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The way in which multiple cell types organize themselves into a carefully sculpted, 3D labyrinth of vessels that regulate blood flow throughout the body has been a longstanding mystery. Clinicians familiar with congenital cardiovascular disease recognize how genetic variants and modest perturbations in this complex set of spatiotemporal interactions and stochastic processes can result in life-threatening anomalies. Although the mystery is not yet fully solved, we are poised at an exciting juncture, as insights from murine disease models are converging with advances in human genetics to shed new light on puzzling clinical phenotypes of vascular disease. The study by High et al. in this issue of the JCI establishes a model system that mimics clinical features of congenital cardiovascular disease and further defines the role of the Notch signaling pathway in the neural crest as an essential determinant of cardiovascular structure (see the related article beginning on page 353).
In the embryo, endothelial precursors initially assemble into a primitive plexus of channels that expand by sprouting and remodeling into a highly organized arborization of vessels that ramify throughout the body. Studies in various knockout mouse models suggest that VEGF, angiopoietins, and PDGF provide local cues that promote vascular morphogenesis and the investment of the endothelial channels with a VSMC layer. The architecture of the vascular tree is further refined to very precise dimensions in accordance with biomechanical parameters of shear and radial stresses (1, 2).
In a classic series of seminal studies involving quail-chick chimeras and tissue ablation experiments, Kirby and colleagues established that the cardiac neural crest plays an essential role in establishing the pattern of the vertebrate vascular system (3). The molecular mechanisms underlying these phenomena are gradually being elucidated (Table (Table1).1). The cardiac neural crest (an ectodermal cell population arising from the dorsal neural tube) migrates to populate the aortic arch arteries and cardiac outflow tract. The migrating cardiac neural crest cells contribute to the septation of the truncus arteriosus into a separate pulmonary artery and aorta. Similarly, a subpopulation of these neural crest cells becomes part of the mass of VSMCs that contribute to the formation of the pulmonary trunk, ductus arteriosus, carotid arteries, and proximal subclavian arteries (3–5). The study reported by High et al. in this issue of the JCI (6) provides the first demonstration to our knowledge that the Notch transcriptional cascade within the neural crest plays an essential mediator role in VSMC differentiation during the formation of the aortic arch and pharyngeal arteries.
The 4 mammalian Notch receptors (Notch 1–4) and 5 ligands (Jagged1 and -2; Delta-like1, -3, and -4) all contain transmembrane domains such that ligand-receptor signaling occurs between adjacent cells (Figure (Figure1).1). The ligand-receptor binding triggers a γ-secretase–dependent cleavage that releases the intracellular domain of Notch to the nucleus and facilitates an association with the transcription factor CBF-1 (also known as RBP-Jκ or CSL). The subsequent recruitment of the coactivator, mastermind-like (MAML) protein, promotes transcriptional activation of downstream effectors such as hairy and enhancer of split (HES) and the HES-related repressor protein (HERP) family of transcription factors (7). Given that MAML is a common signaling node employed by all Notch receptors, High and colleagues’ use of the dominant-negative MAML construct (6) is an effective strategy for inducing broad-based inhibition of the Notch transcriptional cascade.
In murine models, selective knockout of the ligands Jagged1 and Delta-like4 or the receptor Notch1 results in striking abnormalities in vascular development (8). Similarly, the combined deletion of the downstream Notch effectors Herp1 and Herp2 leads to similar vascular malformations. Thus, there is compelling evidence that the Notch signaling pathway plays a key mediator role in vascular morphogenesis (4, 7). However, these previous studies failed to isolate and clarify the specific role of Notch signaling in VSMC differentiation. By focusing on neural crest–derived VSMCs, the study by High and colleagues is among the first that addresses the direct role of Notch in VSMC differentiation in vivo (6).
As progress is made in the dissection of the gene regulatory networks that govern vascular morphogenesis, it is important to translate these insights into an understanding of genetic factors that increase susceptibility to clinical phenotypes of vascular malformation. DiGeorge syndrome is a clinically heterogeneous disease caused by a multigene deletion on chromosome 22q11 that is manifested by cardiovascular anomalies reminiscent of neural crest ablation such as tetralogy of Fallot and interrupted aortic arch. A systematic series of complementation experiments in mice have characterized a T-box transcription factor, T-box 1 (Tbx1), as a major mediator in the neural crest during cardiovascular development (9). Indeed, patients that lack the chromosome 22q11 deletion, yet have mutations in the TBX1 gene, retain features of DiGeorge syndrome (10, 11). Thus, an important paradigm of this unfolding mystery is that highly penetrant clinical phenotypes appear to emerge in the context of mutations of factors (e.g., TBX1) that play a central role in the vascular development gene regulatory network.
Alagille syndrome is a heritable multisystem disease that is clinically characterized by bile duct insufficiency and cardiovascular manifestations that are reminiscent of other neural crest deficiency phenotypes (e.g., pulmonary stenosis, tetralogy of Fallot, and coarctation of the aorta). In accordance with the emerging paradigm, the vast majority of patients with Alagille syndrome have mutations in the Notch ligand Jagged1 (7, 11). Moreover, a recent report suggests that the small percentage of Alagille patients that are negative for Jagged1 mutations have significant mutations in the gene coding for Notch2 (12). Thus, the animal model of neural crest–selective Notch inactivation demonstrated by High et al. (6) appears to translate to the clinical context with high fidelity.
Although the study by High et al. (6) focuses on the role of the Notch pathway in cardiovascular development, the implications of this model extend beyond the realm of congenital heart disease to the context of vascular disease in adulthood. Unlike skeletal and cardiac muscle cells, VSMCs are not terminally differentiated and exhibit substantial plasticity in phenotypic modulation within adult vessels. An emerging pathobiological paradigm suggests that the vascular development gene regulatory network is often reactivated in the context of vascular remodeling and repair in adult vascular disease (2).
Studies in our laboratories and by others have demonstrated that elements of the Notch transcriptional cascade are activated in the context of vascular lesion formation and that the Notch pathway is coupled to the regulation of growth, apoptosis, migration, and differentiation of adult VSMCs (2, 13–15). The essential mediator role of the Notch pathway in vascular disease is evidenced by the finding that Herp2-knockout mice exhibit attenuated neointima lesion formation in response to vascular injury (15).
The role of the Notch pathway in adult vascular disease is also indicated by the observation that mutations in the gene coding for Notch3 results in CADASIL syndrome, an autosomal-dominant arteriopathy characterized by increased susceptibility to stroke and dementia in middle-age adults (16). Moreover, recent animal studies suggest that the Notch signaling cascade is activated during ischemic stroke, such that the inhibition of Notch signaling attenuates brain injury after stroke (17). It is exciting to consider the potential clinical utility of selective inhibition of the Notch pathway to ameliorate the course of adult vascular disease.
The study by High et al. (6) provides compelling evidence that neural crest–directed blockade of Notch signaling with a dominant-negative MAML gene construct inhibits VSMC differentiation and results in aortic patterning defects reminiscent of clinical phenotypes. However, a limitation of this approach is the potential for forced expression of dominant-negative constructs to yield spurious results due to “off-target” effects that may extend beyond the Notch pathway. In fact, recent reports indicate that MAML can also act as a coactivator of myocyte enhancer factor 2C (MEF2C) (18), a well-established mediator of cardiovascular morphogenesis. It remains an open question which additional pathways are engaged in parallel to Notch and what downstream effectors mediate the direct coupling of Notch signaling to VSMC differentiation programs. In addition, the current model fails to reconcile the conflicting data derived from in vitro models that suggest that Notch signaling may inhibit myocardin-induced VSMC differentiation (19, 20). Unfortunately, a common limitation of these in vitro studies is the failure to capture the subtle physiologic interplay among epigenetics, coactivator/corepressor complexes that exist in vivo, and the combinatorial effect of several downstream Notch effectors working in concert to orchestrate the VSMC differentiation program. It is becoming clear that the capacity of Notch to elicit lineage specification in a wide spectrum of developmental processes reflects the exquisite sensitivity of the pathway to the local milieu and the combinatorial interplay with other elements of the gene regulatory network peculiar to a given cellular context. The in vivo model used by High and colleagues captures the complex nuances of these gene regulatory circuits and provides a way forward toward the identification of novel downstream mediators of the Notch pathway in VSMC differentiation.
It is anticipated that the growing application of genomic approaches to define signature patterns in gene expression profiles during lineage commitment will lead to the discovery of new members of the vascular development gene regulatory network, advancing our understanding of human disease (21). This convergence of genomic strategies is exemplified by the recent discovery that mutations in the TGF-β signaling pathway (a key mediator in the vascular development gene circuitry) result in a newly defined form of aortic disease (Loeys-Dietz syndrome) (22), and may foster a novel therapeutic strategy for adult vascular disease (23). Likewise, the growing integration of systems biology approaches (21) into the analysis of cardiovascular development holds promise for unlocking the remaining mysteries of the complex gene regulation circuitry governing vascular morphogenesis.
Nonstandard abbreviations used: HERP, HES-related repressor protein; HES, hairy and enhancer of split; MAML, mastermind-like; Tbx1, T-box 1.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J. Clin. Invest. 117:299–302 (2007). doi:10.1172/JCI31288.
See the related article beginning on page 353.