Notch Signaling Regulates Platelet-Derived Growth Factor Receptor-β Expression in Vascular Smooth Muscle Cells
Notch signaling is critically important for proper architecture of the vascular system, and mutations in NOTCH3 are associated with CADASIL, a stroke and dementia syndrome with vascular smooth muscle cell (VSMC) dysfunction. In this report, we link Notch signaling to platelet-derived growth factor (PDGF) signaling, a key determinant of VSMC biology, and show that PDGF receptor (PDGFR)-β is a novel immediate Notch target gene. PDGFR-β expression was upregulated by Notch ligand induction or by activated forms of the Notch receptor. Moreover, upregulation of PDGFR-β expression in response to Notch activation critically required the Notch signal integrator CSL. In primary VSMCs, PDGFR-β expression was robustly upregulated by Notch signaling, leading to an augmented intracellular response to PDGF stimulation. In newborn Notch3-deficient mice, PDGFR-β expression was strongly reduced in the VSMCs that later develop an aberrant morphology. In keeping with this, PDGFR-β upregulation in response to Notch activation was reduced also in Notch3-deficient embryonic stem cells. Finally, in VSMCs from a CADASIL patient carrying a NOTCH3 missense mutation, upregulation of PDGFR-β mRNA and protein in response to ligand-induced Notch activation was significantly reduced. In sum, these data reveal a hierarchy for 2 important signaling systems, Notch and PDGF, in the vasculature and provide insights into how dysregulated Notch signaling perturbs VSMC differentiation and function.
The vasculature is formed by an initial aggregation of angioblasts during vasculogenesis, followed by remodeling of the primitive vascular plexus through angiogenesis.1,2 Recruitment of mural cells, which differentiate to vascular smooth muscle cells (VSMCs) and pericytes, to the endothelial tube is required for stabilization of the vessels.3 Endothelial and mural cell differentiation is controlled by several key signaling pathways, including PDGF and Notch signaling,2 and, in this study, we addressed the interrelationship between Notch and PDGF signaling in VSMCs.
PDGF signaling is critical for several steps in vascular development and for the homeostasis of blood vessels. There are 4 different genes encoding PDGF ligands (PDGFA through -D), and 2 genes encoding PDGF receptors (PDGFRs) (PDGFR-α and -β). PDGFRs are receptor tyrosine kinases that, on interaction with ligand, activate several intracellular signaling pathways, including phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling.4 Loss-of-function analysis has revealed the importance for PDGF-BB/PDGFR-β signaling in vascular development. Mice in which the PDGF-B or PDGFR-β gene has been targeted are embryonic lethal, and the phenotypes support a model where endothelial cells, through secretion of PDGF-BB, stimulate proliferation and recruitment of PDGFR-β positive mural cells during embryonic development.5,6 PDGF signaling plays an important role also in restenosis in response to angioplasty.7
Notch signaling is, like PDGF signaling, required for several distinct steps in vascular development. The signal-sending cell expresses membrane-bound ligands of the DSL family that bind to and activate Notch receptors on juxtaposed, signal-receiving cells. Notch receptors (Notch1 to -4 in mammals) are transmembrane proteins that, in response to ligand activation, are proteolytically cleaved, resulting in liberation of the Notch intracellular domain (Notch ICD), which constitutes the activated form of the receptor. The Notch ICD translocates to the cell nucleus, where it interacts with the DNA-binding protein CSL. Notch ICD converts CSL from a repressor to an activator of Notch downstream target genes.8 Hes and Hey genes are the most well-characterized immediate downstream genes, but additional immediate downstream genes have been characterized,9,10 suggesting the existence of a larger immediate Notch transcriptome.
Notch signaling is important for development of both endothelial and mural cells. At early stages of vascular development, Notch plays an important role in specifying arterial and venous fates of angioblasts, and remodeling of the primary vascular network does not occur properly in Notch1-, Jagged1-, or CSL-deficient mice (reviewed elsewhere11). Notch induces 10T1/2 cells to smooth muscle differentiation,12 and Notch3 and Jagged1 are expressed in arterial smooth muscle cells13–15 (reviewed elsewhere11). In keeping with the VSMC expression of Notch3, Notch3-deficient mice exhibit a VSMC phenotype and lack the specialized VSMC layers in the wall of muscular arteries, leading to poor control of blood pressure.16 Furthermore, mutations in the human NOTCH3 gene result in CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; Online Mendelian Inheritance of Man no. 125310), a stroke and dementia syndrome characterized by recurrent white matter infarcts attributable to an obliteration of the penetrating arteries of the brain.17 VSMCs are gradually destructed in CADASIL patients,18 but the downstream consequences of the NOTCH3 mutations, ie, whether the mutations lead to gain or loss of Notch signaling, have not yet been established.17 In Notch3−/− mice, expression of the canonical downstream genes Hes and Hey is not altered,16 indicating that expression of novel, yet uncharacterized, downstream genes is affected in CADASIL.
In this report, we show that Notch acts upstream of PDGF signaling and that PDGFR-β is a new immediate Notch downstream gene. Furthermore, PDGFR-β expression is downregulated in VSMCs of Notch3-deficient mice, and Notch-induced PDGFR-β upregulation is reduced in VSMCs from CADASIL patients, suggesting that derailed Notch signaling leading to reduced PDGF signaling plays a role in the pathogenesis of CADASIL.
Materials and Methods
C2C12 cells were infected with adenovirus expressing Notch1 ICD-IRES-EGFP, Notch3 ICD-IRES-EGFP, Hes1, Hey1, or EGFP (Ad-Notch1 ICD, Ad-Notch3 ICD, Ad-Hes1, Ad-Hey1, and Ad-EGFP, respectively) at a multiplicity of infection of 200. VSMCs were infected with Ad-Notch1 ICD, Ad-Notch3 ICD, and Ad-EGFP at a multiplicity of infection of 10. mRNA was harvested 6 hours after infection (see below).
Cell lines, Cell Culture, and Cell Migration Assays
All primary cells, cell lines, cell culture conditions, and cell migration assays, as well as the generation of mouse Notch3lacZ/lacZ embryonic stem (ES) cells and human CADASIL VSMCs, are described in the online data supplement, available at http://circres. ahajournals.org.
Activation of Notch Signaling
Induction with immobilized ligand and conditions for coculture of C2C12 cells and VSMCs with ligand-expressing cells are described in the online data supplement.
RNA Isolation and Quantitative RT-PCR
RNA was isolated from cultured cells using the RNeasy mini (Qiagen) or NucleoSpin RNA II (Macherey-Nagel) kits. Reverse transcription and real-time PCR conditions are described in the online data supplement.
The primary antibodies used in the study are described in the online data supplement.
For detection of PDGFR-β, cells were washed with PBS, directly lysed in Laemmli sample buffer (Invitrogen), and subjected to SDS-PAGE (4% to 12% Nu-PAGE Bis-Tris gels [Invitrogen]) and Western blot analysis. For detection of Notch3, cells were lysed in radioimmunoprecipitation assay buffer (50 mmol/L Tris–HCl pH 7.4, 150 mmol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L EDTA, 1% Triton X-100, 1% Na-deoxycholate, 0.1% SDS), and 30 μg of protein lysates were subjected to SDS-PAGE (6% gels) and Western blot analysis.
Histology, X-Gal Staining, and Immunohistochemistry
Cryosections (12 μm each) from mouse tails were collected on Superfrost glass slides. Hematoxylin/eosin staining was performed according to standard procedures. X-gal staining, immunohistochemistry, and quantification of immunohistochemistry data are described in the online data supplement.
Transfections and Reporter Assays
Transfections and reporter assays were performed essentially as previously described.19 The generation of PDGFR-β promoter constructs is described in the online data supplement.
The chromatin immunoprecipitation (ChIP) assay was performed according to the protocol of the manufacturer (Upstate), and is further described in the online data supplement.
Data from the experiments in which VSMCs were stimulated with immobilized Notch ligand were analyzed using 2-way ANOVA (grouping factors; cell type and stimulation). Post hoc analysis (Tukey test) was used for multiple comparisons between the groups. Probability of <0.05 were considered statistically significant. All other numeric data were analyzed by 2-tailed, 2 sample equal variance Student’s t test. Probability values <0.05 were considered statistically significant.
This work has been approved by the North Stockholm Committee for Animal Experimentation (permit no. 156/03). Experiments comparing heterozygous and homozygous mice were performed on littermates. Tails from newborn mice were dissected in ice cold PBS and fixed in 4% paraformaldehyde for 2 hours at room temperature. Tails were then washed repeatedly in PBS, cryopreserved in 30% sucrose in PBS at 4°C overnight, mounted in Tissue-Tek, and kept at −70°C until sectioning was performed. Genotyping was performed as described in Figure IIA in the online data supplement.
PDGFR-β Is a Novel Notch Immediate Downstream Gene
To explore the relationship between Notch and PDGF signaling, we first asked whether Notch signaling acts upstream of PDGF signaling. We focused on PDGFR-β, because PDGFR-β is expressed in VSMCs and thus could potentially be under direct control of activated Notch receptors in these cells. We first analyzed whether activation of Notch signaling resulted in upregulated expression of PDGFR-β in C2C12 cells, a myogenic cell line in which Notch signaling controls differentiation.19–21 Introduction of Notch1 ICD or Notch3 ICD via adenoviral vectors (Ad-Notch1 ICD and Ad-Notch3 ICD, respectively) led to upregulation of PDGFR-β mRNA levels, as compared with an adenoviral control virus expressing only enhanced green fluorescent protein (Ad-EGFP) (Figure 1A). As a control for Notch induction, the canonical Notch target genes Hes1 and Hey1 were, as expected, induced by Ad-Notch1 ICD and Ad-Notch3 ICD (supplemental Figure IA). Although the induction of PDGFR-β expression was observed already 6 hours after Notch induction, indicating a direct effect by Notch ICD, we wanted to test that PDGFR-β activation was not a result of activities further down the canonical Notch signaling cascade. In keeping with this notion, expression of Hes1 and Hey1 from adenoviral vectors (Ad-Hes1 and Ad-Hey1, respectively) in C2C12 cells did not increase PDGFR-β expression above the level seen after Ad-EGFP expression (Figure 1A).
To activate Notch via ligand stimulation of endogenous full-length Notch receptors, we cocultured C2C12 cells stably expressing full-length Notch1 receptor with 293T cells stably expressing the Jagged1 ligand (293T-Jagged1).19,21 Activation of immediate Notch downstream target genes requires the γ-secretase–mediated liberation of the Notch ICD from the membrane but does not depend on protein synthesis. Coculture experiments, in which protein synthesis was blocked by cycloheximide or generation of Notch ICD was blocked by the γ-secretase inhibitor DAPT, showed that upregulation of PDGFR-β mRNA expression does not require protein synthesis but is dependent on generation of the Notch ICD (Figure 1B). The efficiency of ligand induction was verified by upregulation of Hes1 and Hey1 expression following coculture with Jagged1-expressing cells (supplemental Figure IB). The cycloheximide-mediated block of protein synthesis was validated by examining levels of the unstable Pen-2 protein (data not shown).22 The upregulation observed at the mRNA level was paralleled by increased amounts of PDGFR-β protein in response to Ad-Notch1 ICD or Ad-Notch3 ICD or activation by immobilized Jagged1 ligand in the C2C12 cells (Figure 1C and 1D).
We next turned to primary VSMCs to validate whether the Notch–PDGFR-β link also functions in a vascular cell type in which Notch signaling is known to be important.17 Coculture of VSMCs with 3T3-Jagged1 or 3T3-Babe cells upregulated PDGFR-β mRNA expression only in VSMCs cocultured with ligand-expressing cells. A significant upregulation was observed after 6 hours and further increased after 12 hours (Figure 1E). Moreover, following infection with Ad-Notch1 ICD or Ad-Notch3 ICD, PDGFR-β mRNA expression was increased (Figure 1F). Upregulation was specific to PDGFR-β, because PDGFR-α levels were, in fact, downregulated in both VSMCs and C2C12 cells (data not shown). As for C2C12 cells, PDGFR-β protein levels were elevated by activated Notch signaling in VSMCs (Figure 1G). Collectively, these data show that PDGFR-β is a Notch downstream gene.
Notch1 ICD and Notch3 ICD Upregulate PDGFR-β Expression by Distinct Mechanisms
Because both Notch1 and Notch3 ICD upregulated PDGFR-β expression, we asked whether they activated PDGFR-β expression by similar or distinct mechanisms. We first tested their potential to elicit a transcriptional response from a 1.5-kb proximal PDGFR-β promoter containing a putative CSL-binding site from −256 to −249, linked to a luciferase gene (pPDGFR-β-luc) (Figure 2A). The 1.5-kb promoter element was sufficient to mediate a Notch1 ICD–induced transcriptional response (Figure 2B). Mutation of the −256/249 CSL-binding site (pPDGFR-β-ΔCSL-luc) resulted in a considerably lower level of activation (Figure 2B). To learn whether Notch1 ICD was directly recruited to the CSL-binding site–containing region of the proximal PDGFR-β promoter, we performed ChIP experiments. Notch1 ICD has previously been shown to be recruited to CSL-binding sites in the Hes1 and Hey2 genes,21,23 and immunoprecipitation with an antibody recognizing Notch1 ICD following Ad-Notch1 ICD infection demonstrated an interaction between Notch1 ICD and the part of the PDGFR-β promoter containing the −256/249 CSL-binding site (Figure 2C).
In contrast to the data for Notch1 ICD, the same 1.5-kb PDGFR-β promoter element did not elicit a Notch3 ICD–induced response (Figure 2D). Similarly, ligand activation of a 293T cell line carrying a full-length Notch3 receptor did not upregulate the PDGFR-β promoter reporter gene activity (Figure 2E). To verify that activation of another, more general, Notch-responsive promoters indeed occurred, we showed that the 12XCSL-luciferase reporter construct, which reflects immediate Notch downstream signaling,19,21 was robustly upregulated in both cases (Figure 2D and 2E).
To test whether CSL was required for the Notch-mediated activation of PDGFR-β activation, we analyzed PDGFR-β upregulation in ES cells deficient for CSL. In CSL−/− ES cells upregulation by either Notch1 ICD, Notch3 ICD or ligand-activation was completely blunted whereas in CSL+/− ES cells, PDGFR-β mRNA expression was upregulated (Figure 2F and 2G). Collectively, this shows that CSL is critically required in the PDGFR-β activation process but that Notch1 and 3 ICD differ with regard to how they activate PDGFR-β expression.
Notch Activation in VSMCs Leads to an Elevated Intracellular Response to PDGF-BB Stimulation
We next analyzed whether the Notch-induced increase in PDGFR-β protein levels in VSMCs (Figure 1G) also resulted in an augmented intracellular response to PDGF activation. PDGF stimulation results in phosphorylation of specific tyrosine residues in the ICD of PDGFR-β and activation of the Ras/MAP kinase pathway, leading to increased phosphorylation of Akt.4 We analyzed the effect of PDGF-BB stimulation in VSMCs expressing Notch1 ICD, Notch3 ICD, or EGFP. In both Notch1 and Notch3 ICD–expressing cells, we observed increased amounts of total and phosphorylated PDGFR-β, whereas for Akt, only the amount of phosphorylated Akt, but not total Akt levels, were elevated (Figure 3A and 3B). This shows that the elevation in signaling following Notch1 or Notch3 ICD stimulation is approximately proportional to the increase in the amount of PDGFR-β protein (Figure 3C), suggesting that Notch signaling affects the amount of functional PDGFR-β receptor rather than augmenting the activity of downstream signaling per se.
Notch and PDGF Signaling Exert Opposite Effects on VSMC Migration
The increase in the amount of PDGFR-β protein and downstream signaling in response to Notch activation poses an interesting question with regard to cell migration. PDGF signaling has been shown to enhance6 and Notch signaling to reduce24,25 VSMC migration. To test which effect dominates, we analyzed VSMC migration in a transchamber migration assay under various combinations of Notch and PDGF signaling. Stimulation by PDGF-BB, as expected, resulted in increased VSMC migration (Figure 4A and 4B) but Notch activation, despite the fact that it increased PDGFR-β protein levels and intracellular PDGF signaling (Figure 3), inhibited migration in all situations tested, ie, both in the absence and presence of PDGF-BB stimulation (Figure 4A and 4B). These data corroborate and extend a previous report that Notch signaling can reduce VSMC migration.24
PDGF-BB Stimulation Negatively Regulates Notch3 and PDGFR-β Expression
PDGF-BB has previously been shown to downregulate Notch3 expression,24 and to learn whether this is the case in VSMCs and whether PDGF-BB stimulation then also downregulates PDGFR-β expression, we measured Notch3 and PDGFR-β mRNA levels at different time points after PDGF-BB stimulation. No difference was observed after 6 hours of stimulation (data not shown), but after 18 hours of PDGF-BB stimulation, expression of both Notch3 and PDGFR-β mRNA was downregulated (Figure 4C). PDGFR-β protein levels were also reduced (Figure 4D). The Notch3 downregulation was observed at a later time point as compared with the previous report,24 and whether it depends on extracellular signal-regulated kinase 1/ 2 signaling, which was upregulated in the VSMCs in response to PDGF-BB signaling, but not further upregulated by Notch signaling (data not shown), remains to be established.
Reduced PDGFR-β Expression in VSMCs in Arteries of Notch3-Deficient Mice
Notch3-deficient mice exhibit marked arterial defects and a VSMC phenotype.16 We therefore wanted to learn whether the VSMC phenotype in mice homozygous for a Notch3 loss-of-function allele coincided with altered PDGFR-β expression. In cross-sections of the tail from a newborn (P0) Notch3−/− mouse, in which both alleles of the Notch3 gene were interrupted by a lacZ insertion (Notch3lacZ/lacZ; for genotyping, see supplemental Figure IIA),26 we observed punctuated β-galactosidase (β-gal) expression in cells surrounding the tail lateral artery and vein (Figure 5A). There were no overt morphological differences in the P0 mice (Figure 5A), whereas the adult Notch3lacZ/lacZ mice exhibited structural defects (supplemental Figure IIB), in keeping with a previous study.16 To identify the cells expressing Notch3, we compared the β-gal expression pattern with the distribution of platelet endothelial cell adhesion molecule (PECAM) and smooth muscle actin (SMA), as markers for endothelial cells and VSMCs, respectively. PECAM staining was confined to the endothelial cell layer in both the tail lateral artery and vein in the P0 Notch3lacZ/lacZ mice but did not overlap with β-gal immunoreactivity (Figure 5B). In contrast, coexpression of β-gal and SMA was observed in both arteries and veins (Figure 5B), indicating that in the vessel wall, VSMCs, and not endothelial cells, express Notch3.
We observed PDGFR-β immunoreactivity in the artery in the Notch3+/lacZ mouse and a significant codistribution of PDGFR-β and Notch3 immunoreactivity (Figure 5C). Importantly, the PDGFR-β expression was reduced in the Notch3lacZ/lacZ mouse (Figure 5C and supplemental Figure III). Quantification revealed that PDGFR-β expression was reduced by 46% in the Notch3lacZ/lacZ compared with Notch3+/lacZ arteries (Figure 5D), whereas SMA levels were not significantly altered (Figure 5C and 5D and supplemental Figure III). The low level of PDGFR-β expression in the vein was not noticeably altered but was too low to be quantified. The localization of reduced PDGFR-β expression coincided with Notch3 activity rather than with overall distribution of the Notch3 receptor, as demonstrated by Joutel and colleagues, who have shown that Notch3 signaling occurs in tail lateral arteries but not in veins, although the receptor is expressed in both arteries and veins (see supplemental figure 3 in the previously published article27).
To corroborate the role of endogenous Notch3 in regulation of PDGFR-β expression, we analyzed the extent of PDGFR-β upregulation in Notch3-deficient ES cells following ligand activation. In response to ligand induction, upregulation of PDGFR-β expression was considerably higher in Notch3+/lacZ ES cells than in 3 Notch3lacZ/lacZ ES cell lines (Figure 5E).
PDGFR-β Expression in Response to Notch Activation Is Reduced in VSMCs From CADASIL Patients
CADASIL is caused by missense mutations or small deletions in the NOTCH3 receptor,28,29 leading to VSMC degeneration, but differences in gene expression have not yet been identified in CADASIL patients. We therefore tested whether NOTCH regulated PDGFR-β expression in immortalized VSMCs from the umbilical cord of a CADASIL patient carrying an arginine-to-cysteine substitution at position 133 (R133C) and, as control, cells immortalized in parallel from 2 healthy, non-CADASIL control individuals. Stimulation by immobilized Jagged1 ligand resulted in robust upregulation of PDGFR-β expression in the 2 control cell lines but a considerably more modest upregulation in the CADASIL R133C cells (Figure 6A). Culturing CADASIL and control VSMCs on immobilized Jagged1 ligand revealed that CADASIL VSMCs also exhibited a blunted increase in PDGFR-β protein expression following Notch receptor activation (Figure 6B). Upregulation of the HEY1 and HES1 genes was also analyzed. HEY1 expression was increased 10- and 9-fold in the control cell lines but only 2.1-fold in the CADASIL R133C cell, and HES1 expression was increased 8-, 8.5-, and 2-fold, respectively. The difference in PDGFR-β, HEY1, and HES1 upregulation was not a consequence of different levels of NOTCH3 receptor in the R133C and control cells, because both the amount of the full-length and the S2-processed, membrane-tethered forms of the receptor were very similar in the 3 cell lines (Figure 6C).
We provide evidence for a novel link between Notch and PDGF signaling, where Notch directly controls expression of the PDGFR-β gene. Activation of Notch led to a rapid increase in PDGFR-β mRNA and protein expression, and upregulation was blunted in CSL−/− ES cells, indicating that upregulation is controlled at the Notch ICD:CSL level. Both Notch1 and 3 ICD robustly upregulated PDGFR-β expression but, interestingly, differed in how they accomplish the upregulation. Thus, Notch1 ICD, but not Notch3 ICD, activated expression from a 1.5-kb proximal PDGFR-β promoter, whereas both required the presence of CSL to mediate upregulation. This suggests that Notch3 ICD uses alternative CSL-binding sites, located outside the 1.5-kb promoter element, for activation of the PDGFR-β gene. This is in line with the finding that different Notch ICDs have different target sequence selectivity30 and is reminiscent of the situation for the proximal Hes1 and Hes5 promoters, where Notch1 ICD is a considerably more potent activator than Notch3 ICD.31
The Notch-induced increase in PDGFR-β protein expression, and the proportionally augmented intracellular PDGF response, suggests that Notch is epistatic over PDGF signaling. However, the situation is more complex and dynamic, because PDGF-BB stimulation, in turn, downregulated expression of the Notch3 gene in VSMCs, in keeping with a previous report,24 and also reduced PDGFR-β expression. An initial transient upregulation of PDGFR-β by Notch may therefore lead to an elevated intracellular PDGF response, resulting in a subsequent reduction of Notch3 and PDGFR-β expression. The dynamic interplay between Notch and PDGF signaling may also be of importance for control of VSMC cell migration, because the migration-promoting capacity of PDGF-BB was found to be counteracted by a Notch-induced inhibition of VSMC migration, in line with an earlier study.24
We found that PDGFR-β expression was reduced in VSMCs in the tail lateral arteries in newborn Notch3lacZ/lacZ mice, ie, in the cells showing an aberrant phenotype in the Notch3-deficient mice. Given the importance of PDGF signaling for VSMC differentiation, it is tempting to speculate that the reduced PDGFR-β expression contributes to the observed phenotype, and this notion is supported by the notion that the reduction in PDGFR-β expression takes place well before the morphological changes are observed. It is also of note that the reduction of PDGFR-β expression is observed specifically in the cells, which, in the Notch3 wild-type mouse, have active Notch3 signaling.27 This observation, combined with the reduction of PDGFR-β upregulation in Notch3-deficient ES cells in response to Notch activation, further supports a role for Notch3 in regulation of PDGFR-β expression.
The CADASIL R133C cell line exhibited a reduced upregulation of PDGFR-β, HES1, and HEY1 expression in response to ligand activation. This is the first observation of deregulated gene expression linked to a CADASIL mutation and suggests that NOTCH downstream signaling is generally attenuated in the CADASIL cells. Whether the attenuation in the NOTCH downstream response reflects a strong haploinsufficiency or a dominant negative effect of the mutant receptor remains to be investigated. Similarly, it is an open issue whether expression of other Notch receptors can substitute for the NOTCH3 receptor in regulation of PDGFR-β expression in VSMCs in vivo. With regard to substitution, NOTCH4 is a potential candidate, because NOTCH4 ICD has been demonstrated to induce PDGFR-β expression in endothelial cells.32 In conclusion, the linking of the Notch and PDGF signaling pathways in VSMCs opens new perspectives for understanding how signaling through these key signaling pathways is synchronized for controlling vascular differentiation and how this can be derailed in disease.
We are grateful to Susanne Bergstedt for technical assistance and to Dr Tasuku Honjo for the kind gift of the CSL−/− ES cells.
Sources of Funding
This work was supported by the Swedish Foundation for Strategic Research (Center of Excellence in Developmental Biology and Strategic Research Center in Organic BioElectronics), the Swedish Cancer Society, the Swedish Research Council, Swedish Brain Power, the Nordic Center of Excellence for Neurodegenerative Disease, and the European Union project EuroStemCell.
↵*Both authors contributed equally to this work.
Original received November 20, 2007; revision received April 28, 2008; accepted May 1, 2008.
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