In this study, we found that NICD, the active form of Notch-1, is present in both endothelial and smooth muscle cells of human brain AVMs, but not in control human cerebral blood vessels. The Notch-1 ligands Jagged-1 and Dll4, and the downstream Notch-1 target (Hes-1), were also increased, suggesting that Notch-1 signalling is activated in human brain AVMs. This observation is consistent with the previously demonstrated role of Notch signalling in the developing circulation (Weinmaster and Kopan, 2006
; Gridley, 2007
) We also found that continuous intraventricular administration of a Notch-1 activator in normal rats stimulates the proliferation of both endothelial and vascular smooth muscle cells, suggesting that enhanced Notch-1 signalling can independently induce a pro-angiogenic state. However, it remains unclear whether more prolonged administration, or higher doses, of a Notch-1 activator would lead to the formation of arteriovenous shunts or vascular malformations. Our data imply that the enhanced Notch signalling in vascular malformations may have effects not only during development, but also in post-natal brain.
The significance of this work is several-fold. First, although the activation of Notch-1 signalling in surgical specimens cannot address the necessity or sufficiency of this pathway in the aetiology or pathogenesis of brain AVMs, it suggests that this pathway is involved at some stage of the natural history. The functional studies in rats are important in this regard because they establish that, in post-natal brain, there is a pro-angiogenic effect of Notch-1 signalling. Second, even if Notch signalling is not the primary cause of AVM development, its involvement at any stage of disease progression makes it a plausible target for therapy development. Other than surgical extirpation, endovascular embolization and radiotherapy, there is no treatment to prevent bleeding from cerebral AVMs, and each of these modalities carries the risk of disability or death. Approximately 20% of AVM patients cannot be offered these options because of excessive risk (Choi and Mohr, 2005
The genesis of AVMs has been enigmatic, and we cannot determine from our findings whether Notch-1 signalling is necessary or sufficient for brain AVMs to occur. Brain AVMs may form independently of Notch-1 pathways, and haemodynamic stresses caused by high flow arteriovenous shunting may secondarily activate biomechanical responses that involve Notch-1 activation. Unlike the association of antecedent head trauma or other injuries with the pathogenesis of dural arteriovenous fistulae, overt environmental risk factors for AVMs are unknown. Considering the high utilization of pre-natal ultrasound, there is remarkably little evidence for the common belief that true AVMs—which do not include Vein of Galen lesions—arise during embryonic development. In fact, the mean age at detection is roughly 40 years, with a normal distribution (Kim et al.
). Furthermore, there have been multiple reports of AVMs that grow or regress, as well as of local re-growth of AVMs after treatment (Du et al.
). The scarce data available on longitudinal assessment of AVM growth after detection suggest that ~50% of cases display interval growth (Hashimoto et al.
), but the relationship of such growth to clinical consequences remains unknown. Since post-natal growth of AVMs occurs, one plausible aim for therapy might be to reduce the rate of growth over time.
The Notch signalling pathway is activated through direct cell–cell interactions that facilitate binding between Notch ligands (Dll1, Dll3, Dll4, Jagged-1 and Jagged-2) on a signalling cell and the Notch receptor on a responding cell (Lai, 2004
). Signals exchanged between these cells can amplify and consolidate molecular changes that eventually dictate cell fate. The importance of this signalling pathway is demonstrated by the fact that targeted disruption of Notch ligands in mice results in embryonic lethality with vascular defects (Xue et al.
; Gale et al.
). The importance of Jagged-1 in human disease is also suggested by its role in Alagille syndrome, a congenital disorder linked to mutations in the Jagged-1 gene (Oda et al.
). Additionally, Dll4 has emerged as the critical ligand in Notch signalling-mediated vascular malformations in mice (Gale et al.
; Gridley, 2007
). Transgenic mice that conditionally overexpress Dll4 exhibit profound abnormalities in the developing vasculature, including fusion of large arteries and veins, enlargement of arteries with excess fibronectin accumulation and decreased vessel branching (Trindade et al.
). Similarly, vascular malformations in Notch4
-overexpressing transgenic mice are reversible if expression of an activated Notch4
transgene is repressed (Carlson et al.
Notch signalling in adult endothelium is sufficient to confer arterial characteristics and lead to vascular malformations, since endothelial expression of constitutively active Notch-4 and Notch-1 causes hepatic (Carlson et al.
; Murphy et al.
) and cerebral (Murphy et al.
) vascular malformations. These findings suggest that brain vascular malformations might be treatable with Notch inhibitors. Our data indicate that both Jagged-1 and Dll4 are prominently expressed in the vasculature, and that Dll4 expression is increased in brain AVMs, suggesting that Dll4 may be a major ligand involved in Notch-1 signalling in human brain AVMs. Our finding that continuous intraventricular infusion of a Notch-1 activator in rat brain promotes angiogenesis further suggests such a link.
Upon ligand binding, Notch is proteolytically cleaved by γ-secretase to release NICD, which translocates to the nucleus, where it binds to the transcription factor CSL (Core binding factor-1 in humans, Suppressor of Hairless in Drosophila
, LAG in Caenorhabditis elegans
) (Kato et al.
). The NICD/CSL interaction converts CSL from a transcriptional repressor to a transcriptional activator by displacing the co-repressor complex and recruiting co-activators, which regulate expression of Notch target genes (Artavanis-Tsakonas et al.
). The most widely accepted Notch/CSL targets are members of the Hes
(Kageyama and Ohtsuka, 1999
) and HRT
(Nakagawa et al.
) gene families. Although there are seven Hes and HRT
genes, not all are clear Notch targets. As observed for Notch receptor- or ligand-deficient embryos, embryos deficient in Notch downstream signalling targets also display vascular defects. For example, RBP-Jk
embryos fail to express several artery-specific endothelial cell markers and exhibit vascular defects (Krebs et al.
). Both Hey1
knockout mice display a lethal vascular defect and recapitulate most of the known cardiovascular phenotypes of disrupted Notch pathway mutants, including defects in arteriovenous specification, septation and cushion formation (Fischer et al.
; Kokubo et al.
). Our data show that expression of Hes1 protein is increased in the nucleus of both endothelial and smooth muscle cells of human cerebral AVMs, suggesting that Hes-1 is a downstream target of Notch-1 signalling in this setting. Whether other Notch target genes are activated in human brain AVMs remains to be explored.
Taken together with previous observations, our findings that Notch-1 signalling is activated in human brain AVMs and that Notch-1 signalling may promote abnormal angiogenesis suggest that aberrant Notch-1 signalling may have a role in the pathogenesis of brain AVMs. Further study is essential to determine if this is the case. Another important area for future work will be to examine the overlap between Notch signalling and tumour growth factor (TGF)-β signalling in the context of brain AVMs. Hereditary haemorrhagic telangiectasia (Osler–Rendu–Weber disease), which is associated with AVMs in brain and other solid organs, is most commonly caused by loss-of-function mutations in endoglin or in activin A receptor-like kinase, both of which are involved in TGF-β signalling (Azuma, 2000).