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Published as doi: 10.1096/fj.05-4723fje.
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(The FASEB Journal. 2006;20:1182-1184.)
© 2006 FASEB

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

Maike Schmelter*, Bernadette Ateghang*, Simone Helmig*, Maria Wartenberg{dagger} and Heinrich Sauer*,1

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

{dagger} Department of Cell Biology, GKSS Research Institute, Teltow, Germany

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

ABSTRACT

Growing stem cells are subjected to mechanical forces, which may initiate differentiation programs. Mechanical strain stimulated cardiovascular differentiation of mouse embryonic stem (ES) cells as evaluated by quantification of contracting cardiac foci and capillary areas, respectively. Mechanical strain rapidly elevated intracellular reactive oxygen species (ROS). After 24 h up-regulation of NADPH oxidase subunits p22-phox, p47-phox, p67-phox, and Nox-4 as well as Nox-1 and Nox-4 mRNA was observed. In parallel, mechanical strain increased hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) and vascular endothelial growth factor (VEGF) mRNA and protein as well as MEF2C and GATA-4 mRNA, which are involved in cardiovascular development. Furthermore, phosphorylation of extracellular-regulated kinase 1,2 (ERK1,2), p38, and c-jun N-terminal kinase (c-Jun NH2-terminal kinase (JNK)) was observed. Stimulation of cardiovascular commitment, HIF-1{alpha}, VEGF, and MEF2C expression as well as MAPK activation were abolished by free radical scavengers, whereas GATA-4 expression was increased. Cardiomyogenesis was inhibited by the p38 inhibitor SB203580, the ERK1,2 inhibitor UO126, and the JNK inhibitor SP600125. Vasculogenesis/angiogenesis was blunted following inhibition of ERK1,2 and JNK, whereas p38 inhibition was ineffective. Our data outline a role of ROS as mechanotransducing molecules in mechanical strain-stimulated cardiovascular differentiation of ES cells, and point toward a microenvironment of elevated ROS required for signaling cascades initiating cardiovascular differentiation programs.—Schmelter, M., Ateghang, B., Helmig, S., Wartenberg, M., Sauer, H. Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain-induced cardiovascular differentiation.


Key Words: cardiomyogenesis • hypoxia-inducible factor-1{alpha} • mechanical stretch • NADPH-oxidase • vasculogenesis

PHYSICAL FORCES, which are either exerted toward cells and tissues or are generated through themselves, are essential signals that mediate cell structure, survival, proliferation, and differentiation. It has been known for a long time that a large number of physiological processes, e.g., maturation and branching of the fetal lung (1 , 2) , morphogenesis of the heart (3 4 5) , arteriogenesis (6) , microvascular remodelling (7 , 8) , and maturation of bone (9) and cartilage (10) , are critically dependent on mechanical factors. Embryologic investigations performed on chick embryos have shown that embryonic heart development and heart rotation are at least partially dependent on the local biomechanic environment which is determined by the bloodstream within the splanchnopleuric and omphalomesenteric veins (3 , 4 , 11) . The impact of mechanical forces for the development of the left-right asymmetry of the vertebrate embryo (mouse) was recently described (12) . In this elegant study, it was demonstrated that an artificial rightward-directed stream that was opposed to the leftward laminar flow at the primitive node disturbed the left-right asymmetry in the body plan of the embryo.

Besides the physiological effects of mechanical forces on cells, tissues and organs of various pathophysiological alterations have been reported. These include diseases like cardiac hypertrophy (13) or arteriosclerosis (14) , which develop under conditions of pathological mechanical strain. It is generally assumed that all cells of the body respond toward mechanical stimulation. However, the means by which the cells sense the mechanical stimulus are largely unknown.

Currently localized and decentralized models of mechanotransduction have been discussed. In the localized model, the cellular signal is generated in the spatial vicinity of the cell membrane. In the decentralized model, mechanical forces that are generated at the cell membrane are transduced to more distant locations via the cytoskeleton (15) . In addition to affecting mechanosensitive ion channels and the integrin cytoskeleton, ROS have been proposed to play a role in mechanotransduction (15) . ROS are generated mainly via a cell membrane-associated NADPH oxidase or within the respiratory chain of mitochondria. NADPH oxidases have been found to be involved in the regulation of vascular tone, smooth muscle growth, inflammatory responses, and matrix metalloproteinase activity. They have been implicated in hypertension, atherosclerosis, heart failure, diabetic vascular disease, and restenosis (16 , 17) . Possible mechanotransduction via ROS has been discussed to be mediated via mitochondria, which are linked via actin-binding complexes to the cytoskeleton, thereby allowing mechano-activation via the cytoskeleton. In this respect, it was shown that mechanical strain induced ROS generation in HUVEC cells via mitochondria, since this effect was not observed in a mitochondria-deficient cell line. Furthermore, the generation of ROS was abolished when cells were incubated with cytochalasin D, which disrupts the actin cytoskeleton (18) .

In mouse embryonic stem (ES) cells, ROS have been shown to be predominantly generated via NADPH oxidase (19, 20); however, their activation and expression in response to physical forces has not been elaborated. Possible effects of physical stimuli on differentiation of ES cells have been discussed for some time (21) . Recently, the effects of laminar shear stress on ES cell differentiation were investigated, and it was found that shear stress enhanced lysine acetylation of histone H3 at position 14 as well as serine phosphorylation at position 10 (S10) and lysine methylation at position 79 (K79). Furthermore, cardiovascular markers were up-regulated (22) . In a further study that used Flk-positive ES cells, phosphorylation of Flk-1 (VEGFR2) on exposure to laminar shear stress was observed (23) .

In the present study, the effects of static mechanical strain on cardiovascular differentiation of ES cells were investigated. We observed that promotion of cardiovascular differentiation by mechanical strain is transduced through the generation of ROS. Hence, our study gives novel insights into basal mechanisms of ES cell differentiation toward the cardiovascular cell lineage and sheds light on the principles by which mechanical strain may steer heart formation in the early postimplantation embryo.

MATERIALS AND METHODS

Reagents
N-(2-mercapto-propionyl)-glycine (NMPG) and water-soluble vitamin E (Trolox) were obtained from Sigma (Deisenhofen, Germany). Sytox Green was observed from Molecular Probes (Eugene, OR). The MAPK inhibitors UO126, SB203580, and SP600125 were purchased from Calbiochem (Bad Soden, Germany).

Spinner-culture technique for cultivation of embryoid bodies
The ES cell line CCE was grown on mitotically inactivated feeder layers of primary murine embryonic fibroblasts for a maximum of eight passages in Iscove’s medium (Life Technologies, Inc., Rockville, MD) supplemented with 18% heat-inactivated (56°C, 30 min) fetal calf serum (FCS) (Sigma), 2 mM Glutamax (PAA, Cölbe, Germany), 100 µM ß-mercaptoethanol (Sigma), 1% (v/v) NEA nonessential amino acids stock solution (100x) (Biochrom, Berlin, Germany), 0.8% (v/v) MEM amino acids (50x) (Biochrom), 1 mM Na+-pyruvate (Biochrom), 0.25% (v/v) penicillin/streptomycin (200x) (Biochrom) and 1000 U/ml LIF (Chemicon, Hampshire, UK) in a humidified environment containing 5% CO2 at 37°C and passaged every 2–3 d. At day 0 of differentiation, adherent cells were enzymatically dissociated using 0.2% trypsin and 0.05% EDTA in PBS (Life Technologies, Inc.) and seeded at a density of 1·107 cells ml–1 in 250 ml siliconized spinner flasks (Integra Biosciences, Fernwald, Germany) containing 100 ml Iscove’s medium supplemented with the same additives as described above. After 24 h, 150 ml medium was added to give a final vol of 250 ml. The spinner flask medium was stirred at 20 r.p.m. using a stirrer system (Integra Biosciences), and 150 ml cell culture medium was exchanged every day.

Cell stretching system
Cells were subjected to mechanical strain using the Flexercell Strain Unit (model FX-4000T, Flexercell International Corporation, McKeesport, PA). In this system, mechanical strain is obtained using 6-well, flexible-bottomed, collagen-coated culture plates (Bioflex culture plates) with a hydrophilic surface, capable of up to 20% stretch. Frequency, strain rate and degree of elongation of the culture substratum are controlled with a computer by regulating the rate of evacuation (vacuum concentration) and rate of air influx to the cell culture plate bottoms. For the experiments 10–15 3-day-old embryoid bodies were plated in complete cell culture medium into 6-well, flexible-bottomed culture plates. After 24 h, outgrown embryoid bodies were subjected to mechanical strain by elongation of the flexible membranes by either 5, 10, or 20%. Cell viability after mechanical strain application was controlled by the lethal cell dye Sytox green (Molecular Probes, Eugene, OR). It was found that the applied mechanical strain conditions did not affect cell viability of ES cells (data not shown).

Immunohistochemistry
Immunohistochemistry was performed with whole mount embryoid bodies. As primary antibodies, the rat monoclonal anti-PECAM-1 (CD31) (Chemicon) (dilution 1: 100), the rabbit polyclonal anti-ERK1,2, anti-c-Jun NH2-terminal kinase, and antip38 antibodies, directed against the active (phosphorylated form of the proteins) (New England Biolabs, Frankfurt, Germany) (dilution 1:100), the monoclonal anti-HIF-1{alpha} (ABRm, Golden, CO) (dilution 1:100), the monoclonal anti-vascular endothelial growth factor (Santa Cruz Biotechnology, Santa Cruz, CA) (dilution 1:100)), the polyclonal goat antimouse Nox-4, p22-phox, p47-phox, and rabbit antimouse p67-phox antibodies (dilution 1:50) (all from Santa Cruz) were used. For PECAM-1 and NADPH oxidase subunit staining the respective tissues were fixed in ice-cold methanol for 30 min at –20°C, and washed with PBS containing 0.1% Triton X-100 (PBST) (Sigma). For VEGF, HIF-1{alpha}, ERK1,2, JNK, p38 the tissues were fixed for 1 h at 4°C in 4% formaldehyde in PBS. Blocking against unspecific binding was performed for 60 min with 10% FCS dissolved in 0.01% PBST. The tissues were subsequently incubated for 90 min at room temperature with primary antibodies dissolved in PBS supplemented with 10% FCS in 0.1% PBST. The tissues were thereafter washed three times with PBST (0.01% Triton) and reincubated with either a Cy5-conjugated goat anti-rat IgG (H+L) (PECAM-1), a Cy5-conjugated goat anti- mouse IgG (H+L) (VEGF, HIF-1{alpha}), or a Cy5-conjugated goat anti-rabbit IgG (ERK1,2, JNK, p38) (all from Dianova, Hamburg, Germany) at a concentration of 3.8 µg/ml in PBS containing 10% FCS in 0.01% PBST. After washing three times in PBST (0.01% Triton), the tissues were stored in PBS until inspection. Fluorescence recordings were performed by means of a confocal laser scanning setup (Leica TCS SP2, Bensheim, Germany) connected to an inverted microscope (DMIRE2, Leica). The confocal setup was equipped with a 5 mW helium/neon laser single excitation 633 nM (excitation of Cy5). Emission was recorded at > 665 nM. The pinhole settings of the confocal setup were adjusted to give a full width half maximum (FWHM) of 10 µm. Fluorescence was recorded in a depth of 80–120 µm in the depth of the tissue, and the fluorescence values in the respective optical section were evaluated by the image analysis software of the confocal setup.

Quantitative RT-PCR
Total RNA from CCE S103 embryoid bodies treated with mechanical strain was prepared using Trizol (Invitrogen) according to manufacturer’s recommendations followed by genomic DNA digestion using DNase I (Invitrogen). Total RNA concentration was determined by the optical density260 nM method. cDNA synthesis was performed using 2 µg RNA in a total vol of 20 µl with MMLV RT (Gibco BRL).

Primer sequences for quantitative RT-PCR were:

HIF1-{alpha}: fwd.: 5'-TCA CCA GAC AGA GCA GGA AA-3'

rev.: 5'-CTT GAA AAA GGG ACC (AGC) ACC (CAT) CA-3'

VEGF: fwd.: 5'-TCC ACC ATG CCA AGT GGT-3'

rev.: 5'-TCG GGG TAC TCC TGG AAG AT-3'

GATA4: fwd.:5'-TCA AAC CAG AAA ACG GAA GC-3'

rev.: 5'-GTG GCA TTG CTG GAG TTA CC-3'

MEF2C: fwd.: 5'-TCA TCT CTG TCT GGC TTC AAC-3'

rev.: 5'-GGT GGT GGT ACG GTC TCC-3'

Nox-1: fwd.: 5'-AAT GCC CAG GAT CGA GGT-3'

rev.: 5'-GAT GGA AGC AAA GGG AGT GA-3'

Nox-4: fwd: 5'-GAT CAC AGA AGG TCC CTA GCA G-3'

rev.: 5'-GTT GAG GGC ATT CAC CAA GT-3'

Housekeeping genes:

BACT: fwd.: 5'-GAT GAC CCA GAT CAT GTT TGA G-3

rev.: 5'-CCA TCA CAA TGC CTG TGG TA-3'

GAPDH: fwd.: 5'- TCG TCC GGT AGA CAA AAT GG-3'

rev.: 5'-GAG GTC AAT GAA GGG GTC GT-3'

The primer concentration was 10 pM/20 µl. Amplifications were performed in an Icycler Optical Module (Bio-Rad) using absolute Sybr Green Fluorescein Mix (Abgene, Absom, UK). The following programs were used:

93°C for 15:00 min hot start step/denaturation

93°C for 30s, AT 30s, 72°C 30s, 45 times

50°C for 10 min

Annealing temperatures were:

60°C for HIF-1{alpha}, Nox-1, GATA4 and BACT

62°C for VEGF, MEF2c

64°C for Nox-4 and GAPDH

Fluorescence increase of SYBR Green was automatically measured after each extension step.

Amplified transcripts were loaded on a 2% agarose gel. CT values were automatically obtained. Relative expression values were obtained by normalizing CT values of the tested genes in comparison with CT values of the housekeeping genes using the {Delta}{Delta} CT method.

Measurement of ROS generation
Intracellular ROS levels were measured using the fluorescent dye 2'7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Molecular Probes), which is a nonpolar compound that is converted into a nonfluorescent polar derivative (H2DCF) by cellular esterases after incorporation into cells. H2DCF is membrane impermeable and is rapidly oxidized to the highly fluorescent 2',7'-DCF (DCF) in the presence of intracellular ROS. For the experiments, embryoid bodies were incubated in serum-free medium, and 20 µM H2DCF-DA dissolved in dimethyl sulfoxide (DMSO) was added. Intracellular DCF fluorescence was recorded either directly after mechanical strain application or after 20 min as indicated. DCF fluorescence (corrected for background fluorescence) was evaluated in 3600 µm2 regions of interest using an overlay mask. For fluorescence excitation, the 488-nm band of the argon ion laser of the confocal setup was used. Emission was recorded using a longpass LP515-nm filter set. In parallel experiments, ROS were analyzed with the redox-sensitive fluorescence dye dihydrorhodamine-123 (Molecular Probes) after 10 min incubation with 10 µM of the substance. Fluorescence excitation was performed with the 543 nM band of a He/Ne laser. Emission was recorded at 570–630 nM.

Statistical analysis
Data are given as mean values ± SD, with n denoting the number of independent samples within one treatment group. In each experiment 15–20 embryoid bodies were analyzed. Statistical analysis was performed by one way ANOVA, followed by adjusted t tests with P values corrected by the Bonferroni method. A value of P < 0.05 was considered significant.

RESULTS

Stimulation of cardiovascular differentiation of ES cells by mechanical strain
ES cell-derived embryoid bodies differentiate within 8 d toward the cardiovascular cell lineage. The underlying molecular mechanisms are thus far unknown but may include physical stimuli. In the present study, 4-day-old undifferentiated embryoid bodies outgrown on flexible membranes were subjected for 2 h to mechanical strain resulting in 5%, 10%, and 20% extension of the membranes. Following further 4 d in cell culture, the formation of capillary-like structures was evaluated on day 8 of cell culture by quantification of cell areas staining positive for the endothelial cell-specific marker PECAM-1 (Fig. 1 ). Extension of the flexible membranes by either 5, 10, 15, or 20% resulted in an increase of the capillary area to 159 ± 16%, 207 ± 28%, 150 ± 20%, and 105 + 15%, respectively (n=6) (control set to 100%), which clearly indicates that mechanical strain stimulates vasculogenesis/angiogenesis in ES cells.


Figure 1
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  		Figure 1. Stimulation of vasculogenesis/angiogenesis after application of static mechanical strain to ES cell-derived embryoid bodies. 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. 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 staining in control (a), 5% (b), 10% (c), 15% (d), and 20% (e) strain-treated embryoid bodies. The graph gives the means ± SD of 6 independent experiments. The bar represents 200 µm. *P < 0.05, significantly different from the untreated control.

The effect of mechanical strain on cardiomyogenesis was evaluated by counting spontaneous contracting embryoid bodies 4 d after mechanical strain treatment (Fig. 2 A, B). It was apparent that mechanical strain dose-dependently increased the number of spontaneously contracting embryoid bodies to 158 ± 22%, 189 ± 54%, and 221 ± 67%, when embryoid bodies were treated with either 5, 10, or 20% mechanical strain, respectively (n=7) (Fig. 2A ). To investigate whether mechanical strain increased the area of beating cardiac cells, control and mechanically strained embryoid bodies were immunostained with cardiac-specific {alpha}-actinin, and the area of immunolabeled cardiac cells was analyzed by confocal laser scanning microscopy (Fig. 2B ). In parallel to the number of spontaneously contracting embryoid bodies, the area covered with cardiomyocytes was significantly increased to 152 ± 39, 325 ± 64, and 345 ± 74% when embryoid bodies were treated with 5, 10, and 20% mechanical strain, respectively (n=4). When cyclic strain (60 cycles/min, 15% elongation of the membrane) was applied, comparable effects on cardiovascular differentiation as compared to static mechanical strain were observed (data not shown).


Figure 2
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  		Figure 2. Stimulation of cardiomyogenesis after application of static mechanical strain to ES cell-derived embryoid bodies. 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%, respectively. On days 7–9 of cell culture, beating embryoid bodies in control and mechanical strain-treated samples were counted by microscopical inspection (A). For investigation of the beating areas, the tissues were fixed and stained for sarcomeric {alpha}-actinin (B). The images in (B) 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.

Effects of mechanical strain on the generation of ROS and expression of NADPH oxidase
Static and cyclic mechanical strain has been shown to increase ROS in a variety of preparations (24) . In previous studies, we have demonstrated that exogenously applied low concentrations of ROS can stimulate cardiomyogenesis of ES cells (19, 20). In contrast, high concentrations of ROS inhibit cardiomyogenic differentiation of ES cells (25) . To evaluate ROS generation, embryoid bodies were labeled with the ROS-sensitive indicator H2DCF-DA and the generation of oxidized fluorescent DCF was monitored either directly or at different times as indicated after 10% mechanical strain application (Fig. 3 A–C). Already after ~100 s an increase in ROS generation was observed, which was inhibited in the presence of the NADPH oxidase inhibitors DPI (10 µM) and apocynin (10 µM) (Fig. 3A ; n=3). ROS levels remained elevated for several hours as assessed by determination of DCF fluorescence 2, 6, 12, and 24 h after mechanical strain treatment (Fig. 3B ; n=3). At 20 min after strain application DCF fluorescence in embryoid bodies was increased to 178 ± 55%, which was totally inhibited in the presence of the free radical scavenger vitamin E (93±24%) (control set to 100%) (n=5; Fig. 3C ). A comparable increase in intracellular ROS levels (176±31%, n=5) was found when ROS was determined with the redox-sensitive fluorescence indicator dihydrorhodamine-123. The generation of ROS in embryoid bodies has been previously shown by us to occur predominantly via the activity of a NADPH oxidase (19) . To assess up-regulation of NADPH oxidase following mechanical strain of embryoid bodies, semiquantitative immunohistochemistry of NADPH subunits as well as quantitative RT-PCR of Nox-1 and Nox-4, which are homologues of gp91-phox, was performed (Fig. 4 A–C). It was found that 10% mechanical strain applied for 2 h resulted in a significant increase of p22-phox, p47-phox, p67-phox, and Nox-4 protein expression to 148 ± 25%, 159 ± 13%, 139 ± 17%, and 136 ± 16% after 24 h (n=3) (control set to 100%) (Fig. 4A, B ). Quantitative RT-PCR revealed an increase in Nox-1 (n=4) and Nox-4 (n=5) mRNA expression to 176 ± 21% and 628 ± 15% 24 h after mechanical strain application, respectively (control set to 100%) (Fig. 4C ). In contrast, no expression of Nox-2 mRNA was observed (data not shown).


Figure 3
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  		Figure 3. Generation of ROS following treatment of embryoid bodies with mechanical strain. Embryoid bodies were either treated for 2 min (indicated by the arrow) with 10% mechanical strain and ROS generation was evaluated immediately thereafter by use of the redox-sensitive dye H2DCFDA (A), or they were treated for 2 h with mechanical strain, and intracellular ROS were assessed after 2, 6, 12, and 24 h (B). C), intracellular ROS generation (evaluated 20 min after a 2 h period of 10% mechanical strain) was significantly inhibited in the presence of the free radical scavenger vitamin E (100 µM). *P < 0.05, significantly different as indicated.


Figure 4
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  		Figure 4. Up-regulation of NADPH oxidase subunits (A, B) and Nox-1, Nox-4 mRNA (C) following mechanical strain application to ES cell-derived embryoid bodies. Four-day-old embryoid bodies were treated for 2 h with 10% mechanical strain. After 24 h they were either fixed for immunolabeling with antibodies directed against NADPH oxidase subunits, or mRNA was isolated for RT-PCR. A) representative images of embryoid bodies, which remained either untreated (left panel) or were mechanically strained and subsequently labeled with antibodies for Nox-4, p22-phox, p47-phox, and p67-phox (the bar represents 300 µm). B) Semiquantitative plot of changes in NADPH oxidase subunit expression on mechanical strain. C) Real-time RT-PCR analysis of Nox-1 and Nox-4 mRNA expression. *P < 0.05, significantly different from the untreated control.

Effects of free radical scavengers on mechanical strain-induced cardiovascular differentiation
To investigate the association of ROS generation and stimulation of cardiovascular differentiation, embryoid bodies were preincubated for 2 h prior to mechanical strain treatment with either the free radical scavenger (NMPG) (100 µM) or vitamin E (100 µM). As shown in Fig. 5 A pretreatment with either NMPG or vitamin E resulted in complete inhibition of mechanical strain-induced stimulation of cardiomyogenesis as evaluated by counting the percentage of spontaneously contracting embryoid bodies (n=3). Comparably, the area of capillary-like structures in mechanically-strained embryoid bodies was significantly reduced, when embryoid bodies were mechanically strained in the presence of either the free radical scavengers NMPG (n=3) or vitamin E (n=4) (Fig. 5B ).


Figure 5
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  		Figure 5. Effects of free radical scavengers on mechanical strain-stimulated cardiomyogenesis (A) and vasculogenesis/angiogenesis (B). Four-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 was significantly inhibited, pointing toward an involvement of ROS. *P < 0.05, significantly different as indicated.

Role of mechanical strain-induced ROS on HIF-1{alpha} and VEGF expression
Vasculogenesis/angiogenesis in ES cell-derived embryoid bodies is critically dependent on VEGF expression, as we have previously shown that treatment with the Flk-1 antagonist SU5416 totally abolished endothelial cell differentiation (26) . The expression of VEGF is regulated by the transcription factor HIF-1{alpha}, which is highly expressed in avascular, undifferentiated embryoid bodies and down-regulated on vascularization (26) . Mechanical strain increased protein expression of HIF-1{alpha} to 150 ± 16%, 160 ± 11%, 190 ± 15%, and 220 ± 23%; 2, 4, 8, and 24 h after mechanical strain treatment (10% strain, 2 h) (n=5; Fig. 6 ). In further experiments the effects of mechanical strain on HIF-1{alpha} (Fig. 7 A, B) and VEGF (Fig. 8 A, B) expression in embryoid bodies treated with free radical scavengers were investigated 24 h after mechanical strain (10% strain, 2 h) application by quantitative RT-PCR and semiquantitative immunohistochemistry. It was apparent that mechanical strain significantly increased HIF-1{alpha} protein expression to 169 ± 24% (control set to 100%) (n=5), whereas in the presence of vitamin E (100 µM) the mechanical strain-induced HIF-1{alpha} increase was significantly inhibited (109±18%, n=5; Fig. 7A ). In the absence of mechanical strain, vitamin E treatment resulted in a decrease in HIF-1{alpha} protein expression to 83 ± 15% (n=3).


Figure 6
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  		Figure 6. Time course of HIF-1{alpha} induction after mechanical strain. 4-day-old embryoid bodies were treated for 2 h with 10% mechanical strain and were fixed and stained for HIF-1{alpha} protein after 2, 4, 8, and 24 h. Upper panel) Representative images of HIF-1{alpha} labeled embryoid bodies (the bar represents 400 µm). Lower panel) Plot of semiquantitative analysis of HIF-1{alpha} protein at different times after mechanical strain application. *P < 0.05, significantly different as indicated.


Figure 7
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  		Figure 7. Inhibition of mechanical strain-stimulated HIF-1{alpha} protein (A) and mRNA (B) expression by the free radical scavenger vitamin E. Four-day-old embryoid bodies were preincubated for 2 h prior to mechanical strain (10%) application with vitamin E (100 µM). HIF-1{alpha} expression was evaluated 24 h following mechanical strain application. *P < 0.05, significantly different as indicated.


Figure 8
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  		Figure 8. Inhibition of mechanical strain-stimulated VEGF protein (A) and mRNA (B) expression by the free radical scavenger NMPG. Four-day-old embryoid bodies were preincubated for 2 h prior to mechanical strain (10%) application with NMPG (100 µM). VEGF expression was evaluated 24 h following mechanical strain application. *P < 0.05, significantly different as indicated.

Mechanical strain likewise induced an increase in HIF-1{alpha} mRNA expression to 202 ± 10% (control set to 100%), whereas in the presence of vitamin E (100 µM) (n=5), the observed increase in mRNA expression was significantly inhibited (97±12%; Fig. 7B ). In the absence of mechanical strain free radical scavengers did not significantly change HIF-1{alpha} mRNA expression (n=5).

Comparable results were obtained for VEGF (Fig. 8A, B ). Mechanical strain (10%, 2 h) resulted in an increase of VEGF protein expression to 156 ± 30% (control set to 100%) (n=5), which was reduced to 101 ± 18% in the presence of NMPG (100 µM) (n=3) (see Fig. 8A ). In the absence of mechanical strain an insignificant reduction of VEGF expression to 80 ± 12% of the untreated control was observed. RT-PCR revealed an increase in VEGF mRNA expression to 483 ± 16%, which was significantly inhibited in the presence of 100 µM NMPG (77±7%; n=4). No significant change of VEGF expression was observed in NMPG-treated embryoid bodies in the absence of mechanical strain application (Fig. 8B ).

Role of mechanical strain-induced ROS on the expression of the transcription factors MEF-2C and GATA-4
The transcription factor MEF-2C has been previously shown to be critical for early embryonic cardiogenesis (27) as well as vasculogenesis (28) . GATA-4 is a cardiac-specific member of the GATA family of zinc finger transcription factors and is a potent transcriptional activator of several cardiac muscle-specific genes and a key regulator of the cardiomyocyte gene program (29) . Since the data of the present study demonstrate that mechanical strain stimulation of cardiovascular differentiation is dependent on the generation of intracellular ROS, the effect of free radical scavengers on MEF-2C (Fig. 9 A) as well as GATA-4 (Fig. 9B ) mRNA expression were investigated. Mechanical strain increased MEF-2C expression 24 h following mechanical strain to 273 ± 7% (control set to 100%; n=3), which was completely abolished in the presence of the free radical scavenger NMPG (100 µM) (87±15%) (n=3). In the absence of mechanical strain application, no significant change in MEF-2C expression in NMPG-treated embryoid bodies was observed. Mechanical strain likewise increased GATA-4 mRNA expression to 162 ± 16%. However, in contrast to MEF-2C, preincubation with NMPG (100 µM) in the absence of mechanical strain resulted in increased expression of GATA-4 to 227 ± 14% as compared to the untreated control. Application of mechanical strain further increased GATA-4 expression to 408 ± 11%, which suggests a negative regulation of GATA-4 expression by ROS (n=4). Comparable results where observed when vitamin E was applied as free radical scavenger (data not shown).


Figure 9
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  		Figure 9. Inhibition of mechanical strain-stimulated MEF2C (A) and GATA-4 (B) expression by the free radical scavenger NMPG. Four-day-old embryoid bodies were preincubated for 2 h prior to mechanical strain (10%) application with NMPG (100 µM). MEF2C and GATA-4 expression was evaluated 24 h following mechanical strain application. *P < 0.05, significantly different as indicated.

Involvement of MAPKs in mechanical strain-stimulated cardiovascular differentiation of ES cells
Mechanical strain may be transduced via the MAPKs ERK1,2, p38 and JNK to the cell nucleus where they activate transcription factors that mediate the transcription of genes related to the cardiovascular cell lineage. MAPKs have been demonstrated to be regulated via ROS (30) . It was, therefore, hypothesized that ROS may act as mechanotransducers of mechanical strain and relay MAPK activity to the transcription of genes involved in cardiovascular commitment. Indeed, mechanical strain induced phosphorylation of ERK1,2 (maximum after 10 min; Fig. 10 A, B), JNK (maximal after 10 min; Fig. 10A, C ), and p38 (maximal after 30 min; Fig. 10A, D ), which was significantly inhibited in the presence of the free radical scavenger vitamin E (100 µM), indicating regulation of MAPK activity by ROS.


Figure 10
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  		Figure 10. Inhibition of mechanical strain-stimulated ERK1,2 and JNK and p38 activation by the free radical scavenger vitamin E. Four-day-old embryoid bodies were preincubated for 2 h prior to mechanical strain (10%) application with vitamin E (100 µM). ERK1,2 (B), JNK (C), and p38 (D) activation was evaluated after either 10 min (ERK1,2, JNK) or 30 min (p38) of mechanical strain application. A) representative images of embryoid bodies which remained either untreated or were treated with mechanical strain in the absence and presence of vitamin E (the bar represents 300 µm) and labeled with antibodies for active ERK1,2, JNK and p38. B–D) plots of semiquantitative analysis of MAPK activation after mechanical strain in the absence and presence of the free radical scavenger vitamin E. *P < 0.05, significantly different as indicated.

To investigate the impact of MAPK activity on cardiomyogenic as well as vascular differentiation, embryoid bodies were preincubated with either the ERK1,2 inhibitor UO126 10 µM), the p38 inhibitor SB203580 (1 µM), or the JNK inhibitor SP600125 (1 µM) prior to mechanical strain application (Fig. 11 A, B). Subsequently, cardiovascular differentiation was assessed by either counting the number of spontaneously beating embryoid bodies (Fig. 11A ) or determining the PECAM-1-positive cell area (Fig. 11B ). It was shown that cardiomyogenesis was significantly inhibited by SB203580, UO126 and SP600125, indicating the involvement of p38, ERK1,2 and JNK in the signaling cascade (n=4). Notably, SP600125 did not impair cardiomyogenesis in the control sample, indicating that JNK activation only plays a role under conditions of mechanical strain. The increase in the PECAM-1-positive cell area following mechanical strain was significantly inhibited in the presence of SP600125 and UO126, whereas SB203580 was without effect (n=5). This points toward the notion that p38 signaling is not required for mechanical strain-stimulated vasculogenesis/angiogenesis in ES cell-derived embryoid bodies.


Figure 11
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  		Figure 11. Effects of inhibition of MAPK pathways on cardiomyogenesis (A) and vasculogenesis/angiogenesis (B). Embryoid bodies were treated from day 4 to day 10 with either the ERK1,2 inhibitor UO126 (10 µM), the p38 inhibitor SB203580 (1 µM), or the JNK inhibitor SP600125 (1 µM). Cardiomyogenesis was assessed by counting the number of spontaneously contracting cardiomyocyte foci; vasculogenesis/angiogenesis was assessed by evaluating the extension of the area of PECAM-1-positive capillary-like structures. Note that inhibition of p38 significantly impaired cardiomyogenesis, whereas vasculogenesis/angiogenesis was not affected. *P < 0.05, significantly different as indicated.

DISCUSSION

Although numerous studies in the past years have reported on cardiomyogenesis as well as vasculogenesis of ES cells (31 32 33) , the underlying molecular mechanisms are largely unknown. Recently, it was pointed out that embryonic cardiovascular development involves a dynamic interaction of genetic and epi-genetic (environmental) factors, the latter including mechanics as a key stimulus during development of the heart and blood vessels (11) . Comparable mechanisms may prevail during ES cell differentiation. It is evident that mechanical forces are generated when ES cells grow and differentiate within the three-dimensional tissue of embryoid bodies. During the time course of differentiation, embryoid bodies augment in size, resulting in an increasing tissue tension exerted on every single cell, which possibly initiates differentiation programs. The significance of these mechanical forces in developmental biology has been largely neglected, however, although these forces may be of primary significance not only for ES cell cardiovascular differentiation but also for embryonic heart formation. Additionally, mechanical strain may be integral to the success of cell therapy approaches, since it should be assumed that physical forces control the behavior of stem cells after cell transplantation into the recipients’ tissues. These forces critically influence the cells’ survival, differentiation and proliferation capacity (e.g., after transplantation into the infarcted tissue of a beating heart).

The data of the present study conclusively demonstrate that mechanical strain stimulates cardiovascular differentiation of ES cells and decipher molecular mechanisms by which the physical forces of mechanical strain are transduced in biological responses. Our experiments point toward a primary role of ROS generated by a NADPH oxidase as signaling molecules in mechanical strain stimulation of cardiovascular differentiation. Notably, ROS generation in embryoid bodies was induced at times when the cells were still undifferentiated (i.e., at day 4 of cell culture) whereas differentiation of the cardiovascular cell lineage occurs between day 6 and day 9 of cell culture (33) . Furthermore, increased ROS generation was apparently a general event occurring in all cells prior to the initiation of differentiation programs. However, since ROS, i.e., hydrogen peroxide, are known to freely diffuse within tissues and are highly cell membrane permeable, it may be assumed that individual cells differ in their antioxidative capacity, which may critically determine their fate of cell differentiation. Whereas the present study for the first time reports on strain-induced ROS formation in ES cells, the generation of ROS following cyclic mechanical strain is a well-documented feature in a variety of preparations, including endothelial cells (34 35 36 37) , smooth muscle cells (38 39 40) , fibroblasts (41) , and ventricular cardiomyocytes (42 , 43) . A correlation between application of cyclic mechanical strain of isolated ventricular cardiomyocytes, generation of ROS, and induction of hypertrophic cell growth was conclusively demonstrated (44) . Consequently, treatment with antioxidants (45 , 46) as well as inhibition of ROS-generating NADPH oxidase (47) , have been successfully applied to prevent cardiac hypertrophy in animal models.

The data of the present study suggest that ROS generation following mechanical strain of ES cells apparently involves a two-step mechanism consisting of an immediate response within a few minutes of mechanical strain application and a long-term response that is mediated by up-regulation of a ROS-generating NADPH oxidase. Up-regulation of several NADPH oxidase subunits was demonstrated on the protein concentration, and increased expression of Nox-1 and Nox-4 was shown on the mRNA concentration. The precise isoform involved has to be investigated in future studies. We previously documented the continuous generation of ROS during the time course of differentiation of ES cells, and a NADPH oxidase-like enzyme was identified as source of ROS generation in ES cell-derived embryoid bodies (20) . The generated ROS may interfere with a variety of signaling cascades, thereby affecting the activity of transcription factors and genes involved in cardiovascular differentiation. In the present study, it was shown that HIF-1{alpha} was up-regulated on the gene and protein levels within 24 h, which was accompanied by up-regulation of VEGF. We have previously shown that expression of VEGF is critical for vasculogenesis of ES cells (26) and is induced under conditions of increased ROS generation (e.g., following application of direct current electrical fields to ES cell cultures (48) ). The increase in HIF-1{alpha} as well as VEGF following mechanical strain is clearly redox-dependent, since preincubation with free radical scavengers abolishes the observed effects and consequently inhibits the stimulation of capillary-like area formation following mechanical strain application. Interestingly, maximal effects were achieved with 10% strain, whereas the increase in capillary area with 20% strain was not significantly different from the control. Presumably higher rates of mechanical strain increase intracellular ROS to levels causing oxidative stress, which is detrimental to endothelial cell differentiation. A comparable redox control was observed for mechanical strain-induced cardiomyogenesis. We have previously shown that low concentrations of exogenous ROS (in the nanomolar range) stimulated cardiomyogenesis, whereas an adverse effect was already exerted with 1 µM hydrogen peroxide (20) .

In contrast to vasculogenesis/angiogenesis where significant evidence for a role of ROS in vascular cell growth is available (for review, see (49 , 50) ) almost nothing is known about the role of ROS in cardiac cell differentiation. In previous studies, we demonstrated that low levels of ROS in the nanomolar range applied either by exogenous addition of H2O2 or following application of electrical fields significantly increased cardiomyogenesis of ES cells (19 , 20) . Furthermore, we recently demonstrated that the effects of cardiotrophin-1 (computed tomography (CT)-1) on ES cell-derived cardiomyocyte proliferation are mediated by ROS, which regulate the activity of several CT-1-induced signaling cascades (51) . In addition to the phenomenological investigation of mechanical strain-stimulated cardiomyogenesis by quantification of beating embryoid bodies and cardiac cell areas, we assessed the expression of transcription factors known to be involved in cardiomyogenesis of ES cells and embryonic heart development. In the present study, it was found that mechanical strain robustly increased the expression of MEF2C, which was inhibited in the presence of free radical scavengers. In parallel, up-regulation of GATA-4 m-RNA was observed, albeit at lower levels, which was paradoxically further up-regulated by free radical scavengers in untreated as well as mechanical strain-treated samples. Obviously, ROS generation is not required for mechanical strain-induced GATA-4 expression, indicating that a fine-tuned interplay between redox-dependent and -independent transcription factors may be required for proper cardiomyogenic differentiation. Furthermore, these findings suggest that an experimental increase of GATA-4 expression with free radical scavengers is not sufficient to enhance cardiomyogenesis of ES cells, since under these experimental conditions no stimulation of MEF2C expression nor increase in the number of beating embryoid bodies was observed.

Transcription factors involved in cardiomyogenesis as well as vasculogenesis/angiogenesis may be regulated by MAPK signaling cascades. In this respect, it has been previously shown that p38 MAPK is involved in MEF2C activity (52) . Inhibition of p38 prevented the differentiation program in myogenic cell lines and human primary myocytes, and activation of p38 enhanced the transcriptional activities of MEF2C by direct phosphorylation (53) . In the same study, it was demonstrated that activation of ERK was inhibitory toward myogenic transcription in myoblasts but contributed to the activation of myogenic transcription and regulated postmitotic responses (i.e., hypertrophic growth) in myotubes (53) . In the present study, it was shown that cardiomyogenesis was significantly down-regulated in the presence of the p38 inhibitor SB203580, which was, however, ineffective in inhibition of vasculogenesis/angiogenesis. This points toward the notion that p38 is specifically involved in differentiation of cardiomyogenic precursor cells and is not acting on a common mesoderm-derived precursor of the cardiac and vascular cell lineage. Besides p38, ERK1,2 and JNK are apparently involved in mechanical strain-stimulated cardiomyogenesis of ES cells, since differentiation was significantly inhibited in the presence of an ERK1,2 and JNK inhibitor. JNK inhibition only blunted the stimulation of cardiomyogenesis under conditions of mechanical strain, whereas cardiomyogenesis under control conditions remained unimpaired, which suggests an additional stress signaling pathway required for the stimulation of cardiomyogenesis following mechanical strain. Recently, ERK1,2 activation as well as p38 have been shown to mediate the wall stress-induced activation of GATA-4 binding in adult heart (54) . This indicates that mechanical strain elicits comparable signaling pathways in ES cells differentiating into the cardiovascular cell lineage and in the intact adult heart.

In contrast to its effects on cardiomyogenesis, p38 appeared not to be involved in mechanical strain-induced vasculogenesis/angiogenesis, which was significantly inhibited after application of ERK1,2 and JNK inhibitors. These results corroborate previous investigations of our group that demonstrated that electrical field-induced vasculogenesis/angiogenesis of ES cells was ERK1,2 and JNK dependent with no participation of p38 (48) .

The requirement of ROS for the functioning of MAPK pathways has been demonstrated in a variety of studies (30 , 55) . The present study clearly shows redox sensitivity of ERK1,2, JNK and p38, which links the observed generation of ROS to the activation of signaling cascades involved in initiation of cardiovascular differentiation of ES cells. The present study demonstrated for the first time the importance of mechanical strain for cardiovascular differentiation of ES cells and the role of NADPH oxidase-derived ROS within the signaling cascade. A microenvironment consisting of an elevated intracellular redox state and mechanical strain may be required for survival, proper integration, differentiation, and proliferation of stem cells after cardiomyoplasty. Increased generation of ROS following cardiac infarction is a well-known feature, and sophisticated procedures have been elaborated to reduce oxidative stress-induced myocardial reperfusion injury (56) . It should kept in mind, however, that it is just this microenvironment of elevated intracellular ROS generation that may provide the differentiation stimulus that directs transplanted stem cells into the cardiovascular cell lineage and, therefore, may be inevitably necessary for successful cell transplantation.

ACKNOWLEDGMENTS

This work was supported by the German Research Foundation (DFG) graduate college 534, Biological Basis of Vascular Medicine.

Received for publication August 4, 2005. Accepted for publication January 17, 2006.

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