Endothelial Notch4 signaling induces hallmarks of brain arteriovenous malformations in mice

Pacific Vascular Research Laboratory, Division of Vascular Surgery, Department of Surgery, University of California, San Francisco, CA 94143, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 09/2008; 105(31):10901-6. DOI: 10.1073/pnas.0802743105
Source: PubMed


Brain arteriovenous malformations (BAVMs) can cause devastating stroke in young people and contribute to half of all hemorrhagic stroke in children. Unfortunately, the pathogenesis of BAVMs is unknown. In this article we show that activation of Notch signaling in the endothelium during brain development causes BAVM in mice. We turned on constitutively active Notch4 (int3) expression in endothelial cells from birth by using the tetracycline-regulatable system. All mutants developed hallmarks of BAVMs, including cerebral arteriovenous shunting and vessel enlargement, by 3 weeks of age and died by 5 weeks of age. Twenty-five percent of the mutants showed signs of neurological dysfunction, including ataxia and seizure. Affected mice exhibited hemorrhage and neuronal cell death within the cerebral cortex and cerebellum. Strikingly, int3 repression resolved ataxia and reversed the disease progression, demonstrating that int3 is not only sufficient to induce, but also required to sustain the disease. We show that int3 expression results in widespread enlargement of the microvasculature, which coincided with a reduction in capillary density, linking vessel enlargement to Notch's known function of inhibiting vessel sprouting. Our data suggest that the Notch pathway is a molecular regulator of BAVM pathogenesis in mice, and offer hope that their regression might be possible by targeting the causal molecular lesion.

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    • "The molecular identity of AVMs is unknown and, given that different genetic lesions can lead to AVMs, the expression of AV markers is likely to differ among AVMs. Notch is a crucial regulator of AV specification and is both necessary and sufficient to promote arterial and repress venous EC identity (Krebs et al., 2004; Murphy et al., 2012, 2008). Notch activity positively regulates arterial Efnb2 and negatively regulates venous Ephb4 in zebrafish (Lawson et al., 2001; Thurston and Yancopoulos, 2001; Zhong et al., 2001) and mouse (Kim et al., 2008) embryos. "
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    ABSTRACT: Arteriovenous malformations (AVMs) are tortuous vessels characterized by arteriovenous (AV) shunts, which displace capillaries and shunt blood directly from artery to vein. Notch signaling regulates embryonic AV specification by promoting arterial, as opposed to venous, endothelial cell (EC) fate. To understand the essential role of endothelial Notch signaling in postnatal AV organization, we used inducible Cre-loxP recombination to delete Rbpj, a mediator of canonical Notch signaling, from postnatal ECs in mice. Deletion of endothelial Rbpj from birth resulted in features of AVMs by P14, including abnormal AV shunting and tortuous vessels in the brain, intestine and heart. We further analyzed brain AVMs, as they pose particular health risks. Consistent with AVM pathology, we found cerebral hemorrhage, hypoxia and necrosis, and neurological deficits. AV shunts originated from capillaries (and possibly venules), with the earliest detectable morphological abnormalities in AV connections by P8. Prior to AV shunt formation, alterations in EC gene expression were detected, including decreased Efnb2 and increased Pai1, which encodes a downstream effector of TGFβ signaling. After AV shunts had formed, whole-mount immunostaining showed decreased Efnb2 and increased Ephb4 expression within AV shunts, suggesting that ECs were reprogrammed from arterial to venous identity. Deletion of Rbpj from adult ECs led to tortuosities in gastrointestinal, uterine and skin vascular beds, but had mild effects in the brain. Our results demonstrate a temporal requirement for Rbpj in postnatal ECs to maintain proper artery, capillary and vein organization and to prevent abnormal AV shunting and AVM pathogenesis.
    Full-text · Article · Sep 2014 · Development
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    • "In cultured cells, NOTCH4 is reported to signal in response to ligand [38] [39] but little else is known about its mechanism of signal transduction. Studies in mice show that endothelial specific expression of constitutively active NOTCH4 (encoded by the int3 allele) causes dramatic vascular changes including lack of small branched vessels, loss of vessel integrity [40] [41] and arterial shunts [42] [43]. Induction of int3 expression in mature vessel endothelium (postnatally), causes expression of the Notch target gene EphrinB2 and increased smooth muscle layers, resulting in arterialisation of venous vessels [40]. "
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    ABSTRACT: Notch4 is a divergent member of the Notch family of receptors that is primarily expressed in the vasculature. Its expression implies an important role for Notch4 in the vasculature; however, mice homozygous for the Notch4(d1) knockout allele are viable. Since little is known about the role of Notch4 in the vasculature and how it functions, we further investigated Notch4 in mice and in cultured cells. We and found that the Notch4(d1) allele is not null as it expresses a truncated transcript encoding most of the NOTCH4 extracellular domain. In cultured cells, NOTCH4 did not signal in response to ligand. Moreover, NOTCH4 inhibited signalling from the NOTCH1 receptor. This is the first report of cis-inhibition of signalling by another Notch receptor. The NOTCH4 extracellular domain also inhibits NOTCH1 signalling when expressed in cis, raising the possibility that reported Notch4 phenotypes may not be due to loss of NOTCH4 function. To better address the role of NOTCH4 in vivo, we generated a Notch4 null mouse in which the entire coding region was deleted. Notch4 null mice exhibited slightly delayed vessel growth in the retina, consistent with our novel finding that NOTCH4 protein is expressed in the newly formed vasculature. These findings indicate a role of NOTCH4 in fine-tuning the forming vascular plexus.
    Preview · Article · Mar 2014 · Biochimica et Biophysica Acta
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    • "These mice show abnormal connections between arteries and veins associated with ectopic expression of the arterial marker ephrin B2 in veins. Activation of constitutively active Notch4 in the blood vessels of the developing mouse brain induces vessel enlargement followed by hemorrhages in the cerebellum and the neocortex, neurological damage and death [86]. "
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    ABSTRACT: The blood–brain barrier (BBB) is a complex physiological structure formed by the blood vessels of the central nervous system (CNS) that tightly regulates the movement of substances between the blood and the neural tissue. Recently, the generation and analysis of different genetic mouse models has allowed for greater understanding of BBB development, how the barrier is regulated during health and its response to disease. Here we discuss: 1) Genetic mouse models that have been used to study the BBB, 2) Available mouse genetic tools that can aid in the study of the BBB, and 3) Potential tools that if generated could greatly aid in our understanding of the BBB.
    Full-text · Article · Jan 2013 · Fluids and Barriers of the CNS
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