Notch-1 signalling is activated in brain arteriovenous malformations in humans

doi:10.1093/brain/awp246 Brain 2009: 132; 3231–3241 | 3231 BRAIN A JOURNAL OF NEUROLOGY Notch-1 signalling is activated in brain arteriovenous malformations in humans Qichuan ZhuGe,1,* Ming Zhong,1,* WeiMing Zheng,1 Guo-Yuan Yang,2,3 XiaoOu Mao,4 Lin Xie,4 Gourong Chen,5 Yongmei Chen,2 Michael T. Lawton,3 William L. Young,2,3,6 David A. Greenberg4 and Kunlin Jin1,4 1 Department of Neurosurgery, First Affiliated Hospital, Wenzhou Medical College, Wenzhou, China 2 Department of Anesthesia and Perioperative Care, Center for Cerebrovascular Research, University of California, San Francisco, CA 94121, USA 3 Department of Neurological Surgery, University of California, San Francisco, CA 94121, USA 4 Buck Institute for Age Research, 8001 Redwood Blvd., Novato, CA 94945, USA 5 Department of Pathology, First Affiliated Hospital, Wenzhou Medical College, Wenzhou, China 6 Department of Neurology, University of California, San Francisco, CA 94121, USA Correspondence to: Kunlin Jin, MD, PhD, Buck Institute for Age Research, 8001 Redwood Blvd., Novato, CA 94945, USA E-mail: *These authors contributed equally to this work. 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 Notch1 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. Keywords: Notch-1; AVM; human; brain; signalling; angiogenesis Abbreviations: AVM = arteriovenous malformations; Dll4 = anti-Delta-like-4; NICD = intracellular domain of Notch; TGF = tumour growth factor Introduction Brain arteriovenous malformations (AVMs) are abnormal vascular structures consisting of tortuous arteries and dilated veins, which arise from developmental failure of the intervening capillary beds, and are thought in most cases to be congenital. AVMs are distinct from other vascular malformations of the brain, including venous angiomas and cavernous haemangiomas. AVMs may be asymptomatic, or may cause intracerebral haemorrhage, seizures, headache or focal neurological deficits. The molecular mechanisms Received January 15, 2009. Revised July 28, 2009. Accepted August 21, 2009. Advance Access publication October 7, 2009 ß The Author (2009). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: 3232 | Brain 2009: 132; 3231–3241 Q. ZhuGe et al. that underlie the formation and growth of brain AVMs are poorly understood. Notch signalling, a fundamental pathway controlling cell fate acquisition in development (Artavanis-Tsakonas et al., 1999), plays a critical role during vascular development in zebrafish, mice and humans (Gridley, 2007). Mutations in Notch-3 or Jagged-1, a Notch ligand, lead to human cardiovascular disease: cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy, and Alagille syndrome (paucity of intrahepatic bile ducts with cholestasis, cardiac disease, skeletal abnormalities, ocular abnormalities and characteristic facies), respectively (Joutel et al., 1996). A role for Notch signalling in the development of vascular malformations has been suggested based on abnormalities that result from Notch pathway mutations (Weinmaster and Kopan, 2006; Gridley, 2007). For example, Notch signalling-deficient zebrafish embryos lose expression of markers such as Ephrin B2 from arteries, where Eph B4, an Ephrin B2 receptor normally associated with veins, is ectopically expressed (Lawson et al., 2001). Changes in the arteriovenous gene expression profile of these animals are accompanied by arteriovenous shunts, a hallmark of AVMs, between the dorsal aorta and posterior cardinal vein (Lawson et al., 2001). Similar findings are observed in Notch-1–/– mouse embryos (Krebs et al., 2004). Although the survival of Notch-4-deficient mice shows that Notch-4 is dispensable for vascular development (Krebs et al., 2000), expression of an activated form of Notch-4 within the endothelium disrupts normal vascular development (Uyttendaele et al., 2001; Carlson et al., 2005). Interestingly, the inducible expression of an activated Notch-4 transgene in adult mice causes vascular malformations in an apparently tissue-specific fashion, along with vessel arterialization, ectopic venous expression of Ephrin B2 and increased numbers of vascular smooth muscle cells (Carlson et al., 2005). Mice with constitutively active Notch-4 in endothelial cells develop cerebral arteriovenous shunting and vessel enlargement by 3 weeks of age (Murphy et al., 2008). Surprisingly, these malformations are reversible if Notch-4 transgene expression is repressed (Carlson et al., 2005), suggesting that the involvement of Notch signalling in the development of dysplastic vasculature can extend to the post-natal period. Although Notch signalling plays a critical role in arteriovenous cell fate determination during vascular development and is implicated in vascular malformations, its function in normal adult vascular physiology and in the pathogenesis of AVMs in humans has not been established. In this study, we found that Notch-1 signalling was activated in smooth muscle and endothelial cells of human brain AVMs. The Notch-1 ligands Jagged-1 and Delta-like 4, and the downstream Notch-1 target Hes-1, also showed increased abundance and activation in brain AVMs. Moreover, angiogenesis was increased in adult rats given a Notch-1 activator. Our findings suggest that activation of Notch-1 signalling is a phenotypic feature of brain AVMs, and that activation of Notch-1 in normal vasculature induces a pro-angiogenic state, which may contribute to the development of vascular malformations. Material and methods Human brain specimens Thirteen brain specimens from patients with brain AVMs were obtained from the University of California, San Francisco Brain AVM Study Project and the First Affiliated Hospital, Wenzhou Medical College by surgical (...truncated)