1 AG Molecular Cardiology and Stem Cell Research, Clinic for Internal Medicine I Friedrich Schiller University Jena Prof. Dr. Maria Wartenberg Erlanger Allee 101, Jena Our research interests focus on signalling pathways leading to cardiovascular differentiation. We like to develop a new approach to generate cardiomyocytes from stem cells of different origin. Also we are interested to characterise cardiovascular progenitor cells and study which microenvironment and which signalling cascades conduct progenitor cells to differentiate into functional cardiomyocytes and endothelial cells as well. Our major research fields are: 1. Cardiovascular differentiation from embryonic stem cells / Redox-sensitive signalling pathways / Function of NAD(P)H oxidases in blood vessel and cardiomyocyte differentiation 2. Stem cell derived in vitro model systems/ replacement of animal experiments / in vitro models of cellular defence and inflammation / leucocyte differentiation from ES-cells 3. Induced pluripotent stem cells (ips-cells)/ generating ips-cells from fibroblasts or mesenchymal stem cells / isolation and characterisation of ips clones / regulation the embryonic gene expression. 4. Metabolic processes and differentiation/ effect of PPARγ on differentiation and proliferation Research Projects in detail: 1.1 Redox sensitive signalling pathways leading to cardiovascular differentiation The project was supported by SFB 604 "Multifunctional Signalling Proteins - Oligomeric Protein Complexes as Mediators of Cellular Regulation Processes" Embryonic stem (ES) cells spontaneously differentiate into blood vessels and cardiomyocytes. We and other groups pointed out that reactive oxygen species (ROS) act as second messengers in signalling cascades leading to cardiovascular differentiation. We study the interaction of NAD(P)H oxidases with receptor tyrosine kinase induced signalling cascades which influence vasculogenesis/angiogenesis and cardiomyocyte differentiation in a particularly controlled mechanism. Angiogenesis assay on Matrigel showed formation of capillary networks Immunofluorescence staining for Vasculogenesis, angiogenesis and Isolated cardiomyocytes detected by laser scanning confocal microscope (C-LSM ) ROS levels were measured using the fluorescent dye H2DCF-DA and recorded by C-LSM
2 1.2 Membrane potential changes and external electromagnetic field application control angiogenesis and pacemaker/cardiomyocyte differentiation in stem cells The project was supported by Deutsche Stiftung Herzforschung External modulation of the membrane potential induces signalling cascades in ES cells which control the differentiation of blood vessels cardiomyocytes and chondro-osteogenesis as well. We study the effect of external electromagnetic field pulses on stem cell differentiation. Induced membrane potential changes at multicellular spheroids following application of external electromagnetic field pulses; di-8-anneps and Confocal laser scanning microscopy. Induced membrane potential changes: Hyperpolarisation, 1,600 1,550 1,500 Ratio - Intensity 1,450 1,400 1,350 1,300 1,250 1,200 18,920 31,530 44,150 56,760 69,370 81,990 Time [s] 1.3 NAD(P)H -oxidases regulate angiogenesis and leukocyte differentiation The project was supported by the IZKF Jena ROS generated by the NADPH -oxidase enzyme complex may act as second messenger and can thus control cellular processes such as proliferation, differentiation, and gene expression. Aim of this study is to assess the impact of the NADPH-oxidases NOX1, NOX2, and NOX4 on the vascular and leukocyte differentiation using the murine in-vitro-differentiation model of the Embryoid Body (EB). Therefore, we established stem cell lines repressing NOX1, NOX2, or NOX4 expression by shrna technique. 2. In vitro models for cellular defence and inflammation: Testing the biocompatibility of biodegradable polymers in vitro The project was supported by Stiftung Forschung 3R, Swizerland Artificial implants are more and more common in clinical daily routine and were used in large diversity. This includes for instance dental prosthetics, bone reconstruction, artificial joints, vascular prosthetics and artificial heart valves. Recently biocompatible polymers and biodegradable polymers were developed. They have enormous potential in processing of artificial heart valves, artificial blood vessels, occluders etc. Biodegradable Polymers support healing process; they are able to degrade in a biological surrounding. In this project we use ES cell to cultivate an immuno- competent vascularised tissue (INFLAPLANTtissue) which is able to show a cellular response to specific materials which allows conclusions about the biocompatibility. Our INFLAPLANT-tissue approves the study of angiogenesis, differentiation of inflammatory cells and their migration next to the polymer material. Leukocytes indicated by CD68 immunohistochemistry (yellow) Angiogenesis indicated by PECAM-1 immunohistochemistry (red) in
3 interact with biodegradable polymers (blue). INFLAPLANT-tissue in co- culture with biodegredable polymers (blue) 3.1 Generation of induced pluripotent stem cells (ips-cells) We generate ips cells from human fibroblasts and adult mesenchymal stem cells. Our aim is to develop protocols for the induction of endothelial cells and cardiomyocytes from ips cells. PECAM-1 immunohistochemistry on a layer-culture from cells differentiated from human ips-cells Human Induced pluripotent stem (ips) cell colony cultured on mouse embryonic feeders 3.2 Effect of microenvironment on embryonic gene expression in ES-cells, human ASC and induced pluripotent stem cells. Embryonic genes (oct3/4, Nanog, Lin28, Sox) characterise pluripotency and plasticity in embryonic, adult stem cells and ips-cells after reprogramming. We intend to explain how the microenvironment of different stem cell types controls the expression of embryonic genes. 4. The effect of PPARγ (Peroxisome proliferator-activated receptor-γ) on differentiation and proliferation of endothelial cells and smooth muscle cells PPARγ affects pathways important in a variety of human diseases. Moreover, PPARγ agonists exert anti-inflammatory, anti-oxidative and anti-proliferative effects on vascular wall cells. We conclude that the activation or inhibition of PPARγ may have effects of the differentiation of endothelial cells, smooth muscle or cardiomyocytes. Furthermore, PPARγ agonists are likely to have atheroprotective properties. In a previous project we took part in a study investigating the effect of PPARα on cardiomyogenesis from murine ES-cells. In the actual project, which represents a translational collaboration between basic and clinical research, we seek to determine the effect of PPARγ agonists on endothelial and smooth muscle cell differentiation and proliferation in vitro and to compare this data with experimental findings from blood vessels in vivo. Other study objectives are: to identify the sites were PPARγ interacts with receptor tyrosine kinase pathways, to investigate if PPARγ inhibits NAD(P)H -oxidases and how this interaction takes place.
4 Anti-PECAM1 (red) and Anti-alpha smooth muscle (green) immunohistochemistry for 20 days differentiated EBs plated in petri dishes Our projects are conducted in collaboration with: 1. Collaborative Projects at our University Prof. Dr. Reinhard Wetzker, CMB Prof. Dr. Regine Heller, CMB Prof. Dr. Heinemann, CMB PD Dr. Jörg Müller, CMB PD Dr. Markert, Plazentalabor, Frauenklinik Prof. Dr. Ralf Claus, Anesthesiologie Prof. Dr. Schultze-Mosgau, Klinik für Gesichts- und Kieferchirurgie 2. External Collaborative Projects Prof. Dr. Heinrich Sauer, Universitätsklinikum Gießen Prof. Dr. Anna Wobus, IPK, Gatersleben Prof. Dr. Fouad A. Abou-Zaid, Tanta University, Egypt PD Dr. Kleger, Universitätsklinikum Ulm Prof. Dr. med. A.H. Jacobs European Institute for Molecular Imaging - EIMI University of Münster Dr. Schnabelrauch INNOVENT e.v. Jena Our group welcomes all those who are interested to cooperate with us. Publications : 2010 The 5-lipoxygenase pathway regulates vasculogenesis in differentiating mouse embryonic stem cells. Finkensieper A, Kieser S, Bekhite MM, Richter M, Mueller JP, Graebner R, Figulla HR, Sauer H, Wartenberg M. Cardiovasc Res Jan 6. [Epub ahead of print] Glycolytic pyruvate regulates P-Glycoprotein expression in multicellular tumor spheroids via modulation of the intracellular redox state. Wartenberg M, Richter M, Datchev A, Günther S, Milosevic N, Bekhite MM, Figulla HR, Aran JM, Pétriz J, Sauer H. J Cell Biochem Feb 1;109(2): Platelet-derived growth factor BB stimulates vasculogenesis of embryonic stem cell-derived endothelial cells by calcium-mediated generation of reactive oxygen species. Lange S, Heger J, Euler G, Wartenberg M, Piper HM, Sauer H. Cardiovasc Res Jan 1;81(1):
5 Static electromagnetic fields induce vasculogenesis and chondro-osteogenesis of mouse embryonic stem cells by reactive oxygen species-mediated upregulation of vascular endothelial growth factor. Bekhite MM, Finkensieper A, Abou-Zaid FA, El-Shourbagy IK, Omar KM, Figulla HR, Sauer H, Wartenberg M. Stem Cells Dev Sep 29. [Epub ahead of print] 2008 Reactive oxygen species and upregulation of NADPH oxidases in mechanotransduction of embryonic stem cells. Sauer H, Ruhe C, Müller JP, Schmelter M, D'Souza R, Wartenberg M. Methods Mol Biol. 2008;477: Phosphatidylinositol 3-kinase-dependent membrane recruitment of Rac-1 and p47phox is critical for alphaplatelet-derived growth factor receptor-induced production of reactive oxygen species. Bäumer AT, Ten Freyhaus H, Sauer H, Wartenberg M, Kappert K, Schnabel P, Konkol C, Hescheler J, Vantler M, Rosenkranz S. J Biol Chem Mar 21;283(12): Peroxisome proliferator-activated receptor alpha agonists enhance cardiomyogenesis of mouse ES cells by utilization of a reactive oxygen species-dependent mechanism. Sharifpanah F, Wartenberg M, Hannig M, Piper HM, Sauer H. Stem Cells Jan;26(1): Epub 2007 Oct 18. Direct current electrical fields induce apoptosis in oral mucosa cancer cells by NADPH oxidase-derived reactive oxygen species. Wartenberg M, Wirtz N, Grob A, Niedermeier W, Hescheler J, Peters SC, Sauer H. Bioelectromagnetics Jan;29(1): Control of leukocyte differentiation from embryonic stem cells upon vasculogenesis and confrontation with tumour tissue. Hannig M, Figulla HR, Sauer H, Wartenberg M. J Cell Mol Med Jul 4. [Epub ahead of print] How does intracellular Ca2+ oscillate: by chance or by the clock? Skupin A, Kettenmann H, Winkler U, Wartenberg M, Sauer H, Tovey SC, Taylor CW, Falcke M. Biophys J Mar 15;94(6): Epub 2007 Dec Polyphenols prevent cell shedding from mouse mammary cancer spheroids and inhibit cancer cell invasion in confrontation cultures derived from embryonic stem cells. Günther S, Ruhe C, Derikito MG, Böse G, Sauer H, Wartenberg M. Cancer Lett May 18;250(1): Epub 2006 Oct 30. In vitro differentiation of murine embryonic stem cells into keratinocyte-like cells. Haase I, Knaup R, Wartenberg M, Sauer H, Hescheler J, Mahrle G. Eur J Cell Biol Dec;86(11-12): Epub 2007 Aug 22. The acute phase protein alpha2-macroglobulin induces rat ventricular cardiomyocyte hypertrophy via ERK1,2 and PI3-kinase/Akt pathways. Padmasekar M, Nandigama R, Wartenberg M, Schlüter KD, Sauer H. Cardiovasc Res Jul 1;75(1): Epub 2007 Mar 12. Stimulation of ES-cell-derived cardiomyogenesis and neonatal cardiac cell proliferation by reactive oxygen species and NADPH oxidase. Buggisch M, Ateghang B, Ruhe C, Strobel C, Lange S, Wartenberg M, Sauer H. J Cell Sci Mar 1;120(Pt 5): Epub 2007 Feb Embryonic stem cells: a novel tool for the study of antiangiogenesis and tumor-induced angiogenesis. Wartenberg M, Dönmez F, Budde P, Sauer H. Handb Exp Pharmacol. 2006;(174): Review. Confrontation cultures of embryonic stem cells with multicellular tumor spheroids to study tumor-induced angiogenesis. Wartenberg M, Finkensieper A, Hescheler J, Sauer H. Methods Mol Biol. 2006;331: Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain-induced cardiovascular differentiation. Schmelter M, Ateghang B, Helmig S, Wartenberg M, Sauer H. FASEB J Jun;20(8):
6 Regulation of cardiotrophin-1 expression in mouse embryonic stem cells by HIF-1alpha and intracellular reactive oxygen species. Ateghang B, Wartenberg M, Gassmann M, Sauer H. J Cell Sci Mar 15;119(Pt 6): Epub 2006 Feb 28. Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation. ten Freyhaus H, Huntgeburth M, Wingler K, Schnitker J, Bäumer AT, Vantler M, Bekhite MM, Wartenberg M, Sauer H, Rosenkranz S. Cardiovasc Res Jul 15;71(2): Epub 2006 Mar 20.
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