hsp60) proteins. These highly conserved intracellular protein chaperones, commonly seen in atherosclerotic plaques, appear to participate in protection against cellular stress. Interestingly, oxLDL also modulated several F-Actin capping proteins involved in Actin polymerization and motility: gelsolin, CapG, and CapZ. In conclusion, we have demonstrated the effects of oxLDL in the modulation of several proteins in human macrophages and established a functional profile of the human macrophage during the atherosclerotic process.

Application of saturation dye 2D-DIGE proteomics to characterize proteins modulated by oxidized low density lipoprotein treatment of human macrophages.

Abdominal aortic aneurysms (AAA) are defined by an increased aortic diameter and characterized by impairment of the extracellular matrix, macrophages infiltration and decreased density of smooth muscle cells. Our aim is to identify the key molecules involved in the pathogenesis of AAAs. This study investigated transcriptomic and proteomic profiles of macrophages from AAA patients (>50 mm aortic diameter) (n = 24) and peripheral arterial occlusion (PAO) patients without AAA detected (n = 18), who both needed a surgery. An antibody protein microarray, generated by printing antibodies onto membranes against proteins selected from the transcriptomic and proteomic analysis, was performed to validate the proteins differentially expressed specifically in macrophages and plasma from the same patients. We found a restricted number of proteins differentially expressed between AAA and PAO patients: TIMP-3, ADAMTS5, and ADAMTS8 that differ significantly in plasma of AAA patients compared to PAO patients, as found in the macrophages. In contrast to plasma MMP-9, soluble glycoprotein V (sGPV) and plasmin-antiplasmin complex levels, plasma TIMP-3 levels were not correlated to AAA size but interestingly correlated to sGPV, a platelet activation marker. Combining transcriptomic and proteomic is a valid approach to identify diseases causing proteins and potential biomarkers.

Profile of macrophages in human abdominal aortic aneurysms: a transcriptomic, proteomic, and antibody protein array study.

The aim of our study was to analyze the proteomic pattern of human macrophages obtained over a 4 year period from blood donors. The purpose was to simulate a long-term clinical study to assess the application of 2-D DIGE technique for differential proteomic analysis of these scarce samples. Bioinformatic analysis of 2-D DIGE gels of 19 different cultures of macrophages assessed whether they did or did not contain at least specific five spots identified by MS as being or containing bovine deoxyribonuclease I (DNase I). Bovine DNase I was used during sample treatment to remove nucleic acids from protein extracts. Macrophages were classified in two groups, which appeared to be differentiated by the completeness of DNase I treatment. Further detailed analysis revealed a different proteomic pattern of macrophage protein samples according to the completeness of this treatment. The major group of proteins affected, accounting for one third of the differentially expressed proteins, included proteins involved in cell motion and actin cytoskeleton reorganization. The use of DNase I for the removal of nucleic acids from protein samples must be avoided in proteomic studies since it can generate bias in the analysis of protein expression patterns.

Impact of incomplete DNase I treatment on human macrophage proteome analysis.

Abdominal aortic aneurysm (AAA) is characterized by increased aortic vessel wall diameter (>1.5 times normal) and loss of parallelism. This disease is responsible for 1-4% mortality occurring on rupture in males older than 65 years. Due to its asymptomatic nature, proteomic techniques were used to search for diagnostic biomarkers that might allow surgical intervention under nonlife threatening conditions.

Quantitative mass spectrometry analysis using PAcIFIC for the identification of plasma diagnostic biomarkers for abdominal aortic aneurysm.

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Thoracic aortic aneurysm is a pathological local dilatation of the aorta, potentially leading to aortic rupture or dissection. The disease is a common complication of patients with bicuspid aortic valve, a congenital disorder present in 1-2% of the population. Using 2 dimensional fluorescence difference gel electrophoresis proteomics followed by mRNA expression, and alternative splicing analysis of the identified proteins, differences in dilated and non-dilated aorta tissues between 44 patients with bicuspid and tricuspid valves was examined. The pattern of protein expression was successfully validated with LC-MS/MS. A multivariate analysis of protein expression data revealed diverging protein expression fingerprints in patients with tricuspid compared to the patients with bicuspid aortic valves. From 302 protein spots included in the analysis, 69 and 38 spots were differentially expressed between dilated and non-dilated aorta specifically in patients with tricuspid and bicuspid aortic valve, respectively. 92 protein spots were differentially expressed between dilated and non-dilated aorta in both phenotypes. Similarly, mRNA expression together with alternative splicing analysis of the identified proteins also showed diverging fingerprints in the two patient groups. Differential splicing was abundant but the expression levels of differentially spliced mRNA transcripts were low compared with the wild type transcript and there was no correlation between splicing and the number of spots. Therefore, the different spots are likely to represent post-translational modifications. The identification of differentially expressed proteins suggests that dilatation in patients with a tricuspid aortic valve involves inflammatory processes whereas aortic aneurysm in patients with BAV may be the consequence of impaired repair capacity. The results imply that aortic aneurysm formation in patients with bicuspid and tricuspid aortic valves involve different biological pathways leading to the same phenotype.

A combined proteomic and transcriptomic approach shows diverging molecular mechanisms in thoracic aortic aneurysm development in patients with tricuspid- and bicuspid aortic valve.

An&eacute;vrisme de l&#39;aorte thoracique est une dilatation locale pathologique de l&#39;aorte, pouvant d&eacute;boucher sur une rupture de l&#39;aorte ou de dissection. La maladie est une complication fr&eacute;quente des personnes atteintes de la valve aortique bicuspide, une affection cong&eacute;nitale pr&eacute;sente dans 1 &agrave; 2 % de la population. &Agrave; l&#39;aide de 2 dimensions fluorescence diff&eacute;rence gel &eacute;lectrophor&egrave;se prot&eacute;omique suivie d&#39;expression de l&#39;ARNm et l&#39;analyse des prot&eacute;ines identifi&eacute;es d&#39;&eacute;pissage alternatif, diff&eacute;rences dans les tissus de l&#39;aorte dilat&eacute;e et non dilat&eacute; entre 44 patients atteints de pr&eacute;molaire et valves de tricuspid a &eacute;t&eacute; examin&eacute;e. Le mod&egrave;le d&#39;expression de la prot&eacute;ine a &eacute;t&eacute; valid&eacute; avec succ&egrave;s par LC-MS/MS. Une analyse multivari&eacute;e des donn&eacute;es d&#39;expression de prot&eacute;ine a r&eacute;v&eacute;l&eacute; divergentes empreintes d&#39;expression de prot&eacute;ine chez les patients avec tricuspide par rapport aux patients avec des valves aortiques bicuspides. 302 Taches de prot&eacute;ines incluses dans l&#39;analyse, points 69 et 38 ont &eacute;t&eacute; diff&eacute;rentiellement exprim&eacute;s entre aorte dilat&eacute;e et non dilat&eacute; sp&eacute;cifiquement chez les patients avec une valve aortique tricuspide et pr&eacute;molaire, respectivement. 92 taches de prot&eacute;ines ont &eacute;t&eacute; exprim&eacute;s de fa&ccedil;on diff&eacute;rentielle entre l&#39;aorte dilat&eacute;e et non dilat&eacute; dans les deux ph&eacute;notypes. De m&ecirc;me, les ARNm expression ainsi qu&#39;une analyse &eacute;pissage alternative des prot&eacute;ines identifi&eacute;es ont montr&eacute; aussi divergentes empreintes digitales dans les deux groupes de patients. &Eacute;pissage diff&eacute;rentiel &eacute;tait abondant, mais les niveaux d&#39;expression des transcriptions d&#39;ARNm &eacute;piss&eacute; diff&eacute;rentiellement &eacute;taient faibles par rapport &agrave; la transcription de type sauvage et il n&#39;y n&#39;avait aucune corr&eacute;lation entre l&#39;&eacute;pissage et le nombre de taches. Par cons&eacute;quent, les diff&eacute;rents spots sont susceptibles de repr&eacute;senter des modifications post-traductionnelles. L&#39;identification des prot&eacute;ines diff&eacute;rentiellement exprim&eacute;es sugg&egrave;re cette dilatation chez les patients avec un tricuspide valve aortique implique des processus inflammatoires alors que l&#39;an&eacute;vrisme de l&#39;aorte chez les patients avec BAV peut &ecirc;tre la cons&eacute;quence de la capacit&eacute; de r&eacute;paration avec facult&eacute;s affaiblies. Les r&eacute;sultats indiquent que la formation d&#39;an&eacute;vrisme de l&#39;aorte chez les patients atteints de pr&eacute;molaire et valves aortiques tricuspides impliquent diff&eacute;rentes voies biologiques conduisant au m&ecirc;me ph&eacute;notype.

Un combin&eacute; prot&eacute;omique et transcriptomique approchent montre divergentes des m&eacute;canismes mol&eacute;culaires dans le d&eacute;veloppement d&#39;an&eacute;vrisme de l&#39;aorte thoracique chez les patients avec valve aortique tricuspide - et pr&eacute;molaire.

Aortenaneurysma ist eine pathologische lokalen Dilatation der Aorta, potenziell zu Aortenaneurysma-Ruptur oder Dissektion f&uuml;hrt. Die Krankheit ist eine h&auml;ufige Komplikation von Patienten mit bikuspide, eine angeborene St&ouml;rung bei 1-2 % der Bev&ouml;lkerung. Mit 2 dimensionale Fluoreszenz Unterschied Gel Elektrophorese Proteomics gefolgt von mRNA Ausdruck und Alternative Splei&szlig;en Analyse der identifizierten Proteine, 44 Patienten mit Pr&auml;molaren und Tricuspid Ventile Unterschiede bez&uuml;glich dilatative und nicht-dilatative ascendens Gewebe wurde untersucht. Das Muster der Proteinexpression wurde erfolgreich mit LC-MS/MS &uuml;berpr&uuml;ft. Eine multivariate Analyse von Protein-Ausdruck-Daten offenbart divergierende Protein Ausdruck Fingerabdr&uuml;cke bei Patienten mit Chordae im Vergleich zu Patienten mit bicuspid Aorten Ventile. Von 302 Protein-Spots in die Analyse einbezogen wurden 69 und 38 Spots differentiell zwischen dilatative und nicht-dilatative ascendens speziell bei Patienten mit Trikuspidalklappe und bicuspid Aortenklappe, bzw. ge&auml;u&szlig;ert. 92 Proteinspot wurden zwischen dilatative und nicht-dilatative ascendens in beiden Ph&auml;notypen differenziell ausgedr&uuml;ckt. In &auml;hnlicher Weise mit Fingerabdr&uuml;cken mRNA Expression zusammen mit alternativen Splei&szlig;en Analyse der identifizierten Proteine auch divergierende zeigte bei beiden Patientengruppen. Differenzielle Splei&szlig;en war reichlich aber die Stufen der differentiell gesplei&szlig;ten mRNA Transkripte niedrig waren, verglichen mit den Wildtyp-Transkript und gab es keine Korrelation zwischen Splei&szlig;en und der Anzahl der Spots. Daher d&uuml;rften die verschiedenen Spots &Uuml;bersetzungs&auml;nderungen darstellen. Die Identifizierung der differentiell exprimierten Proteinen suggeriert, dass Dilatation bei Patienten mit einer Chordae Aortenklappe umfasst entz&uuml;ndliche Prozesse Aortenaneurysma bei Patienten mit BAV kann die Folge gest&ouml;rter Reparatur-Kapazit&auml;t sein. Die Ergebnisse implizieren, dass Aortenaneurysma-Bildung bei Patienten mit Pr&auml;molaren und Trikuspidalklappe Aorten Ventile beinhalten verschiedene biologische &Uuml;bertragungswege bei der gleichen Ph&auml;notyp.

Eine kombinierte Proteomic und Transcriptomic Ansatz zeigt unterschiedliche Molekulare Mechanismen bei Aortenaneurysma Entwicklung bei Patienten mit Chordae und Pr&auml;molaren Aortenklappe.

Aneurisma dell&#39;aorta toracica &egrave; una dilatazione patologica locale dell&#39;aorta, potenzialmente portando a rottura aortica o dissezione. La malattia &egrave; una complicanza comune dei pazienti con valvola aortica bicuspide, una malattia congenita presente nel 1-2% della popolazione. Differenze nei tessuti aorta dilatata e non dilatate tra 44 pazienti con bicuspide e tricuspide valvole mediante fluorescenza dimensionale 2 differenza gel elettroforesi proteomics espressione del mRNA e alternative splicing analisi delle proteine identificate in seguito, &egrave; stato esaminato. Il pattern di espressione della proteina &egrave; stato convalidato con LC-MS/MS. Un&#39;analisi multivariata di dati dell&#39;espressione della proteina ha rivelato divergenti delle impronte di espressione della proteina nei pazienti con tricuspide rispetto ai pazienti con valvola aortica bicuspide. Da 302 proteine punti inclusi nell&#39;analisi, punti 69 e 38 erano differenzialmente espressi tra aorta dilatata e non dilatate in particolare nei pazienti con valvola aortica bicuspide e tricuspide, rispettivamente. 92 punti proteine differenzialmente espresse tra aorta dilatata e non dilatate in entrambi fenotipi. Allo stesso modo, il mRNA di espressione insieme ad analisi di splicing alternativo di proteine identificate ha mostrato anche divergenti le impronte digitali nei due gruppi di pazienti. Splicing differenziale era abbondante, ma i livelli di espressione dei trascritti di mRNA differenzialmente impiombato erano bassi rispetto con la trascrizione di tipo selvatico e non non c&#39;era alcuna correlazione tra lo splicing e il numero di macchie. Pertanto, i diversi punti sono suscettibili di rappresentare traslazionali. L&#39;identificazione di proteine differenzialmente espresse suggerisce che dilatazione in pazienti con una tricuspide valvola aortica coinvolge processi infiammatori considerando aneurisma aortico nei pazienti con BAV pu&ograve; essere la conseguenza della capacit&agrave; di riparazione alterata. I risultati implicano che formazione di aneurisma aortico in pazienti con bicuspide e tricuspide valvole aortiche coinvolgono diverse vie biologiche che porta lo stesso fenotipo.

Un combinato proteomic e trascrittomica approccio spettacoli divergenti meccanismi molecolari nello sviluppo di aneurisma dell&#39;aorta toracica in pazienti con valvola aortica bicuspide e tricuspide.

&#x80F8;&#x90E8;&#x5927;&#x52D5;&#x8108;&#x7624;&#x6F5C;&#x5728;&#x7684;&#x5927;&#x52D5;&#x8108;&#x7834;&#x88C2;&#x307E;&#x305F;&#x306F;&#x90ED;&#x6E05;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B;&#x5927;&#x52D5;&#x8108;&#x306E;&#x75C5;&#x7406;&#x5B66;&#x7684;&#x30ED;&#x30FC;&#x30AB;&#x30EB;&#x62E1;&#x5F35;&#x3067;&#x3059;&#x3002;&#x3053;&#x306E;&#x75C5;&#x6C17;&#x306F;&#x4E00;&#x822C;&#x7684;&#x306A;&#x5408;&#x4F75;&#x75C7;&#x60A3;&#x8005;&#x306E;&#x5927;&#x52D5;&#x8108;&#x4E8C;&#x5C16;&#x5927;&#x52D5;&#x8108;&#x5F01;&#x3001;&#x5148;&#x5929;&#x6027;&#x969C;&#x5BB3;&#x306E;&#x4EBA;&#x53E3;&#x306E; 1-2 &#xFF05; &#x306B;&#x5B58;&#x5728;&#x3067;&#x3059;&#x3002;MRNA &#x306E;&#x767A;&#x73FE;&#x3068;&#x89E3;&#x6790;&#x8B58;&#x5225;&#x3055;&#x308C;&#x305F;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x9078;&#x629E;&#x7684;&#x30B9;&#x30D7;&#x30E9;&#x30A4;&#x30B7;&#x30F3;&#x30B0;&#x306E;&#x4EE3;&#x66FF;&#x304C;&#x7D9A;&#x304F; 2 &#x6B21;&#x5143;&#x86CD;&#x5149;&#x5DEE;&#x30B2;&#x30EB;&#x306E;&#x96FB;&#x6C17;&#x6CF3;&#x52D5;&#x30D7;&#x30ED;&#x30C6;&#x30AA;&#x30DF;&#x30AF;&#x30B9;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;44 &#x75C7;&#x4F8B;&#x5C0F;&#x81FC;&#x6B6F;&#x3068;&#x4E09;&#x5C16;&#x5F01;&#x306E;&#x62E1;&#x5F35;&#x304A;&#x3088;&#x3073;&#x975E;&#x62E1;&#x5F35;&#x5927;&#x52D5;&#x8108;&#x7D44;&#x7E54;&#x306E;&#x76F8;&#x9055;&#x306B;&#x3064;&#x3044;&#x3066;&#x691C;&#x8A0E;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x8868;&#x73FE;&#x306E;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x306F; LC MS/MS &#x3092;&#x6B63;&#x5E38;&#x306B;&#x691C;&#x8A3C;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x767A;&#x73FE;&#x91CF;&#x30C7;&#x30FC;&#x30BF;&#x306E;&#x591A;&#x5909;&#x91CF;&#x89E3;&#x6790;&#x767A;&#x6563;&#x578B;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x767A;&#x73FE;&#x6307;&#x7D0B;&#x60A3;&#x8005;&#x306B;&#x304A;&#x3051;&#x308B;&#x5927;&#x52D5;&#x8108;&#x4E8C;&#x5C16;&#x5927;&#x52D5;&#x8108;&#x5F01;&#x3068;&#x60A3;&#x8005;&#x306B;&#x6BD4;&#x8F03;&#x3057;&#x3066;&#x4E09;&#x5C16;&#x3067;&#x660E;&#x3089;&#x304B;&#x306B;&#x3002;&#x5206;&#x6790;&#x306B;&#x542B;&#x307E;&#x308C;&#x3066;&#x3001;302 &#x306E;&#x86CB;&#x767D;&#x30B9;&#x30DD;&#x30C3;&#x30C8;&#x304B;&#x3089; 69 &#x3068; 38 &#x30B9;&#x30DD;&#x30C3;&#x30C8;&#x5DEE;&#x52D5;&#x62E1;&#x5F35;&#x304A;&#x3088;&#x3073;&#x975E;&#x62E1;&#x5F35;&#x5927;&#x52D5;&#x8108;&#x5177;&#x4F53;&#x7684;&#x306B;&#x4E09;&#x5C16;&#x5F01;&#x30FB;&#x5927;&#x52D5;&#x8108;&#x4E8C;&#x5C16;&#x5927;&#x52D5;&#x8108;&#x5F01;&#x3001;&#x60A3;&#x8005;&#x9593;&#x305D;&#x308C;&#x305E;&#x308C;&#x8868;&#x73FE;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;92 &#x86CB;&#x767D;&#x30B9;&#x30DD;&#x30C3;&#x30C8;&#x306F;&#x4E21;&#x65B9;&#x306E;&#x8868;&#x73FE;&#x578B;&#x3067;&#x62E1;&#x5F35;&#x304A;&#x3088;&#x3073;&#x975E;&#x62E1;&#x5F35;&#x5927;&#x52D5;&#x8108;&#x9593;&#x7279;&#x7570;&#x7684;&#x306B;&#x8868;&#x73FE;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x540C;&#x69D8;&#x306B;&#x3001;&#x5206;&#x5C90;&#x306F;&#x307E;&#x305F;&#x8B58;&#x5225;&#x3055;&#x308C;&#x305F;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x4EE3;&#x66FF;&#x30B9;&#x30D7;&#x30E9;&#x30A4;&#x30B7;&#x30F3;&#x30B0;&#x5206;&#x6790;&#x3068;&#x5171;&#x306B;&#x767A;&#x73FE;&#x3092;&#x793A;&#x3057;&#x305F; mRNA &#x306F; 2 &#x3064;&#x306E;&#x60A3;&#x8005;&#x30B0;&#x30EB;&#x30FC;&#x30D7;&#x306E;&#x6307;&#x7D0B;&#x3057;&#x307E;&#x3059;&#x3002;&#x5DEE;&#x52D5;&#x30B9;&#x30D7;&#x30E9;&#x30A4;&#x30B7;&#x30F3;&#x30B0;&#x306F;&#x8C4A;&#x5BCC;&#x3060;&#x3063;&#x305F;&#x304C;&#x3001;&#x91CE;&#x751F;&#x578B;&#x30C8;&#x30E9;&#x30F3; &#x30B9;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066;&#x7279;&#x7570;&#x7684;&#x30B9;&#x30D7;&#x30E9;&#x30A4;&#x30B7;&#x30F3;&#x30B0;&#x306E; mrna &#x306E;&#x8868;&#x73FE;&#x306E;&#x30EC;&#x30D9;&#x30EB;&#x306F;&#x4F4E;&#x304B;&#x3063;&#x305F;&#x3057;&#x3001;&#x30B9;&#x30D7;&#x30E9;&#x30A4;&#x30B7;&#x30F3;&#x30B0;&#x3068;&#x30B9;&#x30DD;&#x30C3;&#x30C8;&#x306E;&#x6570;&#x3068;&#x306E;&#x76F8;&#x95A2;&#x306F;&#x3042;&#x308A;&#x307E;&#x305B;&#x3093;&#x3067;&#x3057;&#x305F;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x5225;&#x306E;&#x30B9;&#x30DD;&#x30C3;&#x30C8;&#x306F;&#x3001;&#x30DD;&#x30B9;&#x30C8;&#x7FFB;&#x8A33;&#x306E;&#x4FEE;&#x6B63;&#x3092;&#x8868;&#x3059;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;&#x767A;&#x73FE;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x540C;&#x5B9A;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x308B;&#x305D;&#x306E;&#x62E1;&#x5F35;&#x75C7;&#x60A3;&#x8005;&#x306F;&#x4E09;&#x5C16;&#x306E;&#x5927;&#x52D5;&#x8108;&#x5F01;&#x306B;&#x306F;&#x5927;&#x52D5;&#x8108;&#x7624;&#x60A3;&#x8005;&#x306E; BAV &#x969C;&#x5BB3;&#x4FEE;&#x5FA9;&#x80FD;&#x529B;&#x306E;&#x7D50;&#x679C;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x4E00;&#x65B9;&#x3001;&#x708E;&#x75C7;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x304C;&#x542B;&#x307E;&#x308C;&#x307E;&#x3059;&#x3002;&#x305D;&#x306E;&#x5927;&#x52D5;&#x8108;&#x7624;&#x5F62;&#x6210;&#x5C0F;&#x81FC;&#x6B6F;&#x75C7;&#x4F8B;&#x306B;&#x304A;&#x3051;&#x308B;&#x793A;&#x5506;&#x3068;&#x5C16;&#x5927;&#x52D5;&#x8108;&#x5F01;&#x3092;&#x542B;&#x3080;&#x69D8;&#x3005; &#x306A;&#x751F;&#x7269;&#x5B66;&#x7684;&#x7D4C;&#x8DEF;&#x306B;&#x540C;&#x3058;&#x8868;&#x73FE;&#x578B;&#x3092;&#x30EA;&#x30FC;&#x30C9;&#x3057;&#x307E;&#x3059;&#x3002;

&#x8907;&#x5408;&#x30D7;&#x30ED;&#x30C6;&#x30AA;&#x30FC;&#x30E0;&#x3001;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30AF;&#x30EA;&#x30D7;&#x30C8;&#x30FC;&#x30E0;&#x5927;&#x52D5;&#x8108;&#x5F01;&#x5C16;&#x3068;&#x5C0F;&#x81FC;&#x6B6F;&#x75C7;&#x4F8B;&#x306B;&#x304A;&#x3051;&#x308B;&#x80F8;&#x90E8;&#x5927;&#x52D5;&#x8108;&#x7624;&#x306E;&#x958B;&#x767A;&#x306B;&#x304A;&#x3051;&#x308B;&#x5206;&#x5B50;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x767A;&#x6563;&#x30B7;&#x30E7;&#x30FC;&#x3092;&#x30A2;&#x30D7;&#x30ED;&#x30FC;&#x30C1;&#x3057;&#x307E;&#x3059;&#x3002;

&#xD749;&#xBD80; &#xB300;&#xB3D9;&#xB9E5; &#xB3D9;&#xB9E5; &#xB958;&#xB294; &#xB300;&#xB3D9;&#xB9E5; &#xD30C;&#xC5F4; &#xB610;&#xB294; &#xD574; &#xBD80;&#xC5D0; &#xC7A0;&#xC7AC;&#xC801;&#xC73C;&#xB85C; &#xC774;&#xC5B4;&#xC9C0;&#xB294; &#xB300;&#xB3D9;&#xB9E5;&#xC758; &#xBCD1; &#xC801;&#xC778; &#xC9C0;&#xC5ED; &#xD33D;&#xCC3D;. &#xC9C8;&#xBCD1; bicuspid &#xB300;&#xB3D9;&#xB9E5; &#xBC38;&#xBE0C;, &#xC778;&#xAD6C;&#xC758; 1-2%&#xC5D0; &#xC874;&#xC7AC; &#xD558;&#xB294; &#xC120; &#xCC9C; &#xC131; &#xC7A5;&#xC560; &#xD658;&#xC790;&#xC758; &#xC77C;&#xBC18;&#xC801;&#xC778; &#xD569;&#xBCD1;&#xC99D; &#xC774;&#xB2E4;. 2 &#xCC28;&#xC6D0; &#xD615;&#xAD11; &#xCC28;&#xC774; &#xC824; &#xC804;&#xAE30; &#xC774;&#xB3D9; &#xBC95; proteomics &#xB4A4;&#xC5D0; mRNA &#xD45C;&#xD604;&#xACFC; &#xD655;&#xC778; &#xB41C; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xBD84;&#xC11D;&#xC744; &#xC811;&#xD569; &#xD558;&#xB294; &#xB300;&#xC548;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; bicuspid 44 &#xD658;&#xC790;&#xC640; tricuspid &#xBC38;&#xBE0C; &#xD655;&#xB300; &#xBC0F; &#xBE44; &#xB113;&#xD790; &#xB300;&#xB3D9;&#xB9E5; &#xC870;&#xC9C1;&#xC5D0;&#xC11C; &#xCC28;&#xC774;&#xC810; &#xC870;&#xC0AC; &#xB418;&#xC5C8;&#xB2E4;. &#xB2E8;&#xBC31;&#xC9C8; &#xBC1C;&#xD604; &#xD328;&#xD134; &#xC131;&#xACF5;&#xC801;&#xC73C;&#xB85C; LC-MS/MS&#xC640; &#xD568;&#xAED8; &#xC720;&#xD6A8;&#xC131;&#xC744; &#xAC80;&#xC0AC; &#xD569;&#xB2C8;&#xB2E4;. &#xB2E8;&#xBC31;&#xC9C8; &#xC2DD; &#xB370;&#xC774;&#xD130;&#xC758; &#xBCC0;&#xC218; &#xBD84;&#xC11D; tricuspid bicuspid &#xB300;&#xB3D9;&#xB9E5; &#xBC38;&#xBE0C;&#xC640; &#xD658;&#xC790; &#xB4E4;&#xC5D0; &#xBE44;&#xD574; &#xAC00;&#xC9C4; &#xD658;&#xC790;&#xC5D0;&#xC11C; diverging &#xB2E8;&#xBC31;&#xC9C8; &#xC2DD; &#xC9C0;&#xBB38; &#xACF5;&#xAC1C;. 302 &#xB2E8;&#xBC31;&#xC9C8; &#xBD84;&#xC11D;&#xC5D0; &#xD3EC;&#xD568; &#xB41C; &#xAD00;&#xAD11; &#xBA85;&#xC18C;&#xC5D0;&#xC11C; 69 &#xBC0F; 38 &#xC810;&#xC774; &#xB41C; &#xCC28;&#xB3D9; &#xAC01;&#xAC01; tricuspid bicuspid &#xB300;&#xB3D9;&#xB9E5; &#xBC38;&#xBE0C;&#xB97C; &#xAC00;&#xC9C4; &#xD658;&#xC790;&#xC5D0;&#xC11C; &#xD2B9;&#xD788; &#xB3D9; &#xACF5;&#xC774; &#xD655;&#xC7A5; &#xB418;&#xC5B4; &#xBC0F; &#xBE44; &#xB113;&#xD790; &#xB300;&#xB3D9;&#xB9E5; &#xC0AC;&#xC774;&#xC758; &#xD45C;&#xD604; &#xB429;&#xB2C8;&#xB2E4;. 92 &#xB2E8;&#xBC31;&#xC9C8; &#xBC18;&#xC810; &#xB450; &#xACE0;&#xAE30;&#xC5D0; &#xB3D9; &#xACF5;&#xC774; &#xD655;&#xC7A5; &#xB418;&#xC5B4; &#xBC0F; &#xBE44; &#xB113;&#xD790; &#xB300;&#xB3D9;&#xB9E5; &#xC0AC;&#xC774; &#xCC28;&#xB3D9; &#xD45C;&#xD604; &#xD588;&#xB2E4;. &#xB9C8;&#xCC2C;&#xAC00;&#xC9C0;&#xB85C; &#xD655;&#xC778; &#xB41C; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xB300;&#xCCB4; &#xC811;&#xD569; &#xBD84;&#xC11D; &#xD568;&#xAED8; &#xC2DD; &#xB610;&#xD55C; &#xBCF4;&#xC5EC;&#xC8FC;&#xC5C8;&#xB2E4; &#xBD84;&#xAE30; mRNA &#xB450; &#xD658;&#xC790; &#xADF8;&#xB8F9;&#xC5D0; &#xC9C0;&#xBB38;. &#xCC28;&#xB3D9; &#xC811;&#xD569;&#xB294; &#xD48D;&#xBD80; &#xD558;&#xC9C0;&#xB9CC; &#xC2DD; &#xC218;&#xC900;&#xC758; &#xCC28;&#xB3D9; spliced mRNA &#xC131;&#xC801;&#xD45C; &#xD588;&#xB2E4; &#xC57C;&#xC0DD; &#xD0C0;&#xC785; &#xB300; &#xBCF8;&#xC5D0; &#xBE44;&#xD574; &#xB0AE;&#xC740; &#xACE0; &#xC811;&#xD569; &#xBC0F; &#xAD00;&#xAD11; &#xBA85;&#xC18C;&#xC758; &#xC218; &#xAC04;&#xC5D0; &#xC0C1;&#xAD00; &#xAD00;&#xACC4;&#xAC00; &#xC5C6;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, &#xB2E4;&#xB978; &#xAD00;&#xAD11; &#xBA85;&#xC18C; &#xD3EC;&#xC2A4;&#xD2B8; &#xBC88;&#xC5ED; &#xC0C1; &#xC218;&#xC815; &#xB098;&#xD0C0;&#xB0BC; &#xAC00;&#xB2A5;&#xC131;&#xC774; &#xB192;&#xB2E4;. &#xCC28;&#xB3D9; &#xD45C;&#xD604;&#xB41C; &#xB2E8;&#xBC31;&#xC9C8; &#xC2DD;&#xBCC4; &#xB098;&#xC654;&#xB2E4;&#xB294; tricuspid &#xD658;&#xC790;&#xC5D0;&#xC11C; &#xADF8; &#xD33D;&#xCC3D; &#xB300;&#xB3D9;&#xB9E5; &#xBC38;&#xBE0C; BAV &#xD658;&#xC790;&#xC5D0;&#xC11C; &#xB300;&#xB3D9;&#xB9E5; &#xB3D9;&#xB9E5; &#xC7A5;&#xC560; &#xBCF5;&#xAD6C; &#xB2A5;&#xB825;&#xC758; &#xACB0;&#xACFC; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xBC18;&#xBA74; &#xC5FC;&#xC99D; &#xC131; &#xD504;&#xB85C;&#xC138;&#xC2A4;&#xB97C; &#xC218;&#xD589; &#xD574;&#xC57C; &#xD569;&#xB2C8;&#xB2E4;. &#xACB0;&#xACFC; bicuspid &#xAC00;&#xC9C4; &#xD658;&#xC790;&#xC5D0;&#xC11C; &#xB300;&#xB3D9;&#xB9E5; &#xB3D9;&#xB9E5; &#xB958; &#xD615;&#xC131;&#xC744; &#xC554;&#xC2DC; tricuspid &#xB300;&#xB3D9;&#xB9E5; &#xBC38;&#xBE0C; &#xD3EC;&#xD568; &#xB2E4;&#xB978; &#xC0DD;&#xBB3C;&#xD559; &#xD1B5;&#xB85C; &#xAC19;&#xC740; &#xD45C;&#xD604; &#xD615;&#xC744; &#xC120;&#xB3C4; &#xD558; &#xACE0; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xACB0;&#xD569;&#xB41C; proteomic &#xBC0F; transcriptomic &#xC1FC; bicuspid &#xBC0F; tricuspid-&#xB300;&#xB3D9;&#xB9E5; &#xBC38;&#xBE0C;&#xB97C; &#xAC00;&#xC9C4; &#xD658;&#xC790;&#xC5D0;&#xC11C; &#xD749;&#xBD80; &#xB300;&#xB3D9;&#xB9E5; &#xB3D9;&#xB9E5; &#xB958; &#xAC1C;&#xBC1C;&#xC5D0; &#xBD84;&#xC790; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xBD84;&#xAE30;&#xC5D0; &#xC811;&#xADFC; &#xD55C;&#xB2E4;.

Aneurisma de la aorta tor&aacute;cica es una dilataci&oacute;n patol&oacute;gica del local de la aorta, puede conducir a la ruptura a&oacute;rtica o disecci&oacute;n. La enfermedad es una complicaci&oacute;n frecuente de los pacientes con v&aacute;lvula a&oacute;rtica bic&uacute;spide, un desorden cong&eacute;nito presente en 1-2% de la poblaci&oacute;n. Utilizando 2 fluorescencia dimensional diferencia gel de electroforesis prote&oacute;mica seguido de la expresi&oacute;n del mRNA y alternativa que empalma el an&aacute;lisis de las prote&iacute;nas identificadas, diferencias en los tejidos de la aorta dilatada y no dilatado 44 pacientes con bic&uacute;spide y tric&uacute;spide v&aacute;lvulas fue examinado. El patr&oacute;n de expresi&oacute;n de la prote&iacute;na fue validado con &eacute;xito con LC-MS/MS. Un an&aacute;lisis multivariante de datos de la expresi&oacute;n de prote&iacute;na revel&oacute; divergentes las huellas de expresi&oacute;n de prote&iacute;nas en pacientes con tric&uacute;spide en comparaci&oacute;n con los pacientes con v&aacute;lvulas a&oacute;rticas bic&uacute;spide. De 302 puntos de prote&iacute;na incluidas en el an&aacute;lisis, 69 y 38 puntos diferencialmente expresaron entre aorta dilatada y no dilatado espec&iacute;ficamente en pacientes con v&aacute;lvula a&oacute;rtica bic&uacute;spide y tric&uacute;spide, respectivamente. 92 puntos de prote&iacute;na fueron expresados diferencialmente entre aorta dilatada y no dilatado en ambos fenotipos. Del mismo modo, mRNA expresi&oacute;n junto con an&aacute;lisis de empalme alternativo de las prote&iacute;nas identificadas tambi&eacute;n mostraron divergentes huellas digitales en los dos grupos de pacientes. Empalme diferencial fue abundante pero los niveles de expresi&oacute;n de las transcripciones de mRNA diferencialmente empalmado fueron bajos en comparaci&oacute;n con la transcripci&oacute;n de tipo salvaje y no hab&iacute;a correlaci&oacute;n entre el empalme y el n&uacute;mero de puntos. Por lo tanto, los diferentes lugares suelen representar modificaciones post-traduccionales. La identificaci&oacute;n de prote&iacute;nas expresadas diferencialmente sugiere que la dilataci&oacute;n en pacientes con un tric&uacute;spide v&aacute;lvula a&oacute;rtica implica procesos inflamatorios que aneurisma a&oacute;rtico en pacientes con BAV puede ser la consecuencia de la alteraci&oacute;n de la reparaci&oacute;n. Los resultados implican que formaci&oacute;n de aneurisma de la aorta en pacientes con bic&uacute;spide y tric&uacute;spides v&aacute;lvulas a&oacute;rticas implican distintas v&iacute;as biol&oacute;gicas hacia el mismo fenotipo.

Un combinado prote&oacute;micos y transcript&oacute;micos enfoque muestra divergentes mecanismos moleculares en el desarrollo de aneurisma de aorta tor&aacute;cica en pacientes con v&aacute;lvula a&oacute;rtica bic&uacute;spide y tric&uacute;spide.

In this study, we developed a novel computational approach based on protein-protein interaction networks to identify a list of proteins that might have remained undetected in differential proteomic profiling experiments. We tested our computational approach on two sets of human smooth muscle cell protein extracts that were affected differently by DNase I treatment. Differential proteomic analysis by saturation DIGE resulted in the identification of 41 human proteins. The application of our approach to these 41 input proteins consisted of four steps: (i) Compilation of a human protein-protein interaction network from public databases; (ii) calculation of interaction scores based on functional similarity; (iii) determination of a set of candidate proteins that are needed to efficiently and confidently connect the 41 input proteins; and (iv) ranking of the resulting 25 candidate proteins. Two of the three highest-ranked proteins, beta-arrestin 1, and beta-arrestin 2, were experimentally tested, revealing that their abundance levels in human smooth muscle cell samples were indeed affected by DNase I treatment. These proteins had not been detected during the experimental proteomic analysis. Our study suggests that our computational approach may represent a simple, universal, and cost-effective means to identify additional proteins that remain elusive for current 2D gel-based proteomic profiling techniques.

Identification of additional proteins in differential proteomics using protein interaction networks.

The incidence of carcinoma increases greatly with aging, but the cellular and molecular mechanisms underlying this correlation are only partly known. It is established that senescent fibroblasts promote the malignant progression of already-transformed cells through secretion of inflammatory mediators. We investigated here whether the senescent fibroblast secretome might have an impact on the very first stages of carcinogenesis. We chose the cultured normal primary human epidermal keratinocyte model, because after these cells reach the senescence plateau, cells with transformed and tumorigenic properties systematically and spontaneously emerge from the plateau. In the presence of medium conditioned by autologous senescent dermal fibroblasts, a higher frequency of post-senescence emergence was observed and the post-senescence emergent cells showed enhanced migratory properties and a more marked epithelial-mesenchymal transition. Using pharmacological inhibitors, siRNAs, and blocking antibodies, we demonstrated that the MMP-1 and MMP-2 matrix metalloproteinases, known to participate in late stages of cancer invasion and metastasis, are responsible for this enhancement of early migratory capacity. We present evidence that MMPs act by activating the protease-activated receptor 1 (PAR-1), whose expression is specifically increased in post-senescence emergent keratinocytes. The physiopathological relevance of these results was tested by analyzing MMP activity and PAR-1 expression in skin sections. Both were higher in skin sections from aged subjects than in ones from young subjects. Altogether, our results suggest that during aging, the dermal and epidermal skin compartments might be activated coordinately for initiation of skin carcinoma, via a paracrine axis in which MMPs secreted by senescent fibroblasts promote very early epithelial-mesenchymal transition of keratinocytes undergoing transformation and oversynthesizing the MMP-activatable receptor PAR-1.

Senescent fibroblasts enhance early skin carcinogenic events via a paracrine MMP-PAR-1 axis.

Heart failure (HF) following myocardial infarction (MI) is characterized by progressive alterations of left ventricular (LV) structure and function, named LV remodelling. Although several risk factors such as infarct size have been identified, HF remains difficult to predict in clinical practice. Recently, using phosphoproteomic technology, we found that serine(208)-phosphorylated troponin T (P-Ser(208)-TnT) decreases in LV of HF rats. Our aim was to determine the performance of P-Ser(208)-TnT as plasma biomarker of HF compared to conventional cardiac biomarkers such as B-type natriuretic peptide (BNP), cardiac troponin I (cTnI), C-reactive protein (CRP) or tissue inhibitor of metalloproteinase I (TIMP-1) measured by x-MAP technology, as well as its capacity to reflect a pharmacological improvement of HF. We observed a significant increase of BNP, TnT and cTnI levels and a significant decrease of P-Ser(208)-TnT and TIMP-1 in the plasma of 2-month-MI rats compared with control rats with no modulation of CRP level. Circulating levels of P-Ser(208)-TnT were shown to be associated with most of the echocardiographic and haemodynamic parameters of cardiac function. We verified that the decrease of P-Ser(208)-TnT was not because of an excess of phosphatase activity in plasma of HF rats. Two-month-MI rats treated with the heart rate reducing agent ivabradine had improved LV function and increased plasma levels of P-Ser(208)-TnT. Thus, circulating phosphorylated troponin T is a highly sensitive biological indicator of cardiac dysfunction and has the potentiality of a new biomarker of HF post-MI, and of a surrogate marker for the efficacy of a successful treatment of HF.

Circulating plasma serine208-phosphorylated troponin T levels are indicator of cardiac dysfunction.

Left ventricular (LV) remodeling following myocardial infarction (MI) is characterized by progressive alterations of structure and function, named LV remodeling. Although several risk factors such as infarct size have been identified, LV remodeling remains difficult to predict in clinical practice. Changes within the extracellular matrix, involving matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), are an integral part of left ventricular (LV) remodeling after myocardial infarction (MI). We investigated the temporal profile of circulating MMPs and TIMPs and their relations with LV remodeling at 1 year and clinical outcome at 3 years in post-MI patients.

Serum MMP-8: a novel indicator of left ventricular remodeling and cardiac outcome in patients after acute myocardial infarction.

Remote ischemic preconditioning (RIPC) has emerged as an attractive strategy in clinical settings. Despite convincing evidence of the critical role played by circulating humoral mediators, their actual identities remain unknown. In this study, we aimed to identify RIPC-induced humoral mediators using a proteomic approach.

Apolipoprotein a-I is a potential mediator of remote ischemic preconditioning.

Remote ischemic preconditioning's (RIPC) ability to render the myocardium resistant to subsequent prolonged ischemia is now clearly established in different species, including humans. Strong evidence suggests that circulating humoral mediators play a key role in signal transduction, but their identities still need to be established. Our study sought to identify potential circulating RIPC mediators using a proteomic approach. Rats were exposed to 10-min limb ischemia followed by 5- (RIPC 5') or 10-min (RIPC 10') reperfusion prior to blood sampling. The control group only underwent blood sampling. Plasma samples were isolated for proteomic analysis using surface-enhanced laser desorption and ionization - time of flight - mass spectrometry (SELDI-TOF-MS). A total of seven proteins, including haptoglobin and transthyretin, were detected as up- or down-regulated in response to RIPC. These proteins had previously been identified as associated with organ protection, anti-inflammation, and various cellular and molecular responses to ischemia. In conclusion, this study indicates that RIPC results in significant modulations of plasma proteome.

Maggy Chwastyniak

Inserm UMR744

University Lille Nord de France

Proteomic Profiling of Macrophages by 2D Electrophoresis

Maggy Chwastyniak

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University Lille Nord de France

Proteomic Profiling of Macrophages by 2D Electrophoresis

Inserm UMR744