Published online before print January 15, 2009, doi: 10.1161/CIRCRESAHA.108.184846
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© 2009 American Heart Association, Inc.
Molecular Medicine |
NOTCH3 Expression Is Induced in Mural Cells Through an Autoregulatory Loop That Requires Endothelial-Expressed JAGGED1
From the Vascular Biology Center and Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta.
Correspondence to Brenda Lilly, Vascular Biology Center and Department of Obstetrics and Gynecology, Medical College of Georgia, 1459 Laney Walker Blvd, Augusta, GA 30912. E-mail blilly{at}mcg.edu
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Abstract |
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Endothelial cells and mural cells (smooth muscle cells, pericytes, or fibroblasts) are known to communicate with one another. Their interactions not only serve to support fully functional blood vessels but also can regulate vessel assembly and differentiation or maturation. In an effort to better understand the molecular components of this heterotypic interaction, we used a 3D model of angiogenesis and screened for genes, which were modulated by coculturing of these 2 different cell types. In doing so, we discovered that NOTCH3 is one gene whose expression is robustly induced in mural cells by coculturing with endothelial cells. Knockdown by small interfering RNA revealed that NOTCH3 is necessary for endothelial-dependent mural cell differentiation, whereas overexpression of NOTCH3 is sufficient to promote smooth muscle gene expression. Moreover, NOTCH3 contributes to the proangiogenic abilities of mural cells cocultured with endothelial cells. Interestingly, we found that the expression of NOTCH3 is dependent on Notch signaling, because the
-secretase inhibitor DAPT blocked its upregulation. Furthermore, in mural cells, a dominant-negative Mastermind-like1 construct inhibited NOTCH3 expression, and endothelial-expressed JAGGED1 was required for its induction. Additionally, we demonstrated that NOTCH3 could promote its own expression and that of JAGGED1 in mural cells. Taken together, these data provide a mechanism by which endothelial cells induce the differentiation of mural cells through activation and induction of NOTCH3. These findings also suggest that NOTCH3 has the capacity to maintain a differentiated phenotype through a positive-feedback loop that includes both autoregulation and JAGGED1 expression.
Key Words: endothelial cells • mural cells • fibroblasts • smooth muscle cells • NOTCH3 • JAGGED1
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Introduction |
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Within the vasculature, endothelial cells and mural cells (defined here as vascular support cells that include smooth muscle cells, pericytes, and fibroblasts) are closely associated and can regulate the activity of each other throughout development and into adulthood.1–3 Several groups have shown that mural cells influence blood vessel assembly by controlling such events as endothelial cell proliferation, migration, sprouting, and regression.4–10 Later, in intact vessels, these cells influence how endothelial cells respond to humoral and hemodynamic cues.11 Likewise, endothelial cells are known to modulate mural cell phenotype and function. In addition to proliferation and migration, endothelial cells can promote smooth muscle differentiation and influence contractile activity.12,13 Despite their intimate association and obvious abilities to respond to one another in intact vessels, there is still much to be learned about the nature of their interactions, particularly during blood vessel formation.
A handful of signaling mediators form the basis of our understanding about how these 2 cell types communicate during vasculogenesis and angiogenesis. Growth factor/receptor families, including platelet-derived growth factor (PDGF)-B/PDGF receptor (PDGFR)-β, transforming growth factor-β, and angiopoietin-Tie2, have established roles in their interactions.1,2 For these, secreted factors uniquely expressed from 1 cell type bind to a receptor on the other cell type to trigger downstream events that influence behavior. Additionally, cell-specific extracellular matrix deposition, matrix metalloproteinases, and their inhibitors (TIMPs) can directly affect the activities of these cellular neighbors.2,4,10,14 These modes of communication act independently of cell contact; however, direct cell–cell contact has also been shown to play a role in endothelial/mural cell crosstalk.15 Recently, the Notch signaling pathway has emerged as an interesting candidate that may have a vital function in modulating the interactions of endothelial cells and mural cells.16,17
The Notch family of membrane-bound receptors have a prominent place in vascular development.18–20 Notch receptors (NOTCH1 to 4) and their ligands (Delta-like and Jagged) and downstream mediators (HES/HEY) are highly expressed in both endothelial cells and mural cells (smooth muscle cells) in distinct combinations. A host of studies have helped to define their specific activities within the vasculature that include regulation of angiogenic remodeling, arterial/venous specification and tip cell differentiation.18,19 For example, loss of Notch1 in mice, which is abundantly present in endothelial cells, results in an array of vessel abnormalities such as disorganized intersomitic vessels, an absence of remodeling, and defects in arteriogenesis.21–23 In vascular smooth muscle cells, Notch3 is the predominant Notch receptor and is the causal gene for the neurovascular disorder CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy).24,25 Targeted inactivation of the mouse Notch3 gene revealed defects in smooth muscle maturation,26 indicating a potential role in differentiation, among other things. In support of this, neural crest ablation of Notch activity similarly prevents smooth muscle differentiation.27 Several independent studies examining the role of Notch on smooth muscle gene expression have shown that an overexpressed Notch intracellular domain can promote the expression of smooth muscle genes.28–31 Yet, these reports are in contrast to others that have demonstrated robust inhibition of smooth muscle gene expression by Notch.32–34 A more recent report has hinted that these discrepancies might be explained by context-dependent activity that is controlled by the ratio of the Notch activator relative to its downstream repressor proteins (HES/HEY).31
Given that Notch signaling is initiated by cell–cell contact of membrane bound receptor–ligand combinations, it is reasonable to assume that these proteins regulate the communication between endothelial and mural cells within the vasculature. Data supporting this hypothesis come from an endothelial-specific knockout of Jagged1 that gives rise to an embryonic lethal phenotype with an absence of smooth muscle gene expression in the vasculature.16 These findings strongly support a role for Notch signaling in the association of these 2 cell types, but the extent and the precise mechanisms underlying Notch-dependent interactions remain to be elucidated.
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Materials and Methods |
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Cell culture methods, including angiogenesis assays, Transwell culture experiments, transfections, and lentiviral infections were performed with primary cultures of human endothelial cells, dermal fibroblasts, and smooth muscle cells. Plasmid constructs for transfections and lentiviral infections were generated using standard cloning strategies. Expression analysis of protein, by Western blotting and immunohistochemistry, and RNA, by quantitative RT-PCR (qPCR), were carried out with common established methods. An expanded Materials and Methods is available in the online data supplement at http://circres.ahajournals.org.
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Results |
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Endothelial Cells and Mural Cells Interact in a Three-Dimensional Model of Angiogenesis
Endothelial cells and mural cells (pericytes, smooth muscle cells, and fibroblasts) are known to communicate with one another. Several groups, including ours, have demonstrated that angiogenesis is enhanced in the presence of mural cells, indicating they are important mediators of endothelial cell function during blood vessel assembly.4–10 As shown in Figure 1A through 1D, a 3D angiogenesis assay using human umbilical vein endothelial cells (HUVECs) cultured alone or with human dermal neonatal fibroblasts (HDFNs), umbilical artery smooth muscle cells (HUASMCs), or coronary artery smooth muscle cells (HCASMCs) revealed robust enhancement of endothelial-derived blood vessel formation by cocultured mural cells. In this model, communication is bidirectional, because mural cells also responded to endothelial cells and were seen surrounding nascent vessels in an apparent mimic of the in vivo circumstance (Figure 1E). The inference from these observations is that these cells are programmed to respond to one another in defined ways, of which we still have a limited understanding.
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Endothelial Cells Increase NOTCH3 Expression in Mural Cells
The obvious physical and functional interactions of these cell types in this in vitro model of angiogenesis led us to ask what genes may be regulated by their unique association. To address this, we performed a microarray to identify genes that were specifically modulated in this 3D coculture assay using HUVECs and HDFNs. Fibroblasts were used to emulate naive mural cells, with the intent of identifying endothelial-regulated specification or differentiation genes. In doing so, we identified NOTCH3 as 1 gene that was robustly increased in fibroblasts when cocultured with endothelial cells. Western blot analysis revealed low levels of NOTCH3 protein in HDFNs cultured by themselves, whereas much higher levels of NOTCH3 were observed in fibroblasts cocultured with endothelial cells (Figure 2A). Endothelial cells and fibroblasts were separated after coculture using endothelial-specific anti–platelet/endothelial cell adhesion molecule (PECAM)1-conjugated beads. The expression and induction of NOTCH3 were specific to HDFNs, because no protein could be detected in endothelial cells cultured alone. In cocultured endothelial cells, a very small amount of NOTCH3 was detected after separation and likely reflects contaminating fibroblasts resulting from incomplete separation.35
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Although our initial observation was in a 3D assay, NOTCH3 protein expression was also increased in 2D coculture conditions, with peak levels occurring at 48 hours (Figure 2B) and remaining for up to 96 hours, the longest time tested (data not shown). Western blot and qPCR indicated that NOTCH3 could be induced in fibroblasts, smooth muscle cells, and bovine retinal pericytes. The increase in NOTCH3 transcript expression ranged from 6- to 10-fold in dermal fibroblasts and umbilical artery smooth muscle cells (Figure 2C). The increase in coronary artery smooth muscle cells was the least dramatic at both RNA and protein levels; however, as can be seen in Figure 2, the basal level of NOTCH3 was considerably higher in these cells. In contrast, no induction of NOTCH3 by endothelial cells was observed in HepG2 liver cells, suggesting that this activation is specific to mural cells. We further tested whether other endothelial cells could affect NOTCH3 expression. Using human microvascular endothelial cells (HMECs), we demonstrated that NOTCH3 was increased in fibroblasts and umbilical artery smooth muscle cells (Figure 2D); however, HepG2 cells were not able to produce a similar enhancement (data not shown). Taken together, these results indicate that endothelial cells can specifically increase the expression of NOTCH3 in mural cells, which may explain how endothelial cells modulate their function.
Endothelial Cell Activation of Notch Signaling in Mural Cells Is Dependent on NOTCH3
The induction of NOTCH3 implies that endothelial cells are activating Notch signaling through an increase in receptor expression. To examine Notch activity in mural cells, we transfected luciferase reporter constructs with or without 5 Notch-sensing CBF1 binding sites upstream of an SV40 promoter into fibroblasts. In the absence of endothelial cells, the CBF1 reporter had no activity above the basal level of the SV40 promoter; however, in the presence of endothelial cells, the CBF1 reporter exhibited a 
10-fold increase in activity (Figure 3A). Thus, these results indicate that in the absence of endothelial cells, there is little or no CBF-dependent Notch signaling in fibroblasts, and HUVECs can strongly activate this, which is consistent with a previous report.31 To examine whether Notch activity was augmented as a result of an increase in NOTCH3 expression, we examined downstream targets of Notch. As described for previous experiments, cells were cocultured for 48 hours and separated using anti–PECAM1-conjugated beads to measure expression exclusively in mural cells. Indeed, HES1 and HEYL/HRT3 RNA levels were increased in fibroblasts and smooth muscle cells when cocultured with endothelial cells (Figure 3B and 3C). Similar increases were seen for HRT1, HRT2, and HES4 (data not shown). Furthermore, the expression of HEYL/HRT3 was reduced when NOTCH3 levels were blocked by small interfering (si)RNA, indicating a direct effect of NOTCH3 in the upregulation of this factor (Figure 3D through 3F).
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NOTCH3 Is Essential for Endothelial-Induced Smooth Muscle Gene Expression
Given that Notch signaling has been shown to regulate the expression of smooth muscle genes,28–31 we next examined whether stimulation of NOTCH3 by endothelial cells facilitated endothelial-induced smooth muscle gene expression. Coculture of endothelial cells with fibroblasts and smooth muscle cells resulted in a significant increase in smooth muscle marker gene expression in all but 2 instances, as determined by qPCR of transcript levels (Figure 4A). Significant increases were observed for smooth muscle 
-actin and calponin in HDFNs, HCASMCs, and HUASMCs. SM22 expression was increased in HDFNs and HCASMCs, but the increase in HUASMCs was not statistically different. Smooth muscle myosin heavy chain expression showed increases in both smooth muscle cell types but was not significantly increased in fibroblasts, which is indicative of a myofibroblast transition rather than smooth muscle differentiation per se. Protein expression of these genes was confirmed by Western blot analysis and exhibited similar increases relative to RNA levels (Figure I in the online data supplement). Under coculture conditions, siRNA knockdown revealed that the expression of these genes was largely contingent on NOTCH3 (Figure 4B), with 2 cell-specific exceptions that imply the presence of additional regulators. Overall, these data show for the first time that endothelial-induced smooth muscle differentiation is dependent on NOTCH3 expression.
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NOTCH3 Expression Depends on Notch Transcriptional Activity
As a means to define the signal from endothelial cells that was responsible for causing NOTCH3 expression, we cultured cells together or prevented their contact using a 0.4-µm Transwell filter. Shown in Figure 5A, separation of the endothelial cells and fibroblasts by a porous filter blocked the upregulation of NOTCH3, suggesting that direct cell–cell contact is critical for the inductive abilities of endothelial cells. Accordingly, endothelial cell-conditioned media did not induce expression of NOTCH3 (data not shown). In testing various pathway inhibitors that could abolish NOTCH3 induction, we observed that the -secretase inhibitor, DAPT, robustly abrogated the ability of HUVECs to increase NOTCH3 in HDFNs (Figure 5B and 5C). Similar results were also observed in HUASMCs and HCASMCs (data not shown). -Secretase facilitates Notch signaling by a cleavage event that releases the Notch intracellular domain.36 Based on this, Notch signaling could be responsible for the upregulation of NOTCH3 and further supports the notion that it could regulate itself. To address this hypothesis, we performed experiments to determine whether Notch receptor signaling via its transcriptional activity was necessary for NOTCH3 expression in fibroblasts.
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Using a dominant-negative Mastermind-like1 (DN-MAML) construct,27,37 we tested whether inhibition of this Notch coactivator would affect NOTCH3 induction by endothelial cells. The DN-MAML was introduced into fibroblasts by viral transduction, and 48 hours later, the cells were cocultured with endothelial cells or cultured by themselves as a control. Whereas the control virus harboring green fluorescent protein did not block an increase in NOTCH3, DN-MAML significantly blunted this response, exhibiting a reduced level of endothelial-induced NOTCH3 protein and RNA (Figure 5D and 5E). HEYL/HRT3 expression showed a similar profile, with attenuated expression in the presence of DN-MAML (Figure 5F). Thus, inhibition of Notch signaling at 2 independent steps within the pathway show that Notch activity is an important component in the upregulation of NOTCH3 by endothelial cells.
NOTCH3 Induction Requires JAGGED1 on Endothelial Cells
Because Notch receptor signaling and transcriptional activity were required in mural cells for the expression of NOTCH3, we reasoned that Notch ligands on endothelial cells were responsible for NOTCH3 induction by these cells. The Notch ligand JAGGED1 is strongly expressed by endothelial cells, and therefore we targeted this ligand by siRNA to determine its role in endothelial-induced NOTCH3 expression. Endothelial cells were transiently transfected with JAGGED1 siRNA and 24 hours later cocultured with fibroblasts for 48 hours. Examination of NOTCH3 expression by Western blot and qPCR revealed that knockdown of JAGGED1 in endothelial cells significantly abrogated the induction of NOTCH3 in fibroblasts (Figure 6). Consistent with this, HEYL/HRT3 RNA expression was also blocked by the loss of endothelial-expressed JAGGED1. Although endothelial-expressed JAGGED1 has previously been shown to transduce Notch signaling,16,28 our data have extended these findings by demonstrating that an increase in NOTCH3 expression is a JAGGED1-dependent consequence of vascular cell heterotypic interactions.
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NOTCH3 Promotes Its Own Expression and That of JAGGED1
Given that inhibition of Notch signaling prevents NOTCH3 induction, we next tested whether an activated form of NOTCH3 could promote its own transcription. A human NOTCH3 intracellular domain (NICD3) was introduced into fibroblasts and smooth muscle cells by viral transduction, and NOTCH3 transcript expression was examined by qPCR using primers that recognized the extracellular region of NOTCH3 to distinguish endogenous expression from the virally produced intracellular domain. Interestingly, NICD3 strongly induced NOTCH3 transcripts in fibroblasts and smooth muscle cells (supplemental Figure II). It is important to note that the infection efficiency of fibroblasts was 80% to 90%, whereas the infection percentage in both smooth muscle cell types was considerably lower (between 40% and 60%). Hence, we do not know whether the reduced induction in smooth muscle cells is a cell-specific difference or a reflection of reduced amounts of NICD3 in these cells. As expected, the expression of HEYL/HRT3 was also robustly increased by NICD3. Furthermore, consistent with previous studies,28–31 NICD3 was able to increase transcript expression of smooth muscle genes in fibroblasts and smooth muscle cells (supplemental Figure II).
Because NOTCH3 was able to autoregulate its expression, we wondered whether it also could regulate other Notch signaling mediators, particularly Notch ligands. Examination of JAGGED1 expression in cells expressing NICD3 revealed that JAGGED1 transcript and protein levels were increased in each cell type (supplemental Figure III). Moreover, like NOTCH3, JAGGED1 was also increased in mural cells cocultured with endothelial cells. We believe these results are the first to demonstrate that NOTCH3 can control its own expression, and corroborates findings showing its ability to regulate one of its ligands.38,39 To determine whether NOTCH3 regulates JAGGED1 expression in vivo, we examined Jagged1 protein levels in blood vessels of Notch3-null and heterozygous mice.26 In the mouse retina, Jagged1 is robustly expressed in pericytes and smooth muscle cells surrounding mature arteries.40 We measured Jagged1 expression by immunostaining of retinas isolated from mice at postnatal day 15. The level of Jagged1 in the retinal arteries of Notch3 null mice was significantly less compared to heterozygous animals (Figure 7), thus indicating that the absence of Notch3 in vivo results in reduced expression of the Jagged1 ligand.
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Taken together, these data strongly support a role of NOTCH3 as a conduit of smooth muscle differentiation promoted by endothelial cells. Endothelial cells, through the JAGGED1 ligand induce NOTCH3 expression, which autoregulates itself by a positive feedback loop that in turn activates smooth muscle-specific gene expression and maintains vascular support cells in a differentiated phenotype.
NOTCH3 Modulates Angiogenesis
The ability of mural cells to enhance angiogenesis likely depends on multiple factors that cooperate to facilitate blood vessel formation. Because NOTCH3 expression was strongly induced in mural cells by endothelial cells, we asked whether NOTCH3 might facilitate fibroblast-enhanced angiogenesis. To address this, we knocked down NOTCH3 expression by lentivirally transduced short hairpin RNA in fibroblasts. Angiogenesis assays were performed by coculturing endothelial cells with NOTCH3-deficient or control fibroblasts to examine potential differences in blood vessel formation (supplemental Figure IV). Interestingly, we observed a significant decrease in vessel structures in the presence of NOTCH3-deficient fibroblasts compared to control by measuring total vessel area. These results convincingly demonstrate that NOTCH3 has a role in fibroblast-mediated angiogenesis.
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Discussion |
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Endothelial/mural cell interactions are an important component of blood vessel formation and function. Their functional and physical crosstalk is evident under 3D coculture conditions in which mural cells can enhance vessel assembly, while being recruited to encase the newly formed vessels. This striking in vitro interaction prompted us to ask what genes might be regulated by their close association. In doing so, we discovered that endothelial cells can promote the expression of NOTCH3 in mural cells, and this requires direct cell–cell contact between the 2 cell types. This was an intriguing result considering what is already known about NOTCH3 in vascular smooth muscle cells. In a carotid artery balloon injury model, Notch3 was shown to be acutely downregulated in denuded smooth muscle, with expression reappearing 7 days after injury.41–43 The reduction in expression was attributed to release of PDGF-BB, which was shown to directly decrease Notch3 levels. However, in the context of our results, loss of endothelial cell contact may be the main reason for the reduction in Notch3 expression within these injured arteries. In accordance with this, Lindner et al reported that Notch3 expression was only observed where endothelial cells were still present in balloon injured vessels.42
Solid evidence has linked Notch3 to the control of smooth muscle maturation. The smooth muscle cells of the small arteries and arterioles in Notch3-null mice exhibit reduced expression of smooth muscle differentiation genes, show defects in elongation and orientation, and display impairments in arteriolization, whereas large elastic and major muscular arteries are unaffected.26 Given that endothelial cells are known to promote differentiation, our initial result hinted that endothelial-induced differentiation might be dependent on NOTCH3. Indeed, knockdown of NOTCH3 inhibited endothelial-induced smooth muscle gene expression, providing a novel mechanism by which endothelial cells regulate this process in neighboring mural cells. These data help to bridge a gap in our understanding of endothelial-induced differentiation and Notch signaling. High et al nicely showed the importance of endothelial-expressed JAGGED1 on smooth muscle differentiation,16 whereas other groups have used overexpressed Notch intracellular domains to activate smooth muscle gene expression.28–31 Here, we show that JAGGED1 specifically activates NOTCH3 in mural cells, and this activity is both necessary and sufficient for the expression of smooth muscle genes in three distinct mural cell populations. We did observe some differences in the ability of HUVECs and NOTCH3 to regulate certain smooth muscle genes in the different cells (Figure 4). In particular, HCASMCs, which showed robust basal expression of NOTCH3, appeared less responsive to endothelial-induced Notch signaling, possibly because the Notch pathway was already highly activated. Although the reasons for this are unknown, it points to additional complexities in the role of Notch and the transcriptional control of these key smooth muscle markers.
Our data show that the expression of NOTCH3 requires the activity of the Notch signaling mediators -secretase and Mastermind-like1 in mural cells. In addition, we demonstrate that JAGGED1 is necessary for NOTCH3 expression. Although Notch receptors and their ligands have been shown to use various positive and negative-feedback mechanisms to control availability and ultimately signaling,20,44,45 there are limited reports examining their effect on the expression of one another. Most described mechanisms use reciprocal regulation of a receptor/ligand pair that acts to contain and amplify the signal.21 Our results not only show that mural cell expression of NOTCH3 is dependent on endothelial-expressed JAGGED1 but also demonstrate that activated NOTCH3 can promote its own expression as well as that of its ligand JAGGED1 in the same mural cell. Consistent with this, using Notch3 knockout mice, we show that Jagged1 protein is decreased in the retinal vasculature in vivo. To our knowledge, these results represent the first direct evidence of a positive autoregulatory loop that involves the upregulation of a Notch receptor/ligand combination in the same cell. Although NOTCH3 expression exists in an autoregulatory loop, our data also show that endothelial cells may use mural cell–expressed NOTCH3 to modulate their own function. Ablation of NOTCH3 in fibroblasts results in a decrease in their proangiogenic abilities, which suggests that endothelial cells promote NOTCH3 expression as a means to facilitate their vessel-forming potential.
The ability of NOTCH3 to regulate its own expression and that of JAGGED1 offers evidence for a model in which endothelial cells initiate differentiation of mural cells that can maintain or amplify this signal through a positive feedback loop. As shown in Figure 8, the physical association of endothelial cells and mural cells allows for Notch signaling to be initiated by a JAGGED1/NOTCH3 interaction. In mural cells, this augments smooth muscle gene transcription and causes an increase in NOTCH3 and JAGGED1 expression, leading to more NOTCH3 receptors on the cell surface, as well as JAGGED1 ligands. With an increase in both the receptor and ligand, mural cells can interact more efficiently with endothelial cells or can activate Notch signaling through homotypic interactions of neighboring mural cells presenting NOTCH3 and JAGGED1. This model suggests that once endothelial cells initiate the cascade, mural cells are then able to maintain the signal and distribute it to other mural cells through continued activation and expression of NOTCH3. Whether the continued presence of endothelial cells is required and if "activated" mural cells are able to pass on this Notch signal remains to be determined. Irrespective of these possibilities, our data conclusively show the importance of Notch signaling in the interactions of these 2 cell types and demonstrate 1 mechanism through which these cells likely communicate with each other within the vasculature.
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Acknowledgments |
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We are grateful to Wenbo Zhang and Irfan Ali for critical reading of the manuscript. The support of the Metabolic Vascular Disease Group of the Vascular Biology Center is greatly appreciated.
Sources of Funding
This work was supported by NIH grant R01 HL076428 (to B.L.).
Disclosures
None.
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Footnotes |
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Original received August 7, 2008; revision received December 12, 2008; accepted January 6, 2009.
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References |
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  J. N. Regan and M. W. Majesky Building a Vessel Wall With Notch Signaling Circ. Res., February 27, 2009; 104(4): 419 - 421. [Full Text] [PDF]
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