optimized-high-quality-dna-extraction-from-formalin-fixed-paraffin

Optimized High Quality DNA Extraction from Formalin-Fixed Paraffin-Embedded Human Atherosclerotic Lesions

Formalin-fixed paraffin-embedded (FFPE) tissues represent a valuable source for molecular analyses and clinical genomic studies. These tissues are often ...

Technical University Munich

University Hospital Zurich

Rimodellamento della cromatina gioca un ruolo essenziale nella regolazione della trascrizione del gene. Di conseguenza, mirati cambiamenti nella cromatina possono anche aumentare la pluripotenza delle cellule somatiche. Lo scopo del presente studio era di valutare l&#39;effetto della droga epigenetico BIX-01294 (BIX), un inibitore dell&#39;istone G9a, sulla metilazione del DNA, espressione di geni pluripotenza POU5F1 (isoforma un), NANOG, KLF4 e CMYC in cellule staminali mesenchimali e la capacit&agrave; di aumentare il loro potenziale di differenziazione in cellule endoteliali (ECs). Umane derivato da adiposo cellule staminali mesenchimali (AdMSCs) sono state isolate da tessuto adiposo addominale. Le cellule sono state pretrattate con BIX per 48 h e ulteriormente differenziato in mezzo endoteliale per 7 e 14 giorni. Metilazione globale del DNA &egrave; stata determinata dall&#39;applicazione di MethyLight, espressione di geni per la pluripotenza, endoteliale e marcatori angiogenic da PCR in tempo reale basati su SYBRGreen, immunocitochimica e immunobloting. A seguito di trattamento con BIX, lo stato di metilazione del DNA di AdMSCs era significativamente ridotto del 53% (p = 0,008), l&#39;espressione di POU5F1 e NANOG &egrave; stata aumentata da 2.2-fold (p = 0.016) e 1.5-fold (p &lt; 0.001), rispettivamente. Inoltre, il pretrattamento BIX migliorato la capacit&agrave; di differenziazione dei AdMSCs in ECs e significativamente aumentata espressione di diversi marcatori endoteliali e fattori coinvolti nella formazione del vaso sanguigno: VCAM-1, PECAM-1, fattore von Willebrand, VEGFR-2, PDGF e ANG-1 rispetto a AdMSCs senza pretrattamento BIX. In questo studio dimostriamo che droga modifica epigenetica che Bix-01294 &egrave; in grado di aumentare la capacit&agrave; di differenziarsi in ECs coinvolgente metilazione del DNA e dell&#39;istone AdMSCs. Quindi, BIX-01294 potrebbe servire come un semplice strumento per aumentare il potenziale di differenziazione delle AdMSCs.

Endoteliali differenziazione delle cellule staminali mesenchimali derivate adiposo &egrave; migliorato da droga modifica epigenetica BIX-01294.

&#x67D3;&#x8272;&#x8D28;&#x91CD;&#x5851;&#x57FA;&#x56E0;&#x8F6C;&#x5F55;&#x8C03;&#x8282;&#x4E2D;&#x53D1;&#x6325;&#x91CD;&#x8981;&#x4F5C;&#x7528;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x6709;&#x9488;&#x5BF9;&#x6027;&#x7684;&#x67D3;&#x8272;&#x8D28;&#x53D8;&#x5316;&#x4E5F;&#x80FD;&#x589E;&#x52A0;&#x4F53;&#x7EC6;&#x80DE;&#x7684;&#x591A;&#x529F;&#x80FD;&#x6027;&#x3002;&#x672C;&#x7814;&#x7A76;&#x7684;&#x76EE;&#x7684;&#x662F;&#x8BC4;&#x4F30;&#x8868;&#x89C2;&#x9057;&#x4F20;&#x836F;&#x7269; BIX-01294 (BIX)&#xFF0C;&#x4E00;&#x7EC4; G9a &#x86CB;&#x767D;&#x6291;&#x5236;&#x5242;&#xFF0C;&#x5BF9; DNA &#x7532;&#x57FA;&#x5316;&#x3001; &#x591A;&#x6F5C;&#x80FD;&#x57FA;&#x56E0; POU5F1 &#x8868;&#x8FBE;&#x7684;&#x5F71;&#x54CD; (&#x9F3B;&#x606F;&#x8089;)&#xFF0C;NANOG&#x3001; KLF4 &#x548C; CMYC &#x5728;&#x9AA8;&#x9AD3;&#x95F4;&#x5145;&#x8D28;&#x5E72;&#x7EC6;&#x80DE;&#xFF0C;&#x5E76;&#x589E;&#x52A0;&#x5176;&#x5206;&#x5316;&#x4E3A;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7EC6;&#x80DE; (ECs) &#x6F5C;&#x5728;&#x7684;&#x80FD;&#x529B;&#x3002;&#x4EBA;&#x7C7B;&#x8102;&#x80AA;&#x6E90;&#x6027;&#x95F4;&#x5145;&#x8D28;&#x5E72;&#x7EC6;&#x80DE; (AdMSCs) &#x88AB;&#x9694;&#x7EDD;&#x4E86;&#x4ECE;&#x8179;&#x90E8;&#x8102;&#x80AA;&#x3002;&#x7EC6;&#x80DE;&#x9884;&#x5904;&#x7406;&#x4E0E; BIX &#x4E3A; 48 h&#xFF0C;&#x8FDB;&#x4E00;&#x6B65;&#x5728;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x4E2D;&#x533A;&#x5206;&#x4E86; 7 &#x548C; 14 &#x5929;&#x3002;&#x5168;&#x7403; DNA &#x7532;&#x57FA;&#x5316;&#x786E;&#x5B9A;&#x7684; MethyLight &#x5E94;&#x7528;&#x7A0B;&#x5E8F;&#x4E2D;&#xFF0C;&#x4E3A;&#x591A;&#x6F5C;&#x80FD;&#x5185;, &#x76AE;&#x7EC6;&#x80DE;&#x548C;&#x8840;&#x7BA1;&#x751F;&#x6210;&#x6807;&#x8BB0;&#x7531; SYBRGreen &#x57FA;&#x4E8E;&#x5B9E;&#x65F6;&#x8367;&#x5149;&#x5B9A;&#x91CF; PCR&#x3001; &#x514D;&#x75AB;&#x7EC6;&#x80DE;&#x5316;&#x5B66;&#x548C; immunobloting &#x7684;&#x57FA;&#x56E0;&#x7684;&#x8868;&#x8FBE;&#x3002;BIX &#x4E0E;&#x6CBB;&#x7597;&#x4E4B;&#x540E;, DNA &#x7532;&#x57FA;&#x5316;&#x72B6;&#x6001;&#x7684; AdMSCs &#x5927;&#x5927;&#x51CF;&#x5C11;&#x4E86; 53%(p = 0.008)&#xFF0C;POU5F1 &#x548C; NANOG &#x7684;&#x8868;&#x8FBE;&#x7684;&#x589E;&#x52A0;&#x4E86; 2.2-fold (p = 0.016) &#x548C; 1.5-fold (p &lt; 0.001&#xFF09;&#xFF0C;&#x5206;&#x522B;&#x3002;&#x6B64;&#x5916;&#xFF0C;BIX &#x9884;&#x5904;&#x7406;&#x6539;&#x8FDB;&#x6210; ECs AdMSCs &#x7684;&#x5206;&#x5316;&#x80FD;&#x529B;&#x548C;&#x51E0;&#x4E2A;&#x5185;&#x76AE;&#x6807;&#x5FD7;&#x7269;&#x548C;&#x6240;&#x6D89;&#x53CA;&#x7684;&#x8840;&#x7BA1;&#x5F62;&#x6210;&#x56E0;&#x7D20;&#x5927;&#x5927;&#x589E;&#x52A0;&#x7684;&#x8868;&#x8FBE;&#x5F0F;&#xFF1A; &#x7C98;&#x9644;&#x5206;&#x5B50;-1&#x3001; PECAM 1&#x3001; &#x8840;&#x53CB;&#x75C5;&#x56E0;&#x5B50;&#x3001; &#x751F;&#x957F;&#x56E0;&#x5B50;&#x53D7;&#x4F53;-2&#x3001; &#x8840;&#x5C0F;&#x677F;&#x884D;&#x751F;&#x7684;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x548C;&#x6602;-1 &#x6BD4; AdMSCs &#x672A;&#x7ECF; BIX &#x9884;&#x5904;&#x7406;&#x3002;&#x5728;&#x672C;&#x7814;&#x7A76;&#x4E2D;&#x6211;&#x4EEC;&#x8868;&#x73B0;&#x51FA; BIX-01294 &#x662F;&#x80FD;&#x591F;&#x589E;&#x52A0; AdMSCs &#x80FD;&#x591F;&#x5206;&#x5316;&#x6210; ECs &#x4ECE;&#x4E8B; DNA &#x548C;&#x7EC4;&#x86CB;&#x767D;&#x7532;&#x57FA;&#x5316;&#x8868;&#x89C2;&#x9057;&#x4F20;&#x4FEE;&#x6539;&#x836F;&#x7269;&#x3002;&#x56E0;&#x6B64;&#xFF0C;BIX-01294 &#x53EF;&#x80FD;&#x4F5C;&#x4E3A;&#x4E00;&#x4E2A;&#x7B80;&#x5355;&#x7684;&#x5DE5;&#x5177;&#x6765;&#x63D0;&#x9AD8; AdMSCs &#x7684;&#x5206;&#x5316;&#x6F5C;&#x529B;&#x3002;

&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x5206;&#x5316;&#x7684;&#x8102;&#x80AA;&#x95F4;&#x5145;&#x8D28;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x8868;&#x89C2;&#x9057;&#x4F20;&#x4FEE;&#x6539;&#x836F;&#x7269; BIX-01294 &#x5F97;&#x5230;&#x6539;&#x8FDB;&#x3002;

Chromatin remodeling plays an essential role in regulation of gene transcription. Consequently, targeted changes in chromatin may also augment pluripotency of somatic cells. The aim of the present study was to evaluate the effect of epigenetic drug BIX-01294 (BIX), a histone G9a inhibitor, on DNA methylation, expression of pluripotency genes POU5F1 (isoform a), NANOG, KLF4, and CMYC in mesenchymal stem cells, and the ability to increase their differentiation potential into endothelial cells (ECs). Human adipose-derived mesenchymal stem cells (AdMSCs) were isolated from abdominal adipose tissue. Cells were pre-treated with BIX for 48h and further differentiated in endothelial medium for 7 and 14 days. Global DNA methylation was determined by MethyLight application, expression of genes for pluripotency, endothelial and angiogenic markers by SYBRGreen-based real-time PCR, immunocytochemistry, and immunobloting. Following treatment with BIX, DNA methylation status of AdMSCs was significantly reduced by 53% (p=0.008), the expression of POU5F1 and NANOG was increased by 2.2-fold (p=0.016) and 1.5-fold (p<0.001), respectively. Furthermore, BIX pre-treatment improved the differentiation capacity of AdMSCs into ECs and significantly increased expression of several endothelial markers and factors involved in blood vessel formation: VCAM-1, PECAM-1, von Willebrand factor, VEGFR-2, PDGF, and ANG-1 in comparison with AdMSCs without BIX pre-treatment. In the present study we demonstrate that epigenetic modifying drug BIX-01294 is able to increase the ability of AdMSCs to differentiate into ECs engaging DNA and histone methylation. Hence, BIX-01294 might serve as a simple tool to increase the differentiation potential of AdMSCs.

Endothelial differentiation of adipose-derived mesenchymal stem cells is improved by epigenetic modifying drug BIX-01294.

Remodelage de la chromatine joue un r&ocirc;le essentiel dans la r&eacute;gulation de la transcription du g&egrave;ne. Par cons&eacute;quent, des changements cibl&eacute;s dans la chromatine pourraient augmenter &eacute;galement la pluripotence des cellules somatiques. Le but de cette &eacute;tude &eacute;tait d&#39;&eacute;valuer l&#39;effet de la drogue &eacute;pig&eacute;n&eacute;tique 01294-BIX (BIX), un inhibiteur de l&#39;histone G9a, sur la m&eacute;thylation de l&#39;ADN, l&#39;expression des g&egrave;nes de pluripotence POU5F1 (isoforme un), NANOG, KLF4 et CMYC en cellules souches m&eacute;senchymateuses et la capacit&eacute; d&#39;augmenter leur potentielle de diff&eacute;renciation en cellules endoth&eacute;liales (CE). D&eacute;riv&eacute;s adipeux m&eacute;senchymateuses cellules souches humaines (AdMSCs) ont &eacute;t&eacute; isol&eacute;es du tissu adipeux abdominal. Les cellules ont &eacute;t&eacute; pr&eacute;-trait&eacute;es avec BIX pendant 48 h et plus diff&eacute;renci&eacute;e en endoth&eacute;liale moyenne pendant 7 &agrave; 14 jours. Globale m&eacute;thylation de l&#39;ADN a &eacute;t&eacute; d&eacute;termin&eacute;e par application de MethyLight, expression des g&egrave;nes de la pluripotence, endoth&eacute;liale et marqueurs angiog&eacute;niques par PCR en temps r&eacute;el bas&eacute; sur SYBRGreen, immunocytochimie et immunobloting. Apr&egrave;s le traitement avec BIX, statut de m&eacute;thylation de l&#39;ADN de AdMSCs a &eacute;t&eacute; significativement r&eacute;duit de 53 % (p = 0,008), l&#39;expression de POU5F1 et NANOG a &eacute;t&eacute; augment&eacute;e de 2.2-fold (p = 0,016) et 1,5 fois (p &lt; 0,001), respectivement. En outre, le pr&eacute;traitement BIX am&eacute;lior&eacute; la capacit&eacute; de diff&eacute;renciation des AdMSCs dans ECs et expression significativement accrue de plusieurs marqueurs endoth&eacute;liales et des facteurs impliqu&eacute;s dans la formation de vaisseaux sanguins : VCAM-1, PECAM-1, facteur de von Willebrand, VEGFR-2, PDGF et ANG-1 par rapport &agrave; AdMSCs sans traitement pr&eacute;alable BIX. Dans la pr&eacute;sente &eacute;tude, nous d&eacute;montrons cette drogue modification &eacute;pig&eacute;n&eacute;tique BIX-01294 est capable d&#39;augmenter la capacit&eacute; des AdMSCs &agrave; se diff&eacute;rencier en ECs s&#39;engageant la m&eacute;thylation de l&#39;ADN et les histones. Par cons&eacute;quent, BIX-01294 pourrait servir d&#39;un outil simple pour augmenter le potentiel de diff&eacute;renciation des AdMSCs.

Endoth&eacute;liale diff&eacute;renciation des cellules souches m&eacute;senchymateuses issues d&#39;adipeux est am&eacute;lior&eacute;e par la drogue modification &eacute;pig&eacute;n&eacute;tique 01294-BIX.

&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x306B;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x8EE2;&#x5199;&#x306E;&#x8ABF;&#x7BC0;&#x306B;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x305D;&#x306E;&#x7D50;&#x679C;&#x3001;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x306E;&#x5BFE;&#x8C61;&#x3068;&#x306A;&#x308B;&#x5909;&#x66F4;&#x3082;&#x4F53;&#x7D30;&#x80DE;&#x306E;&#x591A;&#x80FD;&#x6027;&#x3092;&#x5F37;&#x5316;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3002;&#x672C;&#x7814;&#x7A76;&#x306E;&#x76EE;&#x7684;&#x306F;&#x3001;&#x30A8;&#x30D4;&#x30B8;&#x30A7;&#x30CD;&#x30C6;&#x30A3;&#x30C3;&#x30AF;&#x306A;&#x85AC;&#x30D3;&#x30C3;&#x30AF;&#x30B9; 01294 (BIX)&#x3001;&#x591A;&#x80FD;&#x6027;&#x907A;&#x4F1D;&#x5B50; POU5F1 &#x306E;&#x767A;&#x73FE;&#x3001;DNA &#x306E;&#x30E1;&#x30C1;&#x30EB;&#x5316;&#x4E0A;&#x306E;&#x30D2;&#x30B9;&#x30C8;&#x30F3; G9a &#x963B;&#x5BB3;&#x5264;&#x306E;&#x52B9;&#x679C;&#x3092;&#x8A55;&#x4FA1;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3055;&#x308C;&#x305F; &#xFF08;&#x30A2;&#x30A4;&#x30BD; &#x30D5;&#x30A9;&#x30FC;&#x30E0;&#x306F;)&#x3001;NANOG&#x3001;KLF4&#x3001;CMYC &#x9593;&#x8449;&#x7CFB;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x5185;&#x76AE;&#x7D30;&#x80DE; (Ec) &#x306B;&#x6F5C;&#x5728;&#x7684;&#x306A;&#x5206;&#x5316;&#x3092;&#x4FC3;&#x9032;&#x3059;&#x308B;&#x3053;&#x3068;&#x3002;&#x3072;&#x3068;&#x8102;&#x80AA;&#x7D44;&#x7E54;&#x7531;&#x6765;&#x9593;&#x8449;&#x7CFB;&#x5E79;&#x7D30;&#x80DE; (AdMSCs) &#x306F;&#x3001;&#x8179;&#x90E8;&#x306E;&#x8102;&#x80AA;&#x7D44;&#x7E54;&#x304B;&#x3089;&#x5206;&#x96E2;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x7D30;&#x80DE;&#x3092;&#x30D3;&#x30C3;&#x30AF;&#x30B9;&#x3068; 48 h &#x3092;&#x524D;&#x51E6;&#x7406;&#x3057;&#x3055;&#x3089;&#x306B;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x4E2D;&#x306E; 7 &#xFF5E; 14 &#x65E5;&#x533A;&#x5225;&#x3057;&#x307E;&#x3059;&#x3002;&#x5168;&#x4F53;&#x7684;&#x306A; DNA &#x30E1;&#x30C1;&#x30EB;&#x5316;&#x306F; MethyLight &#x30A2;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x3001;&#x591A;&#x80FD;&#x6027;&#x3001;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x306E;&#x8840;&#x7BA1;&#x65B0;&#x751F;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x306B;&#x3088;&#x308B; SYBRGreen &#x30D9;&#x30FC;&#x30B9; &#x30EA;&#x30A2;&#x30EB;&#x30BF;&#x30A4;&#x30E0; PCR, &#x514D;&#x75AB;&#x67D3;&#x8272;, &#x304A;&#x3088;&#x3073; immunobloting &#x907A;&#x4F1D;&#x5B50;&#x306E;&#x767A;&#x73FE;&#x306B;&#x3088;&#x3063;&#x3066;&#x6C7A;&#x5B9A;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;BIX &#x306E;&#x6CBB;&#x7642;&#x306E;&#x5F8C;&#x3001;AdMSCs &#x306E; DNA &#x30E1;&#x30C1;&#x30EB;&#x5316;&#x72B6;&#x614B;&#x5927;&#x5E45;&#x306B; 53% &#x6E1B;&#x5C11;&#x3057;&#x305F; (p = 0.008&#xFF09;&#x3001;POU5F1 &#x304A;&#x3088;&#x3073; NANOG &#x306E;&#x767A;&#x73FE;&#x306B;&#x3088;&#x3063;&#x3066; 2.2 &#x5897;&#x8CC7; (p 0.016 =) &#x3068; 1.5 &#x500D; (p &lt; 0.001)&#x3001;&#x305D;&#x308C;&#x305E;&#x308C;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x30D3;&#x30C3;&#x30AF;&#x30B9;&#x524D;&#x51E6;&#x7406; AdMSCs ECs &#x3078;&#x306E;&#x5206;&#x5316;&#x80FD;&#x529B;&#x3068;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x30DE;&#x30FC;&#x30AB;&#x30FC;&#x304A;&#x3088;&#x3073;&#x8840;&#x7BA1;&#x306E;&#x5F62;&#x6210;&#x306B;&#x95A2;&#x4E0E;&#x3059;&#x308B;&#x56E0;&#x5B50;&#x306E;&#x5927;&#x5E45;&#x306B;&#x5897;&#x52A0;&#x3057;&#x305F;&#x767A;&#x73FE;&#x6539;&#x5584;: VCAM 1&#x3001;PECAM 1&#x3001;&#x30D5;&#x30A9;&#x30F3;&#x30F4;&#x30A3;&#x30EC;&#x30D6;&#x30E9;&#x30F3;&#x30C9;&#x56E0;&#x5B50;&#x3001;VEGFR 2&#x3001;PDGF&#x3001;&#x304A;&#x3088;&#x3073; ANG-1 &#x30D3;&#x30C3;&#x30AF;&#x30B9;&#x524D;&#x51E6;&#x7406;&#x306A;&#x3057; AdMSCs &#x3068;&#x6BD4;&#x8F03;&#x3057;&#x3066;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x30D3;&#x30C3;&#x30AF;&#x30B9; 01294 &#x306F; DNA &#x3068;&#x30D2;&#x30B9;&#x30C8;&#x30F3;&#x306E;&#x30E1;&#x30C1;&#x30EB;&#x5316;&#x9B45;&#x529B;&#x7684;&#x306A; ECs &#x306B;&#x533A;&#x5225;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B; AdMSCs &#x306E;&#x80FD;&#x529B;&#x3092;&#x9AD8;&#x3081;&#x308B;&#x3053;&#x3068;&#x304C;&#x305D;&#x306E;&#x5F8C;&#x6210;&#x7684;&#x5909;&#x66F4;&#x85AC;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x30D3;&#x30C3;&#x30AF;&#x30B9; 01294 AdMSCs &#x306E;&#x5206;&#x5316;&#x3055;&#x305B;&#x308B;&#x30B7;&#x30F3;&#x30D7;&#x30EB;&#x306A;&#x30C4;&#x30FC;&#x30EB;&#x3068;&#x3057;&#x3066;&#x5F79;&#x7ACB;&#x3064;&#x304B;&#x3082;&#x3057;&#x308C;&#x306A;&#x3044;&#x3002;

&#x8102;&#x80AA;&#x7531;&#x6765;&#x9593;&#x8449;&#x7CFB;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x5185;&#x76AE;&#x3078;&#x306E;&#x5206;&#x5316;&#x306F;&#x3001;&#x30A8;&#x30D4;&#x30B8;&#x30A7;&#x30CD;&#x30C6;&#x30A3;&#x30C3;&#x30AF;&#x306A;&#x5909;&#x66F4;&#x85AC;&#x30D3;&#x30C3;&#x30AF;&#x30B9; 01294 &#x306B;&#x3088;&#x3063;&#x3066;&#x6539;&#x5584;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#xAC1C;&#xC7A5; &#xD558;&#xB294; chromatin, &#xC720;&#xC804;&#xC790; &#xC804;&#xC0AC; &#xC870;&#xC808;&#xC5D0; &#xD544;&#xC218;&#xC801;&#xC778; &#xC5ED;&#xD560;&#xC744; &#xD569;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, chromatin &#xD0C0;&#xAC9F;&#xB41C; &#xBCC0;&#xACBD; pluripotency &#xCCB4;&#xC138;&#xD3EC;&#xC758; &#xC99D;&#xB300; &#xB610;&#xD55C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xD604;&#xC7AC; &#xC5F0;&#xAD6C;&#xC758; &#xBAA9;&#xD45C;&#xB294; epigenetic &#xB9C8;&#xC57D; BIX 01294 (BIX), DNA &#xBA54; &#xD2F8; &#xD654;, &#xD788;&#xC2A4;&#xD1A4; G9a &#xC5B5;&#xC81C;&#xC81C;, pluripotency &#xC720;&#xC804;&#xC790; pou5f1&#xC758; &#xC2DD;&#xC758; &#xD6A8;&#xACFC; &#xD3C9;&#xAC00; &#xD588;&#xB2E4; (isoform&#xB294;), NANOG, KLF4, &#xBC0F; CMYC &#xC911;&#xAC04; &#xC5FD; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC640; &#xB0B4; &#xD53C; &#xC138;&#xD3EC; (ECs)&#xB85C; &#xC7A0;&#xC7AC;&#xC801;&#xC778; &#xADF8;&#xB4E4;&#xC758; &#xBD84;&#xD654;&#xB97C; &#xC99D;&#xAC00; &#xD558;&#xB294; &#xB2A5;&#xB825;. &#xC778;&#xAC04; &#xC9C0;&#xBC29; &#xC720;&#xB798; &#xAC04; &#xC5FD; &#xC904;&#xAE30; &#xC138;&#xD3EC; (AdMSCs) &#xBCF5; &#xBD80; &#xC9C0;&#xBC29; &#xC870;&#xC9C1;&#xC5D0;&#xC11C; &#xACE0;&#xB9BD; &#xB418;&#xC5C8;&#xB2E4;. &#xC140; 48 h BIX&#xB85C; pre-treated &#xB418;&#xC5C8;&#xACE0; &#xCD94;&#xAC00; 7 ~ 14 &#xC77C;&#xC5D0; &#xB300; &#xD55C; &#xB0B4; &#xD53C; &#xC131;&#xC7A5; &#xB9E4;&#xCCB4;&#xC5D0; &#xCC28;&#xBCC4;&#xD654; &#xB41C;. &#xAE00;&#xB85C;&#xBC8C; DNA &#xBA54; &#xD2F8; &#xD654; MethyLight &#xC751;&#xC6A9; &#xD504;&#xB85C;&#xADF8;&#xB7A8;, pluripotency, &#xB0B4; &#xD53C; &#xBC0F; SYBRGreen &#xAE30;&#xBC18;&#xC758; &#xC2E4;&#xC2DC;&#xAC04; PCR, immunocytochemistry, &#xBC0F; immunobloting&#xC5D0; &#xC758;&#xD574; angiogenic &#xB9C8;&#xCEE4;&#xC5D0; &#xB300; &#xD55C; &#xC720;&#xC804;&#xC790;&#xC758; &#xD45C;&#xD604;&#xC5D0; &#xC758;&#xD574; &#xACB0;&#xC815; &#xB418;&#xC5C8;&#xB2E4;. &#xCE58;&#xB8CC; BIX, &#xB2E4;&#xC74C; Admscs&#xC758; DNA &#xBA54; &#xD2F8; &#xD654; &#xC0C1;&#xD0DC; &#xC0C1;&#xB2F9;&#xD788; 53%&#xB85C; &#xAC10;&#xC18C; &#xD588;&#xB2E4; (p = 0.008), POU5F1 &#xBC0F; NANOG &#xC2DD; 2.2-fold&#xC5D0; &#xC758;&#xD574; &#xC99D;&#xAC00; &#xD588;&#xB2E4; (p = 0.016) &#xBC0F; 1.5-fold (p &lt; 0.001), &#xAC01;&#xAC01;. &#xB610;&#xD55C;, BIX &#xC804;&#xCC98;&#xB9AC; &#xD5A5;&#xC0C1; &#xB41C; Ecs&#xC5D0; Admscs&#xC758; &#xCC28;&#xBCC4;&#xD654; &#xC6A9;&#xB7C9; &#xBC0F; &#xC5EC;&#xB7EC; &#xB0B4; &#xD53C; &#xB9C8;&#xCEE4; &#xBC0F; &#xD608;&#xAD00; &#xD615;&#xC131;&#xC5D0; &#xAD00;&#xC5EC; &#xC694;&#xC778; &#xD06C;&#xAC8C; &#xC99D;&#xAC00; &#xC2DD;: VCAM 1, PECAM-1, &#xD3F0; Willebrand &#xC778;&#xC790;, VEGFR 2, PDGF, &#xBC0F; &#xC911;&#xC559;-1 BIX &#xC804;&#xCC98;&#xB9AC; &#xC5C6;&#xC774; Admscs&#xC640; &#xBE44;&#xAD50; &#xD558; &#xC5EC;. &#xD604;&#xC7AC; &#xC5F0;&#xAD6C; BIX 01294 ECs DNA&#xC640; &#xD788;&#xC2A4;&#xD1A4; &#xBA54; &#xD2F8; &#xD654; &#xB9E4;&#xB825;&#xC73C;&#xB85C; &#xCC28;&#xBCC4;&#xD654; &#xD558;&#xB294; Admscs&#xC758; &#xB2A5;&#xB825;&#xC744; &#xC99D;&#xAC00; &#xC2DC;&#xD0AC; &#xC218; &#xC788;&#xB2E4; &#xADF8; &#xD6C4; &#xC218;&#xC815; &#xB9C8;&#xC57D; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, BIX 01294 Admscs&#xC758; &#xBD84;&#xD654; &#xAC00;&#xB2A5;&#xC131;&#xC744; &#xB192;&#xC774;&#xAE30; &#xC704;&#xD574; &#xAC04;&#xB2E8;&#xD55C; &#xB3C4;&#xAD6C; &#xC5ED;&#xD560;&#xC744; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xC9C0;&#xBC29; &#xC720;&#xB798; &#xC911;&#xAC04; &#xC5FD; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; &#xB0B4; &#xD53C; &#xBD84;&#xD654; &#xD6C4; &#xC218;&#xC815; &#xB9C8;&#xC57D; BIX 01294 &#xD5A5;&#xC0C1; &#xB429;&#xB2C8;&#xB2E4;.

Remodelaci&oacute;n de la cromatina juega un papel esencial en la regulaci&oacute;n de la transcripci&oacute;n del gene. En consecuencia, los cambios en la cromatina tambi&eacute;n pueden aumentar la pluripotencia de c&eacute;lulas som&aacute;ticas. El objetivo del presente estudio fue evaluar el efecto de f&aacute;rmacos epigen&eacute;ticos BIX-01294 (BIX), un inhibidor de la histona G9a, en la metilaci&oacute;n del ADN, la expresi&oacute;n de los genes de la pluripotencia POU5F1 (isoforma una), NANOG, KLF4 y CMYC en c&eacute;lulas madre mesenquimales y la capacidad de aumentar su potencial de diferenciaci&oacute;n en c&eacute;lulas endoteliales (ECs). Humanas derivado de adiposo c&eacute;lulas madre mesenquimales (AdMSCs) fueron aisladas de tejido adiposo abdominal. Las c&eacute;lulas fueron tratadas previamente con BIX durante 48h y m&aacute;s distinguen en medio endotelial por 7 y 14 d&iacute;as. Global metilaci&oacute;n del ADN se determin&oacute; mediante la aplicaci&oacute;n MethyLight, expresi&oacute;n de los genes para la pluripotencia, endotelial y marcadores angiog&eacute;nicos por PCR en tiempo real basado en el SYBRGreen, inmunocitoqu&iacute;mica y immunobloting. Despu&eacute;s del tratamiento con BIX, estatus de metilaci&oacute;n de ADN de AdMSCs se redujeron significativamente en un 53% (p = 0.008), la expresi&oacute;n de POU5F1 y NANOG aument&oacute; con el 2.2-fold (p = 0.016) y sufrir (p &lt; 0,001), respectivamente. Adem&aacute;s, tratamiento previo de BIX mejor&oacute; significativamente mayor expresi&oacute;n de varios marcadores endoteliales y factores que intervienen en la formaci&oacute;n de vasos sangu&iacute;neos y la capacidad de diferenciaci&oacute;n de AdMSCs en ECs: VCAM-1, PECAM-1, factor de von Willebrand, VEGFR-2, PDGF y ANG-1 en comparaci&oacute;n con AdMSCs sin tratamiento previo de BIX. En este estudio demostramos f&aacute;rmaco epigen&eacute;tico que Bix-01294 es capaz de aumentar la capacidad de diferenciarse en ECs participar la metilaci&oacute;n del ADN y las histonas de AdMSCs. Por lo tanto, BIX-01294 podr&iacute;a servir como una simple herramienta para incrementar el potencial de diferenciaci&oacute;n de AdMSCs.

Endotelial diferenciaci&oacute;n de c&eacute;lulas mesenquimales derivadas de adiposo es mejorada por f&aacute;rmaco epigen&eacute;tico BIX-01294.

Polyneuropathy organomegaly endocrinopathy M-protein skin changes (POEMS) syndrome is a rare cause of polyneuropathy. Calciphylaxis, a severe disease leading to necrotic ulcers of the skin, is associated with POEMS syndrome and also with renal disease. This case report describes a patient with POEMS syndrome plus primary focal segmental glomerulosclerosis.

POEMS syndrome, calciphylaxis and focal segmental glomerulosclerosis - VEGF as a possible link.

Signal integration between IFNγ and TLRs in immune cells has been associated with the host defense against pathogens and injury, with a predominant role of STAT1. We hypothesize that STAT1-dependent transcriptional changes in vascular cells involved in cross-talk between IFNγ and TLR4, reflect pro-atherogenic responses in human atherosclerosis. Genome-wide investigation identified a set of STAT1-dependent genes that were synergistically affected by interactions between IFNγ and TLR4 in VSMCs. These included the chemokines Cxcl9, Ccl12, Ccl8, Ccrl2, Cxcl10 and Ccl5, adhesion molecules Cd40, Cd74, and antiviral and antibacterial genes Rsad2, Mx1, Oasl1, Gbp5, Nos2, Batf2 and Tnfrsf11a. Among the amplified genes was also Irf8, of which Ccl5 was subsequently identified as a new pro-inflammatory target in VSMCs and ECs. Promoter analysis predicted transcriptional cooperation between STAT1, IRF1, IRF8 and NFκB, with the novel role of IRF8 providing an additional layer to the overall complexity. The synergistic interactions between IFNγ and TLR4 also resulted in increased T-cell migration and impaired aortic contractility in a STAT1-dependent manner. Expression of the chemokines CXCL9 and CXCL10 correlated with STAT1 phosphorylation in vascular cells in plaques from human carotid arteries. Moreover, using data mining of human plaque transcriptomes, expression of a selection of these STAT1-dependent pro-atherogenic genes was found to be increased in coronary artery disease (CAD) and carotid atherosclerosis. Our study provides evidence to suggest that in ECs and VSMCs STAT1 orchestrates a platform for cross-talk between IFNγ and TLR4, and identifies a STAT1-dependent gene signature that reflects a pro-atherogenic state in human atherosclerosis.

STAT1-dependent signal integration between IFNγ and TLR4 in vascular cells reflect pro-atherogenic responses in human atherosclerosis.

Proinflammatory adaptive immune responses are recognized as major drivers of atherosclerotic lesion formation. Although CD8(+) T cells have recently been proposed as a proatherogenic cell subset, their full scope of actions remains to be elucidated.

CD8+ T Cells Regulate Monopoiesis and Circulating Ly6C-high Monocyte Levels in Atherosclerosis in Mice.

Ischemic stroke of undetermined cause is a major health issue because of its high frequency and clinical relevance. Histopathologic analysis of human thrombi, retrieved from stroke patients with large-vessel occlusion during mechanical thrombectomy, may provide information about underlying pathologies. This study examines the relationship between stroke causes and histological clot composition to identify specific patterns that might help to distinguish causes of cryptogenic stroke.

Thrombus Histology Suggests Cardioembolic Cause in Cryptogenic Stroke.

In the search for markers and modulators of vascular disease, microRNAs (miRNAs) have emerged as potent therapeutic targets.

MicroRNA-210 Enhances Fibrous Cap Stability in Advanced Atherosclerotic Lesions.

Proefferocytic Therapy Promotes Transforming Growth Factor-β Signaling and Prevents Aneurysm Formation.

miRNAs are potential regulators of carotid artery stenosis and concordant vulnerable atherosclerotic plaques. Hence, we analyzed miRNA expression in laser captured micro-dissected fibrous caps of either ruptured or stable plaques (n = 10 each), discovering that miR-21 was significantly downregulated in unstable lesions. To functionally evaluate miR-21 in plaque vulnerability, miR-21 and miR-21/apolipoprotein-E double-deficient mice (ApoemiR-21) were assessed. miR-21 mice lacked sufficient smooth muscle cell proliferation in response to carotid ligation injury. When exposing ApoemiR-21 mice to an inducible plaque rupture model, they presented with more atherothrombotic events (93%) compared with miR-21Apoe mice (57%). We discovered that smooth muscle cell fate in experimentally induced advanced lesions is steered via a REST-miR-21-REST feedback signaling pathway. Furthermore, ApoemiR-21 mice presented with more pronounced atherosclerotic lesions, greater foam cell formation, and substantially higher levels of arterial macrophage infiltration. Local delivery of a miR-21 mimic using ultrasound-targeted microbubbles into carotid plaques rescued the vulnerable plaque rupture phenotype. In the present study, we identify miR-21 as a key modulator of pathologic processes in advanced atherosclerosis. Targeted, lesion site-specific overexpression of miR-21 can stabilize vulnerable plaques.

Local Delivery of miR-21 Stabilizes Fibrous Caps in Vulnerable Atherosclerotic Lesions.

Long noncoding RNAs have emerged as critical molecular regulators in various biological processes and diseases. Here we sought to identify and functionally characterize long noncoding RNAs as potential mediators in abdominal aortic aneurysm development.

H19 Induces Abdominal Aortic Aneurysm Development and Progression.

Atherosclerosis is a chronic inflammatory and lipid-depository disease that eventually leads to acute cardiovascular events. Emerging evidence supports that epigenetic processes such as DNA methylation, histone modification, and noncoding RNAs play an important role in plaque progression and vulnerability, highlighting the therapeutic potential of epigenetic drugs in cardiovascular therapeutics.

Atherosclerosis Is an Epigenetic Disease.

Atherosclerotic cerebrovascular disease underlies the majority of ischemic strokes and is a major cause of death and disability. While plaque burden is a predictor of adverse outcomes, plaque vulnerability is increasingly recognized as a driver of lesion rupture and risk for clinical events. Defining the molecular regulators of carotid instability could inform the development of new biomarkers and/or translational targets for at-risk individuals.

Article	Year
Endothelial differentiation of adipose-derived mesenchymal stem cells is improved by epigenetic modifying drug BIX-01294. European journal of cell biology\| PubMed ID: 23246144	2013
POEMS syndrome, calciphylaxis and focal segmental glomerulosclerosis - VEGF as a possible link. BMC neurology\| PubMed ID: 25369758	2014
STAT1-dependent signal integration between IFNγ and TLR4 in vascular cells reflect pro-atherogenic responses in human atherosclerosis. PloS one\| PubMed ID: 25478796	2014
CD8+ T Cells Regulate Monopoiesis and Circulating Ly6C-high Monocyte Levels in Atherosclerosis in Mice. Circulation research\| PubMed ID: 25991812	2015
Thrombus Histology Suggests Cardioembolic Cause in Cryptogenic Stroke. Stroke; a journal of cerebral circulation\| PubMed ID: 27197854	2016
MicroRNA-210 Enhances Fibrous Cap Stability in Advanced Atherosclerotic Lesions. Circulation research\| PubMed ID: 27895035	2017
Proefferocytic Therapy Promotes Transforming Growth Factor-β Signaling and Prevents Aneurysm Formation. Circulation\| PubMed ID: 29440201	2018
Local Delivery of miR-21 Stabilizes Fibrous Caps in Vulnerable Atherosclerotic Lesions. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 29503197	2018
H19 Induces Abdominal Aortic Aneurysm Development and Progression. Circulation\| PubMed ID: 29669788	2018
Atherosclerosis Is an Epigenetic Disease. Trends in endocrinology and metabolism: TEM\| PubMed ID: 29753613	2018
Chitinase 3 like 1 (CHI3L1) is a regulator of smooth muscle cell physiology and atherosclerotic lesion stability. Cardiovascular research\| PubMed ID: 33471078	2021

Jaroslav Pelisek

Biography

JoVE Journal Publications

Other Publications