Brain arteriovenous malformations (BAVM)s are characterized by a nidus of coiled and tortuous and enlarged vascular lesions that shunt blood directly from feeding arteries to veins1
. They often rupture, resulting in hemorrhagic stroke in young people, most commonly between 20 – 40 years of age1
. BAVMs contribute to half of the hemorrhagic stroke in children2
, and 2% of all stroke1
. Currently, surgical resection is the primary treatment, but the efficacy is questionable3
. Most BAVMs are sporadic, making it difficult to identify the molecular cause by genetic association1
. To date, the cellular and molecular basis for BAVM pathogenesis remains largely unknown. This limited knowledge of BAVM etiology has impeded the rational design of molecular interventions.
Fundamentally, AVMs are a disruption of normal arteriovenous (AV) hierarchy, which was historically thought to be governed by hemodynamic forces4
. The discovery of genes with arterial or venous specific expression in the developing mouse embryo has catalyzed advances in our understanding of the genetic control of AV specification and the establishment of AV hierarchy5
. Notch, a transmembrane receptor first identified in fruit-fly, and involved in cell fate determination and tissue patterning throughout metazoans, has emerged as a critical mediator of AV differentiation6
. Studies in zebrafish and mouse development demonstrated that Notch signaling was necessary and sufficient for the expression of arterial-specific genes6, 7
. Furthermore, we have demonstrated that endothelial Notch signaling regulates the luminal size of developing mouse arteries by promoting of arterial specification, and increasing the arterial allocation of endothelial cells8
. Abnormal Notch signaling induced enlarged AV connections and shunting in both mouse and zebrafish embryo, suggesting a link between AV specification and arteriovenous malformations (AVMs)6, 7
Among the four mammalian Notch receptors and five ligands, Notch receptors 1 and 4 and their ligands Dll1, Dll4, and Jag1 are preferentially expressed in the arterial and not venous endothelium9
. Cell-cell mediated activation of the Notch receptor, by ligand binding to the extracellular domain, results in sequential cleavage events and release of an active intracellular domain (ICD)10
. Once cleaved, the ICD translocates to the nucleus, where it must form a complex with the sequence-specific DNA binding protein Rbpj to promote the transcription of downstream genes10
. Transcription factors of the Hairy/Enhancer of Split and Hes-related families of proteins, such as Hes1, are canonical target genes, and mediate many of Notch’s downstream functions10
. Therefore, Notch-ICD is a constitutively-active mutant. Likewise, the Notch4 mutant that lacks the extracellular domain is constitutively cleaved and constitutively-activated (Notch4*)11
. Thus, nuclear localization of cleaved Notch-ICD and expression of Hes1 are features of active Notch signaling.
To investigate whether upregulation of endothelial Notch signaling can disrupt AV hierarchy and cause AVMs in adult mice, we used a tetracycline-regulated transgenic system to express Notch4* transgene specifically in the endothelium of adult mice (Notch4*-Tet
), and reported AVMs in liver, skin and uterus12
. Expression of the transgene in immature Notch4*-Tet
mice, during post-natal brain growth, resulted in hallmarks of BAVM in all mice, including enlarged and tortuous AV connections, shunting and hemorrhagic stroke13
. In both adult and immature Notch4*-Tet
mice, the disease progression was reversed when the Notch4* transgene was turned off, demonstrating that Notch4* is critical to sustain the disease12, 13
. The urgent question that arose out of this fundamental research is whether increased Notch signaling underlies the development and maintenance of human BAVM.
Notch loss-of-function mutations in Jag1, Notch3 and Notch1 are known to cause Alagille syndrome14
, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)14
, and aortic valve anomalies15
respectively, but it is not clear whether Notch signaling is involved in human BAVM pathogenesis. In this study, we test the hypothesis that Notch signaling is upregulated in human BAVMs by examining Notch signaling activity in the endothelium of human BAVM relative to autopsy and surgical biopsy controls. We demonstrate increased levels of the activated-Notch1 receptor and canonical Notch target Hes1 in BAVM tissue. We reveal similar increases in our Notch4*-Tet
mouse model of BAVM-like abnormalities. Our work puts forward the hypothesis that Notch activation causes and maintains human BAVM, and provides molecular validation of our Notch4*-Tet
model of BAVM as valuable system to dissect the molecular and cellular basis of BAVM pathogenesis.