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: 9.81). 09/2008; 105(31):10901-6. DOI: 10.1073/pnas.0802743105
Source: PubMed

ABSTRACT 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.
    Development 09/2014; 141(19). DOI:10.1242/dev.108951 · 6.27 Impact Factor
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    • "Similarly, vascular malformations in Notch4-overexpressing transgenic mice are reversible if expression of an activated Notch4 transgene is repressed (Carlson et al., 2005). 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., 2005; Murphy et al., 2008) and cerebral (Murphy et al., 2008) vascular malformations. These findings suggest that brain vascular malformations might be treatable with Notch inhibitors. "
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    ABSTRACT: A role for the Notch signalling pathway in the formation of arteriovenous malformations during development has been suggested. However, whether Notch signalling is involved in brain arteriovenous malformations in humans remains unclear. Here, we performed immunohistochemistry on surgically resected brain arteriovenous malformations and found that, compared with control brain vascular tissue, Notch-1 signalling was activated in smooth muscle and endothelial cells of the lesional tissue. Western blotting showed an activated form of Notch-1 in brain arteriovenous malformations, irrespective of clinical presentation and with or without preoperative embolization, but not in normal cerebral vessels from controls. In addition, the Notch-1 ligands Jagged-1 and Delta-like-4 and the downstream Notch-1 target Hes-1 were increased in abundance and activated in human brain arteriovenous malformations. Finally, increased angiogenesis was found in adult rats treated with a Notch-1 activator. Our findings suggest that activation of Notch-1 signalling is a phenotypic feature of brain arteriovenous malformations, and that activation of Notch-1 in normal vasculature induces a pro-angiogenic state, which may contribute to the development of vascular malformations.
    Brain 12/2009; 132(12):3231-3241. DOI:10.1093/brain/awp246 · 10.23 Impact Factor
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    • "Recently, genetic mutant mice were used in research to generate BAVM that mimics human BAVM, including mice with endoglin (ENG) (Satomi et al, 2003; Xu et al, 2004), activin-like kinase 1 (ALK1) (Park et al, 2008), or Notch (int3) (Carlson et al, 2005; Krebs et al, 2004; Murphy et al, 2008) haploinsufficiency. With brain VH in combination with genetic manipulation, a BAVM model that mimics human BAVM will likely be generated. "
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    ABSTRACT: Cerebral venous hypertension (VH) and angiogenesis are implicated in the pathogenesis of brain arteriovenous malformation and dural arteriovenous fistulae. We studied the association of VH and angiogenesis using a mouse brain VH model. Sixty mice underwent external jugular vein and common carotid artery (CCA) anastomosis (VH model), CCA ligation, or sham dissection (n=20). Hypoxia-inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF) and stromal-cell-derived factor-1alpha (SDF-1alpha) expression, and matrix metalloproteinase (MMP) activity were analyzed. We found VH animals had higher (P<0.05) sagittal sinus pressure (8+/-1 mm Hg) than control groups (1+/-1 mm Hg). Surface cerebral blood flow and mean arterial pressure did not change. Hypoxia-inducible factor-1alpha, VEGF, and SDF-1alpha expression increased (P<0.05). Neutrophils and MMP-9 activity increased 10-fold 1 day after surgery, gradually decreased afterward, and returned to baseline 2 weeks after surgery. Macrophages began to increase 3 days after surgery (P<0.05), which coincided with the changes in SDF-1alpha expression. Capillary density in the parasagittal cortex increased 17% compared with the controls. Our findings suggest that mild nonischemic VH results in a pro-angiogenic stage in the brain by upregulating HIF-1 and its downstream targets, VEGF and SDF-1alpha, increasing leukocyte infiltration and MMP-9 activity.
    Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 06/2009; 29(8):1482-90. DOI:10.1038/jcbfm.2009.67 · 5.34 Impact Factor
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