Blood vessels form de novo (vasculogenesis) or upon sprouting of capillaries from pre-existing vessels (angiogenesis). Using high resolution imaging of zebrafish vascular development we discovered a third mode of blood vessel formation whereby the first embryonic artery and vein, two unconnected blood vessels, arise from a common precursor vessel. The first embryonic vein formed by selective sprouting of progenitor cells from the precursor vessel, followed by vessel segregation. These processes were regulated by the ligand EphrinB2 and its receptor EphB4, which are expressed in arterial-fated and venous-fated progenitors, respectively, and interact to orient the direction of progenitor migration. Thus, directional control of progenitor migration drives arterial/venous segregation and generation of separate parallel vessels from a single precursor vessel, a process essential for vascular development.

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.angiogenesis ͉ cell signaling ͉ endothelial cell ͉ stroke ͉ cerebrovascular

Background & Aims: Mechanical forces contribute to portal hypertension (PHTN) and fibrogenesis. We investigated the mechanisms by which forces are transduced by liver sinusoidal endothelial cells (LSECs) into pressure and matrix changes. Methods: We isolated primary LSECs from mice and induced mechanical stretch with a Flexcell device, to recapitulate the pulsatile forces induced by congestion, and performed microarray and RNA-sequencing analyses to identify gene expression patterns associated with stretch. We also performed studies with C57BL/6 mice (controls), mice with deletion of neutrophil elastase (NE-/-) or PAD4 (Pad4-/-) (enzymes that formation of neutrophil extracellular traps [NETs]), and mice with LSEC-specific deletion of Notch1 (Notch1 iΔEC). We performed partial ligation of the suprahepatic inferior vena cava (pIVCL) to simulate congestive hepatopathy-induced portal hypertension in mice; some mice were given subcutaneous injections of sivelestat or underwent bile-duct ligation. Portal pressure was measured using a digital blood pressure analyzer and we performed intravital imaging of livers of mice.

β1 integrin (encoded by Itgb1) is established as a regulator of angiogenesis based upon the phenotypes of complete knockouts of β1 heterodimer partners or ligands and upon antibody inhibition studies in mice. Its direct function in endothelial cells (ECs) in vivo has not been determined because Itgb1-/- embryos die before vascular development. Excision of Itgb1 from ECs and a subset of hematopoietic cells, using Tie2-Cre, resulted in abnormal vascular development by embryonic day(e) 8.5 and lethality by e10.5. Tie1-Cre mediated a more restricted excision of Itgb1 from ECs and hematopoietic cells and resulted in embryonic lethal vascular defects by e11.5. Capillaries of the yolk sacs were disorganized, and the endothelium of major blood vessels and of the heart was frequently discontinuous in mutant embryos. We also found similar vascular morphogenesis defects characterized by EC disorganization in embryonic explants and isolated ECs. Itgb1-null ECs were deficient in adhesion and migration in a ligand-specific fashion, with impaired responses to laminin and collagens, but not to fibronectin. Deletion of Itgb1 reduced EC survival, but did not affect proliferation. Our findings demonstrate thatβ1 integrin is essential for EC adhesion, migration and survival during angiogenesis, and further validate that therapies targeting β1 integrins may effectively impair neovascularization.

A mutual coordination of size between developing arteries and veins is essential for establishing proper connections between these vessels and, ultimately, a functional vasculature; however, the cellular and molecular regulation of this parity is not understood. Here, we demonstrate that the size of the developing dorsal aorta and cardinal vein is reciprocally balanced. Mouse embryos carrying gain-of-function Notch alleles show enlarged aortae and underdeveloped cardinal veins, whereas those with loss-offunction mutations show small aortae and large cardinal veins. Notch does not affect the overall number of endothelial cells but balances the proportion of arterial to venous endothelial cells, thereby modulating the relative sizes of both vessel types. Loss of ephrin B2 or its receptor EphB4 also leads to enlarged aortae and underdeveloped cardinal veins; however, endothelial cells with venous identity are mislocalized in the aorta, suggesting that ephrin B2/EphB4 signaling functions distinctly from Notch by sorting arterial and venous endothelial cells into their respective vessels. Our findings provide mechanistic insight into the processes underlying artery and vein size equilibration during angiogenesis.