Key Words: mouse models of cardiovascular disease Ⅲ angiogenesis Ⅲ endothelial cells A rteriovenous malformations (AVMs) are the most frequent cause of hemorrhagic stroke in young adults. 1,2 The factors leading to AVM formation are unknown, but a number of inherited diseases leading to vascular malformations have now been identified and the causal mutations mapped. 3 Of these familial disorders, hereditary hemorrhagic telangiectasia (HHT) patients have a strikingly high frequency of AVMs. Because HHT is most frequently associated with mutations in the endoglin or ACVRL1 (activin receptor-like kinase 1) gene, 4,5 it is likely that these genes have important roles in preventing AVMs during normal development. HHT is an autosomal dominant disorder affecting approximately 1 in 10 000 people and is characterized by bleeding from small superficial AVMs (known as telangiectases) in the nose and gastrointestinal tract, as well as larger AVMs that may occur in major organs including lung, brain and liver. 6 At present, there is limited understanding of how deficiencies in endoglin or ACVRL1 lead to disease pathology, although their role in transforming growth factor (TGF)␤ family signaling has been the subject of considerable investigation. 7,8 Endoglin is an auxiliary receptor for members of the TGF␤ family of ligands and is expressed primarily on vascular endothelial cells (ECs). It has no signaling kinase domain itself, but can promote TGF␤ signaling through the ACVRL1 receptor to promote cell proliferation and migration. 9,10 ACVRL1 signals by phosphorylating Smad1/5/8 transcription factors which then translocate to the nucleus to regulate expression of downstream genes. 7 Recently, endoglin and ACVRL1 have been shown to respond to bone morphogenetic protein (BMP)9 and BMP10 ligands of the TGF␤ family to promote endothelial cytostasis, even in the presence of angiogenic growth factors. [11][12][13] How these different in vitro responses link to the normal role of these genes in averting AVM formation in development is not yet clear. To address this issue we, and others, have previously derived mouse models with endoglin mutations and found that Endoglin null embryos die halfway through gestation from developmental defects in the cardiovasculature. 14 -16 Mice that are heterozygous for endoglin-null mutations survive and model some features of HHT, but AVMs occur at an extremely low frequency. 17 With the aim of developing a more reproducible model of AVM formation, and bypassing embryonic lethality of the endoglin null mouse, we have taken a conditional knockout approach combining our recently generated endoglin-floxed mouse model 18 with an endothelial specific Cdh5(PAC)-Cre ERT2 transgenic line. 19 As Cre ERT2 is inactive until exposed to tamoxifen, this combination of alleles allows endoglin depletion in ECs at different stages of development and adult life, permitting a high degree of manipulability with which to investigate the role of endoglin in vivo. 2fl/2fl and included both tamoxifen-treated and tamoxi...

Rare inherited cardiovascular diseases are frequently caused by mutations in genes that are essential for the formation and/or function of the cardiovasculature. Hereditary Haemorrhagic Telangiectasia is a familial disease of this type. The majority of patients carry mutations in either Endoglin (ENG) or ACVRL1 (also known as ALK1) genes, and the disease is characterized by arteriovenous malformations and persistent haemorrhage. ENG and ACVRL1 encode receptors for the TGFβ superfamily of ligands, that are essential for angiogenesis in early development but their roles are not fully understood. Our goal was to examine the role of Acvrl1 in vascular endothelial cells during vascular development and to determine whether loss of endothelial Acvrl1 leads to arteriovenous malformations. Acvrl1 was depleted in endothelial cells either in early postnatal life or in adult mice. Using the neonatal retinal plexus to examine angiogenesis, we observed that loss of endothelial Acvrl1 led to venous enlargement, vascular hyperbranching and arteriovenous malformations. These phenotypes were associated with loss of arterial Jag1 expression, decreased pSmad1/5/8 activity and increased endothelial cell proliferation. We found that Endoglin was markedly down-regulated in Acvrl1-depleted ECs showing endoglin expression to be downstream of Acvrl1 signalling in vivo. Endothelial-specific depletion of Acvrl1 in pups also led to pulmonary haemorrhage, but in adult mice resulted in caecal haemorrhage and fatal anaemia. We conclude that during development, endothelial Acvrl1 plays an essential role to regulate endothelial cell proliferation and arterial identity during angiogenesis, whilst in adult life endothelial Acvrl1 is required to maintain vascular integrity.

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