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(Circulation Research. 2007;101:1139.)
© 2007 American Heart Association, Inc.

Cellular Biology

Notch Signaling Contributes to the Expression of Cardiac Markers in Human Circulating Progenitor Cells

Masamichi Koyanagi^*, Philipp Bushoven^*, Masayoshi Iwasaki, Carmen Urbich, Andreas M. Zeiher, Stefanie Dimmeler

From the Department of Molecular Cardiology, Internal Medicine III, J. W. Goethe University, 60590 Frankfurt, Germany.

Correspondence to Stefanie Dimmeler, PhD, Department of Molecular Cardiology, Internal Medicine III, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. E-mail dimmeler{at}em.uni-frankfurt.de

	Abstract

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It has been demonstrated that adult human circulating endothelial progenitor cells (EPCs) can differentiate to a cardiomyogenic phenotype. Notch signaling promotes epithelial-to-mesenchymal transformation and plays a role in heart and vessel development. Here, we investigated the role of Notch activation for cardiac differentiation of EPCs in a coculture system with neonatal cardiomyocyte. After coculture, Notch activation was transiently detected in EPCs, as determined by immunhistochemical detection of NICD (the intracellular cleavage fragment of Notch-1) and expression of human Notch target genes. Inhibition of -secretase blocked Notch cleavage and NICD translocation. Furthermore, the expression of the cardiac marker protein -sarcomeric actinin and troponin T was significantly suppressed by -secretase inhibition or addition of soluble recombinant Jagged-1, indicating that Notch activation facilitates cardiac marker gene expression. Because noncanonical Wnts have previously been shown to promote cardiac differentiation, we additionally determined the influence of Notch activation on the expression of Wnt5a and Wnt11. Wnt5a and Wnt11 expression in the human cells was induced by the coculture and was blocked by -secretase inhibition. Likewise, stimulation of Notch signaling by immobilized Jagged-1 promoted Wnt5a expression in EPCs. These data suggest that Notch is activated upon coculture of EPCs with neonatal rat cardiac myocytes. -Secretase–dependent Notch activation is required for cardiac gene expression in human cells and induces the expression of noncanonical Wnt proteins, which may act in a paracrine manner to further amplify cardiac differentiation.

Key Words: Notch • EPC • cardiac differentiation • Wnt

	Introduction

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Stem or progenitor cells might be useful to regenerate the failing heart. Previous studies have demonstrated that various different types of stem or progenitor cells can differentiate to a cardiomyogenic phenotype. Whereas the differentiation of embryonic stem cells to cardiomyocytes (CMs) has been clearly established, the capacity of adult hematopoietic progenitor cells to differentiate to functionally active CMs has been questioned.¹ However, several studies have provided solid evidence that adult progenitor cells from the bone marrow (including hematopoietic and mesenchymal stem cells) or tissue-resident cardiac stem cells can acquire a cardiac phenotype.^2–4 Using rigorous experimental testing, we and others have demonstrated that circulating blood-derived cultured endothelial progenitor cells (EPCs) or CD34⁺ hematopoietic cells express cardiac marker proteins after coculture with CMs in vitro and in experimental models of cardiac ischemia in vivo.^5–8 Although the incidence of cardiac differentiation of EPCs is rather low, we believe that the elucidation of the mechanism, by which the expression of cardiac genes is induced, may help to design strategies to further enhance the repair capacity of the cells.

In vitro studies established that the presence of CMs was required to induce cardiac differentiation of EPCs, suggesting that cell-to-cell contact would be indispensable for this process.^5,9 Because Notch signaling occurs by physical interaction of the transmembrane ligands (Delta like-1, -3, and -4 and Jagged-1 and -2) and the Notch receptor (Notch-1 to -4) between 2 cells, the Notch signaling pathway may be a candidate mediating the cell-to-cell communication between CMs and EPCs. Notch signaling promotes epithelial-to-mesenchymal transformation^10,11 and plays an important role in heart and vessel development.¹² Notch is activated by binding of Notch ligands, resulting in proteolytic cleavage by -secretases and leading to the formation of the 80-kDa NICD (intracellular cleavage fragment of Notch-1), which translocates to the nucleus and interacts with recombination signal sequence binding protein-J transcription factor and then activates transcription of downstream genes.

Here, we investigated the role of Notch signaling in cardiomyogenic commitment using an in vitro coculture model to stimulate the cardiac differentiation of adult progenitor cells. Our data indicate that Notch activation and cleavage by the -secretase facilitates the expression of cardiac marker proteins in adult progenitor cells.

	Materials and Methods

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Cell Culture
EPCs were cultivated from human peripheral blood mononuclear cells as described previously. After 3 days in culture, adherent cells were characterized by the coexpression of myeloid markers (CD14^low, CD45) and endothelial markers (eg, eNOS, von Willebrand factor).^13,14 Cardiac differentiation was induced by coculturing of EPCs with neonatal CMs as described previously⁵ and was demonstrated by the expression of human cardiac marker proteins -sarcomeric actinin by fluorescence-activated cell-sorting analysis and troponin (Tn)T expression by RT-PCR. In detail, neonatal ventricular CMs were isolated from 0- to 1-day-old Wistar rats. Non-CMs (primarily fibroblasts) were separated from the CMs by differential plating onto plastic dishes. Then EPCs (1.5x10⁵) and freshly isolated CMs were plated onto gelatin-coated dishes at a ratio of 1:3 in medium (DMEM [68%], medium 199 [17%], horse serum [10%], and FBS [5%]). In some experiments, CMs were fixed with 1% paraformaldehyde for 15 minutes, washed with PBS 5 times, and used for coculture.

For inhibitor experiments, the soluble form of Jagged-1 (sJagged) (5 µg/mL; R&D) or -secretase inhibitor X (1 µmol/L; L-685, 458; Calbiochem) were incubated and the medium was changed every 2 days. Recombinant sJagged and -secretase inhibitors did not reveal a toxic effect and the total number of cells in coculture was not changed. For some of the experiments, EPCs were cultured on fibronectin-precoated (1:100; Sigma) or Jagged-1-precoated (5 µg/mL; R&D) dishes.

Flow Cytometric Analysis
After 6 days of the coculture, cells were stained with phycoerythrin-conjugated antibodies recognizing human leukocyte antigen (HLA)-DR and HLA class I (both from Caltag Laboratories, Burlingame, Calif), followed by permeabilization using the Cytofix/Cytoperm kit (BD Pharmingen) and staining with fluorescein isothiocyanate–conjugated (Pierce, Rockford, Ill) anti--sarcomeric actinin antibody (clone EA-53, Sigma) as described. Cells (20 000) were analyzed on a BD FACS Calibur cell sorter (BD Biosciences, San Jose, Calif).

Immunostaining
For immunostaining, cells were fixed with 4% paraformaldehyde for 15 minutes. After permeabilization with 0.2% saponin (Sigma), cells were incubated with anti–cleaved Notch-1 (Chemicon), followed by Alexa Flour 488 anti-rabbit IgG (Molecular Probes). For the detection of cardiac differentiation, an -sarcomeric actinin antibody (Sigma) was used, followed by staining with Alexa Fluor 647–conjugated anti-mouse IgG (Molecular Probes). Nuclei were counterstained with To-pro-3 iodide or Sytox Blue (both from Molecular Probes) according to the protocol of the manufacture.

Immunoblot Analysis
EPCs and CMs were lysed with lysis buffer (Cell Signaling) containing 1 mmol/L phenylmethanesulfonyl fluoride. After centrifugation, the supernatants were collected and subjected to electrophoresis in 10% SDS–polyacrylamide gels. Proteins were transferred to poly(vinylidene difluoride) membrane and incubated with anti-NICD antibody (Cell Signaling and Chemicon) or anti–-tubulin (Dianova) overnight at 4°C. Bound antibody was visualized by using horseradish peroxidase–conjugated sheep anti-mouse or donkey anti-rabbit antibody (both from Amersham).

RNA Isolation and RT-PCR Analyses
Total RNAs from EPCs and human heart and rat CMs were isolated by using TRIzol (Invitrogen). RNA was subjected to RT-PCR by using SuperScript First Strand Synthesis System (Invitrogen). Primer sequences are provided as in Table I in the online data supplement at http://circres.ahajournals.org.

Statistical Analysis
Data are expressed as means±SEM. Unpaired, 2-tailed Student’s t test was used for the comparison between groups based on the original data.

	Results

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Notch Activation in EPCs After Coculture With Neonatal Rat CMs
To assess whether Notch signaling can be stimulated by the coculture, we investigated the expression of Notch receptors and the Notch ligands by RT-PCR. EPCs revealed high levels of the receptors Notch-1 and -2 (Figure 1A), whereas the expression of the Notch ligands Jagged-1 and -2 was only barely detectable (Figure 1A). Neonatal rat CMs, however, expressed Jagged-1 and Jagged-2 (Figure 1A), indicating that CMs may activate Notch signaling in EPCs.

View larger version (30K): [in this window] [in a new window]   Figure 1. Expression and activation of Notch. A, Expression of Notch receptors and Notch ligands was detected by RT-PCR in EPCs and CMs. B, Notch activation was detected in EPCs cultured on fibronectin-coated dishes by immunostaining. A representative image of NICD is shown. C, NICD immunostaining in cocultures of EPCs and CMs. Human EPCs were labeled by DiI-ac-LDL (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine–labeled acetylated-LDL) (red) and CMs were detected by -sarcomeric actinin (blue) after 2 or 6 days of coculture. NICD is shown in green color. A representative confocal image is shown. The green arrow indicates NICD nuclear translocation in EPCs after coculture; white arrow, EPCs without evidence for translocation or differentiation; yellow arrow, differentiated EPCs after 6 days of coculture. D, Negative control using secondary antibodies. Only Dil-ac-LDL is detected. E, Expression of human-specific Hey-2 and Hes-1 and human and rat GAPDH was detected by RT-PCR at different days after initiation of the coculture.

To assess the activation of the Notch signaling pathway, we performed immunohistochemical staining of NICD in EPCs. Under control conditions, in which EPCs were plated on fibronectin without CMs, Notch was preferentially detected at the plasma membrane, indicating that Notch signaling was not active (Figure 1B). After 2 days of coculture with CMs, Notch staining was detectable intracellularly in endosomal structures and in the nucleus of EPCs, indicating cleavage and activation of the Notch pathway (Figure 1C). The activation of Notch signaling was transient and was reduced after 6 days of coculture (Figure 1C). To confirm transient Notch activation, we performed RT-PCR of the well-known Notch target genes Hey-2 and Hes-1 using human specific primers. Indeed, human Hey-2 and Hes-1 were transiently increased after coculture (Figure 1E).

The cleavage of Notch to its intracellular active fragment occurs via the -secretase.¹⁵ Therefore, we added the pharmacological -secretase inhibitor L-685, 458¹⁶ to the coculture. Notch cleavage in the coculture was significantly blocked by the -secretase inhibitor (Figure 2A).

View larger version (28K): [in this window] [in a new window]   Figure 2. Activation of Notch in EPCs. A, NICD localization was detected in EPCs alone or after 6 days of coculture (CM+EPC) in the presence or absence of -secretase inhibitor. A representative Western blot is shown (left). Quantification is shown in the graph (right) (n=4). B, NICD (green) was detected in EPCs cultured for 24 hours on Jagged-1 (JAG1)–coated dishes in the presence or absence of -secretase inhibitor. Arrows indicate cells showing NICD translocation to the nucleus. Higher magnification is shown in the images labeled as nuclei, NICD and Alexa Flour 488 anti-rabbit IgG (for experimental and control conditions, respectively), and merge. Red indicates nuclear staining. Quantification is shown in the graph (top right) (n=3). C, Western blot of NICD on Jagged-1–coated dishes in the presence or absence of -secretase inhibitor is shown (left). Quantification is shown in the graph (right) (n=4). *P<0.05 vs control, #P<0.05 vs Jagged-1.

To specifically assess the Notch activation in EPCs, EPCs were plated on dishes, which were coated with the immobilized recombinant Notch ligand Jagged-1. The immobilized ligand Jagged-1–stimulated Notch cleavage and translocation in a -secretase–dependent manner (Figure 2B and 2C).

Having demonstrated that Notch signaling is activated on coculture of EPCs with CMs in vitro, we determined the role of Notch in the cardiomyogenic differentiation of EPCs. After coculture, the expression of -sarcomeric actinin in human cells was detected in 0.40±0.03% of all cells. The percentage of human cells which expressed -sarcomeric actinin was 4.2±0.49%. Pharmacological inhibition of Notch cleavage by the -secretase inhibitor significantly decreased the number of -sarcomeric actinin⁺/human HLA⁺ cells after 6 days of coculture (Figure 3A and 3B). Moreover, the upregulation of human TnT expression was blocked by the -secretase inhibitor (Figure 3D). To further confirm the involvement of the Notch signaling pathway in the upregulation of cardiac marker protein expression, we added recombinant sJagged-1 to the coculture, which blocks Notch activation.¹⁷ sJagged-1 significantly inhibited the expression of -sarcomeric actinin in human cells (Figure 3C). In a previous study, we demonstrated that 50% of the cardiac marker–expressing human cells after coculture derive from fusion events preferentially mediated by a transient cell-to-cell communication via nanotubes.¹⁸ Therefore, we determined the influence of -secretase on the formation of nanotubes. However, the -secretase inhibitor did not affect nanotube formation (93.0±3.9% compared with control), indicating that Notch signaling stimulates cell differentiation rather than fusion. Furthermore, we used fixed CMs for the coculture assay to exclude the possibility of cell fusion. Human TnT and the human Notch target Hey-2 were increased after coculture of EPCs with fixed cardiac myocytes, indicating that Notch activation and cardiac differentiation can occur independent on cell fusion events (Figure 3E and 3F). This is consistent with the finding that blocking of Notch signaling only resulted in a half-maximal blockade of cardiac marker gene expression (Figure 3B and 3C), leaving the fusion events unaffected. Of note, activation of Notch signaling in the absence of coculture by adding EPCs to Jagged-coated dishes was not sufficient to induce the expression of cardiac marker genes (data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 3. Notch signaling contributes to cardiac marker gene expression. A through C, EPC and CM cocultures were incubated with or without -secretase inhibitor (1 µmol/L) (A and B) or sJagged-1 (5 µg/mL) (C) for 6 days, and EPC differentiation was detected by FACS analysis using -sarcomeric actinin and human HLA. A, Representative FACS histograms. B and C, Summaries of data (n=5 to 8). *P<0.05 vs control. D, RT-PCR of human-specific cardiac TnT in cocultures of EPCs and CMs (CM+EPC) in the presence or absence of -secretase inhibitor. Human heart, EPCs, and CMs alone served as controls. Human GAPDH was used as loading control. Quantification is shown in the graph (right) (n=3). *P<0.05 vs control. E and F, Paraformaldehyde-fixed CMs were cocultured with EPCs. RT-PCR of human-specific cardiac TnT (E) and Hey-2 (F) are shown.

Notch Activation Stimulates Expression of Noncanonical Wnts
Wnt proteins regulate cardiac differentiation via canonical or noncanonical pathways. In particular, noncanonical Wnt proteins (such as Wnt5a or Wnt11) have been shown to regulate cardiogenesis in EPCs and mesenchymal stem cells.^6,19 To determine whether Notch activation interferes with Wnt expression, we detected the expression the canonical Wnt3a and the noncanonical Wnts, Wnt5a, and Wnt11. The expressions of noncanonical Wnt5a and Wnt11 were significantly increased after the coculture, whereas the canonical Wnt3a was not regulated (Figure 4A). The upregulation of Wnt5a was blocked by the -secretase inhibitor (Figure 4B). Likewise, when EPCs were plated on immobilized Jagged-1 to stimulate Notch activation, Wnt5a expression was significantly increased (Figure 4C). Inhibition of -secretase significantly blocked Jagged-1–induced expression Wnt5a (Figure 4D), indicating that activation of Notch stimulates Wnt5a expression in a -secretase–dependent manner.

View larger version (22K): [in this window] [in a new window]   Figure 4. Notch activation and expression of Wnts. A, Expression of human specific Wnt5a, Wnt11, Wnt3a, and GAPDH was detected by RT-PCR at different days after initiation of the coculture. B, Wnt5 expression at day 1 in cocultures of EPCs with CMs in the presence or absence of -secretase inhibitor. A representative RT-PCR of 2 different donors (EPC A and EPC B) is shown. Quantification is prov- ided in the graph (bottom) (n=3). *P<0.05 vs control. C and D, Expression of Wnt5a in EPCs was detected 24 hours after culture on fibronectin or Jagged-coated dishes in the absence (C) or presence (D) of -secretase inhibitor. Representative RT-PCR is shown (left). Quantification is provided in the graph (right) (n=5). *P<0.05 vs fibronectin, #P<0.05 vs Jagged-1.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although extensive reports have provided evidence that adult stem/progenitor cells can acquire a cardiac phenotype, the precise mechanisms still need to be elucidated. Based on our previous work demonstrating the importance of the physical interaction of neonatal CMs with adult progenitor cells to induce cardiac gene expression,^9,18 we investigated the role of Notch signaling, which is activated by a ligand-expressing neighboring cell. Indeed, coculture of EPCs with CMs induced the cleavage of Notch-1, leading to the formation of NICD, which was subsequently translocated to the nucleus. Blocking Notch signaling by -secretase inhibitors reduced this process and inhibited the expression of cardiac marker proteins in EPCs. The contribution of Notch signaling for cardiac marker gene expression at first glance may be surprising in light of various reports demonstrating a Notch-mediated suppression of cardiomyogenesis in Xenopus, Drosophila, and embryonic stem cells.^20–22 In addition, Notch signaling was originally described as a cellular signaling mechanism maintaining cells in an undifferentiated state. However, this view may be oversimplified as Notch signaling also has been shown to direct cells toward alternate differentiation fates²³ and Notch activation precedes heart regeneration in zebrafish.²⁴ Interestingly, activation of Notch in endothelial cells suppressed the expression of endothelial marker proteins and induced a concomitant increase in mesenchymal markers such as -smooth muscle actin.²⁵ The data of the present study are consistent with the concept that Notch induces an endothelial to mesenchymal transformation by demonstrating that endothelial progenitor cells can acquire mesenchymal characteristics in the coculture system. The underlying mechanism requires further elucidation. Although we could rule out an influence of Notch signaling on cell fusion in the present experimental setting, the mechanisms by which Notch contributes to cardiac marker gene expression are not known. Notch activation may directly affect the transcription of mesenchymal marker genes (as has been shown for the transcriptional activation of smooth muscle actin²⁵) or stimulate the expression of cardiac transcription factors. However, the transient activation of Notch signaling and the failure of Notch stimulation by Jagged-1 to induce cardiac marker gene expression in the absence of cardiac myocyte coculture indicates that Notch activation does not directly induce cardiac commitment but rather facilitates the complex process of cell differentiation. This might be mediated via the induction of a mesenchymal transition, as discussed above, or even by resetting the cells to a more immature state. Additionally, the stimulation of factors acting in a paracrine manner by Notch may contribute to the facilitation of cardiac commitment. Indeed, the results of the present study demonstrate that Notch activation induces the expression of the noncanonical Wnts, Wnt5a and Wnt11, in EPCs. Based on these findings, one may speculate that the Notch signaling system cross-talks with noncanonical Wnts, which are well established to enhance the expression of cardiac markers in adult stem and progenitor cells^6,19 and contribute to cardiogenesis in model organisms.²⁶ Interestingly, the canonical Wnt Wnt3a, which exhibits distinct and even opposing roles during cardiac development, was not regulated by Notch.²⁷

In summary, the present study supports the concept that cell-to-cell communication contributes to dictate cell fate. Notch receptor activation in EPCs by adjacent cardiac myocytes expressing the Notch ligand facilitates cardiac marker gene expression. Obviously, the activation of Notch signaling is only one of multiple possible cell-to-cell communication pathways and the acquirement of a different phenotype of adult progenitor cells requires a complex set of signaling events.

	Acknowledgments

 
We thank Marion Muhly-Reinholz, Ariane Fischer, Tino Röxe, and Britta Kluge for excellent technical assistance.

Sources of Funding

This work was supported by the Deutsche Forschungsgemeinschaft (grant Di600/6-3), the Leducq Foundation (to S.D.), and a Japanese Heart Foundation/Bayer Yakuhin Research Grant Abroad (to M.K. and M.I.).

Disclosures

None.

	Footnotes

 
*Both authors contributed equally to this work.

Original received February 28, 2007; revision received September 29, 2007; accepted October 12, 2007.

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