A Novel Mechanism of Vascular Smooth Muscle Cell Regulation by Notch
Platelet-Derived Growth Factor Receptor-β Expression?
See related article, pages 1483–1491
The Notch family of receptors, Notch1 to -4, are heterodimer transmembrane proteins, consisting of an extracellular domain and a noncovalently linked intracellular domain (ICD). Upon interaction with the DSL family of proteins (Jagged, Delta-like) on neighboring cells, Notch undergoes proteolytic cleavage, which frees the ICD from the plasma membrane. This results in translocation of the ICD into the nucleus, where it forms a complex with the CSL family of transcriptional repressors (CBF1/RBP-Jk), removing the repression and allowing for target gene (Hes, Hey) transcription.1,2
Tissue distribution of the Notch proteins varies widely. Notch1 and -4 are predominantly endothelial, prominent in both arteries and veins, and present in all stages of development (embryonic to adult); the expression of Notch2 is typically confined to pulmonary endothelium, but Notch3 is primarily expressed in adult arterial vascular smooth muscle cells (VSMCs) in large conduit, pulmonary, and systemic resistance arteries.3 This specific pattern of temporal and spatial distribution correlate to diverse functions of the Notch family in vascular development and physiology in vertebrates reported to date.4
In the cardiovascular system, Notch signaling plays a role in several aspects of vascular development, including vasculogenesis, angiogenesis, differentiation, vascular remodeling, and VSMC maturation. Notch1 and -4 signaling appears critical in vasculogenesis and angiogenesis during early development, when it interacts with vascular endothelial growth factor signaling to specify artery–vein differentiation of endothelial cells (ECs). Transgenic mice deficient for Notch1 fail to undergo embryonic angiogenic remodeling, and vascular development is arrested at the primitive undifferentiated plexus, resulting in an embryonic lethal phenotype. Notch2-null mutations likewise result in embryonic lethality characterized by multiple large and small vessel aneurysms. Notch4 deletion results in no obvious phenotypic alteration. Notch3−/− mice are viable; however, they fail to develop proper arterial VSMC phenotype, resulting in vein-like arterial walls and poor autoregulation of blood pressure.5 Although multiple studies have addressed the critical role for Notch signaling in vascular remodeling and cell fate determination during development, much less is known concerning the role for Notch in adults and/or VSMC physiology.1,2,4
Furthermore, controversy exists regarding the effect of Notch signaling on VSMC phenotype. Several in vivo studies have reported that Notch signaling promoted VSMC differentiation.1 Shear stress upregulation of differentiation-specific VSMC markers is associated with increased Notch1 and -3 expression. The opposite has been reported in vitro.6,7 Additional cell culture studies offer further conflicting findings, because Notch family members have been reported to either promote or inhibit cell migration and proliferation.1,2 These disparate findings may be related to cell-specific expression and temporal regulation of Notch isoforms, as well as to the significance of complex cell–cell interactions in vivo in the modulation of Notch signaling. Notch3 appears to be specifically expressed in healthy adult arterial VSMCs, with a marked lack of expression in the venous vasculature. Notch3−/− mice, unlike Notch1−/− and Notch2−/− mice, do not exhibit gross vascular abnormalities during embryonic development. Instead, their arteries have normal endothelium but fail to mature properly postnatally, resulting in thinner arterial walls lacking the arterial-specific VSMC marker smoothelin, proper dense bodies, and VSMC orientation, particularly in the resistance arteries. This is consistent with arterial VSMC-specific expression of Notch3.5
Several observations suggest that temporal regulation of Notch signaling may be equally important. All 4 Notch isoforms were observed in VSMCs after vascular injury. Interestingly, several studies reported downregulation of all members of the Notch signaling pathway, including Notch3, 2 days after vascular injury, but an upregulation 7 to 14 days postinjury. The possible interaction between platelet-derived growth factor (PDGF)-BB and Notch signaling is particularly interesting in this setting. Although both Notch and PDGF expression are upregulated in response to injury, it is well known that PDGF induces VSMC dedifferentiation and loss of contractile phenotype, but Notch3 appears to promote it.2 Because the early vascular response to injury involves VSMC dedifferentiation and acquisition of a proliferative and migratory phenotype, and VSMC death is prominent in later phases of injury-induced remodeling, these observations are consistent with the critical role for temporally specific Notch3 signaling in promotion and/or maintenance of vascular cell phenotypic stability (differentiated phenotype), as well as with the requirement for Notch3 in VSMC survival (Akt, extracellular signal-regulated kinase [ERK]1/2 activation) reported previously.8
Whereas a body of evidence has clearly established a role for Notch1 and -4 in the determination of cell fate during development, and emerging studies have identified Notch3 as an important regulator of VSMC phenotype, little is known about the mechanism by which Notch3 regulates this in adult arterial VSMCs. The expression of canonical downstream targets of Notch3 (Hes, Hey) is not altered in Notch3−/− mice, suggesting that Notch3-mediated regulation of the differentiated VSMC phenotype occurs via alternate signaling pathways.1 For example, Morrow et al reported that cyclic strain-induced Notch3 downregulation was Gi- and ERK1/2-dependent.6
Alterations in Notch3 signaling have clinical implications. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult-onset disease characterized by progressive arterial degeneration, especially of cerebral vasculature, linked to a variety of mutations in the Notch3 allele. Although crucial to VSMC regulation, surprisingly, Notch3 mutations associated with CADASIL have failed to alter the classic Notch-mediated signaling (CSL).9 This observation further points to crosstalk between Notch and other signaling involved in VSMC regulation or the existence of yet undefined pathways activated by Notch.
In this issue of Circulation Research, Jin et al10 propose that Notch signaling in VSMCs is linked to PDGF signaling. This is a logical interplay because PDGF, which acts via binding to the membrane-bound tyrosine kinase PDGF receptor (PDGFR), is a well-established mediator of vascular development, VSMC phenotype, and blood vessel homeostasis. Using the expression of the PDGFR-β as a readout, Jin et al completed a well-executed series of experiments that confirmed that PDGFR-β was a direct target of activated Notch1 and Notch3 receptors. Mechanistic investigation of Notch-mediated upregulation of PDGFR-β expression revealed that Notch1 and Notch3 ICDs bind alternative CSL sites in the PDGFR-β promoter, suggesting distinct mechanisms of activation. In further support of the pathway linking Notch to PDGF signaling, transfection of Notch1 or Notch3 ICDs into primary human VSMCs resulted in potentiated PDGF-induced activation of ERK1/2 and Akt. This indicated that Notch-induced PDGFR-β upregulation translates to a functional increase in intracellular responses consistent with increased VSMC growth and migration. Agonist stimulation with PDGF-BB decreased the expression of both Notch3 and PDGFR-β, with this effect on receptor expression potentially a result of Notch3 downregulation. This is consistent with previous studies and may be indicative of negative feedback between Notch3 and PDGF signaling because the overexpression of either Notch1 or 3-ICD attenuated PDGF-induced VSMC migration. To translate this novel pathway to in vivo studies, Jin et al used a Notch3-deficient mouse in which both alleles of the Notch3 gene were interrupted by lacZ insertion. The expression pattern of Notch3 and PDGFR-β were assessed in tail artery and vein in newborn mice, revealing significant overlap between β-galactosidase, PDGFR-β, and smooth muscle actin suggestive of VSMC-specific localization of Notch3 and PDGFR-β. Importantly, PDGFR-β expression was significantly attenuated in Notch3-deficient mice, corroborating with cell culture data that established Notch-dependent regulation of PDGFR-β expression in VSMCs. Because PDGFR-β expression has been reported to be upregulated in mice deficient for endothelial Notch1, the present findings further implicate the importance of cell-specific Notch isoform expression and signaling. Because missence mutations or deletions in NOTCH3 receptor are associated with CADASIL, VSMCs from a patient carrying a mutation at position 133 (R133C) were used to assess the Notch–PDGFR signaling interaction. Compared with VSMCs isolated from healthy non-CADASIL patients, PDGFR-β expression in response to stimulation with Jagged1 was significantly attenuated in VSMCs from the CADASIL patient, as was HEY1 and HES1 expression. The implications of these key findings suggest that Notch-mediated activation of PDGFR-β may be a novel mechanism worthy of consideration as a contributor to underlying vascular degeneration associated with CADASIL.
Although these studies clearly advance our understanding of Notch signaling in VSMCs, they also raise several provocative questions. First, they indicate the necessity for further study of Notch3 localization in adult vasculature, because previous studies report Notch3 to be specifically localized to arteries,5 but Jin et al10 observe expression in both the tail artery and vein. Second, the relevance of investigating Notch3 signaling in large conduit vessels (tail artery) may have limited clinical applicability to CADASIL, because CADASIL is mainly an arteriopathy of resistance arteries.5 Interestingly, the experimental approach by Jin et al, in which either the Notch1 or -3 ICD was transfected into cultured VSMCs and resulted in similar signaling profiles, suggests a potential redundancy between the Notch isoforms in VSMCs. This could be of importance, for instance, in vascular remodeling following injury, where all Notch isoforms are expressed in VSMCs. However, these results should be interpreted somewhat cautiously because cell culture experiments do not allow for consideration of cell–cell interactions. Furthermore, in the present study, Jin et al examined smooth muscle actin expression in Notch3−/− animals only on postnatal day 0 to localize Notch3-dependent PDGFR-β expression to VSMCs. If Notch3-mediated regulation of the PDGFR-β is a key mechanism underlying progressive vascular degeneration, such as occurs in CADISIL, it will be crucial to investigate the influence of this mechanism on VSMC differentiation state at several time points during postnatal vascular development. Addressing this would allow for assessment of the potential influences of healthy endothelium and endothelial-specific Notch isoforms on VSMC phenotype. Lastly, Jin et al make an interesting and provocative observation that whereas Notch3 is required for PDGFR-β expression, PDGF-BB downregulates both Notch3 and its own receptor. It is tempting to speculate that a potential function of Notch3 in postnatal vascular maturation and preservation may be to temper the promigratory and antidifferentiation effect of PDGF-BB. This stresses the importance of the interaction between PDGF and Notch signaling in postnatal development and arterial maturation, which is further emphasized by the lack of an effect of PDGF-BB deletion on Notch3 expression in mouse embryos.11
In summary, this thought-provoking study by Jin et al10 clearly identifies a novel mechanism by which Notch3 participates in the regulation of adult VSMC phenotype and function, while simultaneously identifying several important questions. Future answers to these questions will extend our understanding of both Notch signaling and the mechanistic basis of clinical pathologies associated with its disruption, including CADASIL and repair following vascular injury.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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