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Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain-induced cardiovascular differentiation

Maike Schmelter^*, Bernadette Ateghang^*, Simone Helmig^*, Maria Wartenberg and Heinrich Sauer^*,1

^* Department of Physiology, Justus-Liebig-University, Giessen, Germany; and

Department of Cell Biology, GKSS Research Institute, Teltow, Germany

¹Correspondence: Department of Physiology, Justus-Liebig-University, Giessen Aulweg 129 Giessen 35392, Germany. Email: heinrich.sauer{at}physiologie.med.uni-giessen.de

SPECIFIC AIMS

Growing stem cells within three-dimensional tissues are subjected to mechanical forces, thereby initiating differentiation programs. The mechanical stimulus may be transduced to the cell nucleus via highly diffusible reactive oxygen species (ROS) generated by NADPH oxidase activity. We examine how a mechanical strain of embryonic stem (ES) cells initiates the cardiovascular differentiation program, and we analyze the role of NADPH oxidase and ROS in the activation of mitogen-activated protein kinase (MAPK) pathways as well as cardiac/vascular-specific genes involved in the commitment of the cardiovascular cell lineage.

PRINCIPAL FINDINGS

1. Mechanical strain of ES cell-derived embryoid bodies results in stimulation of vasculogenesis/angiogenesis as well as cardiomyogenesis
To analyze the effects of mechanical strain on cardiovascular differentiation of ES cells 4-day-old undifferentiated embryoid bodies grown on flexible membranes were subjected to 2 h of mechanical strain resulting in 5%, 10%, 15%, and 20% elongation of the membrane. Following further 4 d of cell culture, cardiovascular differentiation was assessed by computer-assisted image analysis of PECAM-1-positive capillary areas, counting of the numbers of spontaneously contracting cardiac foci, and examination of the size of sarcomeric -actinin-positive cell areas of cardiac muscle. Mechanical strain significantly increased the capillary area in embryoid bodies with maximum effects at 10% membrane elongation (207±28% of the untreated control, n=6), indicating stimulation of vasculogenesis/angiogenesis in embryoid bodies (Fig. 1 A). In parallel, a significantly increased number of spontaneously contracting cardiac foci (maximum at 20% membrane elongation with 221±67% of the untreated control) and increased areas covered with cardiac muscle were observed (Fig. 1B ), which clearly shows that mechanical strain stimulated cardiomyogenesis of ES cells.

View larger version (29K): [in this window] [in a new window]   Figure 1. Stimulation of vasculogenesis/angiogenesis (A) and cardiomyogenesis (B) after application of static mechanical strain to ES cell-derived embryoid bodies. A) Embryoid bodies were treated on day 4 for 2 h with mechanical strain resulting in extension of the flexible membrane to 5, 10, 15, and 20%. On day 8 of cell culture the tissues were fixed and stained for PECAM-1 to analyze endothelial cell differentiation. The areas of PECAM-1-positive capillary-like structures were analyzed by the use of the image analysis software of the confocal setup. The images show representative PECAM-1 stainings in control (a), 5% (b), 10% (c), 15% (d), and 20% (e) strain-treated embryoid bodies. B) Embryoid bodies were treated on day 4 for 2 h with mechanical strain resulting in extension of the flexible membrane to 5, 10, and 20%. For investigation of the extension of the beating cardiac areas, tissues were fixed and stained against sarcomeric -actinin. The images show representative cardiac areas in control samples (a) as well as samples treated with 5% (b), 10% (c) and 20% (d) mechanical strain. The bar represents 200 µm. *P < 0.05, significantly different from the untreated control.

2. Mechanical strain results in increased ROS generation and induces NADPH-oxidase expression
ROS may be utilized to transduce the mechanical stimulus into cardiovascular gene programs. To verify this assumption, generation of ROS was investigated by use of the redox-sensitive fluorescent dye H₂DCF-DA and analysis of protein as well as mRNA expression of NADPH oxidase subunits. Mechanical strain (10% membrane elongation) resulted in significantly increased generation of ROS after 2 h. After 24 h, up-regulation of the NADPH oxidase subunits p22-phox, p47-phox, p67-phox, and Nox-4 as well as Nox-1 and Nox-4 mRNA was observed.

3. Mechanical strain-induced cardiovascular differentiation is dependent on ROS generation
To investigate whether ROS generated following mechanical strain in ES cell-derived embryoid bodies are involved in the observed stimulation of cardiovascular differentiation, tissues were incubated with either the free radical scavengers vitamin E (100 µM) or N-(2-mercapto-propionyl)-glycine (NMPG), (100 µM) (Fig. 2 A, B). This treatment significantly inhibited the increase in the capillary area on mechanical strain as well as the observed increase in the number of spontaneously contraction cardiac foci. Consequently, treatment with free radical scavengers abolished the increase in hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) observed in response to mechanical strain. Furthermore, treatment with free radical scavengers inhibited mRNA expression of the transcription factor MEF2C, which is a key regulator of the cardiomyocyte gene program. In contrast, mRNA expression of the cardiogenic transcription factor GATA-4 following mechanical strain was potentiated after preincubation with free radical scavengers, which suggests negative regulation of GATA-4 by ROS.

View larger version (13K): [in this window] [in a new window]   Figure 2. Effects of free radical scavengers on mechanical strain-stimulated cardiomyogenesis (A) and vasculogenesis/angiogenesis (B). 4-day-old embryoid bodies were preincubated for 2 h prior to mechanical strain (10%) application with either NPMG (100 µM) or vitamin E (100 µM). Free radical scavengers were present in the cell culture medium until inspection. Note that in the presence of free radical scavengers, mechanical strain-stimulated cardiomyogenesis as well as vasculogenesis/angiogenesis were significantly inhibited, pointing toward an involvement of ROS. *P < 0.05, significantly different as indicated.

4. Mechanical strain activates MAPKs involved in cardiovascular commitment via ROS generation
On mechanical strain application, phosphorylation/activation of the MAPKs ERK1,2, JNK, and p38 was observed that was abolished in the presence of free radical scavengers, suggesting that ROS interact with MAPK signaling pathways involved in the regulation of cardiovascular gene programs. To assess the role of MAPK pathways in cardiovascular differentiation of ES cells, embryoid bodies were incubated with the ERK1,2 inhibitor UO126, the JNK inhibitor SP600125, and the p38 inhibitor SB203580. Vasculogenesis/angiogenesis after mechanical strain application was significantly impaired on inhibition of ERK1,2 and JNK, whereas p38 inhibition was without effect. In contrast mechanical strain-induced cardiomyogenesis was abolished on inhibition of ERK1,2, p38, and JNK.

CONCLUSIONS AND SIGNIFICANCE

Organs within embryos as well as differentiating stem cells within tissues are subjected to significant mechanical strain, which may represent one key stimulus for the initiation of differentiation programs. In this respect, it has been known for a long time that ES cells require the three-dimensional tissue microenvironment of embryoid bodies for proper differentiation of the cardiovascular cell lineage. The impact of physical stimuli for stem cell differentiation has been largely neglected thus far. The requirement for a proper biomechanical microenvironment should be taken into consideration, however, when cell therapies based on stem cells are designed for future patient treatment.

The data of the present study conclusively demonstrate that experimentally applied mechanical strain significantly stimulate cardiovascular differentiation of ES cells that is related to an increase of ROS generation and expression of NADPH oxidase. Increased ROS generation in response to mechanical strain has been previously reported to occur in endothelial and smooth muscle cells under conditions of shear stress and has been discussed to play a role in smooth muscle cell alignment within blood vessels. Furthermore, angiogenesis in response to ischemia has been shown to require NADPH oxidase-derived ROS for endothelial cell growth and VEGF-mediated signaling cascades. In the heart, the involvement of ROS in the development of cardiac hypertrophic cell growth is a well-known feature. This is significant also for the understanding of hyperplastic fetal cardiac cell growth, since comparable signaling pathways are presumably utilized within the developing embryo.

The molecular mechanisms of mechanotransduction resulting in the activation of cell lineage-specific gene programs are still unknown. Recently, an involvement of ROS as signaling molecules has been discussed in models of mechanotransduction, since an increasing number of publications have reported on ROS elevation following experimental mechanical strain application to a variety of different cell types. ROS generated by NADPH oxidase activity may interfere with signal transduction cascades on different levels and may activate/inactivate redox-sensitive transcription factors. Furthermore, ROS have been demonstrated to be involved in many mechanical strain-associated biological functions, e.g., in the regulation vascular tone, the formation and tension of the cytoskeleton, and the modulation of myofibrillar calcium sensitivity, thereby enhancing force generation. The data of the present study demonstrate that ROS are involved in the activation of MAPKs following mechanical strain, since the observed effects were blunted in the presence of free radical scavengers. Furthermore, expression of HIF-1 and consequently of VEGF were clearly activated by strain-induced ROS generation, which may be directly linked to phosphorylation of upstream ROS-regulated MAPK members. Interestingly, our experiments demonstrated ROS-sensitive activation of the transcription factor MEF2C by mechanical strain, which is known to be involved in embryonic cardiac development, whereas the cardiac-specific transcription factor GATA-4 was apparently negatively regulated by ROS since its expression was increased in the presence of free radical scavengers in the controls as well as in the mechanical strain-treated samples. This points toward a fine-tuned interplay of pro-oxidant- vs. antioxidant-induced transcription factors that may orchestrate the time course of cardiac gene expression patterns.

A properly correlated pro-oxidative microenvironment and appropriate mechanical tissue architecture may be a key factor for the initiation of cardiovascular cell differentiation programs, however, and may therefore represent a prerequisite for successful future cell therapies.

View larger version (20K): [in this window] [in a new window]   Figure 3. Diagram of the involvement of ROS in signaling cascades resulting in cardiovascular commitment of ES cells. ROS are generated through the activity of a presumably membrane-bound NADPH oxidase that is up-regulated following mechanical strain application. ROS initiate phosphorylation of the MAPKs ERK1,2, JNK and p38. Vasculogenesis/angiogenesis requires the activation of ERK1,2 and JNK, whereas the activity of ERK1,2, JNK and p38 is necessary for cardiomyogenesis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4723fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-4723fjev1 20/8/1182    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schmelter, M. Articles by Sauer, H. Search for Related Content PubMed PubMed Citation Articles by Schmelter, M. Articles by Sauer, H.