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Die epigenetische Substanz BIX-01294 verbessert die Differenzierung mesenchymaler Zellen aus humanem Fettgewebe in Endothelzellen

The epigenetic substance BIX-01294 improves differentiation of mesenchymal cells from human fatty tissue into endothelial cells

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Zusammenfassung

Hintergrund

Patienteneigene Endothellzellen zur Auskleidung von Gefäßprothesen würden die Überlebenschance der betroffenen Patienten deutlich erhöhen und die Komplikationsrate signifikant reduzieren. Entsprechende Zellen sind jedoch schwierig in ausreichender Zahl zu gewinnen. Epigenetik spielt eine essenzielle Rolle in Regulation der Genexpression und Organogenese. Folglich könnte eine gezielte epigenetische Veränderung adulter somatischer Zellen zur Verbesserung deren Differenzierungspotenzials maßgeblich beitragen. Das Ziel der vorliegenden Studie lag deshalb in der Untersuchung der epigenetischen Substanz BIX-01294 (BIX), einem Inhibitor der Histon G9a Methyltransferase, im Bezug auf die Beeinflussung der DNA-Methylierung, der Expression der Pluripotenzgene OCT4, NANOG, KLF4, und CMYC in mesenchymalen Zellen und der Steigerung deren Differenzierungsfähigkeit in endotheliale Zellen.

Methoden

Humane mesenchymale Zellen wurden aus abdominalem Fettgewebe von Patienten gewonnen, die einem chirurgischen Eingriff in der Unfallchirurgie unterzogen wurden. Anschließend wurden die Zellen für 48 h mit BIX und danach mit Endothelzellmedium für weitere 7 und 14 Tage behandelt. Globale DNA-Methylierung wurde mittels PCR und der wiederholenden DNA-Sequenz LINE1 bestimmt. Die Expressionsanalyse wurde mit SYBR-Green RT-PCR, Immunhistochemie und Immunblotting durchgeführt. Die Differenzierung in Endothelzellen wurde über die Expression der endothelialen und angiogenetischen Marker analysiert.

Ergebnisse

Nach der Behandlung der mesenchymalen Zellen mit BIX reduzierte sich die globale DNA-Methylierung signifikant auf 53 % (p = 0,008), die Expression der Pluripotenzmarker PU5F1 und NANOG erhöhte sich 2,2- und 1,5-fach (p = 0,016 und p < 0,001). Weiterhin führte die Zugabe von BIX zu den mesenchymalen Zellen zur Erhöhung deren Differenzierung in Endothelzellen, nachgewiesen durch verstärkte Expression von VCAM-1, PECAM-1, von Willebrand-Faktor, VEGFR-2, PDGF und Angiopoietin 1.

Schlussfolgerung

In der vorliegenden Studie wurde zum ersten Mal gezeigt, dass eine Behandlung von geeigneten, mesenchymalen Zellen mit epigenetischen Substanzen, wie z. B. BIX-01294, zur Verbesserung ihres Differenzierungspotenzials beitragen kann. Folglich können Substanzen, die gezielt die Epigenetik beeinflussen, als Hilfsmittel dienen, um die Pluripotenz adulter Zellen zu erhöhen.

Abstract

Background

Autologous endothelial cells covering the inside of vascular grafts would markedly improve the survival of affected patients and would significantly reduce potential complications. However, it is difficult to obtain a sufficient yield of such cells. Epigenetic changes play an essential role in the regulation of gene transcription and organogenesis. Consequently, targeted changes in chromatin may also augment the pluripotency of somatic cells. The aim of the present study was to evaluate the effect of the epigenetic drug BIX-01294 (BIX) a histone G9a inhibitor, on DNA methylation, expression of the pluripotency genes POU5F1 (isoform a), NANOG, KLF4 and CMYC in mesenchymal stem cells and the ability to increase the differentiation potential into endothelial cells (EC).

Methods

Human mesenchymal stem cells were isolated from abdominal adipose tissue. Cells were pretreated with BIX for 48 h and further differentiated in endothelial medium for 7 and 14 days. Global DNA methylation was determined by PCR of the repetitive DNA sequence LINe1. Expression analysis was performed using SYBRgreen-based real-time PCR, immunocytochemistry and immunoblotting. Differentiation into endothelial cells was analyzed by detection of endothelial and angiogenic markers.

Results

After treatment of the mesenchymal cells with BIX, DNA methylation was significantly reduced by 53 % (P = 0.008), the expression of the pluripotency genes for POU5F1 and NANOG was increased 2.2-fold (P = 0.016) and 1.5-fold (P < 0.001), respectively. Furthermore, BIX pretreatment improved the differentiation capacity of mesenchymal cells into ECs and significantly increased expression of several endothelial markers and factors involved in blood vessel formation, such as VCAM-1, PECAM-1, von Willebrand factor, VEGFR-2, PDGF and ANG-1.

Conclusion

In the present study it was shown for the first time that treatment of mesenchymal cells with an appropriate epigenetic modifying drug, e.g. BIX-01294 increases the ability of these cells to differentiate into ECs. Consequently, targeted epigenetic modifications might be considered implemental to increase the differentiation potential of adult cells.

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Einhaltung ethischer Richtlinien

Interessenkonflikt. M. Culmes, A. Zernecke, H.-H. Eckstein und J. Pelisek geben an, dass kein Interessenkonflikt besteht. Dieser Beitrag beinhaltet keine Studien an Menschen oder Tieren.

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  1. Klinik und Poliklinik für Vaskuläre und Endovaskuläre Gefäßchirurgie, Klinikum rechts der Isar der Technischen Universität München, Ismaninger Str. 22, 81675, München, Deutschland

    M. Culmes, A. Zernecke, H.-H. Eckstein &  J. Pelisek

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  1. M. Culmes
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  2. A. Zernecke
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  3. H.-H. Eckstein
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Correspondence to J. Pelisek.

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Culmes, M., Zernecke, A., Eckstein, HH. et al. Die epigenetische Substanz BIX-01294 verbessert die Differenzierung mesenchymaler Zellen aus humanem Fettgewebe in Endothelzellen. Gefässchirurgie 19, 69–76 (2014). https://doi.org/10.1007/s00772-013-1271-0

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